Clinical Immunology_ Principles and Practice ( PDFDrive )

86









Glucocorticoids



Anthony J. Frew












Genomic Actions of Glucocorticoids
Glucocorticoids (GCs) are among the most commonly prescribed
1
drugs and are used for a wide range of medical conditions. More The antiinflammatory and immunomodulatory effects of GCs are
than 60 years after their introduction into clinical practice, they mainly mediated by genomic mechanisms (Figs. 86.1 and 86.2).
remain the most important and most frequently employed class Their lipophilic structure and low molecular mass allow GCs
of antiinflammatory drugs, and their use continues to increase, to pass easily through the cell membrane and bind to cytosolic
with about 10 million new prescriptions issued for oral GCs each glucocorticoid receptors (cGCRs). Ultimately this either induces
2
year in the United States. Community survey data suggest that the synthesis of regulatory proteins (“transactivation”) or inhibits
5
0.5% of the general population and 1.75% of women aged over their synthesis (“transrepression”). About 10–100 genes per
3,4
55 years are taking oral GCs. About 56–68% of patients with cell are directly regulated by GCs, but many other genes are
rheumatoid arthritis (RA) are treated more or less continuously regulated indirectly through interaction with transcription factors
1
6
with GCs. Although GCs are relatively inexpensive, total market and coactivators (see below). It has been estimated that GCs
volume is valued at ≈US$10 billion per year. 2 influence the transcription of approximately 1% of the entire
GCs are widely used because they are the most effective (and genome.
cost-effective) antiinflammatory and immunomodulatory drugs
available. However, GCs can cause serious adverse effects, Structure of the Cytosolic Glucocorticoid Receptor
especially when used incorrectly. The nonactivated cGCR (cGCRα) is a 94-kilodalton (kDa) protein
held in the cytoplasm as a multiprotein complex, consisting of
MECHANISMS OF ACTION several heat shock proteins (hsps), including hsp90, hsp70, hsp56,
and hsp40 (chaperones) (Fig. 86.3). The cGCR interacts with
The way that GCs are used in different clinical conditions is immunophilins, p23, and several kinases of the mitogen-activated
essentially empirical, as there is only limited evidence to support protein kinase (MAPK) signaling system, including Src, which
1,7
1
current practice in specific clinical settings. In general, GC dosages also act as molecular (co)chaperones (see Figs. 86.1 and 86.3).
are increased in parallel with the clinical activity and severity of The general function of molecular (co)chaperones is to bind
the disease under treatment. The rationale for this approach is and stabilize proteins at intermediate stages of folding, assembly,
that higher dosages increase GC receptor saturation in a dose- translocation, and degradation. With regard to cGCR, they also
dependent manner (Table 86.1), which intensifies the therapeuti- regulate cellular signaling, which includes (i) stabilizing a high-
cally relevant, genomic actions of GCs. Moreover, with increasing affinity conformational state of cGCR; (ii) opening the GC-
dosages, additional and qualitatively different, nonspecific, binding cleft to be accessed by GCs; and (iii) stabilizing the
nongenomic actions of GCs come into play (see Table 86.1). binding of the GCR to the promoter. 1
The first step in assembling the multiprotein cytosolic complex
is adenosine triphosphate (ATP)–dependent and hsp40(YDJ-1)–
KEY CONCEPTS dependent formation of a cGCR–hsp70 complex that primes
Characteristics Applying to Genomic Actions the receptor for subsequent ATP-dependent activation by hsp90,
8
Hop, and p23. The GCR consists of different domains with
• Physiologically relevant distinct functions: an N-terminal domain; a DNA-binding domain
• Therapeutically effective at all dosages, even very small ones (low
dose therapy). (DBD); and a ligand-binding domain (LBD) (see Fig. 86.3). The
• Slow; significant changes in the regulator protein concentrations are N-terminal domain serves transactivation functions, especially
not seen within less than 30 minutes because of the time required within the “τ1” region. A zinc finger motif, a sequence common
for cytosolic glucocorticoid receptor (cGCR) activation/translocation, to many DNA-interacting proteins, is found twice within the
transcription, and translation effects. DBD. The LBD consists of 12 α helices, several of which help
• The glucocorticoid (GC)–induced synthesis of regulator proteins can form a hydrophobic ligand-binding pocket. The cGCR contains
7
be prevented by inhibitors of transcription (e.g., actinomycin D) or another major transactivation region (“τ2”) that can interact
translation (e.g., cycloheximide).
with the above-mentioned cofactors (see Fig. 86.3). Following
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1166 ParT TEN Prevention and Therapy of Immunological Diseases



TABLE 86.1 Current Knowledge on the relationship Between Clinical Glucocorticoid (GC)
Dosing and Cellular GC actions
Terminology Cytosolic Glucocorticoid
(Mg Prednisone Genomic actions Unspecific receptor (cGCr)–Mediated
Equivalent Per Day) Clinical application (receptor Saturation) Nongenomic actions Nongenomic actions
Low dose (≤7.5) Maintenance therapy for many + (<50%) − ?
rheumatic diseases
Medium dose Initially given in primary ++ (>50% – <100%) (+) (+)
(>7.5 – ≤30) chronic rheumatic diseases
High dose (>30 – ≤100) Initially given in subacute ++(+) (almost 100%) + +
rheumatic diseases
Very high dose Initially given in acute and/or +++ ([almost] 100%) ++ +(+?)
(>100 mg) potentially life-threatening
exacerbations of rheumatic
diseases
Pulse therapy (≥250 mg Particularly severe and/or +++ (100%) +++ +(++?)
for 1 or a few days) potentially life-threatening
forms of rheumatic diseases
From Buttgereit F, Straub RH, Wehling M, Burmester GR. Glucocorticoids in the treatment of rheumatic diseases. An update on mechanisms of action. Arthritis Rheum 2004;50:
3408–17, with permission.)

Genomic mechanisms Glucocorticoid Nongenomic mechanisms





mGCR
Plasma membrane
Cytosol

HSP HSP 3 4
cGCR
mGCR-mediated nonspecific
Src nongenomic m. nongenomic m.
Src 2
HSP
cGCR-mediated
nongenomic m.
Nucleus
HSP
1
cGCR-mediated Antiinflammatory, immunomodulatory
genomic m. and other (including unwanted
cGCR adverse) effects
FIG 86.1 Mechanisms of the Cellular Action of Glucocorticoids (GCs). As lipophilic substances,
GCs pass very easily through the cell membrane into the cell, where they bind to ubiquitously
expressed cytosolic glucocorticoid receptors (cGCRs). This is followed by either the classic
cGCR-mediated genomic effects (1) or by cGCR-mediated nongenomic effects (2). Moreover,
the GC is very likely to interact with cell membranes, either specifically via membrane-bound
glucocorticoid receptors (mGCRs) (3) or via nonspecific interactions with cell membranes (4).
(From Buttgereit F, Straub RH, Wehling M, Burmester GR. Glucocorticoids in the treatment of
rheumatic diseases. An update on mechanisms of action. Arthritis Rheum 2004;50:3408–17.)

9
GC/cGCR binding, the hsp90 molecules and other molecular of FKBP52 and dynein to the GCR. Depending on the target
chaperones are rapidly shed. This allows translocation into the gene, transcription is then either activated (transactivation via
cell nucleus, where the GC/cGCR complex binds as a homodimer positive GRE) or inhibited (transrepression via negative GRE).
to consensus palindromic DNA sites (glucocorticoid-responsive
elements [GREs]). 5 Interactions With Transcription Factors
As well as the interactions of GC/cGCR complexes with GRE,
Translocation Into the Nucleus a further important genomic mechanism of GC action is the
Nuclear translocation of the GC/cGCR complex occurs within interaction of activated cGCR monomers with transcription
20 minutes. This may be caused by hormone-directed recruitment factors. Accordingly, although the GC/cGCR complex does

CHaPTEr 86 Glucocorticoids 1167


Ligand-binding domain

Transcription
cGCR p23
A GRE COOH

Transcription τ hinge HSP90
cGCR NH 2 2 τ 2 HSP90 HSP70
B nGRE C C C C HSP56
Zn Zn SRC
C C C C and other
chaperones
cGCR
P65 P50 N-terminal DNA-binding
Transcription
No binding domain
C κB site FIG 86.3 Structure of the Cytosolic Glucocorticoid Receptor
(cGCR). The nonactivated (unligated) cGCR is a 94-kilodalton
Co-activator cGCR (kDa) protein retained in the cytoplasm as a multiprotein complex
consisting of several heat shock proteins (hsps), including hsp90,
Co-activator hsp70, hsp56, and hsp40 (chaperones). Furthermore, the cGCR
interacts with immunophilins, p23, and several kinases of the
AP-1 mitogen-activated protein kinase (MAPK) signaling system,
No binding Transcription including Src, which also act as molecular (co)chaperones. An
D AP1 site important function of molecular (co)chaperones is to stabilize a
FIG 86.2 Genomic Mechanisms of Glucocorticoids (GCs). specific conformational state of the GC which binds ligand with
This figure illustrates the different mechanisms by which the high affinity (see text). The receptor protein itself consists of
activated glucocorticoid receptor (GCR) complex leads to the different domains: an N-terminal, a DNA-binding domain (DBD),
induction or to the inhibition of transcription and finally translation/ and a ligand-binding domain (LBD). The N-terminal harbors
synthesis of specific regulator proteins. Details are given in the transactivation functions, especially within the so-called τ1 region.
text. (From Buttgereit F, Straub RH, Wehling M, Burmester GR. Another major transactivation region is τ2, which can interact
Glucocorticoids in the treatment of rheumatic diseases. An update with the above-mentioned cofactors. (From Buttgereit F, Straub
on mechanisms of action. Arthritis Rheum 2004;50:3408–17.) RH, Wehling M, Burmester GR. Glucocorticoids in the treatment
of rheumatic diseases. An update on mechanisms of action.
Arthritis Rheum 2004;50:3408–17.)



not inhibit their synthesis, it modulates the activity of AP-1 is thought to function as a negative inhibitor of cGCRα, and it
(activator protein-1), nuclear factor (NF)-κB (nuclear factor- may play a role in the clinical phenomenon of GC resistance.
κB) and NFAT (nuclear factor for activated T cells). This leads cGCRβ lacks the GC-binding domain that is needed for activation,
to inhibition of nuclear translocation and/or function of these and as it does not undergo ligand-dependent downregulation,
transcription factors and, thus, to inhibition of expression of it has a longer half-life than the active form (cGCRα). It is thought
many immunoregulatory and inflammatory cytokines. Possible that the likely mechanism of the dominant negative activity of
5
mechanisms include : cGCRβ is through the formation of inactive heterodimers with
• Synthesis of IκB (a specific inhibitor of NF-κB) induced cGCRα. 10
through GC/cGCR complex–GRE interaction (see Fig. 86.2A)
• Protein–protein interaction of the GC/cGCR complex with Posttranscriptional and Posttranslational Mechanisms
transcription factors through binding to their subunits (see GCs also act through posttranscriptional and posttranslational
Fig. 86.2C), which prevents their DNA binding mechanisms, including reduction of the half-life of cytokine
• Competition for nuclear coactivators between the GC/cGCR messenger RNA (mRNA) and downregulation of the GCR,
complex and transcription factors (see Fig. 86.2D) via reduced mRNA levels and reduced stability of the GCR
Inhibition of transcription factor function and the resultant protein.
inhibition of protein expression are referred to as transrepression.
Numerous genes are regulated by this mechanism. Many adverse
effects of GCs are caused by transactivation (induced synthesis
of regulator proteins), whereas most antiinflammatory effects KEY CONCEPTS
are mediated by transrepression (inhibited synthesis of regula- Glucocorticoid (GC) Effects on Immune Cells
tor proteins). This differential molecular regulation underlies
current drug-discovery programs aimed at developing dissociated • Inhibit leukocyte traffic and access of leukocytes to the site of
cGCR-ligands. 2 inflammation
• Interfere with functions of leukocytes, fibroblasts, and endothelial
The cGCRβ Isoform cells
• Suppress the production and actions of humoral factors involved in
The cGCRβ isoform is an alternative splicing variant of cGCRα the inflammatory process
that does not bind GCs or activate gene expression. This isoform

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For example, pulsed intravenous (IV) methylprednisolone is
Glucocorticoid Receptor Resistance effective in the treatment of systemic lupus erythematosus
Several mechanisms may explain the clinical finding of GCR (SLE) and causes rapid immunosuppression in patients with
resistance, including alterations in the number, binding affinity, or organ- and/or life-threatening manifestations of SLE. However,
phosphorylation status of GCR. Other possible explanations are the standard regime (1 g/day for 3 consecutive days) is associated
polymorphic changes and/or overexpression of (co)chaperones, with significant risks of infection, and lower doses may be just
increased expression of inflammatory transcription factors, as effective. 15
overexpression of the GCRβ isoform, the multidrug resistance High-dose GCs are also often used in immune thrombo-
pump, and altered membrane-bound (mGCR) expression. 11 cytopenia associated with SLE, although comparative studies
16
are lacking. It has been calculated that in such situations,
Nongenomic Actions of Glucocorticoids the concentrations achieved in vivo are high enough (around
Some regulatory effects of GCs occur within seconds or minutes. 10–5 mol/L) to cause immediate nonspecific nongenomic effects
1
These are too rapid to result from genomic actions and must, on immune cells. Intraarticular injections also bring high
therefore, be caused by nongenomic mechanisms of action. concentrations of GCs into contact with inflammatory cells,
Three different rapid nongenomic actions of GCs have been although it is difficult to assess locally achieved concentrations
described. 1,12,13 because crystal suspensions are most often used.
cGCR-Mediated Nongenomic Actions Specific Nongenomic Actions
Dexamethasone can rapidly inhibit epidermal growth factor- GCs can also induce specific nongenomic actions, mediated
stimulated cPLA2 (cytosolic PLA2) activation with subsequent through membrane-bound glucocorticoid receptors (mGCRs).
12
arachidonic acid release. This effect is thought to be mediated by The existence and function of membrane-bound receptors
occupation of cGCR, rather than changes in gene transcription, have been demonstrated for various steroids (including min-
as the observed effect is RU486 sensitive (i.e., GCR dependent) eralocorticoids, gonadal hormones, vitamin D, and thyroid
but actinomycin insensitive (i.e., transcription independent). hormones). 1,17 Small numbers of mGCRs can be demonstrated
Chaperones or (co)chaperones of the multiprotein complex may by immunofluorescence on human peripheral blood mononuclear
act as signaling components to mediate this effect. Following cells (PBMCs; monocytes and B cells) obtained from healthy
17
GC binding, the cGCR is released from this complex to mediate controls. The monoclonal antibody (mAb) used to detect mGCRs
classic genomic actions. However, there is also a rapid release also recognizes cGCRs, suggesting that mGCRs are probably
of Src and other (co)chaperones of the multiprotein complex, variants of cGCRs produced by differential splicing or promoter
which may cause rapid inhibition of arachidonic acid release. switching. Immunostimulation with lipopolysaccharide increases
+
Similarly, dexamethasone has been reported to have cardiovas- the percentage of mGCR monocytes, and this can be prevented
cular protective effects, which are neither genomic (because by inhibiting the secretory pathway with brefeldin A. This suggests
they occurred too quickly and were actinomycin insensitive) that mGCRs are actively upregulated and transported through the
nor nonspecific–nongenomic effects (because they occurred at cell after immunostimulation. These in vitro findings are consistent
+
13
a very low concentration [100 nM]). These may involve binding with observations that the frequency of mGCR monocytes is
of GCs to cGCRs, leading to nontranscriptional activation of increased in patients with rheumatic disorders and is positively
17
phosphatidylinositol 3-kinase, protein kinase Akt, and endothelial correlated with disease activity in RA. It remains unclear
NO synthase. whether mGCRs are involved in pathogenesis. Alternatively,
and perhaps more likely, they may cause negative feedback
Nonspecific Nongenomic Actions regulation.
GCs are sometimes administered at very high doses. Systemic
daily dosages >100 mg prednisone equivalent are regarded as
“very high dose.” “Pulse therapy” is the daily administration of GLUCOCORTICOID EFFECTS ON IMMUNE CELLS
≥250 mg prednisone equivalent for 1 day or a few consecutive
14
days (see Table 86.1). At a daily dose of 100 mg prednisone Through the above mechanisms, GCs mediate a wide range of
equivalent, almost all cGCRs are completely saturated, which antiinflammatory and immunomodulatory effects, with virtually
means that specificity (i.e., the exclusivity of receptor-mediated all primary and secondary immune cells affected to some extent
effects) is lost. Nonspecific nongenomic actions occur in the (Table 86.2). 18
form of physicochemical interactions with biological membranes,
1
which probably contribute to the therapeutic effect. Intercala-
tion of GC molecules into cell membranes is thought to alter
cell function by influencing cation transport and increasing
mitochondrial proton leak. The resulting inhibition of calcium KEY CONCEPTS
and sodium cycling across the plasma membrane of immune
cells is thought to contribute to rapid immunosuppression and Definition of Conventional Terms for
to reduced inflammation. Glucocorticoid (GC) Dosages
Such high GC doses are only used in a few clinical specialties, Low dose ≤7.5 mg prednisone equivalent per day
and this practice has been criticized by some endocrinologists Medium dose >7.5 mg, but ≤30 mg prednisone equivalent per day
and pharmacologists. Unfortunately, there are no randomized High dose >30 mg, but ≤100 mg prednisone equivalent per day
controlled trials of high-dose GC therapy, but it is often used with Very high dose > 100 mg prednisone equivalent per day
clinical success in acute exacerbations of life-threatening diseases Pulse therapy ≥ 250 mg prednisone equivalent per day for 1 or a
few days
and various clinical conditions resistant to other therapies.

CHaPTEr 86 Glucocorticoids 1169



TABLE 86.2 Important Effects of Proinflammatory cytokines, such as interleukin-1β (IL-1β)
Glucocorticoids (GCs) on Primary and and tumor necrosis factor-α (TNF-α), stimulate 11βHSD1 and
Secondary Immune Cells downregulate 11βHSD2 expression. Hence, specific proinflamma-
tory cytokines can modulate local intracellular GC metabolism,
Monocytes/Macrophages which may affect their own proinflammatory effects. More
↓ Number of circulating cells (↓ myelopoiesis, ↓ release) recently, substantial GC metabolism has been shown in joints,
↓ Expression of major histocompatibility complex (MHC) class II since TNF-α and IL-1β induce 11βHSD1 activity in primary
molecules and Fc receptors
↓ Synthesis of proinflammatory cytokines (e.g., interleukin [IL]-2, IL-6, cultures of synovial fibroblasts isolated from synovial tissue biopsy
20
tumor necrosis factor [TNF]-α) and prostaglandins specimens from patients with RA.
In a rodent model of immune-mediated arthritis, targeted
T Cells disruption of GC signaling in osteoblasts attenuates joint inflam-
21
↓ Number of circulating cells (redistribution effects) mation and cartilage destruction. These results suggest that
↓ Production and action of IL-2 (most important) under the control of endogenous GCs, osteoblasts modulate
immune-mediated inflammatory responses and, as a consequence,
Granulocytes inflammation-induced cartilage damage and bone integrity. These
↓ Number of eosinophil and basophil granulocytes findings are supported by evidence suggesting that the effects
↑ Number of circulating neutrophils
of GCs follow a dose–response curve, with permissive or even
Endothelial Cells stimulatory effects at physiological concentrations and suppressive
20
↓ Vessel permeability effects at pharmacological concentrations.
↓ Expression of adhesion molecules
↓ Production of IL-1 and prostaglandins THERAPEUTIC USE

Fibroblasts A wide range of GC molecules are available for clinical use: The
↓ Proliferation common basic structure has been modified to improve their
↓ Production of fibronectin and prostaglandins usefulness in various clinical applications (Fig. 86.4). Despite
their widespread use, the designation of GC treatment regimens
(From Buttgereit F, Saag K, Cutolo M, et al. The molecular basis for the
effectiveness, toxicity, and resistance to glucocorticoids: focus on the treatment of is often imprecise. Recommendations for a standardized nomen-
rheumatoid arthritis. Scand J Rheumatol 2005; 34: 14–21, with permission.) clature for GC therapy are summarized below. 14
Terminology
Although the term steroid is widely used to describe this class of
The Role of Endogenous Glucocorticoids in drugs, it is too broad, as it simply describes chemical compounds
characterized by a common multiple-ring structure (including
Inflammatory Arthritis cholesterol, vitamin D, and sex hormones). Similarly, the terms
Exogenous (therapeutic) and endogenous (physiological) GCs corticosteroid or corticoid are not sufficiently precise, as the adrenal
differ in several respects. The most important differences are in cortex synthesizes not only GCs but also mineralocorticoids
their relative mineralocorticoid and GC (antiinflammatory) and androgens. For these reasons, the terms glucocorticoid or
activities. Exogenous and endogenous GCs also differ in their glucocorticosteroid are preferred; however, glucocorticoid is the
pharmacological characteristics, such as plasma kinetics, metabo- more widely used term.
lism, biological half-life, lipophilicity, drug–receptor interactions, When describing the use of GCs, it is necessary to define the
and nongenomic potencies. 19 drug, the dosage, the route of administration, and the timing,
The actions of exogenous GCs as described above are well frequency, and duration of treatment.
established. In contrast, we know relatively little about the role Different GC drugs have different potencies, and they differ
of endogenous GCs in arthritis. Although GC actions on target in their ability to produce the distinct therapeutic effects discussed
tissues are thought to be determined by GC plasma concentrations above. Drug potencies are usually described by the equivalent
and the tissue-specific density of GCRs, it seems that endogenous dosages (relative potencies) to produce classic genomic effects
GCs are subject to extensive prereceptor metabolism. Within target (Table 86.3). These values have been used for decades, although
cells or tissues, GC action depends not only on plasma hormone the experimental and clinical evidence for their precise relative
levels, receptor expression, and receptor-effector coupling but potency is weak. In practice, relative potencies are useful as a
also on local GC metabolism. Specifically, 11β-hydroxysteroid general therapeutic guideline in daily clinical practice, as long
dehydrogenases appear to govern access of GCs to their cognate as they are not used dogmatically. It has, therefore, been suggested
receptors by changing the balance between active and inactive that we should continue to use relative potencies until more
19
GCs within the cell (reviewed in ). Thus the predominant exact data are available and that doses of different GCs should
reductase activity of 11β-hydroxysteroid dehydrogenase type be expressed in terms of “prednisone equivalent” (i.e., doses of
1 (11βHSD1) catalyzes the formation of bioactive cortisol different GCs are expressed as equivalent to milligrams of
from inactive cortisone (in humans) and corticosterone from prednisone [or prednisolone, as the potency of prednisone is
11-dehydrocorticosterone (in rodents). This nicotinamide adenine equal to that of prednisolone]).
dinucleotide phosphate (NADPH)–dependent enzyme is present However, data indicate that the concept of equivalent dosages
in many tissues and usually increases the intracellular availability is only valid for doses less than 100 mg prednisone equivalent
of active GCs. In contrast, 11βHSD2 only possesses dehydrogenase because nongenomic effects come into play at higher doses. The
activity: It inactivates active GCs and therefore decreases the reason this is important is that the relative potencies of different
intracellular concentration of bioactive GCs. GCs to produce these nongenomic effects are completely different

1170 ParT TEN Prevention and Therapy of Immunological Diseases


21
CH OH CH OH CH 2 OH
2
2
20
18 CO CO CO
12 CH 3 CH 3 CH 3
O OH O OH HO OH
19 11 13 17 16 CH 3
CH 3 9 C D CH 3 CH 3
2 14 15
A B 8 F
3
O 5 7 O O
4 6
Cortisone Prednisone Dexamethasone
CH OH CH OH CH OH
2
2
2
CO CO CO
CH 3 CH 3 CH 3
HO OH HO OH HO OH
CH 3 CH 3 CH 3
O O O
CH 3
Hydrocortisone (cortisol) Prednisolone Methylprednisolone
FIG 86.4 Molecular Structures of Cortisone and Commonly Used Glucocorticoids (GCs).
Carbon and ring nomenclature is indicated on cortisone molecule.


