Mawahib Elmutasim Hassan Abdalla Elfeil

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International University of Africa Repository

محیرلا نحمرلا هللا مبس   International University of Africa Graduate College  Pharmacological Investigations and LD50 Determination of New Amide Prodrug of Ibuprofen  A thesis submitted to the department of pharmacology and toxicology, faculty of pharmacy, for the fulfillment of requirement for the degree of M. Pharm. in pharmacology By  Mawahib Elmutasim Hassan Abdalla Elfeil (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.)  November, 2016   بسم هللا الرحمن الرحيم  ُ الهِذيَن آََمنُوا ِمْنُكْم َوالهِذيَن ﴿  يَْرفَعِ َّللاه ُ  بَِما تَْعَملُوَ  أُوتُوا اْلِعْلَم َدَرَجاٍت َوَّللاه﴾(11) َخبِير   صدق هللا العظيم(11) سورة المجادلة االية         Dedication  I dedicate this work To my lovely mother WAFA for always encourage and   for staying on my side. To my lovely father ELMUTASIM for his unconditional support  To my sisters and brothers who have never left my side and are very special  To my big family, friends and colleges who supported me throughout the process Finally, this work is dedicated to all those who believe in the richness of learning.  MAWAHIB, 2016        ACKNOWLEDGMENT I thank the almighty God for giving me the courage and the determination as well as guidance in conducting this research. Many people should be thanked for their contribution in this study. First and foremost, I would like to thank my supervisors Dr. Aimun Abdelgaffar and Dr. Tilal Elsaman for their encouragement, patience, valuable comments and support given throughout the study. Second I would like to thank Dr.Tarig Alhadiah. Dr. Tasnim Omer, Dr. Ammar Alhafiz, Dr. Salma Yousif, Dr. Mazin Yousif and Dr. Samreen Khalil without their assistance the completion of this work would have been impossible. We are very thankful to the Dean of the Faculty of Pharmacy, International University of Africa Dr. Asma Nur Aldaim. Our thanks also go to the Technical staff at the Faculty of Pharmacy, IUA, especially Mr. Salah Abd Eljbar.  So many people, so little room to write about them. Of course our dear colleagues and families were always there for us and should not be overlooked. BIOGRAPHICAL SKETCH  Mawahib EHA. Elfeil was born on Jun. 26, 1991 in Marawy, Sudan. B. Pharm. degree in pharmacy, International University of Africa on Aug. 2013 in Khartoum. And she will obtain her M. Pharm. degree in Pharmacology, International University of Africa at 2016. I  CONTENTS  CONTENTS .............................................................................................. I List of Tables ............................................................................................ II List of Figures .......................................................................................... III LIST OF ABBREVIATIONS ................................................................... V ENGLISH ABSTRACT.......................................................................... VI  VIII ................................................................................................ المستخلص1. INTRODUCTION AND LITERATURE REVIEW ............................... 1 1.1. Introduction ..................................................................................... 1 1.2. Literature review ............................................................................ 10 1.3. The study rationale ......................................................................... 23 1.4. Study objectives ............................................................................. 24 2. MATERIALS AND METHODS ......................................................... 25 2.1. Materials ........................................................................................ 25    2.2. Methods ......................................................................................... 30 3. RESULTS ............................................................................................ 42 3.1. Effect of TAI-Pd  on carrageenan induced paw edema .................. 42 3.2. Effect of TAI-Pd on albumin denaturation .................................... 47 3.3. In vitro pharmacological effect(s) for the TAI-Pd ......................... 