بسم ه الرحمن الرحيم International University of Africa Postgraduate college

Pharmacological Evaluation of Synthetic New Amide Pro-drugs

A thesis submitted to the department of pharmacology and Toxicology, faculty of pharmacy, International University of Africa, for the full fulfillment of requirement for the degree of Master of pharmacology

By Samrin Khalil Nogd Alla Ahmed (B. Pharm., 2013) Supervisor Dr. Aimun Abdelgaffar Elhassan Ahmed (B. Pharm., M. Pharm., Ph.D.) Co-supervisor Dr. Talal Elsaman Elemam Elbashir (B. Pharm., M. Pharm., Ph.D.)

October, 2016

~@DEDICATION@~

I dedicate this work

To my parents

To my brothers and sisters

To my teachers

To my families

To my friends

To all whom I love

With my deepest love and respect

~@Samrin @~ ACKNOWLEDGEMENT

I thank the almighty God for giving us the courage and the determination as well as guidance in conducting this research.

Many people should be thanked for their contribution in this study. This research is made possible through the help and support from everyone, including: parents, teachers, family, friends, and in essence, all sentient beings. Especially, please allow me to dedicate my acknowledgment of gratitude toward the following significant advisors and contributors:

First and foremost, I would like to thank Dr. AIMUN AE.AHMED for the continuous support of my master study and research, for his patience, motivation, enthusiasm, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor.

Second, I would like to thank Dr. TALAL EE.ELBASHIR for his support by ready synthetic compounds and encouragement. He kindly read my paper and offered invaluable detailed advices on grammar, organization, and the theme of the dissertation.

I would like to thank my colleagues Dr. MAWAHIB ELMUTASIM and

Dr. MAZIN YOUSIF for support and co-operation to complete this thesis.

I

Our thanks also go to the Lecturers and Technical staff at the Department of

Pharmacology, Faculty of Pharmacy, IUA.

Finally, I sincerely thank to my parents, family, and friends, who provide the advice and support. The product of this research paper would not be possible without all of them.