TABLE 86.3 Drug Potencies of Selected dependent, both quantitatively and qualitatively. The standardized
Glucocorticoids (GCs) nomenclature for GC dosages and GC treatment regimens, as
discussed below, is now recommended. 14
Cortisol 1 (per definition)
Prednyliden 3.5 Low Dose
Predniso(lo)ne 4.0
Methylprednisolone 5.0 Treatment with doses of up to 7.5 mg prednisone equivalent
Dexamethasone 25 per day is considered low-dose GC therapy because these doses
Betamethasone 25 occupy less than 50% of the receptors. Such courses are frequently
used for maintenance therapy and have relatively few adverse
Drug potencies describe the potency of the respective drug to produce classical
genomic (antiinflammatory) effects relative to cortisol. These potencies provide the effects (e.g., osteoporosis). As there may be relative hypocorti-
basis for calculating equivalent dosages. solism in chronic inflammatory conditions, such as RA and
(Data from Lipworth BJ. Therapeutic implications of non-genomic glucocorticoid activity.
Lancet 2000;356:87–9.) polymyalgia rheumatica, low-dose GCs act, in part, as replacement
therapy for reduced adrenal GC production.
Medium Dose
from their classic genomic effects (see Table 86.3). For example, GC doses of more than 7.5 mg but less than 30 mg prednisone
methylprednisolone is used for pulse therapy of exacerbations equivalent per day are considered medium-dose therapy because
of immunologically mediated disorders. Prednisolone and they lead to a significantly higher receptor engagement between
methylprednisolone have similar genomic potencies, but when 50% and <100%. These doses are effective in modulating disease
used for high-dose therapy, the nonspecific nongenomic effect activity in various rheumatic diseases but may have considerable
of methylprednisolone is more than three times stronger. This and dose-dependent adverse effects if given for longer periods.
may explain the apparent superiority of high-dose methyl-
prednisolone. In contrast, betamethasone has very low nonge- High Dose
nomic potency, which may be why this drug is considered less Treatment with doses of more than 30 mg and up to 100 mg of
effective systemically, even though it has the same genomic prednisone equivalent per day is considered high-dose therapy
potency as that of dexamethasone. In summary, the clinical usage because these doses significantly increase receptor saturation, in
of different GCs is clearly determined by the magnitude of their a dose-dependent manner. At approximately 100 mg prednisone
clinical efficacy, but another important factor in selecting which equivalent per day, receptor saturation is almost complete, and
one to use is their nongenomic potency. it is likely that genomic GC effects are fully exerted (see Table
86.1). High-dose therapy can be successfully given as initial
Glucocorticoid Treatment Regimens: General Aspects treatment for subacute diseases, such as non-life-threatening
For many decades, there has been confusion surrounding the exacerbations or visceral complications of RA or other connective
terms used to describe dosage (very low, low, mild, mild to tissue diseases, but cannot be administered over the long term
moderate, moderate, high, very high, ultrahigh, and mega) and because of the danger of severe adverse effects.
by loose usage of such terms as “low-dose therapy,” “high-dose
therapy,” and “pulse therapy.” A 2002 consensus statement has Very High Dose
clarified this situation, partly to achieve scientific consistency Doses above 100 mg of prednisone equivalent per day are
and partly to recognize that GC actions are strongly dose considered “very high.” At this level, there is virtually 100%

CHaPTEr 86 Glucocorticoids 1171


saturation of cytosolic receptors, so any further increase in dose than 14 days in duration. With longer periods of treatment, the
may affect the pharmacodynamics (e.g., receptor off-loading and dose should be reduced gradually to allow the HPA axis to recover,
reoccupancy), receptor synthesis and expression. At these doses, but the rate of reduction is usually faster than for rheumatological
nongenomic effects may deliver additional therapeutic benefit, conditions.
although it remains unclear whether these effects have any direct
therapeutic relevance. Experimental data suggest that these Glucocorticoids in Rheumatoid Arthritis: an Example
differential effects come increasingly into play above ≈100 mg/ GCs are crucially important in the management of RA and are
day (see Table 86.1). Doses of >100 mg of prednisone equivalent used in various dosages at different disease stages. RA is, therefore,
per day are frequently (and successfully) given as initial treatment a useful example to discuss GC therapy in more detail.
for acute or life-threatening exacerbations of connective tissue
diseases, vasculitis, and RA. These doses cannot be administered Low-Dose Maintenance Therapy
over the long term because of their severe adverse effects. In early RA, GC in doses <10 mg are highly effective for relieving
symptoms in patients with active arthritis. Many patients are
Pulse Therapy functionally dependent on this low-dose therapy and continue
22
Pulse therapy involves administration of ≥250 mg prednisone it over the long term. Improvement has been documented not
equivalent per day (usually intravenously) for a short period only in all clinical parameters, including pain scales, joint scores,
(typically 1–5 days, rarely longer). At such high doses, the morning stiffness, and fatigue, but also in acute inflammatory
nongenomic potencies of GCs come into play. It is likely that markers, such as erythrocyte sedimentation rate (ESR) and
these explain the success of very high doses and pulse therapy C-reactive protein (CRP). After 6 months of therapy, the beneficial
in acute exacerbations of immunologically mediated diseases. effects of GCs seem to diminish, but if therapy is then tapered
The immediate effects of very high doses may be additive to the and stopped, patients often experience an exacerbation of
genomic effects mediated by cGCRs. These additional effects symptoms within a few months.
may make a crucial contribution to the therapeutic effect by The disease-modifying properties of GCs were first described
terminating acute exacerbations. Circumstances where very high in 1995, in a 2-year trial of 7.5 mg prednisolone in patients
doses or pulse therapy can be successful include acute episodes who had RA of short or intermediate disease duration and
or particularly severe forms of rheumatic diseases, such as SLE, were treated with NSAIDs (95%) and disease-modifying
23
vasculitis, polymyositis, and RA (see below). antirheumatic drugs (DMARDs) (71%). Nowadays, GCs are
considered to have disease-modifying properties in early RA,
Alternate-Day Regimens but it is less clear whether they inhibit the progression of erosions
Alternate-day regimens were introduced for long-term oral GC in RA of longer duration. A meta-analysis of 15 studies that
therapy with the aim of minimizing undesirable adverse effects, included 1414 patients concluded that GCs given in addition
such as suppression of the hypothalamo–pituitary–adrenal (HPA) to standard therapy can substantially reduce the rate of erosion
24
axis. Instead of daily dosing, a single dose is administered every progression in RA. Another meta-analysis of 70 randomized
other morning, usually in a dose equivalent to or somewhat placebo- or drug-controlled studies found similar effects of
higher than twice the usual daily dose. The idea behind this DMARDs, GCs, and biologicals on radiographic progression
regimen is to allow the HPA axis to remain active by exposing in RA. 25
the body to exogenous GC on alternate days instead of suppressing
it every day. This strategy only works if the HPA axis is still Glucocorticoid Pulse Therapy
active, and, unfortunately, patients often experience breakthrough GC pulse therapy is used to treat some serious complications
symptoms on the second day of treatment. Alternate-day regimens of RA and to induce remission in active disease, for example,
are used rarely these days except in patients with juvenile idio- when initiating second-line antirheumatic treatment. Pulse
pathic arthritis, in whom alternate-day GC regimens cause less therapy with methylprednisolone 1 g/day, dexamethasone 200 mg/
inhibition of body growth. day, or equivalent, given intravenously for 1–3 days, was shown
to be effective in most studies, demonstrating a beneficial effect
Glucocorticoid Withdrawal Regimens that generally lasted for about 6 weeks, albeit with large variations.
Because of their significant adverse effects, GCs are usually reduced This means that it is not wise to apply pulse therapy in active
or stopped as soon as the disease is under control. This needs RA, unless the therapeutic strategy is changed (e.g., DMARD
to be done carefully to avoid a relapse in disease activity and to treatment introduced to maintain the remission induced by pulse
permit recovery of adrenal function. There are no controlled therapy).
comparative studies to support a specific regimen for weaning
patients off GCs, as this process needs to be adjusted according Intraarticular Glucocorticoid Injections
to disease activity, dose/duration of therapy, and clinical response. Intraarticular injections with GCs are often used in RA. The
When patients with RA are treated with up to 10 mg/day of benefit achieved varies, depending on several factors, such as
prednisone, the daily dose can be reduced by 2.5 mg every month the joint treated (size, weight-bearing or non–weight-bearing),
until 5 mg/day is reached. Thereafter, doses can often be reduced inflammatory activity, the volume of synovial fluid in the joint,
by 1 mg per month. When higher doses have been used, a reduc- whether or not synovial fluid was aspirated before the injection,
tion by 5 mg every 1–2 weeks down to 20 mg/day is often well the choice and dose of GC preparation, injection technique,
26
tolerated, followed by further reductions of 1–2.5 mg/day every and whether the joint is rested after the injection. To prevent
2–3 weeks. Addition of immunomodulatory drugs, such as GC-induced joint damage, it is recommended that intraarticular
methotrexate and azathioprine, may allow further dose reductions. GC injections should not be repeated more often than once every
Short courses of GCs for some conditions, such as asthma, may 3–4 weeks, and no more than 3–4 times a year in a weight-bearing
be ceased without tapering if the total treatment course is less joint.

1172 ParT TEN Prevention and Therapy of Immunological Diseases


KEY CONCEPTS and growth hormone production, induce muscle wasting, and
2
Glucocorticoid (GC) Therapy in Rheumatoid modulate RANKL/OPG, NF-κB, and AP-1 signaling in bone.
All of these changes lead to enhanced osteoclast function and
Arthritis (RA) lifespan and thus to increased bone resorption. Consequently,
Our view of the risk–benefit ratio of low-dose GC has shifted in recent markers of bone resorption are often increased in patients treated
2
years: with GCs. However, reduced bone formation as a result of
• GCs can now be considered as disease-modifying antirheumatic drugs, reduced osteoblast function is likely to be a more important
especially in early RA. effect of GCs on skeletal health. Oral doses of prednisone as low
• Adverse effects of low-dose GCs are less abundant and less severe as 2.5 mg/day have been shown to suppress serum osteocalcin, a
than previously suggested, and some (e.g., osteoporosis) can be well marker of bone formation. Histologically, mean wall thickness
managed.
• The goal of antirheumatic treatment in early RA is to induce remission is reduced, reflecting the reduced amount of bone replaced in
of disease by aggressive management: GCs are part of this aggressive each remodeling unit. In vitro, osteoblasts and their precursors
strategy. are highly GC responsive. Here, the predominant effect is to
promote osteoprogenitor proliferation, lineage commitment,
and osteoblast differentiation, resulting in the formation of
bone nodules of increased size and numbers. However, GCs
also inhibit type I collagen expression and reduce preosteoblastic
replication. Finally, GCs promote apoptosis of osteoblasts and
osteocytes. The inhibitory effects of GCs on bone formation
Adverse Effects may partly be a result of downregulation of insulin-like growth
Studies of GC toxicity tend to be retrospective and observational. factor 1 (IGF-1) expression by osteoblasts. Fortunately, we now
This can make it difficult to differentiate unfavorable outcomes have effective strategies for the prevention and treatment of
attributable to GCs from those occurring as a result of the GC-induced osteoporosis, using calcium, vitamin D, and specific
underlying disease or other comorbidities. Furthermore, there osteotropic agents, such as bisphosphonates or parathyroid
is a strong selection bias for GC use, as physicians are more hormone. 28
inclined to use them in patients with more severe disease. Fre-
quent, but less serious, adverse effects (e.g., skin thinning, Osteonecrosis
Cushingoid appearance) may be of great concern to patients, Osteonecrosis has long been considered an important consequence
whereas more debilitating toxicities, such as osteoporosis, cata- of high-dose GC use. In a Japanese study of femoral head
racts, and GC-induced hypertension, may initially go unrecognized osteonecrosis, 35% of cases were related to GC treatment. Higher
or be asymptomatic. Interpretation of toxicity data is further average dose may be a more important predictor of avascular
confounded by the use of GCs at variable points in the disease necrosis of bone compared with cumulative dose. Osteonecrosis
course, limited data defining “threshold” doses for particular is particularly noted in SLE but rarely occurs in patients with
adverse events, and the fact that toxicity reports cover a hetero- RA receiving low-dose therapy, affecting <3% of patients.
geneous group of GC-treated diseases. Osteonecrosis rarely occurs in patients with SLE on prednisone
Compared with other antirheumatic agents, GCs have a low doses <20 mg/day.
incidence of short-term symptomatic toxicity, and patients rarely
discontinue therapy for this reason. Despite >60 years of use, Myopathy
we still lack robust data on the longer-term toxicities of GCs As with osteonecrosis, GC-induced myopathy is rare in
from large randomized controlled trials with long-term follow-up. patients receiving low-dose GCs. In small studies, myopathy
The most common GC toxicities are summarized below. appears more closely associated with fluorinated GC prepara-
Some progress has been made by formulating recommenda- tions, such as triamcinolone, than with prednisone. Notably,
tions on which adverse effects of GC treatment should be myopathy has been reported after only 3 months’ treatment with
monitored in RA, how to monitor them, and how often. Two triamcinolone 8 mg/day. In general, myopathy attributable to
levels of monitoring GC adverse events have been proposed: (i) prednisone only occurs after higher doses and longer durations of
for routine clinical practice, details are given on how to identify treatment.
adverse events in a systematic and practical way, and this should
result in preventive and therapeutic measures to minimize the Cardiovascular Adverse Effects
risks of GC therapy; and (ii) for clinical trials, recommendations GC-induced hypertension seems to be, at least in part, mediated
have been made on how to accurately assess the frequency and via fluid retention (as a result of mineralocorticoid effects); it
severity of a wider range of adverse events. 27 is dose related and less likely with medium-dose or low-dose
therapy. Individual variation in susceptibility and other factors,
Osteoporosis such as the starting level of blood pressure, dietary salt intake,
Glucocorticoid-induced osteoporosis (GIOP) is the most impor- functional renal mass, associated diseases, and drug therapy, may
tant potential complication of prolonged GC therapy. Chronic also play a role.
GC treatment results in rapid and profound reductions in bone Another troublesome potential toxicity of low-dose GC is
mineral density, with most bone loss occurring during the first the development of premature atherosclerotic vascular disease.
6–12 months of treatment. 2,28 This has proven difficult to investigate: studies evaluating the
GIOP initially affects trabecular bone, but cortical bone is also effects of GCs on lipids and atherosclerosis in patients with RA
affected with more chronic use, at such sites as the femoral neck. have yielded mixed results, with some studies suggesting that
Precisely how GCs affect bone remains obscure. GCs decrease GCs may, in fact, reverse unfavorable lipid changes. At present,
calcium absorption, increase renal calcium loss, diminish sex there is no evidence of a strong association between low-dose

CHaPTEr 86 Glucocorticoids 1173


GCs and cardiovascular disease in RA, even though atherosclerotic
vascular disease is known to be accelerated in patients with Other Adverse Effects
Cushing syndrome. Systemic GC use during pregnancy may also Adverse effects on the HPA axis and on glucose metabolism are
have epigenetic effects leading to adult hypertension in the next reviewed elsewhere, together with neuropsychiatric and oph-
generation. 29 thalmological adverse effects. 30,32,33
Dermatological Adverse Effects IMPORTANCE OF TIMING OF
Skin thinning and ecchymoses are common adverse events with GLUCOCORTICOID ADMINISTRATION
GCs, even at low doses. Cutaneous atrophy results from the
catabolic effects of GCs on keratinocytes and fibroblasts. Purpura In patients with RA, major symptoms, such as pain, inflammation,
and easy bruising in GC-treated patients probably result from and stiffness, vary during the course of the day, usually with
decreased vascular structural integrity. A Cushingoid appearance the highest severity in the morning hours. These symptoms are
is very troubling to patients but is uncommon at doses below preceded by elevated levels of proinflammatory cytokines. On the
the physiological range. One study reported facial fullness (“moon basis of these considerations, it has been proposed that altering the
facies”) in 13% of patients receiving 4–12 mg triamcinolone for timing of GC administration may help optimize RA therapy. 19,34
up to 60 days. These adverse effects are observed in over 5% of
patients exposed to ≥5 mg prednisone equivalent for ≥1 year. The NEW GLUCOCORTICOID RECEPTOR LIGANDS ON
incidence of iatrogenic Cushing syndrome is dose dependent THE HORIZON
30
and generally becomes evident after >1 month of GC therapy.
Alternate-day therapy may reduce the incidence, although there The various mechanisms of GC action provide interesting
are only limited data supporting this concept. GC-induced acne, opportunities for developing optimized GCs and GCR ligands.
hirsutism, and striae are other undesirable dermatological effects
that occur even with lower doses. Selective Glucocorticoid Receptor Agonists
The genomic component mechanisms of transactivation and
Gastrointestinal Adverse Effects transrepression offer the opportunity to develop GCR ligands
GCs are considerably less toxic to the upper gastrointestinal (GI) that predominantly cause transrepression rather than trans-
tract compared with NSAIDs. If GCs independently increase the activation. This concept is based on the proposition that the
risk of GI events (e.g., gastritis, ulceration, bleeding), the effect antiinflammatory properties of GCs mostly result from repression
is slight, with estimated relative risks varying from 1.1 (not of AP-1–stimulated and NF-κB–stimulated synthesis of inflam-
significant) to 1.5 (marginally significant). There have also been matory mediators, whereas their adverse effects are associated
anecdotal reports of intestinal rupture, diverticular perforation, with transactivation of genes involved in metabolic processes.
and pancreatitis attributed to low-dose GC therapy. GCs are Investigators have, therefore, sought novel GCR ligands with high
frequently used concurrently with NSAIDs in RA, and meta- transrepression activity but low effect on transactivation. One
analyses have confirmed that the combination of the two drugs such compound, A276575, exhibits a high affinity for the GCR
synergistically increases the risk of adverse GI events. In a large- and potently represses IL-1α–induced IL-6 production, similar
scale study based on the UK General Practice Research Database, to dexamethasone. However, unlike dexamethasone, A276575
the risk of upper GI complications was 1.8 (95% confidence induces little aromatase activity. Other novel nonsteroidal GCR
interval [CI] 1.3–2.4) times higher in GC users than in nonusers. ligands that possess high repression activities against inflammatory
The risk tended to be greater with higher GC doses, but the dose mediator production but have lower transactivation activities
gradient was not statistically significant. The risk was >12 times than traditional GC are being developed. Substances that cause
higher in those taking both GCs and NSAIDs than in those not a receptor conformation preferring a GCR–protein interaction
using either. No studies have yet looked at the GI effects of as opposed to a GCR/DNA binding–dependent mechanism
combining GCs with cyclooxygenase 2 (COX-2)–selective NSAIDs. are called selective glucocorticoid receptor agonists (SEGRA) or
Preventive strategies for the adverse effects of GC on the GI tract “dissociated glucocorticoids.” The SEGRA concept has, however,
have been reviewed by Caplan et al. 30 been challenged by studies on a mouse knock-in strain with
a dimerization-deficient GCR, which demonstrated that some
Infectious Diseases inflammatory processes can be suppressed by GCs and others
35
Medium- to high-dose GC therapy may increase the risk of cannot. Also, these mice exhibited the classic adverse effects
serious infections leading to hospitalization or surgery, particularly of GCs, such as GC-induced osteoporosis. Thus depending on
when administered for prolonged periods. However, there is no the process being treated, SEGRA could be therapeutically more
evidence that infection rates are increased in patients on doses effective or less effective; moreover, not all adverse effects of GC
of prednisone below 10 mg/day or cumulative doses below therapy may be reduced. 35,36 As of now, whether SEGRA will
700 mg. In those taking higher doses, the risk of infection appears become clinically relevant in practice remains uncertain.
to be lessened with alternate-day therapy.
In GC-treated patients, physicians should be aware of the Nitrosteroids
risk of infections with typical and atypical organisms, recognizing Nitrosteroids are another new class of GC drugs that have been
that GCs may blunt classic clinical features and delay diagnosis. tested in RA and inflammatory bowel disease (IBD). In addition
However, it is difficult to separate the independent effects of GC to their enhanced GC properties, nitrosteroids release low levels
use from those of other commonly used antirheumatic agents, of NO and have fewer undesirable effects. The prototype of these
31
such as methotrexate and anti-TNF-α agents. At present, the new steroids, 21-NO-prednisolone (NCX-1015), is much more
independent role of GCs in facilitating herpes zoster virus (HZV) potent than prednisolone in models of acute and chronic inflam-
infection in patients with RA remains uncertain. mation, including experimental arthritis induced by collagen II.

1174 ParT TEN Prevention and Therapy of Immunological Diseases


38
In contrast, NCX-1015 did not activate primary osteoclast activity morning stiffness. Further clinical developments seem likely
in vitro, whereas prednisolone did. This lack of adverse effect of to follow (e.g., new GCR ligands and liposome encapsulation),
NCX-1015 seems to be chiefly caused by NO. It has been suggested all with a view to improving the risk–benefit ratio of GC therapy
that posttranslational modification of GCR (tyrosine nitration) and the well-being of patients.
by NCX-1015 may explain its enhanced antiinflammatory activity.
Moreover, NCX-1015 potently stimulates IL-10 production, sug- ON THE HOrIZON
gesting that nitrosteroids induce Tregs that negatively modulate
inflammation. However, further work is needed to confirm the • Selective activators of transrepression (SEGRA)
• Nitrosteroids (with effect of nitric oxide on endothelium)
utility of nitrosteroids in clinical practice. • Modified release steroids
Long-Circulating Liposomal Glucocorticoids • Liposomal encapsulation
• Optimized combinations with biologicals
The antiinflammatory efficacy of GCs can be improved by the
additional benefits of nongenomic actions at high GC concentra- ACKNOWLEDGMENTS
tions. This has led to the use of long-circulating liposomal GCs
in experimental models. In rats with experimental autoimmune This chapter has been revised and updated from the version in
encephalitis, GC-containing liposomes accumulated at sites of the previous edition. The editors are grateful to Frank Buttgereit,
inflammation, reaching concentrations >10–5 mol/L for ≥18 Hans Bilsma and Markus Seibel for their contributions to previous
hours. These liposomes may be therapeutically superior to editions and for their permission to let us update the chapter.
conventional intravenous high-dose GC therapy, as evidenced
by their successful use in rats with adjuvant-induced arthritis. A Please check your eBook at https://expertconsult.inkling.com/
single injection of 10 mg/kg liposomal prednisolone phosphate for self-assessment questions. See inside cover for registration
resulted in complete remission of the inflammatory response details.
for almost a week. In contrast, the same dose of unencapsulated
prednisolone phosphate did not reduce inflammation and had
only a slight effect after repeated daily injections. It may be that REFERENCES
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CHaPTEr 86 Glucocorticoids 1175.e1


MULTIPLE-CHOICE QUESTIONS

1. Genomic effects of glucocorticoids (GCs) include ALL of the 3. Promising future prospects for steroid development include:
following except: A. Liposomal encapsulation
A. Induction of antiinflammatory cytokines b. Nitrogenated steroids
B. Inhibition of arachidonic acid release C. Slow release preparations
C. Interaction with transcription factors AP-1 and nuclear D. Selective inducers of transactivation
factor-κB
D. Competition for nuclear coactivators
2. The most important complication of GC use is:
A. Increased risk of infections
B. Diabetes mellitus
C. Osteoporosis
D. Glaucoma

87









Immunomodulating Pharmaceuticals



Gideon P. Smith, Edwin S.L. Chan







Excitement over biologic agents and their capacity to regulate proliferation (Table 87.1). These actions are dependent on
immunological reactions that significantly impact on such inhibition of dihydrofolate reductase; hence toxicities arising
immunologically mediated diseases, such as rheumatoid arthritis from high-dose methotrexate therapy can be treated with folic
(RA) and inflammatory bowel disease (IBD), has overshadowed acid derivatives, such as leucovorin. However, folic or folinic
the older, small-molecule therapeutic agents. Nonetheless, when acid, which are often given in conjunction with methotrexate
tested head-to-head, some small-molecule agents (most notably in inflammatory diseases to reduce the incidence of mucositis
methotrexate) have proven to be almost as effective as biologics; and bone marrow suppression, do little to inhibit its antiinflam-
moreover, combining small-molecule therapies with biological matory efficacy. Decreases in purine and pyrimidine concentra-
agents generally leads to significantly better outcomes than use tions in the serum have been observed following a single dose
1
of either agent alone. Thus it is likely that small-molecule of methotrexate, along with decreased proliferation of antigen-
immunomodulatory drugs will continue to be in wide use. Here, stimulated lymphocytes. However, these changes are transient
we review the most widely accepted and commonly used immu- and insufficient to explain the antiinflammatory effectiveness
nomodulators currently in clinical use. of once-weekly dosing. This, as well as the low doses of methotrex-
ate required to produce an antiinflammatory effect, suggests
METHOTREXATE that the antiinflammatory actions are mediated via different
mechanisms.
Methotrexate (Fig. 87.1) was employed in the treatment of RA Methotrexate also blocks intracellular transmethylation reac-
as early as 1951, but its popularity with regard to RA did not tions and inhibits production of S-adenosylmethionine. Since
come until the 1980s. Over the years, extensive experience with S-adenosylmethionine is necessary for formation of the toxic
its use in inflammatory diseases as diverse as RA (Chapter 52), polyamine metabolites spermine and spermidine, their accumula-
psoriasis (Chapter 64), and IBD (Chapter 75) has taught us a tion at the inflammatory site is prevented. This inhibition of
great deal about its safety, efficacy, and toxicity, as well as its transmethylation is associated with an impairment of monocyte
antiinflammatory mechanisms of action. In this respect, metho- and lymphocyte function and thus potentially the synthesis of
trexate, much as corticosteroids, can be justly regarded as a reactive oxygen species. However, diminution of transmethylation
cornerstone of immunomodulatory therapy. by the use of the S-adenosylhomocysteine hydrolase inhibitor,
deaza-adenosine, has failed to produce any beneficial clinical
Pharmacokinetics of Methotrexate effects in RA.
As an antiinflammatory agent, methotrexate is administered at Methotrexate and its long-acting polyglutamate metabolites
low doses (usually 10–25 mg/week) once weekly, usually orally, also exert antiinflammatory effects by releasing the endog-
3
but it can also be given subcutaneously or intramuscularly. enous autocoid adenosine. As potent inhibitors of the enzyme
At these doses, oral bioavailability is high (60–70%), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)
although transporters are responsible for its absorption from the transformylase, methotrexate polyglutamates promote accumula-
gastrointestinal (GI) tract, saturation effect does not occur. A small tion of AICAR in tissues. Since AICAR inhibits catabolizing
portion of methotrexate is metabolized by hydroxylation into enzymes for both adenosine and adenosine monophosphate
7-hydroxymethotrexate. Both compounds have a serum half-life (AMP), which can be dephosphorylated to adenosine, the net
of no more than 8 hours. The much longer antiinflammatory effect is intracellular and extracellular increases in adenosine
action, which allows for once-weekly dosing, must therefore be levels. These metabolic pathways are pharmacologically relevant,
mediated by other longer-lasting metabolites, such as polygluta- since aminoimidazole carboxamide and adenosine have been
mates. Excretion occurs principally via the urinary tract but also shown to be increased in urine following low-dose methotrexate
4
via the biliary tract. Therefore renal function is an important treatment in patients with psoriasis. Adenosine causes diminution
consideration in methotrexate dosing, and any medication that of neutrophil accumulation, adhesion, phagocytosis and genera-
impairs glomerular filtration may also potentiate methotrexate’s tion of reactive oxygen species, inhibition of adhesion molecule
effectiveness and toxicity. 2 expression, suppression of proinflammatory cytokines, and
induction of antiinflammatory cytokines, as well as modulation
Mechanisms of Action for Methotrexate of macrophage and endothelial function. Indeed, blockade of
2
As an analogue of folic acid, methotrexate is an inhibitor of adenosine receptors reversed the antiinflammatory effects of
purine and pyrimidine synthesis and thereby suppresses cellular methotrexate on animal models. It has also been suggested that

