ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ Cephradine Bioequivalence and its Interaction with Khat and Food (Al-Sayadeyah) in Yemen

A thesis submitted By

Abdulkarim Kassem Yehia Kassem (B. Pharm., University of Khartoum) In fulfillment of the requirements of University of Khartoum for Master degree

Under the supervision of

Dr. Sumia Sir Elkhatim Mohamed (B. Pharm., M. Pharm., Ph.D.) Associate professor of pharmaceutics Department of Pharmaceutics Faculty of pharmacy University of Khartoum

Co - supervision of

Dr. Adnan Abdu Hussein Al-Adhal (B. Pharm., M. Pharm., Ph.D.) Assistant Professor of Pharmacology Department of Pharmacology Faculty of pharmacy University of Sana’a

May, 2004

Dedication

To my Brother, Abdu Al-Rahman Mohammad Nser Al-Shamiri Acknowledgments

I would like to express my gratitude to my supervisor associate Professor Sumia Sir Elkhatim Mohamed, Head Department of Pharmaceutics, Faculty of Pharmacy, University of Khartoum for her valuable advise, continued assistance and guidance since my enrollment as postgraduate in the Graduate College, University of Khartoum, up to this stage of the work.

My thanks also go to my external supervisor assistant Professor Adnan Al-Adhal, Department of Pharmacology, Sana'a University for his supervision, guidance, valuable suggestions and continued evaluation.

My thanks are extended to Dr. Ahmad Al-Meklafi for his valuable advice, and to Dr. Yehya Ragaa for his support in statistical evaluation of my data. I would like to express my appreciation to my friends Rafied who prepared common Yemeni food (Al-Sayadeyah), Gamdan who prepared Khat also Yaser, Mohammed, Abdullah Taha and Qais who helped me for samples analysis. Finally many thanks to all volunteers and every body who helped me in this research. Abstract The pharmacokinetic parameters and bioequivalence of three oral brands of cephradine capsules were estimated and compared to that of reference standard product. The study was carried out in eight healthy, adult, male Yemeni volunteers after fully understood the purpose of the study and give their approval in a written consent form. Four – way crossover trials were performed using a single dose (500 mg) of cephradine on an empty stomach preceded by an overnight fast. Urine samples were collected at specified time intervals following the drug administration. Volunteers were allowed to take only water to enhance dieresis until 2 hours after medication. Standard breakfast was then served. A 7 days washout period was allowed between trials. The urine samples were kept at 4 C0 only one day till analysis. Samples were analyzed by HPLC system. The pharmacokinetic parameters (Mean ± SD) of the percentage dose excreted unchanged in urine over 12 hrs (PDE%) and the maximum peak of the urinary excretion rate (MPE, mg/min) together with the time taken to reach this peak (TTP, hrs) were used to describe the extent and rate of bioavailability respectively. The extent of cephradine bioavailability, expressed as PDE%, for brands A, B, C, and D was in the order D > A > B > C (75.3 ± 13.9, 74.2 ± 10.6, 72.8 ± 17.4 and 69.8 ± 13.6, respectively). The rate of cephradine bioavailability, expressed as MPE, for the four brands was in the order D > A > B > C (4.3 ± 0.8, 4.1 ± 1.37, 3.98 ± 0.86 and 3.7 ± 1.4, respectively). The values of TTP for the four brands (C > A = B = D) were found to be highly comparable (1.3 ± 0.56, 0.84 ± 0.25, 0.84 ± 0.25and 0.84 ± 0.25, respectively). The half lives for brand A, B, C and D are 0.71, 0.94, 0.98 and 0.84, respectively and the elimination rate constants for brand A, B, C and D were 0.98, 0.73, 0.71 and 0.83, respectively. The relative bioavailability of each of the three brands B, C and D compared to the reference brand A is in the range 80 - 120 %. Statistical analysis using student’s t-test showed that there are no statistically significant differences in all pharmacokinetic parameters between the three brands B, C and D and the reference standard product A (Pֿ> 0.05). However, TTP of brand C was > found to be significantly different compared to brand A (Pֿ 0.05). Studies on the effect of Khat and common Yemeni food (Al- Sayadeyah) on the bioavailability of reference brand A were performed in the same way as the bioequivalence studies, except that the drug was administered with khat in the first trial and with food in the second one. The results of these studies are summarized in the following table:

Parameter Brand A Brand A Brand A Fasting with Khat with Al-Sayadeyah PDE (%) 74.2 ± 10.6 51.6±10 55.3±19.8 MPE, (mg/min) 4.1 ± 1.37 2.6± 0.6 1.9 ± 0.8 TTP (h) 0.84 ± 0.25 2.2 ±0.7 3.1 ± 0.9 t½ (hr) 0.71 0.5 0.72 -1 Ke (hr ) 0.98 1.4 0.96

Statistical analysis using student’s t-test showed that there are statistically significant differences in all pharmacokinetic parameters when Brand A is given in fasting condition and with either Khat or Al-Sayadeyah (P < 0.05). Khat and Al-Sayadeyah effect in the bioavailability of cephradine and decrease absorption of cephradine 50 %. The study concluded that the four brands A, B, C and D are bioequivalent and they can be used interchangeable. Bioequivalence studies were recommended to be performed for all oral dosage forms locally marketed and manufactured before issuing their certificate of registration. The study recommended the avoidance of chewing Khat and eating Al-Sayadeyah for at least three hours following cephradine and similar drugs administration.

ﻣﻠﺨﺺ اﻟﺒﺤﺚ

ﺗﺸﺘﻤﻞ هﺬﻩ اﻷﻃﺮوﺣﺔ ﻋﻠﻰ ﺣﺴﺎب وﻣﻘﺎرﻧﺔ اﻟﺤﺮآﻴﺔ اﻟﺪواﺋﻴﺔ واﻟﺘﻜﺎﻓﺆ اﻟﺤﻴﻮي ﻷرﺑﻌﺔ أﺻﻨﺎف ﻣﻦ اﻟﺴﻴﻔﺮادﻳﻦ ﻋﻦ ﻃﺮﻳﻖ اﻟﻔﻢ. ﺗﻤﺖ هﺬﻩ اﻟﺪراﺳﺔ ﺑﻤﺸﺎرآﺔ ﺛﻤﺎﻧﻴﺔ ﻣﻦ اﻟﻤﺘﻄﻮﻋﻴﻦ، اﻟﺒﺎﻟﻐﻴﻦ، اﻷﺻﺤﺎء، اﻟﺬآﻮر اﻟﻴﻤﻨﻴﻴﻦ. ﺷﺎرك اﻟﻤﺘﻄﻮﻋﻴﻦ ﺑﻌﺪ ﻓﻬﻤﻬﻢ اﻟﻬﺪف واﻟﻔﺎﺋﺪة اﻟﻤﺮﺟﻮة ﻣﻦ إﻗﺎﻣﺔ هﺬﻩ اﻟﺪراﺳﺔ وﺳﺠﻠﻮا ﻣﻮاﻓﻘﺘﻬﻢ آﺘﺎﺑﺔ. اﺗﺒﻌﺖ اﻟﺘﺠﺎرب اﻹآﻠﻴﻨﻴﻜﻴﺔ ﻃﺮﻳﻘﺔ اﻻﺧﺘﻴﺎر اﻟﻌﺸﻮاﺋﻲ ﻟﺠﺮﻋﺔ واﺣﺪة ﻣﻦ اﻟﻌﻘﺎر ﺗﻌﻄﻰ ﻟﻠﻤﺘﻄﻮع ﺻﺒﺎﺣﺎ ﻋﻠﻰ ﻣﻌﺪة ﻓﺎرﻏﺔ ﺑﻌﺪ ﺻﻴﺎم ﻃﻮل اﻟﻠﻴﻞ . أﺟﺮﻳﺖ هﺬﻩ اﻟﺘﺠﺎرب ﻓﻲ أرﺑﻊ ﻓﺘﺮات زﻣﻨﻴﺔ (أرﺑﻌﺔ ﺗﺠﺎرب إآﻠﻴﻨﻴﻜﻴﺔ)، ﺗﺒﻌﺪ آﻞ ﺗﺠﺮﺑﺔ ﻋﻦ اﻷﺧﺮى أﺳﺒﻮع وهﻲ اﻟﻔﺘﺮة اﻟﻜﺎﻓﻴﺔ ﻟﺘﻨﻘﻴﺔ اﻟﺠﺴﻢ ﻣﻦ ﺟﺮﻋﺔ اﻟﺴﻴﻔﺮادﻳﻦ اﻟﺴﺎﺑﻘﺔ. ﺳﻤﺢ ﻟﻠﻤﺘﻄﻮﻋﻴﻦ ﺑﻌﺪ ﺗﻌﺎﻃﻲ اﻟﻌﻘﺎر ﺑﺘﻨﺎول اﻟﻤﺎء ﻓﻘﻂ ﻟﺘﺤﻔﻴﺰ إدرار اﻟﺒﻮل ﺣﺘﻰ ﻣﺮورﺳﺎﻋﺘﻴﻦ ﻣﻦ أﺧﺬ اﻟﺠﺮﻋﺔ ﺣﻴﺚ ﻳﺤﻴﻦ ﻣﻮﻋﺪ ﺗﻨﺎول وﺟﺒﺔ اﻹﻓﻄﺎر. ﺗﻢ أﺧﺬ اﻟﺠﺮﻋﺔ ﻣﻦ اﻟﺴﻴﻔﺮادﻳﻦ وﺗﻢ أﺧﺬ ﻋﻴﻴﻨﺎت اﻟﺒﻮل ﻓﻲ اﻟﺰﻣﻦ اﻟﻤﺤﺪد وﺗﻢ ﺣﻔﻈﻬﺎ ﻓﻲ درﺟﺔ ﺣﺮارة أرﺑﻌﺔ درﺟﺔ ﻣﺌﻮﻳﺔ إﻟﻰ اﻟﻴﻮم اﻟﺜﺎﻧﻲ ﻣﻦ أﺧﺬهﺎ ﺣﻴﺚ ﺗﻢ ﺗﺤﻠﻴﻠﻬﺎ ﺑﻮاﺳﻄﺔ اﻟﺘﺤﻠﻴﻞ اﻟﻜﺮوﻣﺎﺗﻮﻏﺮاﻓﻲ ﺗﻢ ﺣﺴﺎب ﻣﺘﻮﺳﻂ اﻟﻨﺴﺒﺔ اﻟﻤﺌﻮﻳﺔ ﺑﺸﻜﻠﻬﺎ ﻏﻴﺮ اﻟﻤﺴﺘﻘﻠﺐ ﻓﻲ اﻟﺒﻮل ﺧﻼل 12 ﺳﺎﻋﺔ واﻟﺤﺪ اﻷﻋﻠﻰ ﻟﻺﻃﺮاح اﻟﺒﻮﻟﻲ ﻣﻊ اﻟﺰﻣﻦ اﻟﻼزم ﻟﻠﻮﺻﻮل إﻟﻰ هﺬا اﻟﺤﺪ. ﻗﺪ أﻇﻬﺮت اﻟﺪراﺳﺔ أن ﻣﺘﻮﺳﻂ اﻟﻨﺴﺐ اﻟﻤﺌﻮﻳﺔ ﻟﻠﺘﻮاﻓﺮ اﻟﺤﻴﻮي ﻟﻠﺴﻴﻔﺮادﻳﻦ آﺎﻧﺖ ﻋﻠﻰ اﻟﺸﻜﻞ اﻟﺘﺎﻟﻲ:D > A > B > C (and 69.8 ± 13.6 17.4 ± 72.8 ,10.6 ± 74.2 ,13.9 ± 75.3 ﻋﻠﻰ اﻟﺘﻮاﻟﻰ). آﻤﺎ أن اﻟﺤﺪ اﻷﻋﻠﻰ ﻟﻺﻃﺮاح هﻮ: D > A > B > C ) (4.3 ± 0.8, 4.1 ± 1.37, 3.98 ± 0.86 and 3.7 ± 14) آﻤﺎ أن ﻣﺘﻮﺳﻂ اﻟﺰﻣﻦ اﻟﻼزم ﻟﻠﻮﺻﻮل إﻟﻰ اﻟﺤﺪ اﻷﻋﻠﻰ ﻟﻺﻃﺮاح هﻲ: C > A = B = D 1.3 ± 0.56 >0.84 ± 0.25 = 0.84 ± 0.25 = 0.84 ± 0.25 وﻗﺪ أﻇﻬﺮ اﻟﺘﺤﻠﻴﻞ اﻹﺣﺼﺎﺋﻲ ﻟﻠﻨﺘﺎﺋﺞ أﻧﻪ ﻻ ﺗﻮﺟﺪ ﻓﻮارق ذات دﻻﻻت إﺣﺼﺎﺋﻴﺔ ﺑﻴﻦ آﻞ ﻣﻦ اﻟﻤﺴﺘﺤﻀﺮات اﻟﺜﻼﺛﺔ ﻣﻘﺎرﻧﺔ ﻣﻊ اﻟﻤﺴﺘﺤﻀﺮ اﻟﻘﻴﺎﺳﻲ ﻣﺎﻋﺪا ﻓﻲ اﻟﺰﻣﻦ اﻟﻼزم ﻟﻠﻮﺻﻮل ﻟﻠﺤﺪ اﻷﻋﻠﻰ ﻟﻺﻃﺮاح ﻟﻠﻤﺴﺘﺤﻀﺮ C اﻟﺬى أﻋﻄﻰ ﻓﺎرق إﺣﺼﺎﺋﻲ ﻣﻘﺎرﻧﺔ ﻣﻊ اﻟﻤﺴﺘﺤﻀﺮ اﻟﻘﻴﺎﺳﻲ. آﺬﻟﻚ ﺧﻠﺼﺖ اﻟﺪراﺳﺔ إﻟﻰ أهﻤﻴﺔ دراﺳﺔ اﻟﺘﻜﺎﻓﺆ اﻟﺤﻴﻮي ﺑﺎﻟﻨﺴﺒﺔ ﻟﻸدوﻳﺔ اﻟﻔﻤﻮﻳﺔ اﻟﻤﺴﺘﻮردة و اﻟﻤﺼﻨﻌﺔ ﻣﺤﻠﻴﺎ ﻗﺒﻞ إﺻﺪار ﺷﻬﺎدة ﺗﺴﺠﻴﻠﻬﺎ.

آﻤﺎ ﺗﻢ دراﺳﺔ ﺗﺄﺛﻴﺮ اﻟﻘﺎت وآﺬﻟﻚ اﻟﻮﺟﺒﺔ اﻟﺸﻌﺒﻴﺔ (اﻟﺼﻴﺎدﻳﺔ) ﻋﻠﻰ اﻟﺘﻮاﺟﺪ اﻟﺤﻴﻮى ﻟﻠﺼﻨﻒ A. وﻣﻦ ﺧﻼل اﻟﺪراﺳﺔ ﻟﻠﻨﺘﺎﺋﺞ وﻣﻘﺎرﻧﺘﻬﺎ ﻣﻊ ﻧﺘﺎﺋﺞ اﻟﺼﻨﻒ A ﺑﺪون اﺧﺬ اﻟﻘﺎت أو اﻟﻮﺟﺒﺔ اﻟﺸﻌﺒﻴﺔ (اﻟﺼﻴﺎدﻳﺔ) آﺎﻧﺖ اﻟﻨﺘﺎﺋﺞ آﻤﺎ ﻳﻠﻲ:

وﺟﻪ اﻟﻤﻘﺎرﻧﺔ اﻟﺼﻨﻒ A اﻟﺼﻨﻒ A ﻣﻊ اﻟﻘﺎت اﻟﺼﻨﻒ A ﻣﻊ اﻟﺼﻴﺎدﻳﺔ 1.9 ± 0.8 2.6± 0.6 4.1 ± 1.37 MPE 55.3±19.8 51.6±10 74.2 ± 10.6 PDE% 3.1 ± 09 2.2 ±0.7 0.84 ± 0.25 TTP

0.72 0.5 0.71 t½ (hr) -1 0.96 1.4 0.98 Ke (hr )

وﻗﺪ أﻇﻬﺮ اﻟﺘﺤﻠﻴﻞ اﻹﺣﺼﺎﺋﻲ ﻟﻬﺬﻩ اﻟﻨﺘﺎﺋﺞ ﺑﺄﻧﻪ ﺗﻮﺟﺪ ﻓﻮارق ذات دﻻﻻت إﺣﺼﺎﺋﻴﺔ ﻟﻠﻤﺴﺘﺤﻀﺮ A ﻣﻊ اﻟﻘﺎت وأﻳﻀﺎ ﻣﻊ اﻟﻮﺟﺒﺔ اﻟﻴﻤﻨﻴﺔ اﻟﺼﻴﺎدﻳﺔ وﻋﻠﻴﻪ ﻳﻨﺼﺢ ﺑﻌﺪم ﺗﻨﺎول اﻟﻘﺎت واﻳﻀﺎ اﻟﺼﻴﺎدﻳﺔ أﺛﻨﺎء ﺗﻨﺎول آﺒﺴﻮﻻت اﻟﺴﻴﻔﺮادﻳﻦ واﻷدوﻳﺔ اﻟﻤﺸﺎﺑﻬﺔ ﻟﻬﺎ و ذﻟﻚ ﺧﻼل ﻓﺘﺮة ﻻ ﺗﻘﻞ ﻋﻦ ﺛﻼﺛﺔ ﺳﺎﻋﺎت ﺑﻌﺪ ﺗﻨﺎول اﻟﺪواء. ﺧﻠﺼﺖ اﻟﺪراﺳﺔ اﻟﻰ أن اﻟﻤﺴﺘﺤﻀﺮات اﻷرﺑﻌﺔ (A, B, C and D) ﻣﺘﻜﺎﻓﺌﺔ ﺣﻴﻮﻳﺎ.وأن اﻟﻘﺎت وآﺬﻟﻚ اﻟﻮﺟﺒﺔ اﻟﺸﻌﺒﻴﺔ (اﻟﺼﻴﺎدﻳﺔ) ﻳﻌﻤﻼ ﻋﻠﻰ ﻋﺪم اﻣﺘﺼﺎص %50 ﻣﻦ اﻟﺴﻴﻔﺮادﻳﻦ. Table of Contents

