مجلة صيدلة_Vol 2 Issue 1

Editor-in-Chief
Prof. Dr. Mokhtar M. Mabrouk

Managing Editor
Prof. Dr. Fotouh R. Mansour

All rights reserved @ Faculty of Pharmacy, Tanta University, Egypt

https://jampr.journals.ekb.eg/ ISSN: 2636-4158

CONTENT Page
1
Copeptin: Promising Biomarker for Nephropathy in Type II Diabetes
Dina Adam Ali, Ghada Mohammad Al-Ashmawy, Sabhaa Mahmoud Aboelnasr,
Ola Abd El-Salam El-Feky

Preventive Role of Soy Protein in Fructose Induced Metabolic 8
Dysfunction in Rats via Inhibition of Nuclear Factor kappa B Pathway
Nahla El-Ashmawy, Eman Khedr, Hoda Elbahrawy, Reham Abdelhamid

Green Simultaneous Determination of Amlodipine Besylate and 16
Celecoxib by Dual wavelength and Simultaneous Equation
Spectrophotometric Methods
Mokhtar M. Mabrouk, Mohamed A. Abdel Hamid, Mary A. Michael

A Pilot Study Comparing the Effectiveness of Three Combined 24
Therapeutic Regimens in Egyptian Patients with Moderate to Severe
Chronic Obstructive Pulmonary Disease
Tarek M. Mostafa, Gamal A. El-Azab, Ghada A. Atia, Noran S. Lotfy

C-X-C Chemokine Receptor-3 as a Diagnostic Biomarker in Patients 33
with Rheumatoid Arthritis
Mohamed R. Salama, Hanan H. Omar, Samah I. Nasef, Amany H. Hamed,
Azza M. Abdallah

Research Article

Received 2nd October 2020 Copeptin: Promising Biomarker for Nephropathy in Type
Accepted 13th October 2020 II Diabetes
Published 13th October 2020
Dina Adam Ali1, Ghada Mohammad Al-Ashmawy2*, Sabhaa Mahmoud
Aboelnasr3, Ola Abd El-Salam El-Feky2

jampr.journals.ekb.eg 1Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta 31111, Egypt
2Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta 31111, Egypt
3Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta 31111, Egypt

Online ISSN: 2636-4158 ABSTRACT

Kidney injury is a dangerous diabetic microvascular complication responsible for the mortality of
diabetics, which would require biomarkers for early detection of diabetic nephropathy. Copeptin, the
C-terminal portion of vasopressin prohormone, is rapidly released in severe endogenous stress. So,
the current work was carried out to evaluate and estimate copeptin as a marker for diabetic
nephropathy. This cross-sectional work was conducted on 60 female and male type II diabetic cases.
Diabetic cases were divided as normoalbuminuric (urinary albumin was <30 mg/24 h) without
nephropathy and macroalbuminuric (urinary albumin was >300 mg/24 h) with nephropathy. The
Control group was designated from fifteen matched healthy subjects. Controls and patients were
evaluated for fasting blood glucose, glycosylated hemoglobin (HbA1c), urinary albumin, serum
creatinine, and serum copeptin. Copeptin concentrations were significantly increased in type II
diabetics with macroalbuminuria comparing to healthy controls and diabetics with
normoalbuminuria. Serum copeptin concentrations were correlated positively with gold standard
urinary albumin, serum creatinine, and HbA1c. Higher serum copeptin concentration in type II
diabetics particularly in diabetics with nephropathy and its correlation with urinary albumin and
HBA1c reflect the potential role of copeptin as a predictor of diabetes mellitus and development of
diabetic nephropathy among type II diabetics considering other risk factors.

Keywords: Diabetes, Macroalbuminuria, Copeptin, Creatinine, Nephropathy

1. INTRODUCTION resistance to insulin action. The metabolic complications
include carbohydrate, fat, and protein metabolism. Diabetes
Diabetes mellitus (DM) is a chronic metabolic disease with mellitus disturbs all ages with a higher incidence in adults.
serious health complications. Patients with DM are presented Kidney injury is a serious diabetic microvascular disorder that
with hyperglycemia due to insulin release lack and/or may cause mortality of diabetics.1 Blood glucose elevation
develops chronic renal disease and renal injury via formation
*Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta, of active protein kinase C, augmented generation of
Egypt. 31111, Tel.: (202) 040-3336007; fax: (202) 040-3335466. diacylglycerol, and synthesis of advanced glycosylation end
E-mail address: [email protected] products. Also, hyperglycemia is accountable for
hemodynamic changes like stress, glomerular hyperfiltration,

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 1-7 | 1

J Adv Med Pharm Res Research Article

and microalbuminuria.2 The changes donate to irregular Informed consent was requested and collected from controls
activation of resident renal cells which increase glucose and patients. The study was approved by the Ethical
transporter-1 and upregulate intracellular transport and uptake Committee, Faculty of Medicine, Tanta University, Egypt.
of glucose as results of transforming growth factor beta-1
(TGFβ-1) generation. TGFβ-1 results in extracellular matrix 2.3. Laboratory investigations
protein, such as collagen types I, IV, V, and VI, fibronectin,
and laminin, deposition at the glomeruli, producing mesangial Early morning fasting blood samples were obtained. The
expansion and thickening of glomerular basement collected blood was obtained by standard venipuncture in
membrane.3 VACUETTE® blood collection tubes. Samples of serum were
prepared by standing the samples at room temperature to clot
Copeptin, the COOH-terminal stable part of vasopressin for 5–10 minutes and were centrifuged at 1,000× g for 10
precursor, is a simply quantifiable substitute biomarker of minutes and were kept at –20°C till the time of biochemical
vasopressin.4 Researches on healthy subjects have reported analysis.8 Also, 24-hour urine samples were collected. Blood
that plasma vasopressin and copeptin levels strongly correlate glucose, serum creatinine, and 24-hour urine albumin were
over a different osmolalities range.5 Christ-Crain, 2019 found assessed by commercial kits purchased from Biodiagnostic
that vasopressin level is increased in diabetics and selective Co., Egypt, using Shimadzu® (Japan) Spectrophotometer.
vasopressin V2-receptor antagonist treatment abolished the Hemoglobin A1c was determined for glycemic control by
albuminuria elevation in diabetics.1 Early diabetic measuring the HbA1c% according to the method described by
nephropathy diagnosis is crucial and supports to decrease Hanas et al.,9 using a commercial kit obtained from
diabetic death. The current research designed to inspect the Biosystems (Barcelona, Spain). Serum copeptin levels were
clinical implication of serum copeptin in patients of type II assayed using human copeptin ELISA kit, obtained from
diabetes with and without nephropathy and to assess the Shanghai Sunred Biological Technology Co., China, using
relation of copeptin with other laboratory and clinical markers Awareness Technology® (USA) ELISA reader. Serum
as a risk parameter for diabetic nephropathy prediction and copeptin concentration was expressed as ng/mL.
also to examine serum copeptin association with elevating
serum creatinine concentration risk in patients with DM type 2.4. Statistical analysis
II and normoalbuminuria or macroalbuminuria from our
study. Data were processed using SPSS statistical package version
22.0, IBM Corporation Software Group, USA. Quantitative
2. PATIENTS AND METHODS results were examined for normality using one sample
Shapiro-Wilk test. Normally distributed data as mean ±SD.
2.1. Patients Analysis of variance (ANOVA) was used to compare
normally distributed quantitative data. For non-normally
Our study was conducted on type II diabetics presented to distributed data; Mann-Whitney U test was performed to
Diabetes and Endocrinology Unit, Internal Medicine compare differences between groups. Spearman correlation
Department, Tanta University Hospital, Egypt. The work coefficient was used to assess the strength and direction of
involved 60 type II diabetic cases. The diagnosis of diabetes association that exists between variables. All P values were
was considered as fasting blood glucose ≥126 mg/dL two-tailed and P<0.05 was considered statistically significant.
according to WHO.6 The exclusion criteria include patients Receiver operating characteristic (ROC) curves were operated
with diabetes type I, type II diabetics with cancer, end-stage to evaluate the accuracy of copeptin and other biomarkers to
renal failure, liver disease or heart disease. The diagnosis of diagnose diabetic nephropathy. The area under the receiver
nephropathy was evaluated according to measuring operating characteristic curve (AUC) is a summary measure
albuminuria >300 mg/24 h in at least two of three successive over criteria and cut-point points.
assessments on 24-h sterile collected samples of urine.
Urinary microalbumin was used to classify patients to type II 3. RESULTS
diabetics without nephropathy (having normoalbuminuria
<30 mg/24 h) and type II diabetics with nephropathy (having 3.1. Main findings
albuminuria >300 mg/24 h).7
Table 1 displays the patient baseline data which include; age,
The Control group consisted of fifteen healthy age- body mass index (BMI), and duration of diabetes. The study
matched subjects. The history was engaged for participants involved 15 healthy volunteers and 60 type II diabetic
with a precise recording of diabetes duration, urinary patients. Thirty patients (50%) were diagnosed with diabetic
symptoms, therapeutic history and history of any other nephropathy. Diabetic groups without and with nephropathy
associated disorder. Table (1) illustrated the studied groups were age-matched in age compared to healthy control group.
distribution regarding age, body mass index (BMI) and BMI exhibited a significant increase in both diabetic patient
duration of diabetes. groups with and without nephropathy compared to control
group (P<0.001), also, BMI showed a significant increase in
2.2. Ethical statement diabetic group with nephropathy (P<0.05) compared to
diabetic group without nephropathy. Moreover, the duration

2 | J Adv Med Pharm Res., 2021, 1, 1-7 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

of diabetes was significantly (P<0.05) increased in diabetic blood glucose, HbA1c%, urinary microalbumin, creatinine,
patients with nephropathy compared to diabetic patient and copeptin, which was analysed using Mann–Whitney test.
without nephropathy (Table 1). As presented in (Table 2) and (Figure 1d) serum levels of
Table 1: Baseline characteristics of healthy controls and diabetes copeptin in diabetic patients with nephropathy group shows a
mellitus groups significant increase [10.06 (IQR, 9.94-10.63) ng/mL]
compared to diabetic patients without nephropathy and
Characteristics Control DM without DM with control groups [7.10 (IQR, (6.69-7.20) and 5.99 (IQR, 3.99-
nephropathy nephropathy 6.81) ng/mL, respectively, P < 0.001].

N 15 30 30 Table 2: Biomarkers of healthy controls and diabete0s mellitus
56.20±1.78 56.63 ± 2.23 57.10 ±2.94 groups
Age (years) 19.17 ±2.31 24.29 a**± 1.88 25.57 a**b*±

Body mass index 2.10
(BMI) (Kg/m2)

Biomarkers Control DM without DM with

Duration of --- 6.30 ± 1.68 9.09 b*± 2.15 FBG (mg/dL) 104.20 nephropathy nephropathy
diabetes (years) 116.52 a 148.50 a

(99.96- (110.1-151.02) (115.30-

Data are presented as mean ± SD Hb A1c% 110.00) 7.11 a 203.98)
Body mass index (BMI) was calculated as [mass (kg)]/[height (m)]2 5.16 8.11 ab
***: significant P<0.001, *: Significant P<0.05
DM: Diabetes mellitus, N: number of subjects in each group Urinary (4.23-6.48) (5.39-7.64) (8.40-8.69)
a: Significant versus the control group 7.25 9.20 a 235.69 ab
b: Significant versus type II DM without nephropathy group
P values were compared by of analysis of variance (ANOVA) test Microalbumin (5.36-8.69) (8.76-10.99) (89.70-319.01)

(mg/ 24 hr) 0.95 0.89 2.8 ab
Creatinine

(mg/dL) (0.63-0.98) (0.70-1.10) (2.61-3.14)
Copeptin 5.99 7.10 a 10.06 ab

3.2. Relation between serum copeptin and blood (ng/mL) (3.99-6.81) (6.69-7.20) (9.94-10.63)
glucose
Data are presented as median (IQR), P<0.05 was set as significant.
Statistical difference between groups was according to Mann–Whitney test.

FBG: fasting blood glucose, DM: diabetes mellitus

We reported a significant increase in serum levels of copeptin a: Significant versus the control group
in diabetic patients without nephropathy group compared to
normal controls [7.10 (IQR, 6.69–7.20) ng/mL] vs. [5.99 b: Significant versus DM without nephropathy group
(IQR, 3.99-6.81) ng/mL; P<0.001; (Table 2) and (Figure
1d)]. The ROC curve analysis of serum copeptin levels Table 3: Performance of copeptin, urinary microalbumin, and
(Figure 2a) showed a sensitivity of 90.0% and a specificity of HbA1c compared to FBG as predictors of DM risk
66.7%, with the area under the curve at 0.841 (95% CI, 0.708-
0.974). As illustrated in (Table 3) and (Figure 2); Biomarker AUC P- Sensitivity Specificity Cut off
measurement of serum copeptin levels exhibited a
significantly higher discriminatory ability for diagnosis of (95% value point
hyperglycemia as compared with urinary microalbumin
(AUC, 0.828; 95% CI, 0.703-0.953; P<0.001, Figure 2b), CI)
HbA1c% (AUC, 0.803; 95% CI, 0.672-0.935; P<0.01,
(Figure 2c)), and FBG (AUC, 0.872; 95% CI, 0.773-0.971; Copeptin 0.841 0.000 90.0% 66.7% 6.11
P<0.001). (ng/mL) (0.708- 0.000 80.0% 66.7% 8.17
0.974 0.001 73.3%
Furthermore, in the present study, we found a significant Urinary 0.828 73.3% 6.21
positive correlation (non-parametric Spearman correlation micro- (0.703-
test) between serum levels of copeptin and each of fasting albumin 0.953)
blood glucose, HbA1c%, urinary microalbumin, and (mg/ 24 h)
creatinine (P<0.001 for each) which confirmed ROC-AUC HbA1c% 0.803
results and indicated that an increased risk of DM was
associated with increased serum levels of copeptin. FBG (0.672- 0.000 --- --- ---
(mg/dL) 0.935)
0.872
(0.773-
0.971)

Healthy controls and diabetic patients without nephropathy group are included
to perform receiver operating curve (ROC) analysis. AUC: Area under curve,

CI: Confidence interval, FBG: Fasting blood glucose

3.3. Serum copeptin for diagnosis of DM 3.4. Diagnostic performance of copeptin for
nephropathy diabetic nephropathy detection

(Table 2) illustrated a significant increase in fasting blood The ROC-AUC analysis for copeptin to predict diabetic
glucose, HbA1c%, and urinary microalbumin among diabetic nephropathy at the cut-off point of 6.40 ng/mL shows a
patients with and without nephropathy compared to control sensitivity of 90.0% and a specificity of 73.3%, with the area
group, while a non-significant change in creatinine was under the curve (AUC) at 0.921 (95% CI, 0.851-0.991)
observed in diabetic patients without nephropathy compared (Figure 3a) which was almost comparable to urinary
to normal controls. (Figure 1) shows the statistical differences microalbumin (AUC, 0.914, 95% CI, 0.847-0.980) (Figure
between control group and diabetic patient groups for fasting 3b) and higher than that of HbA1c (AUC, 0.899,0 95% CI,
0.826-0.971) (Figure 3c) and serum creatinine (AUC, 0.760,
95% CI, 0.649-0.871) (Figure 3d). The ROC-AUC analysis,

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 1-7 | 3

J Adv Med Pharm Res Research Article

Figure 1: Levels of different biomarkers in different groups. (a) serum levels of fasting blood glucose, (b) HbA1c%, (c) serum levels of creatinine, (d)
serum levels of copeptin. Mann–Whitney U test. All data are medians and interquartile ranges (IQR). DM: Diabetes mellitus. NS: non-significant;
***P <0.001; ** P<0.01. The outlier’s values are represented by circles

Figure 2: Comparison of diagnostic values of copeptin, urinary microalbumin, and HbA1c to discriminate Type II DM (without nephropathy) from
healthy controls. (a) ROC curves of copeptin for differential diagnosis of Type II DM (without nephropathy) (n = 30) from healthy controls (n = 15). (b) ROC
curves of urinary microalbumin for differential diagnosis of Type II DM (without nephropathy) (n = 30) from healthy controls (n = 15). (c) ROC curves of
HbA1c for differential diagnosis of Type II DM (without nephropathy) (n = 30) from healthy controls (n = 15). DM: diabetes mellitus, AUC: area under curve,
ROC: Receiver operating characteristic.

sensitivity, and specificity of measured parameters were (Table 5) illustrated a significant positive correlation between
summarized in (Table 4). serum copeptin and urinary microalbumin. Also, each of
copeptin and urinary microalbumin had a significant positive
Moreover, we found that the serum copeptin improved the correlation with fasting blood glucose, HbA1c, and creatinine.
diagnostic performance of HbA1c as showed in the combined These results confirmed the ROC curve analysis of copeptin
ROC curve model (Figure 4) (AUC of HbA1c in the and indicated that measurement of serum copeptin could be a
combined model: 0.913, P<0.001). predictor of diabetes mellitus risk and a diagnostic for diabetic
nephropathy (Figure 5). Also, measurement of serum
3.5. Correlation of serum copeptin with the copeptin have a better patient connivance compared to 24-h
other biomarkers used for diabetic nephropathy urine samples collection which needed for urinary
detection microalbumin determination.

4 | J Adv Med Pharm Res., 2021, 1, 1-7 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

Figure 3: Performance of Copeptin, urinary microalbumin, HbA1c, and creatinine in diagnosis of Type II DM patients (with and without
nephropathy). ROC curves of copeptin (a), urinary microalbumin (b), HbA1c (c), and creatinine (d), showing that copeptin had the highest ROC-AUC for
diabetic nephropathy detection. AUC: Area under curve, ROC: Receiver operating characteristic.

Table 4: Diagnostic performance of copeptin, urinary microalbumin,
and HbA1c compared to creatinine in diagnosis of diabetic nephropathy

Biomarkers AUC P- Sensitivity Specificity Cut

value off

point

Copeptin 0.921 0.000 90.0% 73.3% 6.40
(ng/mL) (0.851- 0.000 86.7% 66.7% 5.94
0.991) 0.000 90.0% 66.7% 8.17
HbA1c% 0.899
(0.826-
Urinary 0.971)
microalbumin 0.914
(0.847-

(mg/24 h) 0.980)

Figure 4: Combined ROC curve of copeptin and HbA1c for differential Creatinine 0.760 0.002 63.3% 86.70% 0.99
diagnosis of Type II DM complicated with nephropathy. Combined ROC (mg/dL) (0.649-
curve of copeptin and HBA1c for differential diagnosis of Type II DM (with 0.871)
nephropathy) (n = 30) from Type II DM (without nephropathy) (n = 30). DM:
diabetes mellitus, AUC: area under curve, ROC: Receiver operating Healthy controls and diabetic patients without and with nephropathy groups
characteristic. are included to perform receiver operating curve (ROC) analysis. AUC: Area
under curve, CI: Confidence interval.

