University of Nigeria
Research Publications
OBONGA, Wilfred Ofem
Author Author
PG/Ph.D/98/25429
Evaluation of Phyto-Chemical and Medicinal Properties of Cannabis Sativa Linn. Title Title (Family: Moraceae).
Pharmaceutical Sciences Faculty Faculty
Pharmaceutical Chemistry
Department Department
Date Date June, 2005
Signature Signature
Evaluation of Phyto-Chemical and Medicinal Properties of Cannabis sativa Linn. (Family: Moraceae).
A THESIS SUBMITTED
OBONGA, WILFRED OFEM PGlPh.Dl98125429
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D) IN THE DEPARTMENT OF PHARMACEUTICAL CHEMISTRY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA
If - ..
JUNE, 2005 CERTIFICATION
Obonga, Wilfred Ofem a postgraduate student in the Department of Pharmaceutical Chemistry with the Registration Number PG/Ph.D/98/25429, has satisfactorily completed the requirements for the degree of Doctor of Philosophy in Pharmaceutical Chemistry. The work embodied in the thesis is original and has not been submitted in part or full for any other diploma or degree of this or any other university.
(Late)
Dr. G. B. Okide Dr. (Mrs.) P. 0.0kadebe (Supervisor) (Supervisor)
-p , , , , ...... -a
Ag. Head, Department of Pharmaceutical Chemistry. DEDICATION
This work is dedicated to God almighty and my entire family. ACKNOWLEDGEMENT First, my sincere gratitude goes to God Almighty for his guidance, protection support, inspiration and other countless blessings. My unquantifiable appreciation and immense contributions goes to my supervisor Late Dr. G.B. Okide whose guidance, research experience, expertise, tolerance and patience in no small measure contributed to the success of this work. It is unfortunate that death facilitated by an assassin could not allow him to live to see the end of this work. God knows everything. I acknowledged with gratitude my indebtedness to my co-supervisor, Dr (Mrs.) P.O. Osadebe for her invaluable guidance, motherly assistance and thorough supervision amidst ever increasing academic responsibility in the department. Then, of course, there were the combined efforts of the outstanding corrections/editing of the Work by Prof. P. A. Akah, Prof. Obioma Njoku, Dr. V. C. Okore, Dr. S. Ofoefule, Dr. 0. F. C. Nwodo. At various stages of the progress of this work, the suggestions and contributions of Dr. C.O. Esimone, Dr. A. A. Attamah, Dr. B. C. Nwanguma. Dr. L. U. S. Ezeanyika, Dr. F. C. Chilaka, Dr. L. Ezugwu, Dr. C. 0. Okoli, Dr. G. C. Onunkwu, Dr. J. Ihediohar and Dr. V. 0. Shoyinka were extremely valuable. The efforts of Prof. Nze Aguwa, Prof. M.U. Adikwu, Dr. K. I. Chukwu, Prof. A. Chukwu, Mr. G. Ebi and Mr. 0:E~Ibd''u are'appreciated. I am not unmindful of your contributions, my colleagues- Dr. Ajali, P. Udeogaranya, Dr. Kenneth Oforkansi, Dr. Ngozi Nwodo, Dr. Uche Odo, Dr. 0. Okorie, Mr. Mathew Okonta, Mr. Madu Ezejiofor, Mr. Okechukwu Ndu, Mr. Ikem Uzochukwu, Mr. David Uzuegbu, Mr. ~rhze-akoli,Dr S. V. Nwafor (late) Mr. Itoro Ebebe (late) Mr. Arunsi. May I express my appreciation to all the laboratory staff of the Faculty of Pharmaceutical Sciences: Mr. Justus Nwoga, Mrs. J. C. Eleanya, Mr. W. C. Eya, Mr Ogboso Kalu, Mr. Awa Uka, Madam Rose Anyaoha and a host of others. I am grateful to, Prof. A. A. Ngokere and Dr. Shu both of UNTH's Pharmacology and Histopathology department for the detailed analysis of my histopathological slides. I am greatly indebted to Prof. and Prof. (Mrs). I. T. K. Egunu for providing me with a home away from my home and such wonderful support and encouragement. My profound appreciation to all my dear friends, too numerous to list here. You guys remain close to my heart and also to those people I met who shared with me their ups and downs during the course of my study and without whom I would have accomplished very little. To my dad and mum (Chief (Sir) & Lady Mrs. W.I. Obonga), my only sister and brothers (Anita, Iborbor, Obonga and Okim) I lack the words to appreciate all your love for me and words encouragement. All the same I am indebted to all of you. Special thanks to Nigerian Drug Law Enforcement Agency (NDLEA) for providing me with the Cannabis samples, UNTH Pathology Laboratory, UNN Histopathology Laboratory of Veterinary Medicine, UNN Science and Training Center (STC), Bioresource and Development Center Programme (BDCP), UNN Animal Research House. May the almighty God grant each and every one of you everlasting peace and happiness in all your endeavours. TABLE OF CONTENTS