Page

i Acknowledgments Abstract (English) ii Abstract (Arabic) v Table of contents vii List of tables xi List of figures xiii Appendices xvii Abbreviations xviii Chapter 1: Introduction 1.1. Introduction and literature review 1 1.2. History of β-lactam 2 1.2.1. β - lactamase inhibitors 3 1.3. History and Sources 4 1.4 Chemistry of Cephalosporins 5 1.4.1. Chemical Degradation 6 1.5. Structure activity relationship (SAR) 8 1.6. Mechanism of action of cephalosporins 9 1.6.1. Inhibition of synthesis 9 1.7. Mechanism of Bacterial resistance 10 1.8. Cephradine 11 1.8.1. Chemistry 11 1.8.2. Physical properties 12 1.8.3. Pharmacological properties 12 1.8.4. Antimicrobical activities 13 1.8.5. Cephradine drugs interaction 13 1.9. of Drugs 14 1.9.1. Absorption 14 1.9.1.1 Factors affecting absorption 14 1.9.1.2. The gastrointestinal tract and drug absorption 26 1.9.1.3. Effects of food on drug Absorption 30 1.9.1.4. Mal Absorption 32 1.9.1.5. Bioavailability 32 1.9.1.6 Assessment of bioavailability 33 1.9.2. Distribution 34 1.9.3. 35 1.9.4. Drug excretion 37 1.10. Bioequivalence 40 1.11. Measurement of cephradine in biological fluids 40 1.12. Clinical uses of cephalosporins 42 1.13. Khat 43 1.13.1. Historical background 43 Pharmacological action of the Khat on the gastrointestinal 1.13.2. tract 44 1.13.3. Tannins 45 1.13.3.1 Physicochemical properties of tannins 45 1.13.3.2. Pharmacological properties of tannins 45 1.14 Drug-food interactions 47 1.14.1. AL-Sayadeyah 48 β-lactam distribution in the Republic of Yemen 1.15. (ROY) 49 Chapter 2: Aims and Objectives 2.1 Aims 51 2.2 Objectives 52 Chapter 3: Materials and Methods 3.1. Subject and study site 53 3.2. Study design 54 3.3. Clinical protocol 54 3.4. Khat Constituents 56 3.5. Chemical Composition of Fish used in the studies 56 3.6. Cephradine Khat studies 58 3.7. Cephradine Al-Sayadeyah studies 58 3.8 HPLC analysis of cephardine 58 3.8.1 Chemicals 58 3.8.2 Instruments 58 3.9 Method of analysis 59 3.9.1 Chromatographic conditions 60 3.9.2. Stock solution preparation 60 3. 9. 3. Calibration curves in urine 60 3.9.4. Validation of the HPLC analytical method 61 3.10. Data analysis 61 3.11. Calculations 61 3.12. Statistical analysis 62 Chapter 4: Results and discussion 4.1. Analytical procedure 63 4.1.1 Specificity 63 4.1.2 Limit of detection 63 4.1.3 Absolute recovery 65 4.1.4 linearity 66 4.1.5 Inter-day reproducibility 66 4.1.6 Accuracy, precision and intra-day reproducibility 68 4.2. Cephradine comparative bioequivalence studies 69 4.3 Cephradine-Khat interaction studies 92 4.4 Cephradine-common Yemeni food interactions 101 Chapter 5: Conclusion and

Recommendations

5. Conclusion and Recommendations 110

Chapter 6: References 6. References 112 Chapter 7: Appendices 7. Appendices 127

List of Tables

Page Table 1.1 Percentage of imports of to the total imports of drugs 50 Table 1. 2 Distribution of cephradine available in the market of Yemen 50 Table 3.1. Demographic data of the volunteers 53 Table3.2. Study design and treatment schedule 54 Table 3.3. Cephradine (500 mg) brands used in the study 55 Table 3.4. Khat contents 56 Table 3. 5. Major components of tuna fish 57 Table 3.6 Mineral components of tuna fish 57 Table 3.7. Vitamins A, D, C and E components of tuna fish 57 Table 4.1. Sensitivity test of cephradine in urine by HPLC analysis 65 Table 4.2 Absolute recovery of cephradine in urine compared to mobile phase. 65 Table 4.3 Inter-day reproducibility of cephradine in mobile phase 66 Table 4.4. Inter-day reproducibility of cephradine in urine 68 Table 4.5. Accuracy, Precision and within day reproducibility for cephradine in urine 68 Table 4.6 Mean ± SD urinary excretion rates (mg / min) at various mean times of cephradine following oral administration of 500 mg capsules of brands A, B, C and D to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 70 Table 4.7 Mean ± SD Cumulative urinary excretion (PDE %) of cephradine following oral administration of 500mg capsules of brands A, B, C and D to eight healthy, adult, male Yemeni volunteers under fasting conditions 79

Table 4.8 Mean ± SD of pharmacokinetic and bioavailability parameters of cephradine following its administration as 500 mg capsules of brands A, B, C and D to eight healthy volunteers under fasting conditions. 89 Table 4.9. Mean ± SD urinary excretion rates (mg / min) at various mean times of cephradine following oral administration of 500 mg capsules of brand A to eight healthy, adult, male, Yemeni volunteers under fasting conditions and with Khat. 93 Table 4.10 Mean ± SD cumulative urinary excretion of cephradine following oral administration of 500 mg capsules of brand A to eight healthy, adult, male, Yemeni volunteers under fasting conditions and with chewing of Khat. 96 Table 4.11 Bioavailability parameters (Mean ± SD) of cephradine following administration of 500 mg cephradine (Brand A) under fasting condition and with chewing the Khat 97 Table 4.12 Mean ± SD urinary excretion rates (mg/min) at various mean times of cephradine following oral administration of a 500 mg capsule of brand A to eight healthy, adult, male Yemeni volunteers under fasting conditions and with Al- Sayadeyah 102 Table 4.13 Mean ± SD cumulative urinary excretion of cephradine following oral administration of 500 mg capsule of brand A to eight healthy, adult, male, Yemeni volunteers under fasting conditions and with administration of Al- Sayadeyah 105 Table 4.14 Pharmacokinetic and bioavailability parameters (Mean ± SD) of cephradine following administration of 500 mg capsule under fasting condition and with Al-Sayadeyah 106 List of Figures

Page Figure 1.1 Structures of cephalosporins 6 Figure 1.2 Chemical degradation of 7 Figure 4.1 Chromatograms of blank urine (A) and urine sample of a volunteer following oral administration of 500 mg cephradine capsule (B, RT = 1.658 minutes). 64 Figure 4.2 Calibration curve of Cephardine in urine 67 Figure 4.3 Mean ±SD urinary excretion rate (mg/min) at various mean times of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers under fasting condition. 71 Figure 4.4 Mean ± SD urinary excretion rate (mg/min) at various mean times of cephradine following oral administration of 500 mg capsule of brands (B) to 8 healthy, adult, male Yemeni volunteers under fasting condition. 72 Figure 4.5 Mean ± SD urinary excretion rate (mg/min) at various mean times of cephradine following oral administration of 500 mg capsules of brand (C) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 73 Figure 4-6 Mean ± SD urinary excretion rate (mg / min) at various mean times of cephradine following oral administration of 500 mg capsules of brand (D) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 74 Figure 4.7 Mean ± SD urinary excretion rate (mg / min) at various mean times of cephradine following oral administration of 500 mg capsules of brands (A and B) to 8 healthy, adult, male Yemeni volunteers under fasting condition. 75 Figure 4.8 Mean ± SD urinary excretion rate (mg / min) at various mean times of cephradine following oral administration of 500 mg capsules of brands (A and C) to 8 healthy, adult, male Yemeni volunteers under fasting condition. 76 Figure 4.9 Mean ± SD urinary excretion rate (mg / min) at various mean times of cephradine following oral administration of 500 mg capsules of brands (A and D) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 77 Figure 4.10 Mean ± SD urinary excretion rate (mg/min) at various mean times of cephradine following oral administration of 500 mg capsules of brand A (reference) and brands, B, C and D (testes) to 8 healthy, adult, male Yemeni volunteers under fasting condition. 78 Figure 4.11 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 81 Figure 4.12 Mean ± SD cumulative urinary excretion profile of cephradine Following oral administration of 500 mg capsules of brand (B) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 82 Figure 4.13 Cumulative urinary excretion profile of cephradine (PDE%) following oral administration of 500 mg capsule of brand (C) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 83 Figure 4.14 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (D) to 8 healthy, adult, male, Yemeni volunteers under fasting conditions. 84

Figure 4.15 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brands (A and B) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 85 Figure 4.16 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsule of brands (A and C) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 86 Figure 4.17 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsule of brands (A and D) to 8 healthy, adult, male, Yemeni volunteers under fasting conditions. 87 Figure 4.18 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand A (reference) and brands B, C and D (tests) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 87 Figure 4.19 Mean ± SD rate of excretion versus-time profiles of cephradine (Brand A) after administration of 500 mg capsule to 8 healthy, adult, male Yemeni volunteers with Khat. 94 Figure 4.20 Mean ± SD Urinary excretion rate versus-times profiles of cephradine (Velosef®) after administration of 500 mg capsules to (8) healthy, adult, male Yemeni volunteers under fasting condition and with Khat 95 Figure 4.21 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers with Khat. 98

Figure 4.22 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers under fasting conditions and with administration of Khat. 99 Figure 4.23 Mean ± SD Urinary excretion rate of versus-times profiles of cephradine (Velosef®) after administration of 500 mg capsules to (8) healthy, adult, meal volunteers with administration of AL-Sayadeyah. 103 Figure 4.24 Mean ± SD Urinary excretion rate of versus-time profiles of cephradine (Velosef®) after administration of 500 mg capsules to (8) healthy, adult, male volunteers under fasting condition and with administration. 104 Figure 4.25 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers with administration of Al-Sayadeyah. 107 Figure 4.26 Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers under fasting conditions and with administration of Al-Sayadeyah. 108 Appendices

Appendix (A) Table 7.1. t-test for brand B and brand A 127 Table 7.2. t-test for brand C and brand A 127 Table 7.3. t-test for brand D and brand A 128 Appendix (B) Table 7.4. t-test for brand A alone and with Khat 129 Appendix (C) Table 7.5. t-test for brand A alone and with Al-Sayadeyah 130 7.4. Appendix (D):Informed consent form 131 7.5. Appendix (E) Bioavailability study data sheet 132

Abbreviations

AUC : The Area Under the Curve. BP :British Pharmacopoeia. C.V. : Confidence of Variance or Coefficient of Variation.

Cmax : The Maximum drug concentration in Blood.

F : Relative bioavailability. HPLC : High Performance Liquid Chromatography.

Ke : Elimination rate constant. MPE : The Maximum Peak of Excretion. PDE% : Percentage Dose of Excretion.

2 R : Correlation coefficient. RBC : Red Blood Cell. RT : Retention Time S.D : Standard Deviation.

T 1/2 : Half - life.

Tmax : The Time required to observe Cmax in the blood. TTP : Time to reach the maximum Peak of excretion. USP : United State Pharmacopoeia. UV : Ultra Violet. WHO : World Health Organization. 1.1. Introduction and literature review

Antibiotics are extensively used in the Republic of Yemen (ROY), consistently occupying the number one position of pharmaceutical imports and representing 23-26% of the total expenditure of pharmaceutical supplies (Al-Meklafi, 1998). The availability of substandard medicine to the general public possesses many problems both clinically and economically. It is widely believed that such sub-standard preparations are readily available in many developing countries. This may be due to poor manufacturing of products, poor storage conditions or deliberate counterfeiting of branded or generic products (Shakhor et al, 1995). Previous comparative bioequivalence studies carried out on different brands of showed that one of these brands failed to be bioequivalent. This indicates that some biopharmaceutical factors failed to achieve the requirements of bioavailability which are the rate and extent of absorption of active ingredient. The recommendation of this study suggested that bioequivalence studies on different brands of different oral dosage forms marketed in Yemen shall be continued (AL- Mekhlafi, 1998). Consequently four cephradine brands were chosen for this study. This research work will also deal with one of the major problem in quality control of drugs in Yemen, which is the study of the effect of the Khat (Catha edulis, family celastaceaes) on the bioavailability of drugs. It is known that the leaves of the Khat which are chewed contain cathinone, cathine and nor pseudoephedrine. These are some of the possible structural isomers of phenyl propanol amine and are identical to the β- hydrox metabolite of dexamphetamine. Khat leaves also contain tannins 0(7-14 %). The tannins in Khat may damage health by causing gastrointestinal upsets (Bowman, 1980). Yemeni food is different in contents and consistency in comparison with other types of food. Therefore it is of paramount importance to study the effect of common Yemeni food on the bioavailability of different drugs. Cephradine will be investigated in this project for Khat-drug interaction and Yemeni food (Al-Sayadeyah) interaction.

1.2. History of β-lactam The history of β-lactam goes back to the discovery of . In 1929 Alexander Fleming observed that a mould contained in one of his cultures caused the bacteria in the vicinity to undergo lysis. A broth in which the fungus was grown was markedly inhibitory for many organisms. The mould belongs to the genus penicillin, so Fleming named the antibacterial substance penicillin. A decade later Penicillin was developed as a systemic therapeutic agent by a group of investigators at Oxford University headed by Florey in 1949. Also Chain and Abraham by May 1940. The crude material was found to produce dramatic therapeutic effects when administered parentrally to mice with experimentally produced streptococcal infection. Despite great obstacles to its laboratory production, enough penicillin was accumulated by 1941 to conduct therapeutic trials in several patients desperately ill with staphylococcal and streptococcal infection refractory to all other therapy. Herrel in 1945 recorded that bed pans were actually used by Oxford groups for growing culture of Penicillin notatum. A vast research program was soon initiated in the United States. During 1942, 122 million units of Penicillin were made available. By the spring of 1943, 200 patients had been treated with the drug. The results were so impressive that the surgeon general of the US army authorized the trial of the antibiotic in a military hospital. Soon thereafter penicillin was adopted throughout the medical services of the US armed forces. The deep fermentation, a procedure for biosynthesis of penicillin, marked a crucial advance in the large-scale production units. The quantity manufactured rose to over 200 trillion units (nearly 150 tons) by 1950. The first marketable penicillin cost several dollars per 100,000 units; today the same dose costs few cents (Goodman and Gilman 1993). More than 4000 antibiotics have been isolated from microbial sources and reported in the literature. More than 30,000 semi synthetic antibiotic have been prepared; of these only about 100 are used clinically. Utility not only depend on a high antibiotic activity but also on other important properties such as good tolerance, selective toxicity favorable pharmacokinetics (Reiner, 1983). These antibiotics are today among the most efficient weapons in the armoury of the physicians in their fight against infectious diseases. Thereafter, they were used to a large extent and constitute the largest class of medicaments with respect to the turnover values. Beside what was mentioned above, in the development of semisynthetics penicillin and cephalosporins one of the primary goals was to find compounds that are insensitive to the β-lactamase activity.

1.2.1. β-lactamase inhibitors Certain molecules can bind to β-lactamases and inactivate them; thus preventing the destruction of β-lactam antibiotics that are substrates for these enzymes e.g. and , both of them have poor intrinsic antibacterial activities but are inhibitors of β-lactamases produced by wide range of Gram-positive and Gram-negative microorganisms. (Goodman and Gilman, 1993). Thus their principle uses are likely to extend the antibacterial spectra of penicillin and cephalosporins to include bacterial infections that are resistant to the latter by virtue of β-lactamases production (Burger’s, 1979). This effect of β-lactamase inhibitors reinforced the use of antibiotics in future.

1.3.Cephalosporins History and Sources Cephalosporium acremonium the first source of the cephalosporins was isolated by Brotzu (1948) from the sea near a sewer out let of the Sardinian coast. Crude filtrates from cultures of this fungus were found to inhibit the in vitro growth of Staphylococcal aureus and to cure staphylococcal infections, and typhoid fever in man. In 1951 Gottshall and co-workers reported antibiotic activities in culture fluids of Tilachildium and named the active agent Synnematin B. Culture fluids in which the Sardinian fungus was cultivated were found to contain three distinct antibiotics: 1) Cephalosporin P, active only against Gram-positive

microorganism. 2) Cephalosporin N, a new type of penicillin with a side chain derived from D-7-aminoadipic acid and similar to synnemantin B was effective against both Gram-positive and Gram-negative bacteria. 3) , present in partially purified preparations of cephalosporin N but possessing the same range of antimicrobial effectiveness. With the isolation of the active nucleus of cephalosporin C, 7- aminocephalosporinic acid and with the addition of side chains it becomes possible to produce semi synthetic compound, with antibacterial activities very much greater than that of the parent substance.

1.4. Chemistry of Cephalosporins Cephalosporin P is a compound related chemically to helvolic acid and to fusidic acid; asteroid antibiotic elaborated by Fusidium cocuneum. Cephalosporin N (Penicillin N) is an N-acyl derivative of 6-amino penicillanic acid and is inactivated by pencillanase. It has a polar side chain not previously demonstrated in antibiotics, extremely hydrophilic because of the zwitter ionic side chain and yields penicillamine when hydrolyzed. Cephalosporin C resembles cephalosporin N in containing a side chain derived from D-α-aminoadipic acid but differs from it because the side chain is condensed with a dihydrothiazine-β-lactam ring system (7- amino cephalosporanic acid) instead of thiazolidine-β-lactam ring complex. It is much more stable than cephalosporin N in aqueous solution at pH 2 to 3 and is highly resistant to penicillanase. Cephalothin is a semisynthetic derivative of Cephalosporin C. Other congeners are cephaloglycin, cephaloridine and cephalexin. The structural formulae of these compounds as will as that of the central nucleus, 7-aminocephalosporinic acid are shown in figure 1-1. Despite the similarities between the two families of the cephalosporins, the N-acyl derivatives of 7-aminocephalosporinic acid are distinctly different from those of 6-aminopenicillanic acid (cephalosporin N), due to the presence of the dihydrothiazine-β-lactam ring system in the former. Compound containing 7-amino cephalosporinic acid are relatively stable in dilute acid and highly resistant to penicillanase, regardless of the nature of their side chains and their affinity for the enzyme (Goodman and Gilman, 1993).

O H H S R1 C NH N O R2 COOH Cephalosporins S S O N CH OCCH N O 2 3 O COOH Cepham Cephalosporanic Acid

S CH CO NH CH CH CH2 + NH3 CO N C C CH3 COO- Cephradine

Figure 1.1 Structures of cephalosporins

1.4.1. Chemical Degradation Cephalosporins experience a variety of hydrolytic degradation reactions, the specific nature of which depends on the individual structure among 7-acylamino-cephalosporanic acid derivatives; the 3- acetoxylmethyl group is the most reactive site. In addition to its reactivity to nucleophilic displacement reactions, the acetoxyl function of this group readily undergoes solvolysis in strongly acidic solutions to form the desacetylcephalosporin derivatives. The latter lactonize to form the desacetylcephalosporin lactones, which are virtually inactive. The 7- acylamino group of some cephalosporins can also be hydrolyzed under enzymatic (acylases) and possibly nonezymatic conditions to give 7-ACA (or/and-ADCA) derivatives. Following hydrolysis or solvolysis of the 3- acetoxymethyl group, 7-ACA also lactonizes under acidic conditions (Fig. 1-2). Hydrolysis of the β-lactam of cephalosporins is believed to give initially cephalosporic acids (in which the R = H or S heterocycle) or possibly anhydrodesacetyl cephalosporonic acids. It has not been possible to isolate either of these initial hydrolysis products in aqueous systems. Apparently, both types of cephalosporanic acid undergo fragmentation reactions that have not been characterized fully (Wilson and Gisvold, 1982).