Table 5: Correlation between measured biomarkers in different
groups

Parameters Copeptin level Urinary
r values microalbumin

FBG (mg/dL) 0.425* r values
HbA1c% 0.712* 0.433*
0.772* 0.703*
Urinary Microalbumin
(mg/24 hr) 0.712* ---

Creatinine (mg/dL) 0.691*

Figure 5: Combined ROC curve of copeptin, urinary microalbumin, Copeptin (ng/mL) --- 0.772*
HbA1c, and creatinine for differential diagnosis of Type II DM from
healthy controls. DM: diabetes mellitus, AUC: area under curve, ROC: FBG: fasting blood glucose, *: significant P<0.001. Non-parametric
Receiver operating characteristic. Spearman correlation analysis was performed

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 1-7 | 5

J Adv Med Pharm Res Research Article

4. DISCUSSION regulation of inflammation, adipocyte development, and
glucose homeostasis. Elevated glucocorticoid level in blood
Diabetic nephropathy is one of the commonly occurring is clinically characterized by the development of diabetes,
microvascular complications of DM and is considered the insulin resistance and visceral adiposity.20
main cause of end-stage renal failure in Middle East
countries.10 Protein glycosylation with advanced glycated end ROC curve analysis in the present study revealed that
products (AGEs) is the main pathological source of elevated copeptin could be an excellent undependable predictor of DM
urinary albumin excretion, which resulted in hypertrophy of risk with a sensitivity of 90.0% and specificity of 66.7%
glomerular and renal systems. The continuous leakage of (AUC; 0.841) compared to fasting blood glucose and HbA1c
albumin and other proteins into urine in turn lead to diabetic (AUC; 0.872 and 0.803, respectively). These results were in
nephropathy.11 line with Zhu et al.15 Moreover, copeptin shows high
sensitivity and specificity (90.0% and 73.3%, respectively)
In our work, diabetic nephropathy was evident in Type II (AUC; 0.921) for diagnosis of nephropathic complications of
DM patients and characterized by a significant increase in type II DM. while other biomarkers such as urinary
serum creatinine as well as urinary microalbumin compared microalbumin and HbA1c exhibited decreased diagnostic
to DM patients without nephropathy. Our findings agreed values (AUC; 0.914 and 0.899, respectively) compared to
with El-Ashmawy et al.11 who reported a significant positive copeptin.
correlation between microalbuminuria and serum creatinine We also revealed that the addition of copeptin to HbA1c
levels. improved the diagnostic value of HbA1c as shown in the
combined ROC-AUC curve which was consistent with the
Diabetic nephropathy is associated with presence of large previous work.21
amount of urinary proteins, mainly albumin.12 We reported a
significant increase in microalbuminuria in diabetic patients Moreover, we reported a significant positive correlation
with nephropathy, which was in line with Satchell and with urinary microalbumin as a gold standard biomarker of
Tooke.13 Moreover, we reported a normoalbuminuric range in DM nephropathy.
diabetic patients without nephropathy which was consistent
with Viswanathan et al.,14 who suggested that the risk of In this study, the increased levels of serum copeptin in
diabetic nephropathy development starts even when urinary diabetic patients with/without nephropathy compared to
excretion of albumin is within the normoalbuminuric range, controls strongly indicate the potential role of copeptin in the
and the progression from normoalbuminuria into development of DM and progressive decline of renal
micro/macroalbuminuria occurs more frequently in type II functions.
DM patients with baseline urinary albumin > 2.5 mg/24 h.
5. CONCLUSIONS
In the present study, microalbuminuria showed a
significant positive correlation with fasting blood glucose and The present study suggests the strong correlation of copeptin
HbA1c%. Furthermore, ROC curve analysis revealed that with DM development and declined renal functions in patients
microalbuminuria has a greater efficacy as a biomarker of of Type II DM. Increased levels of copeptin in diabetic
diabetic nephropathy (AUC; 0.914) compared to HbA1c% patients without nephropathy strongly indicates the potential
(AUC; 0.899) and serum creatinine (AUC; 0.760).15 So, the use as an early predictor of diabetic nephropathy. Future
study of new diagnostic tools and markers for prediction and studies are warranted to investigate the role of copeptin as
diagnosis of diabetic nephropathy is an urgent need. early biomarker for diagnosis of diabetic nephropathy on
larger sample size.
Arginine vasopressin (AVP) has a significant role in the
pathophysiology of DM and its associated complications such CONFLICTS OF INTEREST
as diabetic nephropathy. Copeptin is an inactive analogue of
AVP which secreted in an equimolar quantity to AVP and is The authors declare that they have no competing interests.
considered one of the most reliable biomarkers of AVP as it
has a relatively longer half-life and stable structure than 6. REFERENCES
AVP.16 Furthermore, copeptin; unlike other biomarkers such
as creatinine, HbA1c, and urinary microalbumin excretion; is 1 M. hrist-Crain, Rev. Endocr. Metab. Disord., 2019,
sensitive only to DM and its related complications.17 20, 283–294.

In this work, we reported for the first time a significant 2 Y. Wu, Y. Ding, Y. Tanaka and W. Zhang, Int. J.
increase in serum levels of copeptin among Type II DM Med. Sci., 2014, 11, 1185–1200.
patients without and with nephropathy compared to healthy
controls. Consistent with our findings, Bjornstad et al. (2016) 3 C. Hills, G. W. Price, M. J. Wall, T. J. Kaufmann, S.
reported that copeptin was strongly associated with diabetic Chi-Wai Tang, W. H. Yiu and P. E. Squires, Cell.
kidney disease and coronary atherosclerosis in adults with Physiol. Biochem., 2018, 45, 2369–2388.
Type I DM.18
4 N. G. Morgenthaler, J. Struck, S. Jochberger and M.
High level of copeptin may play a critical role in the W. Dünser, Trends Endocrinol. Metab., 2008, 19, 43–
process of DM progression. Binding of AVP to vasopressin 49.
V1b receptor in the anterior hypophysis leads to release of
adrenocorticotrophic hormone and increase the glucocorticoid 5 G. Velho, N. Bouby, S. Hadjadj, N. Matallah, K.
levels in blood.19 Glucocorticoids are involved in the Mohammedi, F. Fumeron, L. Potier, N. Bellili-

6 | J Adv Med Pharm Res., 2021, 1, 1-7 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

Munoz, C. Taveau, F. Alhenc-Gelas, L. Bankir, M.
Marre and R. Roussel, Diabetes Care, 2013, 36,
3639–45.
6 L. Bellamy, J.-P. Casas, A. D. Hingorani and D.
Williams, Lancet, 2009, 373, 1773–1779.
7 B. C. Martin, J. H. Warram, A. S. Krolewski, J. S.
Soeldner, C. R. Kahn and R. N. Bergman, Lancet,
1992, 340, 925–929.
8 N. E. El-Ashmawy, F. Z. Hussien, O. A. El-Feky, S.
M. Hamouda and G. M. Al-Ashmawy, Life Sci., 2020,
259, 118193.
9 R. Hanas and G. John, in Diabetes Care, American
Diabetes Association, 2010, vol. 33, pp. 1903–1904.
10 T. W. C. Tervaert, A. L. Mooyaart, K. Amann, A. H.
Cohen, H. T. Cook, C. B. Drachenberg, F. Ferrario,
A. B. Fogo, M. Haas and E. de Heer, J. Am. Soc.
Nephrol., 2010, 21, 556–563.
11 N. E. El-Ashmawy, E. A. El-Zamarany, N. F. Khedr,
A. I. Abd El-Fattah and S. A. Eltoukhy, Int. J.
Diabetes Dev. Ctries., 2015, 35, 431–438.
12 N. K. Chowta, P. Pant and M. N. Chowta, Indian J.
Nephrol., 2009, 19, 53.
13 S. C. Satchell and J. E. Tooke, Diabetologia, 2008,
51, 714.
14 G. Viswanathan and A. Upadhyay, Adv. Chronic
Kidney Dis., 2011, 18, 243–248.
15 F.-X. Zhu, H.-L. Wu, K.-S. Tu, J.-X. Chen, M. Zhang
and C. Shi, J. Diabetes Complications, 2016, 30,
1566–1570.
16 G. Luckner, M. W. Dünser, S. Jochberger, V. D.
Mayr, V. Wenzel, H. Ulmer, S. Schmid, H. Knotzer,
W. Pajk and W. Hasibeder, Crit. Care Med., 2005, 33,
2659–2666.
17 K. S. Evers and S. Wellmann, Front. Pediatr., 2016,
4, 75.
18 P. Bjornstad, D. M. Maahs, T. Jensen, M. A. Lanaspa,
R. J. Johnson, M. Rewers and J. K. Snell-Bergeon, J.
Diabetes Complications, 2016, 30, 1093–1096.
19 E. S. van der Valk, B. van der Voorn, A. M. Iyer, S.
van den Berg, M. Savas, Y. B. de Rijke, E. van den
Akker, O. Melander and E. F. C. van Rossum, Eur. J.
Endocrinol.
20 S. Canivell, M. Mohaupt, D. Ackermann, M. Pruijm,
I. Guessous, G. Ehret, G. Escher, A. Pechère-
Bertschi, B. Vogt and O. Devuyst, J. Endocrinol.
Invest., 2018, 41, 799–808.
21 C. Then, B. Kowall, A. Lechner, C. Meisinger, M.
Heier, W. Koenig, A. Peters, W. Rathmann and J.
Seissler, Acta Diabetol., 2015, 52, 103–

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 1-7 | 7

Research Article

Received 11th October 2020 Preventive Role of Soy Protein in Fructose Induced
Accepted 30th October 2020 Metabolic Dysfunction in Rats via Inhibition of Nuclear
Published 30th October 2020 Factor kappa B Pathway

Nahla El-Ashmawy1, Eman Khedr1, Hoda Elbahrawy1, Reham Abdelhamid1*

1Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta 31111, Egypt

jampr.journals.ekb.eg ABSTRACT
Online ISSN: 2636-4158
Soy protein is an important component of soybeans that has a beneficial role in improving insulin
resistance. Nuclear factor-kappa B (NF-κB) is a crucial pathway that has been implicated in the
development of metabolic syndrome. Our study aimed to examine the protective effect of soy protein
isolate - as a natural NF-κB inhibitor - and its possible mechanism of action in amelioration of
inflammatory and metabolic disorders induced in male albino rats by high fructose diet (10% w/v)
via using synthetic NF-κB inhibitor (IMD-0354). Rats were randomized into normal control group,
soy group, NF-κB inhibitor (IMD-0354) group, high fructose group, high fructose with soy group,
high fructose with NF-κB inhibitor group, high fructose with soy and NF-κB inhibitor group. Serum
glucose, serum insulin, serum-free fatty acids, insulin resistance (HOMA-IR), NF-κB,
phosphorylated insulin receptor (pISR), carbohydrate-responsive element-binding protein
(ChREBP) were determined and histopathological examination of liver tissue was performed. The
concurrent administration of IMD-0354 and/or soy protein with high fructose significantly increased
pISR and decreased FFAs, NF-κB, glucose, insulin, HOMA-IR, and ChREBP, as well as improved
the pathological conditions of the livers. The metabolic and inflammatory disorders induced by
chronic consumption of fructose could be inhibited by co-administration of soy protein through the
regulation of the NF-κB signaling pathway.

Keywords: fructose; metabolic dysfunction; soy protein; NF-κB; insulin resistance.

1. INTRODUCTION proinflammatory processes. Additionally, there are many

Fructose is a highly lipogenic, ketonic monosaccharide that is metabolic disorders induced by high fructose consumption
found in several fruits and is used as a sweetener in the food
industry. The over-consumption and chronic consumption of due to increased levels of FFAs that activate the inhibitor
fructose results in increased levels of reactive oxygen species kappa kinase β (IKK-β) and protein kinase C theta (PKCθ),
(ROS) and free fatty acids (FFAs) as well as reduced which are related to the dysregulation of insulin signalling.2
antioxidant capacity.1 Fructose consumption is associated
with the dysregulation of adipokines and augmentation of Soy protein is a protein isolated from the soybean. It

*Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta, is a rich source of dietary protein which provides the cells with
Egypt. 31527, Tel.: (202) 040-3336007; fax: (202) 040-3335466.
E-mail address: [email protected] essential amino acids and valuable macronutrients as shown
in Table (1).3 The unique importance of soy protein could be

attributed to that it contains isoflavones that responsible for
many biological and beneficial properties for maintenance of
health4. Isoflavones are known as phytoestrogens that have

estrogen-like structures and effects that protect

postmenopausal women from bone loss and maintain a

8 | J Adv Med Pharm Res., 2021, 1, 8-15 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

healthy heart.5 Consumption of soy protein has been found to rats were utilized. The rats were purchased from the National
Research Center (NRC) Dokki, Giza, Egypt. The rats were
reduce serum concentrations of total cholesterol, low-density weighed and housed in separated aluminium cages for two
weeks under identical environmental conditions and a 12-hour
lipoproteins (LDLs), and triglycerides. Soy protein also light-dark cycle. The animals were allowed free access to a
standard pellet diet and water ad libitum. After the
contributed to the control of hyperglycemia, body weight, acclimatization period, rats were weighed and randomly
divided into seven groups: Group 1 [the normal control group;
hyperlipidemia, and hyperinsulinemia. n=10], the rats were given a normal diet with a daily i.p.
injection of the vehicle for eight weeks. Group 2 [the soy
Genistein, one of the phytochemicals found in soy protein group; n=10], the rats fed on a diet containing 20%
soy protein isolate powder (Solae Company®, USA) daily for
protein, has a role in cancer prevention by preventing the eight weeks.10 Group 3 [the inhibitor (IMD) group; n=10], the
angiogenesis process.6 Much research has been conducted to rats received a daily i.p. injection of 10 mg/kg IMD-0354
(Selleckchem®, USA) for eight weeks.11 Group 4 [the high
study the effect of soy protein and its isoflavones in the fructose (HF) group; n=10], the rats were given 10% w/v
fructose in their drinking water (UNIPHARMA®, Egypt)
reduction of body fat mass and weight. Isoflavones could daily for eight weeks.12 Group 5 [the high fructose with soy
(HF-S) group; n=10], the rats received 10% w/v fructose in
reduce the obesity through lowering triglycerides and their drinking water along with a diet containing 20% soy
decrease the insulin resistance.7 protein isolate powder daily for eight weeks. Group 6 [the
high fructose with inhibitor (HF-IMD) group; n=10], the rats
A novel NF-κB inhibitor, IMD-0354 (N-(3,5-bis- received 10% w/v fructose in their drinking water along with
trifluoromethylphenyl) – 5 - chloro -2-hydroxybenzamide), a daily i.p. injection of 10 mg/kg IMD-0354 for 8 weeks.
inhibits IKK-β and blocks IκB phosphorylation in the NF-κB Group 7 [the high fructose with soy and inhibitor (HF-S-IMD)
pathway. The NF-κB signalling pathway plays an essential group; n=10], the rats received 10% w/v fructose in their
drinking water along with a diet containing 20% soy protein
role in both inflammation and angiogenesis. Inhibition of NF- isolate powder and a daily i.p. injection of 10 mg/kg IMD-
κB by IMD-0354 has been studied in several preclinical 0354 for eight weeks.

models, including models of cancer, reperfusion injury, After the experiment (8 weeks), the rats were
allergy, and lung fibrosis.8,9 weighed, anaesthetized by halothane (Delta Pharma®,
Egypt), and sacrificed. Blood was drawn from the heart
Accordingly, the present work aimed to study the through the cardiac puncture, and serum was separated for
determination of glucose, insulin, and FFAs. Serum samples
different metabolic disorders induced by chronic consumption were stored at -20°C until the analytical methods were
performed. The rats were dissected under completely sterile
of fructose. This work also aimed to evaluate the role and conditions to collect liver samples. The fresh liver was washed
twice with ice-cold saline, dried on a clean paper towel, and
possible mechanism of action of soy protein in amelioration weighed. The liver index was calculated as liver weight
(g)/final body weight (g) × 100. The liver was divided into
of inflammatory and metabolic disorders induced by over- four portions: one portion was preserved in 10% formalin for
histopathological examination, and the other parts were
consumption of fructose in rat models. immediately frozen in liquid nitrogen and stored at -80°C for
biochemical analysis.
Table (1): Components of soy protein
2.2. Biochemical analysis of blood and serum
Composition or element Amount determined in soy
protein isolate The fasting blood glucose level was determined in rat blood
determined 93.45 using an Accu-Chek glucometer (Roche diagnostics
<0.1 Deutschland GmbH®, Germany), and the level of blood
Protein (weight percent) glucose is expressed as mg/dL. Fasting serum insulin levels
2.32 were measured using a rat insulin enzyme-linked
Crude fat (weight percent) immunosorbent assay (ELISA) kit (Glory Science®, USA).
5.51 The concentration of serum insulin was determined according
Fat by acid hydrolysis (weight 21.165 to the manufacturer's procedure. The level of insulin was
percent) 1461 obtained from the standard curve and is expressed as mIU/L.
Insulin resistance (HOMA-IR) was calculated using the
Ash (weight percent) 856 following formula: HOMA-IR = [fasting insulin level
11.684 (μIU/mL) × fasting glucose level (mg/dL)] / 405.13 Serum
Sodium (ppm)
Potassium (ppm) 516

Calcium (ppm) 121.1

Phosphorus (ppm) 14.62
Magnesium (ppm) 24.02
2.09
Isoflavones 4.18
25.06
(microgram/gram total dry matter) 36.55
Daidzin 5.22
6’’-O-malonyldaidzin 4.18
6’’-O-acetyldaidzin 2.09
Daidzein 2.09
1.04
Genistin 81.2
6’’-O-malonylgenistin
6’’-O-acetylgenistin 86

Genistein

Glycitin
6’’-O-malonylglycitin

Glycitein

Nitrogen Solubility Index (NSI) (%)
Viscosity (10% Dispersion) (cP)

2. METHOD

2.1. Experimental design

This study was performed in accordance with the guidelines
for the care and use of laboratory animals and was approved
by the Research Ethics Committee (Faculty of Pharmacy,
Tanta University, Egypt). In our study, 140-160 g male albino

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 8-15 | 9

J Adv Med Pharm Res Research Article

FFAs were assayed using a rat FFA ELISA kit (Sunred Fig. 1: Effects of fructose, soy protein, and IMD-0354 on the percent
Biological Technology®, China). The concentration of serum change in rat weight in the studied groups. HF: high fructose diet, IMD:
FFAs was determined according to the manufacturer's inhibitor of NF-κB, HF-S: high fructose diet with soy protein, HF-IMD: high
procedure using a standard curve and is expressed as μmol/L. fructose diet with NF-κB inhibitor, HF-S-IMD: high fructose diet with soy
protein and NF-κB inhibitor. a: significant versus normal control. b:
2.3. Biochemical analysis of liver tissue significant versus HF. c: significant versus soy protein. d: significant versus
IMD. e: significant versus HF-S. f: significant versus HF-IMD. g: significant
NF-κB concentration was determined in liver tissue using a versus HF-S-IMD. Significance was set at p < 0.05.
rat NF-κB ELISA kit (Sunred Biological Technology®,
China) according to the manufacturer's procedure. The level 3.2. Liver weight and liver index
of NF-κB was obtained from a standard curve and is expressed
as ng/mL. Phosphorylated insulin receptor (pISR) Liver weight and liver index were significantly increased in
concentration was determined in liver tissue using a rat pISR the HF group (1.50- and 1.42-fold increase, respectively,
ELISA kit (Sunred Biological Technology®, China) p<0.001) compared to the normal control group. Liver weight
according to the manufacturer's procedure. The level of pISR and liver index were significantly decreased in the HF-S
was obtained from a standard curve and is expressed as
ng/mL. Carbohydrate-responsive element-binding protein Table 2: Changes in rat weight, liver weight, and liver index in
(ChREBP) concentration was determined in liver tissue using different studied groups
a rat ChREBP ELISA kit (Sunred Biological Technology®,
China) according to the manufacturer's procedure. The level Data are presented as mean ± SD, n=10 rats. HF: high fructose diet, IMD:
of ChREBP was obtained from a standard curve and is
expressed as ng/mL. Groups Weight of Weight of Liver Liver index
rats (g) rats (g)
2.4. Histopathological examination (before) (after) weight (g) (%)

The liver was fixed in 10% formalin and embedded in paraffin Normal 152.00 165.00 4.36 2.64
to form blocks that were serially sectioned (3-5 mm thick) and control ±2.54 ±2.94 ±0.25 ±0.0018
stained with haematoxylin and eosin (H&E). The slides were
examined blindly by a pathologist under a light microscope. HF 152.00 174.10 6.55 3.76
Images were viewed and recorded using an Olympus ±2.49 ±5.10 acdeg ±0.45 acdefg ±0.0018 acdefg
microscope equipped with a spot digital camera using the Soy
computer program MATLAB software in the Histochemistry protein 152.70 162.90 4.69 2.88
and Cell Biology Department, Medical Research Institute, IMD ±3.16 ±2.56b ±0.58b ±0.0033bg
Alexandria University, Egypt. HF-S
151.87 164.13 4.44 2.70
2.5. Statistical analysis HF-IMD ±2.59 ±4.32b ±0.29be ±0.0062b

All measured biochemical parameters were analyzed using 152.40 165.70 5.07 3.06
SPSS software version 20.0. Quantitative data are presented ±2.84 ±2.11b ±0.22abdf ±0.0027abfg
as the mean ± standard deviation (SD) of the mean. Statistical
comparisons between more than two groups were performed 152.50 170.30 4.43 2.60
by one-way analysis of variance (ANOVA) followed by post ±.90 ±0.79 ±0.55be ±0.0014be
hoc Tukey's test. The significance of the obtained results was
obtained at p<0.05. HF-S- 153.10 163.20 4.04 2.48
IMD ±3.28 ±7.79b ±0.38b ±0.0031bce
3. RESULTS
inhibitor of NF-κB, HF-S: high fructose diet with soy protein, HF-IMD: high
3.1. Body weight fructose diet with NF-κB inhibitor, HF-S-IMD: high fructose diet with soy
protein and NF-κB inhibitor. a: significant versus normal control. b:
Final rat weight was significantly increased in the HF group
(1.05-fold increase, p<0.05) compared to the normal control significant versus HF. c: significant versus soy protein. d: significant versus
group, while concurrent use of soy protein with a high
fructose diet significantly decreased the weight of rats (1.05- IMD. e: significant versus HF-S. f: significant versus HF-IMD. g: significant
fold decrease, p<0.05) compared to the weight of rats in the
HF group. Additionally, in the HF-S-IMD group, rat weight versus HF-S-IMD. Significance was set at p < 0.05.
significantly decreased (1.06-fold decrease, p<0.05)
compared to the HF group (Table 2). The change in rat weight
is expressed as the percentage change in weight, and the
results are shown in Figure (1).