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CONTENTS

ACKNOWLEDGEMENT ...... I

CONTENTS ...... III

LIST OF TABLES ...... VII

LIST OF FIGURES ...... IX

LIST OF ABBREVIATIONS ...... XIV

ENGLISH ABSTRACT ...... XV

XVII ...... لص لعبي

1.INTRODUCTION AND LITRETURE REVIEW ...... 1

1.1. Introduction ...... 1

1.1.1. The pain ...... 1

1.1.2. The inflammation process ...... 2

1.1.3. The available agents ...... 7

1.1.4. Non steroidal anti-inflammatory drugs (NSAIDs) ...... 8

1.1.5. Need for new Drugs, Why? ...... 11

1.1.6. Drug sources ...... 11

III

1.1.7. The pre-clinical Studies in Drug discovery ...... 12

1.1.8. Animal models in drug discovery ...... 13

1.1.9. The models for anti-inflammatory and studies ...... 13

1.1.10. Drug safety and toxicological studies for new drug entity ...... 14

1.1.11. Methods for LD50 determination ...... 15

1.1.12. Protocol 425 as a best available one for LD50 determination ...... 15

1.2.Literature Review ...... 17

1.2.1. Drug Discovery and Development for synthetic compounds ...... 17

1.2.2. The selected compounds for the study ...... 17

1.2.3. Fenamate derivatives ...... 18

1.3. The rationale of the Study ...... 26

1.4. Study Objectives ...... 26

2. MATERIALS AND METHODS ...... 28

2.1. Materials ...... 28

2.1.1. The standard drugs ...... 28

2.1.2. The synthetic compounds ...... 28

2.1.3. Chemicals ...... 29

IV

2.1.4. Experimental Animals ...... 30

2.1.5. Equipments ...... 30

2.1.6. Study schematic diagram……………………………………………31 2.2. Methods ...... 32

2.2.1. Pharmacological investigations ...... 32

2.2.2. In vivo anti-inflammatory activity method ...... 33

2.2.3. In vitro anti-inflammatory activity using albumin denaturation ...... 33

2.2.4. In vitro isolated preparations studies ...... 34

2.2.5. The LD50 determination from acute oral toxicity, in vivo on rats ..... 38

2.2.6. Statistical analysis methods ...... 39

3. RESULTS ...... 41

3.1. Effect of tested compounds on tail flick latency ...... 41

3.2. Anti-inflammatory activity of TFAD-01 ...... 44

3.3. In vitro effects on isolated Rabbit intestine ...... 50

3.4. Acute oral toxicity of the most active compound using Protocol 425 ... 65

4- DISCUSSION ...... 68

4.1. Effect of analgesic activity of Synthetic new Fenamic Acids Amide Pro- drugs in vivo ...... 69

V

4.2. Anti-inflammatory activity of the selected most active Compound

(TFAD-01) ...... 70

4.3. Effect of TFAD-01 synthetic compound on isolated rabbit intestine .... 72

4.4. Investigation of mechanism of action ...... 72

4.5. Possible Pharmacodynamic Interactions ...... 73

4.6. Acute oral toxicity of TFAD-01 ...... 74

5. CONCLUSION AND RECOMMENDATIONS ...... 75

5.1. Conclusion ...... 75

5.2. Recommendations ...... 77

REFERENCES ...... 78

VI

LIST OF TABLES

Table 2.1: Chemicals used in the experiment ...... 29

Table 3.1: Summary of the main pharmacological parameters EC50 and

Emax of TFAD-01 synthetic compound and vehicle control presented as

Mean ± S.E.M in both conditions...... 51

Table 3.2: Summary of the main pharmacological parameters EC50 and

Emax of TFAD-01 synthetic compound and standard acetylcholine presented as Mean±S.E.M in both conditions...... 53

Table 3.3: Summary of the main pharmacological parameters EC50 and

Emax of TFAD-01 synthetic compound and standard 5-hydroxy tryptamine presented as Mean±S.E.M in both conditions...... 54

Table 3.4: Summary of the main pharmacological parameters EC50 and

Emax of TFAD-01 synthetic compound and standard Barium chloride presented as Mean±S.E.M in both conditions...... 56

-8 - Table 3.5: pA2 values of the action of in a dose of [10 M and 10

10M] on contraction activity of TFAD-01synthetic compound and standard

Acetylcholine on isolated rabbit intestine...... 58

VII

-8 Table 3.6: pA2 values of the action of cyprohepatadine in a dose of [10 M and 10-10M] on contraction activity of TFAD-01 synthetic compound and standard 5-hydroxy tryptamine on isolated rabbit intestine...... 61

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LIST OF FIGURES

Fig.1.1: Prostanoid mediators derived from arachidonic acid and sites of drug action. ASA, acetylsalicylic acid (); LT, leukotriene; NSAID, nonsteroidal anti-inflammatory drug ...... 9

Fig.1.2: The First synthetic compound, N-cyclohexyl-N-(cyclohexyl carbamoyl)-2-(2, 3-dimethylphenylamino) benzamide ( derivative), with Mwt of 447, Mp 141-143oC and soul in MeOH, DMSO,

CHCl3...... 21

Fig.1.3: The Second synthetic compound, N-cyclohexyl-N-(cyclohexyl carbamoyl)-2-(2, 6-dichloro-3-methylphenylamino) benzamide

( derivative), with Mwt of 501, Mp 132-134oC and soul in

MeOH, DMSO, CHCl3...... 22

Fig.1.4: The Third synthetic compound, N-cyclohexyl-N-(cyclohexyl carbamoyl)-2-(3-(trifluoromethyl) phenylamino) benzamide ( derivative), with Mwt of 487, Mp106-110oC and soul in MeOH,

DMSO, CHCl3...... 23

Fig.3.1: The effects of the vehicle control (corn oil) and TFAD-01 in different doses at different time using tail flick method (1A) and standard

Mefenamic acid (control) (2A)...... 41

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Fig.3.2: The effects of the vehicle control (corn oil) and TFAD-02 in different doses at different time using tail flick method (1B) and standard

Mefenamic acid (control) (2B)...... 42

Fig.3.3: The effects of the vehicle control (corn oil) and TFAD-03 in different doses at different time using tail flick method (1C) and standard

Mefenamic acid (control) (2C)...... 43

Fig.3.4: The effects of the different doses of TFAD-01(green), TFAD-02

(red), TFAD-03 (blue) and a single dose of the standard Mefenamic acid on the tail flick method...... 44

Fig.3.5: The effects of the different doses of TFAD-01 and a single dose of the standard Mefenamic acid on the Carrageenan-induced rat Paw edema at time 1 of TFAD-01 administration ...... 45

Fig.3.6: The effects of the different doses of TFAD-01 and a single dose of the standard Mefenamic acid on the Carrageenan-induced rat Paw edema at time 2 of TFAD-01 administration...... 46