S S R CONH CH CH CH2 R CONH CH CH CH2 CO N C CO N C CH OH C CH3 R` C 2

CO2H CO2H H O Cephalosporin Desacetylcephalosporin 2 Acylase + H , -H2O S S H2N CH CH CH2 R CONH CH CH CH2 CO N C β-Lactamase C CH2 R` CO N C OH or H O+ C 3 CH2 CO2H C 7-Aminocephalosporanic Acid O O Desacetylcephalosporin Lacton

H+ , S S R CONH CH CH CH2 -H2O R CONH CH CH CH2 CO2H N C C CH CO2H N C 2 C CH2 R` S CO2H CH CH CH CO H H2N 2 2 Anhydrodesacetylcephalosporoic CO N C Cephalosporoic Acid Acid C CH2 C O O Desacetyl-7-aminocephalosporanic Fragmentation and Rearrangement Acid Lactone Products

Figure 1.2 Chemical degradation of cephalosporin

1.5. Structure activity relationship (SAR) The structure activity studies on cephalosporins have largely paralleled work in semisynthetic pencillins. Indeed, the primary objective of cephalosporin SAR studies has been the opposite; to increase relatively low intrinsic activity of the natural cephalosporins as compared to the natural . Studies involving 7-acyl group modification have been the most profitable source of clinically useful antibiotics. Alterations of this side chain have played a significant role in influencing level and breadth of antibacterial activity. At 3-position variations comprise the second most important type of alteration of the cephalosporins structure (Burger’s 1979). Infact most cephalosporins analogues have relatively poor activity against Gram- negative bacteria. Only a selected few ones combine high intrinsic activity with spectrum of susceptibility broad enough to make them candidates for clinical use. Some of cephalosporin acyl group is associated with significant broad spectrum, especially Gram-negative activity. They include the side chain of the cephalosporanic acids that are significant to qualify for clinical use (Burger’s 1979). Lack of oral activity: an almost universal characteristic of the cephalosporins is unexpected since these compounds have distinctly better acid stability than the penicillins, which are absorbed orally except when limited by instability to gastric acid. Good oral absorption of cehpalosporins in humans has been ascribed to the presence in the structure of both the α-amino group on the 7-acyl substituent, and small-uncharged group at the 3-position. Indeed the cephlosporins that exhibit the highest absorption efficiencies are derivatives of 7-Anhydro Desacetyl Cephalosporic Acid (ADCA) with methyl group at the 3-position (Burger’s 1979).

1.6. Mechanism of action of cephalosporins The β-Lactam antibiotics can kill susceptible bacteria. Although, knowledge of mechanism of action is incomplete, numerous researches have supplied information to wards understanding of the basic phenomenon (Goodman and Gilman, 1993). However, it has been found that it is convenient to present this mechanism of action under four headings: 1- Inhibition of cell wall synthesis. 2- Alteration of cell membrane. 3- Inhibition of protein synthesis. 4- Inhibition of nucleic acid synthesis (Russel, 1977).

1.6.1. Inhibition of cell wall synthesis The initial step in cephalosporin action is binding of the drug to cell receptors. These receptors are cehpalosporin-binding proteins (CBPS), at least some of them are enzymes involved in transpeptidation reactions. From three steps (or more) CBPs can be present per cell. After the cephalosporins molecules have been attached to the receptors this attachment eventually become covalent. synthesis inhibition at the final transpeptidation is blocked. A final bactericidal event is removal or inactivation of an inhibitor of the autolytic enzyme in the cell wall. This activates the autolytic enzyme and results in cell lysis or forming filamentous cells, elongations and release of the cell contents as aspheroplast, which burst unless osmotic protection is provided (Reiner, 1982). On the other hand, structural analogy between cephalosporin and the D-alanyl-D-alanine end of the pentapeptide in the uncross linked precursor of the cell wall have been involved to explain the molecular basis for antibacterial action of cephalosporins. The label CO-N bond in the β-lactam ring of cephalosporins lies in the same position as the peptide bond involved in the transpeptidation. The inhabitation of the biosynthetic reactions by β-lactam drugs is accompanied by distinctive morphologic changes. The nature of these changes depends on the specific organism, the antibiotic used and its concentrations (Zinsser, 1988).

1.7. Mechanism of Bacterial resistance The microorganism may be intrinsically resistant because of structural differences in the CBPS that are targets of the drugs. Furthermore, it is possible for sensitive strains to acquire resistance of this type by mutation. This mechanism for the acquisition of resistance while uncommon has been described for Gram-positive cocci (Goodman and Gilman, 1993). Others resistances of bacteria to the β-lactam antibiotics are caused by the inability of the agent to penetrate to its site of action. This commonly occurs with Gram-negative bacteria. There is an exception, where small hydrophilic antibiotics diffuse through aqueous channels in the outer membrane that are formed by proteins called porin. Bacteria can destroy β-lactam antibiotics enzymatically causing resistance. Increased production of biochemical intermediates that is competitively antagonized by the drug in sensitive cells is also considered as a mechanism of bacterial resistance.

1.8. Cephradine Cephradine is the only cephalosporin derivative available in both oral and parenteral dosage forms. It closely resembles cephalexin chemically (it may be regarded as a partially hydrogenated derivative of cephalexin) and has very similar antibacterial and pharmacokinetic properties to cephalexin (Wilson, and Gisvold, 1982).

1.8.1. Chemistry

S CH CO NH CH CH CH2 + NH3 CO N C C CH3 COO- Cephradine

Cephradine is 7-[α-D-(cyclohexa-1,4-dienyl)-glycylamino]-3- methyl-3-cepham-4-carboxylic acid. Cephradine contains the nucleus, which is common to other cephalosporins. It has a D-cyclohexa-1, 4 dienyl glycyl group at the 7-amino position, as is distinguished from all other cehpalosporins. Cephradine is a zwitterions, i.e. the molecule contains both a basic group (the amino function on the acyl side chain) and an acidic group (the carboxyl at the position 4 of the thiazin ring). Because of the presence of both groups cephradine exists essentially as inner salt at physiological pH. On other side of these values it begins to form a salt with external acids or bases (Griffith and Black, 1970).

1.8.2. Physical properties Cephradine is a white to cream crystalline powder that is slightly soluble in water, insoluble in ethanol 96%, chloroform and ether. It is soluble in 70 parts of methanol and freely soluble in propan,1,2-diol. The British Pharmacopoeia (B.P.) specifies that a 0.5% solution in water has pH of 4.0 to 5.5. The United States Pharmacopoeia (U.S.P) specifies that a 0.5% suspension in water has a pH of 3 to 5.5. Cephradine is recommended to be stored at a temperature not exceeding 30 C0 in airtight containers, protected from light.

1.8.3. Pharmacological properties Cephradine is stable in gastric acid (Griffith and Black, 1970) when given orally under fasting conditions, peak blood levels are reached within one hour. Plasma concentration is 16 µg/ml after a dose of 0.5 g, this concentration is adequate for the inhibition of many Gram-positive and Gram-negative pathogens that are sensitive to cephalosporins (Goodman and Gilman, 1993). Cephradine is not absorbed from the stomach but is totally and rapidly absorbed in the upper intestine (Griffith, 1983). It is widely distributed in the tissues and high concentrations are found in all organs especially the liver and the kidney. Cephradine is reported to be about 15 to 20 % protein bound (Wise, 1990) and excreted unchanged in urine in the range of 69 to 100 % (Griffith, 1983 and Wise, 1990). Goodman and Gilman also reported that the drug is excreted unchanged in the range of 70 to 100 %. Approximately 26 % is excreted through glomerular filtration and about 33 % is execrated by tubular secretion (Foord et al, 1969). One percent of the drug is recovered in the bile (Wise, 1990). Cephradine half-life is reported to range from 0.6 to 1.8 hours (Wise, 1990).

1.8.4. Antimicrobical activities Cehpradine is a first generation cephalosporin antibiotic. Its activity includes Gram-positive bacteria and Gram-negative cocci, actinomyces and spirocheates. Cephradine has the advantage of being active against penicillinase-producing staphylococci although not against -resistant strains or penicillin- resistance Streptococcus pneumonia among Gram-negative bacteria. Cephradine has activity against some entero bacteria including strains of E coli, Klebsiella pneumonia, proteus mirabilis, salmonella and shegella species. It is also active against influenza, moraxella and neisseria species (Martindale, 1993).

1.8.5. Cephradine drugs interaction Angiotensin converting enzyme inhibitors e.g. captopril and anopril and substrates of the peptide carrier competitively inhibit the uptake of cephradine (Yuasa et al, 1994 and Kitagawas et al, 1997). Valacyclovir (L-valayl ester prodrug of cyclovir) interactions with peptides or peptides analogues e.g cephradine, cephalexin and amoxicillin significantly reduced its absorption, (Sinko-pj et al, 1998). Interaction of cephradine with the intestinal absorption of D- gluctose is competitive. Both compounds compete for dipeptidase for absorption in the intestine (Idoate et al, 1996). Concomitant administration of cehphalosporins and aminoglycosides e.g. amikacin may results in an increased risk of nephrotoxicity than use of either drug alone (Bobrow et al, 1972).

1.9. Pharmacokinetics of Drugs Pharmacokinetics is the study and characterization of the time course of drug absorption, distribution, metabolism, and excretion, and the relationship of these processes to the intensity and time course of therapeutic and toxicologic effect of drugs.

1.9.1. Absorption 1.9.1.1 Factors affecting absorption Absorption of drugs from gastrointestinal tract (GIT) is affected by various factors essentially a) Physiological factors. b) Physicochemical factors. c) Pharmaceutical factors.

a) Physiological factors The cell membrane is considered to be composed of lipoprotein structures which act as semi-permeable lipid membranes, so that one major factor is the lipid solubility of the molecule. Drugs which are more lipid-soluble tend to traverse the cell membrane more easily than less lipid soluble or more water-soluble molecules. There are also numbers of physiological transport phenomena, which influence the mechanism by which a drug traverses the cell membrane. Absorption mechanisms can be summarized in the following:

I- Passive diffusion It is the major trans membrane process for most drugs. The driving force for passive diffusion is the difference in drug concentration on either side of the cell membrane. According to Fick’s law of diffusion drug molecules diffuse from a region of high drug concentration to a region of low drug concentration. dQ DAK = ()Cgi − Cp dt h Where: dQ/dt = rate of diffusion. D = diffusion coefficient. K = partition coefficient. A = Surface area of membrane. h = membrane thickness. (Cgi-Cp) = difference between the concentration of drug in the gastrointestinal tract and the plasma. Since the drug is distributed rapidly into a large volume after entering the blood, the concentration of drug in blood will be quite low with respect to the concentration at the site of drug administration. If the drug is given orally, then Cgi >> Cp and a large concentration gradient is maintained, acting as a “maintained driving force” during absorption. Since D, A, K and h are constants under usual condition for absorption. Combined P = permeability coefficient may be defined as: DAK p = h Furthermore in Fick’s law equation, if the drug concentration in the plasma (CP) is << than concentration in GIT, CP will be negligible and the following relationship for Fick’s law is obtained. dQ = p(Cgi) dt This equation is for a first order process. In practice, the extra vascular absorption of most drugs tends to be first order absorption process. Moreover, due to the large concentration gradient between Cgi and CP the rate of drug absorption is usually more rapid than the rate of drug elimination (Gibaldi, 1991).

II- Active transport It is a carrier medicated transmembrane process, which plays an important rule in gastrointestinal tract for many drugs and metabolites. Active transport is characterized by the fact that the drug is transported against concentration gradient i.e. from regions of low drug concentration to regions of high concentration. Therefore this is energy-consuming system. In addition, active transport is a specialized process requiring carrier that binds to the drug to form carrier-drug complex, which shuttles the drug across the membrane and then dissociate the drug on the other side of the membrane. Examples include dopamine, methyldopa some antimetabolites such as methotrexate, 5-fluorouracil, lithium and Iodine (Al-Adhal, 1995).

III- Facilitated diffusion It take place when the intestinal tract transports certain solutes down hill but at rate much greater than would be anticipated based on the polarity of solute and its molecular size (Glibaldi, 1977). Facilitated absorption is usually explained by assuming that carriers in the lipoprotein membranes of the intestinal epithelial cells are responsible for shuttling these solutes in a mucosal-to-serosal direction. The major substances that are believed to be actively transported are sodium, other ions such as calcium and iron, glucose and gluctose, amino acids, bile salts, and vitamin B12. A large number of substances, including other vitamins such as riboflavin, thiamine and certain drugs, are believed to be absorbed by facilitated diffusion. The number of apparent carriers in the intestinal membranes is limited. The rate of carrier-mediated transport is described by the following: V x C Absorption rate = max K m + C C = is the solute concentration at the absorption site

Vmax and Km are constants

At low solute concentration, such that Km >> C V Absorption rate = max C = KC K m Apparent first order kinetics is observed. Under these conditions there is sufficient number of carriers so that a constant proportion of solute molecules presented to the epithelial surface are transported. When

C >> Km

Absorption rate = V max Further increases in solute concentration are not associated with any increase in the rate of absorption (zero-order kinetics) (Gibaldi,

1990).

IV- Pinocitosis (vesicular transport) It is the process of engulfment of large macromolecules like sabin polio vaccine, fats, starch and some vitamins A, D, E and k (Ritschel, 1976).

V- Pore (convective) transport Very small molecules such as urea, water and sugars are able to rapidly cross the cell membrane as if the membrane contained channels or pores. b) - Physiochemical Factors I- Crystal form Many drugs can exist in more than one physical form, a property known as polymorphism. Although the drugs are chemically indistinguishable, each polymorph may differ substantially with respect to certain physical propriety such as density, melting point, solubility and dissolution rate. At any one temperature and pressure only one crystal form will be stable. Any other polymorph formed under these conditions is metastable and will eventually convert to the stable form, but the conversion may be slow. The metastable polymorph is a higher energy form of the drug and usually has a lower melting point, greater solubility and greater dissolution rate than the stable form. Accordingly, the absorption rate and clinical efficacy of a drug may depend on which crystal form is administered. Some drugs also occur in an amorphous form, which shows little crystallinity. The energy required for a drug molecule to be transferred from the lattice of crystalline solid to a solvated one is much greater than that required from amorphous solid. For these reasons, the amorphous form of a drug is always more soluble than the crystalline form e.g. two polymorphs of novobiocin have been identified, the amorphous material is at least 10 times more soluble than the crystalline form (Gibaldi, 1984). Studies in dogs failed to detect any absorption of novobiocin after oral administration of the crystalline solid, while the amorphous form was rapidly absorbed. Chloramphenicol has several crystal forms, and when given orally as a suspension, the drug concentration in the body was found to be dependent on percent β-polymorph in the suspension. The β-form is more soluble and better absorbed in general; the crystal form with the lowest free energy is the most stable polymorph. The other polymorph is metastable and may be converted to the more stable form over time. A change in the crystal form causes cracking in a tablet or even inability for granulation to be compressed to form a tablet. Some drugs interact with solvent during preparation to produce crystalline forms called solvates. Water may form a special crystal with drugs called hydrates, which may have quite different solubility compared to the anhydrous form of the drug. The anhydrous form of caffeine, theophylline and glutethimide dissolve more rapidly than do the hydrous form in water (Gibaldi, 1984). Polymorphous may be stabilized by the addition of viscosity increasing agent in order to retard transition of one polymorph into another (Ritschel, 1976). It has been proven that there is no difference in the extent and rate of the antibiotic absorption with the use of trihydrate and anhydrous ampicillin capsules and ampicillin trihydrate tablets.

II- Surface Area and Particle Size A drug dissolves more rapidly when its surface area is increased, which is usually accomplished by reducing the partial size of the drug. Particle size reduction usually results in more rapid and complete absorption and also increases the bioavailability of the product. Particle size may also be important factor in the bioavailability of many drugs e.g. digoxin powder (widely used in the United Kingdom for tablet manufacture) was found to have a mean particle size diameter of 20 to 30 µm. This material was slowly and incompletely absorbed. Reduction in digoxin particle size by ball milling to a mean diameter of 3.7 µm leads to an increase in the rate and extent of absorption of the drug (Shaw et al, 1974). The influence of both particle size and gastrointestinal motility on digoxin was clinically investigated. In this study, ten healthy subjects received 0.5 mg of digoxin as 2 standard tablets or tablets containing micronized digoxin or large particle size digoxin. The tablets were given 30 minutes. After 15 mg propantheline (which increases the residence time in the small intestine) and 10 mg metoclopramide (which decrease the residence time in the small intestine) or placebo and following an over night fast, the extent of absorption was estimated and these results were obtained: assigning a value of 100 % for the bioavailability or digoxin in subjects who took the micronized tablet. The relative bioavailability of digoxin taken with placebo was 94 % but only 43 % after the large particle size tablets. Propantheline improved the absorption of digoxin form these particles by about 15 % whereas metoclopramide had an effect on the absorption of digoxin after standard or micronized tablets (Johnson et al, 1978). A general principle derived from these studies is that the bioavailability of slowly dissolving drugs may be sensitive to normal variation and other changes in GIT motility. The gastrointestinal absorption of medroxy- acetate from tablets showed two fold increase in the extent of absorption when micronized compared to non- micronized substance was used. In clinical study of pyrivinium pamoate in the treatment of 93 patient infested with pinworms, the cure rate (100 %) for the suspension form of the drug was significantly higher than the cure rate (61 %) for the existing tablet form. In further studies with 209 infested patients, the efficacy of a new tablet formulation in which pyrivinium pamoate particles were less than 10 µm in diameter, in contrast to the 50 to 90 µm diameter of these in the original tablet, was compared to that of suspension cure rates with both tablet and suspension were similar and exceeded 90 %. The effective surface area of hydrophobic drug particles may be increased also by the addition of a wetting agent to the formulation. In an investigation, volunteers received 1.5 g of phenacetine as a fine suspension with and without polysorbate 80, as medium suspension and as coarse suspension. Drug absorption was assessed by determining phenacetin concentration and of the drug metabolites. The maximum phenacetin concentration in the plasma and urinary recovery of its metabolites after the administration of different forms of the drug are presented below (Prescott et al, 1970).

