This document provides information about a PhD scholar named Haseeb Ahsan who is exploring the therapeutic potential of Naproxen derivatives in treating rheumatoid arthritis under the supervision of Dr. Alamgeer. It introduces rheumatoid arthritis and issues with current treatments. The scholar hypothesizes that newly synthesized Naproxen derivatives will have anti-arthritic effects and safety. The document outlines plans to evaluate the anti-inflammatory effects of compounds in vitro and in animal models of arthritis, and to assess toxicity.

2. Scholar name:
Haseeb Ahsan
PhD Scholar ( Pharmacology)
Supervisor name:
Dr. Alamgeer
PhD Pharmacology
Incharge
Department of Phamacology
Faculty of Pharmacy
University of Sargodah

3. Exploration of therapeutic
potential of Naproxen derivatives
in Rheumatoid Arthritis

4. Incidence of disease.
 During last decades’ various incidence studies on
Rheumatoid arthritis have been performed, showing
variation of this disease in different communities.
 The annual incidence rates of RA vary between 20 and
50 cases per 100,000 inhabitants in North American
and North European countries.
 However, there are no studies on RA incidence from
developing countries
(Alamanos, Y & Drosos, A. A. ,2005).
INTRODUCTION

5. Rheumatoid Arthritis
 Rheumatoid arthritis, characterized by Pain, swelling
and stiffness in joints, is a chronic disease.
 In the progress of disease, macrophage activation and
dysfunction play a central role that is indicated by
increased levels of mainly macrophage derived pro-
inflammatory cytokines, i.e., tumor necrosis factor-α,
interleukin-1 (IL-6), IL-1β, and COX-2 in swelled
synovial joints
(Ulfgren AK et al., 1995)

6. Research Problem
 Moreover, all the available drugs with anti-inflammatory
activities cause various adverse effects as well as these
drugs are not always sufficiently effective, that requires a
search for alternative therapy of inflammation. (Bello, A. E.
,2014; Chan, F. K. L.,2007).
 Currently available NSAIDs such as Naproxen produces
gastric toxicity due to carboxylic acid moiety and less
prostaglandins secretions that lead to decreased
physiological muco-protective function of prostaglandins.
(Smith CJ et al., 1998; Hawkey C et al., 2004).

7. Hypothesis
 Newly synthesized drugs with chemicals modifications
of Naproxen have anti-arthritic potentials and safety
profiles in rheumatoid arthritis

8. Aims and objectives
 To evaluate anti arthritic potential of newly synthesized
naproxen derivatives
 To evaluate the effect of these synthesized drugs on pro-
inflammatory mediators
 To find safety profile of these synthesized pro-drugs
 To compare pharmacological effectiveness of these
compounds with standard parent drug

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10. Methodology
Naproxen Derivatives
Pharmacological evaluation Toxicological Studies
In vitro studies In vivo studies Acute toxicity
Protein denaturation Formaldehyde induced
by egg albumin arthritis Subchronic toxicity
Protein denaturation CFA induced arthritis
by BSA
Membrane stabilization

11. 1-Protein de-naturation by using Egg
albumin
 The reaction mixture will be prepared that will contains 0.2ml of egg
albumin, 2.8 ml of phosphate buffered saline of PH 6.4 and 2ml of
prepared solution of variable concentrations of 6400ug/ml,
3200ug/ml, 1600ug/ml, 800ug/ml, 400ug/ml, 200ug/ml,1ooug/ml
and 50ug/ml of newly synthesized drugs in dilute DMSO
respectively.
 Similar volume of dilute DMSO will serve as control. The mixtures
will be incubated at (37±2) °C in incubator for 15 minutes then placed
in oven at 70°C for 5 min then cool down it for 5 mins.
 After that this will be run on UV-Visible spectrophotometer at
660nm wavelength to measure absorbance. By using the absorbance
of treated and control group percentage inhibition will be
determined
(Prakash et al., 2013)
In-Vitro Activities