48 3.4. The LD50 estimation using protocol 425, in vivo…………..……....74 4. DISCUSSION ...................................................................................... 77 5. CONCLUSION AND RECOMMENDATIONS .................................. 81 5.1. Conclusion ..................................................................................... 81 5.2. Recommendations .......................................................................... 82  REFERENCES ....................................................................................... 83   II  List of Tables  Table 1.1: Actions of the principal plasma protein–derived mediators of inflammation............................................................................................. 9 Table. 2.1: Drugs used in the experiment, with their manufacturer and country. ....................................................................................................25 Table. 2.2: Chemicals, solvents and reagents used in the experiment. .....26 Table 3.1: The main pharmacological parameters of TAI-Pd and the vehicle control DMSO .............................................................................49 Table 3.2: The main pharmacological parameters of TAI-Pd and ACh ..51 Table 3.3: The main pharmacological parameters of TAI-Pd and 5-HT .53 Table 3.4: The main pharmacological parameters of TAI-Pd and BaCl2.55 Table 3.5: pA2 values of the action of atropine on contracting activity of TAI-Pd and the standard Acetylcholine...................................................58 Table 3.6: pA2 values of the action of cyproheptadine on contracting activity of TAI-Pd and the standard 5-HT ...............................................61 Table.3.7: pA2 values of the action of [TAI-Pd] on the contracting activity of acetylcholine compared with that produced by standard atropine on ACh on isolated rabbit intestine .......................................................................70 Table 3.8: pA2 values of the action of TAI-Pd on the contracting activity of 5-HT compared with that produced by standard cyproheptadine on standard 5-HT on isolated rabbit intestine………………………………..73   III  List of Figures  Fig.1.1: A primer of the inflammatory cascade. ........................................ 4 Fig.1.2: Structural formula of Ibuprofen ..................................................17      Fig.1.3: Synthesis pathway of TAI-Pd…………………….……………...21 Fig.1.4: Structural formula and three dimension structure of TAI-Pd. ....22 Fig.2.1: The study schematic diagram summarize the main study steps. ..30 Fig.3.1: The effects of the different doses of TAI-Pd and a single dose of Ibuprofen on the carrageenan -induced rat Paw edema at 1 hour- time interval. ....................................................................................................42 Fig.3.2: The effects of doses of TAI-Pd and a single dose of Ibuprofen on the carrageenan -induced rat Paw edema at 2 hours- time interval ...........43 Fig.3.3: The effects of doses of TAI-Pd and a single dose of Ibuprofen on the carrageenan -induced rat Paw edema at 3 hours- time interval. ..........44 Fig.3.4: The effects of doses of TAI-Pd and a single dose of Ibuprofen on the carrageenan -induced rat Paw edema at 4 hours- time interval. ..........45 Fig.3.5: Comparison of the anti-inflammatory activity of Ibuprofen and TAI-Pd at different time intervals. ..........................................................46 Fig.3.6: Comparison of the inhibition of protein denaturation of TAI-Pd and Ibuprofen in a three different doses. ...................................................47 Fig.3.7: Cumulative dose-response curve of TAI-Pd and DMSO on isolated rabbit intestine. ........................................................................................48 Fig.3.8: Cumulative dose-response curve of TAI-Pd and the standard acetylcholine on isolated rabbit intestine. .................................................50 Fig.3.9: Cumulative dose-response curve of TAI-Pd and the standard 5-HT on isolated rabbit intestine. ......................................................................52 Fig.3.10: Cumulative dose-response curve of TAI-Pd and the standard BaCl2 on isolated rabbit intestine. ............................................................54 