Average plasma Urinary No. Preparation phenacetine (µg /ml) recovery Fine suspension with 1 13.5 75 polysorbate 80

2 Fine suspension 9.6 51

3 Medium suspension 3.3 57

4 Coarse suspension 1.4 48

Polysorbate 80 significantly enhances the rate and extent of absorption of phenacetin, probably by increasing the wetting and solvent penetration of the particles and minimizing aggregation of the suspended particles. Physiologic surface-active agents, like bile salts and lysolecithin, probably facilitate the dissolution and absorption of poorly water-soluble drugs in the small intestine (Miyazaki, et al, 1980). It is clear from the above that the reduction in the particle size and the addition of a wetting agent improve the rate and the extent of the absorption of the drugs which have poor water solubility (Gibaldi, 1991).

III- Drug stability and hydrolysis in the GIT Acidic and enzymatic hydrolysis of drugs in the GIT is not uncommon. Poor bioavailability results if degradation is extensive. The hydrolysis and in activation of penicillin G occurs in less than 1 minute at pH 1.0 and about 9 minute at pH 2.0. The stability of methicillin is comparable. Other penicillins, notably ampicillin, which is considerably more resistant to acid hydrolysis and therefore, results in better bioavailability. The degradation rate of penicillin G decreases sharply with increasing pH. Drugs are generally stable in the small intestine, however, certain prodrugs require hydrolysis to the parent drug in the GIT fluids to produce clinical effects, and e.g. chloramphenicol is sometimes given as a palmitate or stearate ester, particularly in pediatric practice. The low water solubility of these esters minimizes the objectionable taste of the drug base and facilitates its use in oral suspension. However, the esters are poorly absorbed. Adequate drug absorption requires conversion of the prodrug to chloramphenicol base in the small intestine. Clinical studies in children with serious bacterial infections, who were given the palmitate ester, suggested that the conversion is about 70 % complete in average, and results in satisfactory serum levels of chloramphenicol (Pickering et al, 1979).

C) Pharmaceutical factors

I- Adsorption Certain insoluble substances may adsorb coadministred drugs; this often leads to poor absorption. Charcoal is a good example, which significantly reduces both the rate and the extent of drug absorption. Also charcoal has been used for various gastrointestinal tract disorders. More important, charcoal is considered to be an efficient antidote in many drug intoxications. Other adsorptive substances include kaolin-pectin mixture and cholestyramine; significantly reduce the absorption of lincomycin and warfarin (Gibaldi, 1984).

II- Dissolution Dissolution is the process by which a chemical or a drug becomes dissolved in a solvent. In the biological system drug dissolution in an aqueous medium is an important step to the process of systemic absorption. The rate at which drugs with poor aqueous solubility dissolve from an intact or disintegrated solid dosage form in the gastrointestinal tract often controls the rate of systemic absorption of the drugs. In 1897 Noyes and Whitney and other investigators studied the rate of dissolution of solid drugs according to their observations, the steps in dissolution include the process of drug dissolution of the surface of the solid particle, thus forming what is known as the "stagnant layer" which diffuses to the bulk of the solvent from the regions of high drug concentration to the regions of low drug concentration. The overall rate of drug dissolution may be described by the Noyes Whitney equation which resembles Fick’s law of diffusion: − dc DAK = ()Cs − C dt h Where dc/dt = rate of drug dissolution D = diffusion rate constant A = surface area of the particle Cs = concentration of drug in the stagnant layer C = concentration of drug in the bulk solvent K = oil/water partition coefficient h = thickness of stagnant layer. The rate of dissolution, (dc/dt) (1/A), is the amount of drug dissolved per unit area per time (e.g. g/cm2min) it can be seen from the Noyes Whitney equation that dissolution kinetics may be influenced by the physiochemical characteristics of the drug, the formulation and the solvent. A drug in the body, particularly in the gastrointestinal tract, is considered to be dissolving in an aqueous environment. Since dissolution is thought to take place at the surface of the solute, the greater the surface area the more rapid the rate of drug dissolution. The degree of aqueous solubility of the drug also affects the rate of dissolution. Generally, the ionizable salt of the drug is more water- soluble than the free acid or free base, e.g. Novobiocin a weak acid when administered as such or in the form of a salt (Shargel, 1980). The bioavailability of the drug after administration of the sodium salt is twice that of the calcium salt and 50 times that of the free acid (Gibaldi, 1984). Also formulation factors can affect drug dissolution e.g. excipient such as suspending agent increases the viscosity of the drug vehicle and thereby diminishes the rate of drug dissolution of suspension. Tablet lubricant such as magnesium stearate may repel water and reduce dissolution when used in large quantities.

III- Complexation Complexation of a drug in the gastrointestinal fluids may alter the rate and in some cases the extent of absorption. The complexing agent may be a substance normal to the gastrointestinal tract, a dietary component, or a component of the dosage form. Intestinal mucus, which contains the polysaccharide mucin, can avidly bind streptomycin and dihydrostereptomycin. This binding may contribute to the poor absorption of these antibiotics. Bile salts in the small intestine interact with certain drugs including tubocurarine, neomycin and kanamycin to form insoluble, non- absorbable complexes. Incorporation of dicalcium phosphate as filler in dosage forms also reduces the availability of the drug. Furthermore tetracycline forms insoluble complexes with calcium ion. Absorption of this antibiotic is substantially reduced if they are taken with milk, certain food or other sources of calcium such as antacids. Complexation probably occurs often in pharmaceutical dosage forms. Complex formation between drugs and gum, cellulose derivatives, polyols or surfactants is common. The physiochemical properties of these complexes suggested poor absorption. Fortunately, most of these complexes are freely soluble in the fluids of GIT and dissociate rapidly thus in most instances little or no effect on absorption is noted. There are, however some exceptions, amphetamine interacts with carboxy methyl cellulose to form a poorly soluble complex that leads to reduced absorption of the drug (Gibaldi, 1984). 1.9.1.2. The Gastrointestinal tract and drug absorption The major components of the GIT are the stomach, duodenum, small intestine and large intestine or colon. They differ from one another anatomically and morphologically as well as with respect to secretions and pH. The stomach is a pouch-like structure lined with a relative smooth epithelial surface. Extensive absorption of mainly weakly acidic or non- ionized and certain weakly basic drugs can be demonstrated under experimental conditions. However, under normal conditions, when gastric emptying is not impeded, the stomach’s role in drug absorption is much more modest. The absorption of Aspirin from human stomach after oral administration of aqueous solutions to healthy subjects has been estimated to be about 10 % of the dose (Cooke et al, 1970). The small intestinal is the most important site for drug absorption in the GIT. The epithelial surface area through which absorption can take place in the small intestine is extraordinary large because of the presence of the villi and microvilli, finger-like projections arising from and forming folds in the intestinal mucosa. The irregularities in the mucosal surface caused by the villi and microvilli, and submucosal folds increase the area available for absorption more that thirty times than, which would be present if the small intestine is a smooth tube. The effective surface area of the small intestine is about ten times that of the stomach as estimated from studies in rates (Crouthamel, 1971). The large intestine, like the stomach, has considerably less irregular mucosa than that of small intestine. This segment serves as a reserve area for the absorption of drugs that have escaped absorption proximally because of their physiochemical properties or their dosage form e.g. enteric coated tablets and sustained released products. On the other hand the large intestine may play an important role in the efficacy of orally administrated drugs, such as sulphasalazine that require metabolism by intestinal bacterial in the ileum and colon for bioactivities (Gibaldi, 1984). Bacterial enzymes may also be responsible for hydrolysis of glycoside bond of anthraquinone purgatives and the artificial sweetening agent, cyclamate can be converted by a bacterial enzyme to cyclohexylamine. Azobonds in dyes used as coloring agents for food and preparations can also be reduced and split by bacterial enzymes (Bowman, and Rand 1980).

I- blood perfusion of the Gasteroinstinal tract The blood flow to the GIT is important in carrying the drug to the systemic circulation and hence to the site of action. The intestinal area is perfused by the mesenteric blood vessel. The drug is delivered into the liver via the hepatic portal vein and then to the systemic circulation. Any decrease in the mesenteric blood flow as in congestive heart failure will decrease the rate of removal of drugs from the intestinal tract and thereby the rate of bioavailability (Shargel, 1980).

II- Major GIT physiological properties affecting drug absorption It is known than 10 millions-fold and 10 thousands-fold difference in hydrogen ion concentration exist between the stomach, the colon, and the duodenum respectively. The pH at the absorption site is an important factor in drug absorption because many drugs are either weak organic acids or bases. On the other hand it is known that the GIT barrier (as well as many other barriers and membranes in the body) is much more permeable to unchanged, lipid-soluble solute. A drug may be well absorbed from one segment of the GIT, where a "favorable" pH exists, but poorly absorbed from another segment of a less favorable pH. The fraction of the drug in solution that exists in the nonionized form is a function of both the pKa of the drug and the pH of the environment. In theory, weakly acidic drugs should be better absorbed from the stomach than from the intestine, because large fraction of the dose would be in a nonionized, lipid soluble form. However, the limited residence of the drug in stomach and the relatively small surface area of the stomch more than balance the influence of pH in determining the optimal site of absorption. Thus, factors that promote gastric emptying tend to increase the absorption rate of all drugs. The converse is equally true and equally important. Slow gastric emptying can seriously delay the onset of action. Prompt gastric emptying is also important for drugs that are unstable in stomach fluids because of low pH or enzyme activity e.g. the extent of degradation of penicillin G after oral administration depends on its residence time in the stomach fluids. Gastric empty often appears to be an exponential process. Standard low bulk meals and liquids are transferred from the stomach to the duodenum in an apparent first order fashion, with a half-life of 20 to 60 minute in the healthy adult. However, many factors can influence the rate of this process. Gastric Empting is enhanced by: 1- Fasting or hunger. 2- Alkaline buffer solutions. 3- Anxiety and lying on the right side. 4- Hyperthyroidism. 5- Cholinergic drugs such as metoclopramide. 6- Gastric emptying of liquids is much faster than that of solid food or solid dosage forms On the other hand gastric empting is retarded by: 1- Fats and fatty acids in the diet. 2- High concentration of electrolytes or hydrogen ions. 3- High viscosity, bulk or cold beverages. 4- Diseases such as mental depression, hypothyroidism and diseases of the GIT. 5- Anticholinergic drugs such as atropine and propantheline as well as narcotic, analgesics, amitriptyline, impiramine, desipramine, chloropromazine and aluminium hydoxide.

There are numerous examples of the influence of drugs that affect gastric emptying on the absorption rate of other drugs administered concomitantly. Propantheline has been found to significantly reduce the absorption rate of sulfamethoxazole, ethanol, and acetaminophen. On the other hand metoclopramide significantly increases the absorption rate of ethanol, acetaminophen, tetracycline and in most cases there is little effect on the extent or completeness of absorption (Gibbon et al, 1975 and Nimmo, 1975). Propantheline and similar drugs (anticholinergics) significantly increase small bowel transit time, whereas metoclopromide accelerates transit through small intestine so the first drug increases the absorption of riboflavin by more than two fold in healthy subject, hydrochlorothiazide by about one third and nitrofurantion by about 50 % and markedly increases the steady-state serum concentration of digoxin in patients (Manninem, 1973). On maintenance digoxin therapy in another group of patients, the concomitant administration of metoclopromide significantly reduced the steady-state serum concentration of digoxin. The effect of propantheline and metoclopramide on digoxin levels in serum are the result of changes in the extent of absorption of digoxin.

1.9.1.3. Effects of Food on drug Absorption In general gastrointestinal absorption is favored by an empty stomach. However, one should not give all drugs on empty stomach; some are irritating to the stomach and should be administered with or after a meal to reduce adverse effects. The absorption rate but not the extent of absorption of most drugs is reduced in the presence of food. Examples include digoxin, acetaminophen, sodium and various sulfonamides. The effect of food on absorption rate of drugs is probably the result of a delay in gastric emptying. Administration of certain antibiotics after meal frequently results in a significant decrease in both the rate and extent of absorption. This has been observed with , penicillin, cephalosporins, lincomycin and . Food seems to have less effect on the absorption of doxycycline than on that of tetracycline. Studies in healthy human subjects indicated that serum levels of tetracycline are uniformly reduced by about 50 % by various test meals where as doxycycline are reduced by only 20 %. The absorption of tetracycline is also markedly reduced when they are taken with milk or milk products. Presumably because of the interaction of the drug with calcium resulting in poorly absorbed complex (Rosenblatt, 1966). The absorption of , an a gent, is considerably impaired when it is given after a meal. Also there are different studies on as a base or its ester. These studies indicated that food markedly impaired the absorption of erythromycin stearate but had no effect on the absorption of erythromycin base (Welling, 1978). The absorption of few drugs is actually promoted when administered following meals. The absorption of riboflavin is significantly greater than usual when the vitamin is given after standard breakfast as shown below (Levy, and Jusko, 1966).

Dose (mg) Percent of absorbed Fasting Non fasting

5 48 62 10 30 63 15 16 61

The absorption of griseoflavin can be doubled under certain condition by administering the drug after a high fat content breakfast. The bioavailability of chlorothiazide is doubled when taken immediately following a meal compared to that in fasting subjects (Crounse, 1961) .The administration of nitrofurantoin in commercial capsule macro crystalline drug after standard breakfast result in more complete absorption of the antibacterial agent compared to that obtained after administration to fasting subjects. The effect of food however is much more pronounced with the macro crystalline form of the drug. Clinical investigations convincingly demonstrated that the bioavailability of certain drugs subject to first-pass hepatic metabolism during absorption, is increased after meal.

1.9.1.4. Mal Absorption Mal absorption may be defined as any disorder with impaired absorption of fat, carbohydrate, protein, vitamins, electrolytes, minerals and water. Drug-induced mal absorption has been observed after administration of different drugs including neomycin, , aminosalicylate and certain anti neoplastic agent such as methotrexate (Rahman et al, 1973). 5-fluorouracil (5-FU) damages the gastrointestinal epithelium and impairs the function of the mucosa to serve as a barrier to large polar molecules. The absorption of polyvinylpyrolidine (PVP) and tobramycin, both of which are poorly absorbed, is substantially enhanced in patients receiving a course of (5-FU) therapy. Also surgical resection of the small intestine can result in impaired absorption of digoxin as well as of other drugs. Reduced bioavailability of digoxin was found in 5 of 9 patients who had undergone jejunal by-pass surgery. The bioavailability of hydrochlorothiazide is reduced by 50 % in patients who had under gone intestinal shunt operation for obesity (Gibaldi, 1984). Furthermore, previous study indicated mal absorption of oral antibiotics in gardia lambia infection e.g. Amoxicillin, Cephalexin, Cephradine (Hugh et al, 1987).

1.9.1.5 Bioavailability In order that a drug can exert a pharmacological effect it must reach the general circulation in an active form. Bioavailability is defined as the rate and extent of absorption of a drug from specific dosage form e.g. tablets, capsules, suspensions…etc) which enters the systemic circulation. It may be in therapeutic level for low therapeutic levels because the absorption is incomplete or because the drug is metabolized in the gut wall or liver before reaching the systemic circulation (Magaret et al, 1982). Bioavailability is affected by the same factors affecting gastrointestinal absorption plus metabolism in the gut and liver. Examples of drugs that undergo extensive first-pass effect in the gut wall include isoprenaline, dopamine and , and those that undergo extensive first-pass effect in the liver include propranolol, aspirin and imipramine (Al-Adhal, 1995).

1.9.1.6 Assessment of bioavailability 1. Blood level data. 2. Urinary Excretion data 3. Pharmacologic data 4. Clinical data. The most commonly used method of assessing the bioavailability of a drug involves the construction of a blood or plasma concentration- time curve. Two pharmaceutical products or more are considered bioequivalent if there are no significant differences in their AUC, Cmax and Tmax. Other methods include drug urinary excretion data which is used in this study. Here, two pharmaceutical products or more are considered bioequivalent if there are no significant differences in their maximum peak of excretion (MPE), time to each the peak (TTP) and percentage dose of excretion (PDE %). Whenever possible blood level studies should be carried out and are preferable to all other studies. If such studies are not feasible they can be substituted by urinary excretion studies (Ritschel, 1980). Ali (1981) investigated the bioavailability of , following oral administration of two commercially available products to healthy volunteers. The rate and extent of bioavailability were determined using urinary excretion method. Bacampicillin was excreted as ampicillin. The two brands were found to be bioequivalent. Ali (1985) also described ampicillin bioavailability following oral coadministration with chloroquine. The combined actions of chloroquine on the gastrointestinal tract reduced the rate and extent of ampicillin bioavailability. The study concluded that the two drugs are to be administered separately at different times with not less than 2.0 hrs in between. Ruiz (2000) carried out a pharmacokinetic and absolute bioavailability study of oral axetil in the rat. The result of the study indicates that the first pass effect in the intestine and liver reduce oral bioavailability when the drug is administered orally. Cefuroxime bioavailability after oral and IP administration estimated from the plasma levels was nearly 24 and 75% respectively.

1.9.2. Distribution Distribution is the transfer of drug from blood to the extravascular fluids (extracellular and intracellular) and tissues. Drug molecules are distributed throughout the body by means of the circulating blood. The entire blood volume (6 L) is pumped through the heart each minute, within minutes after drug enters the bloodstream it is diluted into the total blood volume. A drug that is restricted to the vascular space and can freely penetrate erythrocytes has a volume of distribution of 6 L. If the drug cannot permeate the red blood cells (RBCs), the available space is reduced to about 3 L (plasma volume). All drugs easily cross capillaries and are rapidly diluted to a much larger volume, the extracellular space. Capillaries, except those in the brain, are more like filters than lipid membranes in term of permeability. Drugs with molecular weights of up to 500 or 600 Daltons quickly diffuse out of the vascular system and reach the interstitial fluid bathing the cells. Drugs concentration in body fluids depends on the degree of drug binding in the fluid. Drug concentration in extracellular fluid (ECF) is frequently less than that in plasma, because the ECF has a lower albumin concentration than plasma (Gibaldi, 1991). The volume of distribution depends on many factors such as blood flow rate in different tissues (between < 2 to > 500 ml blood / 100 ml tissue/minute), lipoid solubility of the drug, partition coefficient of drug and different types of tissue, pH, and binding to biological material and body weight (Ritschel, 1980).