1177

1178 Part tEN Prevention and Therapy of Immunological Diseases


COOH
NH 2 CH 3 Sulfasalazine
N CH 3 N C NHCH HOOC
N
O CH 2 HO N N SO 2 NH

H N N CH 2 N
2
N
COOH
Methotrexate HOOC
FIG 87.1 Methotrexate—chemical structure.
HO N H 2 N SO 2 NH
N
TABLE 87.1 Methotrexate: Mechanisms 5-Aminosalicylic acid Sulfapyridine
of action
FIG 87.2 Sulfasalazine—chemical structure.
Suggested Mechanism rationale
Folate antagonism Prevents purine and pyrimidine
synthesis required for the TABLE 87.2 Methotrexate: adverse Effects
proliferation of actively dividing
immune cells, such as Gastrointestinal Cardiovascular
lymphocytes Stomatitis Pericarditis
Inhibition of spermine and Reduces formation of polyamines Anorexia Thrombosis
spermidine production harmful to tissues Nausea
Alteration of cellular redox state Reversible inhibition of lymphocyte Vomiting Pulmonary
and macrophage functions Diarrhea Pulmonary fibrosis
Release of adenosine Generation of a potent Cirrhosis Interstitial pneumonitis
endogenous antiinflammatory Pancreatitis
mediator through inhibition of Others
catabolism of both adenosine Hematological
and adenosine monophosphate Leukopenia Skin rashes
Renal failure
Anemia Abortion
Thrombocytopenia Impotence
Hypogammaglobulinemia Headache
caffeine, itself a nonselective antagonist of adenosine receptors, Lymphoma Opportunistic infections
may both reduce the effectiveness of methotrexate in RA and
protect against the development of cirrhosis of the liver, a major
side effect of methotrexate. 5
less than 1 in every 1000 patients with RA but may be more
Adverse Effects common among those with psoriasis. Risk factors, such as ethanol
consumption, hepatitis B and C, diabetes, obesity, and alpha-
tHEraPEUtIC PEarLS 1-antitrypsin deficiency, identify patients most likely to develop
Methotrexate methotrexate-induced hepatic injury. However, other serious
side effects, such as pneumonitis, may be overlooked, since early
Proven safety profile symptoms (mild cough or shortness of breath) are often ignored.
Concomitant administration of folic acid advisable Early identification allows for prompt discontinuation. The risk
Antiinflammatory effects may be reduced by heavy use of caffeine of developing solid tumors is debated, since the risk of malignan-
Hepatotoxicity a rare but real concern
Risk of hepatotoxicity increased with: cies is intrinsic to some of the conditions, such as RA, for which
Alcohol use methotrexate is used. It is likely the risk of drug-induced
Hepatitis malignancy is real, since reports have documented tumor regres-
Diabetes sion following discontinuation of methotrexate, but the risk
Obesity remains extremely small.
Alpha-1-antitrypsin deficiency
SULFASALAZINE
Over the years, methotrexate has proven to be one of the safest
disease-modifying antirheumatic drugs (DMARDs) in use. Serious Sulfasalazine (Fig. 87.2) was originally introduced in the late
side effects such as cirrhosis are much less common than previ- 1930s for the treatment of RA but is now used in a wide range
ously thought (Table 87.2). The use of folic acid has decreased of inflammatory diseases, in particular, IBD and the seronegative
the occurrence of mucosal and GI side effects, without limiting arthritides. It consists of a derivative of the antiinflammatory
its antiinflammatory activity, and cytopenias are managed salicylic acid, 5-amino-salicylic acid, and the antimicrobial
adequately with regular blood counts. Although side effects, such sulfapyridine. These two moieties are joined together by an azo
as nausea and vomiting, may resolve spontaneously or respond bond. Which component is responsible for the drug’s antiinflam-
to dose reduction or folic acid supplementation, mild transami- matory actions is unclear, but it appears to vary according to
nasemia has rarely led to discontinuation of the medication. disease states. For instance, in IBD, 5-amino-salicylic acid is likely
Risk of serious hepatotoxicity over 5 years of use is likely to be the main active component, as it is poorly absorbed following

CHaPtEr 87 Immunomodulating Pharmaceuticals 1179


metabolism by intestinal flora. In inflammatory arthritides, the imidazole moiety occurs rapidly within erythrocytes, both
sulfapyridine is likely to play a more important role, as it is rela- enzymatically by glutathione transferase and nonenzymatically.
tively well absorbed and has a bioavailability in the region of Several enzymes (Table 87.3) participate in the metabolism of
60%. Since acetylation is the principal route of metabolism of 6-mercaptopurine into active and inactive compounds. One of
sulfapyridine following absorption, acetylator status is a major these, thiopurine methyltransferase, is associated with genetic
determinant of the plasma half-life. For the same reason, slow polymorphisms; inherited changes in its activity may impact
acetylators are more liable to develop side effects. patient response to azathioprine. Xanthine oxidase inactivates
6-mercaptopurine by converting it to 6-thiouric acid. Since this
Mechanisms of Action for Sulfasalazine occurs mainly in the liver, toxicity from azathioprine therapy is
a danger in enzyme deficiency states, as a result of disease or
KEY CONCEPt use of drugs, such as allopurinol.
Sulfasalazine: Mechanism of Action Proposed Mechanisms of Action for Azathioprine
Suppresses the proliferation of lymphocytes The immunomodulatory mechanism of azathioprine remains
Suppresses proinflammatory cytokine production unclear. As purine analogues, the active metabolites interfere
Inhibits activation of nuclear factor (NF)-κB with the salvage pathway and de novo synthesis of purines, and
Promotes adenosine accumulation
they are incorporated into RNA and DNA. Proliferation of T
and B lymphocytes is inhibited, and function of natural killer
Sulfasalazine has a number of immunomodulatory effects. (NK) cells is suppressed without any change in cell numbers.
Lymphocyte proliferation is suppressed in vitro and involves The production of antibodies is also suppressed, although it is
both B-cell and T-cell populations. In vivo, a decrease in acti- not known which of these effects predominate in vivo. Cellular
vated lymphocytes in the peripheral blood is also seen. Tumor responses to chemoattractants are altered, and the production
necrosis factor-α (TNF-α) production is suppressed, and receptor of cytokines, such as interleukin-6 (IL-6), is also affected.
binding is inhibited. Sulfasalazine also inhibits activation of
the transcription factor, nuclear factor (NF)-κB. Like methotrexate, Adverse Effects
sulfasalazine inhibits AICAR transformylase and thus promotes Azathioprine is generally well tolerated. The most common side
accumulation of adenosine and its antiinflammatory actions via effects are mild and affect the GI system. Pancreatitis can occur
the adenosine A 2A receptor. Indeed, treatment of animals with as an idiosyncratic reaction. Hepatotoxicity and cholestasis are
an adenosine A 2A receptor antagonist reversed sulfasalazine’s not uncommon, and hepatic peliosis and nodular regenerative
reduction of leukocyte accumulation in an air-pouch model hyperplasia occur rarely. There have been reports of a possible
of inflammation. heightened risk for non-Hodgkin lymphoma, but because of
the rarity of these events, no definite link has been established
Adverse Effects between azathioprine and malignancies. Bone marrow suppression
In a large series, a quarter of those treated over 11 years stopped and opportunistic infections pose far greater threats.
6
treatment because of toxicity. Most toxicities occurred early
and were both trivial and resolved following therapy withdrawal. CYCLOPHOSPHAMIDE
Most common are nausea, vomiting, anorexia, and rash. Serious
cutaneous reactions, such as exfoliative dermatitis or Stevens- Alkylating agents were used in the treatment of inflammatory
Johnson syndrome, are rare. Transaminasemia and drug-induced diseases after promising reports on using nitrogen mustard in
hepatitis can occur. Blood dyscrasias with megaloblastic anemia, RA. Cyclophosphamide (Fig. 87.4) is metabolized to produce
neutropenia, aplastic anemia, and myelodysplastic syndrome the alkylating agent phosphoramide mustard as well as acrolein,
may arise. Neurological adverse effects include headache and which, although inactive, results in the hemorrhagic cystitis
dizziness or, more seriously, peripheral neuropathy, Guillain-Barré associated with cyclophosphamide. Cyclophosphamide can be
syndrome, or transverse myelitis. Sulfasalazine should be avoided given intravenously, but bioavailability following oral administra-
in patients with sulfa allergy, and glucose-6-phosphate dehydro- tion is high (>75%). Toxicity has severely limited its use in
genase (G6PD) deficiency screening should be performed before inflammatory diseases, although its contribution to the manage-
prescribing. ment of lupus nephritis cannot be denied. The alkylating actions
occur at guanine residues (principally on DNA but also on RNA)
AZATHIOPRINE resulting in cross-linkable strands and disruption of transcription
and translation.
Azathioprine, an imidazolyl derivative of 6-mercaptopurine (Fig.
87.3), has been widely used in RA and IBD as well as in solid
organ transplantations. Cleavage into 6-mercaptopurine and TABLE 87.3 Principal Enzymes Involved in
the Metabolism of azathioprine

Enzyme action
Azathioprine N
Glutathione transferase Cleaves azathioprine into 6-mercaptopurine
and imidazole moieties
H C N N NH Thiopurine Metabolism of 6-mercaptopurine
3
methyltransferase
N NO 2 N N Xanthine oxidase Conversion of 6-mercaptopurine to
6-thiouric acid
FIG 87.3 Azathioprine—chemical structure.

1180 Part tEN Prevention and Therapy of Immunological Diseases


Cyclophosphamide CH CH CL Leflunomide O
2
2
O N C NH CF
P CH CH CL 2
2
2
O N
N O CH 3
FIG 87.4 Cyclophosphamide—chemical structure. FIG 87.5 Leflunomide—chemical structure.

Mechanisms of Action of Cyclophosphamide A77 1726 O
This alkylating process has immunomodulating effects on resting N C C C NH CF 3
and actively dividing cells. The numbers of circulating CD4 T
lymphocytes and, to a lesser extent, CD8 T lymphocytes are CH 3 OH
reduced, thus reducing the CD4/CD8 ratio. Despite an apparent FIG 87.6 A77 1726—chemical structure.
increase in immunoglobulin-secreting cells, B-cell function is
suppressed, and overall immunoglobulin synthesis is reduced.
synthesis. Because of this long half-life, therapy with leflunomide
Adverse Effects is usually started with a loading dose to quickly achieve therapeutic
The best known toxicity is hemorrhagic cystitis. Since this occurs levels. A77 1726 is highly plasma-protein bound and undergoes
more frequently following oral dosing, this route of adminis- enterohepatic recirculation.
tration is rarely used. This may relate to continuous exposure
of the bladder to acrolein, so the acrolein-neutralizing agent Mechanisms of Action of Leflunomide
2-mercaptoethane sulfonate (Mesna) is used prophylactically By inhibiting pyrimidine synthesis, pyrimidine nucleotide avail-
along with copious hydration. Hemorrhagic myocarditis can also ability becomes insufficient for proliferation of immune-response
occur and may cause myocardial necrosis, hemopericardium, cells. This deficiency is inadequately replenished by the salvage
and congestive cardiac failure. However, survivors of acute pathways, rendering cell proliferation inefficient and limiting
cardiac toxicity do not show any residual electrocardiographic the clonal expansion of T cells. B-cell proliferation is similarly
or echocardiographic abnormalities. suppressed with reduction of Cdk2, a cyclin-dependent kinase.
Besides bone marrow suppression, reduction of fertility, and Leflunomide also inhibits NF-κB activation. Although the effects
a heightened risk of infection, cyclophosphamide therapy has of moderate concentrations are reversed by uridine in vitro, this
been associated with secondary malignancies, which may occur reversal does not occur at higher concentrations, suggesting
years after drug cessation. Malignancies of the bladder, often of possible involvement of other mechanisms. Leflunomide is known
a transitional cell type, tend to occur only in those with a history to inhibit tyrosine kinase activity at higher concentrations,
of treatment-related hemorrhagic cystitis. Myeloproliferative and although the relevance of this effect to therapeutic concentrations
lymphoproliferative disorders have also been associated with achievable in vivo remains questionable.
cyclophosphamide use.
Adverse Effects
Other Nitrogen Mustard Derivatives GI symptoms are the most common side effect, and hepatic
Chlorambucil, or 4-[bis(2chlor-ethyl)amino]benzenebutanoic damage is the most important toxicity. Although there are
acid, has wide distribution in tissue and 87% oral bioavailability, similarities to the toxicity profile of methotrexate, clinical trials
but unlike cyclophosphamide, it does not require metabolism have shown that leflunomide and methotrexate can be safely
7
by the liver to become metabolically active. The only indication and effectively given together to patients with RA, but transami-
approved by the US Food and Drug Administration (FDA) is nasemia occurs more often than with methotrexate alone. 9,10
for treatment of chronic lymphocytic leukemia (CLL), but like Fulminant hepatic failure is rare, but fatal cases have occurred.
cyclophosphamide, chlorambucil has been reported to be used Skin reactions are mostly minor; however, more serious manifesta-
in treatments for the same wide range of inflammatory conditions. tions, such as Stevens-Johnson syndrome and toxic epidermal
The mechanism of action and side effect profile is also similar necrolysis, have been reported.
to those of cyclophosphamide, but with a higher risk of permanent
aplasia. 8 MYCOPHENOLATE MOFETIL
Melphalan is another phenylalanine derivative of nitrogen
mustard, mainly used in treating multiple myeloma. It has been Mycophenolate mofetil (Fig. 87.7) is widely used in solid organ
less widely adopted but has been used off-label in a variety of transplantation and has also been increasingly employed in
inflammatory conditions. Adverse effects and mechanism are treatment of autoimmune diseases. It is rapidly absorbed and
the same as those of cyclophosphamide, as described above. hydrolyzed into the active compound, mycophenolic acid, which
is a reversible inhibitor of inosine monophosphate dehydrogenase.
LEFLUNOMIDE Since inosine monophosphate dehydrogenase is a key enzyme
in the de novo synthesis of guanine nucleotides, its inhibition is
Leflunomide is an inhibitor of de novo pyrimidine synthesis. most significant in T and B lymphocytes, which are reliant on
Leflunomide (Fig. 87.5) is converted into the long-acting active this pathway, as they lack the hypoxanthine-guanine phospho-
compound A77 1726 (2-cyano-3-hydroxy-N-[4-trifluoromethyl]- ribosyl transferase salvage pathway. The immunological effects
butenamide) (Fig. 87.6), a reversible inhibitor of the enzyme are numerous. DNA synthesis in lymphocytes requires the
dihydroorotate dehydrogenase that is involved in pyrimidine incorporation of guanine nucleotides so that proliferation of

CHaPtEr 87 Immunomodulating Pharmaceuticals 1181


Mycophenolatemofetil CH 3 CH 2
CH 3 CH
O OH 2
CH 2 CH C CH 2 CH 2 CH 2
O CH 3 CH 3 CH 3 OH
O C C
(CH ) 2 3 CH 3 (CH ) (CH ) O C CH 3 O CH 3
O O 2 3 N 2 2
CH 3 CH 3 CH 2 C C N CH C N C C N CC N CO
O OCH CH O CH
CH 2 3 3 2
N CH 3
Cyclosporine

O N C C NH C C NH C C N C C NH C N CH 3
FIG 87.7 Mycophenolate mofetil – chemical structure. CH 3 O CH 3 O CH 3 O (CH ) CH 3 O (CH ) O C
2 3
2 3
CH 3 CH 3 (CH )
2 3
lymphocytes is suppressed. Antibody production and NK-cell CH
activity are also reduced, and in vitro cytokine production by 3
11
activated human mononuclear cells is affected. In addition, FIG 87.8 Cyclosporine—chemical structure.
delayed-type hypersensitivity responses are suppressed. Although
effective in a subset of patients with psoriasis and RA, myco- Tacrolimus CH
phenolate mofetil has not been widely used in these conditions 2
because other more effective medications are available. It is,
however, becoming more popular in the treatment of some H CO CH
3
diseases, such as myositis, systemic contact dermatitis, severe
atopic dermatitis, chronic urticaria, refractory pyoderma gan- C CH 3 C OH
grenosum, bullous pemphigoid, pemphigus vulgaris, and C O
pemphigus foliaceus, where it is effective with a low risk of side CH 2
effects. N O CH O
Adverse Effects O C C CH 2 CH CH 2
Absolute contraindications for mycophenolate mofetil are drug O C CH H O
2
3
allergy and pregnancy (Category D). Relative contraindications H C OH C CH 3
include lactation; renal, hepatic, or cardiopulmonary disease; O
and peptic ulcer. It is generally well tolerated when used in
autoimmune diseases, such as RA. The most common side effects H CO C CH 2 C CH 2
are nausea, vomiting, abdominal discomfort, diarrhea, fever, 3 OCH 3 CH 3
headache, skin rash, back pain, and tremor, but these do not FIG 87.9 Tacrolimus—chemical structure.
usually lead to discontinuation. Rarely reported side effects include
leukopenia and other cytopenias, cutaneous and noncutaneous
malignancies, and pancreatitis. Toxic doses have not been ORAL CYCLOSPORINE AND TACROLIMUS (FK506)
established for this medication. One patient suffered only moder-
ate leukopenia with no significant GI side effects after ingesting Cyclosporine (Fig. 87.8) and tacrolimus (Fig. 87.9) are structur-
25g of mycophenolate mofetil. Up to 4 g/day have been used in ally similar drugs that have been widely used in solid organ
cardiac and up to 5 g/day in hepatic transplantation patients. transplantation as well as in the treatment of immunological
However, increased efficacy was not observed above 2 g/day, and diseases. Cyclosporine has potent inhibitory effects in dampening
patients were more likely to experience GI symptoms and the production of proinflammatory mediators, such as IL-2,
neutropenia at higher doses. For this reason, doses up to 2 g/ by immunocompetent cells, most importantly T lymphocytes.
day are usually employed to treat inflammatory conditions. It does so through binding cyclophilin, which produces a
cyclosporine–cyclophilin complex. This complex binds the
13
HYDROXYUREA serine/threonine phosphatase calcineurin. This disrupts the
phosphorylation of regulatory proteins for which the nucleated
Hydroxyurea is urea with one additional hydroxyl group. It inhibits factor of activated T cells (NF-ATs) is a critical component,
ribonucleotide reductase, which catalyzes the reduction of preventing these proteins from translocating into the nucleus.
ribonucleotides to deoxyribonucleotides, and is thus essential Thus the transcription of genes, such as IL-2, which induces
12
in DNA synthesis. It is effective in the treatment of psoriasis. mitogenesis in activated T cells, cannot be effectively activated.
Hydroxyurea is well tolerated, with the most common side effects A number of other cytokines are affected, including IL-3, IL-6,
being hematological, usually megaloblastic anemia, but also transforming growth factor (TGF)-β, and interferon (IFN)-γ.
leukopenia and thrombocytopenia. Other significant but rare Another T cell–specific immunophilin, FK506-binding protein
adverse effects include renal and GI toxicity, a dermatomyositis- (FKBP), binds tacrolimus to form a FK506-FKBP complex with
like syndrome, leg ulcers, radiation recall, and leukemias. similar resultant inhibitory activity on calcineurin. 14

1182 Part tEN Prevention and Therapy of Immunological Diseases



KEY CONCEPt Imiquimod
Cyclosporine: Mechanism of Action NH
N 2
Association with cyclophilin
Formation of cyclosporine–cyclophilin complex
Binds calcineurin
Inactivates calcineurin N
Regulatory proteins unable to translocate into nucleus
Transcription of proinflammatory genes affected CH 2
CH
Adverse Effects CH 3 CH 3
The most common side effects of cyclosporine are hypertension, FIG 87.10 Imiquimod—chemical structure.
hyperkalemia, hypomagnesemia, and hyperlipidemia. More
importantly, cyclosporine has well-documented short-term and
15
long-term adverse effects on kidney function. These data come
from patients who had received solid organ transplants, most SIROLIMUS
notably renal transplants. In such patients, initial doses of
15–25 mg/kg/day led to a reduction in the glomerular filtration Sirolimus is a macrolide that binds the cytosolic protein FK-binding
rate (GFR) and rise in serum creatinine in a percentage of patients, protein 12 (FKBP12). In contrast to the tacrolimus–FKBP12
16
as well as histologically proven nephropathy. The pathogenic complex, which inhibits calcineurin, the sirolimus–FKBP12
mechanism of kidney damage is poorly understood but is believed complex directly binds the mammalian target of rapamycin
17
to consist of two phases. The first is a period of partial ischemia (mTOR) complex1 (mTORC1), thereby inhibiting the mTOR
secondary to vascular contraction, which is reversible with dose pathway. Thus it inhibits the response to IL-2, blocking activa-
reduction or drug discontinuation. The later, irreversible phase tion of T and B cells. It has shown promise in the treatment
19
results from chronic scarring of the glomeruli. Treatment recom- for systemic lupus erythematosus (SLE); Sjögren syndrome ;
20
mendations for some diseases, such as psoriasis, have therefore RA ; psoriasis; genetic disorders, such as tuberous sclerosis;
21
been targeted at much lower maximum daily doses of 5 mg/kg/ and neoplastic disorders, such as Kaposi sarcoma. The main
day, with a reduced dose if creatinine rises 30% above baseline. advantage of sirolimus over tacrolimus or cyclosporine is reduced
Otherwise healthy patients treated at these dosage levels have renal toxicity.
been successfully managed for many years on cyclosporine with
18
no impact on the GFR. Nephrotoxicity has also been a concern IMIQUIMOD
during tacrolimus therapy. Other adverse effects include increased
rate of infections, malignancy, hepatotoxicity, GI upset, rash, Imiquimod (Fig. 87.10), an imidazoquinoline drug, activates
tremor, headache, and insomnia. Toll-like receptor (TLR)-7 and possesses both antiviral and
22
antitumor activities. As a cream preparation, it is effective in
TOPICAL PIMECROLIMUS AND the treatment of external genital warts caused by infection with
TACROLIMUS (FK506) human papillomavirus (HPV). Immune amplification responses
are induced through stimulation of inflammatory cytokines. 23,24
Tacrolimus is also available in topical formulation. Pimecrolimus Production of IFN-α is stimulated, and this suppresses replication
is an alternative topical calcineurin inhibitor with similar structure of viruses in infected keratinocytes. NK-cell activity is also
and identical mechanisms of action. The cyclosporine molecule increased, partly through the induction of oligoadenylate synthase.
is too large to penetrate skin (1203 Da), whereas tacrolimus and The increase of dermal IFN-α transcript levels is rapid and
25
pimecrolimus can, as they are much smaller molecules (804 and dramatic. Other cytokines modulated by imiquimod include
80 daltons [Da], respectively). Both have been approved by the TNF-α and IL-12, especially in peripheral blood monocytes. 26,27
FDA for the treatment of atopic dermatitis but have found The overall effect is a shift from a T-helper-2 (Th2)-cytokine–
widespread use in many other conditions (psoriasis, oral and predominant profile toward a Th1-cytokine–predominant profile.
cutaneous lichen planus, vitiligo, pemphigoid, and pemphigus). Other conditions where imiquimod is effective include actinic
They are most often used when long-term topical corticosteroids keratosis, herpes simplex, basal cell carcinoma, and molluscum
are contraindicated. contagiosum.
Adverse Effects Adverse Effects
The most common side effect is local irritation at the site of Inflammation at the site of application represents the most
application in severely inflamed skin. Therefore initial short-term common adverse reaction. However, nondermatological side
treatment is often combined with topical steroids. An association effects, such as fever, fatigue, myalgia, and headache, have also
with malignancy remains controversial. In 2005, 17 case reports been described.
of malignancy in patients using these topicals resulted in a
black-box warning, although further studies suggested that the 5-FLUOROURACIL
rate of lymphoma from these case reports was lower than the
rate observed in the general population in the United States. In 5-Fluorouracil is a uracil analogue that has two modes of
2006, the FDA revised the wording, but a black-box warning action. First, it inhibits cell proliferation via direct incorpora-
about these products remains in place. tion into RNA causing abnormal base pairing. Second, it binds

CHaPtEr 87 Immunomodulating Pharmaceuticals 1183


thymidylate synthetase, blocking the conversion of deoxyuridine CONCLUSIONS
monophosphate to deoxythymidine monophosphate, which is
essential to DNA synthesis. It may also increase expression of p53, The field of small molecule immunomodulators contains both
28
a frequently mutated gene in nonmelanoma skin cancers. It can traditional and new molecules. The traditional molecules come
be administered topically, intramuscularly, or intravenously. In with a long history of efficacy and side-effect data, and this
topical form, its main uses are to treat actinic keratosis, superficial allows us to safely and accurately utilize these therapies. Despite
basal cell carcinoma, Bowen disease, keratoacanthoma, porokera- the introduction of biologics, traditional small-molecule agents,
tosis, and verruca vulgaris. However, intravenous treatment of such as methotrexate and cyclosporine, are both clinically effica-
recalcitrant psoriasis, mycosis fungoides, and scleroderma has cious and cost-effective and thus remain the workhorses of our
also been reported. modern pharmaceutical armamentarium. However, as our
understanding of the pathways involved in inflammation evolve,
Adverse Effects our therapeutics are also refined, allowing the production now,
Topical application is associated with an irritant dermatitis, but for example, of topically effective calcineurin inhibitors, such as
this is also seen as a marker of clinical efficacy. Parenteral tacrolimus and pimecrolimus, which avoid the adverse effects
administration for inflammatory conditions is not widespread; of systemic cyclosporine or topical steroids, and sirolimus, which
adverse effects from parenteral administration are more severe reduces toxicity profiles of systemic FKBP2 signaling. In addition,
and include clinically significant bone marrow suppression, GI newer agents, such as glatiramer and fingolimod, allow treatment
toxicity, and cutaneous reactions. of MS, a disease recalcitrant to more traditional therapies, and,
although not currently being used in other inflammatory dis-
GLATIRAMER orders, have shown promise in animal models. The field of
small-molecule immunomodulators therefore remains one of
Glatiramer acetate is a random polymer of glutamic acid, lysine, both current clinical relevance and continuing, exciting new
tyrosine, and alanine, the four amino acids found in myelin developments.
basic protein (MBP). The mechanism of action for glatiramer
is unknown, but its similarity in structure to MBP may allow it ON tHE HOrIZON
to act as a decoy for immune targeting of myelin. It may also
induce expression of glatiramer acetate–specific suppressor T Refinement of our understanding of the pathways and actions of receptors
cells, and these have been shown to be present in animal models. targeted by our current medications is the key to producing more
elegant therapies.
In contrast to imiquimod, glatiramer shifts the population of T Separating therapeutic effects from the pathways leading to side effects
cells from proinflammatory Th1 cells to regulatory Th2 cells would enable much safer and more tolerable small-molecule
that suppress the inflammatory response. It is FDA approved medications.
for the treatment of adults with relapsing–remitting multiple Better understanding of the dysregulation that occurs in inflammatory
sclerosis (MS), even after only one event. conditions, and the activation of the immune system will be essential
to ensuring the efficacy of such new modalities.
Adverse Effects
Absolute contraindications include allergy to glatiramer or to The challenge in the next 5–10 years is to continue this
mannitol. It is pregnancy Category B, but it is unknown if the refinement in the targeting of small molecules. Many of the
drug is secreted in breast milk. The drug is given via subcutaneous agents we currently use target early parts of pathways or indis-
injection, and the most common adverse effects are injection criminately trigger multiple receptors. This leads to a greater
site reaction, flushing, rash, dyspnea, and transient chest pain. range of side effects and thus limits our ability to reach adequate
dosing levels. A prime example of this is methotrexate, which
FINGOLIMOD (FTY720) leads to increased adenosine levels and thus triggers all four
adenosine receptors. By developing more selective molecules
Fingolimod is a relatively new immunomodulator for treating targeting, for instance, just the adenosine A 2A receptor, not only
MS. It is a structural analogue of sphingosine and is phosphory- would we avoid the side effects attributable to other adenosine
29
lated intracellularly by sphingosine kinases. Signaling via one receptors, we might also avoid the effects of methotrexate on
of the sphingosine 1 phosphate receptors, S1PR1, it is believed bone marrow and mucous membranes via inhibition of dihy-
to prevent migration of lymphocytes by halting their ability to drofolate reductase. In other medications, more specific targeting
leave lymph nodes. However, fingolimod has also been reported of pathways may similarly reduce side effects and make for more
30
31
to act as a cPLA2 inhibitor, a cannabinoid receptor antagonist, effective therapeutics. This will require a better understanding
32
and a ceramide synthase inhibitor. So far, the FDA has only of the immune system and its dysregulation in disease, as well
approved it for use in MS. However, it has also shown promise as development and application of these medications to clinically
in murine models of SLE and RA, with reduced mortality from relevant models
34
33
lupus nephritis and improvement in arthritis, respectively. It
also has a potential role in the treatment of cutaneous inflam- Please check your eBook at https://expertconsult.inkling.com/
matory conditions, such as psoriasis and atopic dermatitis. 35 for self-assessment questions. See inside cover for registration
details.
Adverse Effects
The most common side effects are minor and include headache REFERENCES
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CHaPtEr 87 Immunomodulating Pharmaceuticals 1184.e1