10 | J Adv Med Pharm Res., 2021, 1, 8-15 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

group (1.29- and 1.23-fold decrease, respectively, p<0.001), significantly decreased in the HF-IMD (1.80- and 4.08-fold
the HF-IMD group (1.48- and 1.45-fold decrease, decrease, respectively, p<0.001) and HF-S-IMD groups
respectively, p<0.001) and the HF-S-IMD group (1.62- and (1.79- and 4.12-fold decrease, respectively, p<0.001)
1.52-fold decrease, respectively, p<0.001) compared to the compared to the HF group (Table 3).
HF group (Table 2).
3.5. Serum free fatty acids
3.3. Fasting blood glucose
The level of serum FFAs was significantly increased in the
The level of fasting blood glucose was significantly increased HF, HF-S, HF-IMD, and HF-S-IMD groups (↑169.28%,
in the HF group (2.25-fold increase, p<0.001) compared to the 149.38%, 142.77%, and 144.19% respectively, p<0.001)
normal control group. Blood glucose levels were significantly compared to the normal control group. Serum FFAs were
decreased in the soy protein group (1.07-fold decrease, significantly decreased in the HF-S, HF-IMD, and HF-S-IMD
p<0.001) compared to the normal control group. Fasting groups (↓7.39%, 9.84%, 9.31% respectively, p<0.001)
blood glucose levels in the HF-S, HF-IMD, and HF-S-IMD compared with the HF group, (Table 4).
groups were significantly decreased compared with the levels
in the HF group [1.84-, 2.27- and 2.29-fold decrease, 3.6. Carbohydrate responsive element binding
respectively, p<0.001, (Table 3). protein (ChREBP) in liver tissue

3.4. Fasting serum insulin and HOMA-IR The level of ChREBP in liver tissue was significantly
increased in the HF, HF-S, HF-IMD, and HF-S-IMD groups
Levels of fasting serum insulin and HOMA-IR were (↑158.53%, 154.86%, 154.86%, and 155.38% respectively,
significantly increased in the HF group (1.77- and 3.98-fold p<0.001) compared to the normal control group. The soy
increase, respectively, p<0.001) compared to the normal protein group showed an insignificant decrease in ChREBP
control group. Fasting serum insulin and HOMA-IR were level (↓22.57%, p<0.05) compared to the normal control
significantly decreased in the soy protein group (1.44- and group. The decrease in levels of ChREBP in liver tissue was
1.09-fold decrease, p<0.001) compared to the normal control insignificant in the HF-S, HF-IMD, and HF-S-IMD groups
group. However, fasting serum insulin and HOMA-IR were (↓1.42%, 1.42%, and 1.22% respectively, p<0.05) compared
decreased in the HF-S group (1.58- and 2.91-fold decrease, to the HF group (Table 4).
respectively, p<0.001) compared to the HF group.
Additionally, fasting serum insulin and HOMA-IR were 3.7. Nuclear factor-kappa B in liver tissue

Table 3: Changes in fasting blood glucose, fasting serum insulin, In the HF group, there was a significant increase in NF-κB in
and HOMA-IR in different studied groups liver tissue (2.17-fold increase, p<0.001) compared to the
normal control group. There was a significant decrease in NF-
Groups FBG FSI HOMA-IR κB level in IMD group (1.26-fold decrease, p<0.001) and in
(mg/dL) (mIU/L) the soy group (1.25-fold decrease, p<0.001) compared to the
normal control group. On the other hand, NF-κB in liver tissue
Normal 92.00 17.36 3.94 was significantly decreased in the HF-S, HF-IMD, and HF-S-
control ±2.40 ± 0.40 ± 0.17 IMD groups (2.43-, 2.97- and 2.95-fold decrease respectively,
p<0.001) compared to the HF group (Table 4).
HF 206.90 30.69 15.69
±5.82acdefg ±1.36acdfg ± 0.98acdfg 3.8. Phosphorylated insulin receptor (pISR) in
Soy protein liver tissue
86.00 12.02 2.55
±2.67abef ±0.46abe ± 0.10abefg The level of pISR measured in liver tissue was significantly
decreased in the HF group (1.64-fold decrease, p<0.001)
IMD 89.88 17.16 3.81 compared to the normal control group. pISR in liver tissue
±3.91be ± 0.41be ± 0.14be was significantly increased in the HF-S, HF-IMD, and HF-S-
IMD groups (1.98-, 1.76- and 1.66-fold increase respectively,
HF-S 112.10 19.45 5.39 p<0.001) compared to the HF group. However, the pISR level
HF-IMD ±5.52abcdfg ±0.89abcdfg ±0.44acdfg was significantly higher in the HF-S group than in the normal
control group (1.21-fold increase, p<0.01) (Table 4).
91.30 17.02 3.84
±2.41bce ± 0.40be ± 0.12abce 3.9. Histopathology

HF-S-IMD 90.40 17.07 3.81 Histopathological analysis of liver sections in the normal
±1.84be ± 0.44be ± 0.11abce control group revealed normal hepatocytes arranged in cords
around the portal area, as shown in Figure (2A), and a normal
Data are presented as mean ± SD, n=10 rats. HF: high fructose diet, IMD: histological structure of the hepatic lobule (Figure 2B). The
inhibitor of NF-κB, HF-S: high fructose diet with soy protein, HF-IMD: high
fructose diet with NF-κB inhibitor, HF-S-IMD: high fructose diet with soy
protein and NF-κB inhibitor, FBG: fasting blood glucose, FSI: fasting serum

insulin. a: significant versus normal control. b: significant versus HF. c:

significant versus soy protein. d: significant versus IMD. e: significant versus

HF-S. f: significant versus HF-IMD. g: significant versus HF-S-IMD.

Significance was set at p < 0.05.

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 8-15 | 11

J Adv Med Pharm Res Research Article
within the normal limits (Figure 5A). Histopathological
livers of animals fed a soy protein-containing diet showed examination of the livers of rats fed with 10% fructose and
normal hepatocytes arranged in cords (Figure 3A). The livers injected with IMD showed normal-sized hepatocytes arranged
of animals injected with IMD showed normal hepatic in hepatic cords and a normal degree of hepatic vacuolation
vacuolation associated with quiescent Ito cells (Figure 3B). (Figure 5B). Histopathological changes induced by
Microscopic examination of liver sections of rats concurrent administration of fructose with soy protein and
supplemented with 10% fructose showed swollen hepatocytes IMD showed a normal degree of hepatic vacuolation and
associated with vacuolation of their cytoplasm, consistent steatosis (Figure 6A, B).
with increased glycogen storage and TGs accumulation
(Figure 4A). Fig. 2: Photomicrographs of liver sections (H&E, X200) from the normal
control group (normal diet with i.p. injection of the vehicle showing A:
Table 4: Changes in free fatty acids, Carbohydrate responsive normal hepatocytes arranged in cords (arrow) around the portal area
element binding protein, Nuclear factor-kappa B, and (arrowhead). B: normal histological structure of hepatic lobules. CPV:
Phosphorylated insulin receptor in different studied groups Cytoplasmic vacuolation, CV: Central vein, KC: Kupffer cell, SD: Sinusoidal
dilatation.
Groups FFA ChREBP NFκB PIR
(µmol/L) (ng/mL) (ng/mL) (ng/mL) Fig. 3: Photomicrographs of liver sections (H&E, X200) from (A): the soy
group (20% soy protein-containing diet) showing normal hepatocytes
Normal 275.40 3.81 6.97 4.96 arranged in cords (arrowhead). (B): the IMD group (i.p. injection of 10 mg/kg
control ± 9.87 ± 0.77 ±0.64 ±0.64 IMD-0354) showing normal hepatic vacuolation (arrow) associated with
quiescent Ito cells (arrowhead).
HF 741.60 9.85 15.11 3.03
±56.77acdefg ±0.48acd ±0.70acdef ±0.27acdefg Fig. 4: Photomicrographs of liver sections (H&E, X200) from the HF group
Soy (10% w/v fructose in drinking water) showing (A): swelling of the
protein g 5.76 hepatocytes associated with vacuolation of their cytoplasm, consistent with
±0.39bg increased glycogen storage (arrow). (B): periportal inflammatory cell
265.40 2.95 5.56 (macrophage and lymphocyte) infiltration (arrow) associated with hepatic
±11.05befg ±0.201abdefg ±0.49abdfg steatosis (round fat vacuoles within the hepatocytes) (arrowhead). (C): a
marked appearance of a mosaic pattern in the liver, which is associated with
IMD 270.62 4.32 5.54 5.30 periportal increased glycogen storage (arrowheads).
±3.11befg ±0.66befg ± 0.38abc ±0.48b

HF-S 686.80 9.71 6.21 6.00
±25.86abcd ±0.61acd ±0.77abfg ±0.82b
HF-
IMD 721.60 9.71 5.13 5.33
HF-S- ±11.82acd ±0.35acd ±0.27abce ±0.33b
IMD
672.50 9.73 5.08 5.02
±9.61abcd ±0.52acd ±0.23abce ±0.40bc

Data are presented as mean ± SD, n=10 rats. HF: high fructose diet, IMD:
inhibitor of NF-κB, HF-S: high fructose diet with soy protein, HF-IMD: high
fructose diet with NF-κB inhibitor, HF-S-IMD: high fructose diet with soy
protein and NF-κB inhibitor. FFA: free fatty acids, ChREBP: carbohydrate
responsive element binding protein, NFκB: nuclear factor kappa B, PIR:

phosphorylated insulin receptor. a: significant versus normal control. b:

significant versus HF. c: significant versus soy protein. d: significant versus

IMD. e: significant versus HF-S. f: significant versus HF-IMD. g: significant

versus HF-S-IMD. Significance was set at p < 0.05.

Additionally, periportal inflammatory cell infiltration was
associated with hepatic steatosis (Figure 4B). Liver sections
of animals supplemented with 10% fructose showed a marked
appearance of a mosaic pattern in the liver, which is
associated with increased periportal glycogen storage (Figure
4C).

The livers of rats fed with 10% fructose and soy protein Fig. 5: Photomicrographs of liver sections (H&E, X200)
showed a mild degree of hepatic vacuolation but were mostly

12 | J Adv Med Pharm Res., 2021, 1, 8-15 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

from (A): the HF-S group (10% w/v fructose along with 20% The histopathological changes induced by a high
soy protein) showing a mild degree of hepatic vacuolation and fructose diet in the current work were partially corrected by
mostly within the normal limits (arrow). (B): the HF-IMD the administration of soy protein and/or IMD-0354, where the
group (10% w/v fructose along with i.p. injection of 10 mg/kg liver sections showed an area of recovering hepatocytes with
IMD-0354) showing a normal degree of hepatic vacuolation a mild degree of hepatic vacuolation that was mostly within
(arrow). the normal limits and normal-sized hepatocytes arranged in
hepatic cords. Our results were in accordance with the
Fig. 6: Photomicrographs of liver sections (H&E, X200) from the HF-S- findings of Zhou et al.19 They observed a significant decrease
IMD group (10% w/v fructose in drinking water along with 20% soy protein- in the severity of hepatocellular vacuolation and improved
containing diet and i.p. injection of 10 mg/kg IMD-0354) showing (A): a pathological conditions in the livers of rats that received a soy
normal degree of hepatic vacuolation (arrow). (B): a normal degree of hepatic protein-containing diet.
steatosis (arrow)
The body weight, liver weight, and liver index were
4. DISCUSSION significantly increased in the HF group compared to those in
the normal control group because fructose enhances the
Non-alcoholic fatty liver disease (NAFLD) is the most accumulation of fats in the liver, resulting in higher liver
common liver disease worldwide, with a prevalence ranging weight. These findings were in line with the results by Toop
from 25% to 45%, increasing in parallel with obesity and et al.20 21 who reported that a high fructose diet induces
diabetes.14 Because of the disease burden, effective metabolic disorders with fatty liver in animal models.
prevention is a significant unmet need.15 However, the concurrent use of soy protein with a high
fructose diet significantly decreased the body weight, liver
This work aimed to examine the protective effect of weight, and liver index compared to the use of a high fructose
soy protein isolate as a natural NF-κB inhibitor in male rats diet alone as it reduces hepatic lipid accumulation and
exposed to induction of NAFLD by a high fructose diet (10% improves liver function. These results were in agreement with
w/v) and to evaluate the anti-inflammatory, anti- the findings of Won et al.21, who reported that the use of soy
hyperlipidaemic, and anti-diabetic properties of soy protein. protein could maintain normal liver weight.
High fructose diet-induced NAFLD has been used in
experimental rat models because fructose is a highly lipogenic Determination of serum FFAs is regarded as a good
sugar, and fructose is phosphorylated by fructokinase, method to quantify NAFLD and to evaluate the effectiveness
forming fructose-1-phosphate, which can then be converted to of new anti-inflammatory agents.22 FFAs represent the major
several three-carbon molecules, including glyceraldehyde, source of hepatic fat accumulation in NAFLD. FFAs are also
dihydroxyacetone phosphate, and glyceraldehyde-3- involved in the pathogenesis of different metabolic disorders
phosphate. Some of these three-carbon molecules can be that are associated with insulin resistance by activation of the
converted to glucose through gluconeogenesis, or they can be c-Jun-terminal kinase (JNK) pathway, contributing to the
used to generate other products such as triglycerides (TGs). development of hepatic steatosis and insulin resistance.23
TGs can be hydrolyzed by lipoprotein lipase to form FFAs
and monoacylglycerol.16 Excessive fructose intake leads to Our results showed that serum FFAs were
high levels of FFAs, causing fatty liver, inflammation, and significantly increased in the HF group compared to the
metabolic disorders.17 normal control group and the other groups that received either
soy protein alone or IMD-0354 alone. Serum FFAs were
In the present study, NAFLD was manifested by a significantly decreased in the HF-S, HF-IMD, and HF-S-IMD
significant increase in body weight, an enlarged liver, a groups compared to the HF group. These results were in
greater liver index, and elevated levels of blood glucose, agreement with those by Deol et al.24
serum insulin, serum FFAs, NF-κB, and ChREBP, as well as
decreased levels of pISRs in liver tissue. This effect was also Free fatty acids can activate the NF-κB pathway,
manifested histologically by swelling of the hepatocytes which is a vital mediator of NAFLD and insulin resistance in
associated with cytoplasm vacuolation, consistent with fructose-fed models. NF-κB activation regulates a variety of
increased glycogen storage and TG accumulation in the HF cytokines involved in inflammation and transcription
group. Moreover, the HF group showed periportal initiation of many genes such as TNF-α, IL-1, IL-6, and IL-
inflammatory cell infiltration associated with hepatic steatosis 8.25 These findings are supported by our results, which
and a marked mosaic pattern in the liver, which is associated revealed higher levels of NF-κB in the HF group compared
with increased periportal glycogen storage. The present with the normal control group. NF-κB activation by the high
findings were in agreement with those by Sreeja et al.18 who fructose diet may be due to the accumulation of lipids in the
reported that high fructose diet-induced metabolic disorders liver, resulting in increasing levels of fatty acid oxidation, thus
were associated with significant histopathological changes in stimulating inhibitor kappa kinase β (IKK-β), which activates
liver tissue. NF-κB. On the other hand, concurrent administration of soy
protein and/or IMD-0354 with fructose showed a marked
reduction in NF-κB when compared with the administration
of the high fructose diet alone, supporting the anti-
inflammatory effect of soy protein.