1.9.3. Metabolism Drug metabolism or biotransformation refers to the biochemical (enzymatic) conversion of a drug to another chemical form. Many tissues in the body are capable of metabolizing drugs, but most drugs are mainly metabolized in the liver by enzymes localized in hepatic microsomes. Drug metabolizing enzymes oxidize, reduce, hydrolyze, or conjugate compounds. Reduction, oxidation and hydrolytic reactions result in metabolites with functional groups (e.g. hydroxyl, amine, or carboxyl) that can be conjugated. In man most common conjugations of drugs or metabolites occur with acetate, sulfate, glycine and glucuronic acid. Many drugs, as will as steroidal hormones are oxidized by the microsomal system. Oxidation of certain drugs, such as xanthines, may be catalyzed by non-microsomal enzymes; mercaptopurine and azothioprine are examples. This process of metabolism does not mean the end of the product which is removed from the body via fecal or renal excretion, but in some cases it produces active metabolites e.g. prednisone and cortisone are reduced to active metabolites, prednizolone and hydrocortisone respectively. Sulfasalzine is also cleaved by intestinal bacteria to form aminosalicylate, the active component, and sulfa pyridine (Gibaldi, 1984). Glucuronide formation is the most common conjugation process of drug metabolism. It involves the reaction between uridine diphosphate glucuranic acid and drugs containing hydroxyl, carboxyl or amino groups. Also glucuronides are water-soluble acids that are easily excreted in urine and bile. Some ester glucuronides are labile and can be hydrolyzed in urine or plasma to the parent drug. High blood levels of clofibrate in patients with renal disease are the result of accumulation and hydrolysis of the glucuronide conjugate in plasma. Generally the metabolism of drug decreases its lipid solubility by making it more polar and therefore more hydrophilic there by facilitating its excretion. Drug, which is water-soluble, but not lipid soluble are usually more excreted unchanged in urine and have short durations in the body. On the other hand, drugs that are lipid-soluble, which are not metabolized stay for a longer time in the body. Striking examples being suramine and isophenoxic acids (Bowman, 1980).

1.9.4. Drug excretion The renal excretion of a drug is a complex phenomenon involving glomerular filtration, active tuber secretion and passive reabsorption.

I- Glomerular filtration The kidneys receive about 25 % of the cardiac output or 1.2 to 1.5 L of blood per minute. About 10 % of this volume is filtered at the glomeruli; therefore about 130 ml of plasma water is filtered each minute. Although the pores of the glomerular capillaries are sufficiently large to permit the passage of most drug molecules, the glomeruli effectively restrict the passage of blood cells and plasma proteins. Accordingly, only the free drug can be filtered. Although about 180 L of protein free filtrate pass through the glomeruli each day, only about 1.5 L is excreted as urine, the remainder is reabsorbed in the renal tubules. This often results in high urinary concentrations of certain solutes, including drugs that are not similarly reabsorbed. Many drugs, however, are efficiently reabsorbed from the distal portion of the nephron. In most instances, tubular reabsorption of drug is a passive phenomenon. Non - ionized, lipid-soluble drugs are rapidly and extensively reabsorbed, whereas polar compounds and ions are unable to diffuse across the renal epithelium and excreted in urine. For drugs that are principally eliminated by renal excretion, the more efficient the reabsorption of the drug, the longer is its biological half-life (Gibaldi, 1984).

II- Tubular secretion Tubular secretion is an active transport process whereby the drug diffuses against a concentration gradient from the blood capillaries across the tubular membrane to the renal tubules. This active process accounts for the fact that certain drugs like , although extensively bound to plasma protein and not subject to hepatic metabolism are rapidly eliminated. Plasma protein binding does not affect the rate of tubular secretion because there is rapid transport of unbound drug and rapid dissociation of the drug protein complex. The secretion process shares many of the characteristics of the specialized transport (absorption) systems of the intestine. This process exhibits some degree of structural specificity. Transport systems specific for organic acids (thiazide diuretics) and organic bases (e.g. triametrene) have been identified. Another similarity of tubular secretion to active intestinal absorption is competitive inhibition of one drug by another. This characteristic has been used to prolong the half - life of drugs like penicillin that are eliminated to a considerable extent by tubular secretion. Probenicid, a weak organic acid, competitively inhibits the tubular secretion of penicillin G and other penicillins reduce the rate of urinary excretion. Probenicid has been used clinically to increase the duration of action of penicillins. Parenteral penicillin G or ampicillin, in high doses with probenicid, is considered to be an effective treatment for gonorrhea (Gibaldi, 1991).

III- Tubular reabsorption Most drugs are subject to tubular reabsorption, which is usually a passive process. Tubule membranes favor the transport of lipid-soluble drugs. Compounds that are poorly lipid-soluble of ionized drugs are poorly reabsorbed. The reabsorption of drugs that are weak acids or bases depends on the pH of the tubular fluids. The pH of fluids in proximal tubules approximates that of the plasma (7.4) whereas the pH in the distal tubules approximates that of urine, which may vary from 4.5 to 8.0, on average urine pH is 6.3. Urine pH is affected by diet, drugs and condition of the patient. Some drugs like acetazolamide and sodium bicarbonate produce alkaline urine; ammonium chloride and ascorbic acid produce acidic urine. According to the pH-partition hypothesis, acidification of urine promotes the renal reabsorption of weak acids but retards the reabsorption of weak bases. Renal clearance of weak acids is increased if urine is made alkaline because more drugs are in the ionized form and can not be reabsorbed. On the other hand, the renal clearance of weak bases is decreased in alkaline urine but may be increased dramatically if the urine is acidified. The influence of pH on tubular reabsorption also depends on the pka of the drug. Relatively strong acids or bases are virtually completely ionized over the entire range of pH and undergo little reabsorption. Generally, Perry et al (2001) described the clinical use and pharmacokinetics of which is a third generation cephalosporin that is used for a variety of infections such as meningitis, gonorrhea and community acquired pneumonia. The most important aspect of its pharmacokinetics includes a long half life, excellent tissue penetration and saturable serum protein binding. Ehinger (2002) determined the pharmacokinetics of cephalexin from two oral formulations in dogs. Cephalexin from the tested preparations reached a mean area under the plasma concentration time curve of 115.3 and 102.4 micrograms h/ml, respectively. The plasma concentration decreased rapidly with a mean half life of 1.4 hrs.

1.10. Bioequivalence Equivalence is more a relative term that compares one drug product with another or with a set of established standards. Equivalence may be defined in several ways: - Chemical equivalence: Indicates that two or more dosage forms contain the labeled quantities (Plus or minus specified range limits) of the drug. - Clinical equivalence: Occurs where the same drug from two or more dosage forms gives identical in vivo effects as measured by a pharmacology response or by a control of symptoms or disease. - Therapeutic equivalence: Implies that one structurally different chemical can yield the same clinical result as another chemical. Bioequivalence indicates that the drug in two or more similar dosage forms reaches the general circulation with the same relative rate and the relative extent (Shargel and Andrew, 1980).

1.11. Measurement of cephradine in biological fluids Measurement of cephradine and other cephalosporins levels in biological fluids has been described using several analytical methods. Cowdrey et al, (1998) and Lin et al (2000) used electrophoresis technique in their determinations. Cephradine was determined microbiologically as shown by Bretchneider et al, (1999). A UV radiation method has also been described (Pandey et al, 2002). Steppe et al (2002) carried out a determination of cephalexin in oral suspensions by micellar electrokinetic chromatography. The method showed good selectivity and was found to useful for the study of cephalexin stability in pharmaceutical preparations.

High performance liquid chromatography (HPLC) has been used extensively for the analysis of antibiotics in general and cephalosporins in particular. Johnson and his associates (2000) determined cephalosporin antibiotics, including cephradine, in human plasma by HPLC with ultraviolet detection. Cephradine and cephaloridine (internal standard) were extracted from human plasma by perchloric acid protein precipitation followed by centrifugation and the extracts were analyzed by HPLC. This method has been successfully applied to clinical studies including an oral bioequivalence study. Gallo et al (2002) carried out a comparison of several methods used for the determination of cephalosporins. The precision of UV absorbance of intact and acid degraded cephalosporins, ninhydrin were compared with HPLC and iodometric methods used for analysis of , , cephazolin and cephalexin. Their results showed that the HPLC technique is the most sensitive and precise method. Abreu and his coworkers (2003) used HPLC for the determination of beta lactam antibiotics (Amoxicillin) following a comparative bioavailability study in healthy volunteers after a single dose administration. The study concluded that the validated HPLC method employed was proved to be simple, fast, reliable, selective and sensitive enough to be used in clinical pharmacokinetic studies of amoxicillin in humans. The study showed that an amoxicillin 500 mg capsule was bioequivalent to reference standard product capsules 500 mg, in term of their rate and extent of absorption. According to the above and supported by other authors (Farag, 1998; Hassanzadeh et al, 1999; Jandik et al, 2002 and Kai et al, 2003) the HPLC method was considered to be the best method for the determination of cephalosporins in biological fluids. It is more sophisticated, very sensitive, highly selective and precise compared to other methods.

1.12. Clinical uses of cephalosporins Mattila (1982) treated 37 patients with acute pulmonary or urinary tract infection with intramuscular cephradine for 5 days followed by oral cephradine for 2 weeks in doses of 0.5 to 1 g, 4 times a day. Cephradine was used plus metronidazole in the prophylaxis of post-operative infection in vascular surgery (Kester et al, 1999). Cephradine was shown to be effective in the treatment of acute pyelonephritis in pregnancy (Ovalle et al, 2000). Hasui et al (2001) carried out studies of clinical and bacteriological effects of ceftriaxone in pediatric patients with respiratory infections and suggested that a once daily regimen of ceftriaxone is useful. Singh and his coworkers (2001) described that penicillins and cephalosporins continue to be a mainstay of antimicrobial therapy because of their broad spectrum of activity, clinical efficacy and favorable tolerability profile. Scott et al, (2001) described an updated review of the use of cefuroxime in the management of bacterial infections against several Gram-positive and Gram-negative organisms. Geroulanos in 2001 described the use of cephalosporins in surgical prophylaxis. Cephalosporins are considered the drugs of choice because they offer fewer allergic reactions. , a first generation cephalosporin has been widely recommended with success. From the second generation cephalosporins; cefuroxime, and cefoxitin are increasingly recommended. However, their antistaphylococcal activity is less strong but their activity against Gram-negative bacteria is stronger. In addition cefoxitin has good activity against anaerobes. Third generation cephalosporins such as cefotaxime, , ceftriaxone, and are the most commonly used drugs in surgical prophylaxis. Pelaez et al (2003) described the uses of penicillins and cephalosporins in upper respiratory tract infections on patients under 16 years old and decided to support specific campaign for rational use of antibiotics. Duke and associates (2003) described the use of ceftriaxone as the first line treatment effective in reducing meningitis.

1.13. Khat 1.13.1. Historical background Khat was probably known and used on the Ethiopian uplands, where it seems that it originated in very ancient times. However it is impossible to fix accurately its original habitat and the region where question seems to be in the first half of it first developed. The first historical reference, as far as could be determined from the literature consulted occurs in a medieval Arab manuscript (ms. 143 Bibloiotheque rationale, Paris) where it is stated that king of Ifat, Sabr-ad-din decided to plant Khat in the town of Marad in the period of the fourteenth century. According to Rochet, Sheikh Abu Zebin introduced Khat from Ethiopia into the Yemen in 1424. Another reference to its cultivation, in the fourteenth century in the region of Aden and Yemen, is found in a sixteenth century Arab chronicler, Abdul-kader. Its cultivation in that region is thought to be earlier than that of coffee. Khat was not known to the scientific world until the end of the eighteenth century. During an expedition organized by King Fredrick V of Denmark, the physician and botanist Peter Forsskal collected, among many other plants, specimens of Khat, which is described under the name of catha edulis family celastaceae. The only survivor of the five members of the expedition, the Hanoverian geographer Karsten Niebular, published a botanical paper in 1775, and in memory of his friend called catha edulis (Catha edulis Forssk) (ODCCP, 1956). The total cultivated Khat area, in the Republic of Yemen, is 11.75% of the total cultivated land. The chewing persons in the ROY are determined as 60% between male and 34 % between female (Abdul Rab, 2000). Catha edulis, or Khat, is attractive glossy evergreen plant that can reach a height of 20 feet. Khat is more than a psychotropic plant. In Yemen, Ethiopia and east Africa it is the basis of a life style and plays a dominant role in celebrations, marriages, and political meetings. Leaves are used in Ethiopia, Somalia, and Yemen to make tea or chewed for their stimulant properties.

1.13.2. Pharmacological action of the khat on the gastrointestinal tract The Khat causes gastritis, colitis, and constipation. It also causes a hypotonic and a-tonic stomach probably due to the sympathomimetic action of khatamines, which inhibits the peristaltic movement and delays the emptying time of the stomach (Abdul Rab, 2000). As has been previously mentioned, the leaves of Khat contain cathinone, cathine and nor pseudoephedrine. Khat leaves also contain tannins (7-14 %, Bowman, 1980).

1.13.3. Tannins Tannins are high molecular weight poly phenols that have the ability to form insoluble complexes with proteins. They tend to interfere with utilization of dietary proteins and, therefore, with digestion process by in activation of enzymes in the alimentary canal.

1.13.3.1. Physicochemical properties of tannins They are non-crystalizable molecules, which form colloidal solution with water. They possess an acidic reaction and a sharp astringent taste. Any polyphenolic compound that has the ability to precipitate proteins from aqueous solution can be regarded as tannin. However, it is often specified that tannins range in molecular weight from 500-3000 Dalton. It is generally agreed that tannins precipitate proteins because they contain a number of functional grumps (at least two) which complex strongly with two or more protein molecules building up a large cross- linked protein-tannin complex (Uro 1992).

1.13.3.2. Pharmacological properties of tannins Tannins are locally acting substances that precipitate proteins but have so little penetrability that only the surface of the cell is affected. Consequently the permeability of the cell membrane is greatly reduced, but the cell itself remains viable. Tannic acid was formerly used orally for symptomatic treatment of diarrhea, topically for management of extensive burns and rectally for various rectal disorders. The longer time which is thus allowed for absorption of fluid results in the formation of hard stool. The dry rough sensation in the mouth and throat experienced when tannic acid is swallowed is a result of the superficial precipitation that occurs (Uro 1992). Several studies regarding the physicochemical, medical, pharmacological and social aspects of Khat and its effect on the pharmacokinetics and bioavailability of different drugs has been published. Hattab et al (2000) described fluoride content in Khat chewing leaves. The total fluoride was found to be 0.93 microgram fluoride /gram in dried leaves, and 2.07 microgram fluoride /gram in ash.

Al-Motareb and his group (2002) described pharmacological and medical aspect of Khat and its social use in Yemen. They showed that cathinone gives the same actions as amphetamine which are central stimulation action, cardiovascular effects, increase of blood pressure and heat rate. They concluded that the risk of chewing Khat may increase the incidence of acute myocardial infarction. Hassan and coworkers in 2002 carried out a study on the effect of chewing Khat leaves on human mood. Khat chewing did result in functional mood disorder and this effect caused by sympathomimetic action of cathinone on the central nervous system. In 2003 Saif carried out an investigation on the effect of Khat on plasma glucose and C-peptide in both type 2 diabetics and non diabetics. Chronic Khat chewing does not affect serum glucose and C-peptide in healthy individuals while it increased glucose and C-peptide level during the Khat session in diabetic individuals. Tonnes and associates (2003) assessed the pharmacokinetics of cathinone, cathine and norephdrine after the chewing of Khat leaves. The study concluded that cathinone was eliminated from the central compartment with a mean half-life of 1.5 ± 0.8 hours and the half-life of cathine was 5.2 ± 3.4 hours. The pharmacokinetics of Khat alkaloids in humans explain why chewing is preferred to ingestion of Khat. Subject absorbed a mean dose of 45 mg of cathinone and did not suffer any severe adverse reactions. Al-zubairi et al (2003) determined the effect of Khat chewing on plasma lipid peroxidation. The plasma triglycerides, total cholesterol and LDL-cholesterol were shown to be not significantly affected by Khat. Murugan and coworkers (2003) carried out an investigation on the effect of Khat chewing on gallbladder motility. There was no significant changing in gallbladder motility after chewing the Khat.

1.14. Drug-Food interactions

Drug-food interactions were documented in several reports. Cambria (2002) studied the effect of soymilk on warfarin efficacy. The study concluded that soymilk caused decline in levels in warfarin treated patient. Schmidt, et al (2002) described the effect of food on bioavailability of different drugs. Some drugs interact with food and reduce its bioavailability. Such interactions are frequently caused by chelating with components in food as (alendronic acid, clodronic acid, didanosine, etidronic acid, penicillamine, tetracycline, ciprofloxacin and norfloxacin) or by other direct interactions between drug and certain food components (avitriptan, , itraconazole solution, levodopa and mercaptopurine). In addition the physiological response to food intake in particular gastric acid secretion may reduce the bioavailability of certain drugs (ampicillin, azithromycin, erythromycin and ). For other drugs concomitant food intake result in an increase in drug bioavailability because of a food induced increase in drug solubility (albendazole, atovaquone, griseofulvine, and mefloquine). Shepard et al, (2002) estimated the effect of food on the disposition of oral 5-fluorouracil in combination with eniluracil. Administration of food with oral 5-FU and eniluracil slowed absorption of 5-FU and decreased 5-FU Cmax but no effect on AUC was reported. A formulation dependent food effect was demonstrated for a modified release preparations. The study showed that ® the bioavailability (AUC and Cmax values) was lower for Coral than Adalat® in the fasted state. Under fed conditions differences in bioavailability between both products were markedly increased (Schug, 2002). Fruit juices inhibit organic anion transporting polypeptide (OATP)-mediated drug uptake and decrease the oral availability of fexofenadine. The study concluded that fruit juice and constituents are more potent inhibitors of OATPs than P-glycoprotein activities, which can reduce oral drug bioavailability (Dresser et al 2002).

1.14.1. AL-Sayadeyah

It is a common Yemeni dish taken at lunch especially in the south and west of Yemen. It is a cooked mixture containing fish, potatoes, and tomatoes, which is eaten with bread. The type of fish used in this study is Tuna called in Yemen (Thamad). In the present study, Al-Sayadeyah effect on bioavailability of cephradine was investigated.