Fig.3.7: The effects of the different doses of TFAD-01 and a single dose of the standard Mefenamic acid on the Carrageenan-induced rat Paw edema at time 3 of TFAD-01 administration ...... 47

X

Fig.3.8: The effects of the different doses of TFAD-01 and a single dose of the standard Mefenamic acid on the Carrageenan-induced rat Paw edema at time 4hrs of TFAD-01 administration ...... 48

Fig.3.9: The effects of the different doses of TFAD-01 and a single dose of the standard Mefenamic acid on the Carrageenan-induced rat Paw edema at time intervals 1,2,3,4 hr of TFAD-01 administration ...... 49

Fig.3.10: The effect of the different doses of TFAD-01 and Mefenamic acid on albumin denaturation in vitro...... 50

Fig.3.11: The cumulative dose-response curve of TFAD-01 and vehicle control on isolated rabbit intestine...... 51

Fig.3.12: The cumulative dose-response curve of TFAD-01 and acetylcholine on isolated rabbit intestine...... 52

Fig.3.13: The cumulative dose-response curve of TFAD-01 and 5-hydroxy tryptamine on isolated rabbit intestine...... 54

Fig.3.14: The cumulative dose-response curve of TFAD-01 and Barium chloride on isolated rabbit intestine...... 55

Fig.3.15: The cumulative dose-response curve of the effect of Atropine in a dose of [10-10M and 10-5M] on contraction activity of TFAD-01 (1A) and acetylcholine (1B) on isolated rabbit intestine...... 57

XI

Fig.3.16: The Emax values for TFAD-01 and Acetylcholine with atropine in concentration of 10-8M and 10-10M and without atropine...... 58

Fig.3.17: The cumulative dose-response curve of the effect of

Cyprohepatadine in a dose of [10-10M and 10-5M] on contraction activity of

TFAD-01(2A) and 5-HT (2B) on isolated rabbit intestine...... 59

Fig.3.18: The Emax values for TFAD-01 and 5-HT with cyprohepatadine in concentration of 10-8 M and 10-10 M and without cyprohepatadine...... 60

Fig.3.19: The cumulative dose-response curve of TFAD-01 and acetylcholine and TFAD-01 with Acetylcholine in a ratio (50:50) on isolated rabbit intestine...... 62

Fig.3.20: The Emax values for TFAD-01 (blue) and Acetylcholine alone (red) and together in a ratio (50:50) in concentration of 10-8 M and 10-13 M

(without colour)...... 63

Fig.3.21: The cumulative dose-response curve of TFAD-01 and 5-hydroxy tryptamine and TFAD-01 with 5-hydroxy tryptamine in a ratio (50:50) on isolated rabbit intestine...... 64

Fig.3.22: The Emax values for TFAD-01 (blue) and 5-hydroxy tryptamine alone (red) and together in a ratio (50:50) in concentration of 10-8M and 10-

13M (without colour)...... 65

XII

Fig.3.33: The screen shot of statistical program showing the results and recommendation report of TFAD-01 toxicity limit test...... 66

Fig.3.34: The screen shot of statistical program showing the results and recommendation report of TFAD-01 toxicity main test and the estimated

LD50...... 67

XIII

LIST OF ABBREVIATIONS

AOT425StatPgm Acute Oral Toxicity 425 Statistical Program

COX-1, COX-2 Cyclo-oxygenase type1 and 2

EC50 Half maximum effective concentration

Emax Maximum possible effect of the

IAEC International Animal Ethical Committee

LD50 The median lethal dose

OECD Organization for Economic Cooperation

and Development pAx Negative logarithm of antagonist molar

concentration.

PD2 Negative log (EC50)

PGE2 Prostaglandin subtype E2

TFAD-01 Talal Fenamic acid Derivatives compound

one

TFAD-02 Talal Fenamic acid Derivatives compound

two

TFAD-03 Talal Fenamic acid Derivatives compound

three

XIV

ENGLISH ABSTRACT

Introduction: Non-steroidal anti-inflammatory drugs (NSAIDs) are a chemically heterogeneous group of compounds and commonly used in palliative therapy in musculoskeletal pain. Fenamates includes (mefenamic, meclofenamic and flufenamic acids), which represent an important group of clinically used NSAIDs.

Aim: to investigate the pharmacological effects of novel three fenamic acid derivatives using experimental animals.