Proinflammatory cytokines are the main players in
promoting insulin resistance because they are activators of
signalling pathways, such as the JNK and the inhibitor kappa

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 8-15 | 13

J Adv Med Pharm Res Research Article

kinase (IKK) pathways, which negatively interfere with the 1. M. B. Vos and J. E. Lavine, Hepatology, 2013, 57,
early steps in the insulin signalling cascade. Therefore, 2525–2531.
chronic administration of fructose induces metabolic
disorders and insulin resistance due to increased levels of 2. C. Brennan and B. K. Tiwari, Food Processing and
FFAs and NF-κB.26 This was confirmed in our study by a Preservation, 2018, vol. 42.
significant increase in blood glucose and serum insulin levels,
a higher HOMA-IR score, and a significant decrease in pISRs 3. Paula, J, Fernanda, V. Ribeiro, F. Clasen, J. Pesek, N.
in the HF group compared to the normal control group. Levien, and M. Cardoso, J Food Process Preserv.,
2019, 43, 1–9.
Our results show a significant decrease in blood
glucose and serum insulin levels in the HF-IMD, HF-S, and 4. G. Prinsloo, G. Papadi, M. G. Hiben, L. de Haan, J.
HF-S-IMD groups compared to the HF group. Additionally, Louisse, K. Beekmann, J. Vervoort, and I. Rietjens,
the pISR levels in the liver tissue of these groups were Drug Discov Today. 2017, 22, 8, 1187-1200.
approximately near the normal level in the control group.
These results indicate that receiving soy protein and/or IMD- 5. D. Ramdath, E. Padhi, S. Sarfaraz, S. Renwick, and
0354 can properly maintain pISR levels by their effects on A. Duncan, Nutrients, 2017, 324.
insulin resistance. These results confirm the effects of soy
protein and/or IMD-0354 on NAFLD and insulin resistance. 6. M. Karamali, M. Kashanian, S. Alaeinasab, and Z.
Asemi, J. Hum. Nutr. Diet, 2018, 1, 1–11.
Kim et al.27 demonstrated that ChREBP is a
transcriptional activator of glycolytic and lipogenic genes that 7. S. Mohamed, Trends Food Sci. Technol., 2013, 35,
plays a critical role in insulin resistance and de novo 114–128.
lipogenesis. Fructose consumption was associated with
activating ChREBP production, as fructose ingestion directly 8. A. Lennikov, P. Mirabelli, A. Mukwaya, M.
transactivates glucose-6-phosphatase expression, a Schaupper, M. Thangavelu, M. Lachota, Z. Ali, L.
mechanism by which activating ChREBP might contribute to Jensen, and N. Lagali, Angiogenesis, 2018, 21, 2, 267-
impaired glucose homeostasis, which is supported by our 285.
results, where the level of ChREBP in the liver tissues of the
HF group was significantly increased compared with that in 9. Y. R. Li, C. C. Lin, C.Y. Huang, Y. H. Wong, C. H.
the normal control group. On the other hand, ChREBP was Hsieh, H. W. Wu, J. J. Chen, and Y. S. Wu, Chem.
decreased significantly in the soy group compared with all Biol. Drug Des., 2016, 90, 6, 1307-1311.
other groups, indicating that the use of flavonoids present in
soy protein significantly decreased lipogenic gene expression 10. M. E. Oliva, A. Chicco, and Y. B. Lombardo, Eur. J.
and suppressed ChREBP signalling. Nutr., 2015, 54, 407–419.

5. CONCLUSIONS 11. S. Hosokawa, G. Haraguchi, A. Sasaki, H. Arai, S.
Muto, A. Itai, S. Doi, S. Mizutani, and M. Isobe,
The current study shows that a high fructose diet is associated Cardiovasc. Res., 2013, 99, 35–43.
with metabolic and inflammatory diseases, which could lead
to metabolic syndrome and insulin resistance. After the 12. J. I. Felice, L. Schurman, A. D. McCarthy, C.
addition of an NF-κB inhibitor to the high fructose diet, the Sedlinsky, J. I. Aguirre, and A. M. Cortizo, Diabetes
levels of insulin, insulin receptors, glucose, ChREBP, and Res. Clin. Pract., 2017, 126, 202–213.
FFAs were significantly decreased compared with those in the
control group, which indicates that the mechanism of fructose 13. N. Shaat, C. Ignell, A. Katsarou, and K. Berntorp,
induction of metabolic diseases is NF-κB dependent. The use Acta Obstet. Gynecol. Scand., 2017, 96, 821–827.
of soy protein as a natural NF-κB inhibitor significantly
reduced the deleterious effects of the high fructose diet. Thus, 14. M. E. Rinella, Jama, 2015, 313, 2263.
the use of soy protein could be beneficial in patients with high 15. R. Mastrocola, D. Nigro, A. S. Cento, F. Chiazza, M.
fructose-induced metabolic disorders and may be used in
combination with other medications for decreasing the risks Collino, and M. Aragno, Neurobiol Dis., 2016 , 89,
of metabolic diseases as well as hepatic steatosis. Further 65-75.
studies are warranted to investigate the therapeutics efficacy 16. A. A. Yuruk and R. Nergiz-Unal, Lipids Health Dis.,
of soy protein against hepatic steatosis in clinical settings. 2017, 16, 1–12.
17. S. Yoo, H. Ahn, and Y. Park, Nutrients, 2016, 9, 11.
CONFLICTS OF INTEREST 18. S. Sreeja, R. Geetha, E. Priyadarshini, K. Bhavani,
and C. V. Anuradha, ISRN Inflamm., 2014, 641096,
The authors declare no conflict of interest 1–8.
19. D. Zhou, S. Lezmi, H. Wang, J. Davis, and W. Banz,
6. REFERENCES Nutrition, 2014, 22, 151–158.
20. C. R. Toop, B. S. Muhlhausler, K. O’Dea, and S.
Gentili, J. Physiol., 2017, 595, 4379–4398.
21. S. B. Won, A. Han, and Y. H. Kwon, J. Nutr.
Biochem., 2017, 42, 51–61.
22. F. Tranchida, L. Tchiakpe, Z. Rakotoniaina, V.
Deyris, O. Ravion, A. Hiol, and J. Zhejiang, Univ. Sci.
B, 2012, 13, 307–317.
23. S. Petta, A. Gastaldelli, E. Rebelos, E. Bugianesi, P.
Messa, L. Miele, G. Svegliati-Baroni, L. Valenti, and
F. Bonino, Int J Mol Sci, 2016, 17.
24. P. Deol, J.R. Evans, J. Dhahbi, K. Chellappa, D. S.
Han, S. Spindler, and F. M. Sladek, PLoS One, 2015,
10, 1–31.
25. L. Zeng, W. J. Tang, J. J. Yin, and B. J. Zhou, Int. J.

14 | J Adv Med Pharm Res., 2021, 1, 8-15 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

Clin. Exp. Med., 2014, 7, 1624–1631.
26. M. Baena, G. Sangüesa, A. Dávalos, M. J. Latasa, A.

Sala-Vila, R. M. Sánchez, N. Roglans, J. C. Laguna,
and M. Alegret, Sci. Rep., 2016, 6, 26149.
27. M. S. Kim, S. A. Krawczyk, L. Doridot, A. J. Fowler,
J. X. Wang, S. A. Trauger, H. L. Noh, H. J. Kang, J.
K. Meissen, M. Blatnik, J. K. Kim, M. Lai, and M. A.
Herman, J. Clin. Invest., 2016, 126, 4372–4386,
2016.

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 8-15 | 15

Research Article

Received 11th November 2020 Green Simultaneous Determination of Amlodipine
Accepted 29th January 2021 Besylate and Celecoxib by Dual wavelength and
Published 25th February 2021 Simultaneous Equation Spectrophotometric Methods

Mokhtar M. Mabrouk1, Mohamed A. Abdel Hamid1, Mary A. Michael1*

1Departement of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Tanta University, Tanta 31111, Egypt

jampr.journals.ekb.eg ABSTRACT
Online ISSN: 2636-4158
Two facile, accurate, green, and sensitive spectrophotometric methods; using dual-wavelength
spectrophotometry (method I) and simultaneous equation method (method II), were developed for
the simultaneous determination of amlodipine besylate (AMO) and celecoxib (CEX). Method I was
based on measuring the absorbance difference between 252 nm and 357 nm for the determination of
CEX, where AMO can be determined directly by measuring the absorbance at wavelength 357 nm
as CEX has no absorbance at 357 nm. Method II was based on solving two simultaneous equations
for both drugs at 252 nm and 357nm. The linearity range for AMO was found to be 5-65 µg/mL
while for CEX was found to be 1-25 µg/mL for both methods. The developed methods were
successfully applied for the determination of AMO and CEX in a laboratory prepared mixture
containing all possible excipients present in a tablet dosage form. The mean percentage recovery
values were found to be 101.157 ± 0.750, and 101.765 ± 0.140 for method I, but 101.603 ± 0.750,
and 100.549 ± 1.262 for method II for AMO and CEX, respectively. The methods were found to be
eco-friendly as they were evaluated according to the Green Analytical Procedure Index (GAPI) and
Analytical Eco-Scale.

Keywords: Amlodipine besylate, Celecoxib, Dual wavelength, Analytical Eco-Scale.

1. INTRODUCTION peripheral vascular resistance. AMO is clinically used for the
treatment of hypertension, chronic stable angina, and
Amlodipine besylate (AMO; 3-Ethyl, 5-Methyl (±)-2-[(2- prinzmetal's angina. AMO is official in the British
Aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4- Pharmacopoeia (BP),1 the United States Pharmacopoeia
dihydro-3,5-pyridinedicarboxylate Monobenzenesulfonate) is (USP),2 and the Indian Pharmacopoeia.3
a calcium channel blocker.1,2 It inhibits cardiac and vascular
smooth muscle contraction through inhibition of calcium ions Celecoxib (CEX; 4-[5-(4-methylphenyl)-3-
uptake into myocardial and vascular smooth muscle cells (trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide) is a
resulting in the reduction of coronary artery muscle tone and non-steroidal anti-inflammatory drug that inhibits
cyclooxygenase 2 enzyme (COX-2), resulting in decreasing
*Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, tissue concentrations of prostaglandins to reduce pain.
Tanta University, Tanta, Egypt, 31111, Tel: (202) 040-3336007; fax: (202) 040- Prostaglandins sensitize afferent nerves and potentiate the
3335466. action of bradykinin in inducing pain and are mediators of
Email: [email protected] inflammation. Since CEX is an inhibitor of prostaglandin
synthesis, it has analgesic, anti-inflammatory, and antipyretic

16 | J Adv Med Pharm Res., 2021, 1, 16-23 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

properties which are used for the treatment of the signs and solubilizing agent. A RP-HPLC method for simultaneous
the symptoms of osteoarthritis of the knee and hip.4 CEX is determination of AMO and CEX has been reported.25
official in both British Pharmacopoeia1 and the United States
Pharmacopoeia.2 The chemical structures of AMO and CEX The present work aims to develop two sensitive and
are shown in Figure 1. fully validated spectrophotometric methods for simultaneous
determination of AMO and CEX without prior separation.
The methods were successfully used for the determination of
both drugs in their bulk and in laboratory prepared tablets
without interference from the excipients that may be present
in dosage form. The proposed methods allow high sensitivity
and increasingly fast analysis time. So, the developed
methods have several advantages over other previously
reported spectroscopic methods. These advantages make the
developed methods suitable for routine quality control
analysis.

(a) Celecoxib 2. Experimental
2.1. Apparatus and software

Spectrophotometric measurements were carried out using
Shimadzu (UV-1800) UV-VIS double beam
spectrophotometer equipped with 1 cm quartz cells and
connected to a personal computer loaded with UV-Probe 2.33
software. Absorption spectra were recorded on a wavelength
range of 200-400 nm at a scan rate of 400 nm/min and spectral
bandwidth of 0.1 nm.

(b) Amlodipine besylate 2.2. Material and reagent

Figure 1: Chemical structure of (a) Celecoxib and (b) Amlodipine AMO was kindly supplied by Sigma Company for
besylate. Pharmaceutical Industries, Quesna, Menofia, Egypt. The
purity was found to be 99.4%. CEX was kindly supplied by
Consensi®; is a newly FDA approved fixed-dose combination Amriya Pharmaceutical Industries, Alexandria, Egypt with a
tablet dosage form of AMO and CEX at a ratio of 1:20, purity of 99.1%.
indicated for patients who have hypertension and
osteoarthritis. It can be also used in lowering blood pressure Acetonitrile was purchased from Sigma Aldrich,
and reducing the risk of fatal and non-fatal cardiovascular Germany. Mannitol DC 200, croscarmellose sodium,
events, primarily strokes and myocardial infarctions.5 povidone K-30, sodium lauryl sulfate, magnesium stearate,
and colloidal silicon dioxide were of analytical grade.
Several publications are describing analytical
methods for the determination of AMO either alone or in 2.3. Stock and working solution
combination with other drugs. These methods include
capillary electrophoresis;6,7 adsorptive square-wave anodic 2.3.1. Stock standard solution
stripping voltammetry8 and spectrophotometry with
atorvastatin,9 or olmesartanmedoximil,10 or valsartan and Stock standard solutions of AMO and CEX were prepared in
hydrochlorothiazide.11 Different reverse phase high- acetonitrile to obtain solutions with a concentration of 1.0
performance liquid chromatography (RP-HPLC) methods mg/mL. AMO standard solution was protected from light due
have been developed for the determination of AMO in to its photosensitivity. The standard solutions were found to
combination with other drugs such as metoprolol be stable for 10 days when kept in the refrigerator at 40C
succinate,12,13 benazepril hydrochloride,14 and inhuman during this period the solutions remain suitable for their use
plasma.15-17 in the analysis.

Different methods were developed for the 2.3.2. Working standard solution
determination of CEX including spectrophotometric methods
either alone,18 or with the interleukin-1 beta inhibitor Accurately measured volumes of stock standard solutions of
diacerein using adsorption correction and chemometric AMO and CEX were transferred into two 10 mL volumetric
method.19 Different RP-HPLC methods have been developed flasks and diluted appropriately with acetonitrile to obtain
for the determination of CEX in human plasma.20-23 There is
a spectrophotometric method for simultaneous determination
of AMO and CEX has been reported,24 which uses a

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 16-23 | 17

J Adv Med Pharm Res Research Article

working standard solutions with concentration 100 µg/mL for up to final volume with acetonitrile to prepare solutions
AMO and CEX. having concentrations within the linearity range of both drugs.
The procedures were carried out as mentioned in section (2.4.)
2.4. Construction of calibration curve and then concentrations of both drugs were calculated from
the corresponding regression equations of the two methods.
In a series of 10 mL volumetric flasks, different aliquots of
the working standard solutions of AMO (100 µg/mL) and 3. RESULTS AND DISCUSSION
CEX (100 µg/mL) were transferred separately; several
appropriate dilutions were carried out with acetonitrile to The present work describes the development and validation of
obtain solutions of AMO ranging from 5-65 µg/mL and two simple, precise, and accurate spectrophotometric methods
solutions of CEX ranging from 1-25 µg/mL. UV spectra were (dual-wavelength and simultaneous equation
scanned for these solutions using acetonitrile as blank. spectrophotometry) for the simultaneous determination of
AMO and CEX in their combination at the dosage form ratio
2.4.1. Calibration curve of AMO at λ 357 nm (1:20) for AMO and CEX, respectively. The methods were
successfully applied to laboratory-prepared tablets.
AMO was determined by measuring the absorbance at 357
nm. The calibration curve was obtained by plotting A (357nm) 3.1. Method development
against corresponding concentrations for AMO.
The zero-order UV spectra of AMO and CEX (Figure 2)
2.4.2. Calibration curve of CEX by dual wavelength exhibits certain overlap, where AMO can be determined
method directly by measurement of the absorbance at λ 357 nm
(A357nm), where CEX has no absorbance at 357 nm. CEX is
CEX was determined by measuring the A between 252 nm difficult to be determined from a zero-order spectrum.
and 357 nm; at which A for AMO equals zero. The
calibration curve was obtained by plotting A (252nm- Dual-wavelength spectrophotometry offers an
357nm) against corresponding concentrations for CEX. efficient method for analysing a component in presence of an
interfering component. In this mixture, CEX is considered as
2.4.3. Simultaneous equation spectrophotometric a component of interest and AMO is considered as an
method interfering component. For elimination of interferences, two
wavelengths were selected for CEX at the same time the
It was found that only AMO showed interference at λmax of difference in absorbance is zero for AMO.
CEX, but CEX had no absorbance at λmax of AMO. So,
calibration curves were obtained by plotting the absorbance at 3.1.1. Estimation of AMO at λ 357 nm
λ 252 nm and 357 nm for AMO at λ 252 nm for CEX against
their corresponding concentrations. Two simultaneous By overlaying the zero-order spectrum of AMO and CEX, it
equations were constructed. was found that AMO can be determined at 357 nm, as CEX
has no absorbance at that wavelength. So, AMO can be
2.5. Preparation of laboratory prepared determined directly from the zero-order spectrum. AMO was
mixture determined by measuring the absorbance at 357 nm.

The dosage form Consensi® is not available in the local 3.1.2. Estimation of CEX by dual-wavelength
market, therefore a laboratory prepared mixture simulated to method
tablet dosage form was prepared by mixing of 10 mg AMO,
200 mg CEX, and the following excipients: 87 mg mannitol By overlaying the zero-order spectrum of AMO and CEX, it
DC 200, 16.5 mg croscarmellose sodium, 6.6 mg povidone K- was found that CEX cannot be determined directly in presence
30, 3.3 mg sodium lauryl sulfate, 3.3 mg magnesium stearate of AMO. For CEX, two wavelengths were selected at which
and 3.3 mg colloidal silicon dioxide (aerosol). the absorbance difference (A) at these wavelengths equals
zero for AMO. CEX was determined by measuring the A
The laboratory prepared mixture was transferred to a between 252 nm and 357 nm; at which A for AMO equals
100 mL volumetric flask, dissolved in 50 mL of acetonitrile, zero. AMO exhibits equal absorbance at 252 nm and 357 nm,
and sonicated for 15 minutes. Then, the solution was made up then absorbance difference A(252nm-357nm) for AMO
to the required volume using acetonitrile. The solution was equals zero. The measured A(252nm-357nm) is concerned
filtered, and the first 10 mL of the filtrate was discarded. An as a function of CEX concentration.
aliquot equivalent to 1 mL of the filtrate was transferred to a
100 mL volumetric flask and made up to the final volume with 3.1.3. Simultaneous equation spectrophotometric
acetonitrile. Different aliquots of this solution were Method
transferred to a set of 10 mL volumetric flasks, spiked with 1
mL from a standard solution of AMO (100 μg/mL), and made

18 | J Adv Med Pharm Res., 2021, 1, 16-23 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

Figure 2. Zero-order UV spectra of 20 µg/mL celecoxib (……) and 15 µg/mL amlodipine besylate (___) in acetonitrile showing the
selected wavelength for the dual-wavelength method.

In 1934, Vierordt’s26 developed an equation for the algebraic 3.2. Validation of the developed methods

calculation of the individual absorbance of two components The validity of the methods was studied regarding linearity,
specificity, accuracy, and precision according to International
present in a mixture. This equation, which was later named Council for Harmonisation (ICH) guidelines.27,28

after him, opened a wide field for the solution of 3.2.1. Linearity

spectrophotometric interference using many mathematical Linearity was studied to determine the range over which
analyte response is linear as a function of concentration. This
equations. study was performed by preparing standard solutions at seven
different concentrations and analyses were performed in
The UV absorbance of AMO and CEX were triplicates. The methods were found to be linear over a
concentration range of 5-65 μg/mL for AMO and 1-25 μg/mL
recorded within the wavelength range 200-400 nm at 0.1 nm for CEX. Regression equations were calculated, the results of
interval. The wavelength of maximum absorbance (λmax) was slope, intercept, standard deviation about the slope, and
intercept. The quantitative statistical parameters for the
found to be 357 nm and 252 nm for AMO and CEX, determination of AMO and CEX are summarized in Table 1
The high values of correlation coefficients (r) with negligible
respectively. The original zero-order spectra of AMO and intercepts indicate good linearity of the calibration curves.
CEX in acetonitrile showed a certain overlapping. λmax values
of AMO and CEX. Only AMO showed interference at the λmax 3.2.2. Detection and quantitation limits

of CEX. Calibration curves were generated for AMO at 357

nm and 252 nm and CEX at 252 nm only.

The two simultaneous equations were generated as

follows:

357 = a 357 ……………. (1)
AMO

252 = 252 + a 252 ……….……. (2)
CEX AMO

Where, a 357 and a 252 are the slope of calibration curve of

AMO AMO

AMO at 357 nm and 252 nm respectively,a 252 is the slope of
CEX

the calibration curve of CEX at 252 nm.

Table 1: Quantitative parameters for the determination of AMO and CEX by the proposed spectrophotometric methods.