1.15. β-lactam antibiotic distribution in the Republic of Yemen Antibiotics are extensively used in the ROY. During the past years antibiotics consistently occupied the number one position in the country’s pharmaceutical imports. Antibiotics represent 14 - 24 % of the total expenditure of pharmaceutical supplies as described in the official annual reports of the Supreme Board of Drugs and Medical Appliances (SBDMA) of the Minister of Health, Sana’a, for the years 1995-2002 (table 1.1). The SBDMA registration records for pharmaceutical products show that, antibiotics are a leading therapeutic category. Ampicillin, amoxicillin, cephalexin, and cephradine are the top four antibiotic constituting 24 %, 34 %, 21 % and 19 %, of the total value of antibiotic imports, respectively. The four antibiotics are manufactured by 16 - 39 companies consisting of Non-Research Based form the 3rd world (NRB, 61 %), Research Based from developed countries (RB, 30 %), and Local Manufacturing from ROY (LM, 9 %, Al-Mekhlafi, 1998). Extensive use and high demand for antibiotics in the ROY is rather a result of irrational utilization. Irrational utilization of antibiotics involves distribution, dispensing, prescribing and patient’s compliance. However, under the present situation, it is rather difficult to assess or evaluate the consumption of this agent and/or relate it to actual needs. Demand, in this situation is to a great extent an artifact of actual needs. Table 1.2 illustrates availability of cephradine in the local market. As can be seen in the table a total number of 11 countries are involved in cephradine imports. 21 brands of cephradine different dosage forms were registered in the Yemeni market. 16 companies manufacture these 21 brands.

Table 1.1 Percentage of imports of antibiotics to the total imports of drugs Year Antibiotic imports Total imports Percentage (Yemeni Riyal) (Yemeni Riyal) 1995 258023477 1460509764 18% 2000 2229529893 9286148583 24% 2001 3051316666.71 17019415977.82 18% 2002 3047483175.56 22157414260.41 13.7%

1 US Dollar = 63 Yemeni Riyal in 1995 1 US Dollar = 160 - 170 Yemeni Riyal in 2000 - 2002

Table 1. 2 Distribution of cephradine available in the market of Yemen

Origin Number

Countries 11

Companies 16 Brand name 21 2. Aims and Objectives

2.1. Aims Yemen is an open market for more than 300 pharmaceutical companies, so there are always different brands for the same drug in the local market. Bioequivalence studies are the only criteria by which we can determine the best bioavailability order for the different brands of the same drug. By continuous assessment of drug bioavailability, the quality control would play an important role in the protection of population against substandard drugs and promote rational utilization of drug s in the country. Most people in Yemen chew Khat more than 6 hours per day. Concomitant administration of food and other drugs are among the factors that can alter bioavailability of drugs. Therefore the effect of chewing Khat on bioavailability of drugs is very much needed. In addition, studies on drug-local food interactions are of paramount importance. The aim of this project is to provide information regarding the bioavailability of different brands of cephradine marketed locally and to assess the effect of chewing Khat and concomitant food intake on their bioavailability. Another important aim of this project is providing advanced training opportunity in bioequivalence studies on an area which is not available and very much needed in the ROY.

2.2. Objectives a) To investigate the comparative bioequivalence of four cephradine brands available in the market of Yemen. b) To assess the effect of chewing Khat on rate and extent of cephradine absorption. c) To determine the effect of common Yemeni food (Al-Sayadeyah) on bioavailability of cephradine. 3. Materials and Methods

3.1. Subject and study site The study was carried out in the Faculty of Pharmacy at Sana’a University starting on June 2001 and completed in Modern Pharma. Factory in Yemen October 2002. Eight healthy, adults, male Yemeni volunteers participated in the trials of cephradine. They are of comparable age 25.8 ± 1.64 years (range 22 – 27 years) and weight 57.4 ± 9.95 kilograms (range 50 – 80 kg). Demographic data of the subjects are summarized in table 3.1.

Table 3.1. Demographic data of the volunteers Body Weight Age No Code (Kg) (Years) 1 M 58 27 2 N 52 25 3 J 47 26 4 R 80 25 5 W 53 22 6 H 50 27 7 T 65 27 8 K 54 27 Mean ± SD 57.4 ± 9.95 25.8 ± 1.64

None of the subjects takes alcohol or smoke cigarettes. They did not take cephradine or any other medication during the trials. Chewing the Khat was not allowed, before and during the trials. No abnormalities were found according to the physical and laboratory tests. The volunteers were given a full explanation of the purpose of the study, procedures, duration, discomfort, confidentiality. Informed consent forms were signed by all the volunteers before participation in the study (Appendix D).

3.2. Study design The studies were carried out as an open randomized four-way crossover trials in 8 healthy subjects (table 3.2.).

Table3.2. Study design and treatment schedule

No of Period volunteers I II III IV 1 A B C D 2 B C D A 3 C D A B 4 D A B C 5 A B C D 6 B C D A 7 C D A B 8 D A B C

3.3. Clinical protocol Four different brands of cephradine capsules were used in the different bioequivalence studies as shown in table 3.3. Brand A was used as a reference standard and brands B, C and D as test products. The standard product A was used for Khat and food-interaction studies. Each product was administered as single dose of 500 mg capsules on an empty stomach, preceded by an over night fasting, with 250 ml drinking water. Breakfast was allowed 2 hours post dosing. Uniformity of meals was ensured through out all the trials and a wash out period of one week was ensured between the trials. No drugs were taken during the trials period, also Khat chewing was not allowed a day before and during the trials. The bladder was completely emptied just before taking the antibiotic and at each sample collection. Urine samples were collected at 0.0 hour before dosing and at 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, and 12.0 hours post dosing. The total volume of urine voided at any time is recorded and about 20 ml were transferred into stoppered glass bottles and store at 4 C° awaiting analysis. Following each sample collection the volunteers were instructed to drink 250 - 500 ml of water to induce diurisis. Urine samples were allowed to attain room temperature prior to analysis, which was carried out 24 hours post drug administration. The antibiotic urinary levels were measured by HPLC system. The same protocol was used for Khat and food interaction trials except that the design was a two-way cross-over design.

Table 3.3. Cephradine (500 mg) brands used in the study.

Date Manufacturing Batch Code Country Manufacturing Expiry No. date date A* U.K 5/2002 5/2005 2E091 B UAE 3/2001 3/2004 105 C Korea 8/2002 8/2004 208001 D Yemen 5/2002 5/2005 02018

* Reference product

3.4. Khat Constituents Khat leaves contain psychoactive ingredients known as cathinone, which is structurally and chemically similar to d- amphetamine, and cathine a milder form of cathinone. Fresh leaves contain both ingredients. Leaves left unrefrigerated beyond 48 hours would contain only cathine. Cathinone the most potent active principle of Khat is chemically unstable. Khat also contains tannin (7 to 14% by weight of the dried leaves) vitamin C (150 mg/100 g of fresh leaves),(Kalex, 1986) minute amounts of thiamin, niacin, riboflavin and carotene as well as iron and amino acids. Khat contents are summarized in the table 3.4(Al-Mekhlafi 2003).

Table 3.4. Khat contents

Content Percent

Tannins 7-14% Cathine Variable quantities Cathinone Variable quantities Ascorbic acid 136-324/100 gm Minerals 11% Water content in twigs 60-66% Water content in leaves 8.4-9.2%

3.5. Chemical Composition of Fish used in the studies

The fish (Tuna) contains proteins, fat and water; these contents produce high energy values. Tuna fish is also rich with most of minerals and vitamins (tables 3.5 - 3.7).

Table 3. 5. Major components of tuna fish

Species Scientific Water Fat % Protein % Energy value name % calories/Ib Tuna Thinness 71 % (4 - 1 ) % 25.2 630 (Thamad) sp

Table 3.6. Mineral components of tuna fish

Average value Mineral Range (mg/l00 g ) (mg/100 g) Sodium 72 30 - 134 Potassium 278 19 - 502 Calcium 79 19 - 881 Magnesium 38 4.5 - 452 Phosphorus 190 68 - 550 Sulphur 191 130 - 257 Iron 1.55 1 - 5.6 Chlorine 197 3 - 761 Silicon 4 ------Manganese 0.82 0.0003 - 25.2 Zinc 0.96 0.23 - 2.1 Copper 0.20 0.001 - 3.7 Arsenic 0.37 0.24 - 0.6 Iodine 0.15 0.0001 - 2.73

Table 3.7. Vitamins A, D, C and E components of tuna fish

Specie Vitamin A I.U. / Vitamin D Oil Vitamin C Vitamin E s g I.U. / g content% mg / 100 µg / 100 g g

Tuna 40000 - 800000 16000- 10-35% 3 mg/100g 12 µg/100g (Thamad) 30000

All the above information about the fish constituents were obtained from FAO Organization researches (Murray and Burt, 2001).

3.6. Cephradine-Khat studies Reference product cephradine (Brand A, Velosef®) was given to all subjects. 500 mg capsules were administrated orally under fasting conditions and immediately before starting chewing the Khat. The style of the Khat used in this study is known, as Khatal. The origin of this Khat is from Arhap, Sana’a. Equal amount of the Khat was given to each volunteer.

3.7. Cephradine Al-Sayadeyah studies 500 mg capsules of reference product cephradine (Brand A, Velosef®) were given orally under fasting condition and immediately after administration of the common Yemeni food (AL- Sayadeyah).

3.8. HPLC analysis of cephradine 3.8.1. Chemicals 1. Acetic acid anhydrous, 0.7 N (Merck, India). 2. Sodium acetate, 0.5 M (Merck, India). 3. Methanol, HPLC grade (Merck, India). 4. Cephradine-reference standard powder (ACS DOB FAR, Italy).

3.8.2. Instruments 1. High performance liquid chromatography system (HPLC) system consisted of:

a. Two Pumps. b. UV - VIS detector. c. Oven. d. Degasser. e. System controller.

f. Column C18, length 15 cm and diameter 4.6 mm. g. Printer. h. Computer. All these parts are from Shimadzu, (Kyoto, Japan). 2. pH meter , Jenway company (U.K) 3. Unit filtration (Milli pore) Sartorius company (U.K). 4. Ultrasonic bath (Osworld, India). 5. Explorer (Ohaus) balance. Sartorius Company, (U.S.A.) 6. Distilled water system, (U.S.A). 7. Laboratory oven (250 ºC), (Osworld, India). 8. Scientific calculator Casio model FX– 350TL (China).

3.9. Method of analysis Previous studies used spectrophotometric methods to determine the amount of cephalexin excreted unchanged in urine (Ali, 1981; Al- Mekhlafi, 1998 and Rodenas, 1997). However, the method used in these studies is the HPLC method which is more sophisticated and more sensitive compared to the previously used spectrophotometric methods. This method is described in USP (2000) and established by George et al (1997). 3.9.1Chromatographic conditions

Column: symmetry C18, length 15 cm and diameter 4.6 mm. Mobile phase: water, methanol, 0.5M sodium acetate, and 0.7N acetic acid (782:200:15:3). The mobile phase was filtered through a filtration unit (Milli pore), 1µm fine porosity filter and degassed before use. Detection: UV at 234 nm. Flow rate: 2 ml / min. Temperature: 40 °C Injection volume: 100 µl. Fresh mobile phase was prepared daily. The HPLC system was washed for 0.5 – 1 hour by methanol, then washed for 10 – 15 minutes by mobile phase.

3.9.2. Stock solution preparation An amount of cephradine monohydrate standard powder equivalent to 50 mg of cephradine was accurately weighed by using sensitive balance and transferred to a dry volumetric flask 1000 ml. Mobile phase was added until 100 ml, then shaken and ultrasonic system used until cephradine was completely dissolved to produce a stock solution of a concentration of 50 µg /ml.

3.9.3. Calibration curves in urine Six volumetric flasks (25 ml) were labeled and by using volumetric pipettes, 2 ml of blank urine was added to each volumetric flask. From the stock solution serial dilutions were made to prepare different concentration for calibrations (0, 10, 20, 30, 40 µg/ml). The volumes were completed with mobile phase. HPLC was injected from stock solution and this step was repeated several times until the system was stabilized and the peak was fixed in a specific position (Figure 4.1). Different concentrations of calibration were then injected and the peak areas were recorded. For study, samples the amount of antibiotic per sample was determined with reference to an appropriate calibration curve constructed on every day of analysis.

3.9.4. Validation of the HPLC analytical method The modified analytical procedure was validated before sample analysis. Validation included the determination of absolute recovery, linearity, limit of quantification, precision, accuracy and reproducibility.

3.10. Data analysis The comparative bioavailability studies performed in the present work are based on measuring the amounts of antibiotics excreted unchanged in urine, the percentage dose excreted unchanged over 12 hrs (PDE %) and the maximum peak of excretion (MPE mg/min), together with the time taken to reach this peak (TTP, hrs) are used to describe the extent and rate of antibiotics bioavailability respectively (Ritschel, 1976).

3.11. Calculations A scientific calculator Casio model Fx– 350 TL (China) was used for regression analysis as follows: For Regression equation Y = A + B X Y = Peak area. A = Intercept B = Slope X = Concentration A and B are calculated from the regression line and Y is the peak area of the sample. So X is calculated as: Y − A X = mg/ml (Appendix E). Bx1000 Amount of excretion(mg) = X (mg /ml) x Dilution factor x Sample volume(ml)

Amount excreted (mg) Rate of excretion (mg / min) = Time interval (min)

Cumulative amount excreted X 100 Percentage Dose of excretion = 500 mg 3.12. Statistical analysis The computer software excel was used for statistical analysis where Student’s t-test was performed. All results were expressed as (Mean ± SD).The confidence of variance (C.V.) was calculated for all pharmacokinetic parameters. Significant differences were assumed if P ≤ 0.05. 4. Results and discussion

4.1. Analytical procedure Determination of cephradine excreted unchanged in the urine of healthy volunteers was achieved using a rapid, sensitive and selective HPLC procedure. The analytical method was validated before use. The validation procedure can be summarized in the following:

4.1.1. Specificity: the specificity of the method was determined by comparing the chromatograms obtained from a sample containing cephradine with those obtained from blank samples and a sample containing a mixture of cephalosporin compounds. The HPLC analytical method exhibited good specificity and permitted determination of cephradine in urine with no interference of other compounds. The retention time was 1.7 minutes when flow rate was kept at 2 ml/minute and the analytical run was completed within 3 minutes (Figure 4.1).

4.1.2. Limit of detection: this is a parameter that provides the lowest concentration that can be detected in a sample, but not quantified, under the stated experimental conditions. The limit of detection of cephradine in this study was found to be 1.8 ng. However, the chromatographic conditions used allowed a measurement of concentration down to 0.1 µg /ml. (CV = 0.7, table 4. 1).

Chromatogram A

Chromatogram B

Figure 4.1: Chromatograms of blank urine (A) and urine sample of a volunteer following oral administration of 500 mg cephradine capsule (B, RT = 1.658 minutes).

Table 4.1. Sensitivity test of cephradine in urine by HPLC analysis

Concentration Peak Area Mean ± C.V. No µg/ml 12/2/02 15/3/02 25/4/02 SD % 1122670 1 50 µg/ml 1099312 1156028 1112670 2 ± 24209 899671 2 40 µg/ml 905007 893624 900384 0.5 ± 4674 5485 3 0.2 µg/ml 5489 5480 5487 0.07 ± 3.9 2796 4 0.1 µg/ml 2809 2769 2811 0.7 ± 19

4.1.3. Absolute recovery: The sample preparation procedure was simple, rapid and easy to perform. The absolute recovery was 90 - 93.9 % in the concentration range of 10 – 50 µg/ml (table 4.2).

Table 4.2 Absolute recovery of cephradine in urine compared to mobile phase.

Concentration Mobile phase Urine Absolute No µg/ml (Peak Area) (Peak Area) Recovery % 1 10 µg/ml 244609 220187 90 2 20 µg/ml 484254 451414 93.2 3 30 µg/ml 733167 685799 93.9 4 40 µg/ml 964204 897552 93.1 5 50 µg/ml 1228456 1130645 92

4.1.4. Linearity calibration curve of cephradine in urine is illustrated in (figure 4.2). The calibration curve was constructed by plotting the peak area of cephradine against different concentrations of standard solutions of cephradine in the range of 10 – 50 µg/ml. The calibration line was established by linear regression analysis using the computer software Excell®. The curve showed good linear relationship where a straight line passing through the origin with a correlation coefficient of R2 = 0.9998 was obtained.

4.1.5. Inter-day reproducibility: This was achieved by injecting five different concentrations (10 – 50 µg/ml) in five different days. The method showed good reproducibility both in mobile phase (0.4 - 3.3 %) and in urine (0.2 - 3.3%) (Tables 4.3 and 4.4). The method is sensitive enough and showed higher reproducibility compared to previous reports.

Table 4.3 Inter-day reproducibility of cephradine in mobile phase

Peak area No Concentration Mean C.V. µg/ml ± SD % 11/2/02 12/2/02 2/3/02 28/3/02 25/4/02

1 10 µg/ml 255515 245080 250297 239627 232528 244609 3.3 ± 8028

2 20 µg/ml 485623 486807 486215 479254 483375 484254 0.6 ± 2757

3 30 µg/ml 724776 729903 727339 736251 729567 733167 0.8 ± 5646

4 40 µg/ml 965743 967620 966681 956861 964118 964204 0.4 ± 3849

5 50 µg/ml 1214962 1220979 1230848 1222263 1253231 1228456 1 ± 13385

R2 = correlation coefficient

Figure 4-2: Calibration curve of Cephardine in urine

Table 4.4. Inter-day reproducibility of cephradine in urine

Peak area Concentration C .V No Mean ± µg/ml SD % 11/2/02 12/2/02 28/3/02 25/4/02 2/5/02

1 10 µg/ml 219482 219572 219270 218671 223940 220187 0.9 ± 1902

2 20 µg/ml 449159 433980 441569 472801 459564 451414 3.02 ± 13636

3 30 µg/ml 689056 686794 690659 687355 690135 688799 0.2 ± 1511

4 40 µg/ml 905007 893624 900384 895761 892987 897552 0.5 ± 4540

5 50 µg/ml 1099312 1156028 1112670 1094309 1190907 1130645 3.3 ± 37150

4.1.6. Accuracy, precision and intra-day reproducibility: accuracy and intraday reproducibility for cephradine were determined using three different concentrations of cephradine. Five replicates of each concentration were injected on the same day. The results are depicted in table 4.5. As can be seen in the table the coefficients of variation are satisfactory for measurement of cephradine in urine (CV = 0.96 - 2.1 %).