MUL t IPLE-CHOICE QUES t IONS

1. Which of the following medications’ antiinflammatory effects C. Glatiramer
may be reduced in heavy coffee drinkers? D. Azathioprine
A. Methotrexate E. 5-Florouracil
B. Azathioprine 3. In which of the following medicines should glucose-
C. Mycophenolate mofetil 6-phosphate dehydrogenase (G6PD) screening be considered
D. Cyclosporine before use?
E. Tacrolimus
A. Methotrexate
2. Which of the following medications is pregnancy Category B. Azathioprine
B? C. Sulfasalazine
A. Methotrexate D. Mycophenolate mofetil
B. Mycophenolate mofetil E. Imiquimod

88
CHAPTER 88 Protein Kinase Antagonists in Therapy of Immunological and Inflammatory Diseases 1185





Protein Kinase Antagonists in Therapy of

Immunological and Inflammatory Diseases



Arian Laurence, Massimo Gadina, John J. O’Shea











Reversible phosphorylation is one of the major mechanisms kinase-independent manner but, nonetheless, signal through
controlling protein activity in all eukaryotic cells and, as such, kinase cascades to exert their effects. It is clear that many aspects
is involved in all fundamental cellular processes, including cell of protein phosphorylation are of major importance in immune
cycle and cell growth, cell shape and movement, metabolism, and inflammatory mechanisms.
differentiation, and apoptosis. This covalent modification is a The nonredundant functions of various kinases in different
major means for transmitting information from outside the cell immune cells are exemplified by both studies in knock-out mice
and between the subcellular components within the cell. Phos- and humans with mutations. On the basis of these findings,
phorylation is a major mechanism underlying normal signaling, targeting protein kinases has been proposed to be a useful strategy
as exemplified by insulin and other growth factors, but in addition, in the development of novel immunosuppressant drugs and is
the importance of protein phosphorylation is supported by one of the most active areas of pharmaceutical drug development
evidence that mutations and dysregulation of protein kinases (Table 88.3), with much of the impetus coming from oncology.
play causal roles in human disease. This is especially true in The field is so vast that it is impractical to comprehensively
cancer, in which mutant protein kinases or their upstream review all this information in one chapter; therefore we will
activators function as oncogenes. focus both on important historical precedents in the field and
From the point of view of an immunologist, protein phos- then discuss drugs and targets that are most immunologically
phorylation is also the major mechanism by which immune relevant. We will start by briefly reviewing some of the basics
receptors, including the T-cell receptor (TCR), B-cell receptor, of kinase biochemistry.
and natural killer (NK) cell receptor and Fc receptors, trigger
signaling. As discussed in Chapter 4, the first step in signaling by STRUCTURE AND FUNCTION OF PROTEIN KINASES
multichain immune recognition receptors (e.g., the aforementioned
receptors) is tyrosine phosphorylation of the receptor itself and
adapter molecules, such as linker of activated T cells (LAT), KEY CONCEPTS
mediated by Src family protein tyrosine kinases (PTKs). This Kinase Families
leads to the recruitment of PTK members spleen tyrosine kinase
(Syk) and Zap70 followed by phosphorylation of adapters, such • 518 kinases in the genome
as SH2 domain-containing leukocyte phosphoprotein of 76 kDa • 90 protein tyrosine kinases (e.g., Janus kinases [JAKs])
(SLP-76) and the activation of Tec family PTKs. These initial • 400 protein serine/ threonine kinases:
steps trigger the activation of serine-threonine kinases, including • AGC kinase family (e.g., protein kinase B/AKT)
• CAMK kinase family (e.g., calmodulin-dependent kinase)
the mitogen-activated protein kinases (MAPKs) and protein kinase • CMGC kinase family (e.g., MAP kinases: ERK, JNK, p38)
C (PKC) family (Fig. 88.1). We now know a great deal of details • STE kinase family (e.g., MAPK kinases, MAPKK kinases)
about how the cascade of protein phosphorylation links events • TKL kinase family (e.g., IRAK)
at the plasma membrane to calcium modulation, cytoskeletal • Others: casein kinase family, GYC kinase family, IKK family
rearrangement, gene transcription, and other canonical features
of lymphocyte action (Chapter 12).
Similarly, a critical first step in signaling by many cytokine Protein kinases or phosphotransferases catalyze the transfer of
receptors is the activation of phosphorylation. The receptors for the γ-phosphate from a purine nucleotide triphosphate (i.e.,
classical growth factor cytokines, including stem cell factor and adenosine triphosphate [ATP] and guanosine triphosphate [GTP])
platelet-derived growth factor (PDGF) are receptor tyrosine to the hydroxyl groups of their protein substrates. They generate
kinases (RTKs), whereas the receptors for transforming growth phosphate monoesters by using protein alcohol groups (on serine
factor family cytokines are receptor serine–threonine kinases. and threonine residues) and/or protein phenolic groups (on
Type I and II cytokine receptors signal via the activation of tyrosine residues) as phosphate acceptors. Thus protein kinases
receptor-associated Janus kinases (JAKs; see below, Table 88.1 can be classified by the amino acid substrate preference: serine/
and Table 88.2) (Chapter 9). Other cytokines like interleukin threonine kinases, tyrosine kinases, and dual kinases (i.e., both
(IL)-1 and tumor necrosis factor (TNF) initiate signaling in a serine/threonine and tyrosine residues can be phosphorylated).

1185

1186 PART TEN Prevention and Therapy of Immunological Diseases





Antigen-presenting cell

CD80
MHC


TCR
CD28


CD3 PIP 3 ITK Ras RasGAP Ras PIP 3
Ick GTP GTP
fyn LAT PDK1
P Grb2 Sos RasGAP p110 p85
P SLP-76 GADS P
PLCγ1 P P
P P P P13’K
P
DaG
P
P ZAP-70 P IP 3
P’tn
PKC Raf PKB
T cell
Downstream serine/threonine
kinases and their effectors
FIG 88.1 The Proximal Signaling Events in Response to T-Cell Receptor (TCR) Activation
by an Antigen-Presenting Cell. Tyrosine and lipid kinases are indicated in red, and serine/ threonine
kinases are indicated in blue.




TABLE 88.1 Functions and Signaling of Cytokines Sharing the Common γ Chain
Cytokine
Receptor IL-2R IL-4R IL-7R IL-9R IL-15R IL-21R
Functions Control of peripheral Regulation of B-cell Thymocyte survival Goblet cell CD8 memory T-cell T- and B-cell
self-tolerance (mice) function (in concert and development hyperplasia survival and proliferation
with interleukin factor proliferation factor factor
[IL]-21 and IL-25)
Development and Immunoglobulin Peripheral T-cell Mucus Peripheral T cell Regulates
maintenance of class switching survival factor production survival factor immunoglobulin
regulatory T cells Mediates homeostatic Mediates homeostatic production (with
reconstitution of reconstitution of IL-4)
lymphopenic animals lymphopenic animals
Differentiation of Differentiation of T B-cell progenitor Natural killer (NK) cell NK cell proliferation
helper and cytotoxic helper cells (Th2 survival factor (mice) development, and activation
T cells lineage) differentiation and factor
survival factor
In vitro expansion Costimulant for
and differentiation growth in T, B, and
of antigen-selected mast cells
T and NK cells
Inhibition of Th1
differentiation and
macrophage
activation
Downstream PI3K, Ras MAPK, PI3K, Ras MAPK, PI3K, STAT(1), (3), 5 PI3K, Ras MAPK, PI3K, Ras MAPK, PI3K, Ras MAPK,
signaling STAT1,3,5 STAT6,5 STAT1,3,(5) STAT1,3,5 STAT1,3,(5)
pathways

CHAPTER 88 Protein Kinase Antagonists 1187



TABLE 88.2 The JAK Family of Almost all protein kinases have catalytic domains that belong
Tyrosine Kinases to a single eukaryotic protein kinase (ePK) superfamily. The
common evolutionary ancestry of the kinase domain (also known
Murine Phenotype as the catalytic domain), which consists of 250–300 amino acid
Associated With Gene Associated residues, manifests as a highly conserved three-dimensional
Gene Deletion Receptor structure. In the human genome, there are 518 kinases, which
Jak1 Perinatal lethality, block in Many including IL-7R are divided into eight major groups, in totality, representing
thymocyte development and IFNRs 1.7% of the human genome. The PTK family has 90 members,
Jak2 Embryonic lethality due to anemia Erythropoietin receptor one-third of which are RTKs, and the remainder are cytoplasmic
Jak3 T-, B-, and NK-cell lymphopenia, Common gamma proteins that typically function in close proximity to, and
SCID phenotype chain receptor downstream of, receptor–ligand complexes.
Tyk2 Failure to clear Toxoplasma, IL-12/23 receptor In terms of its catalytic role, the kinase domain has three
reduced arthritis
functions: the binding of the ATP (or GTP) phosphate donor
2+
2+
JAK, Janus kinase; IL, interleukin; IFNR, interferon receptor; NK, natural killer; SCID, as a complex with a divalent cation (usually Mg or Mn ), the
severe combined immune deficiency.

TABLE 88.3 Selected Kinase Inhibitors and Related Drugs

Mechanism Compound Kinase Inhibited Comments
Direct binding Imatinib Kit, platelet-derived growth Approved for treatment of chronic myeloid leukemia (CML), eosinophilic leukemia,
to the factor (PDGFR), and Abl and gastric stromal tumors
kinase Used in the treatment of chronic sclerodermatous graft-versus-host disease (GvHD)
Under evaluation in multiple types of cancer and in combination with other cancer
drugs
Ponatinib US Food and Drug Administration (FDA) approved for the treatment of CML and
PH + acute lymphoblastic leukemia (ALL)
Phase II trial for acute myeloid leukemia (AML) and a number of solid cancers
Nilotinib Approved for Philadelphia chromosome-positive CML; in Phase II trials for AML and
ALL
Radotinib PDGFR and Abl FDA approved for the treatment of CML
Dasatinib Multiple Src family tyrosine Approved for treatment of adult CML and Philadelphia chromosome positive ALL
kinases, including Abl Phase III trial for prostate cancer
Phase II trial for CLL, Sarcomas
Bosutinib Approved for the treatment of imatinib-resistant CML
Phase II trial for glioblastoma
Ibrutinib Btk, Itk FDA approved for the treatment of CLL, mantle-cell lymphoma (MCL),
lymphoplasmacytic lymphoma (LPL)
Phase III trial for the treatment of diffuse large B-cell lymphoma (DLBCL),
posttransplantation lymphoproliferative disease (PTLD), pancreatic cancer
Phase II trial for GvHD
Spebrutinib Phase II trial for RA, Phase I trial for CLL, non-Hodgkin lymphoma (NHL)
Acalabrutinib Phase III trial for CLL, phase II trial for rheumatoid arthritis (RA)
FDA approved for the treatment of mantle cell lymphoma (MCL)
Lapatinib ErbB-2 and epidermal growth Approved for treatment of breast cancer
factor receptor (EGFR) Minor survival improvement in head and neck cancer
kinases Phase III trials for brain metastasis
Afatinib FDA approved for treatment of non–small cell lung carcinoma (NSCLC)
Phase III trial for breast cancer and head and neck cancer
Neratinib Phase III trial for treatment of breast cancer
Phase II trial for NSCLC
Gefitinib Approved for use in NSCLC as a first-line therapy by FDA
Successful phase III study in esophageal cancer
Erlotinib HER1/EGFR, JAK2 Approved for treatment of pancreatic cancer and certain categories of NSCLC
In phase II and III trials for various other cancers, including myeloproliferative
disease
Vandetanib VEGFR-2, EGFR, RET, and Approved in 2011 for certain types of progressive medullary thyroid cancer
ErbB-1 tyrosine kinases Phase II trial for small cell lung cancer
Tivozanib Vascular endothelial growth Phase I trial for solid tumors
Lenvatinib factor receptor (VEGFR) FDA approved for thyroid cancer and under phase III trials for hepatocellular cancer
Regorafenib kinases FDA approved for the treatment of Colorectal cancer and gastrointestinal stromal
tumor (GIST)
Phase III trials for hepatocellular cancer
Brivanib VEGFR-2, FGFR Orphan drug status for the treatment of hepatocellular cancer
(N)intedanib VEGFR, PDGFR, FGFR Approved for the treatment of idiopathic pulmonary fibrosis and NSLCLC
adenocarcinoma
In phase III trials for the treatment of ovarian and colorectal carcinoma

Continued

1188 PART TEN Prevention and Therapy of Immunological Diseases



TABLE 88.3 Selected Kinase Inhibitors and Related Drugs—cont’d
Mechanism Compound Kinase Inhibited Comments
Cabozantinib VEGFR, MET, RET FDA approved for treatment of thyroid cancer
Phase III registration for hepatocellular cancer
Axitinib VEGFR1, 2 and 3, Abl Approved for treatment of renal cancer; has orphan drug status for follicular,
(T315I), PDGFR-β and Kit medullary, and anaplastic thyroid carcinoma and metastatic or locally advanced
papillary thyroid cancer
Effective in imatinib resistant CML associated with the T315I mutation
Sunitinib Inhibits multiple receptor For treatment of gastrointestinal stromal tumor after disease progression on or
tyrosine kinases (RTKs), intolerance of imatinib and for treatment of advanced renal cell carcinoma and
including PDGFR3-alpha, pancreatic neuroendocrine tumors
PDGFR-β, VEGFR1, Completed phase II trials in NSCLC and breast cancer
VEGFR2, VEGFR3, Kit, Flt3, Potential benefit in Flt3 + AML
CSF-1R, and RET
Sorafenib Dual-specific inhibitor FDA approved for treatment of advanced renal cell carcinoma, thyroid cancer, and
blocking both tyrosine and hepatocellular cancer
serine/threonine kinases, In phase II trials for AML, ALL, CML, myelodysplastic syndrome (MDS),
including RAF kinase, neurofibromatosis, portal hypertension
VEGFR-2, VEGFR-3,
PDGFR-B, KIT, Flt3, and
RET
Pazopanib Inhibits multiple RTKs, Approved for treatment of advanced renal cell cancer and soft tissue sarcoma
including PDGFR3-α, Mixed results as a treatment for age related macular degeneration when used
PDGFR-β, VEGFR1 topically
VEGFR2, VEGFR3, cKIT,
Lck, c-FMS, FGFR-1, and
FGFR-3
Entrectinib ALK, TrkA, TrkB, TrkC, ROS1 Phase II trial for colorectal cancer, NSCLC
Phase I trial for neuroblastoma
Crizotinib ALK, c-Met Approved for treatment of ALK-positive NSCLC
Phase II trial for anaplastic lymphoma kinase (ALK)–positive lymphoma
Filgotinib JAK 1 Completed phase IIb studies in RA
Completed phase II studies in Crohn disease
ABT-494 In phase III trial for RA and phase II trial for Crohn disease
Ruxolitinib JAK1 and JAK 2 FDA approved for myeloproliferative diseases
In trials for treatment of GvHD
Baricitinib Completed phase III trials for rheumatoid arthritis
Completed phase IIb trials in psoriasis
Gandotinib In phase II clinical trials for myeloproliferative disorders
(LY2784544)
Oclacitinib FDA approved for the treatment of canine allergic dermatitis
AC-410 In phase I clinical trials for autoimmune and myeloproliferative syndrome
Tofacitinib JAK3, JAK1, (JAK2) FDA approved for the treatment of RA
Completed phase III trials for the treatment of ulcerative colitis (UC)and psoriasis
Peficitinib Phase IIb for RA and UC
Decernotinib JAK 3 Completed phase III studies in the treatment of RA
Momelotinib JAK1, JAK2, IKKe, and TBK1 In phase III trials for pancreatic adenocarcinoma
TG02/ SB-1317 JAK2, Flt3, CDK1, -2, -7, -9 In phase I clinical trials for ALL, AML, CLL
Fedratinib JAK2, Flt3, RET In phase II trial for chronic beryllium disease
AT-9283 JAK2, JAK3, aurora A/B Completed phase I trial for solid tumors in children
kinase, highly active
against the Gleevec-
resistant T315l Abl
mutation
Lestaurtinib Flt3, TrkA, and JAK2 In phase III clinical trials for treatment of patients with AML who have an
Flt3-activating mutation at first relapse from standard induction chemotherapy
In phase II trials for psoriasis and pancreatic cancer

CHAPTER 88 Protein Kinase Antagonists 1189



TABLE 88.3 Selected Kinase Inhibitors and Related Drugs—cont’d
Mechanism Compound Kinase Inhibited Comments
Quizartinib Class III receptor tyrosine Completed phase II clinical trial for drug-resistant AML
kinases (Flt3, Kit, CSF1)
Fostamatinib Syk, Flt3 Phase III for idiopathic thrombocytopenic purpura (ITP)
Phase II for immunoglobulin A (IgA) nephropathy
Failed phase III for RA, phase I for GvHD
PRT-062607/ Syk In phase I clinical trials for RA, CLL, NHL; preclinical for systemic lupus
BIIB057/ erythematosus (SLE)
P505-15
R-348 JAK/Syk In phase II trials for ocular GvHD
Vemurafenib BRAF FDA approved for the treatment of BRAF mutated melanoma; phase II trial for
colorectal cancer and myeloma
Dabrafenib FDA approved for the treatment of BRAF mutated melanoma
Selumetinib MKK family Phase III trial for NSCLC and thyroid cancer
Phase II for colorectal cancer and biliary cancer
Cobimetinib FDA approved for the treatment of melanoma
Phase II trial for the treatment of breast cancer and Langerhans cell histiocytosis
Trametinib FDA approved for the treatment of BRAF mutated melanoma in combination with
dabrafenib
Binimetinib Phase III trial for the treatment of BRAF mutated melanoma
PD-325901 Phase II trial for neurofibromatosis
CC 90003 ERK Phase I trial for solid tumors
XG102 JNK Phase III trial for ocular inflammation and sensorineural hearing loss
RES-529 AKT, MTOR, VEGFR Phase I trial for age-related macular degeneration
Uprosertib AKT Phase II trial for AML, solid tumors
MK-2206 Phase II trial for solid and hematological malignancies
AZD-5363 Phase II trial for breast and gastric cancer
Pexidartinib CSF-1R, Kit Phase II trial for pigmented villonodular synovitis
AZD 2014 MTORC1 and C2 Phase II trial for DLBCL and solid tumors
CC 223 Phase II trial for NHL and solid tumors
Sotrastaurin PKC Phase II trial for DLBCL
Ribociclib CDK4,6 Phase III trial for breast cancer
Phase II trial for gastrointestinal cancer
Palbociclib FDA approved for the treatment of breast cancer
Phase III trial for NSCLC
Buparlisib PI3K Phase III trial for breast cancer
Bardoxolone IKbK, COX2, NOSII, STAT3 Phase II trial for diabetic neuropathy and pulmonary hypertension
Fasudil ROCK1/ ROCK2 Phase III for Raynaud phenomenon and scleroderma completed
KD-025 ROCK2 Phase II for psoriasis vulgaris
Phase II for idiopathic pulmonary fibrosis
Indirect Sirolimus mTORC1 FDA licensed for use in solid organ and bone marrow transplantation
binding to (Rapamycin)
kinase Everolimus FDA licensed for use with cyclosporine in cardiac and renal transplantation
FDA licensed for use in neuroendocrine tumors and renal cell carcinoma
Temsirolimus FDA licensed for use with MCL, renal cancer
Ridaforolimus Phase II trial for coronary artery restenosis
Monoclonal Trastuzumab EGFR FDA licensed for use in HER2/neu positive breast carcinoma and metastatic gastric
antibodies cancer
binding to Cetuximab FDA licensed for use in relapsed colorectal and head/neck cancers
receptor Bevacizumab VEGF—prevents binding to FDA licensed for use in metastatic breast and colorectal cancers, glioblastoma, and
tyrosine its receptor (Flt-1) NSCLC (non-squamous cell histology) in combination with traditional
kinases chemotherapy
Other Barasertib Aurora kinase B (functions in Disrupts mitosis and cellular division in tumor cells; in phase III clinical trials for
mechanisms the attachm ent of the AML
of action mitotic spindle to the
centromere)

1190 PART TEN Prevention and Therapy of Immunological Diseases


binding of the protein substrate, and the transfer of the Phosphorylated active
γ-phosphate from ATP or GTP to the protein substrate. Despite loop tyrosines
the huge number of serine/threonine and tyrosine kinases, there
is evidence of a common ancestor, and this is reflected in structural
similarities, particularly in the active (ATP bound) confirmation.
The major kinase domains of all typical protein kinases consists N-lobe
‘Gatekeeper’
of two lobes (N-lobe and C-lobe) that surround the nucleotide active site residue
1
binding site (Fig. 88.2). The smaller N-lobe consists of a cluster
of β-pleated sheets with a single α helix. The larger C-lobe is Hinge region
made up of α helices. Within the C-lobe lies the substrate-binding Inhibitor
site, typically a groove on the surface. A hinge region connects
the two lobes. The hinge together with two loops emerging from
each lobe form the ATP-binding pocket: the primary target for C-lobe
most kinase inhibitors. In many protein kinases, a loop emerging
from the C-lobe must be phosphorylated for the kinase to be
fully active (see Fig. 88.2). This is known as the activation loop.
Substrates of PTKs often include the activation loop of down-
stream kinases, creating signaling cascades of proteins, which,
in turn, phosphorylate each other; examples include the MAPKs FIG 88.2 Crystal Structure of the Janus Kinase 3 (JAK3)
(Fig. 88.3). Domain Complexed With Staurosporine (pdb Accession Code
1YVJ). This structure captures the active conformation of JAK3
with both active loop tyrosine residues phosphorylated (green).
THE DISCOVERY OF KINASE INHIBITORS The molecule can be described as halves, with the N-terminal
Given that protein kinases bind ATP, the notion that therapeuti- lobe presented in blue and the C-terminal domain in red. These
cally useful kinase inhibitors could be generated was initially are linked by a hinge region that forms part of the active site.
met with some skepticism. First, as there are more than 500 Highlighted in purple within the active site is the gatekeeper
human kinases, many of which serve critical cellular functions, residue. Bound within this site is an analogue of the inhibitor
would it really be possible to attain the specificity needed? Second, staurosporine, and its proximity to the “gatekeeper” residue
protein kinases are not the only kinases—there are lipid kinases highlights why this residue and this region are critical for the
and nucleotide kinases, as well as many other ATP-binding specificity of inhibitors for individual protein kinases.











Activator Ras-GTP TRAF6


MAPKKK c-Raf1 MEKK1 TAK1


MAPKK MKK1 SEK1 MKK6


MAPK ERK1 JNK1 p38 MAPK


Substrates p90-RSK p90-RSK

P P
P P
EIK1 P c-fos P P P P P P
SRF c-fos c-Jun ATF2
SRF ATF2

SRE AP-1 AP-1
FIG 88.3 A Summary of the Mitogen-Activated Protein Kinase (MAPK) Signal Transduction
Pathways. Examples of receptors that activate Ras include the interleukin (IL)-2 and T-cell receptor
(TCR). Examples of receptors that activate tumor necrosis factor receptor–associated factor 6
(TRAF6) include the IL-1 receptor.