Method Drug Linearity Λ r2 A b Sy/x Sa Sb DL QL
µg/mL (nm) µg/mL
(10--4) µg/mL
3
Zero-order AMO 5 – 65 357 0.9995 0.0005 0.0112 0.0053 0.00337 0.98 1
1
Method CEX 1 – 25 252 – 0.9996 0.0018 0.0049 0.0094 0.00493 3.55 0.33
Dual 3
0.75
Wavelength AMO 5 – 65 357 1
Simultaneous 357 0.9995 0.0005 0.0112 0.0053 0.00337 0.98

equation CEX 1 – 25 252 0.9997 0.0032 0.0501 0.0075 0.00381 2.8 0.25

r2: coefficient of determination, a: intercept, b: slope, Sy/x: residual standard deviation of the regression line, Sa: standard error of intercept,
Sb: standard error of slope, DL: detection limit (calculated), QL: quantitation limit (calculated).

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 16-23 | 19

J Adv Med Pharm Res Research Article

Detection (DL) and quantitation (QL) limits can be calculated and not more than 0.182% for CEX, for simultaneous
equation method as shown in Table 3.
depending on the standard deviation of the response and the
3.2.5. Specificity
slope. They may be expressed as:
For testing the specificity of the proposed method; the %
DL = 3.3 σ and QL = 10 σ recovery of AMO and CEX was determined in laboratory
S prepared tablets containing both drugs with the possible
S excipients present in dosage form by the two proposed
methods as shown in Table 4.
where;"" is the standard deviation of the y-intercept of
Mean % recovery ± S.D was 101.157 ± 0.750 for
regression line (Sa) and "S" is the slope of the calibration AMO and 101.765 ± 0.140 for CEX, for the dual-wavelength
method. Mean % recovery ± S.D was 101.603 ± 0.750 for
curve. AMO and 100.549 ± 1.262 for CEX, for simultaneous
equation method. This confirmed that the excipients in the
For dual-wavelength method; calculated DL and QL laboratory prepared tablets did not show any interference.

were found to be 1 g/mL and 3 g/mL for AMO and 0.33 3.2.6. Assessment of the greenness of the proposed
methods using green analytical procedure
g/mL and 1 g/mL for CEX. DL and QL calculated using index (GAPI ) and analytical eco-scale

simultaneous equation method were 1 g/mL and 3 g/mL Analytical eco-scale is a new semi-quantitative mean for
evaluation of analytical practice and educational attributes in
for AMO and 0.25 g/mL and 0.75 g/mL for CEX, green analytical chemistry protocols. Even though it
compares different steps and parameters in the analytical
respectively (Table 1). practice, it does not supply us with comprehensive data about
evaluation protocols.29,30 The ideal green analysis has an
3.2.3. Accuracy analytical Eco-Scale value of 100 and the penalty points of
(energy, reagent, waste, and hazard) were subtracted from the
The accuracy of the proposed methods was evaluated by ideal green analysis. According to that, the developed
triplicate determination of three different concentrations of spectrophotometric methods were found to have an analytical
binary mixtures of AMO and CEX within the linearity range. Eco-Scale value of 84 indicating an excellent green method of
Accuracy was expressed as the % recovery ± S.D. analysis with low laboratory requirements as shown in Table
5.
Mean % recovery ± S.D was 99.082 ± 1.260 for
AMO and 101.303 ± 0.620 for CEX, for the dual wavelength Green Analytical Procedure Index (GAPI) indicates
method. Mean % recovery ± S.D was 99.443 ± 1.280 for the green properties of all over the analytical practice, from
AMO and 99.687 ± 1.407 for CEX, for simultaneous equation sampling to sample preparation, storage, transport, and final
method as shown in Table 2. determination GAPI has the advantage that it supplies data

3.2.4. Precision

Precision was carried out for both methods by triplicate
determination of three different concentrations of laboratory
prepared mixture of AMO and CEX within the linearity range.
Precision was carried out on the same day (intra-day
precision) or in three successive days (inter-day precision).
Standard deviation (S.D.) and % relative standard deviation
(% R.S.D.) values of the results obtained were calculated.

The % R.S.D was not more than 0.285% for AMO
and not more than 0.209% for CEX, for the dual-wavelength
method. The % R.S.D was not more than 0.284% for AMO

Table 2: Evaluation of the accuracy for the determination of AMO and CEX by the proposed spectrophotometric methods.

Dual wavelength Simultaneous equation

Concentration Concentration found Mean % Mean % Concentration found Mean % Mean %
taken (µg/mL) concentration Recovery Recovery (µg/mL) concen- Recovery Recovery
tration
(µg/mL) 10.750 11.000 10.804 found 100.474 ± 10.795 11.045 10.848 100.887 ±
9.830 9.794 9.777 (µg/mL) 98.006 S.D. 9.875 9.839 9.821 found 98.452 S.D.
AMO 10.8 19.929 19.429 19.902 98.765 19.973 19.473 19.946 (µg/mL) 98.988
10.852 99.082 99.443
10 10.896
9.801 ± ±
20 9.845
19.753 1.260 1.280
19.798

CEX 16 16.142 16.478 16.281 16.300 101.877 101.303 16.248 16.166 16.200 16.205 101.279 99.687
10 10.148 10.126 10.142 10.138 101.383 ± 9.854 9.849 9.879 9.861 98.607 ±
20 20.372 20.012 20.004 20.130 100.648 19.701 20.040 19.765 19.835
0.620 99.176 1.407

20 | J Adv Med Pharm Res., 2021, 1, 16-23 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

Table 3: Evaluation of the intra-day and inter-day precision for the determination of AMO and CEX by the spectrophotometric methods

Dual wavelength Simultaneous equation

Intra-day Inter-day Intra-day Inter-day

Concentration Mean % % Mean % % Mean % % Mean % %
taken concent- Recov- R.S.D concentr- Recov- R.S.D concentra- Recov- R.S.D concent- Reco- R.S.D.

(µg/mL) ration ery ation ery tion ery ration very

found* found* found* found*

(µg/mL) (µg/mL) (µg/mL) (µg/mL)

AMO 10.8 10.851 100.474 0.131 10.741 99.454 0.097 10.896 100.887 0.130 10.786 99.868 0.096

10 9.801 98.006 0.028 9.823 98.234 0.022 9.845 98.452 0.028 9.879 98.795 0.015

20 19.753 98.765 0.285 19.815 99.077 0.078 19.798 98.988 0.284 19.860 99.301 0.078

CEX 16 16.300 101.876 0.166 16.232 101.448 0.080 16.205 101.279 0.041 16.182 101.137 0.020

10 10.138 101.383 0.011 10.121 101.215 0.050 9.861 98.607 0.016 9.906 99.062 0.088

20 20.130 100.648 0.209 20.140 100.700 0.063 19.835 99.176 0.182 19.880 99.402 0.065

about quantification of the evaluated procedure.31 GAPI has 3.2.7. Comparison with other reported methods:
five pentagrams which are used to evaluates the
environmental effect on each step of the proposed method, Data presented in Table 6 compares results obtained using the
through three colors: red, yellow, and green representing high, methods hereby proposed with other methods already
medium to low effects, respectively. The additional described in literature.24, 25 This comparison reveals that the
information obtained from GAPI pictograms is about the proposed methods show a fast method that doesn’t need a long
extraction process is included, and if it is included which type time in manipulation and pre-treatment of the sample
of extraction and its scale. preparation as in the reported spectroscopic and HPLC
methods. It is used eco-friendly reagents and solvents without
According to the GAPI pictograms, the same results need of tedious procedure. So, the proposed method has the
obtained from Analytical Eco-Scale analysis were lowest impact on the environment according to the used
determined, as shown in Figure 3, the spectrophotometric reagents and compounds. Moreover, in terms of
methods are direct green methods, without any extraction instrumentation and waste, it has lower environmental impact
procedures, and the use of small volume from non-toxic than the reported methods.
solvents. The waste volume is low. Also, this method is for
quantification and qualification.

Table 4: Recovery data of Amlodipine besylate and Celecoxib from laboratory prepared tablets by the proposed spectrophotometric methods

Dual wavelength Simultaneous equation

Concentration Concentration found Mean % Mean % Concentration found Mean % Mean %
taken(µg/mL) (µg/mL) (µg/mL)
concentration Recovery Recovery concentration Recovery Recovery

found(µg/mL) ± S.D. found(µg/mL) ± S.D.

AMO 0.4 0.405 0.407 0.408 0.407 101.625 101.157 0.407 0.409 0.409 0.408 102.071 101.603
0.8 0.811 0.812 0.815 0.812 101.554 0.816 102.000 ±
100.292 ± 0.815 0.815 0.818 1.209 100.738
1.2 1.196 1.206 1.208 1.204 101.835 0.75 7.938 99.219 0.75
CEX 8 8.087 8.176 8.178 8.146 101.856 101.765 1.202 1.211 1.214 16.112 100.699 100.549
16.297 101.605 7.896 7.955 7.961 24.415 101.730
24.385 ±
16 16.277 16.301 16.313 ± 16.123 16.132 16.080 1.262

24 24.380 24.347 24.428 0.140 24.292 24.491 24.462

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 16-23 | 21

J Adv Med Pharm Res Research Article

Table 5: The penalty points of the proposed methods according
to the analytical Eco-Scale per sample

Reagents Penalty points

Acetonitrile 8

Instrument Penalty points

UV ( 1.5 kWh per sample) 0
Occupational hazard (analytical 0
8
process hermitization)
Waste (10 mL, no treatment)

Total penalty points 16 Figure 3: The Green Analytical Procedure Index profile for the
Analytical eco-scale total 84 proposed spectrophotometric methods.

score a 4. CONCLUSIONS

aIf the score is > 75, it represents excellent green analysis The developed methods are simple, accurate, robust,
If the score is > 50, it represents acceptable green analysis sensitive, and green for the simultaneous estimation of
If the score is <50, it represents inadequate green analysis celecoxib and amlodipine besylate in the ratio (20: 1) as in
their pharmaceutical dosage forms. The excipients present in
Table 6: Comparison of the analytical parameters among the tablets dosage form did not interfere in the analysis, which
proposed and the reported methods proved the specificity of the method for these drugs. The
sample preparation is simple and rapid. Hence, the proposed
Parameters Proposed HPLC method Reported spectrophotometric methods can be used for the routine
quality control analysis in the combined formulation either in
spectroscopic spectroscopic authentic samples or in dosage forms, and they can be applied
to the toxicological and biological work.
methods method
CONFLICTS OF INTEREST
AMO CEX AMO CEX AMO CEX
The authors declare no conflict of interest.
Linearity 5 – 65 1-25 5-30 50-500 2-10 10-50
range (µg/mL) 5. REFERENCES

Mobile Acetonitrile Phosphate 2 M sodium 1. The British Pharmacopoeia, The Stationary Office:
buffer (20mM, benzoate as London, 2017.
phase PH adjusted to hydrotropic
5.6 with diluted solubilizing 2. United States Pharmacopeial Convention: The United
or diluting NaOH): agent States Pharmacopeia 39 ; The National Formulary 34.
acetonitrile and Rockville, Maryland, USA, 2016.
solvents used methanol at a 84
ratio 3. The Indian Pharmacopoeia, The Controller of
Analytical 84 30:55:15(v/v) Publications, New Delhin, 1996, 72.
Eco-Scale Eluted at
value 1.2 mL/min 4. J. V. Faria, P. F. Vegi, A. G. Miguita, M. S. dos Santos,
N. Boechat and A. M. Bernardino, Bioorganic & Med.
77 Chem., 2017, 25, 5891–5903.

Analysis 18 21 1440 5. F. Angeli, M. Trapasso, S. Signorotti, P. Verdecchia and
time(min) G. Reboldi, Expert Rev. Clin. Pharmacol., 2018, 11,
(including 1073–1084.
sample pre-
treatment) 6. M. M. Salim, W. M. Ebeid, N. El-Enany, F. Belal, M.
Walash and G. Patonay, J. Sep. Sci., 2014, 37, 1206–
1213.

22 | J Adv Med Pharm Res., 2021, 1, 16-23 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

7. Y. Wei, H. Wang, S. Sun, L. Tang, Y. Cao and B. Deng, 28. G. ICH-Q2B Validation of Analytical Procedures:
Biosens. Bioelectron., 2016, 86, 714–719. Methodology.International conference on harmonization
of technical requirements for registration of
8. A. A. Gazy, Talanta, 2004, 62, 575–582. pharmaceuticals for human use, Switzerland, 1996.
9. V. B. Patel and R. Sahu, Indian J. Pharm. Sci., 2007, 69,
29. A. Gałuszka, Z.M. Migaszewski, P. Konieczka and J.
110-111. Namieśnik, TrAC, Trends Anal. Chem., 2012, 37, 61-72.
10. P. Mehulkumar, V. Ramesh, V.V. Kumar, R. Srinivas
30. M. Tobiszewski, M. Marć, A. Gałuszka and J.
and P.V. Diwan, Asian J. Res. Chem., 2009, 2, 127-130. Namieśnik, Molecules, 2015, 20, 10928-10946.
11. V.R. Galande, K. Baheti, S. Indraksha and M. Dehghan,
31. J. Płotka-Wasylka, Talanta, 2018, 181, 204-209.
Indian J. Pharm. Sci.,2012, 74, 18-23.
12. V.G. Dongre, S.B. Shah, P.P. Karmuse, M. Phadke and

V.K. Jadhav, J. Pharm. Biomed. Anal., 2008, 46, 583-
586.
13. M. Rao, S. Rahaman, Y.R. Prasad and P.G. Reddy,
International Journal of Pharmaceutical Research and
Development, 2010, 2, 69-76.
14. K.R. Naidu, U.N. Kale and M.S. Shingare, J. Pharm.
Biomed. Anal., 2005, 39, 147-155.
15. S.M. El-Gizawy, O.H. Abdelmageed, M.A. Omar, S.M.
Deryea and A.M. Abdel-Megied, Am. J. Anal. Chem.,
2012, 3, 422-430.
16. M.M. Mabrouk, S.F. Hammad, S.F. El-Malla and E.A.
Elshenawy, Microchem. J., 2019, 147, 635-642.
17. A.K. Sarkar, D. Ghosh, A. Das, P.S. Selvan, K.V.
Gowda, U. Mandal, A. Bose, S. Agarwal, U. Bhaumik
and T.K. Pal, Journal of Chromatography B, 2008, 873,
77-85.
18. R. Saha, C. Sajeev, P. Jadhav, S. Patil and N. Srinivasan,
J. Pharm. Biomed. Anal., 2002, 28, 741-751.
19. N. Patel, V. Nandurbarkar, A. Patel and S. Patel,
Spectrochim. Acta, Part A, 2014, 125, 46-52.
20. H. Jalalizadeh, M. Amini, V. Ziaee, A. Safa, H. Farsam
and A. Shafiee, J. Pharm. Biomed. Anal., 2004, 35, 665-
670.
21. M. Zhang, G.A. Moore, S.J. Gardiner and E.J. Begg,
Journal of Chromatography B, 2006, 830, 245-248.
22. M. Rose, E. Woolf and B. Matuszewski, J. Chromatogr.
B: Biomed. Sci. Appl., 2000, 738, 377-385.
23. A. Zarghi, A. Shafaati, S. Foroutan and A. Khoddam,
Journal of Chromatography B, 2006, 835, 100-104.
24. D. Kushwaha, S. Diwakar, R. Roy, S. Karole, H.
Kushwaha and P. Jain, J. Drug Delivery Ther., 2019, 9,
651-655.
25. M. Attimarad, K.N. Venugopala, N. SreeHarsha, B.E.
Aldhubiab and A.B. Nair, Microchem. J., 2020, 152,
DOI: 10.1016/j.microc.2019.104365.
26. A. H. Kamal, S.F. EL-Malla and S.F. Hammad,
European journal of pharmaceutical and medical
research, 2016, 3,348-360.
27. I. ICH, Q2 (R1): Validation of analytical procedures: text
and methodology, in: International Conference on
Harmonization, Geneva, 2005.

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 16-23 | 23

Research Article

Received 13th January 2021 A Pilot Study Comparing the Effectiveness of Three
Accepted 25th February 2021 Combined Therapeutic Regimens in Egyptian Patients
Published 25th February 2021 with Moderate to Severe Chronic Obstructive Pulmonary
Disease

Tarek M. Mostafa1, Gamal A. El-Azab1, Ghada A. Atia2, Noran S. Lotfy1*

jampr.journals.ekb.eg 1Clinical Pharmacy Department, Faculty of Pharmacy, Tanta University, Tanta 31111, Egypt.
Online ISSN: 2636-4158 2Chest Department, Faculty of Medicine, Tanta University, Tanta 31111, Egypt.

ABSTRACT

Objective: We aimed at comparing the effectiveness of long-acting β-agonist+long acting
muscarinic antagonist (LABA+LAMA) versus both LABA+inhaled corticosteroid (ICS) and
LAMA+ICS in non-asthmatic patients with moderate to severe COPD. Besides, we aimed at
assessing the changes that occur in plasma concentrations of TNF-α, fibrinogen, and IL-6 with the
disease activity. Methods: In this pilot study, 45 non-asthmatic patients with moderate to severe
COPD were randomized into three groups; group I (LABA+ICS) received Formoterol/Budesonide,
group II (LAMA+ICS) received Tiotropium/Budesonide and group III (LABA+LAMA) received
Formoterol/Tiotropium for twelve weeks. The patients were assessed at baseline, four and twelve
weeks after therapeutic intervention through evaluating the changes occur in FEV1% predicted,
mMRC dyspnea scale, and plasma concentrations of TNF-α, fibrinogen, and IL-6. Results: At
baseline, the study groups were statistically concerning the demographic data and disease
characteristics. All study therapeutic options produced an improvement in FEV1% predicted and
mMRC dyspnea scale which was associated with a reduction in plasma concentrations of the
inflammatory markers. The effects produced by the three therapeutic combinations on FEV1%
predicted, plasma TNF-α, IL-6 and fibrinogen concentrations were statistically similar (four weeks
after treatment; p=0.358, p=0.284, p=0.155, p=0.155 respectively) and (twelve weeks after treatment:
p=0.710, p=0.773, p=0.240, p=0.076 respectively). Conclusion: In non-asthmatic patients with
moderate to severe COPD, the three therapeutic combinations showed similar effectiveness.
Furthermore, the results of this pilot study suggest that inflammatory markers can be used to follow
the disease activity.

Keywords: COPD, ICS, LABA, LAMA, TNF-α.