Table 4.5. Accuracy, Precision and within day reproducibility for cephradine in urine Amount Mean C.V. Amount found µg / ml added µg/ml ± SD % 9.6 10 9.7 9.3 9.5 9.6 9.9 2.08 ± 0.2 30.4 30 31 30.7 30.13 30.2 30.16 1.2 ± 0.35 49.74 50 49.2 49.8 49.4 49.7 50.6 0.96 ± 0.48

4.2. Cephradine comparative bioequivalence studies The bioavailability of cephradine from four commercial products (A, B, C and D) marketed in the ROY, was examined following oral administration of 500 mg single dose capsule of each product using the urinary excretion method since the amount of cephradine in the plasma is reflected in the renal tubules (Ali, 1981, Jusco and Lewis 1973). The prerequisites for a valid study were fulfilled. The amounts of cephradine in urine were determined by HPLC technique. The maximum peak of urinary excretion rate (MPE, mg / min) and the time to reach this peak (TTP, hrs) together with the percentage dose excreted unchanged in the urine over 12 hours were used to describe the rate and extent of bioavailability, respectively. The rate and extent of bioavailability were used together to compare different brands of cephradine with a reference brand. The mean ± SD urinary excretion rates (mg / min) at various times (hours) of cephradine after oral administration of 500 mg capsules of brands A, B, C and D to 8 healthy, adult, male Yemeni volunteers under fasting conditions are illustrated in table 4.6. The table reflected individual variations between the subjects, which are satisfactory. As can be seen in table 4.6 the MPE for brands A, B, C and D were 3.8 ± 1.7, 3.68 ± 1.4, 2.84 ± 2.09 and 3.86 ± 1.6, respectively. The time to reach peak was almost identical 0.75 hours for the four brands. This indicates that most of cephradine was absorbed at the first hour.

Table 4.6 Mean ± SD urinary excretion rates (mg / min) at various mean times of cephradine following oral administration of 500 mg capsules of brands A, B, C and D to 8 healthy, adult, male Yemeni volunteers under fasting conditions.

Mean time Brand A Brand B Brand C Brand D (hrs) 0.25 0.43 ± 0.29 0.97 ± 0.84 1.07 ± 1.3 0.4) ± 0.38 0.75 3.8 ± 1.7 3.68 ± 1.4 2.84 ± 2.09 3.86 ± 1.6 1.5 2.7 ± 0.4 2.3 ± 0.99 2.45 ± 0.73 2.88 ± 0.71 2.5 1.04 ± 0.4 1.13 ± 0.61 1.11 ± 0.70 0.96 ± 0.32 3.5 0.34 ± 0.19 0.39 ± 0.21 0.59 ± 0.62 0.42 ± 0.24 0.122 ± 5 0.09 ± 0.06 0.14 ± 0.097 0.123 ± 0.08 0.099 0.017 ± 0.022 ± 7 0.05 ± 0.03 0.03 ± 0.025 0.012 0.008

10 0.03 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Figures 4.3, 4.4, 4.5 and 4.6 represent the mean ± SD of urinary excretion rate (mg / min) versus times of cephradine brands A, B, C and D, respectively. On the other hand, figures 4.7, 4.8 and 4.9 represent a comparison of the mean ± SD of urinary excretion rate (mg / min) versus times of brand B with brand A, brand C with A and brand D with A, respectively. The mean ± SD of urinary excretion rate (mg / min) versus times for the three test brands B, C and D compared with reference brand A are illustrated in figure 4.10. The urinary excretion rate profiles for brands B and D are almost superimposable with brand A. No statistically

6

5

4

3

2 Urinary excretion rate (mg/min) rate excretion Urinary

1

0 012345678910 Mean time(hrs)

Brand B BrandA Figure 4.7: Mean ± SD urinary excretion rate (mg / min) at various mean times of cephradine following oral administration of 500 mg capsules of brands (A and B) to 8 healthy, adult, male Yemeni volunteers under fasting condition.

90

80

70

60

50

40 PDE(%) 30

20

10

0 024681012 Time (hr) BrandA Figure 4.11: Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers under fasting conditions. 70

60

50

40

30

PDE% 20

10

0 024681012 -10

-20 Actual Time (hr)

Brand A with Khat

Figure 4.21: Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers with Khat.

100

80

60

40 PDE (%)

20

0 024681012

-20 Time (hr)

Brand A alone Brand A with Khat

Figure 4.22: Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers under fasting conditions and with administration of Khat. 10

1 024681012 Rate (mg/min)

0.1 Time (hr)

Brand A with Al -Saydih

Figure 3:7:2: Mean Log Rate of excreation versus -time profiles of cephradin (velosef) after administration of 500 mg capsule to (8) volunteers after administration of AL-Syadih.

3

2.5

2

1.5

1

0.5 Urinary excretion rate (mg/min) Urinary

0 024681012 -0.5 Time (hr)

Brand A with Al -Sayadeyah

Figure 4.23: Mean ± SD Urinay excretion rate of versus-times profiles of cephradine (velosef) after administration of 500 mg capsules to (8) healty , adult, meal volunteers with administration of AL-Sayadeyah. 5

4

3

2

1 Urinary excretion rate (mg/min) excretion Urinary 0 024681012 Time (hr) -1 Brand A with Al-Sayadeyah Brand A alone Figure 4.24: Mean ± SD Urinary excretion rate of versus-time profiles of cephradine (velosef) after administration of 500 mg capsules to (8) healthy, adult, male volunteers under fasting condition and with administration of Al-Sayadeyah.

10

1 024681012 Rate (mg/min) 0.1

0.01 Time (hr)

Brand A with Al-Saydih Brand A

Figure 3:7:5: Mean Log Rate of excreation versus-time profiles of cephradin (velosef) after administration of 500 mg capsule to (8) volunteers after administration of Al-Saydih. 70

60

50

40

30 PDE (%) 20

10

0 024681012 -10

-20 actual time (hr)

Brand Awith Al-Sayadeyah

Figure 4.25: Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers with administration of Al-Sayadeyah.

100

80

60

40 PDE (%) PDE

20

0 024681012

-20 Time (hr)

Brand A alone Brand A with Al-Sayadeyah

Figure 4.26: Mean ± SD cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brand (A) to 8 healthy, adult, male Yemeni volunteers under fasting conditions and with adminestration of Al - Sayadeyah .

significant differences (P > 0.05) were observed. On the other hand the urinary excretion profile for brand C showed decreased MPE compared to brand A. The mean ± SD of cumulative urinary excretion profile of cephradine following oral administration of 500 mg capsules of brands A, B, C and D to 8 healthy, adult, male Yemeni volunteers under fasting conditions are illustrated in table 4.7.

Table 4.7 Mean ± SD Cumulative urinary excretion (PDE %) of cephradine following oral administration of 500mg capsules of brands A, B, C and D to eight healthy, adult, male Yemeni volunteers under fasting conditions.

Actual time Brand A Brand B Brand C Brand D (hrs)

0.5 2.6 ± 1.8 4.58 ± 5.45 6.51 ± 7.8 4.6 ± 5.2

26.19 ± 19.35 ± 1 23.6 ± 12.4 23.25 ± 9.05 10.82 14.74

2 56.9 ± 1 4.5 53.56 ± 13 48.5 ± 15.08 57.68 ± 8.42

61.74 ± 69.10 ± 3 69.13 ± 11.53 67 ± 5.6 11.24 10.19 67.67 ± 73.75 ± 4 73.2 ± 10.7 70.53 ± 16.9 1198 12.36 75.18 ± 6 74 ± 10.6 72.6 ± 17.5 69.62 ± 13.5 13.72 69.77 ± 75.31 ± 8 74.2 ± 10.6 72.8 ± 17.4 13.62 13.91 69.77 ± 75.31 ± 12 74.4 ± 10.6 72.8 ± 17.4 13.62 13.91 Figures 4.11, 4.12, 4.13 and 4.14 show the mean ± SD the cumulative urinary excretion profile of cephradine (PDE %) brands A, B, C and D, respectively. No excretion of cephradine after 6 hours was observed. This indicates that the maximum cumulative amount of cephradine is excreted over a period of 6 hours after its administration in various formulations. The figures also indicated that 50 % of the cumulative amount of cephradine was excreted in 1.5 hour. Figures 4.15, 4.16 and 4.17 represent a comparison of the mean ± SD PDE % versus time for brands B, C and D versus brand A, respectively. Figure 4.18 demonstrate the mean ± SD PDE % of the four brands A, B, C and D versus time. No statistically significant differences (P > 0.05) were observed between the four formulations. The PDE % of brand C is lower than that of other brands. This difference was tested statistically and was found to be not significant. A summary of the mean values of pharmacokinetic and bioavailability parameters of cephradine (PDE %, MPE (mg / min) and

TTP (hrs), the elimination rate constants (Ke) and half lives (t½) following oral administration of a single dose of 500 mg capsule to eight healthy, adult, male, Yemeni volunteers under fasting conditions of the four brands A, B, C and D were described in table 4.8.

Table 4.8 Mean ± SD of pharmacokinetic and bioavailability parameters of cephradine following its administration as 500 mg capsules of brands A, B, C and D to eight healthy volunteers under fasting conditions.

Brand MPE TTP (hr) PDE% F F K -1) t code mg /min e (hr ½ ( hr) A* 74.2±10.6 100 4.1±1.37 100 0.84 ± 0.25 0.98 0.71

B 72.8±17.4 98 3.98±0.86 97 0.84 ± 0.25 0.73 0.94

C 69.8±13.6 94 3.7±1.4 90 1.3 ± 0.56 0.71 0.98

D 75.3±13.9 101.5 4.3±0.8 104.9 0.84 ± 0.25 0.83 0.84

* Reference standard product

The Mean ± SD PDE % values for brands A, B, C and D were found to be 74.2 ± 10.6, 72.8 ± 17.4, 69.8 ± 13.6 and 75.3 ± 13.9 %, respectively. The differences between the four brands are not statistically significant (P-values are 0.85, 0.50 and 0.87, Appendix A). Therefore the three tested brands of cephradine B, C and D might be considered bioequivalence to the standard brand A with regard to their extent of bioavailability. Comparison of the mean MPE (mg / min) values arranged the four brands in the order D > A > B > C (4.3 ± 0.8, 4.1 ± 1.37, 3.98 ± 0.86, 3.7 ± 1.4 mg/ml, respectively). Again the differences between mean values of MPE are not statistically significant (P = 0.87, 0.61, 0.0.7, respectively, Appendix A). The mean MPE values of brands B, C and D are bioequivalent to the reference brand A. The TTP values for the brands A, B, C and D were 0.84 ± 0.25, 0.84 ± 0.25, 1.3 ± 0.56, and 0.84 ± 0.25 hours, respectively. The difference between the three brands of cephradine A, B, and D were not statistically significant (P = 1, Appendix A). The mean TTP values were identical for the three brands A, B and D. Brand C showed a significant difference in TTP, 1.3 ± 0.56 compared to 0.84 ± 0.25 hours of the other three brands. This difference was statistically significant (P-value = 0.043, Appendix A). The delay in time to reach the peak of urinary elimination might be due to inter-subject variations. Based on the results described on Table 4.8 the bioavailability of cephradine brands B, C and D was estimated as 98%, 94% and 101.5%, respectively. These values are in good agreement with the recommended FDA range of 80% - 120% for bioequivalence of oral products. Accordingly, the four commercial brands could be judged bioequivalent. A similar bioequivalence study on four brands of cephradine in plasma and in urine was described by Hassanzadeh et al, (1999). The reported mean ± SD PDE% values were in the range from 82.23 to 89.40 % dose compared to our range of 69.77 ± 13.6 – 75.31 ± 13.9%. This difference might be due to ethnic variability between the Yemeni and Iranian Populations. Another bioequivalence study on four brands of Cephalexin was reported by Ali and associates in 1981. The mean PDE % values were 70 ± 3.3, 71 ± 3.5, 71 ± 2.4 and 70.75 ± 0.25. The MPE (mg/min) values were (2.6 ± 0.14, 2.7 ± 0.14, 2.6 ± 0.16 and 2.7 ± 0.21) and the mean TTP (hrs) values were about 1.5 hours for the four brands. Our results are in good agreement with Ali’s results with respect to PDE% and TTP and slightly different regarding MPE. Similar findings have also been noted by Griffith (1983) and Wise (1990). Al-Mekhlafi (1998) performed a bioequivalence study on four brands of a different cephalosporin (cephalexin) in Yemeni volunteers. The results showed mean PDE % values of 73.53 ± 7.8, 73.25 ± 8.24, 71.14 ± 6.9 and 70.76 ± 6.8, mean MPE mg/min values of 4.31 ± 0.73, 3.9 ± 0.74, 3.69 ± 0.67, 3.6 ± 0.8 and TTP, hrs values range from 1.5 ± 0 to 1.75 ± 0.43 hrs. This indicates that the disposition of cephalexin is similar to that of cephradine. Difference between our studies and other reports stated above may be due to individual variability, ethnic variability or cephalosporin brands used. The results of the study indicate clearly the urgent need for the quality control laboratory (QCL) authorities to adopt the FDA and/or WHO List of drugs and dosage forms for which the test of bioequivalence is a must. The urinary excretion method used in the present study is a simple, sensitive enough, not time consuming and non-evasive. Therefore it could be adopted by the QCL for the control of registration and random testing purposes. Need is evident for testing the bioequivalence of all brands currently marketed for cephradine, cephalexin and other antibiotics. The number of brands for each drug tested is just a small fraction of all numbers actually available in the market. This exercise may save the country large sums of money, unnecessary exposure of the population to the adverse effects of antibiotics and reduce the emergence of resistant strains that used to be sensitive. It is rather illogical, irrational and uneconomical to keep the present bioinequivalent brands of antibiotics while few of them of high quality and fulfilling the international standards could do the job and even better. It is not true that bioequivalence and/or other tests are complex, sophisticated high technology control measures that third world countries e.g. Yemen, could not afford to carry out. The training opportunity provided by the present study together with the appropriate equipment, which are currently made available at the ROY can benefit the country with the respect.

4.3. Cephradine-Khat interaction studies Chewing Khat is one of the habits that prevail in Yemen for along time. Khat contains tannins, cathinone, cathine, ascorbic acid and different percentages of minerals. It is not surprising that Khat might affect the bioavailability of concurrently administrated drugs. In this part of the study cephradine-Khat interaction was investigated following the administration of cephradine alone and with Khat to 8 healthy, adult, male, Yemeni volunteers in a two way cross over study. The style of the Khat used in this study is known, as Khatal and the origin of this Khat is from Arhap, Sana’a .The amount of the Khat given to each volunteer is equal. The mean ± SD urinary excretion rate (mg/min) versus time (hour) data of cephradine after oral administration of 500 mg capsules of brands A to 8 health, adult, male Yemeni volunteers under fasting conditions and with Khat is illustrated in table 4.9. The table reflects the effect of chewing Khat in the excretion of brand A. No excretion at the first 1.5 hours was observed. Excretion started at 2.5 hour and continued at least to 10 hours.

Table 4.9. Mean ± SD urinary excretion rates (mg / min) at various mean times of cephradine following oral administration of 500 mg capsules of brand A to eight healthy, adult, male, Yemeni volunteers under fasting conditions and with Khat.

Mean time (hrs) Brand A (Fasting) Brand A with Khat

0.25 0.43 ± 0. 29 N.D.

0.75 3.8 ± 1.7 N.D. 1.5 2.7 ± 0.4 N.D. 2.5 1.04 ± 0.4 1.15 ± 0.95 3.5 0.34 ± 0.19 1.66 ± 1.1

5 0.09 ± 0.06 1.72 ± 1.08 7 0.05 ± 0.03 1.06 ± 0.72 10 0.03 ± 0.0 0.1 ± 0.0

N.D. = Not Detected Figures 4.3 and 4.19 represent the mean ± SD urinary excretion rate (mg/min) versus time for brands A under fasting condition and with chewing the Khat, respectively. Figures 4.20 represents a comparison of mean ± SD urinary excretion rate (mg/min) versus time data of cephradine after oral administration of brand A to 8 healthy, adult, male Yemeni volunteers under fasting conditions and with Khat. Statistically significant differences were observed in PDE% (P = 0.002, Appendix B). The MPE for brand A under fasting condition was 3.8 ± 1.7 (figure 4.3) and MPE for brand A with chewing Khat was 1.72 ± 1.08 (figure 4.19).

The difference in MPE was statistically significant (P = 0.033). The time to reach peak was significantly different, 0.75 hours for brand A under fasting condition compared to 5 hours with chewing Khat (P = 0.0001). Table 4.10 depicts the mean ± SD of cumulative urinary excretion of cephradine following oral administration of 500 mg capsules of brand A, to 8 healthy, adult, male, Yemeni volunteers under fasting condition and with Khat.

Table 4.10 Mean ± SD cumulative urinary excretion of cephradine following oral administration of 500 mg capsules of brand A to eight healthy, adult, male, Yemeni volunteers under fasting conditions and with chewing of Khat.

Actual time Brand A (Fasting) Brand A with Khat (hrs) 0.5 2.6 ± 1.8 N.D. 1 23.6 ± 12.4 N.D. 2 56.9 ± 14.5 N.D. 3 69.13 ± 11.53 7.0 ± 5.6 4 73.2 ± 10.7 21.95 ± 16.19 6 74 ± 10.6 42.46 ± 8.8 8 74.2 ± 10.6 51.58 ± 10.10 12 74.4 ± 10.6 51.86 ± 10.36 Figure 4.11 and 4.21 illustrate the mean ± SD PDE % for brand A under fasting condition and with Khat, respectively. Comparison of mean ± SD PDE % of brand A alone and when administered with Khat is shown in figure 4.22. No excretion of cephradine for brand A after 6 hours under fasting condition and after 7 hour with Khat was observed. This indicates that Khat delays the excretion of cephradine. The figures also illustrated that only 52 % was absorbed from brand A with administration of Khat.

Cephradine was well absorbed in all subjects following oral administration of 500 mg under fasting conditions. In contrast cephradine levels were significantly decreased (P < 0.05) after chewing the Khat (table 4.11).

Table 4.11 Bioavailability parameters (Mean ± SD) of cephradine following administration of 500 mg cephradine (Brand A) under fasting condition and with chewing the Khat

MPE K t Brand Name PDE% F F TTP hr e ½ mg/min (hr-1) ( hr) Brand A 74.2±10.6 100 4.1±1.37 100 0.84±0.25 0.98 0.71

Brand A With 51.6±10 69.5 2.6±0.6 63.4 2.2 ± 0.7 1.4 0.5 Khat

Reference product cephradine was well tolerated in all subjects following oral administration of 500 mg capsule (Velosef®) under fasting condition and immediately before starting chewing the Khat.