Fig.3.11: Cumulative dose-response curve of the effect of atropine on contracting activity of TAI-Pd (1A) and the ACH (1B) in vitro. .............56 IV  Fig.3.12: Emax values for TAI-Pd and Acetylcholine with high and low concentration of atropine and without atropine ........................................57 Fig.3.13: Cumulative dose-response curve of the effect of cyproheptadine in a dose of [10-10M and 10-8M] on contracting activity of TAI-Pd (2A) and the standard 5-HT (2B) in vitro. ...............................................................59 Fig.3.14: Emax values for TAI-Pd and  5-HT with cyproheptadine in concentration of 10-10 M and 10-8 M and without. ....................................60 Fig.3.15: Cumulative dose-response curve of the effect of TAI-Pd and ACh alone and in a combination together in a ratio of 50: 50. ..........................62 Fig.3.16: Emax values for TAI-Pd and ACh alone and in a combination together. ...................................................................................................63 Fig.3.17: Cumulative dose-response curve of the effect of TAI-Pd and 5-HT alone and in a combination together in a ratio of 50: 50. ....................64 Fig.3.18: Emax values for TAI-Pd and 5-HT alone and together in a combination. ............................................................................................65 Fig.3.19: Cumulative dose-response curve of the effect of TAI-Pd and BaCl2 alone and in a combination together in a ratio of 50: 50. ................66 Fig.3.20: Emax values for TAI-Pd and BaCl2 alone and together in a combination. ............................................................................................67 Fig.3.21: Cumulative dose-response curve of the effect of TAI-Pd on the contracting activity of ACh in a dose of 10-8M (3A) and 10-5M (3B). ......68 Fig.3.22: Emax values for acetylcholine alone and with TAI-Pd in concentration of 10-5 M and 10-8 M. .........................................................69 Fig.3.23: Cumulative dose-response curve of the effect of TAI-Pd on the contracting activity of 5-HT in a dose of 10-8M (4A) and 10-5M (4B) ......71 Fig.3.24: Emax values for 5-HT alone and with TAI-Pd in concentration of 10-5 M and 10-8 M. ...................................................................................72 Fig.3.25: Screen shot of statistical program showing the results and recommendation report of the TAI-Pd toxicity limit test. ........................75 Fig. 3.26: Screen shot of statistical program showing the results and recommendation report of the TAI-Pd toxicity main test………………..76  V  LIST OF ABBREVIATIONS   AA   Arachidonic acid  COX   Cyclooxygenase enzyme CV  Cardiovascular EC50 Half maximum effective concentration that indicate the potency Emax maximum possible effect of the agonist that indicate the efficacy Fig                 Figure GI  Gastrointestinal IL  Interleukin  IFN-γ  Interferon-γ LD50  The median lethal dose value LPC   Lyso phosphatidylcholine  NSAIDs Non-steroidal anti-inflammatory drugs OECD Organization for Economic Co-operation and development pA2  Negative logarithm of antagonist concentration that reduces      an agonist effect to Emax/2, indicate the affinity  PAF  Platelet-activating factor pAx   -Log of antagonist conc. PD2   -log (EC50)  PGs   Prostaglandins  ROS   Reactive oxygen species  TAI-Pd Tilal amide Ibuprofen prodrug TG  Test guidelines Th  T-helper cells  TNF-α Tumor necrosis factor-alpha UDP  Up-and-down procedure VI  ENGLISH ABSTRACT  Background: Inflammation is a complex process occurs within the damaged tissue. The severity of the side effects of NSAIDs, which used for inflammation management, encourage researchers to develop new medications with high safety. Literature survey revealed that masking the carboxyl group of NSAIDs improves the therapeutic efficacy and safety. Aims: The study aimed to investigate anti-inflammatory activity of TAI-Pd. In addition, to determine other pharmacological action, possible mechanism(s) of action and pharmacodynamics interactions using isolated rabbit intestine. Moreover, determination of LD50. Methodology: The carrageenan-induced paw edema and the inhibition of protein denaturation were used for the anti-inflammatory activity investigation. A full