1. INTRODUCTION noxious particles and gases, usually from cigarette smoke.

Chronic obstructive pulmonary disease (COPD) is associated Symptomatic COPD patients can be managed with one of the
with abnormal inflammatory response of the lungs towards following inhaled medications; long-acting beta-agonists

*Department of clinical pharmacy, Faculty of Pharmacy, Tanta University, Tanta, (LABA), long-acting muscarinic antagonists (LAMA), and
Egypt. 31111, Tel.: (002) 01286213535; fax: (202) 040-3336007. inhaled corticosteroids (ICS).1 Administration of two or more
E-mail address: [email protected]
medications from different classes seems beneficial when the

disease cannot be controlled adequately with LAMA or
LABA monotherapy.1 LAMAs dilate the airway by
selectively blocking acetylcholine M3 receptors.2 LABAs are

24 | J Adv Med Pharm Res., 2021, 1, 24-32 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

β2-agonists, which provide smooth muscle relaxation by capsule BID plus Tiotropium18 mcg inhaled capsule OD. The
stimulating β2-adrenergic receptors.3 treatment with the study medications was done after a
washout period of two weeks from the entry medications and
Furthermore, LAMAs and LABAs were reported to the only allowed medication during the washout period was
exert anti-inflammatory activity.2,4-5 Although there is still salbutamol. The treatment duration was twelve weeks for all
little evidence for their clinical benefit and increasing groups. Enrolled patients were randomized in a 1:1:1 ratio
evidence that the high doses currently recommended are using a computer-generated code according to the
harmful and costly, ICS are grossly overprescribed secondary Consolidated Standards of Reporting Trials (CONSORT)
to successful marketing.6-7 ICSs were reported to be effective guidelines. The double blindness included the investigator
in bronchial asthma whereas eosinophils play a key role and (physician) and the patients. The main investigator (the
they seem poorly effective in patients with COPD since physician) was provided with a sealed randomization code for
neutrophils play a critical role. Pro-inflammatory cytokines each available treatment generated by an independent
such as TNF-α, IL-1β, and IL-6 appear to amplify researcher. The investigator remained blinded all over the
inflammation in COPD through activation of the transcription study period and till the completion of laboratory analyses.
factor, nuclear factor (NF)-κB, thereby leading to increased The three therapeutic regimens were similar in route of
expression of multiple inflammatory genes. TNF-α was administration, taste, and smell.
reported to be involved in airway inflammation during
COPD.9 Fibrinogen is an acute phase soluble plasma The study was approved by National Research Ethics
glycoprotein, which regulates inflammation in many diseases Committee (CP00011), Tanta University, Egypt, and was
and its plasma concentration may represent a promising registered retroactively as a clinical trial at ClinicalTrials.gov
biomarker to indicate the disease severity.10 Additionally, it identifier: NCT04520230. Eligible patients gave their written
was postulated that IL-6 plays a considerable role in the informed consent. Inclusion criteria were patients with COPD
systemic inflammatory response during COPD.11 aged ≥50 years old, FEV1/FVC < 0.70 and FEV1≥30%
and<80% predicted and both sexes. Exclusion criteria were
There are discrepancies about the most effective patients with very severe COPD (FEV1< 30% predicted),
combined therapeutic strategy for patients with COPD.12 patients with chronic respiratory failure or recent chest
Besides, FEV1% predicted was reported to be poorly infection, and patients with an exacerbation in the past 6
correlated with both symptoms and other measures of the weeks. Patients with a history of asthma, other inflammatory
disease progression; therefore, there is an urgent need for diseases, and patients with clinically significant conditions
other biological biomarkers to follow the disease activity.10,13 such as unstable ischemic heart disease, uncontrolled
Furthermore, the current concepts suggest that an abnormal hypertension, and diabetes were also excluded. The primary
inflammatory response causes disease progression and many outcome was the measure of the effectiveness of the three
inflammatory cytokines were reported to induce airway combinations through evaluating the changes that occur in the
inflammation and to be correlated with the severity of the FEV1% predicted and mMRC dyspnea scale. The secondary
disease.9-11 In this context, we aimed at comparing the outcome was evaluating the changes in plasma concentrations
effectiveness of long acting β2-agonist+long-acting of inflammatory markers.
muscarinic antagonist (LABA+LAMA) versus both LABA+
inhaled corticosteroid (ICS) and LAMA+ICS in patients with 2.2. Demography
moderate to severe COPD through evaluating the changes
occur in FEV1% predicted and mMRC dyspnea scale. Also, All participants were submitted to demography (age, sex, and
we aimed at assessing the changes that occur in plasma smoking habits), physical examination and measurement of
concentrations of inflammatory markers (TNF-α, fibrinogen, weight, height, and calculation of body mass index (BMI).
and IL-6) with the disease activity.
2.3. Assessment of pulmonary function and
2. METHODS dyspnea

2.1. Study design Pulmonary function including FEV1% predicted was assessed
by Spirometry (Chest® Spirometer, Model hl-101, Code
The design of this pilot study was a randomized double-blind 1113088, El-Radwan Company, Egypt). At baseline and
prospective parallel study, which included 45 adult Egyptian twelve weeks after treatment, the modified Medical Research
patients of both sexes with moderate to severe COPD Council scale (mMRC dyspnea scale) was used for the
according to GOLD guidelines. All patients were recruited assessment of dyspnea.
from Chest Disease Department, Tanta University Hospital,
Tanta, Egypt, between November 2016 and December 2018. 2.4. Sample collection and laboratory analyses
The patients were randomly categorized into three groups.
Group I (LABA+ICS) received Formoterol/Budesonide Blood samples were collected at baseline, four and twelve
combination 4.5/160 mcg, 2 inhalations BID. Group II weeks after treatment. Plasma was separated and immediately
(LAMA+ICS) received Tiotropium18 mcg inhaled capsule stored at -80° C until biochemical analyses of plasma tumor
OD plus Budesonide 200 mcg, 2 inhalations BID. Group III
(LABA+LAMA) received Formoterol 4.5 mcg inhaled

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 24-32 | 25

J Adv Med Pharm Res Research Article
necrosis factor-alpha (TNF-α), plasma fibrinogen, and plasma
interleukin 6 (IL-6) concentrations using the commercially The collected data were statistically analyzed using Statistical
available Enzyme-Linked Immunosorbent Assay kits Package for the Social Sciences (SPSS), version 16 (Inc.
(Assaypro, LLC Biotechnology company USA: Catalog No. Chicago, IL, USA). Quantitative data were presented as a
ET2010-1, EF1040-1 and EI1006-1 respectively) using Tecan range, mean ± SD, and median. Qualitative data were
plate reader infinite F 50 (Tecan Group Ltd., Switzerland). All expressed as number and percent. For comparison between
laboratory analyses were carried out at the laboratory of more than two means of parametric data (normally distributed
bioequivalence and pharmaceutical service unit, Faculty of data), the F value of the ANOVA test was calculated. Scheffe
Pharmacy, Tanta University. test was used to compare between each two means if F value
was significant. Paired t-test was also used. For comparison
2.5. Subjective data analysis between more than two means of non-parametric data (non-
normally distributed data), Kruskal-Wallis (χ2) was used.
Through weekly phone calls and biweekly direct meetings, Chi-square test was used for categorical variables. Correlation
patients were followed-up to assess their adherence to the between variables was evaluated using Pearson’s correlation.
study medication and to report any adverse effects. The The significance level was set at p<0.05.
patient was considered non-adherent when he/she underused,
overused, or discontinued the study medications. Patients' 3. RESULTS AND DISCUSSION
adherence to the study medication was assessed by counting
the empty inhalers and through the medications refill rate. The total encounters, screening, randomization, and follow-
Non- adherent participant was excluded from the study as up procedures of the study participants are illustrated in
illustrated in Figure 1. Figure 1. At baseline, the study groups were statistically
similar concerning demographic (age, male sex, weight,
Figure 1: Flow-chart illustrates the participants screening, height, BMI), smoking (duration of smoking, past smokers,
enrollment, and randomization. current smokers, non-smokers), and disease characteristics
(Table 1). There was no group or treatment change during the
2.6. Statistical analysis follow-up of all patients.

Group II showed a significant increase in FEV1%
predicted and significant improvement in the mMRC dyspnea
scale twelve weeks after treatment as compared to baseline
data (p=0.002 and p=0.0001 respectively). Instead, Group I
and group III showed a significant improvement in the
mMRC dyspnea scale twelve weeks after treatment (p=0.0001
and p=0.0001 respectively) which was associated with non-
significant elevation of FEV1% predicted four and twelve
weeks after treatment as compared to baseline data (p>0.05)
as illustrated in Tables 2,3.

Comparing to baseline data, plasma TNF-ɑ
concentration showed a significant decrease four and twelve
weeks after treatment in group I and group III (p=0.019,
p=0.009 respectively for group I and p=0.036, p=0.007
respectively for group III). Concerning Group II, plasma
TNF-ɑ concentration showed significant decrease twelve
weeks after treatment as compared to its baseline value
(p=0.001) as shown in Table 4.

IL-6 plasma concentration showed a statistically
significant decrease within group I four and twelve weeks
after treatment versus baseline data (p=0.026,
p=0.001respectively). Group II showed significant decrease
in serum IL-6 concentration only twelve weeks after treatment
as compared to its baseline data (p=0.043). Group III showed
a non-significant decrease in IL-6 plasma concentration four
and twelve weeks after treatment when compared to baseline
data (p>0.05) as shown in Table 5.

Furthermore, group I showed a significant decrease
in plasma fibrinogen concentration four and twelve weeks
after treatment as compared to its baseline value (p=0.015,
p=0.003 respectively). On the other hand, both group II and

26 | J Adv Med Pharm Res., 2021, 1, 24-32 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

Table 1: Baseline demographics, disease characteristics, pulmonary function test, and dyspnea scale.

Parameters Group1 Group Group 3 P value
LABA+ LAMA+ LABA+
Age (years) LAMA 0.74
Sex (Male) ICS ICS 64.9±9.38 0.44
63.5±9.19 62.5±7.04 11 (73.33%) 0.74
Weight (kg) 10 (66.66%) 13 (86.66%) 0.54
Height 77.57±8.27 0.97
BMI 78.47±5.88 76.43±7.357 172.40±4.12 0.72
Past Smokers 171.33±5.31 170.27±6.03 26.33±3.03 0.76
Current Smokers 26.75±2.36 26.64±2.71 0.71
Never Smoke 11 (73.33%) 0.83
Duration of Smoking 10 (66.66%) 12 (80%) 6 (40%)
(years) 5 (33.33%) 7 (46.66%) 0.30
GOLD Classification 5 (33.33%) 4 (26.66%)
23.9±9.02 3 (20%) 22.27±8.3 0.35
B 21.83±7.25
D 12 (80%) 10 (66.66%) 0.53
FEV1% predicted 3 (20%) 8 (53.33%) 5 (33.33%)
Range 7 (46.66%)
Mean±SD 30.00-79.10 35.90-76.90
mMRC dyspnea scale 61.62±15.14 37.80-75.80 60.24±15.59
Range 54.53±11.57
Mean±SD 2.00-4.00 2.00-4.00
Median 3.07±0.70 2.00-4.00 3.07±0.80
3.33±0.72
Entry Medications 3.00 3.00
SABA 3.00
SAMA 9(60.00%) 7 (46.66%)
Mucolytic 5(33.33%) 7 (46.66%) 7(46.66%)
Expectorant 11 (73.33%) 4(26.66%) 10 (66.66%)
Oral xanthines 7 (46.66%) 8 (53.33 %) 8 (53.33%)
LABA(Formoteol) 5 (33.33%) 5 (33.33%) 4 (26.66%)
ICS(Budesonide) 6 (40.00%) 7 (46.66%)
2 (13.33%) 3 (20%) 2 (13.33%)
8 (53.33%)
4 (26.66%)

The data are expressed as range, mean± SD, and median. LABA: Long acting β2-agonist, ICS: Inhaled corticosteroid, LAMA: Long-acting
muscarinic antagonist, BMI: Body mass index, GOLD: Global Initiative for Chronic Obstructive Lung Disease, FEV1: Forced expiratory
volume in 1 second, mMRC: Modified Medical Research Council, SABA: Short-acting B2-agonist, SAMA: short-acting muscarinic
antagonist.

group III showed a non-significant decline in plasma For group I, there was a significant positive
fibrinogen concentration four and twelve weeks after correlation between plasma TNF-ɑ and IL-6 at baseline and
treatment when compared to its baseline concentration
(p>0.05) as demonstrated in Table 6. twelve weeks after treatment (r=0.61, p=0.015 and r=0.562,
p=0.029 respectively). Four weeks after treatment, TNF-ɑ
There was a non-significant difference between the
three therapeutic strategies four and twelve weeks after showed a significant positive correlation with plasma
treatment regarding FEV1% predicted, plasma TNF-α, Il-6,
and fibrinogen (four weeks after treatment; p=0.358, p=0.284, fibrinogen (r=0.874, p=0.0001). Concerning group II, a
p=0.155, p=0.155 respectively) and (twelve weeks after
treatment: p=0.710, p=0.773, p=0.240, p=0.076 respectively). significant positive correlation was observed between plasma
Besides, twelve weeks after treatment, there was a non- TNF-ɑ and IL-6 at baseline (r=0.599, p=0.018). Additionally,
significant difference in the mMRC dyspnea scale between
the three therapeutic combinations (p=0.749). The four weeks after treatment, a significant negative correlation
comparison between the three groups is illustrated in Tables was observed between plasma TNF-ɑ and FEV1% predicted
2, 3, 4, 5, and 6.
(r= -0.678, p=0.006) as illustrated in Table 7.

Regarding drug-related adverse effects, mild and

manageable adverse effects were reported whereas two

patients in group I (13.33%) and one patient in group II

(6.66%) showed mouth thrush. One patient in group III sho-

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 24-32 | 27

J Adv Med Pharm Res Research Article

Table 2: Mean values of forced expiratory volume in one second (FEV1% predicted) among patients with moderate to severe COPD under
three therapeutic options

Time of assessment Mean values of FEV1 among the studied COPD patients under three F P
options for treatment (n=45) value
Baseline:(a) 0.35
Range Group 1 Group 2 Group 3 1.050 0.35
Mean±SD (LABA+ICS) (LAMA+ICS) (LABA+LAMA) 1.052 0.71
0.346
4 weeks after treatment: (b) (n=15) (n=15) (n=15)
Range
Mean±SD 30.00-79.10 37.80-75.80 35.90-76.90
61.62±15.14 54.53±11.57 60.24±15.59
12 weeks after treatment: (c)
Range 33.50-94.20 46.50-96.50 43.50-87.20
Mean±SD 75.29±21.22 66.10±14.21 70.85±15.89

F value 59.80-116.40 53.80-102.80 52.30-96.50
P 77.61±21.34 72.60±13.31 73.83±15.97
Scheffe test (P)
2.962 7.349 3.060
0.063 0.002* 0.057
a vs b, P=0.064
a vs c, P=0.002*
b vs c, P=0.404

The data are expressed as range, mean± SD. FEV1: Forced expiratory volume in 1 second, LABA: Long acting β2-agonist, ICS: Inhaled
corticosteroid, LAMA: Long acting muscarinic antagonist. *Significant (p<0.05) Scheffe test: Group II: Baseline significantly different from
12 weeks after treatment.

Table 3: m-MRC dyspnea scale at baseline and 12 weeks after treatment among the three studied groups.

Time of m-MRC values among the studied three groups F P
assessment value 0.53
Group 1 Group 2 Group 3 0.74
Baseline: LABA/ICS LAMA/ICS LABA/LAMA 0.644
Range
Mean±SD (n=15) (n=15) (n=15) 0.292
Median
2.00-4.00 2.00-4.00 2.00-4.00
12 weeks after
treatment: 3.07±0.70 3.33±0.72 3.07±0.80
3.00 3.00 3.00
Range
Mean±SD 1.00-3.00 1.00-3.00 1.00-3.00
Median
Paired t-test 1.73±0.80 1.73±0.80 1.93±0.88
P 2.00 2.00 2.00
6.33 12.22 8.50

0.0001* 0.0001* 0.0001*

The data are expressed as range, mean± SD, median. mMRC: Modified medical research council, LABA: Long acting β2-agonist, ICS: Inhaled
corticosteroid, LAMA: Long acting muscarinic antagonist. *Significant difference with ANOVA test (p<0.05)

wed mild palpitation (6.66 %). There was a non-significant changes that occur in plasma concentrations of inflammatory
difference in the reported adverse effects among the three markers (TNF-α, fibrinogen, and IL-6) with the disease
studied groups (P= 0.34 for mouth thrush and P = 0.36 for
palpitation). activity. The sample size used during the current study comes

In this randomized double-blind pilot study, we in accordance with the notion that; a sample size of 12 per
aimed at comparing the effectiveness of long-acting β2- group seems appropriate for a pilot study.14 Furthermore, the
agonist+long-acting muscarinic antagonist (LABA+LAMA)
versus both (LABA+ICS) and (LAMA+ICS) in non- sample size and the follow-up period of the current study were
asthmatic patients with moderate to severe COPD through
evaluating the changes that occur in FEV1% predicted and based on other previous studies conducted on patients with
mMRC dyspnea scale. In addition, we aimed at assessing the COPD.15,16

As compared to baseline data, the results obtained

with group 1 (LABA+ICS) and group III (LABA+LAMA)

four and twelve weeks after treatment revealed the presence

28 | J Adv Med Pharm Res., 2021, 1, 24-32 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

Table 4: Mean values of Tumor necrosis factor alpha (TNF-α) among patients with moderate to severe COPD under three therapeutic options.

Time of assessment Mean values of TNF-α (Pg/ml) among the studied COPD patients F value P

Baseline:(a) under three options for treatment 0.601 0.55
Range 1.297 0.28
Mean±SD (n=45) 0.259 0.77

4 weeks after treatment: (b) Group 1 Group 2 Group 3
Range
Mean±SD (LABA+ICS) (LAMA+ICS) (LABA+ LAMA)

12 weeks after treatment: (c) (n=15) (n=15) (n=15)
Mean±SD
3-44 18-55 18-76
F value 27.53±9.50 30.87±9.29 31.87±14.45
P
Scheffe test (P) 10-30 12-48 14-31
20.07±5.59 24.20±9.10 22.33±5.89

13-30 10-35 11-30
19.33±4.70 18.47±7.06 20.07±6.27

6.453 7.914 6.238
0.004* 0.001* 0.004*
a vs b, P=0.019* a vs b, P=0.114 a vs b, P=0.036*
a vs c, P=0.009* a vs c, P=0.001* a vs c, P=0.007*
b vs c, P=0.959 b vs c, P=0.197 b vs c, P=0.816

The data are expressed as range, mean± SD. TNF-α: Tumor necrosis factor alpha, LABA: Long acting β2-agonist, ICS: Inhaled corticosteroid,
LAMA: Long acting muscarinic antagonist.*Significant (p<0.05) Scheffe test:Group I: Baseline significantly different from both 4 and 12 weeks
after treatment. Group II: Baseline significantly different from 12 weeks after treatment. Group III: Baseline significantly different from both 4
and 12 weeks after treatment.

Table 5: Mean values of Interlukin-6 (IL-6) among patients with moderate to severe COPD under three therapeutic options.