Title page ...... ,.i .. Certification ...... II ... Dedication ...... ,111 Acknowledgement ...... iv Table of contents...... vi .. List of tables ...... XII List of figures ...... xiv .. List of abbreviations ...... XVII
... List of plates ...... XVIII .. Abstract ...... XXII
CHAPTER ONE ...... I
INTRODUCTION ...... 1 Historical Background of Marihuana (Cannabis)...... 2 Cultivation ...... 3 Chemistry ...... 3 Structure - Activity Relationships (SAR) ...... 7 -< ,,.-,'+.. r..;v .I.*.. vi .. Structure - Activity Relationships (SAR) in Man ...... 7 Structure - Activity Relationships (SAR) in Animals ...... ,...... 12 Pharmacology (Therapeutic potential in man and laboratory animals) ...... 28 Health Aspect of Cannabis ...... :...... 28 ,I - .- Acute and Chronic Effects of Cannabis in Humans/Animals ...... 28 1.6.2.1 Acute studies ...... 28 1.6.2.2 Chronic Studies ...... 30 1.6.3 Possible Adverse Effects of Cannabis on Health ...... 30 1.6.3.1 Immunity ...... 30 1.6.3.2 Chromosomal Damage ...... 3 1 1.6.3.3 Pregnancy and Foetal Development ...... 32 Animal Studies ...... 32 Human Studies ...... 33 Cell Metabolism ...... 33 Psychopathology ...... 33 Acute Panic Reaction ...... 34 Toxic delirium ...... 34 Psychoses ...... 35 Flash backs ...... -35 Violence ...... 36 Amotivational Syndrome ...... 36 Residual Psychomotor Impairment ...... 37 Tolerance and Dependence ...... 37 Physical dependence /Withdrawal ...... 38 Lung Problems ...... 39 Bronchial and Pulmonary Damage ...... -39 Bronchial Tissue changes ...... 39 Macrophages ...... 40 Cardiovascular Problems ...... 40 Therapeutic uses ...... 41 . . Multiple SclerosisMeuC~:'ology.~~.:"...... 41 Antiemetic Effects ...... 42 Alcoholism ...... -43 Antiglaucoma Effects ...... 43 ..... Prostaglandin synthesis ...... 44 Anticonvulsant ...... 45 Analgesic ...... 45 Neurotransmitter receptors ...... 46 Opioid receptors ...... -46 Prostaglandins ...... 46 Anti inflammatoiy Activity ...... 47 Anti-asthmatic ...... 48
vii 1.6.4.10 Antibiotic ...... -48 1.6.4.1 1 Appetite Stimulant ...... 48 1.6.4.12 Miscellaneous Uses ...... 49 1.6.4.12.1 Antineoplastic and Immunosuppressant Activity ...... 49 1.6.4.12.2 Anorexia ...... -49 1.6.4.12.3 Antifertility Activity ...... 49 1.6.4.12.4 Gastrointestinal effects ...... 50 The Legal .Illegality of Clinical Cannabis use in Modern Therapy ...... 50 In The Netherlands ...... -51 In Germany ...... 51 In Spain ...... 52 In Italy ...... 52 In Australia ...... 52 In The USA ...... 53 In Nigeria ...... 54 In United kingdom ...... 54 AIM AND SCOPE OF STUDY ...... -55
CHAPTER TWO 2 . 0 Experimental ...... ;,.+...... A...... 56 2.1 Materials ...... 56 2.1 .1 Chemicals and Reagents ...... 56 2.1.2 Instruments1 Equipment ...... 58 2.2 Source and Identity of plant materials."..... : .:": ...... 59 2.3 Preparation of plant extract ...... 59 Preparation of n-Hexane: Chloroform extract ...... 60 Ultraviolet (UV) Absorption Spectra ...... -60 Phytochemcal Tests ...... 60 Test for the presence of reducing sugar ...... 60 Test for the Presence of Flavonoids ...... 60 Test for the Presence of Glycosides ...... 61