CHAPTER 88 Protein Kinase Antagonists 1191


proteins, all of which share structural similarities with PTKs. which binds directly to a methyl group of the phenyl ring of the
4
Third, despite the many potential ways of designing a small- Abl kinase inhibitor imatinib. Across the collective kinase
molecule kinase inhibitor, in practice, the majority work by sitting superfamily, almost any amino acid can appear as the gatekeeper,
2
within the ATP-binding pocket. A priori then, one might conclude although in practice, it is typically a bulky nonpolar residue
5
that it would be impossible to generate an antagonist that did (methionine, tyrosine, phenylalanine, lysine). In principle, as
not target some other essential ATP-dependent process. Fortu- more detailed structural information emerges from the many
nately though, this skeptical view does not reflect reality. protein kinases, capitalizing on subtle differences in structure is
expected to lead to the discovery of novel agents with improved
THERAPEUTIC PRINCIPLES potency and specificity. For instance, cyclin-dependent kinase 2
(CDK2) contains an additional pocket on its C-lobe next to the
Protein Kinase Inhibitors As Drugs ATP-binding pocket. Several CDK2-specific inhibitors exploit
6
this by binding to both pockets.
• Drugs can inhibit protein kinases with a high degree of specificity.
• Multi-kinase inhibitors can be well tolerated and be more efficacious Of further structural significance is the emergence of tumor
than single kinase inhibitors (e.g., second-generation Abl kinase drug resistance in response to the chronic use of protein kinase
inhibitors). inhibitors. Mutant forms of BCR-Abl, Kit, and epidermal growth
• Conversely, unacceptable toxicity has limited the creation of clinical factor receptor (EGFR) have been associated with loss of drug
inhibitors to particular kinase members (e.g., p38 MAPK inhibitors). activity and disease relapse. Interestingly, one of the most common
sites of mutation is the otherwise conserved “gatekeeper residue.”
The appearance of such mutations that cause resistance to imatinib
Imatinib and Other First-Generation Protein Tyrosine have led to the need for other drugs with broader activity against
Kinases Inhibitors a number of kinases. Thus in the setting of oncology, “multikinase”
The first protein kinase inhibitor approved by the US Food and inhibitors, including dasatinib and sunitinib, have now entered
Drug Administration (FDA) is imatinib (see Table 88.3). The clinical practice and have been approved by the FDA. Although
mutated form of the Abl tyrosine kinase BCR-Abl represents a a major problem in the treatment of malignancy, this is less
fusion protein that is the result of a chromosomal translocation likely to be an issue in the treatment of autoimmune disease;
(Philadelphia chromosome) observed in patients suffering from nonetheless, drugs used for oncological indications often end
chronic myeloid leukemia (CML). The pathognomonic presence up being quite useful in the treatment of autoimmunity. Such
of BCR-Abl in CML has led to it becoming one of the most precedents include cyclophosphamide, azathioprine, and metho-
intensively studied PTKs. The fusion protein consists of an trexate. Therefore it is not unreasonable to speculate that a number
oligomerization domain, a PH domain, and a Dbl/cdc24 guanine of the kinase inhibitors developed as anticancer agents may
nucleotide exchange factor homology domain that contains the ultimately be used to treat inflammatory or immunological
N-terminal breakpoint cluster region (BCR) of the protein. The diseases.
Abl half of the fusion protein contains a tyrosine kinase domain, An unexpected finding for a clinically well-tolerated inhibitor,
a Src homology 2 (SH2) domain, and a DNA-binding domain, which was originally thought to be a highly specific inhibitor
together with nuclear localization and nuclear export motifs. for Abl kinase, was the realization that imatinib has activity
3
The Abl kinase is constitutively active within the fusion protein against several other PTKs. Consequently, it has been found to
and has been implicated in initiating numerous signaling pathways be useful in the treatment of a number of cancers that do not
that mediate cell survival and proliferation. In view of this, and have abnormal Abl kinase activity. Imatinib has been used to
the essential requirement for BCR-Abl kinase activity in CML, treat gastrointestinal (GI) stromal tumor and hypereosinophilic
it was thought to be an ideal target despite the aforementioned syndrome through its effects on Kit and PDGFR-FIPIL1 kinases,
caveats with targeting protein kinases. As predicted, imatinib respectively. In spite of efforts to develop highly specific kinase
has revolutionized the treatment of CML. This inhibitor has inhibitors, there is increasing evidence that a partial inhibition
been remarkably successful in arresting the progression of the of multiple kinases is potentially less toxic than originally feared
CML but is also well tolerated with acceptable side effects. 3 and may be important for the efficacy of many inhibitors.
Although conservation of the kinase ATP-binding pocket has Second-generation Abl inhibitors, including dasatinib and
posed a potential problem for designing kinase inhibitors, in bosutinib, are less selective than imatinib. This lack of specificity
practice, this has not happened for several reasons. Although may contribute to their improved response rates in the treatment
7
different kinases may be structurally similar in an active ATP- of CML. With respect to immune-mediated diseases, imatinib
bound conformation, the inactive conformation is substantially is used in the clinic for the treatment of fibrotic diseases, including
4
less frequent and can be used to generate selective inhibitors. skin fibrosis associated with chronic graft-versus-host disease
8
The ATP-binding region is made up of six polar amino acid (GvHD). At the time of writing, there are >20FDA-approved
residues that are invariant across whole families of kinases; small molecule kinase inhibitors, most which are approved for
similarly, there are numerous lipophilic residues that are highly oncological indications. In addition, there are numerous other
conserved. In addition, this critical region contains an amino kinase inhibitors in clinical trial or development (summarized
acid whose amide carbonyl binds to N-6 of adenine in the active in Table 88.3). Besides small-molecule direct inhibitors of kinases,
conformation. The side chain of this amino acid sticks into the which typically block the ATP-binding pocket, there are several
reaction pocket in the inactive state and, for this reason, is referred alternative strategies. These include small inhibiting RNA to
5
to as “the gatekeeper residue.” As the side chain is not involved block protein expression of the kinase Syk (Excellair; ZaBeCor
in direct ATP binding, it varies across kinases, and variation of Pharmaceutical Co., Bala Cynwyd, PA), inhibition of associated
this gatekeeper residue is exploited by a number of inhibitors proteins required to activate the target kinase as in the rapamycin
that are able to bind the inactive conformation of specific kinases. derivatives and the use of monoclonal antibodies (mAbs) that
In the case of Abl kinase, the gatekeeper residue is threonine, inhibit ligand-dependent activation of transmembrane receptor

1192 PART TEN Prevention and Therapy of Immunological Diseases


kinases. These mAbs include and target the designated RTK: identifying mutations in patients with immunodeficiencies.
bevacizumab (vascular endothelial growth factor receptor Specifically, mutation of JAK3 results in a severe combined
[VEGFR]), ranibizumab (VEGFR), cetuximab (epidermal growth immune deficiency (SCID), characterized by an almost complete
factor receptor [EGFR]), pertuzumab (human epidermal growth absence of T cells and NK cells with defective B cells. This
factor receptor [HER]), and trastuzumab (HER2/neu). phenocopies deficiency of the cognate receptor that associates
with JAK3, the IL-2 receptor common γ chain, cγc (encoded by
IL2RG), mutation of which underlies X-SCID (see Table 88.1;
TARGETING CYTOKINE SIGNALING BY INHIBITING Fig. 88.4). The profound, but selective, phenotype associated
JANUS KINASES: TOFACITINIB, RUXOLITINIB, AND with JAK3 deficiency led to the suggestion that targeting JAKs
RELATED COMPOUNDS might be a strategy for the development of a new class of
immunomodulatory drugs. There are now several FDA-licensed
Cytokines regulate growth, survival, development, and differentia- JAK inhibitors for the treatment of immune and neoplastic
tion of immune cells. Their importance in driving inflammatory diseases, and more are in clinical trials.
and immunological responses has already made them attractive Tofacitinib, formerly designated CP-690,550, was one of
targets as antiinflammatory and immunosuppressive agents. As the first JAK inhibitors to enter the clinic. It inhibits JAK3 and
9
indicated above, a large subset of cytokines (roughly 60) signal JAK1 and to a lesser extent JAK2, but has little effect on TYK2.
through Janus kinases (JAKs) (see Table 88.2). The essential Consequently, tofacitinib potently inhibits cγc cytokines but also
function of JAKs was documented by knock-out mice and by blocks interferon (IFN)-γ, interleukin (IL)-6, and to a lesser extent


Daclizumab
IL-2R
αβ γ
Tofacitinib
IL-2
Tipifarnib
Ras Jak 3
Sorafenib PIP 3
GTP Sos Grb2 Jak 1 PDK1
Shc p110
p85
Raf Ick P
P13’K PKB
MEK
P Other substrates -
leading to
STAT P cell survival
MAPK pathway
TSC1 TSC2 AMPK
STAT P P STAT
Rheb
Sirolimus
Cell proliferation
and survival Raptor FKBP12
mLST8 mTOR


4EBP1 S6K1 PDK1



eIF4F S6
Initiation of protein Maintenance of protein
translation translation

Protein translation
leading to cell growth

FIG 88.4 Signal Transduction Pathways Stemming From the Interleukin (IL)-2 Receptor in
T Cells Culminating in the Activation of the Mammalian Target of Rapamycin (mTOR)
Serine/Threonine Kinase. Tyrosine kinases are indicated in red, and serine/ threonine kinases
are indicated in blue.

CHAPTER 88 Protein Kinase Antagonists 1193


IL-12 and IL-23. Functionally, tofacitinib inhibits T-helper 1 (Th1) of kinases is exemplified by the fact that the mutations of BTK
9
cells and Th2 differentiation, as well as pathogenic Th17 cells. underlie Bruton agammaglobulinemia a condition characterized
In addition to inhibiting adaptive immune responses, tofacitinib by the absence of all B cells (Chapter 34). Ibrutinib is the first
9
appears to inhibit innate immune responses as well. Tofacitinib BTK inhibitor approved by the FDA in 2014 for the treatment
passed phase III trials for the treatment of methotrexate-resistant of mantle cell lymphoma and chronic lymphocytic leukemia
14
rheumatoid arthritis (RA) and was FDA approved for this indica- (CLL). Recent work has identified ibrutinib as an inhibitor of
10
tion in 2012. It completed phase III trials in both ulcerative Itk, which suggests it may have value as therapy in autoimmune
11
colitis and psoriasis with successful results. Other diseases in disease. Antigen receptor activation of phospholipase C (PLC)γ1
which tofacitinib is being studied include ankylosing spondylitis appears to require Lck, Zap70, and Tec kinases working in concert.
and juvenile idiopathic arthritis. Tofacitinib has been used in the The action of PLCγ1 leads to elevation in intracellular calcium,
11
treatment of alopecia areata, vitiligo, and atopic dermatitis. which in turn activates the phosphatase calcineurin. Calcineurin
A variety of additional inhibitors that target JAK3, including dephosphorylates and activates nuclear factor of activated T cells
decernotinib, peficitinib, and R-348 (Rigel), are under develop- (NFAT), which translocates to the nucleus and in cooperation
ment or in clinical trials (see Table 88.3). with activator protein-1 (AP-1) transcription factors induces
As gene targeting of Jak2 in mice was embryonically lethal, transcription of IL-2 and other key lymphocyte activation genes. A
it was initially thought that inhibition of JAK2 should be avoided. number of potent clinically successful immunosuppressive drugs
However, the discovery that gain-of-function mutations of JAK2 inhibit calcineurin, including cyclosporine and tacrolimus, drugs
15
underlie primary polycythemia and myelofibrosis led to the idea that have revolutionized organ transplantation. Despite their
that pharmacologically targeting JAK2 could be useful. The JAK1/ success, long-term use of these drugs is limited, as they may
JAK2 inhibitor ruxolitinib was the first JAK inhibitor for the cause renal toxicity
treatment of myelofibrosis to be approved by the FDA in 2011
and was subsequently approved for the treatment of primary CLINICAL PEARLS
11
polycythemia in 2014. As cγc cytokines employ both JAK1 and
JAK3 for signaling, it might be expected that ruxolitinib and Mutations Reveal Key Functions of Kinases in
tofacitinib might block some of the same cytokines. It is therefore Patients With Primary Immunodeficiencies
of interest to note that in a phase II study in RA, ruxolitinib was
12
as efficacious as tofacitinib. A number of novel JAK1/JAK2 • Common γ-chain deficiency, JAK3 deficiency: severe combined
immunodeficiency
inhibitors under development both for the treatment of myelo- • ZAP70 deficiency: severe combined immunodeficiency
proliferative and inflammatory diseases. The JAK1/JAK2 inhibitors • TYK2 deficiency: rare cause of hyper-IgE syndrome
baricitinib, filgotinib, and AC-410 (Daiichi Sankyo, Tokyo, Japan)
all are in trials for the treatment of RA (see Table 88.3).
The side effects of JAK inhibitors include infection, including Protein Kinase C Family and NF-κB
serious infections, anemia, and leukopenia, presumably related TCR signaling also leads to the activation of members of the
to JAK2 inhibition and interference with cytokines, such eryth- PKC family, which, in turn, activate the transcription factor
11
ropoietin, IL-11, and colony-stimulating factors. Little reduction complex nuclear factor (NF)-κB and the Ras guanyl nucleotide-
in CD4 T cells has been seen, but significant reduction in NK releasing proteins (RasGRPs). Genetic studies in mice have
cells and CD8 T cells does occur; just how significant this will identified the importance of one member, PKC-θ in TCR signal-
be in terms of infection risk remains to be determined. 11 ing. Recent work has identified PKC-θ as an inhibitor of regulatory
16
T cell function. Regulatory T cells (Tregs), unlike conventional
TARGETING ANTIGEN RECEPTOR SIGNALING effector T cells, act to constrain the immune system. Enhancing
Treg numbers or function has been suggested as the treatment
17
The first event in TCR signaling is the activation of the Src of a number of autoimmune diseases. For this reason, preserving
family kinase Lck (see Fig. 88.1), making it an attractive target or enhancing Treg function through the inhibition of PKC-θ is
as a therapy for autoimmune diseases and transplant rejection. an attractive strategy. Along with many other receptors, TCR
Several Lck inhibitors have been developed and showed promise signaling leads to the action of NF-κB family transcription factors,
in preclinical models of allograft rejection and autoimmunity, which control the genes involved in cellular activation and
18
although at the cost of inducing progressive lymphopenia. This resistance to apoptosis. In T cells, PKC-θ is the main isoform
is consistent with the finding that induced deficiency of Lck in responsible for NF-κB activation. The novel PKC inhibitor
13
mice leads to a progressive lymphopenia. Despite considerable Sotrastaurin, a potent inhibitor of the PKC-θ, β and α isoforms,
effort, the discovery of a selective Lck inhibitor suitable for use as is currently undergoing phase II trials for the treatment of diffuse
an immunosuppressive agent remains elusive. Lck activation leads large B-cell lymphoma. Early clinical testing on patients with
to the recruitment of a second round of tyrosine kinases to the psoriasis demonstrated that the drug is well tolerated and was
TCR complex; these include Zap70 or Syk. Deficiency of Zap70 able to inhibit T-cell proliferation, IL-2, and IFN-γ secretion.
causes SCID and preferential loss of CD8 T cells, but curiously, However, despite initial positive trial results, it was not effective
a successful Zap70 inhibitor has yet to be obtained. In contrast, in the treatment of psoriasis, ulcerative colitis, or liver transplanta-
Syk inhibitors have been generated; BIIB057 (Biogen, Cambridge, tion. Downstream of several inflammatory signaling pathways,
MA) is being investigated for the treatment of inflammatory including PKC, lies the nuclear factor kappa-light-chain-enhancer
18
diseases, and a second, fostamatinib, is being investigated for the of activated B cells (NF-κB). NF-κB has been implicated as a
treatment of immune thrombocytopenia and immunoglobulin major mediator governing both cancer cell proliferation and
A (IgA) nephropathy. The recruitment of Zap70/Syk results in survival and governing chronic inflammation. It is held in an
activation of another class of PTKs, the Tec family kinases, Rlk inactive state by the binding of the inhibitor of κB (IκB). A
and Itk in T cells and Btk in B cells. The importance of this class cascade of protein kinases that include IκB kinases (IKK) and

1194 PART TEN Prevention and Therapy of Immunological Diseases


23
IKK kinases (IKKKs), of which PKC and PKB are members, of p70 S6K1 and inactivation of 4E-BP1, which are critical for
18
regulate IκB. Several IKK inhibitors have been in development; translation of new proteins.
however, all of them have failed clinical trials. As its name suggests, mTOR is inhibited by the macrolide
rapamycin, now licensed as the drug sirolimus for the treatment
Lipid Kinases and Downstream Signaling of graft rejection. Sirolimus does not inhibit mTOR by directly
Kinases are also important in phosphorylating lipids, and these binding to the ATP-binding pocket but acts indirectly, associating
modifications are very relevant in signal transduction through with FK506-binding protein 12 (FKBP12). This, in turn, inhibits
both the TCR and cytokine receptors. In addition to the produc- the kinase complex made up of mTOR, mLST8, and raptor
23
tion of inositol triphosphate by the action of PLCγ1, there is a (mTORC1). It was hoped that sirolimus would be a potent
second pathway of inositol lipid metabolism regulated by the anticancer drug, but it has met limited success in this regard. In
TCR that is a response shared by costimulatory molecules, such contrast, it has been successfully used as an immunosuppressant,
as CD28, cytokines, and chemokines. This response is mediated typically as part of a combination regimen for allograft rejection
24
by the class I group of phosphatidyl-inositol 3 kinases (PI3Ks), prophylaxis. In view of the ubiquitous expression of mTOR
which is composed of four isoforms (PI3Kα to -δ), which and its role in protein translation, it is not surprising that sirolimus
phosphorylate the 3’-OH position of the inositol ring of would be associated with a number of side effects, including
phosphatidyl-inositol (4,5) bisphosphate (PI(4,5)P 2 ) to produce hyperlipidemia, hypertriglyceridemia, myelosuppression, and
19
PI(3,4,5)P 3 . This lipid and its metabolite PI(3,4)P 2 bind to the delayed wound healing. There is some evidence of renal toxicity,
pleckstrin homology (PH) domains of proteins and either induce but this is minor compared with that caused by the calcineurin
localization of the protein to defined areas of the plasma mem- inhibitors cyclosporine and tacrolimus. Myelosuppression associ-
brane, where activation can occur, or induce conformational ated with sirolimus is typically dose related and rapidly reversible,
changes that allow for allosteric modifications of activity. Targets even in patients receiving the drug for chronic GvHD.
for D-3 phosphoinositides in T cells include a number of There are currently three rapamycin derivatives, namely,
downstream protein serine/threonine kinases and the Rac-1 and temsirolimus, everolimus, and AP23573, undergoing clinical
RhoA guanine nucleotide exchange proteins. Small-molecule trials. Everolimus is licensed for use in the management of heart,
inhibitors of PI3’K, Wortmannin and LY294002, are both potent liver, and kidney transplantation in conjunction with cyclosporine.
inhibitors of T-cell activation, although toxicity prevents either It is under investigation for the treatment of arthritis and a
from being clinically useful. In contrast to the more widely number of solid tumors. Temsirolimus is licensed for the treatment
expressed PI3’Kα and β, PI3’Kγ and -δ are only expressed in of renal cell carcinoma and mantle cell lymphoma. The introduc-
hematopoietic tissue and deletion of PI3’Kγ results in defective tion of novel rapamycin derivatives has been joined by the
migration of neutrophils and macrophages to sites of inflam- introduction of pan-mTOR direct inhibitors AZD2014 (Astra-
mation without other pathology. This limited expression makes Zeneca, Cambridge, UK) and CC-223 (Celgene, Summit, NJ),
PI3’Kγ a potentially useful target, and selective PI3’Kγ inhibitors both of which are in phase II trials for the treatment of non-
have shown to be effective in mouse models of collagen-induced Hodgkin lymphoma and solid tumors.
20
arthritis. For similar reasons, PI3’Kδ is another attractive target
for which the inhibitor idelalisib was FDA-approved for the MAPK Pathways
21
treatment of lymphoma. Over 25 other inhibitors of PI3’K are The MAPK family constitute a complex phospho-relay system
currently being studied in preclinical and clinical trials and include of signal transduction, composed of three sequentially activated
25
buparlisib, which is in a phase III trial for the treatment of breast kinases that are themselves modulated by phosphorylation.
cancer. Substrates of MAPK pathways include transcription factors,
One PI(3,4,5)P 3 -regulated kinase activated by the TCR is phospholipases, cytoskeletal proteins, and other protein kinases.
22
protein kinase B (PKB/AKT). T cells stimulated in the presence Three main MAPK cascades have been identified in mammalian
of the PI3’K inhibitors Wortmannin or LY-294002 fail to activate cells: the extracellular signal regulated kinase (ERK) cascade, the
and proliferate. The ability of cells to take up nutrients and Jun kinase (JNK) cascade, and the p38 MAPK cascade. All start
switch on glycolysis is essential for lymphocyte activation; PKB with a membrane-localized activator followed by three MAPKs
is proposed to be the main effector that mediates the action of that sequentially phosphorylate each other (see Fig. 88.3). The
PI3’K on these fundamentally important metabolic pathways. top level of kinases is termed MAPK kinase kinases (MAPKKKs,
There are three PKB isoforms (PKB-α, -β, -γ, or AKT1, -2, -3, MKKKs, or Map3Ks). The middle level MAPK kinases (MAPKKs,
respectively), and T cells lacking both Akt1 and Akt2 are greatly MKKs, or Map2Ks) phosphorylate a common Thr-Xaa-Tyr motif,
22
impaired. Specific inhibitors of PKB are in clinical trials with where Xaa is any amino acid. The lowest tier consists of the
mixed results; MK-2206 (Merck, Kenilworth, NJ), AZD-5363 MAPKs that phosphorylate Ser/Thr-Pro motifs. Each pathway
(Astra Zeneca, Cambridge, UK), and uprosertib are all in terminates in the phosphorylation of substrate proteins.
phase II trials for the treatment of several malignancies (see
Table 88.3). The ERK Cascade
In addition to glucose metabolism, the PI3’K-regulated serine/ ERK1 and 2 were identified as kinases that were activated in
threonine kinase mammalian target of rapamycin (mTOR) response to growth factor stimulation, which are mimicked by
regulates protein synthesis in response to cellular nutrient and expression of constitutively active Ras. The link between active
23
energy levels. It is activated not only by a number of growth Ras and subsequent phosphorylation of the ERKs was made
factor receptors but also by cytokines, including IL-2. Many both by the discovery of the MAPK kinase MEK1 and its phos-
signaling pathways link growth factor receptors with activation phorylation by the known Ras effector RAF1, now known as a
of mTOR, including the adenosine monophosphate (AMP)– MAPK kinase kinase (M3K). The ERK cascade is ubiquitous in
dependent kinase (AMPK) and phosphatidyl inositol 3’ kinase mammalian cells and is generally considered one of the main
(PI3’K) (see Fig. 88.4). mTOR promotes cell growth by activation effector pathways regulated by the GTPase p21ras. Activation of

CHAPTER 88 Protein Kinase Antagonists 1195


the TCR or the IL-2 receptor is able to trigger ERK signaling in has been frustrated by either unacceptable toxicity or poor efficacy.
25
T cells. Two small-molecule inhibitors of this serine/threonine Currently, a new generation of p38 MAPK inhibitors are being
kinase pathway are undergoing clinical trials: the farnesyl inhibitor assessed in phase II trials for the treatment of chronic obstructive
of Ras tipifarnib, which is in phase III trials for the treatment pulmonary disease (COPD); losmapimod has recently failed
of acute myeloid leukemia; and the multikinase inhibitor phase II testing because of lack of efficacy, and AZD7624
sorafenib, which is FDA approved for the treatment of renal cell (AstraZeneca, Cambridge, UK) and acumapimod are still being
26
carcinoma. Sorafenib is relatively well tolerated despite its ability investigated.
to inhibit numerous kinases, including the M3K RAF, as well as
receptor tyrosine kinases, including platelet-derived growth factor CONCLUSIONS
receptor (PDGFR), VEGFR, Kit, and FLT-3. Its role as an immu-
nosuppressant has yet to be explored. There are three RAF
isoforms: A-RAF, B-RAF, and C-RAF. A number of cancers are ON THE HORIZON
associated with B-RAF mutations resulting in a constitutively • Many kinase inhibitors, initially designed as antitumor agents, have
active kinase, the best known of which is the V600E mutation. been repurposed as immunosuppressants (e.g., ruxolitinib).
Two kinase inhibitors, vemurafenib and dabrafenib, which are • Further understanding of the pathophysiology of autoimmune disease
highly active against the B-RAF V600E kinase, are FDA licensed will lead to the increased use and creation of kinase inhibitors.
for the treatment of melanoma. Their early use was associated • Increasing use of specific kinase inhibitors may improve our understand-
with a partial and short-lived inhibition of the MAPK pathway ing of the pathophysiology of autoimmune disease.
as C-RAF signaling was preserved, which is often hyperactivated
by upstream mutations in Ras signaling or by the presence of Scientific advances in the 1990s have led to the discovery of
B-RAF V600E, which is able to dimerize and activate C-RAF many novel intracellular signaling pathways that link membrane-
27
even in the presence of inhibitors. Moreover, clinical trials are bound receptor and cytokine signaling with alteration of gene
ongoing for the treatment of the histiocytosis known as Erdheim- expression and cellular activation necessary to trigger an immune
Chester disease, for which B-RAF mutations have shown to be cell response. Many of these pathways are interlinked to make
present in the vast majority of the case. up a complex array of networks composed of enzymes, adaptor
The M2K MEK1 and the MAPK ERK1 lie downstream of proteins, and transcription factors, all of which are potential
RAF. Cobimetinib and trametinib are selective MEK inhibitors targets for drug discovery in the quest to make a therapy that
that are FDA licensed for the treatment of melanoma both as a will be able to treat a specific autoimmune disease without an
single agent and in combination with B-RAF V600E inhibitors unacceptable degree of immunosuppression. Now more than a
28
as they are able to block residual C-RAF signaling. Although decade since the identification of many new targets, the first
all of these agents are seen as anticancer drugs rather than generation of drugs designed to interfere with specific immune
immunosuppressants, it is of note that cobimetinib is currently cell signals are being brought to the clinic. The success of the
being assessed in the treatment of Langerhans cell histiocytosis anticancer BCR-Abl inhibitor imatinib and the immunosup-
(LCH). LCH is a clonal disorder characterized both by the B-RAF pressive mTOR inhibitor sirolimus has placed the protein kinases
V600E mutation in histiocytes and by the presence of inflam- center stage as targets of future drug discovery. As many of the
matory lesions that often respond to simple corticosteroid therapy. key steps in the activation of an immune cell are often shared
Selective inhibitors of ERK have been developed: FR180204 has with those that allow a cancer cell to proliferate, many of these
been shown to inhibit the development of collagen-induced agents are being tested as anticancer drugs rather than as
arthritis in mice and is being considered as an agent in the immunosuppressants. Despite this, some agents, such as the
treatment of RA. CC90003 (Celgene, Summit, NJ) is currently mTOR inhibitors, originally intended for the treatment of cancer
being assessed in a phase I trial in patients with solid tumors. have been far more successful in the field of immunology, and
this may continue to be true for future modifiers of cell signaling.
The JNK Cascade Conversely, JAK inhibitors could potentially be used in the
Another limb of the MAPK pathway is the JNK pathway. Many treatment of lymphoma. Either way, we are likely to see a large
inflammatory agents, including lipopolysaccharide (LPS), TNF-α, number of novel immunosuppressants appear both serendipi-
25
and IL-1 are able to activate the JNK pathway. In synoviocytes, tously and intentionally as new protein kinase inhibitors are
this results in secretion of proteases implicated in joint destruction licensed for a wide range of debilitating illnesses.
29
seen in RA. A number of small-molecule inhibitors of JNKs
have been identified and are currently being investigated in the Please check your eBook at https://expertconsult.inkling.com/
treatment of inflammation, cancer, and neurological diseases. for self-assessment questions. See inside cover for registration
XG102 (Xigen Ltd., Bedford, UK) is being assessed in phase III details.
trials for the treatment of ocular inflammation and sensorineural
hearing loss.
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billion or so and call me in the morning. Immunity 2009;30:656–65.