Time of assessment Mean values of Interlukin-6 (IL-6) (pg/ml) among the studied F value P

Baseline:(a) COPD patients under three options for treatment (n=45) 1.270 0.29
Range 1.953 0.15
Mean±SD Group 1 Group 2 Group 3 1.477 0.24

4 weeks after treatment:(b) (LABA+ICS) (LAMA+ICS) (LABA+ LAMA)
Range
Mean±SD (n=15) (n=15) (n=15)

12 weeks after treatment:(c) 3.50-13.10 0.33-19.05 3.60-22.50
Range 6.11±2.90 6.03±4.17 8.17±5.12
Mean±SD
2.15-7.30 1.30-7.60 3.10-21.70
F value 4.01±1.47 4.05±1.80 5.87±4.54
P
Scheffe test (P) 1.50-6.30 0.50-7.10 2.20-13.50
3.07±1.33 3.35±1.81 4.29±2.75

8.809 3.636 3.132
0.001* 0.035* 0.054
a vs b, P=0.026* a vs b, P=0.172
a vs c, P=0.001* a vs c, P=0.043*
b vs c, P=0.452 b vs c, P=0.792

The data are expressed as range, mean± SD. LABA: Long acting β2-agonist, ICS: Inhaled corticosteroid, LAMA: Long-acting muscarinic
antagonist. *Significant (*p<0.05) Scheffe test: Group I: Baseline significantly different from both 4 and 12 weeks after treatment Group II:
Baseline significantly different from 12 weeks after treatment.

of statistically non-significant but clinically important treatment, the three study groups showed significant
improvement in FEV1% predicted. On the other hand, the improvement in the mMRC dyspnea scale as compared to
results obtained with group II (LAMA+ICS) four and twelve their baseline data. The data obtained with the three studied
weeks after treatment showed presence of significant groups can be explained on the basis that, LAMAs are musc-
improvement in FEV1% predicted. Twelve weeks after arinic antagonists which block acetylcholine-mediated bron-

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 24-32 | 29

J Adv Med Pharm Res Research Article

choconstriction by binding to M3 receptors in airway smooth The three therapeutic combinations showed similar
muscles,17 whereas LABAs are β2 agonists which provide efficacy, there was a non-significant difference between the
smooth muscle relaxation by stimulating β2-adrenergic four and twelve weeks after treatment regarding all measured
receptors.3 In addition, it was demonstrated that, LAMA and parameters. In this context, the former result concerning the
LABA combined therapy showed synergistic bronchodilator lack of significant effect of the combinations containing ICS
effect and improved symptoms in COPD patients.17,18 over LAMA+LABA combination may be attributed to the
Furthermore, it was postulated that, ICS may enhance the notion that ICS seems more effective in patients with asthma-
efficacy of LAMA.19 chronic obstructive pulmonary disease overlap syndrome.24
Furthermore, this result may be related to smoking which
During the current study, group I showed a increases airway inflammation and decreases corticosteroids
significant decrease in all inflammatory markers’ responsiveness.25 Our results seem in accordance with a
concentrations while group III showed a significant reduction former study demonstrated that, in real- world clinical
in TNF-ɑ concentrations four and twelve weeks after practice setting of COPD treatment, combined LABA/LAMA
treatment as compared to baseline data. On the other hand, the inhalers appear to be as effective as combined LABA/ICS
results obtained with group II showed a significant reduction inhalers in preventing COPD exacerbations.26 Additionally,
in TNF-α and IL-6 concentrations twelve weeks after our previous data come in agreement with a former study
treatment. These results may be justified on the basis that, reported the absence of a significant difference in transition
LAMA and LABA were reported to exert anti-inflammatory dyspnea index (TDI) focal score between LABA/LAMA and
activity.2, 4, and 5. However, it was postulated that ICS LABA/ICS treated groups.27 In contrast, the data obtained
monotherapy failed to reduce inflammatory markers in with the current study seem incompatible with ENERGITO®
sputum or bronchial biopsies of COPD patients, 20 combining study which proposed that dual bronchodilators can be
drugs with different modes of action may improve outcomes. considered to optimize lung function in patients with COPD
Two-way synergistic activity between ICSs and LABAs has who require maintenance treatment.28 Our result seems in
been demonstrated.21-22 One of the cellular actions of ICSs is contradiction with other authors who reported that, compared
to translocate glucocorticoid receptors from the cytoplasm to to LABA+ICS, LAMA+LABA was associated with greater
the nucleus.21 This action is enhanced in the presence of β- efficacy.29,30
agonists and causes an anti-inflammatory effect greater than
either drug alone.22 In addition, ICSs activate β-receptor genes Regarding the safety and tolerability of the study
to produce more β-receptors, thereby enhancing the medications, the three therapeutic combinations were well
bronchodilator effect of LABA.23 Therefore, the improvement tolerated which comes in agreement with a previously
in both FEV1% predicted and mMRC dyspnea scale may be reported finding.31 Besides, the three therapeutic
attributed also to the reduction of plasma concentrations of combinations showed a statistically similar safety profile, a
inflammatory markers through the anti-inflammatory result seems in accordance with previously reported
properties of the implicated therapeutic regimens. findings.29

Table 6: Mean values of fibrinogen among patients with moderate to severe COPD under three therapeutic options.

Time of assessment Mean values of fibrinogen (µmol/L) among the studied COPD F value P
patients under three options for treatment (n=45)
Baseline:(a)
Range Group 1 Group 2 Group 3
Mean±SD (LABA+ICS) (LAMA+ICS) (LABA+ LAMA)

4 weeks after treatment:(b) (n=15) (n=15) (n=15)
Range
Mean±SD 2.65-16.17 3.23-26.16 3.82-14.73 0.637 0.53
7.14±4.2 9.03±6.14 7.61±3.38
12 weeks after treatment:(c)
1.47-7.05 1.32-20.82 1.76-13.52 1.949 0.15
4.08±1.7 6.94±5.7 5.91±3.59

Range 1.94-6.47 2.03-19.99 0.88-13.82 2.741 0.07
Mean±SD 3.44±1.38 6.82±5.32 5.23±3.99
F value
P 7.771 0.697 1.692
Scheffe test (P) 0.001* 0.504 0.197
a vs b, P=0.015*
a vs c, P=0.003*
b vs c, P=0.822

The data are expressed as range, mean± SD. LABA: Long acting β2-agonist, ICS: Inhaled corticosteroid, LAMA: Long-acting muscarinic
antagonist. *Significant (*p<0.05). Scheffe test: Group I: Baseline significantly different from both 4 and 12 weeks after treatment

30 | J Adv Med Pharm Res., 2021, 1, 24-32 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

Table 7: Correlation between the measured parameters among the STUDY LIMITATIONS
three studied groups

Time of treatment Significant correlations The small sample size represents the major limitation of our
study. In this context, further large-scale studies are still
FEV1% TNF-α needed.
predicted
ACKNOWLEDGMENT
r Pr P
We are so thankful to all participants and the physicians in
COPD patients under the chest department at Tanta University Hospital for their
recommendations.
treatment with
CONFLICT OF INTEREST
LABA+ICS (n=15):
The authors declare that there is no conflict of interest.
Baseline:
5. REFERENCES
IL-6 -0.394 NS 0.61 0.015*
1. Global initiative for chronic obstructive Lung disease
4 weeks after [GOLD]. Global Strategy for the Diagnosis,
Management, and Prevention of Chronic Obstructive
treatment: Pulmonary Disease, 2018.

Fibrinogen -0.200 NS 0.87 0.0001* 2. Alagha K, Palot A, et al., Ther Adv Chronic Dis., 2014,
5, 85-98.
12 weeks after
3. Billington CK, Ojo OO, et al., Pulm Pharmacol Ther.,
treatment: 2013, 26, 112-120.

IL6 -0.272 NS 0.56 0.029* 4. Anderson R, Theron AJ, et al., Mediators Inflamm., 2014,
2014, 105420.
COPD patients under
5. Benfante A, Braido F, et al., Lancet Respir Med., 2018,
treatment with 6, e37.

LAMA +ICS (n=15): 6. Barnes PJ. Am J Respir Crit Care Med., 2000, 161, 342-
344.
At Baseline:
7. Suissa S and Barnes PJ. Eur Respir J., 2009, 34, 13-16.
IL-6 -0.450 NS 0.59 0.018* 8. Barnes PJ. Respiration, 2010, 80, 89-95.
9. Mukhopadhyay S, Hoidal JR, et al., Respir Res., 2006 11,
4 weeks after
7,125.
treatment: -0.678 0.006* 10. Duvoix A, Dickens J, et al., Thorax, 2013, 68, 670-676.
TNF-α 11. He JQ, Foreman MG, et al., Thorax, 2009, 64, 698-704.
12. Horita N, Goto A, et al., Cochrane Database Syst Rev.,
LABA: Long-acting β2-agonist, ICS: Inhaled corticosteroid,
LAMA: Long-acting muscarinic antagonist. *Significant difference 2017, 2, CD012066.
with ANOVA test (p<0.05), r= correlation coefficient. 13. Vestbo J and Rennard S. Am J Respir Crit Care Med.,

During the current study, TNF-α was negatively 2010, 182, 863–864.
associated with FEV1% predicted which seems in accordance 14. Steven A. Julious. Pharmaceut Statist., 2005, 4, 287-291.
with previously reported findings.32 15. Cazzola M, Matera MG, et al., Respir Med., 1995, 89,

Finally, the change that occurred in plasma 357-362.
concentrations of inflammatory markers during the course of 16. Banerji D and Patalano F. Respir Med., 2016, 110, 79-80.
this pilot study can not only suggest their implication in 17. Swinney DC. Nat Rev Drug Discov., 2004, 3, 801-808.
following the disease activity but also can add to treatment 18. Cazzola M, Calzetta L, et al. Respir Med., 2015, 109,
selection. The latter suggestion is based on the current
concepts which postulated that an abnormal inflammatory 1305-1311.
response causes disease progression and drugs with anti- 19. Um SW, Yoo CG, et al., J Korean Med Sci., 2007, 22,
inflammatory activities can modify the underlying disease
mechanisms driving disease progression.33 839-45.
20. Loppow D, Schleiss MB, et al., Respir Med., 2001, 95,
4. CONCLUSIONS
115-121.
The three therapeutic regimens implicated for the treatment of 21. Haque R, Hakim A, et al., J Allergy Clin Immunol., 2013,
non-asthmatic patients with moderate to severe COPD
showed statistically similar safety and efficacy in improving 132, 1166-1173.
FEV1% predicted and mMRC dyspnea scale, and in reducing 22. Usmani OS, Ito K, et al., Am J Respir Crit Care Med.,
the concentrations of inflammatory markers. Furthermore, the
changes that occurred in plasma TNF-α, fibrinogen, and IL-6 2005, 172, 704-712.
concentrations during the treatment course may focus the 23. Barnes PJ. Eur Respir J., 2002, 19, 182–191.
attention on the possibility of implicating these biomarkers to
follow the disease activity.

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 24-32 | 31

J Adv Med Pharm Res Research Article

24. Miravitlles M, Soler-Cataluña JJ, et al., Eur Respir J.,
2013, 41, 1252-1256.

25. Tamimi A, Serdarevic D, et al., Respir Med., 2012, 106,
26. 319–328.
27. Suissa S and Dell'Aniello S, Chest, 2019, 155, 1158-

1165.
28. Mahler DA and Witek TJ Jr, COPD, 2005, 2, 99-103.
29. Beeh KM, Derom E, et al., Int J Chron Obstruct Pulmon

Dis., 2016, 11, 193-205.
30. Rodrigo GJ, Price D, et al., Int J Chron Obstruct Pulmon

Dis., 2017, 12, 907-922.
31. Oba Y, Sarva ST, et al., Thorax, 2016, 71, 15-25.
32. Rennard SI, Tashkin DP, et al., Drugs, 2009, 69, 549-

465.
33. Huang AX, Lu LW, et al., Med Sci Monit., 2016, 22,

2800-2808.
34. Barnes PJ. Chest, 2008, 134, 1278-1286.

32 | J Adv Med Pharm Res., 2021, 1, 24-32 This journal is © Faculty of Pharmacy, Tanta University

Research Article

Received 14th January 2021, C-X-C Chemokine Receptor-3 as a Diagnostic Biomarker
Accepted 16th February 2021 in Patients with Rheumatoid Arthritis
Published 14th March 2021
Mohamed R. Salama1, Hanan H. Omar2, Samah I. Nasef3, Amany H. Hamed1*,

Azza M. Abdallah4

jampr.journals.ekb.eg 1Department of Chemistry, Faculty of Science, Sohag University, Sohag 82111, Egypt
Online ISSN: 2636-4158
2Department of Clinical Pathology, Faculty of Medicine, Suez Canal University, Ismailia 41111, Egypt

3Department of Physical Medicine, Rheumatology, and Rehabilitation, Faculty of Medicine, Suez Canal University, Ismailia 41111, Egypt
4Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia 41111, Egypt

ABSTRACT

Background: Rheumatoid Arthritis (RA) is a chronic and systemic inflammatory disease
characterized by synovial inflammation and the progressive destruction of joint ligaments and
bones. CXCR3 is a seven-transmembrane G-protein-coupled chemokine receptor that has been
appeared to play a vital role in a variety of inflammatory and immunological responses. We aimed
to evaluate the utility of serum C-X-C chemokine receptor 3 (CXCR3) levels in the diagnosis,
monitor, and follow-up of RA patients. Methods: Sixty RA patients were divided into 30 early RA
patients with disease duration < 2 years and 30 longstanding RA patients with disease duration ≥ 2
years. Thirty healthy subjects were recruited as a control group. Medical history and clinical data
were taken. All the patients were assessed for erythrocyte sedimentation rate (ESR), C-reactive
protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP), and CXCR3
serum levels. Results: Serum CXCR3 level was significantly elevated in RA patients compared to
healthy normal controls with a highly statistically significant difference (p< 0.001). The serum
levels of CXCR3 were elevated in long-standing RA more than early RA. Serum level of CXCR3
was only positively correlated with the duration of disease (r= 0.540, p= >0.001) and combined
treatment (r= 0.296, p= 0.022). Receiver operating characteristic (ROC) curves were plotted for the
prediction of serum CXCR3 in RA patients. The best cut-off value that indicates the presence of
early RA disease is 4.026 ng/mL serum CXCR3 with 71.7% sensitivity and 70% specificity (p=
0.005). Conclusion: Serum CXCR3 is an imperative predictive biomarker for the diagnosis of RA,
an indicator for early RA disease, and endorses the established RA disease.

Keywords: Biomarker, Chemokine, Receptor, Prediction, Rheumatoid arthritis.

1. INTRODUCTION and the progressive destruction of joint ligaments and bones.1
RA synovial tissue is regularly described by synovial
Rheumatoid arthritis (RA) is a chronic and systemic hyperplasia, also called pannus, which is penetrated with
inflammatory disease characterized by synovial inflammation inflammatory cells. The synovial pannus in RA produces
proinflammatory cytokines, chemokines, and proteases,
* Department of Chemistry, Faculty of Science, Sohag University, Sohag, Egypt, including monocytes and B and T lymphocytes, attacks and
82511, Tel.: 01150088375. destroys cartilage and bone.2 In addition, fibroblast-like
E-mail address: [email protected] synoviocytes (FLS) have a central role in synovial pannus
formation and joint destruction in RA.3

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 33-38 | 33

J Adv Med Pharm Res Research Article

The process of leukocyte attack into inflammatory erythrocyte sedimentation rate (ESR), patient global health on
sites is basic for the initiation and progression of a variety of a 0 to 10 scale. High disease activity is considered when
inflammatory disorders and is controlled through the disease activity score of 28 joints (DAS 28) is above 5.1,
activation and motioning of particular cell surface chemo- moderate diseases activity between 3.2 and 2.6, and low
attractant receptors by their related protein ligands, named disease activity below 2.6. All the RA patients were under
chemokines.4 Furthermore, the migration of T cells to medical treatment as methotrexate (MTX),
destinations of aggravation is mediated by selectins and their hydroxychloroquine (HCQ), leflunomide, and steroid. There
ligands.5 The guideline of leukocyte relocation is organized were no patients under biological treatment.
by initiating cytokines and adhesion molecules. Other than
that, the enrollment of leukocytes to locales of irritation is Lab examinations included rheumatoid factor (RF)
driven and mediated by the effects of chemo-attractants.6 and anti-cyclic citrullinated peptide (anti-CCP). Quantitative
detection of the CXCR3 level in the serum of the patients and
It has been demonstrated that T helper1 (Th1) and T control was done. Serum was isolated from whole blood via
helper2 (Th2) cells respond differently to several chemokines density gradient centrifugation at 4°C within 4 hours of blood
and express different chemokine receptors. C-X-C chemokine collection and promptly saved into 200 all aliquots at −80°C
receptor 3 (CXCR3) is a seven-transmembrane G-protein- till experimentation.
coupled chemokine receptor that has appeared to play a vital
role in a variety of inflammatory and immunological 2.3. Measurement of serum CXCR3 level
responses. Particularly, the CXCR3 receptor is chiefly
expressed on the activated Th1 cells.7 Serum CXCR3 concentrations were estimated by sandwich
enzyme-linked immunosorbent assay (ELISA) kit, from Bio
RA is a constant immune system illness depicted by kit (Quantikine human CXC3, Shanghai Sunred Biological
steady synovitis. Since chemotactic cytokines (chemokines) Technology Co., Ltd). The outcomes are naturally determined
may play critical roles in the enlistment of leukocytes in RA, to utilize the straight-line regression equation of the standard
investigation for the expression of chemokines and their curve with the standard density and the OD values and are
receptors should provide insight into events in synovial expressed in ng/mL. This examination has high sensitivity and
irritation of RA.8, 9 excellent specificity for the detection of CXCR3. No
significant cross-reactivity or interference among CXCR3 and
Chemokines have a strong significant role in joint analogs was observed. The sensitivity of the CXCR3 level
inflammation, not only by inducing leukocyte chemotaxis but measurement range extends from 0.05 ng/mL to 15 ng/ml.
also by activating immune cells and angiogenesis.10 It was
demonstrated that the expanded articulation of Th1-related 2.4. Statistical analysis
cytokines in cells of synovial liquid and synovial tissue
guessed that Th1 cells may play an active role in the Analysis of data was performed by IBM PC using statistical
development of autoimmune responses in RA.11 package for social science (SPSS) version 25. Data had been
expressed as mean, standard deviation (SD), frequencies, and
Therefore, the purpose of this study was to evaluate percentages were utilized to describe qualitative variables.
the utility of serum CXCR3 levels in the diagnosis, monitor, The comparison between two groups with parametric
and follow-up of RA patients. variables was done using independent sample t-test (t).
ANOVA test was utilized to assess the statistical significance
2. MATERIALS AND METHODS of the difference between more than two study group means.
The correlation coefficient between two parametric
2.1. Patients parameters was calculated by using Pearson and Spearman
correlation coefficient. A Chi-Square test was used to
Patients with RA, who were diagnosed by ACR/EULAR examine the relationship among two qualitative variables. The
(2010) Classification Criteria for RA,12 have been recruited receiver operating characteristic (ROC) curve provides a
from the Rheumatology and Rehabilitation department. The useful way to evaluate the sensitivity and specificity for
study was performed on sixty RA patients. They were divided quantitative diagnostic measures that categorize cases into
into 30 early RA patients with disease duration < 2 years and one of two groups. In all tests if (p> 0.05) it is non-significant,
30 longstanding RA patients with disease duration ≥ 2 years. if (p< 0.05) it is significant and if (p< 0.001) it is highly
Thirty healthy subjects were recruited as a control group. significant.
Informed consents were taken from each patient and control.
The study was approved by the Medical Ethics Committee. 3. RESULTS

2.2. Blood specimen collection and clinical data The studied groups were age and gender-matched. Group 1
included 30 RA patients with disease duration < 2 years, they
Clinical information collection included disease duration, were 27 females (90.0%) and 3 males (10.0%), they had a
medication history, duration of morning stiffness, and body mean age of 40.60±11.99 years. Group 2 included 30 RA
mass index (BMI). Clinical assessment of disease activity was patients with disease duration ≥ 2 years, they were 28 females
done by evaluating the following parameters: swollen joint (93.3%) and 2 males (6.7%), they had a mean age of
includes in 28 joints (SJC 28), tender joint includes in 28
joints status (TJC 28), C-reactive protein (CRP),.or

364 | J Adv Med Pharm Res., 2021, 1, 33-38 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

45.43±9.17 years. The control group included 30 normal 8 CXCR3 (ng/ml)
subjects, 27 females (90.0%) and 3 males (10.0%), they had a
mean age of 42.87±9.31 years. 6

In this study, there was no statistically significant 4
difference between early and long-standing RA patients
regarding family history, BMI, number of tender joints, 2
number of swollen joints, and treatment. Moreover, there was
no statistically significant difference between early and long- 0
standing RA patients regarding RF, Anti-CCP, CRP, ESR, RA patients healthy
DAS28.CRP and DAS28.ESR (Table 1). control

Table 1: Clinical and laboratory data of the studied RA patients

Early RA Long-standing Figure 1: Serum levels of CXCR3 in RA patients and healthy
(n=30) RA control.
(n=30)
Characteristics p-value
10.06±7.25 <0.001*
Duration of disease (yrs)# 1.30±.431 Table 2: serum levels of CXCR3 in early, longstanding RA patients
Mean ± SD 23(76.7%) 0.559 and healthy control.
21(70%) 7(23.3%) 0.488
Family History@ 9(30%) 0.862 Early RA Long-standing Healthy
Negative 29.03±3.99 0.641 (n=30)
Positive 0.451 Variable RA control p-value
10.72±9.82
0.788 (n=30) (n=30)
2.24±4.25 0.243
BMI kg/m2 # 29.92 ±5.79 0.071 CXCR3 5.37 ± 2.39 9.68±6.32 3.89 ±3.57 >0.001*
Mean ±SD 10(33.3%) 0.933 (ng/mL)
10.30 ±8.82 12(40%)
No. of tender joint # 4(13.3%) *p< 0.05 is statistically-significant.
Mean ± SD 1.83±2.11 4(13.3%)
No. of swelling joint # Serum level of CXCR3 was only positively
Mean ± SD 10(36.7%) 46.73± 35.4 correlated with the duration of disease (r= 0.540, p= >0.001)
Treatment No. (%)@ 7(23.3%)
MTX 6(16.7%) 24(80%) and combined treatment (r= 0.296, p= 0.022). The serum level
MTX and HCQ 7(23.3%) 6(20%)
MTX, HCQ and steroid of CXCR3 had no correlation with other studied clinical and
Leflunomide 49.98 ± 55.5 19(63.3%)
11(36.7%) laboratory parameters (Table 3).
Rheumatoid 14.83±15.95
factor (IU/mL) Table 3: Correlation of RA patients’ parameters with - serum
Mean ± SD CXCR3 level.