12. 2- Protein de-naturation by using Bovine
albumin
 The reaction mixtures that contain 0.05ml of different concentrations
of derivatives in dilute DMSO and 0.45 ml of 5% aqueous solution of
bovine albumin will be prepared to run on spectrophotometer.
 This mixture will be incubated in incubator for 30 min at temperature
of 37°C and heated it for 30 mins at 57°C.
 The reaction mixture will be placed on room temperature to cool it and
then 2.5 ml phosphate buffer saline (pH 6.3) will be added to
each tube. After that absorbance will be measured by UV-Visible
spectrophotometer
 Dilute DMSO will serve as control. Naproxen will be considered as a
reference drug. Percentage inhibition of protein denaturation will be
calculated
(Rahman et al., 2012)

13. 3- Membrane stabilization method
 This study will be performed by using Human red blood cells of those
individuals who did not use NSAIDs for 2 weeks before starting the
experiment then mixed equal volume of sterilized Alsevers solution in it,
then centrifuge it at 3000rpm to separate packed cells. After washing it with
hypotonic iso -saline solution, 10% v/v suspension will be prepared by using
isosaline.
 The reaction mixture will be prepared that will contain 1ml of phosphate
buffer, 2 ml of hypo saline and 0.5 ml of HRBC suspension & 0.5 ml
different concentrations of diluted DMSO, reference sample and control.
 All reaction mixture will be placed in incubator for 30 min at 37 degrees
centigrade and centrifuged it 300 rpm.
 The supernatant liquid will be separated and hemoglobin content will be
determined by using absorbanc measured on spectrophotometer at 660nm.
The percentage hemolysis will be estimated by assuming the hemolysis
produced in control as 100%.
(Gautam et al., 2013)

14. IN VIVO ANTI-ARTHRITIC ACTIVITIES
 Immunological arthritis will be assessed using
Complete Freund’s Adjuvant.
 Complete Freund’s Adjuvant (CFA) will be used to
induce arthritis in left hind paw and volume of paw
will be assessed by using plethysmometer at 0, 7, 14, 21
and 28 days’ post induction of phlogestic agent.
Ekambaram et al (2010)
1- Complete Freund’s adjuvant induced arthritis

15.  Spague-Dawely rats having weight of 150-200g of either male or female will
be randomized into group.
 Group I will serve as normal control and will not be administerd Complete
Freund’s Adjuvant.
 Group II as arthritic control and will be given control vehicle.
 Group III as a standard and will be treated with reference dug naproxen in
dose of 30mg/kg body weight.
 Group IV, V and VI will be treated with newly synthesized drugs
 After treatment, on 29 day animals will be sacrificed and blood will be
collected.
 The complete blood count (RBCs, WBC, Hb, ESR and platelet), Liver
Functions (AST , ALT and ALP) and renal functions( Blood Urea and
Creatinine) tests will be performed on all animals.
 However, ELISA test will be performed to evaluate cytokines such as TNF-
α, IL-1β, COX-2 and PGE2 in serum by using kits. Tissue examination will
also be performed
(Chakraborty et al., 2010)

16. Determination of mRNA expression levels of
inflammatory markers
 Total RNA will be extracted from blood samples in accordance to Trizol
method, while nanodrop spectrophotometer will be used to quantify the
purity and yield of RNA.
 500 ng of total RNA per reaction will be reverse transcriptased by with
commercially available kit and cDNA will be synthesized.
 Then product will be amplified in thermal cycler with 45 cycles of
denaturation (95 °C for 10 s), annealing (60 °C for 20 s), and extension (72
°C for 30 s) and quantified by using real-time polymerase chain reaction
(qPCR) through Bio-Rad system.
 Primers of different cytokines will be synthesized
 Ensembl Genome Browser will be observed to pick the genes of different
markers, and only intron-overlapping primers will be chosen to avoid
contamination from genomic DNA. GAPDH will be used as an internal
reference gene.
(Shabbir et al., 2016)

17. 2- Formaldehyde induced arthritis
 This method is used to evaluate chronic anti arthritic
potential.
 In this method, 30 minutes’ post administration of
synthetic derivatives ,2% formaldehyde in the dose of 0.1
ml will be administered by sub planter route on day first, to
induce chronic non immunological arthritis and will be
repeated on day 3.
 Vernier caliper will be used to asses’ arthritis by measuring
the paw diameter after 10 days of treatment. The
percentage inhibition will be measured
Dhage et al (2013)

18. 1- Acute toxicity studies
 This study will be performed in Swiss albino mice of either sex according to
guidelines of OECD (Organization for Economic Co-operation and
Development, Guideline-423, adopted on 17th December, 2001) with slight
variations.
 Forty animals will be selected for such study. They will be divided into four
group
 The newly synthesized drugs will be administered in four higher doses
 Mice will be keenly observed for initial 4hr post treatment and will be
followed the daily observation for 14 days.
 The behavioral changes such as hyperactivity. ataxia, tremors, convulsion,
salivation, diarrhea, sleep and comma and mortality will be observed. The
total study period will be 14 days proposed by
Sim et al (2010).