CHAPTER 88 Protein Kinase Antagonists 1196.e1


MULTIPLE-CHOICE QUESTIONS

1. Specific inhibitors of individual protein kinases are possible 2. The first clinically successful direct tyrosine kinase inhibitor
because: was:
A. Most kinase inhibitors are noncompetitive covalent A. Ruxolitinib
inhibitors. B. Infliximab
B. Protein kinases can be divided into families of serine, C. Imatinib
threonine, and tyrosine kinases that are structurally very D. ZAP70
dissimilar. E. Methotrexate
C. The adenosine triphosphate (ATP)–binding pocket of 3. Patients deficient in the following protein kinases suffer from
kinases are sufficiently distinct to allow generation of a severe combined immunodeficiency (SCID):
selective inhibitor. A. BCR-Abl
D. Most kinase inhibitors avoid the structurally similar ATP- B. JAK3
binding pocket. C. PTEN
E. Distinct kinases occupy different parts of the cell and do D. TYK2
not interact with each other.
E. BTK

89







Biological Modifiers of
Inflammatory Diseases



W. Winn Chatham







During recent decades, the elucidation of specific inflammatory differentiation and proliferation may therefore provide novel
mediators and identification of lymphocyte lineages that con- interventions for managing a variety of inflammatory disorders
tribute to the pathology underlying chronic inflammatory associated with autoimmunity, including SLE. 2
disorders have identified immunological targets amenable to
modification by use of recombinant DNA technologies. The KEY CONCEPTS
resulting biological modifiers of inflammatory diseases have
revolutionized the treatment of rheumatoid arthritis (RA), Interferons: Therapeutic Potential
inflammatory bowel disease (IBD), psoriasis, spondyloarthropa- • Interferon-α (IFN-α) has been used with success to enhance resolution
thies, antineutrophil cytoplasmic antibody (ANCA)–associated of chronic viruses, notably hepatitis C virus (HCV); newer antivirals
vasculitis syndromes, systemic lupus erythematosus (SLE), and are supplanting the need for IFN therapy in HCV.
other autoimmune diseases, as well as allergic disorders. These • IFN-β has immunomodulatory effects that are of benefit in the treatment
targeted therapies, although costly, offer individuals affected by of certain subsets of patients with multiple sclerosis (MS).
these disorders options for better disease control and less of the • Type 1 IFNs (including IFN-α and IFN-β) may unmask or trigger flares
morbidity associated with greater exposure to broader immune of autoimmune disease, most notably systemic lupus erythematosus
(SLE).
suppression and/or corticosteroid use. This chapter summarizes • IFN-γ may be of some benefit in the management of patients with
the biological modifiers of inflammatory diseases that are currently chronic granulomatous disease (CGD) not responding to antifungal
available or in development classed by mechanism of activity and antibacterial prophylactic regimens.
and molecular nature.

IMMUNOMODULATORY CYTOKINES Interferons
IFNs, released primarily by T lymphocytes as well as dendritic
Recombinant Interleukin-2 (Aldesleukin) cells (DCs) in the context of stimulation of Toll-like receptors
First identified as a T-cell growth factor, recombinant interleukin-2 (TLRs) have a wide array of immunomodulatory effects that
(rIL-2) has potent immunomodulatory and antitumor activity, include upregulation of genes governing angiogenesis, cell dif-
promoting the proliferation, differentiation, and recruitment of ferentiation, expression of human leukocyte antigen (HLA)
T cells, B cells, and natural killer (NK) cells (Table 89.1). rIL-2 molecules, and production of inflammatory cytokines. IFN-α
has primarily been used therapeutically in patients with advanced and IFN-β bind to the same cell surface receptor (IFN-1R) and
melanoma and in patients with advanced renal cell carcinoma. are designated type-1 IFNs, whereas IFN-γ binds to a different
IL-2 is a potent inducer of proinflammatory T-cell cytokines, receptor (IFN-2R) and is designated as a type 2 IFN. Recombinant
such as IL-1, tumor necrosis factor-α (TNF-α), and interferon-γ preparations of all three IFNs have been used for the management
(IFN-γ), all of which likely play a major role in dose-related IL-2 of inflammatory disorders associated with chronic viral infection,
toxicity, which may include hypotension, cardiac arrhythmias, primary immunodeficiency (PID), or select autoimmune disorders
increased capillary permeability with pulmonary edema, fever, (most notably multiple sclerosis [MS]). Although there are
and rarely death. The use of aldesleukin in patients with cancer immunomodulatory attributes that render IFNs useful in certain
has been increasingly supplanted by more targeted immune- circumstances, constitutional symptoms and upregulation of a
enhancing “checkpoint” therapies targeting T-cell death receptors wide variety of genes that promote inflammation frequently
(programmed death 1 [PD-1]) and cytotoxic T lymphocyte limit the use of both type 1 and type 2 IFNs.
antigen-4 (CTLA-4), which have comparable antitumor effects
with much less acute toxicity. Interferon-α
More recently, the administration of low-dose rIL-2 has been Recombinant IFN-α2b (rIFN-α2b) has most commonly been
shown to promote preferential growth and proliferation of used in combination with ribavirin for treatment of hepatitis
regulatory T cells (Tregs) without significantly affecting the release C, including hepatitis C virus (HCV)–associated cryoglobulin
3
1
of inflammatory mediators by CD4 T cells. Such low-dose syndromes. Other disorders linked to viral infections, including
regimens of rIL-2 have been used with success in suppressing lymphomatoid granulomatosis (caused by Epstein-Barr virus
manifestations of graft-versus-host disease (GvHD) and may [EBV]) and polyarteritis nodosa (caused by hepatitis B virus
1
also favorably impact the course of type 1 diabetes. The use of [HBV]), have been successfully treated with regimens incorporat-
4
rIL-2 and/or other cytokine therapies promoting enhanced Treg ing IFN-α2b as part of the treatment regimen. Refractory retinal
1197

1198 ParT TEN Prevention and Therapy of Immunological Diseases



TABLE 89.1 recombinant on infection frequency and severity observed with rIFN-γ use
10
Immunomodulatory Cytokines is attributable to its effects on superoxide production.
Fever, myalgias, rash, fatigue, and diarrhea are the most
Molecule Construct Half-Life Dosing common adverse events associated with IFN-γ treatment. During
6
aldesleukin rIL-2 1.5 hours 0.3–3 × 10 IU/m intercurrent infections, treatment with rIFN-γ often enhances
2
subcutaneously (SC) infection-related constitutional symptoms, potentially obscuring
daily responses to antimicrobial therapy. With greater adherence to
IFN-α2b rIFN-α2b 1.7 hours 3–10 × 10 IU 3×/week antimicrobial prophylactic regimens incorporating combination
6
IFN-α2b rIFN-α2b 17 hours 50–150 µg/week therapy with itraconazole and trimethoprim-sulfamethoxazole
pegylated pegylated
IFN-β1a rIFN-β1a 69 hours 8.8–44 µg SC 3×/week (TMP-SMX), the added benefit of treatment with rIFN-γ in
6
INF-β1b rIFN-β1b 0.2–4.3 hours 2–8 × 10 IU every patients with CGD may be marginal.
other day
6
IFN-γ rIFN-γ 6 hours 50 µg (1 × 10 IU)/m2
SC 3×/week KEY CONCEPTS
Tumor Necrosis Factor-α (GR) Inhibitors
• Tumor necrosis factor (TNF)-α inhibitor treatment significantly improves
patient symptoms and function and inhibits structural damage in patients
with rheumatoid arthritis (RA).
vasculitis in the context of Behçet disease, severe flare-ups of • TNF-α inhibitors are very effective in suppressing skin lesions caused
familial Mediterranean fever not responding to treatment with by psoriasis as well as entheseal inflammation in patients with
colchicine, and eosinophilic granulomatosis with polyangiitis seronegative spondyloarthropathies; they do not appear to have any
(EGPA) have also been reported to respond favorably to treatment inhibitory effects on the development of bony ankyloses.
with rIFN-α2b. 5,6 • Vigilance for underlying fungal disease or mycobacterial disease is
Common side effects of rIFN-α2b include flu-like syndrome, prudent prior to commencing with and during treatment courses with
TNF-α inhibitors.
fatigue, anorexia, nausea, weight loss, hair loss, emotional lability • TNF-α inhibitors may trigger or exacerbate certain autoimmune syn-
and depression, cytopenias, and induction of autoantibodies with dromes and should be used with caution or avoided in patients with
enhancement of autoimmune disease. Because of these conse- autoantibody-associated immune disorders, such as systemic lupus
quences of treatment, the inconstant efficacy against certain HCV erythematosus (SLE).
genotypes, and the availability of more highly effective antiviral
agents targeting HCV replication, use of rIFN-α2b in the treat-
ment of HCV has decreased. INHIBITORS OF INFLAMMATORY CYTOKINES
Interferon-β Tumor Necrosis Factor-α Inhibitors
Recombinant IFN-β1a or IFN-β1b (rIFN-β) has been shown to TNF-α is a significant mediator of inflammation associated with
decrease relapse rates, disease severity, and central nervous system psoriasis, RA, spondyloarthropathies, and chronic inflammatory
(CNS) magnetic resonance imaging (MRI) lesions in patients bowel diseases (IBDs; Crohn disease and ulcerative colitis [UC]).
with relapsing MS. However, there are conflicting data with regard TNF-α promotes the ingress of immunocompetent cells at sites
to the efficacy of IFN-β1a or IFN-β1b preparations for patients of inflammation through activation of the vascular endothelium
7
with secondary progressive variants of MS. Immunomodulatory and upregulation of adhesion molecules. TNF-α also stimulates
effects attributed to the beneficial impact of IFN-β1b on MS synthesis of other pro-inflammatory cytokines (IL-1β, IL-6,
include enhancement of suppressor T-cell activity, reduction of granulocyte macrophage–colony-stimulating factor [GM-CSF]),
proinflammatory cytokines, downregulation of antigen presenta- chemokines (IL-8), and inflammatory eicosanoids (prostaglandin
7
11
tion, and reduced trafficking of lymphocytes into the CNS. Side E 1 [PGE 1], leukotriene B 4 [LTB 4 ]). In RA, TNF-α stimulates
effects observed during treatment with both rIFN-β preparations macrophages and monocyte-derived osteoclasts to release media-
include injection site reactions as well as most of the effects tors destructive to bone and cartilage, including matrix metal-
observed during treatment with rIFN-α2b. Reported autoimmune loproteinases (MMPs), such as collagenase and stromelysin. These
complications include SLE-related complications as well as features noted effects of TNF-α have, therefore, prompted the development
often associated with SLE, including immune complex glomeru- of both soluble receptor and monoclonal reagent strategies to
lonephritis, cutaneous vasculitis, and panniculitis. 8 inhibit the effects of TNF-α in disorders, in which it appears to
have a significant role in promoting inflammation and tissue
Interferon-γ injury (Table 89.2).
Recombinant IFN-γ (rIFN-γ) is effective in decreasing the fre- Etanercept is a recombinant soluble p75 TNF receptor (TNFR;
quency and severity of infections in patients with chronic CD120b)-IgG Fc fusion protein that binds soluble TNF-α as
granulomatous disease, an inherited disorder associated with well as lymphotoxin-β. The avidity of the p75 TNFR for soluble
any number of genetic defects that impair assembly of the nico- TNF-α is comparable with that of the p55 TNFR and therefore
tinamide adenine dinucleotide phosphate (NADPH) oxidase and effectively decreases net TNF signaling through either of these
9
production of superoxide anion metabolites in neutrophils. In receptors. The clinical impact of binding lymphotoxin-β is not
permissive X-linked variants of chronic granulomatous disease entirely known, but recent work elucidating the role of
(CGD), treatment with rIFN-γ appears to increase superoxide lymphotoxin-β in maintaining the physiological regulatory
production. However, infection outcomes in murine models of function of macrophages around lymphoid germinal centers
CGD void of any capability of superoxide generation are still suggests that inhibition of this cytokine may enhance the delivery
improved, so it is unclear whether the noted favorable impact of apoptotic body–derived self-antigens to germinal centers, a

CHaPTEr 89 Biological Modifiers of Inflammatory Diseases 1199



TABLE 89.2 recombinant Inhibitors of Pro-Inflammatory Cytokines
Molecule Construct Half-Life Dosing (Maintenance)
Etanercept sTNFR:Fc 3–4 days 50 mg subcutaneously (SC) once a week or 25 mg SC 2x/week
Infliximab aTNF-α (chimeric IgG1κ) 7–12 days 3–10 mg/kg intravenously (IV) q4–8 weeks
Adalimumab aTNF-α (human IgG1κ) 10–20 days 10–40 mg SC q1–2 weeks
Certolizumab aTNF-α (humanized Fab’)-PEG 14 days 200-400 mg SC q2–4 weeks
Golimumab aTNF-α (human IgG1κ) 14 days 50-100 mg SC every 4 weeks 2 mg/kg IV every 8 weeks
Anakinra sIL-1Ra 4–6 hours 1–8 mg/kg SC daily (max 200 mg/day)
Rilonacept IL-1R1:Fc(IgG1):IL-1RAcP 8–9 days 2.2 mg/kg (max 160 mg) SC per week
Canakinumab aIL-1β (human IgG1κ) 26 days 2–8 mg/kg SC (max 600 mg) SC every 8 weeks
Tocilizumab aIL-6R (humanized IgG1κ) 13 days 4-12 mg/kg (max 800 mg) IV every 2–4 weeks 160 mg SC every 1–2 weeks
Ustekinumab aIL-12/23 p40 (human IgG1κ) 15–32 days 0.75 mg/kg (pediatric) or 45–90 mg SC every 12 weeks
Secukinumab aIL-17A (human IgG1κ) 22–31 days 150-300 mg SC every 4 weeks
Ixekizumab aIL-17A (humanized IgG4κ) 13 days 80 mg SC every 2–4 weeks


16
consideration of potential clinical relevance in patients at risk and are approved for treatment of these disorders. Pyoderma
for SLE or related autoimmune syndromes (see below). syndromes and the oral and genital lesions of Behçet disease are
Infliximab, adalimumab, and golimumab are monoclonal reported to respond favorably to TNF-α inhibition therapy. 17,18
antibody (mAb) reagents with comparable avidity for both soluble Although very well tolerated as a class, TNF-α inhibitors may
and cell-bound TNF-α but differ with regard to the derivation impair innate host defenses, resulting in delay of resolution of
of the antigen-binding sequence and half-life. Infliximab is a intercurrent infections. TNF-α inhibitors are associated with
chimeric antibody with a murine-derived variable-region TNF- reactivation of tuberculosis and fungal infections, including
binding domain. Adalimumab and golimumab are both human histoplasmosis and coccidioidomycosis, and viral infections such
sequence–derived mAb reagents. Certolizumab pegol is a construct as hepatitis B. 19,20 In all circumstances of intercurrent infection,
consisting of recombinant human sequence–derived F(ab)’ the standard recommendation is for treatment with TNF-α
anti–TNF-α covalently liked to 40-kilodalton (kDa) polyethylene inhibitors to be withheld until the infection has resolved. No
glycol. The pegylated construct enhances the half-life of the complications have been reported regarding their use in patients
reagent, and absence of the Fc- and complement-binding domains with intercurrent hepatitis C infection or in patients with human
renders certolizumab less likely to engender local injection site immunodeficiency virus (HIV) infection that is well controlled
reactions and precludes placental crossing of certolizumab into with highly active antiretroviral therapy (HAART).
the fetal circulation when administered to pregnant women. In the context of its role in promoting cell apoptosis, TNF-α
In cytokine profile studies of joint tissues and synovial fluids constitutes a component of the host defense against tumor cell
derived from patients with RA, TNF-α is consistently among survival and growth. As such, the incidence and prevalence of
11
the highest expressed inflammatory cytokines. All five TNF-α cancer in populations treated with TNF-α inhibitors has been
inhibitors have been shown to decrease the signs and symptoms emphasized in pharmacovigilance programs surrounding the
of disease activity and to inhibit the progression of structural use of this class of therapeutic agents. Lymphoma has been
damage in RA, with greatest benefits and probability of disease reported in patients treated with TNF-α inhibitors, with most
remission seen among patients with early RA. Inhibition of reported cases occurring in the context of treatment for RA.
radiographic progression of disease, as measured by joint space However, the relative risk for lymphoma in patients with RA in
narrowing and osseous erosions at joint margins, can be dissoci- the pre–biologicals era approximates 3.0, and the prevalence of
ated from clinical efficacy as measured by composite scoring lymphoma in patients with RA exposed to TNF-α inhibitors
measures, such as the American College of Rheumatology has not been shown to exceed the otherwise expected prevalence
20%/50%/70% improvement criteria or the Disease Activity Score among patients with RA. Among patients with RA, IBD, or
(DAS)-28. 12 psoriasis, the risk of cancer has not been shown to be increased
21
TNF-α levels are also notably increased in biopsy samples of in the context of treatment with TNF-α inhibitors. Although
skin and synovial tissues from patients with psoriasis and psoriatic it is often recommended that TNF-α inhibitor therapy be dis-
arthritis, and the TNF-α inhibitors have been shown to be highly continued in the setting of a newly diagnosed cancer and that
effective in suppressing manifestations of plaque psoriasis as initiation of such therapy be avoided in patients with known
well as articular disease in psoriatic arthritis. 13,14 Improvements malignancy, there are no clinical data to confirm whether
in joint symptoms (axial and peripheral) and uveitis manifesta- continued treatment with TNF-α inhibitors impairs therapeutic
tions are observed in patients with other spondyloarthropathies, response to cancer therapy.
including ankylosing spondylitis, during treatment with TNF-α The role of TNF-α inhibition in facilitating apoptosis is also
inhibitors; however, use of these agents has not been shown to of relevance with regard to the development or potentiation of
delay fusion of axial joints in patients with spondylitis. Insights autoimmunity. Although rare, the development of psoriasiform
from rodent models and the known inhibitory effect of TNF-α skin eruptions, demyelinating syndromes, and drug-induced
on Smad pathway–mediated bone formation by osteoblasts suggest SLE are well documented occurrences in patients treated with
that TNF inhibition may, in fact, promote ossification at sights TNF-α inhibitors. 22-24 All of the anti–TNF-α reagents may
of inflammation and increased bone turnover. 15 attenuate TNF-α–mediated apoptosis of autoreactive T cells.
mAb inhibitors of TNF-α as well as certolizumab (but not Infliximab, adalimumab, and golimumab may induce the release
etanercept) have been shown beneficial in the management of of apoptotic products in the context of antibody-dependent
intestinal inflammation in patients with Crohn disease or UC cellular cytotoxicity (ADCC) of cells bearing surface TNF-α,

1200 ParT TEN Prevention and Therapy of Immunological Diseases


25
potentially driving production of antibodies to nucleoproteins. Anakinra is a nonglycosylated recombinant IL-1 receptor
Binding of lymphotoxin-β by etanercept may alter the clearance antagonist (rIL-1Ra) that differs from the endogenous IL-1Ra
of apoptotic products by germinal center adjacent follicular DCs by a single amino acid addition at the amino terminus. Admin-
26
and macrophages. Given these collective considerations, it is istered subcutaneously at daily (or for some indications more
generally recommended that TNF-α inhibitors be discontinued frequent) intervals because of its very short serum half-life,
in patients who develop autoimmune complications during treat- anakinra functions as a competitive inhibitor of IL-1α and IL-1β
ment with this class of biological therapy. Moreover, anti–TNF-α binding to IL-1 receptors.
therapy is not recommended for patients with established SLE Anakinra, presently approved for the treatment of RA,
or other overlap syndromes with lupus-like features associated inhibits joint erosions. However, compared with the clini-
with autoantibody production. cal responses observed with use of TNF inhibitors and IL-6
inhibitors (tocilizumab) with regard to tender and swollen
Interleukin-1β Inhibitors joints, such responses with anakinra are much more modest.
IL-1β is synthesized as an inactive precursor, with activation of Given these observations with biologicals that require much less
IL-1β occurring following engagement of nucleotide-binding frequent dosing, anakinra is used infrequently in the treatment
oligomerization domain (NOD)–like receptors (NLRs) by a variety of RA. With the increased understanding of the role of IL-1
of exogenous or endogenous danger signals, which then trigger inflammasome activation in crystal-induced arthropathies, such
formation of molecular platforms (NLR family domain containing as gout and systemic inflammatory disorders, such as soJIA
protein 3 [NALP] inflammasomes) that facilitate cleavage of and AoSD, anakinra has been gaining renewed interest as a
27
the IL-1β precursor by IL-1–converting enzyme (ICE). IL-1β therapeutic option in the treatment of these disorders. Case
stimulates proliferation of lymphocytes, upregulates the expression reports support use of anakinra in managing acute flare-ups
of adhesion molecules, and triggers the release of a variety of of gout or flare-ups of calcium pyrophosphate–associated
inflammatory mediators from leukocytes, including chemotactic pseudogout when the use of corticosteroids or nonsteroidal
factors, prostaglandins, proteases, and procoagulants. In patients antiinflammatory drugs (NSAIDs) is not favored because of
with RA, IL-1β triggers the release of proteases from phagocytic comorbid conditions, such as uncompensated heart failure or
28
cells and macrophages that are destructive to bone and cartilage. diabetes with significant renal impairment. Prompt resolution
Inflammasome activation has also been shown to mediate of inflammatory markers and clinical manifestations have been
acute flare-ups of arthritis in patients with gout or calcium reported in patients with severe flare-ups of AoSD and soJIA
pyrophosphate deposition. Dysregulation of NLRP3 as a result treated with anakinra, including those associated with MAS. 29,30
of gain-of-function mutations in inflammasome-related genes The relatively short half-life of anakinra may render its use
has been implicated in the pathogenesis of familial cryopyrin- feasible when short-term blockade of IL-1 may be of benefit in
associated periodic syndromes (CAPS, cryopyrinopathies), familial managing severe flare-ups of IL-1–driven inflammation when
Mediterranean fever (FMF), and the pyogenic arthritis, pyoderma there is a concern for intercurrent infection. The potential
gangrenosum, and acne syndrome (PAPA). Increased expression utility of anakinra in this context is supported by a recent
and levels of IL-1β are also noted during flare-ups of inflammation study reexamining the outcomes from a previous sepsis trial,
in patients with adult-onset Still disease (AoSD), systemic-onset where survival was shown to have improved in patients with
juvenile idiopathic arthritis (soJIA), and macrophage activation clinical features of MAS who were randomized to anakinra
syndromes (MAS) associated with these and other autoimmune treatment. 31
27
or infectious disorders. The naturally occurring IL-1 receptor Rilonacept (also referred to as IL-1-Trap) is a recombinant
antagonist (IL-1Ra) prevents the binding of IL-1β and IL-1α to the fusion protein that comprises the extracellular domain of the
IL-1 receptor (IL-1R), but tissue levels of IL-1Ra in in the above IL-1 accessory protein and IL-1 receptor type 1 attached to the
disorders may be insufficient to counteract the effects of IL-1β. Fc portion of immunoglobulin G1 (IgG1). Rilonacept binds to
IL-1α and IL-1β with high affinity and is approved for the
treatment of CAPS. Similar to anakinra, rilonacept is dosed
KEY CONCEPTS subcutaneously, albeit at weekly intervals because of its longer
Interleukin Inhibitors half-life. It is generally well tolerated, with injection site reactions
being the most common adverse events.
• Interleukin (IL)-1β inhibitors are very effective in the treatment of Canakinumab is a human genome sequence–derived mAb
disorders associated with constitutional or secondary activation of the with specificity for IL-1β that also binds to IL-1α. Approved for
IL-1 inflammasome, including cryopyrin syndromes, familial Mediter-
ranean fever flare-ups, and flare-ups of crystal induced arthropathy the treatment of CAPS as well as soJIA, canakinumab has the
(gout and calcium pyrophosphate dihydrate [CPPD] crystal deposition longest half-life of the currently approved therapies that target
disease/pseudogout). IL-1. In a randomized clinical trial comparing the use of
• Use of IL-1β inhibitors with a short half-life may be of benefit in canakinumab with parenteral triamcinolone administration for
managing severe flare-ups of inflammation associated with macrophage acute gout flare-ups, canakinumab was shown to have greater
activation, such as systemic-onset juvenile idiopathic arthritis (soJIA), reductions in joint pain and swelling, decreased needs for rescue
adult-onset Still disease, and severe sepsis syndromes. 32
• IL-6 signaling is complex in the context of signaling that can occur medications, and a greater time to subsequent flare-ups.
through cell membrane bound IL-6R, which mediates primarily IL-6 However, given that the observed rates of serious infections were
homeostatic effects, or through many other cell receptors that engage twice as high in the canakinumab arm, a half-life that well exceeds
IL-6/sIL-6R complexes (trans-signaling), which mediate IL-6 inflammatory the duration of typical flare-ups, and the questionable cost-
effects. effectiveness of using an intervention 5000 times the cost of
• Inhibition of IL-6 binding to IL-6R mediated by monoclonal antibody current therapies that are, by and large, effective for managing
(mAb) reagents, such as tocilizumab, is very effective in suppressing
clinical manifestations of rheumatoid arthritis (RA). acute gout, the use of canakinumab has not found favor in
treatment paradigms for managing gout.