The observed differences in the rate and extent of bioavailability (MPE and PDE %) when the Khat is chewed with the administration of the drug, could be attributed to the various effects of the Khat on the gastrointestinal tract. It is known that the Khat mainly contains tannins (in a remarkable amount equal to 14%), alkaloids and some minerals like iron. The tannins are characterized as chemically active compounds and probably form complex with this drug too. In addition the iron and minerals form minerals-cephradine complexes; both of these complexes, in different mechanisms, cause retardation for the absorption of cephradine in GIT. Tannins have also acidic properties that can change the pH of GIT especially in the small intestine and increase the ionized amount and consequently decrease absorption. The phramacokinetic parameters; half life (t1/2) and elimination rate constant (Ke) in case of chewing Khat compared with under fasting conditions are statistically not different. These results ensure that the major role of the Khat on the absorption of the drug is in the sites of the absorption in GIT. Furthermore tannins decrease cell membrane permeability (Uro, 1992). Besides the alkaloids that are available in the Khat like cathinone and cathine decrease the motility of GIT (Abdu Rab, 2000).Consequently the above-mentioned effects led to decrease in the rate and extent of cephradine absorption in GIT and delayed the time of MPE. Our results are in good agreement with these findings. Previous studies investigating the effect of Khat chewing on bioavailability of ampicillin and amoxicillin showed significant decreases in both rate and extent of absorption (Attef et al., 1997). Evidence of effect of Khat on bioavailability of ciprofloxacin was demonstrated by Al-Adhal (1999) and Al-Meklafi (2003). The present study is in good agreement with above mentioned findings, namely: that Khat interferes with beta lactam antibiotics (cephradine) oral preparations and decreases the rate and extent of their absorption. According to our findings, chewing the Khat immediately after administration of 500 mg cephradine capsule preparations is considered a contraindication during the treatment with cephalosporins. It is necessary to advice the patients who have habit of chewing the Khat to stop the chewing during the course of treatment with cephalosporins products e.g. cephradine, cephalexin, , cefuroxime etc.

4.4 Cephradine-Common Yemeni food interactions Al-Sayadeyah is a common Yemeni dish especially in the southern and western parts of Yemen, which is administered at lunch. It is a cooked mixture containing fish, potatoes and tomatoes, eaten with bread. Evidence that common Yemeni food had reduced both the rate and extent of bioavailability of ciprofloxacin is available (Al-Mekhlafi, 2003). So it is not surprising that administration of Al-Sayadeyah might have an effect on the bioavailability of concurrently administrated drugs. In this part of the study possibility of interaction of cephradine with Al-Sayadeyah was investigated following the administration of cephradine alone and with Al-Sayadeyah to 8 healthy, adult, male Yemeni volunteers in a two way cross over study. The mean ± SD urinary excretion rate (mg/min) versus time (hour) data of cephradine after oral administration of a 500 mg capsule of reference brand A to eight healthy, adult, male, Yemeni volunteers under fasting conditions and with Al-Sayadeyah is illustrated in table 4.12. The table reflects the effect of Al-Sayadeyah in the excretion of brand A. No excretion at the first 1.5 hours was observed.

Table 4.12 Mean ± SD urinary excretion rates (mg/min) at various mean times of cephradine following oral administration of a 500 mg capsule of brand A to eight healthy, adult, male Yemeni volunteers under fasting conditions and with Al-Sayadeyah.

Brand A Mean time (hrs) Brand A (Fasting) with Al-Sayadeyah

0.25 0.43 ± 0.29 N.D.

0.75 3.8 ± 1.7 N.D. 1.5 2.7 ± 0 .4 N.D. 2.5 1.04 ± 0.4 0.88 ± 0.89 3.5 0.34 ± 0.19 1.62 ± 0.9

5 0.09 ± 0.06 1.23 ± 0.6 7 0.05 ± 0.03 0.6 ± 0.5 10 0.03 ± 0.0 0.0 ± 0.0

N.D. = Not Detected

Figures 4.3 and 4.23 represent the mean ± SD urinary excretion rate (mg/min) versus time for brand A under fasting condition and with Al-Sayadeyah, respectively. Figures 4.24 represents a comparison of mean ± SD urinary excretion rate (mg/min) versus time data of cephradine after oral administration of brand A to 8 healthy, adult, male,

Yemeni volunteers under fasting conditions and with Al-Sayadeyah. Statistically significant differences were observed (P = 0.005, Appendix C). The time to reach peak was significantly different, 0.75 hours for brand A under fasting condition and 3.5 hours for brand A with Al- Sayadeyah (P = 0.002). Table 4.13 depicts the mean ± SD of cumulative urinary excretion of cephradine following oral administration of a 500 mg capsule of reference brand A, to 8 healthy, adult, male, Yemeni volunteers under fasting condition and with Al-Sayadeyah.

Table 4.13 Mean ± SD cumulative urinary excretion of cephradine following oral administration of 500 mg capsule of brand A to eight healthy, adult, male, Yemeni volunteers under fasting conditions and with administration of Al-

Sayadeyah.

Actual time Brand A Brand A (hrs) (Fasting) with Al-Sayadeyah 0.5 2.6 ± 1.8 N.D. 1 23.6 ± 12.4 N.D. 2 56.9 ± 14.5 N.D. 3 69.13 ± 11.53 10.68 ± 10.52 4 73.2 ± 10.7 25.4 ± 19.7 6 74 ± 10.6 39.5 ± 17.6 8 74.2 ± 10.6 55.5 ± 18.1 12 74.4 ± 10.6 55.5 ± 18.1

N.D. = Not Detected Figure 4.11 and 4.25 illustrate the mean ± SD PDE % for brand A under fasting condition and with Al-Sayadeyah, respectively. Comparison of mean ± SD PDE % of brand A alone and with Al-Sayadeyah is shown in figure 4.26. No excretion of cephradine after 6 hours for brand A under fasting condition and after 7 hour for brand A with Al-Sayadeyah was observed. This indicates that Al-Sayadeyah delays the excretion of cephradine. The figures also illustrated that only 55 % was absorbed from brand A following concomitant administration with Al-Sayadeyah. Cephradine was well absorbed in all subjects following oral administration of 500 mg under fasting conditions. In contrast cephradine levels were significantly decreased (P < 0.05) following its concurrent administration with Al-Sayadeyah (table 4.14).

Table 4.14 Pharmacokinetic and bioavailability parameters (Mean ± SD) of cephradine following administration of 500 mg capsule under fasting condition and with Al- Sayadeyah.

MPE Ke t½ Brand Name PDE% F F TTP hr -1 mg/min (hr ) (hr) Brand A 74.2±10.6 100 4.1±1.37 100 0.84±0.25 0.98 0.71

Brand A With 55.3±19.8 76.6 1.9±0.8 46.3 3.1± 0.9 0.96 0.72 Al-Sayadeyah

The observed differences in the rate and extent of bioavailability (MPE and PDE %) when the Cephradine capsules are administered immediately after common Yemeni food could be attributed to the various effects of Al-Sayadeyah on gastrointestinal tract. It is known that Al-Sayadeyah is characterized as a bulk food rich with the minerals and

vitamins. Consequently, cephradine interacts with minerals (iron and zinc) available in the fish and form a complex. This mineral- cephradine complex leads to decrease in the absorption of the drug. Vitamins and other compounds also interact with cephradine (Shils, 1999). Furthermore, the bulk of food and high viscosity leads to retardation of cephradine drug (Gibaldi, 1991). Besides, the typical Yemeni food (Bread) is made of wheat or sorghum, which can be considered rich in bran and fibers. This type of food was known to enhance the GIT motility because of its high fibrous contents. Such diets usually shorten the residence of the drug and produce large quantities of feces (Ali and Farouk, 1980). It could be concluded that common Yemeni food interferes with cephradine preparations, and leads to significant decrease in

MPE and PDE% the main two parameters that determine the bioavailability, P = 0.005 and 0.035, respectively (Appendix C).

The phramacokinetic parameters; half life (t1/2) and elimination rate constant (Ke) in case of administration of Al- Sayadeyah compared with under fasting conditions showed no significant differences. These results ensure that the major role of Al-Sayadeyah is in the sites of absorption of drugs in GIT. Consequently it is recommended here, for optimal absorption of cephradine, that the capsule should be swallowed before the administration of common Yemeni food with interval time not less than two hours. This time is enough to prevent interference of common Yemeni food (Al-Sayadeyah) with cephradine absorption. 5. Conclusions and Recommendations

The present study assessed the bioequivalence of three brands of cephradine marketed in the ROY in comparison with a well-known, well-marketed UK brand. In anther part of the study, the effect of both khat and common Yemeni food, Al-Sayadeyah was investigated. Statistical analysis using student’s t-test showed that there are no statistically significant differences P > 0.05 in all pharmacokinetic parameters between the three test brands (B, C and D) and the reference brand A except for the TTP for brand C (P < 0.05). The relative bioavailability of the three test brands B, C and D were 98%, 94% and 101.5% when PDE% were compared, respectively. On the other hand, the relative bioavailability for brands B, C and D were 97%, 90% and 104.9%, upon comparison of MPE, respectively. All these values fall within the FDA specified range of 80-120 % for bioequivalence. Accordingly, all three brands of cephradine used in the study are considered bioequivalent. The study of effect of chewing Khat and common Yemeni food Al-Sayadeyah on the bioavailability of cephradine reference product brand A, gave statistically significant differences (P < 0.05) for all parameters. Chewing Khat reduced the relative bioavailability to 69.5% and 63.4% when comparing PDE% and MPE, respectively. Al-Sayadeyah decreased the relative bioavailability of brand A to 76.6% and 46.3%. The study concluded that Khat and common Yemeni food Al- Sayadeyah decreased both the rate and extent of bioavailability of cephradine. The present data give an indication that only 50% of the dose of cephradine capsules was absorbed when it is administered with Khat or Al-Sayadeyah. In addition to the above, Khat costs as much as 2 – 200 U S Dollars per person per day. It is also associated with several health, social, economical and behavioral problems. Several toxins are known to be used in its cultivation which represents a very serious health hazard. Accordingly the recommendations of this study can be summarized in the following:

1. All three tested brands of cephradine marketed in the ROY and produced by different manufacturers are bioequivalent to the reference brand Velosef®. Therefore the four brands can be used interchangeably. 2. Stop chewing Khat during the whole course of treatment as much as possible or at least avoid taking cephradine and other similar drugs while chewing the Khat simultaneously. 3. Take cephradine and similar cephalosporins 2-3 hours before or after the food (Al- Sayadeyah).

References

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Table 7.1. t-test for brand B and brand A

Parameter Antibiotic N Mean SD t-test P-Value

brand A 8 4.075 1.4665 Rate (MPE) 0.164 0.872 brand B 8 3.975 .9146 brand A 8 74.238 11.3317 PDE% 0.190 0.852 brand B 8 72.775 18.6322 brand A 8 0.84 0.25 Time (TTP) 0.000 1 brand B 8 0.84 0.25

Table 7.2. t-test for brand C and brand A

Parameter Antibiotic N Mean SD t-test P-Value

brand A 8 4.075 1.4665 Rate (MPE) 0.521 0.610 brand C 8 3.693 1.4675 brand A 8 74.238 11.3317 PDE% 0.686 0.504 brand C 8 69.765 14.5586 brand A 8 0.84 0.25 Time (TTP) 2.256 0.043 brand C 8 1.3 0.56

Table 7.3. t-test for brand D and brand A

Parameter Antibiotic N Mean SD t-test P-Value

brand A 8 4.075 1.4665 Rate (MPE) 0.369 0.698 brand D 8 4.313 .8526 brand A 8 74.238 11.3317 PDE% 0.163 0.873 brand D 8 75.313 14.8807 brand A 8 0.84 0.25 Time (TTP) 0.000 1 brand D 8 0.84 0.25

7.2. Appendix (B)

Table 7.4. t-test for brand A alone and with Khat

Parameter Antibiotic N Mean SD t-test P-Value

brand A 8 4.075 1.4665 Rate brand A 2.378 0.033 (MPE) 7 2.629 .6945 with Khat brand A 8 74.238 11.3317 PDE% brand A 3.930 0.002 7 51.586 10.9079 with Khat brand A 8 0.84 0.25 Time brand A 7.000 0.0001 (TTP) 7 2.2 0.7 with Khat

7.3. Appendices (C)

Table 7.5. t-test for brand A alone and with Al-Sayadeyah

Parameter Antibiotic N Mean SD t-test P-Value

brand A 8 4.075 1.4665 Rate (MPE) brand A with 3.409 .005 7 1.934 .8248 Al-Sayadeyah brand A 8 74.238 11.3317 PDE% brand A with 2.347 .035 7 55.271 19.4495 Al-Sayadeyah brand A 8 0.84 0.25 Time (TTP) brand A with 3.898 .002 7 3.1 0.9 Al-Sayadeyah

7.4. Appendix D: Informed consent form اﻟﺸﺮوط اﻟﻤﻄﻠﻮﺑﺔ ﻓﻲ اﻟﺸﺨﺺ اﻟﺮاﻏﺐ ﻓﻲ اﻟﺘﻄﻮع أن ﻳﻜﻮن ﻟﺪى اﻟﻤﺘﻄﻮع اﻟﺮﻏﺒﺔ اﻟﻜﺎﻣﻠﺔ ﻓﻲ اﻟﺘﻄﻮع دون أي إﺣﺮاج أو ﺗﻜﻠﻒ. أن ﻳﻠﺘﺰم اﻟﻤﺘﻄﻮع ﺑﺎﻟﺸﺮوط اﻟﺘﺎﻟﻴﺔ وﻳﻮاﻓﻖ ﻋﻠﻴﻬﺎ ﺧﻄﻴﺎ ﺳﻴﺘﻢ أﺧﺬ اﻟﻌﻴﻴﻨﺎت آﻞ ﻳﻮم أرﺑﻌﺎء ﻣﻦ آﻞ أﺳﺒﻮع ﻟﻤﺪة ﺳﺘﺔ أﺳﺎﺑﻴﻊ. ﻋﺪم ﺗﻨﺎول اﻟﻄﻌﺎم ﻗﺒﻞ وﺑﻌﺪ أﺧﺬ اﻟﻌﻼج ﺑﺜﻼث ﺳﺎﻋﺎت ﻋﻠﻰ اﻷﻗﻞ ﻋ ﻠ ﻤ ﺎُ ﺑﺄن وﺟﺒﺔ اﻟﻔﻄﻮر ﺳﺘﻜﻮن ﻣﻮﺣﺪة ﻟﺠﻤﻴﻊ اﻟﻤﺘﻄﻮﻋﻴﻦ (ﻓﻮل). ﻋﺪم ﺗﻨﺎول أي ﻋﻼج ﺧﻼل اﻷﺳﺎﺑﻴﻊ اﻟﺴﺘﺔ وآﺬﻟﻚ ﻋﺪم ﺗﻨﺎول اﻟﻘﺎت اﻟﻴﻮم اﻷول ﻣﻦ ﺳﺤﺐ اﻟﻌﻴﻨﺎت وآﺬﻟﻚ ﻳﻮم ﺳﺤﺐ اﻟﻌﻴﻴﻨﺎت ﺳﻴﺘﻢ أﺧﺬ اﻟﻌﻴﻴﻨﺎت ﻣﻦ اﻟﺒﻮل ﺧﻼل 12 ﺳﺎﻋﺔ آﺎﻟﺘﺎﻟﻲ: (12,10,8,6,4,3,2,1,0.5,0) ﻳﺒﺪأ أوﻻ ﻗﺒﻞ ﺗﻨﺎول اﻟﻌﻼج ﺑﺈﻓﺮاغ اﻟﻤﺜﺎﻧﺔ ﺗﻤﺎﻣﺎ ﻣﻦ اﻟﺒﻮل وأﺧﺪ ﻋﻴﻨﺔ ﻣﻨﻬﺎ وﺗﻜﻮن هﺬﻩ اﻟﻌﻴﻨﺔ ﻓﻲ اﻟﺰﻣﻦ (ﺻﻔﺮ), ﺛﻢ ﻳﺘﻨﺎول آﺒﺴﻮﻟﺔ ﻣﻦ اﻟﺴﻴﻔﺮادﻳﻦ ﻗﻮة 500ﻣﺠﻢ ﻋﻠﻰ ﻣﻌﺪة ﺧﺎﻟﻴﺔ ﻣﻦ اﻟﻄﻌﺎم ﻣﻊ آﻤﻴﺔ ﻣﻦ اﻟﻤﺎء (500ﻣﻞ) وﺑﻌﺪ ﻧﺼﻒ ﺳﺎﻋﺔ ﻣﻦ أﺧﺬ اﻟﻌﻼج ﻳﺘﻢ إﻓﺮاغ اﻟﻤﺜﺎﻧﺔ ﻣﻦ اﻟﺒﻮل وﻗﻴﺎس آﻤﻴﺔ اﻟﺒﻮل ﺑﻮاﺳﻄﺔ اﻟﻤﺨﺒﺎر اﻟﻤﺪرج وﺗﺴﺠﻴﻠﻬﺎ وأﺧﺬ ﻋﻴﻨﺔ ﻣﻨﻬﺎ. ﻳﺘﻢ ﺗﻜﺮار اﻟﻌﻤﻠﻴﺔ اﻟﺴﺎﺑﻘﺔ ﺧﻼل اﻟﻔﺘﺮات اﻟﺰﻣﻨﻴﺔ اﻟﻤﻄﻠﻮﺑﺔ. اﻷﺳﺒﻮع اﻟﺨﺎﻣﺲ واﻟﺴﺎدس ﺳﻴﻜﻮن دراﺳﺔ ﺗﺄﺛﻴﺮ اﻟﻘﺎت واﻟﻮﺟﺒﺔ اﻟﺸﻌﺒﻴﺔ (اﻟﺼﻴﺎدﻳﺔ)ﻋﻠﻰ اﻟﺼﻨﻒ اﻷﺻﻠﻲ ﻟﻠﺴﻔﺮادﻳﻦ. ﻣﻦ ﻳﺠﺪ ﻓﻲ ﻧﻔﺴﻪ اﻟﺮﻏﺒﺔ ﺑﺎﻻﻟﺘﺰام ﺑﺎﻟﺸﺮوط اﻟﻤﺬآﻮرة أﻋﻼﻩ وﺑﺪون أي إﺣﺮاج اﻟﻤﻮاﻓﻘﺔ ﻋﻠﻴﻬﺎ ﻋ ﻠ ﻤ ﺎً ﺑﺄﻧﻲ ﺳﺄﻗﻮم ﺑﺘﻮﻓﻴﺮآﻞ ﻣﺘﻄﻠﺒﺎت اﻟﻌﻤﻠﻲ.

اﻹﺳﻢ ------

ﻟﺘﻮﻗﻴﻊ------

7.5. Appendix E: Bioavailability study data sheet