19. 2- Sub Chronic Toxicity study
 This study will be carried out in rats that will be randomized
into 2 groups, 12 animals each . The animals will be given newly
synthesized drugs by making suspension in 0.5% carboxy-
methyl cellulose at the dose of 1000 and 2000mg/kg for 30
consecutive days.
 Following parameters such as body weight, food and water
consumption will be observed throughout the study.
 Animals will be fasted 12 hrs prior to collection of blood on day
31. The hematological analysis such as Hemoglobin, total
leukocyte count, differential leukocyte count and biochemical
analysis such as glucose, cholesterol, alkaline phosphatase, AST
and ALT will be done.
 After blood collections, the animals will be sacrificed for
histopathological examination
(Singh et al., 2010).

20. References.
 Ulfgren, A. K., Lindblad, S., Klareskog, L., Andersson, J., & Andersson, U. (1995).
Detection of cytokine producing cells in the synovial membrane from patients with
rheumatoid arthritis. Annals of the rheumatic diseases, 54(8), 654-661.
 Alamanos, Y., & Drosos, A. A. (2005). Epidemiology of adult rheumatoid
arthritis. Autoimmunity reviews, 4(3), 130-136.
 Bello, A. E., & Holt, R. J. (2014). Cardiovascular risk with non-steroidal anti-inflammatory
drugs: clinical implications. Drug safety, 37(11), 897-902.
 Chan, F. K. L., Wong, V. W. S., Suen, B. Y., Wu, J. C. Y., Ching, J. Y. L., Hung, L. C. T., ... &
Wong, G. L. H. (2007). Combination of a cyclo-oxygenase-2 inhibitor and a proton-pump
inhibitor for prevention of recurrent ulcer bleeding in patients at very high risk: a double-
blind, randomised trial. The Lancet, 369(9573), 1621-1626.
 Hawkey, C., Laine, L., Simon, T., Beaulieu, A., Maldonado-Cocco, J., Acevedo, E., ... &
Mortensen, E. (2000). Comparison of the effect of rofecoxib (a cyclooxygenase 2
inhibitor), ibuprofen, and placebo on the gastroduodenal mucosa of patients with
osteoarthritis: a randomized, double-blind, placebo-controlled trial. Arthritis &
Rheumatism, 43(2), 370.
 Smith, C. J., Zhang, Y., Koboldt, C. M., Muhammad, J., Zweifel, B. S., Shaffer, A., ... &
Isakson, P. C. (1998). Pharmacological analysis of cyclooxygenase-1 in
inflammation. Proceedings of the National Academy of Sciences, 95(22), 13313-13318.

21. References
 Prakash, D., Bindal, M. C., Gupta, S. K., & Gupta, A. K. (2013). Vedpal.
Antiarthritic activity of milk extract of Semecarpus anacardium nut. Int
Res J Pharm, 4, 158-60.
 Rahman, H., Eswaraiah, C. M., Vakati, K., & Madhavi, P. (2012). In-Vitro
Studies Suggest Probable Mechanism Of Eucalyptus Oil For Anti-
Inflammatory And Anti-Arthritic Activity. International Journal of
Phytopharmacy, 2(3), 81-83.
 Dhage, P., Kasture, S. B., & Mohan, M. (2013). Analgesic, anti-
inflammatory, antioxidant and antiulcer activity of ethanolic extract of
sterculia scaphigera Hance (sterculiaceae) seeds in mice and rats. IJBPR, 4,
35-45
 Sim, K. S., Nurestri, A. S., Sinniah, S. K., Kim, K. H., & Norhanom, A. W.
(2010). Acute oral toxicity of Pereskia bleo and Pereskia grandifolia in
mice. Pharmacognosy magazine, 6(21), 67.. Pharmacognosy Magazine 6,
67–70.

22. Thank u