CHaPTEr 89 Biological Modifiers of Inflammatory Diseases 1201


clinical development. Sarilumab (human anti–IL-6R) has been
Interleukin-6 Inhibitors shown in phase III trials to have demonstrated efficacy in the
IL-6 mediates the activation of macrophages and osteoclasts, treatment of RA. Vobarilizumab is a nanobody construct consisting
terminal proliferation and differentiation of B cells, differentiation of an IL-6R–specific non-Fc IgG heavy chain covalently linked
of T-helper 17 (Th17) cells, and production of liver acute-phase to serum albumin that blocks binding of IL-6 to both membrane-
33
proteins. However, IL-6 also governs homeostatic processes, bound and soluble IL-6R, currently being evaluated for treatment
including granulopoiesis, enteric epithelial proliferation, and of SLE. Sirukumab and Olokizumab are humanized anti–IL-6
antiinflammatory responses involved in resolution of inflam- constructs that have also been shown in phase II trials to have
mation, including production of the soluble TNFR p55 and the efficacy in RA. Clazakizumab (humanized anti–IL-6) has shown
IL-1Ra. 34 promising results for the treatment of RA as well as musculoskeletal
The biology of IL-6 signaling is complex in that signaling symptoms in patients with psoriatic arthritis. By virtue of
may occur directly through the cell membrane–bound IL-6 engineered aglycosylation, clazakizumab has a longer half-life
receptor (IL-6R)/gp130 protein complex (classic signaling) or than other mAb reagents targeting IL-6 or IL-6R, thereby requiring
through binding of IL-6 to soluble IL-6R (sIL-6R) with the less frequent dosing; however, with higher dose ranges, the
resulting heterodimer and then ligating a variety of other gp130- frequency of serious adverse events and infections appears to
containing membrane receptors (trans-signaling) that mediate be increased.
signaling from cytokines other than IL-6. IL-6R is expressed Of potentially greater interest is the development of soluble
primarily on leukocytes, hepatocytes, and megakaryocytes, whereas gp130 covalently linked to immunoglobulin Fc (sgp130:Fc),
gp130-containing receptor complexes are expressed in almost a construct with specificity for the IL-6/sIL-6R heterodimer
all organs, including the heart, kidneys, spleen, liver, lungs, that mediates IL-6 trans-signaling. Selective inhibition of the
placenta, and brain. In murine models of inflammation employing trans-signaling pathway linked to inflammatory responses
sgp130:Fc constructs that selectively bind and neutralize IL-6/ may be superior to complete IL-6 blockade because important
sIL-6R, trans-signaling appears to mediate many of the observed physiological functions of IL-6 mediated through membrane-
inflammatory consequences of the upregulation of IL-6, whereas bound IL-6R are left intact. These differences have been
classic signaling through membrane IL-6R mediates homeostatic emphasized in a murine model of peritonitis, where selective
processes, such as granulopoiesis, thrombopoiesis, and epithelial inhibition of IL-6 trans-signaling by sgp130:Fc as opposed to
35
cell proliferation. With greater understanding of the different complete blockade of IL-6 signaling by anti–IL-6 mAb was
IL-6 signaling pathways in humans, implementation of therapeutic shown to significantly improve survival and completely prevent
40
strategies targeting IL-6 will require consideration of the impact intestinal epithelial cell apoptosis. Observations in murine
of blocking classic membrane-bound IL-6R signaling and IL-6/ models of tuberculosis have further confirmed the absence of
sIL-6R trans-signaling. any impaired response to disease resolution in the setting of
Tocilizumab is a humanized mAb with specificity for the human either sgp130:Fc transfection or administration of sgp130:Fc
IL-6 receptor. Tocilizumab blocks the binding of IL-6 to IL-6R, constructs. 41
inhibiting both classic signaling by IL-6 through membrane IL-6R
and formation of IL-6/sIL-6R heterodimer ligands that engender Interleukin-12/-23 Inhibitors
36
trans-signaling. Treatment with tocilizumab improves tender IL-12 and IL-23 are heterodimeric molecules that share a common
and swollen joint counts and slows the development of joint p40 subunit but have different immunological effects on T-cell
37
erosions in patients with RA. Treatment with tocilizumab is lineage development. IL-12 comprises p35/p40 subunits and
also reported to be of benefit in patients with soJIA, AoSD, and promotes the development and maturation of Th1 lineage T
Castleman disease. 38,39 cells, a significant source of IFN-γ. IL-23 comprises p19/p40
Presumably related to the role of IL-6 in promoting granu- subunits and promotes the maturation and survival of Th17
lopoiesis, neutropenia may be observed in some patients following lineage T cells. Initial strategies targeting the shared p40 subunit
treatment with tocilizumab, but this is uncommon. Thrombo- of IL-12/IL-23 in murine models of autoimmune inflammation
cytopenia and elevated serum aminotransferase levels have also yielded promising results and prompted human trials in psoriasis,
been observed on rare occasions. A predictable mild increase in RA, and IBD. Subsequent comparative IL12/IL-23 p40, IL-12
serum lipid levels is observed following the initiation of treatment p35, and IL-23 p19 knock-out studies in the murine models
with tocilizumab; the clinical significance of this remains inde- suggest that the observed ameliorative effects of anti-p40 on
terminate, but monitoring of lipid levels is recommended. inflammation in the models studied were by virtue of IL-23
42
Reactivation of tuberculosis and invasive fungal infections can inhibition. As a consequence, selectively targeting IL-23 and
occur, and rare instances of gastric and intestinal rupture have IL-17 (the primary effector cytokine of Th17 cells) has evolved
been reported in patients treated with tocilizumab. These observed as a strategy to treat autoimmune inflammation.
effects on hematopoiesis and loss of intestinal integrity reflect Ustekinumab is a humanized IgG1κ mAb with specificity for
the potential downsides of complete blockade of IL-6–mediated the p40 subunit component of IL-12 and IL-23. Approved for
functions that is inherent in the use of mAb reagents that block the treatment of psoriasis and psoriatic arthritis, ustekinumab
the interaction of IL-6 with the IL-6R and emphasize the potential significantly improves skin lesions as measured by the psoriasis
advantages of more selective IL-6 pathway inhibition (see below, area and severity index (PASI), and has been shown to decrease
sgp130:Fc). both peripheral and axial joint/enthesis manifestations in psoriatic
43
arthritis. Although not yet approved for patients with IBD,
On the Horizon: Other Anti–IL-6R and Anti–IL-6 mAbs and significant improvements in Crohn disease activity have been
Sgp130:Fc Reagents reported in patients treated with ustekinumab, most notably
Several other recombinant mAb-based reagents that inhibit both among patients who failed to respond adequately to anti–TNF-α
classic and trans-signaling mediated by IL-6 are in advanced therapies. 44

1202 ParT TEN Prevention and Therapy of Immunological Diseases


In the reported clinical trials for psoriasis, there were no In clinical trials of secukinumab in patients with psoriasis,
increased occurrences of serious infections or other serious adverse rates of infection are increased over that observed in patients
events in the ustekinumab-treatment arms relative to the placebo- randomized to placebo, but not in excess of the infection rates
treatment arms. Higher than expected rates of infection or noted with anti-TNF therapy. Increased rates of serious infection
malignancy have also not been observed in other controlled have not been observed in relatively short-term RA clinical trials
studies of ustekinumab in spondylitis or IBD. Nonetheless, with secukinumab. However, given the pivotal role of Th17 in
vigilance for mycobacterial, fungal, and Salmonella infections is host responses to chronic and acute infections by bacteria, para-
recommended, given the role of IL-12 and IL-23 in host defense sites, and fungi, it is possible that alterations in the containment
against these pathogens. of microbial pathogens in the enteric mucosa may account for
the failure of targeting IL-17 in IBD.
On the Horizon: IL-23 Specific Inhibitors
Tildrakizumab and Guselkumab are fully human IL-23 p19–specific Ixekizumab
mAbs currently being evaluated in clinical trials for treatment Ixekizumab is a humanized IgG4 mAb targeting IL-17A that has
of psoriasis. been shown to be effective and approved for the treatment of
plaque psoriasis. Similar to what has been observed in clinical
Interleukin-17 Inhibitors trials with secukinumab, rates of infection are slightly increased
IL-17 is a cytokine elaborated primarily by the Th17 lineage of in patients treated with ixekizumab versus placebo, but in short-
+
-
effector T cells, which differentiate from Foxp3 CD4 thymocytes term studies conducted so far, the rate of serious infections has
under the influence of IL-6 and transforming growth factor-β not been observed to be increased relative to that observed in
(TGF-β) and proliferate/survive under the influence of IL-23. patients randomized to placebo or a comparative arm of patients
Th17 cells are prevalent in the inflammatory lesions of a variety randomized to anti-TNF treatment with etanercept. 50
of inflammatory disorders, including RA, IBD, psoriasis, and
spondyloarthropathies. IL-17 is produced by a variety of innate On the Horizon: Il17 Receptor Antagonists
immune cells, including neutrophils, mast cells, keratinocytes, Brodalumab is an anti–IL-17Ra monoclonal antibody. Data from
macrophages, NK cells, NKT cells, and innate lymphoid cells two identically designed phase III randomized trials support the
(ILCs; LLC/LTi), triggering the induction and release of IL-6, efficacy of brodalumab for treatment of moderate to severe plaque
TNF-α, CCL2, CCL3, MMPs from macrophages, inducing psoriasis. Efficacy with regard to clearing skin lesions was noted
activation of osteoclasts at sites of bone resorption, and promoting to be significantly greater than that observed in a comparator
the proliferation, maturation, and chemotaxis of neutrophils. 45,46 group receiving ustekinumab, but Candida infections and
As such, IL-17 is important both in the initial innate host defense neutropenia were more frequent in the brodalumab groups than
to infection and also inflammation driven by acquired immune in the ustekinumab and placebo groups. 51
responses. Given the multitude of effects mediated by IL-17 in
perpetuating inflammation in a number of autoimmune disorders,
mAb reagent–based strategies have been developed targeting Other Immunomodulatory Cytokines and
IL-17 and its receptor (Table 89.2). Cytokine Inhibitors
On the Horizon: Inhibitors of the Type 1 Interferon Pathway
With increasing recognition of the role of type 1 IFNs in the
immunopathogenesis of SLE, strategies to target IFN-α for
KEY CONCEPTS treatment of SLE have emerged. Initial trials with mAb reagents,
Approaches to IL-17 Inhibition such as rontalizumab or sifalimumab with specificity to one or
more of the known IFN-α subtypes (14 known presently), have
• Selective inhibition of pathways promoting differentiation and activation yielded modest results, emphasizing the complexity of the IFN
products of T-helper 17 (Th17) cells is of significant benefit in the
treatment of psoriasis and the seronegative spondyloarthropathies. system in SLE. This strategy is further complicated by the recogni-
• Selective inhibition of interleukin (IL)-17A appears to be of marginal tion that autoantibodies to IFN-α are often prevalent in SLE
benefit in the treatment of inflammatory bowel disease (IBD), whereas sera. The alternative approach of targeting the type 1 IFN receptor
inhibition of both IL-12 and IL-23 by targeting their shared p40 subunit (IFN-1R) in a recent phase II trial with anifrolumab has yielded
appears to be of benefit in both psoriasis and IBD. more encouraging results. Targeting the IFN-1R may also prove
52
efficacious in other autoimmune disorders, such as SLE and
Sjögren syndrome, which are associated with an enhanced type
1 IFN signature. Other approaches targeting the production of
Secukinumab type 1 IFNs currently under investigation include the use of
Secukinumab is a fully human IgG1 mAb that selectively binds mAb reagents to deplete plasmacytoid dendritic cells (pDCs),
to and neutralizes IL-17A. In clinical trials, secukinumab has perceived to be the major source of IFN-1 in SLE.
been shown to significantly decrease the activity of skin lesions In the relatively short-term clinical trials conducted thus far
in patients with psoriasis, decrease tender and swollen joints in with antibodies targeting IFN-α or IFN-1R, increased susceptibil-
patients with psoriatic arthritis, and decrease axial pain and ity to viral pathogens has not been observed. Whether depleting
limitation in patients with ankylosing spondylitis. 47,48 Clinical pDCs, blocking one or more TLRs linked to upregulation of
responses to secukinumab in patients with RA have been less type 1 IFNs, blocking IFN-1R, or using mAb reagents capable
robust. Despite the apparent role of Th17 cells in patients with of neutralizing multiple IFN subtypes critically important in
IBD, no significant improvement and was observed in patients SLE and related autoimmune disorders will prove efficacious
with Crohn disease, and in some of these patients, the disease but not at the expense of susceptibility to viral disease remains
worsened during treatment with secukinumab. 49 to be determined in phase III trials.

CHaPTEr 89 Biological Modifiers of Inflammatory Diseases 1203


C5b-9 terminal attack complex, and failure to generate C5a
On the Horizon: Recombinant Promoters of Treg Function diminishes complement pathway mediated recruitment of
IL-27 may modulate autoimmune inflammation via promotion phagocytic cells. Since both C5a and C5b-9 have potent proco-
of Treg lineage and function; it often opposes the action of IL-6 agulant activity at sites of inflammation, complement activation
and is the only member of the gp130-related cytokine family to is hypothesized to be critical to the generation of intravascular
predominantly signal via the latent transcription factor signal thrombi in thrombotic microangiopathy syndromes, including
transducer and activator of transcription 1 (STAT1) instead of atypical hemolytic–uremic syndrome (aHUS), catastrophic
53
STAT3. In this context, IL-27 acts as a negative regulator of anti-phospholipid syndrome, and thrombotic microangiopathy
Th17 commitment, whereas the p28 subunit of IL-27 antagonizes that may be seen in SLE. Eculizumab is currently approved for
IL-6–STAT3-mediated T-cell responses. Treg function may also use in patients with aHUS but has also been reported to be of
be enhanced by recombinant IL-35. 54 benefit in patients with these other complement activation–
mediated thrombotic syndromes. 55-57
Complement Pathway Inhibitors Eculizumab inhibits terminal complement activation and
In addition to opsonization of microbial pathogens and products therefore renders patients vulnerable to infection with encap-
of apoptosis, products of complement activation play a significant sulated organisms. Life-threatening and fatal meningococcal
role in recruitment and activation of phagocytic cells (C3a, C5a), infections have occurred in patients who received eculizumab,
cell membrane damage (C5b-9), and have potent procoagulant and meningococcal vaccination is recommended at least 2 weeks
activity (C5a, C5b-9) (Table 89.3). Congenital deficiency of C3 prior to receiving eculizumab. In cases where the risks of delaying
or functional C3 impairment is typically associated with severe, eculizumab therapy outweigh the risk of developing a menin-
if not fatal, clinical phenotypes, rendering C3 or its cleavage gococcal infection, meningococcal vaccine should be administered
products unattractive targets for immunomodulation. However, as soon as possible. However, current meningococcal vaccines
inhibition of C5 appears to be well tolerated and may have do not protect against serogroup B strains of Meningococcus,
beneficial effects on disorders in which enhanced cleavage of C5 and thus immunized patients are still at risk of infection by this
contributes to disease manifestations (Table 89.3). strain.
KEY CONCEPTS Adhesion Molecule Inhibitors
Inhibitors of Complement Activation and Trafficking of phagocytic cells and lymphocytes across vascular
Cell Migration endothelium is critical to the development of inflammatory
lesions and associated tissue injury. The transmigration of cells
• Inhibition by eculizumab of C5 cleavage to its active products and across the endothelium is dependent on the upregulation of
resulting assembly of the membrane attack complex is of significant integrin/adhesion molecules, some of which have binding
benefit to patients who have complement-mediated thrombotic specificity for ligands (addressins) in specific organs, such as the
microangiopathy syndromes occurring with atypical hemolytic–uremic CNS or the enteric mucosa. Strategies to decrease the influx of
syndrome, catastrophic antiphospholipid syndrome, or severe flare-ups
of systemic lupus erythematosus (SLE). monocytes and/or T and B lymphocytes using biologicals that
• Immunization against Neisseria species is recommended for patients target adhesion molecules have, therefore, been deployed as
who require dosing with eculizumab. treatments for several inflammatory disorders, including MS
• Antibody targeting the lymphocyte integrin addressin α 4 β 1 (natalizumab) and IBD.
has been shown to be of benefit in managing multiple sclerosis (MS) Natalizumab is a humanized IgG4κ mAb with specificity for
and inflammatory bowel disease (IBD) but is associated with a risk the α 4 subunit of the very late activation antigen-4 (VLA-4)–
of central nervous system (CNS) JC virus activation (progressive
multifocal leukoencephalopathy [PML]). integrin molecule expressed on lymphocytes and monocytes.
• Antibody targeting the α 4 β 7 integrin (vedolizumab) selectively inhibits Natalizumab blocks association of the VLA-4 α 4 β 1 and α 4 β 7
trafficking of lymphocytes into the intestinal lamina propria and is of integrins with their respective vascular receptors, thereby limiting
benefit in the management of IBD. cell transmigration into tissue sites of inflammation in the CNS
and in the intestinal mucosa. On the basis of the premise that
Eculizumab, a humanized IgG2/4κ mAb with specificity for administration of natalizumab blocks the interaction of T-cell
C5, blocks the generation of C5a and C5b by the C5 convertase. α 4 β 1 with its addressin ligand on venules in the CNS, this thera-
The resulting failure to generate C5b impairs assembly of the peutic approach was validated in murine models of encephalo-
myelitis, and subsequent clinical trials confirmed natalizumab
58
to be efficacious in decreasing the frequency of relapses of MS.
TABLE 89.3 recombinant Inhibitors of Natalizumab also inhibits the interaction of the α 4 β 7 integrin
Complement activation and Molecules with mucosal addressin cell adhesion molecule-1 (MAdCAM-1)
Mediating Cell Migration expressed on venules in the enteric mucosa and has been shown
to induce remissions and prevent flare-ups of Crohn disease in
Dosing patients who have failed to have an adequate response to anti–
Molecule Construct Half-Life (Maintenance) TNF-α therapy. 59
Eculizumab anti-C5 (humanized 8–15 days 300–1200 mg Recent additional work in experimental allergic encephalo-
IgG2/4κ) intravenously (IV) myelitis (EAE) models in different strains of mice suggests that
every 1–2 weeks natalizumab affects primarily the transmigration of Th1 cells,
Natalizumab anti-α 4 β 1 (humanized 7–15 days 300 mg IV every with much less of an inhibitory effect on the transmigration of
IgG4κ) 4 weeks Th17 cells. As such, there may be differential efficacy of natali-
60
Vedolizumab anti-α 4 β 7 (humanized 25 days 300 mg IV every
IgG1κ) 8 weeks zumab in the treatment of Th1- versus Th17-driven subtypes
of MS and/or IBD.

1204 ParT TEN Prevention and Therapy of Immunological Diseases


The major concern with regard to the use of natalizumab is increase in the number of circulating memory B cells is observed
the occurrence of reactivation of John Cunningham (JC) polyoma immediately after administration of belimumab, with numbers
virus in patients who are carriers, resulting in progressive multifo- gradually returning to the pretreatment baseline level over the
cal leukoencephalopathy (PML). This complication has a very course of several months of treatment. Total numbers of circulat-
low rate of occurrence, but it is quite debilitating and often fatal, ing B cells are decreased by 20–25% following 1-year treatment
resulting in dampened enthusiasm for the use of natalizumab. periods, with no observed decreases in CD4 and CD8 T lym-
63
However, the risk of PML can be mitigated by limiting the phocytes. Levels of autoantibodies, including anti-dsDNA,
duration of natalizumab treatment to 1 year in known carriers anti-Smith, anti-SSA, and anti-cardiolipin, are decreased by
of JC virus (as defined by seroconversion) and not administering 40–50% percent after the first year of treatment and continue
63
natalizumab concurrent with other immunosuppressive therapy. to decrease over time with long-term treatment. In contrast,
Vedolizumab is a humanized mAb that specifically binds to total antibody levels decrease, on average, by only 15%, with no
α 4 β 7 integrin, blocking its interaction with MAdCAM-1 on significant decreases in measured preexisting antibody titers to
intestinal endothelial cells. Since vedolizumab does not bind to influenza, tetanus toxoid, or pneumococcal serotypes; furthermore,
or block the interaction of the α 4 β 1 integrin to its addressin treatment with belimumab does not appear to have any significant
ligand in the CNS, the risk of developing PML during treatment effects on the primary immune responses to pneumococcal
is substantially lower than that associated with the use of bacterial antigens. 64,65 In two major randomized trials, the fre-
natalizumab. In clinical trials, vedolizumab has been shown to quency of infections was not shown to be increased in the
significantly improve disease activity and reduce flare-ups in belimumab treatment arms.
61
patients with Crohn disease or UC. This strategy may prove Receptors for BLyS (BAFF-R) and BCMA have also recently
to be particularly useful in subsets of patients with IBD as well been identified on murine T-follicular helper (Tfh) cells, with
as clinical features of SLE; in these patients, use of anti–TNF-α ligation of BAFF-R promoting activation of Tfh and ligation of
agents may carry an increased risk of SLE exacerbation. BCMA appearing to play a role in downregulation of Tfh
responses. The significance of these findings in human SLE and
Inhibitors of B-Cell Activation what impact treatment with belimumab has on Tfh responses
Identified targets for regulation of B-cell activation include growth remain to be determined. 66
and survival factors, such as B-lymphocyte stimulator (BLyS;
BAFF) and its respective receptors (BAFF-R, transmembrane On the Horizon: Other Inhibitors of B-Cell Activation
activator and CAML interactor [TACI], and B-cell maturation Atacicept is a recombinant human fusion protein containing the
antigen [BCMA]); costimulatory receptors and their ligands, extracellular, ligand-binding portion of the receptor TACI and a
such as CD40/CD40-ligand; and cell surface receptors, such as modified Fc portion of human IgG. Atacicept binds both BLyS
CD22 or FcγRIIb, which engender inhibitory signaling when (BAFF) and a proliferation-inducing ligand (APRIL), thereby
ligated. Given the prominent role of multiple autoantibodies functioning as an antagonist to the ability of these two ligands
and generalized B-cell activation in SLE, most of the clinical to stimulate B lymphocytes. Amelioration of disease manifesta-
trials, to date, employing strategies targeting B-cell activation tions in murine SLE models employing TACI-Ig transfections
have been in patients with this disorder. provided a mechanistic rationale for pursuing human SLE studies
Belimumab is a recombinant human genome–derived IgG1 using TACI-Ig as a therapeutic intervention; such studies are still
mAb with specificity for soluble (non–membrane-bound) BLyS ongoing. However, the therapeutic window with this approach
(BAFF). Through ligation of the BAFF-R and TACI receptors may be relatively narrow, as membrane-bound TACI is the major
on B lymphocytes, BLyS promotes the maturation of B cells receptor mediating Ig class switching during B-cell maturation,
into antibody-secreting plasmablasts. On the basis of the and excess of the soluble receptor administered over time may
hypothesis that autoreactive B lymphocytes may have greater result in significant humoral immune deficiency as a result of
dependency on BLyS to survive and proliferate, a number of decreases in IgG-secreting B cells and plasma cells. 67
preclinical studies were undertaken to target BLyS using gene Blisibimod is a recombinant fusion polypeptide heterodimer
knock-out strategies or soluble receptor (TACI) transfections in consisting of a BlyS (BAFF)–binding domain covalently linked
murine models of SLE. Following encouraging results in these to the N-terminus of the Fc region of human IgG1. The tetravalent
models, human studies using belimumab were undertaken, and peptibody construct binds to both soluble and cell membrane–
improvement in SLE disease activity was demonstrated by using bound BlyS, with reported higher binding affinities compared
validated disease activity measures (SLE Disease Activity Index with conventional mAbs, inhibiting the interaction of BlyS with
[SLEDAI] and British Isles Lupus Assessment Group [BILAG]) BAFF-R and TACI. Blisibimod and has been shown to have
as well as additional secondary endpoints related to disease biological effects in early-phase human trials, but the clinical
62
flare-ups and sparing of corticosteroid use. Consistent with significance of the membrane-binding attribute of blisibimod
its mechanism of action of targeting a growth and survival has not been determined. 68
factor, noted clinical improvements do not become manifest Anti-CD40-ligand mAb reagents interfere with the interaction
until after 6 months of treatment with belimumab. The majority of T-cell CD40L with B-cell CD40, thereby blocking costimulatory
of observed clinical improvements have been observed in the signals required for cognate T-cell help that promotes antigen-
musculoskeletal, mucocutaneous, and serological domains of specific B-cell proliferative responses. Following favorable results
62
disease activity. The potential effects on more severe neurological with targeting of CD40L in murine SLE models, human trials
or renal domains of disease activity have not been assessed in with anti-CD40L were undertaken but halted in the context
controlled trials. of observed thrombotic complications. Subsequent studies
Significant decreases in the measured numbers of circulating demonstrated upregulation of CD40L on platelets, with platelet
activated B cells and plasmacytoid B lymphocytes are observed aggregation occurring in the context of complement fixation
63
following 6 months’ treatment with belimumab. Transient to platelet membrane–bound anti-CD40L. Newer anti-CD40L