Methodology: the pro-drugs were subjected for analgesic activity through tail flick tests at different doses using albino mice. The most active one was selected for anti-inflammatory assay by carrageenan induced paw edema in albino rat and albumin denaturation method at different doses of TFAD-01.

The cumulative dose-response curves of TFAD-01and various standard spasmogens were constructed using different concentration, alone and in the presence of standard blockers to determine the exact mechanism mediating contracting effect of TFAD-01. The safety of TFAD-01 in animals was confirmed from the acute toxicity studies using limit dose test of up and down method.

XV

Results: The pro-drugs of fenamate showed significant increase in the latency to flick the tail compared to the control group. TFAD-01, which was observed to possess very high analgesic activity than TFAD-02, TFAD-03 in compared with control group. The TFAD-01 pro-drug has similar anti- inflammatory activity to MA in same and higher dose to MA. The confirmation method showed significant inhibition at higher dose of TFAD-

01 in compare with MA. The results revealed that the absence of the vehicle

(DMSO) effect on the isolated rabbit intestine in concentrations used, while the TFAD-01 produced dose-dependent contraction, which is blocked by both antagonists; atropine and cyprohepatadine. In acute oral toxicity some rats survived after administration of a selected high dose 2000 mg/kg/body weight and by using the recommended progression doses of TFAD-01 (175,

550, 2000, 2000, and 2000) mg/kg/body weight.

Conclusion: Fenamate amide pro-drugs have analgesic activity more than

MA and the most potent one is TFAD-01, it has anti-inflammatory similar to

MA. The TFAD-01 produce potential contracting effect on isolated rabbit intestine, but less than produce by standard spasmogens. The possible mechanism of action was found to be cholinergic activity, few serotonergic activities and no any role for direct action to mediate it. TFAD-01 possesses high margin of safety than MA.

XVI

الملخص العربي

مقدمة: م الت غي لستييدي )لسن( هي مجع من لك غي متجنس

كييئي أك ستدم كعا مفض في لم لعا ليي. لفينم تشل حض

ليفينميك، ليفينميك لففينميك، لتي تل مجع هم سيي من م الت

غي استيدي )لسن(.

الهدف: لدس آث لدئي لاث مشت من حض لفينميك بإستد حين لتج.

المنهجية: تم خع ائع حض لفينميك اميد لأي كسن من خا إخت نفض

ليل بجع متف بإستد لف ليء. تم إجء لفحص ل لإلت عن يق

لجين كحفز ل بب لج ي مسخ أليمين بجع متف من مكب

TFAD-01. تم نشء منحني استجب لتكي لكب)TFAD-01( ل ليسي

بجع متف لحده في ج لشا ليسي لتحديد لي لعل. أكد سام TFAD-01

في لحين من س لسي لح بإستد ي لع ل.

النتائج: ائع لفينم أ ي كي في مد نفض ليل من مع مجع ليب

ليسي. لحظ مكب TFAD-01 في متا قد علي ع لتسين من TFAD-02

TFAD-03 من مع مجع لتحم. لع لسعد TFAD-01 لديه نش م لالتب

مثل لحص ليفينميك في نفس ، معف لجع. لي لتأكيدي أ تيط ه في

تسخ ألمين بلجع لعلي من TFAD-01 من مع حض ليفينميك . كشفت لنتئج

خ ليب لع)ثنئي مييل كسيد ليت( من تأثي في معء أنب لعزل

بلتكيز لستدم ، في حين أ مكب TFAD-01 أ لد ع ن عا معء

XVII

انب عت ع لجع لتي تم قف من قل كل من أتبين لسيهيتين.

نتئج لسي لح عن يق لفم بعض لف ع قيد لحي بعد إعء جع علي مت

)2000 مجم / كجم / لجسم( بستد جع متدج من مكب TFAD-01 )175 ،

550 ، 2000 ، 2000 ، 2000 مجم/ كجم/ لجسم(.

الخاصة : مشت ائع لفينم أميدي لأي ل نش مسن أك من ء حض

ليفينم كي ، اك فعلي ه TFAD-01 ل أ نش م لإلت مثل لحض

ليفينميك. لدس مكب TFAD-01 له تأثي نضي ع أمعء أنب

لعزل في لت، ك ه لنش أقل من ل ينتج عن يق لعقي لن ليسي .

آلي لعل لن يحتل ت مث لعل اسيتيل كلين لديه تأثي ضيل ع هم

لسيتنين ليس هن أ لعل لش ع لع لسء. TFAD-01 لديه همش كي

من أم من بحض ليفينميك.

XVIII