Rheumatoid factor Characteristics CXCR3
Positive No. (%)
Negative No. (%) 20(66.7%) Age R p-value
10(33.3%) BMI 0.065 0.543
Anti-CCP (U/mL) Duration of disease (years) 0.02 0.879
Positive No. (%) 12(40%) CRP
Negative No. (%) 18(60%) ESR 0.540 >0.001*
15.19±17.17 DAS28.CRP 0.120 0.361
CRP (mg/L) Mean ± SD DAS28.ESR
Anti-CCP
ESR (ml/hr) Mean ± SD 54.73±27.23 59.07±31.48 0.571 No. of tender joint 0.048 0.715
No. of swelling joint
DAS28.CRP Mean ± SD 4.39±1.52 5.15±1.79 0.082 Rheumatoid factor 0.133 0.309

Treatment (combined) 0.103 0.433

DAS28.ESR Mean ± SD 5.12±1.60 4.91±1.61 0.607 0.019 0.887
0.086 0.518
*#Quantitative data are represented by Mean ± SD and tested by t
test, @Qualitative data represented by chi-Square Test. 0.077 0.565
*Statistically-significant p< 0.05; BMI = Body mass index. -.180 0.168
MTX=Methotrexate; HCQ= Hydroxychloroquine; Anti-CCP:
Antibody to cyclic citrullinated peptide; CRP: C reactive protein; 0.296 0.022*
ESR: erythrocyte sedimentation rate; DAS28: Disease Activity
Score 28 joints. *p< 0.05 is not statistically significant.

Serum CXCR3 level was significantly elevated in ROC curves were plotted for the prediction of serum
RA patients compared to healthy normal controls with a CXCR3 in RA patients. The first ROC curve was plotted for
highly statistically significant difference (p< 0.001) (Figure the prediction of RA disease from healthy persons. The best
1). The serum levels of CXCR3 were elevated in Long- cut-off value that indicates the presence of RA disease was
standing RA more than early RA (Table 2). 5.567 ng/mL serum CXCR3 with 60% sensitivity and 75%

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 33-38 | 35

J Adv Med Pharm Res Research Article

specificity (p=< 0.001). Another ROC curve was plotted for CXCR3 were 0.709, 71% and 70% respectively. The AUC
serum CXCR3 to indicate the presence of early RA sets a value of anti-CCP is higher than that of RF and CXCR3. Anti-
threshold value of 4.026 ng/mL with 71.7% sensitivity and CCP sensitivity is higher than RF and CXCR3, but the
70% specificity (p= 0.005). Additionally, the ROC curve for specificity of RF and anti-CCP is higher than CXCR3.
serum CXCR3 for the prediction of establishing RA in RA Combined, anti-CCP and CXCR3 had the best AUC,
patients was done. The best cut-off value that indicates the sensitivity, and specificity, therefore the combined diagnosis
presence of established RA disease was 6.246 ng/mL serum by RF, anti-CCP, and CXCR3 is an effective model for the
CXCR3 with 60% sensitivity and 67.7% specificity (p= diagnosis of early RA (Figure 3).
<0.035) (Figure 2).
The logistic regression models were followed up by
To establish a logistic regression model to evaluate ROC curves to evaluate their combined diagnostic ability. As
the diagnostic efficacy of RF, anti-CCP, and CXCR3 for early can be observed in (Figure 4), combining RF, anti-CCP, and
RA, we ran binary logistic regressions with early RA and CXCR3 yields an AUC value of 0.859 indicating an acceptable
healthy control as our dependent variable. The area under the discriminating power of the model (Figure 4).
curve (AUC) was established to evaluate the diagnostic ability
of RF, anti-CCP, CXCR3, and their combined diagnostic Finally, in order to investigate which combination of
ability. The AUC, sensitivity, and specificity values of RF RF, Anti-CCP, and CXCR3 values leads to a higher
were 0.699, 66% and 77% respectively, while those of anti- probability of having early RA.
CCP were 0.742, 73% and 77% respectively, and those of

Figure 2: ROC curves for serum CXCR3. A: for prediction of RA disease from healthy persons. B: for prediction of early RA
from healthy persons; C: for prediction of established RA disease in RA patients.

Figure 3: Comparative receiver operating characteristic (ROC) Figure 4: ROC-curve after logistic regression analysis, RF, anti-
analysis for RF, anti-CCP, and CXCR3. The ROC-curves for tests CCP and CXCR3.
were comparable as shown by the area under the curve (AUC) values.

36 | J Adv Med Pharm Res., 2021, 1, 33-38 This journal is © Faculty of Pharmacy, Tanta University

J Adv Med Pharm Res Research Article

4. DISCUSSION DAS28.ESR. On contrary with our result, the study by El-
Barbary et al.,17 reported that CXCR3 has a positive
RA, as an autoimmune disease, causes pain, swelling, and correlation with multiple disease activity measures including
stiffness in the joints, and may cause severe joint damage, loss DAS-28 scores, CRP, ESR, swollen and tender joint counts in
of function, and disability. RA is a condition affecting around 28 joints. Similarly, the study by Lee et al.,24 showed the
2% of the population. 13, 14 CXCR3 has the strongest correlations with disease activity.

Chemokines and their receptors are molecules that Our study reported that there was no correlation
may manage the selective migration of particular T-cell between serum levels of CXCR3 and the number of tender and
subsets. In other investigations, additional expression of swollen joints in RA patients. In contrast to Aldridge et al.,27
CXCR3 was detected in endothelial cells and dendritic cells, who found a negative correlation between CXCR3 and
as well as in eosinophils within Th1 dominated tissues, swollen joint counts of 28 and 66 joints.
including RA synovial tissue.15 CXCR3 plays an important
role during various inflammatory diseases such as psoriasis, In the current study, it was observed that there was no
rheumatoid arthritis, and also during infection.16 significant correlation between serum CXCR3 level in RA
patients with RF and anti-CCP. RF was the primary
In the current study, the measured serum level of autoantibody to be found in people with RA. Despite the name,
CXCR3 was significantly higher in RA patients (early and however, RF is not particular to RA. Almost 20% of those with
long-standing RA) compared to healthy control. Collectively, confirmed RA will not have an abnormal RF test, whereas 5%
our findings were in line with El-Barbary et al.,17 they of individuals who do not have RA will have an abnormal RF
mentioned that CXCR3 levels in peripheral blood were found test. Negative levels do not exclude the disease, and positive
to be increased in RA patients compared with controls. levels do not ensure the diagnosis Song Kang.28
Similarly Motoki et al.,18 they noted that the expression of
CXCR3 on peripheral blood CD4+ T lymphocytes of RA In agreement with our results, El-Barbary et al.,17
patients was significantly higher than healthy controls. In found that RF titer had no significant correlation with the
agreement with the study by Ruschpler et al.,15 who also expression levels of CXCR3 in RA patients. In contrast to our
illustrated that receptor CXCR3 was significantly increased in findings, the study by Paulissen et al.,29 found that CXCR3
RA patients compared to osteoarthritis (OA) patients. level was higher in ACPA+ RA than ACPA− RA patients.
Similarly, Henneken et al.,19 they reported that levels of
CXCR3 were demonstrated to be increased in RA patients The accuracy of serum CXCR3 to discriminate RA
when compared with healthy individuals. cases from the normal population was evaluated using ROC
curve analysis. According to our data, CXCR3 could diagnose
In the current study, there was no correlation between and discriminate RA patients at different points through the
BMI and serum level of CXCR3. Our results differed from course of the disease.
those of the study by Shoda et al.,20 who showed that the
concentration of CXCR3 correlated with BMI. An association This enables the physician to follow up with the
between excess body weight and different patients and choose the appropriate and suitable management.
inflammatory/autoimmune conditions has been proposed in
numerous observational studies.21 Excess body weight was 5. CONCLUSION AND RECOMMENDATIONS
considered as a potential supporter to the development of
RA.22 Serum CXCR3 is an imperative predictive biomarker for the
diagnosis of RA, and is an indicator for early RA disease and
Interestingly, our results also revealed that there was endorses the established RA disease. According to our study,
a positive correlation between the disease duration of RA and we recommend: (1) More studies are needed to clarify that
the serum levels of CXCR3 with a high statistical significant CXCR3 plays a central role in RA inflammation and may
difference, disease duration was used as a predictor of vascular serve as a disease activity marker in established RA. (2)
stiffness in RA patients,23 and in agreement with Lleo et al.,24 Clinical trials are needed to explore the beneficial effects of
who found positive correlation between CXCR3 expression in therapeutic targeting of CXCR3 in RA.
CD4+ T cells and disease duration, as well as with Motoki et
al.,18 who found that the CXCR3 expression in RA patients ETHICS APPROVAL AND CONSENT TO
with long-term disease duration was significantly higher than PARTICIPATE
in those with the short-term disease.
All authors have seen and approved the manuscript,
In our work, there was no correlation between serum contributed significantly to the work, and also that the
levels of CXCR3 and CRP as well as ESR. This was in manuscript has not been previously published in or is not being
accordance with the study done by Motoki et al.,18 who found considered for publication elsewhere. The study was approved
that CXCR3 was not correlated with CRP. In contrast to our by the Ethics Committee.
results, Wang et al.,25 found a positive significant correlation
between The expression levels of CXCR3 mRNA and serum CONFLICTS OF INTREST
levels of ESR as well as CRP in the clinically active RA group.
Similarly, Sun et al.,26 found that the levels of CXCR3 were The authors declare no conflict of interest.
significantly correlated with CRP.

In the present study, serum levels CXCR3 did not
show any significant correlation with DAS28.CRP and

This journal is © Faculty of Pharmacy, Tanta University J Adv Med Pharm Res., 2021, 1, 33-38 | 37

J Adv Med Pharm Res Research Article

FUNDING 19. M. Henneken, T. Dörner, G-R. Burmester, and C. Berek,
Arthritis Res. Ther., 2005, 7, R1001–R1013.
This research did not receive any specific grant from funding
agencies in the public, commercial or not-for-profit sectors. 20. H. Shoda, Y. Nagafuchi, Y. Tsuchida, K. Sakurai, S.
Sumitomo, K. Fujio, and K. Yamamoto, Arthritis Res.
6. REFERENCES Ther., 2017, 19, 111.

1. G. S. Firestein, and I. B. McInnes, Immunity, 2017, 46, 21. M. Versini, P-Y. Jeandel, E. Rosenthal, and Y. Shoenfeld,
183-196. Autoimmun. Rev., 2014, 13, 981-1000.

2. I. Sudoł-Szopińska, E. Kontny, W. Maśliński, M. 22. D. P. M. Symmons, Rheumatology, 2005, 44, iv14-iv17.
Prochorec-Sobieszek, B. Kwiatkowska, K. Zaniewicz- 23. M. Vázquez-Del Mercado, E. Gomez-Bañuelos, E.
Kaniewska, and A. Warczyńska, J. Ultrason., 2012, 12,
202-213. Chavarria-Avila, E. Cardona-Muñoz, C. Ramos-Becerra,
A. Alanis-Sanchez, D. Cardona-Muller, F. Grover-Paez,
3. B. Bartok, and G. S. Firestein, Immunol. Rev., 2010, 233, F. de J Perez-Vazquez, R-E. Navarro-Hernandez, J. M.
233-255. Valadez-Soto, A. A. Saldaña-Millan, L. Gonzalez-Rosas,
G. Ramos-Lopez, M. H Petri, and M. Bäck, Medicine,
4. M. M. Wong, and E. N. Fish, Semin. Immunol., 2003, 15, 2017, 96, e7862.
5-14. 24. A. Lleo, W. Zhang, M. Zhao, Y. Tan, F. Bernuzzi, B. Zhu,
Q. Liu, Q. Tan, F. Malinverno, L. Valenti, T. Jiang, L.
5. P. Rajendran, in Frontiers in Anti-Cancer Drug Tan, W. Liao, R. Coppel, P. Invernizzi, Q. Lu, D. H.
Discovery, ed. Atta-ur-Rahman and M. I. Choudhary, Adams, M. E. Gershwin, and the PBC Epigenetic Study
Bentham Science Publishers Pte. Ltd., Singapore, 2019, Group, Clin. Epigenetics, 2015, 7, 61.
vol. 10, ch. 4, pp. 62-110. 25. G. Wang, L. Shi, Y. Mengxue, L. Shen, W. Gou, L. Guo,
and F. Xue, Chin. J. Rheumatol., 2010, 14, 627-630.
6. S. Nourshargh, and R. Alon, Immunity, 2014, 41, 694- 26. S. Sun, Y. Chen, Z. Zhang, W. Zhang, L. Zuo, and G.
707. Xiang, The Journal of Practical Medicine, 2014, 18,
2902-2904.
7. M-R. Du, S-C. Wang, and D-J. Li, Cell. Mol. Immunol., 27. J. Aldridge, J. M. Pandya, L. Meurs, K. Andersson, I.
2014, 11, 438-448. Nordström, E. Theander, A-C. Lundell, and A. Rudin,
Arthritis Res. Ther., 2018, 20, 150.
8. X. Chen, J. J. Oppenheim, and O. Howard, Cell. Mol. 28. Y. Song, and E. H. Kang, QJM, 2010, 103, 139-146.
Immunol., 2004, 1, 336-342. 29. S. M. J. Paulissen, J. P. van Hamburg, N. Davelaar, H.
Vroman, J. M. W. Hazes, P. H. P. de Jong, and E.
9. S. Sharif, N. Ueda, Y. Nakatani, L. M. Wise, S. Clifton, Lubberts, Arthritis Res. Ther., 2015, 17, 344.
Z. Lateef, A. A. Mercer, and S. B. Fleming, Front.
microbiol., 2019, 10, 1421.

10. J. L. Owen, and M. Mohamadzadeh, Front. physiol.,
2013, 4, 159.

11. A. Jäger, and V. K. Kuchroo, Scand. J. Immunol., 2010,
72, 173-184.

12. D. Aletaha, T. Neogi, A. J. Silman, J. Funovits, D. T.
Felson, C. O. Bingham 3rd, N. S. Birnbaum, G. R.
Burmester, V. P. Bykerk, M. D. Cohen, B. Combe, K. H.
Costenbader, M. Dougados, P. Emery, G. Ferraccioli, J.
M. W. Hazes, K. Hobbs, T. W. J. Huizinga, A.
Kavanaugh, J. Kay, T. K. Kvien, T. Laing, P. Mease, H.
A. Ménard, L. W. Moreland, R. L. Naden, T. Pincus, J. S.
Smolen, E. Stanislawska-Biernat, D. Symmons, P. P. Tak,
K. S. Upchurch, J. Vencovský, F. Wolfe, and G. Hawker,
Arthritis Rheum., 2010, 62, 2569-2581.

13. C. L. Gorman, and A. P. Cope, Best Pract. Res. Clin.
Rheumatol., 2008. 22, 221-238.

14. T. Iwamoto, H. Okamoto, and Y. Toyama, S. Momohara,
FEBS J., 2008, 275, 4448-4455.

15. P. Ruschpler, P. Lorenz, W. Eichler, D. Koczan, C. Hänel,
R. Scholz, C. Melzer, H-J. Thiesen, and P. Stiehl, Arthritis
Res. Ther., 2003, 5, R241-R252.

16. J. E. Ehlert, C. A. Addison, M. D. Burdick, S. L. Kunkel,
and R. M. Strieter, J. Immunol., 2004, 173, 6234-6240.

17. A. El-Barbary, S. Essa, and H. Zaytoun, Egypt.
Rheumatol. Rehabil., 2013, 40, 75-80.

18. Y. Motoki, K. Tani, T. Shimizu, H. Tamiya, K. Hase, Y.
Ohmoto, K. Matsushima, and S. Sone, Mod. Rheumatol.,
2003, 13, 114-120.

38 | J Adv Med Pharm Res., 2021, 1, 33-38 This journal is © Faculty of Pharmacy, Tanta University

Editorial Board

Dr. Nahla E. El-Ashmawy, Professor of Biochemistry & Dean of Faculty of Pharmacy,
Tanta University, Egypt
Dr. Ahmed A. Habib, Emeritus Professor of Analytical Chemistry
Dr. Mokhtar M. Mabrouk, Emeritus Professor of Analytical Chemistry
Dr. Sahar M. Elhaggar, Professor of Clinical Pharmacy & Faculty Vice Dean for
Community Services Affairs
Dr. Sanaa A. El-Gizawy, Emeritus Professor of Pharmaceutics
Dr. Fotouh R. Mansour, Associate Professor of Analytical Chemistry
Dr. Mona El-Aasr, Associate Professor of Pharmacognosy
Dr. Ghada Al-Ashmawy, Lecturer of Biochemistry
PH. Bassant Maher, Drug Information Specialist

Contact Information

Website: jampr.journals.ekb.eg
Email: [email protected]
Publisher: Faculty of Pharmacy, Tanta University, Egypt
Address: Elgeish Street, the medical campus of Tanta University, Tanta, Egypt 31111
ISSN: 2636-4158

All rights reserved @ Faculty of Pharmacy, Tanta University, Egypt

https://jampr.journals.ekb.eg/ ISSN: 2636-4158

Editorial Assistants

PH. Bassant M. Mahboub
PH. Mai A. Mousa.
PH. Nahla H. Eldeep
PH. Marwa E. Mohamed
PH. Mohamed K. Talaat
PH. Amr M. Nowier
PH. Mahmoud H. Aboaisha

All rights reserved @ Faculty of Pharmacy, Tanta University, Egypt

https://jampr.journals.ekb.eg/ ISSN: 2636-4158