CHaPTEr 89 Biological Modifiers of Inflammatory Diseases 1205



TABLE 89.4 recombinant Inhibitors is CD28, which binds CD80/CD86 on antigen-presenting cells
of Lymphocyte Proliferation, Survival (APCs). In the context of T-cell activation, CTLA-4 expression
and activation is upregulated on the surface of T cells. The binding avidity of
CD80/CD86 for CTLA-4 is considerably higher than that for
Dosing CD28, resulting in preferential ligation of CTLA-4, thereby
Molecule Construct Half-Life (Maintenance) disrupting further costimulation as well as engendering inhibitory
Belimumab aBLyS/BAFF 19 days 10 mg/kg intravenously signaling in the T cell as a consequence of CTLA-4 ligation.
(human IgG1λ) (IV) every 4 weeks Soluble CTLA-4 Ig is not bound to the surface of the T cell and
Abatacept CTLA-4:IgG1Fc 13 days 10 mg/kg ; 500–1000 mg therefore does not engender inhibitory signaling; rather, the much
subcutaneously (SC) or higher avidity of the construct for CD80/86 inhibits T cell
IV every 4 weeks costimulation through CD28 (Table 89.4).
Belatacept CTLA-4:IgG1Fc 10 days 10 mg/kg IV every Although the time to maximum clinical response is somewhat
4 weeks
Basiliximab aCD25/IL-2Ra 7–9 days 20 mg IV (repeat ×1 at longer than that observed with TNF-α inhibitors, abatacept is
(chimeric IgG1κ) 4 days) effective in reducing disease activity and inhibiting progression
of structural damage in RA that is unresponsive to treatment
73
with methotrexate. Abatacept has no direct impact on phagocytic
cell responses, and its use in patients with RA may be associated
with fewer bacterial infection complications relative to patients
constructs employing PEGylated anti-CD40L F(ab)’ that appear on anti-TNF therapy. It is, nonetheless, recommended that
to be free of the effect on platelet activation/aggregation are treatment with abatacept be withheld in the context of serious
being evaluated in human SLE trials. 69 intercurrent microbial infections and not be used in conjunction
with other biological therapies targeting inflammation. Abatacept
KEY CONCEPTS may be associated with an increased risk of lung cancer; however,
Inhibitors of B-Cell and T-Cell Activation the occurrence of lymphoma in patients with RA treated with
abatacept has not been shown to exceed the expected occurrence
• Inhibition of BAFF/BLyS (B lymphocyte stimulator) over a period of in patients with RA. In contrast to TNF-α inhibitors, use of
6–12 months decreases the survival and maturation of autoreactive abatacept does not appear to promote autoimmune complications
B cells, decreases autoantibody titers, and decreases lupus disease and may therefore be a preferred option for treatment of patients
activity, with minimal impact on preexisting antibody titers to microbial with RA who need biological therapy and have overlapping
pathogens.
• Inhibition of T-cell costimulation targeting CD80/86 and CD28 via features of SLE or other autoimmune disorders.
exogenous cytotoxic T lymphocyte antigen-4 (CTLA-4):IgFc constructs Belatacept is a second-generation CTLA-4 Ig, which, compared
is effective in suppressing disease activity in rheumatoid arthritis (RA). with abatacept, has higher binding to both CD80 and CD86.
Currently used primarily in organ transplantation, belatacept is
Inhibitors of T-Cell Activation associated with improved patient and renal allograft survival
74
compared with cyclosporine. Although perceived to be of
Given the role of T-cell lymphocytes in orchestrating adaptive potential use in autoimmune disorders, studies of belatacept use
immune responses, selective inhibition of T-cell activation is in disorders, such as SLE and RA, have not yet been reported.
an attractive target for modulating inflammatory disorders
associated with immune responses to autoantigens or allografts. Inhibitors of Mast Cell Activation
Blocking cell receptors for T-cell growth factors, such as IL-2, Omalizumab
has been employed to prevent allograft rejection and therefore Omalizumab is a recombinant humanized mAb that binds to
may be potentially of use in the management of autoimmune the Cε3 domain of IgE. The binding domain is the same site at
disorders. Since productive immune responses are not generated which IgE normally binds to both high- and low-affinity FcεRI
in the absence of effective costimulatory signals, blocking T-cell on mast cells and basophils; as a consequence, free IgE is prevented
costimulation has also been an attractive target for treatment of from binding to the mast cell FcεRI receptor. Omalizumab is
inflammatory diseases driven by autoreactive T cells (Table 89.4). specific to IgE and does not bind to IgG or IgA. Omalizumab
also cannot bind to FcεRI or to IgE already attached to FcεRI
Basiliximab and therefore does not interact with cell-bound IgE or activate
Basiliximab is a chimeric (murine/human) mAb that blocks the mast cells or basophils. Omalizumab is most useful, and presently
α chain of the IL-2R complex expressed on activated T lympho- approved for use in, the treatment of poorly controlled asthma
cytes, inhibiting the binding of IL-2 to IL-2R (CD25). Basiliximab despite inhaled corticosteroid use and in the setting of docu-
is approved for and used primarily in induction regimens for mented sensitization to a perennial allergen in the setting of
75
70
prevention of rejection of transplanted allograft. Uncontrolled serum IgE levels ≥30 IU/mL. It is also approved for use in
small case series have reported manifestations of systemic sclerosis adults and adolescents with chronic idiopathic urticaria who
and pulmonary complications associated with amyopathic remain symptomatic despite H1 antihistamine treatment.
dermatomyositis responding well to (off-label) treatment with Omalizumab may also decrease the severity of asthma in patients
basiliximab. 71,72 with nonatopic (intrinsic) asthma, occupational asthma, virus-
induced asthma, and eosinophilic granulomatosis with polyan-
Abatacept giitis, but its use has not been fully studied in these populations
76
Abatacept (CTLA-4 Ig) is a recombinant human protein consisting to merit approved labeling. Controlled and long-term use studies
of the extracellular domain of CTLA-4 linked to the Fc portion of omalizumab have shown the incidence of adverse events is
of IgG1. Prominent among the T-cell costimulatory molecules not significantly increased (Table 89.5).

1206 ParT TEN Prevention and Therapy of Immunological Diseases



TABLE 89.5 recombinant Inhibitors of TABLE 89.6 recombinant T Cell– and B
Mast Cell and Eosinophil activation Cell–Depleting agents
Dosing Dosing
Molecule Construct Half-Life (Maintenance) Molecule Construct Half-Life (Maintenance)
Omalizumab aIgE (humanized 24–26 days 150–300 mg Rituximab aCD20 (chimeric 18–23 days 375 mg/m 2
IgG1κ) subcutaneously (SC) IgG1κ) intravenously (IV)
every 2–4 weeks per week ×4 (q4–6
Mepolizumab aIL-5 (humanized 16–22 days 100 mg SC every months) 1000 mg
IgG1κ) 4 weeks IV q2 week ×2
Reslizumab aIL-5 (humanized 24 days 3 mg/kg intravenously (q4–6 months)
IgG4κ) (IV) every 4 weeks Ofatumumab aCD20 (human 17 days 1000 mg IV every 4–8
IgG1κ) weeks
Obinutuzumab aCD20 24–36 days 1000 mg IV every 2
(humanized weeks ×2
IgG1κ)
Observed clinicopathological responses to treatment with Alemtuzumab aCD52 1–14 days 12 mg IV daily ×5
omalizumab include a marked downregulation of the surface (humanized days 10–30 mg
IgG1κ)
subcutaneously (SC)
expression of FcεRI on basophils and mast cells and reductions 3 times per week
in FcεR1-mediated production of Th2 cytokines by basophils. Brentuximab- aCD30 (chimeric 4–6 days 0.3–1.8 mg/kg IV
Markers of airway inflammation are significantly reduced, with vedotin IgG1κ):MMAE every 3 weeks
demonstrated reductions in sputum eosinophil counts and
reduced numbers of eosinophils, CD3, CD4, and CD8 T lym-
phocytes, B lymphocytes, IL-4–positive cells, and IgE-positive
cells in the bronchial mucosa. 77 Reslizumab is an mAb that targets IL-5R binding domains of
IL-5. Administered intravenously, reslizumab has been shown
KEY CONCEPTS effective in decreasing the frequency and severity of asthma
Inhibitors of IgE and IL-5R as Treatment for exacerbations and is approved by the FDA for use in adult patients
Severe Allergies with demonstrated eosinophilia and otherwise treatment-resistant
79
asthma. Adverse events include occasional episodes of severe
• Antibodies targeting the Fc binding domain of immunoglobulin E (IgE; hypersensitivity reactions during infusions.
omalizumab) are effective in the treatment of severe asthma and
recurrent mast-cell mediated chronic urticaria. On the Horizon: Anti-IL-5 Receptor Reagents
• Antibodies targeting the interleukin (IL)-5R binding domains of IL-5 The anti–IL-5R antibody benralizumab blocks activation of
are effective in the treatment of resistant asthma associated with eosinophils by IL-5 but has an additional property of depleting
eosinophilia as well as syndromes associated eosinophil-mediated 80
tissue injury. IL-5R–bearing eosinophils and basophils via enhanced ADCC.
The recombinant antibody is afucosylated, rendering a higher
affinity for FcγRIII and enhanced ADCC of IL-5R–bearing cells.
Inhibitors of Eosinophil Activation Studies are currently underway to confirm the clinical efficacy
Eosinophils mediate airway inflammation in patients with asthma, of benralizumab in managing asthma and to determine its utility
contribute to vascular and organ inflammation in patients with in managing other disorders mediated by tissue infiltration of
EGPA, and may mediate organ injury in other hypereosinophilic eosinophils.
syndromes. IL-5 is a potent cytokine mediator of eosinophil
hematopoiesis and has been shown to mediate eosinophilic B Cell– and T Cell–Depleting Agents
inflammation in the airways. Although corticosteroids are potent There are several mAb-based constructs that have been developed
suppressors of eosinophil survival, proliferation, and function, primarily for the treatment of lymphoid malignancies and/or
biologicals targeting IL-5 or the IL-5 receptor may prove useful prevention of transplant rejection. Since B cells have a demon-
as steroid-sparing therapy in managing chronic disorders mediated strated role in the generation of autoantibodies as well as antigen
predominantly by eosinophils. presentation to and costimulatory support for autoreactive T
Mepolizumab is an mAb that binds to IL-5, thereby inhibiting cells, strategies to deplete B lymphocytes have been successfully
its binding to the alpha chain of the IL-5R complex expressed employed in the treatment of autoimmune and inflammatory
on the surface of eosinophils. Mepolizumab has demonstrated diseases. In subsequent randomized trials, some of these agents
efficacy in patients with severe asthma who have blood eosinophil (rituximab) have been shown to have efficacy and are approved
counts of ≥150/µL and has been approved by the US Food and for use in the treatment of both RA and ANCA-associated
Drug Administration (FDA) for add-on, maintenance treatment vasculitis syndromes. Case reports have also documented the
of severe asthma in patients who are age ≥12 years and have successful use of mAb-mediated B cell– and also T cell–depleting
78
demonstrated hypereosinophilia. However, given the impact strategies in the management of patients with severe, refractory
of corticosteroids on peripheral eosinophil counts, limiting flare-ups of SLE, autoimmune inflammatory myopathies, and
selection of patients for treatment based on eosinophil count GvHD (Table 89.6).
thresholds should not be absolute in the setting of required
intercurrent steroid use. Adverse events associated with use of Rituximab
mepolizumab include hypersensitivity reactions and possible Rituximab is a chimeric IgG1 mAb with specificity for the
increased occurrence of herpes zoster. B-lymphocyte surface antigen CD20, a cell-surface molecule

CHaPTEr 89 Biological Modifiers of Inflammatory Diseases 1207


expressed on the surface of pre-B through activated mature B disease of the CNS. Cases of PML have been reported among
+
cells. Rituximab induces lysis of CD20 B cells by several mecha- patients with hematological malignancies, SLE, and RA who
nisms, including complement activation, ADCC, and induction were treated with rituximab, but PML usually occurs in the
of apoptosis. Induction regimens using four weekly doses or context of other therapies impacting lymphocyte survival and
two larger doses administered 2 weeks apart appear to be equally proliferation. 94
+
effective at effecting the depletion of circulating CD20 B cells,
which can last up to ≥9 months after a single course of therapy. Ofatumumab
Treatment with rituximab has been shown to cause significant Ofatumumab is a fully human mAb that binds to an epitope
transient decreases in inflammatory CD4 T cells that express encompassing both small and large loops of the extracellular
IL-17; the extent to which this is caused by an indirect consequence domain of the CD20 cell surface antigen on B lymphocytes. The
+
of CD20 B-cell depletion or depletion of identified subsets of binding epitope of ofatumumab is distinct from that of rituximab,
+
+
CD4 CD20 T cells is uncertain. 81,82 residing more proximal to the cell membrane. In comparative
Approved for use in patients with RA failing to respond to studies with rituximab using chronic lymphocytic leukemia (CLL)
initial disease-modifying antirheumatic drug (DMARD) therapy, cells, ofatumumab elicits similar ADCC but elicits greater
rituximab has been shown to improve the signs and symptoms complement-dependent cytotoxicity (CDC) because of the greater
of disease, functional status, and quality of life and to slow proximity of the binding site to the cell membrane and/or binding
83
radiographic progression of disease in patients with RA. Greater affinity to CD20 epitopes. 95
clinical responses are observed among patients with RA who are
seropositive for rheumatoid factor (RF) and/or anticitrullinated Obinutuzumab
peptide (CCP) autoantibody compared with patients seronegative Obinutuzumab (GA101) is a recombinant, humanized, and
84
for RF or CCP. The optimal treatment schedule for use of glycoengineered type II CD20 mAb of the IgG1 isotype that
rituximab in RA remains to be determined, but retreatment is targets the extracellular loop of the CD20 transmembrane
generally recommended no sooner than 6 months following expressed on the surface of pre-B and mature B lymphocytes.
initial treatment for patients in whom the disease flares up in Relative to rituximab and ofatumumab, glycoengineering of the
association with recovery of B-cell counts and for those who Fc portion of obinutuzumab results in a higher affinity for FcγRIII
had initially responded to rituximab therapy. receptors on immune effector cells, such as NK cells and phago-
Rituximab has also been used with considerable success and cytic cells. In vitro studies with B-cell lymphoma–derived cell
is approved for use in the management of ANCA-associated and lines show that compared with rituximab and ofatumumab,
other vasculitis syndromes. Rituximab plus glucocorticoid therapy obinutuzumab mediates greater induction of direct cell death
was shown to be as effective as cyclophosphamide in the treatment and effector cell–mediated ADCC and cellular phagocytosis.
of patients with granulomatosis with polyangiitis (GPA) or However, compared with rituximab and ofatumumab, CDC is
85
microscopic polyangiitis (MPA). In a subgroup of patients with significantly reduced with obinutuzumab. 96
relapsing ANCA vasculitis, rituximab was, in fact, superior to
85
cyclophosphamide in inducing disease remissions. In patients Alemtuzumab
with cryoglobulin syndromes occurring in the context of either Alemtuzumab is an mAb with specificity for CD52, an antigen
HCV infection or lymphoma, use of rituximab has been shown present on the surface of B and T lymphocytes as well as the
to decrease cryoglobulin and constituent Ig titers and hasten majority of monocytes, macrophages, NK cells, and a subpopula-
resolution of cryoglobulin manifestations, including skin ulcers, tion of neutrophils. Approved for use in the treatment of B-cell
glomerulonephritis, peripheral neuropathy, arthritis, and/or CLL and relapsing-remitting MS, alemtuzumab has also been
hyperviscosity complications. 86 used with success for the treatment of T-cell prolymphocytic
Although not yet formally approved, rituximab has been used leukemia, prevention and treatment of acute GvHD, and preven-
with reported success in the management of other autoantibody- tion of allograft rejection. Alemtuzumab has been used off-label
mediated disorders, including SLE, primary Sjögren syndrome, with reported success in the treatment of patients with severe
inflammatory myopathy, chronic inflammatory demyelinating SLE and Behçet disease refractory to other treatments. 97,98 Despite
polyneuropathy (CIDP), MS, and pemphigus. 87-92 the depletion of T lymphocyte, B lymphocyte, NK cell, and
Despite the potential for immunodeficiency-related to monocyte populations following treatment, reported rates of
+
depletion of CD20 B cells, only minimal increases in serious serious infections following treatment with alemtuzumab are
or opportunistic infections have been reported in patients with not significantly increased compared with other immunosup-
RA or ANCA vasculitis treated with repeated cycles of rituximab. pressive regimens employed to manage the disorders for which
Mild decreases in the overall levels of serum Igs may be observed it is used. However, there is a significant occurrence of secondary
during treatment, but Ig levels are rarely depleted, likely due autoimmunity following use of alemtuzumab, most often manifest
to the preservation of more mature B cells and plasma cells as autoimmune thyroid disease and immune thrombocytopenia.
that have lost surface expression of CD20. However, if used The observed autoimmune complications may be caused by
concomitantly with other immunosuppressive agents impacting homeostatic proliferation of self-reactive memory T cells in the
lymphocyte proliferation, significant hypogammaglobulinemia absence of an effective T regulatory response during immune
may ensue over time as a result of the inability to replenish the reconstitution. 99
93
plasma cell compartment. Use of rituximab carries a safety
warning related to an observed increased risk of viral infections, Brentuximab Vedotin
including CMV, herpes simplex virus (HSV), varicella-zoster Brentuximab vedotin (BTX-v) is an mAb with specificity for
virus (VZV), HBC, and JC virus. Reactivation of JC virus, CD30 that is covalently linked to the antitubulin agent mono-
latent in more than 80% of the general population, can occur methyl auristatin E. Expressed on subpopulations of T lympho-
in immunosuppressed patients to cause PML, a fatal demyelinating cytes, some populations of B cells, and most notably Hodgkin

1208 ParT TEN Prevention and Therapy of Immunological Diseases



KEY CONCEPTS 10. Jackson SH, Miller GF, Segal BH, et al. IFN-gamma is effective in
reducing infections in the mouse model of chronic granulomatous
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11. Ridderstad A, Abedi-Valugerdi M, Möller E. Cytokines in rheumatoid
• Monoclonal antibody (mAb)–mediated depletion of CD20 B lymphocytes arthritis. Ann Med 1991;23(3):219–23.
+
is effective in the treatment of antineutrophil cytoplasmic antibody 12. Aaltonen KJ, Virkki LM, Malmivaara A, et al. Systematic review and
(ANCA)–associated vasculitis, cryoglobulin syndromes, and rheumatoid meta-analysis of the efficacy and safety of existing TNF blocking agents
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significant elevations in rheumatoid factor (RF) and anti–citrullinated
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• There are subsets of T cells that also express CD20, and part of the Opin Rheumatol 2004;16(4):338–43.
clinical efficacy of anti-CD20 mAb reagents may be attributed, in part, 14. Boehncke WH, Prinz J, Gottlieb AB. Biologic therapies for psoriasis. A
to depletion of these subsets of T cells. systematic review. J Rheumatol 2006;33(7):1447.
• mAb reagents with greater binding affinity and binding sites more 15. Li Y, Li A, Strait K, et al. Endogenous TNF-alpha lowers maximum peak
proximate to the cell membrane appear to have greater efficacy bone mass and inhibits osteoblastic Smad activation through NF-κB.
+
depleting CD20 lymphocytes. J Bone Miner Res 2007;22(5):646–55.
• Depletion of CD20 lymphocytes has been associated with reactivation 16. Peyrin-Biroulet L, Deltenre P, de Suray N, et al. Efficacy and safety of
+
of hepatitis B virus (HBV), and vigilance for latent HBV reactivation is tumor necrosis factor antagonists in Crohn’s disease: meta-analysis of
prudent when using anti-CD20 mAb reagents. placebo-controlled trials. Clin Gastroenterol Hepatol 2008;6(6):644.
• mAb reagents targeting CD52 deplete both T-helper cells as well as 17. Hubbard VG, Friedmann AC, Goldsmith P. Systemic pyoderma
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CHaPTEr 89 Biological Modifiers of Inflammatory Diseases 1210.e1


MULTIPLE-CHOICE QUESTIONS

1. A patient has symmetrical inflammatory arthritis and pho- the following biologicals would be MOST appropriate to
tosensitive skin rashes. The serological profile is notable for prescribe?
positive rheumatoid factor (RF), positive anti–citrullinated A. Secukinumab
peptide (CCP) antibody, and positive antinuclear antibody B. Tocilizumab
(ANA) (titer 1 : 640). She is diagnosed with rheumatoid arthritis C. Etanercept
(RA) and systemic lupus erythematosus (SLE) overlap. Treat- D. Natalizumab
ment with weekly dosing with methotrexate and treatment E. Vedolizumab
with an antimalarial (hydroxychloroquine) fails to achieve 4. A 23-year-old patient has a history of asthma, for which an
resolution of the joint inflammation. Which of the following inhaled corticosteroid preparation and a beta-agonist have
biological treatments would be most appropriate to treat the been prescribed, and recurrent urticaria, for which daily doses
ongoing joint inflammation? of an antihistamine (loratadine) and a leukotriene receptor
A. Etanercept antagonist (montelukast) have been prescribed. Although
B. Adalimumab occasional use of a rescue inhaler is required, the asthma is
C. Certolizumab felt to be reasonably well controlled; however, there are recur-
D. Rituximab ring episodes of urticaria and angioedema. Laboratory studies
E. Belimumab
3
are notable for a peripheral eosinophil count of 210/mm
2. A patient with congestive heart failure, chronic kidney disease, and serum immunoglobulin E (IgE) of 110 IU/mL. Comple-
and diabetes is hospitalized with manifestation of volume ment level C4 is 12 mg/dL (normal >14), complement C3 is
overload and fever. Escherichia coli bacteremia from urinary normal, and CH50 is normal. Tests for antinuclear antibodies
sepsis is confirmed. Appropriate parenteral antimicrobial (ANAs) and antineutrophil cytoplasmic antibodies (ANCAs)
therapy and diuresis are initiated, resulting in clinical improve- are negative. Addition of which of the following would be
ment. But on the third hospital day, the patient develops pain, MOST useful in managing this disorder?
swelling, and tenderness in the left great toe, midfoot, and A. Omalizumab
ankle. An ankle arthrocentesis is performed, and it demon- B. Rituximab
strates of urate crystals in the joint fluid; gram staining is C. Mepolizumab
negative for bacterial organisms. Given the reluctance to D. Reslizumab
prescribe nonsteroidal antiinflammatory drugs (NSAIDs) or E. Eculizumab
corticosteroids to manage the acute polyarticular gout because
of this patient’s comorbidities, which of the following bio- 5. A patient with an undifferentiated autoimmune connective
logicals would be MOST appropriate to use in this setting? tissue disease has ongoing severe inflammatory manifestations
A. Canakinumab affecting the central nervous system, lungs, joints, and kidneys
B. Anakinra despite use of standard therapy, prompting consideration of
C. Infliximab more aggressive cell-depleting immunosuppressive therapy.
D. Abatacept Use of which of the following is MOST likely to be associated
E. Mepolizumab with the reemergence of autoimmune manifestations following
immune reconstitution?
3. A patient with inflammatory bowel disease (IBD) has increased A. Rituximab followed by belimumab
weight loss in association with ongoing abdominal pain and B. Obinutuzumab followed by belimumab
diarrhea despite use of adalimumab. Abdominal imaging C. Alemtuzumab
confirms evidence of significant bowel inflammation involving D. Brentuximab vedotin
multiple segments of the ileum. Which of the following of E. Cyclophosphamide followed by low-dose IL-2

90









Vaccines



Sarah Kabbani, Mark J. Mulligan






It might be fair to imagine that the field of vaccinology began That vaccines have a societal benefit in addition to the
with the first recognition that a person who survived an infection individual protection provided to the vaccine recipient is a special
was much less likely to suffer from that disease in the future in and gratifying aspect of this highly valuable biomedical interven-
comparison with a person who had not previously had the disease. tion. Vulnerable community members whose immune systems
That is, the infectious disease, if it did not kill the person, resulted are unable to respond well to vaccines (newborns, immuno-
in an experienced, protected state. Today we would say that the compromised persons, older adults) or are unable to receive
protected survivor had immunity. vaccines (because of allergy or a medical contraindication) depend
Vaccines are clinically simple but immunologically complex on immunization of surrounding community members for
preventive or therapeutic interventions that can dramatically protection against vaccine preventable diseases. The vaccinated
reduce morbidity and mortality caused by infectious diseases in community becomes an immunological cocoon or wall of protec-
children, adolescents, adults, and seniors. In recent years, vac- tion for these vulnerable members of society.
cinations against other categories of disease have become an This chapter first reviews selected events in the history of
increasingly active area of research (e.g., cancer and dementia). vaccination, both distant and recent. The remarkable accomplish-
In addition, vaccines against microbes are increasingly appreciated ments that have resulted from programs of vaccination to
for their potential role in the medical field’s heightening battle date are then highlighted. We also describe important recent
against antimicrobial-resistant bacteria. For example, the protein milestones and changes in vaccine development strategies
conjugate pneumococcal vaccine was shown to reduce the that have the potential to revolutionize the field and offer great
1
incidence of antibiotic-resistant pneumonia. Preventing an illness hope for providing solutions for unmet vaccine needs. Current
through vaccination obviates the need to treat a bacterial infection vaccine recommendations are then summarized. Finally several
with antibiotics, thereby avoiding potential induction of antibiotic present and future challenges for the field of vaccinology are
resistance in either the targeted pathogenic bacterium or the discussed.
patients’ healthy microbiota.
Another important area of recent emphasis has been the HISTORY OF VACCINES
double power of vaccinations for pregnant women, protecting
two—mother and infant—against the targeted pathogen (e.g., The earliest known vaccines for which records have been identified
2
3,4
pertussis or influenza ). In addition, a critical need for pre- were against smallpox and were used in Asia early in the second
15
pregnancy vaccination to protect pregnant women and their millennium. Of course, they were not called “vaccines.” The
fetuses ahead of and during the highly vulnerable first trimester practice was called variolation and involved exposing, usually
of pregnancy is currently underscored by acquisition of Zika through the intranasal route, a smallpox-susceptible (not previ-
virus infections during pregnancy with resulting microcephaly ously infected) person to material from the dried scabs of a
and other birth and developmental anomalies. 5-8 person who had had smallpox. If the recipient survived, he or
Although senior citizens (>65 years of age) experience high she was protected against future smallpox disease. Since natural
proportions of the total morbidity and mortality for several vaccine smallpox had a 30% mortality rate and variolation had a lower
preventable diseases (e.g., seasonal influenza, pneumococcal disease, (≈1%) mortality rate, this ancient practice was an early example
herpes zoster) as a result of immunosenescence, they are least of weighing the risk-to-benefit ratio for a human health interven-
able to mount their own protective immune responses after tion. By 1700, variolation was employed in a number of societies
vaccination. Community protection (also termed herd immunity) in Africa, India, and the Ottoman empire, and it was in use in
16
of seniors by vaccination of children, who are the primary spreaders England and France in the 1700s. The practice of variolation
of many vaccine-preventable infectious diseases, and younger was not without risk and sometimes caused outbreaks of a mild
adults can provide dramatic reductions in infectious disease form of the disease. One of the many who died as a result of
incidence in seniors—for example, the reduction in pneumococcal variolation was a son of England’s King George III.
disease in seniors after the introduction of pneumococcal conjugate In 1796, an English physician was searching for a safer alterna-
vaccine for children. 9,10 Despite suboptimal vaccine responses tive to variolation. This physician was to become known as the
with aging, several vaccines are specifically recommended for father of vaccinology—Edward Jenner. He performed a smallpox
seniors (see Fig. 90.3) and some of the vaccines for seniors now vaccination experiment on James Phipps on May 14, 1796, using
employ novel strategies to enhance the immunosenescent response, as vaccine cowpox pus from the lesions on the hands of Sarah
17
such as high doses of influenza antigen 11,12 or coformulation of Nelmes, a milkmaid. Dr. Jenner then collected lesion material
virus antigen with an adjuvant. 13,14 from a patient with smallpox and used this as the viral challenge
1211