dose-response curves for TAI-Pd and the standard agonists (Ach, 5-HT and BaCl2) were constructed in cumulative manner, in absence and presence of standard antagonists (atropine and cyproheptadine). Also the possible pharmacodynamic interactions were determined for the standard agonists by constructing dose-response curves in the presence of TAI-Pd mixed with each of them in 50: 50 ratio (as agonist), and in high and low dose (as antagonist). In order to determine LD50 in vivo, the OECD test guideline 425 protocol used. Results: The results of the carrageenan-induced paw edema in vivo revealed that TAI-Pd exhibited potent anti-inflammatory effect compared to Ibuprofen, maximum at 4 hours, with % of inhibition of edema [23.25± 2.594] VII  for TAI-Pd at dose 35mg/kg and [19.75± 3.95] for standard Ibuprofen at dose 70mg/kg, and the inhibition of protein denaturation confirmed this findings. The effect on isolated rabbit intestine revealed that TAI-Pd displayed dose dependant contracting effect, that blocked partially by atropine and cyprohepatadine. TAI-Pd potentiate the effect of Ach, while it possesses a neglectable effect on the 5-HT when administered in combination and decreases the agonistic effect of BaCl2. This derivative exhibited a cyprohepatadine like effect on 5-HT when added as antagonist. The LD50 of TAI-Pd is 2000mg/kg. Conclusion: TAI-Pd exhibited potent anti-inflammatory activity when compared to Ibuprofen. TAI-Pd produces partial contracting effect on isolated rabbit intestine in vitro. TAI-Pd represent a better toxicological profile compared with standard Ibuprofen.           VIII   المستخلص انبية  اآلثار الج لخطورةتهاب هو عملية معقدة تحدث داخل األنسجة المصابة. نسبة لاال : خلفية الدراسةلمضادات االلتهاب غير االستيرويدية التي تستخدم عادة لعالج االلتهاب، تشجع الباحثون للبحث عن  إخفاء مجموعة الكربوكسيل الداخلة في  تكوين اثبتت الدراسات السابقة ان عالج اكثرأمانا لاللتهاب.  الفعالية العالجية ومامونية الدواء.مضادات االلتهاب غير االستيرويدية يؤدي الى تحسين )مشتق  TAI-Pdالجديد  تهدف هذه الدراسة إلى التحقق  من الخصائص الدوائية للمركب األهداف:ر هذا اللقاء الضوء على أثااليبوبروفين( ، بالتأكد من فعاليته كمضاد لاللتهابات ، كما تهدف ايضا إلى جانب تحديد الجرعه المميتة    لهيناميكيه  داماكورلفاالمركب و آلية عمله إضافة إلى التداخالت ا ( 50LDلنصف الحيوانات المجربة)ط تثبي و، جرزالتورم الناجم عن الكاراجينان في مخلب ال تاتجرباستخدمت  :سةرالداء اجرإمنھجية  .للتأكد من فعالية المركب كمضاد لاللتهابتمسخ البروتين،والعقاقير القياسية المناهضة )األستيل   TAI-Pdالتراكمي لكل من الستجابة  والجرعة امنحنى ء بناتم د جرعة منخفضة و اخرى في وجود جووفى دة و منفركولين و السيروتونين وكلوريد الباريوم( ، ت لتفاعالاسة األتروبين والسيبروهيبتادين(. كما تم درالقياسية)ت الشاالجرعه عالية من الجرعة امنحنى ء ببنا ،TAI-Pd المحتملة  للعقاقير القياسية المناهضة معيناميكيه داماكورلفاا)كمناهض(، وباضافته قبل كل منهم  50: 50للمركب مختلطا مع كل واحد منهم  بنسبة الستجابة وا بجرعتين عالية و منخفضة )كشال(. .425بروتوكول استخدم  ،الجرعة المميتة لنصف الحيوانات المجربةمن أجل تحديد لديه تأثير قوي  TAI-Pdكشفت نتائج التورم الناجم عن الكاراجينان في المخلب ، أن  نتائج الدراسة:ساعات من الحقن، مع٪ تثبيط   4كمضاد لاللتهابات مقارنة مع  االيبوبروفين ، حيث يبلغ ذروته في [ 3.95±  19.75و ] / كغملغ35في جرعة  TAI-Pd[ ل2.594±  23.25تورم قدرها ]ملغ/ كغ. نتيجة تثبيط تمسخ البروتين في المختبر، جاءت مؤكدة 70لاليبوبروفين القياسي في جرعة  .كمضاد لاللتهابات TAI-Pdللتأثير القوي لثبتت ، و أاألمعاء الدقيقة المعزولة لألرنب على لجرعة وانقباضا يتناسب ا TAI-Pdالمركب  هراظو لقد اظهر المركب تأثيرا  تأزريا مع ين . ديبتاوهلسيبرا وبين وباالترجزئيا له مكانية قفعن إسة رالداIX  األستيل كولين و مثبطا لكلوريد الباريوم عند اضافته مناهضا ومن غير اثر على الفعالية القصوى  للسيروتونين عندما اضيف له كمناهض و اثرا مشابها للسيبروهيبتادين عند اضافته كشال. ملغ/ كغ.2000قيمة  TAI-PDلمركب    المميتة لنصف الحيوانات المجربةالجرعة بلغت على  كما اظهر القدرةاظهر المركب تأثيرا قويا كمضاد لاللتهابات مقارنة بااليبوبروفين،.  :الخالصةبمأمونيه  TAI-Pdيتمتع   .المعزولة لألرنب الملساء األمعاء الدقيقة عضالتالقيام بأثر انقباضي في  اكثر من التي يمتلكها عقار االيبوبروفين. 

Pharmacological Investigations and LD50 Determination of New Amide Prodrug of Ibuprofen

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Pharmacological Investigations and LD50 Determination of New Amide Prodrug of Ibuprofen

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