viii 2.5.3.1 Tests for 0 .and C - glycosides ...... 61 2.5.3.2 Borntagger's test for Anthracene glycosides ...... 61 2.5.3.3 Test for cyanogenic glycosides ...... 62 2.5.4 Test for the presence of alkaloids ...... 62 2.5.5 Test for Starch ...... 62 2.5.6 Test for the Presence of Proteins ...... 62 2.5.7 Test for Tannin ...... 63 2.5.8 Test for Saponins ...... 63 2.5.8.1 Frothing Test ...... 63 2.5.8.2 Emulsion Test ...... -63 2.5.9 Test for Flavonoid ...... 64 2.5.10 Test for Carbohydrate ...... -64 2.6 Microbiological Evaluations ...... 64 2.6.1 Isolation and characterisation of test microorganisms ...... 64 2.6.2 Maintenance and standardisation of stock cultures ...... 64 2.6.3 Preliminary Sensitivity Tests ...... 65 2.6.3.1 Sensitivity test of Cannabis Crude Resin Extracts ...... 65 2.6.4 Minimum Inhibitory Concentrations (MICs) of Extracts ...... 65 2.6.5 Determination of Rate of Kill of the Crude Extracts ...... 66
2.7 Toxicity Tests ...... ,',.c.q ...>.... i...... 66 2.7.1 Cytotoxicity Assay (CDS0)...... 66 2.8 Administration of Cannabis Suspension ...... 67 2.9 Determination of Haematological Parameters ...... 67 2.9.2 Sample Collection ...... 67 2.9.3 Procedure for the haematological determination ...... 68 2.9.4 Data Analysis ...... -69 2.10 Preparation of Cannabis Formulations ...... 69 2.10.2 Materials ...... 69 2.10.2.1 Preparation of Suppositories ...... 69 2.10.3 Evaluation of Suppositories ...... 69 2.10.3.1 Appearance ...... -69 2.10.3.2 Uniformity of Weight ...... 69 2.10.3.3 Liquefaction time ...... 70 2.10.3.4 Construction of Calibration Curve (Beer's plot) ...... 70 2.10.3.5 Release studies ...... 70 2.1 1 Anti inflammatory test ...... 71 2.12 Determination of Biochemical Parameters: ...... 72 2.12.1 Determination of the serum glutamate-oxaloacetate-transaminase (SGOT) ....72 2.12.1.1 Preparation of standard curve for SGOT...... 73 2.12.2 Determination of the serum glutamate-pyruvate-transaminase (SGPT) ...... 73 2.12.2.1 Preparation of standard curve for SGPT...... 74 2.12.3 Determination of urea levels ...... 74 2.13 Histopathological determination ...... 75 2.13.1 Isolation of some vital organs of rats ...... 75 2.14 Stability studies of Cannabis crude resin formulation ...... 77 2.14.1 Preparation of stock solutions ...... 77
2.14.2 Exposure of the Samples to various Temperatures ...... -79 2.14.3 TLC determination ...... 79 2.14.4 Determination of Percentage Degradation ...... 80 2.14.4.1 Determination of Shelf Life ...... 81
?i -< .. .; ...,......
CHAPTER THREE ...... -82 3.0 Results and Discussion ...... 82 3.1 Yield ...... 82 ...... 3.2 Phytochemical Analysis of Cannabis sativa leaves ...... 82 3.3 Cytotoxicity Assay LCSo...... -82 3.4 Pharmacological Parameters ...... 85 3.4.1 Effects of formulation of Cannabis resin on rat paw edema: ...... 85 3.5 Antimicrobial Assay ...... 95 3.6 effect of crude Cannabis resin extract on Haematological Parameters ...... 117 3.7 Cannabis Suppositories formulation and analysis ...... 125 3.7.1 Appearance of the suppositories ...... 125 3.8 Stability studies ...... 138 3.9 BiochemicalParameters ...... 148 3.9.1 Effect of Cannabis Crude Resin on Glutamate-Oxaloacetate ...... 148 3.9.2 Effect of Cannabis Crude Resin on Glutamate-Pyruvate Transaminase Activity in Rats ...... 148 3.9.3 Effect of Cannabis Crude Resin on Urea Activity in Rats ...... 148 3.10 Histopathological effects of Cannabis crude resin ...... 154 3.10.1 Effects of Cannabis Crude Resin on the Liver of Rats ...... 154 3.10.2 Effects of Cannabis Crude Resin on the Brain of Rats ...... 160 3.10.3 Effects of Cannabis Crude Resin on the Kidney of Rats ...... 160 3.10.4 Effects of Cannabis Crude Resin on the Spleen of Rats ...... 169 CONCLUSION ...... 173 REFERENCES ...... 174 APPENDIX ...... -199 List of tables Table 1: Cannabinoids in man ...... 8 Table 2: Natural and synthetic tetrahydrocanabinol (THC) and their pharmacological effects ...... 12
Table 3: Synthetic lea .THC ...... 18 Table 4: Miscellaneous and their pharmacological effects ...... 23 Table 5: ...... 73 Table 6: ...... 74 Table 7: A table showing the formulation ratios of Cannabis resin syrup ...... 79
Table 8: Phytochemical constituents of Cannabis sativa leaves ...... 83 Table 9: Phytochemical constituents of solvent extracts of crude Cannabis resin ...... 83 Table 10: Cytotoxicity of Cannabis crude resin extract singly and in combination ...... 84 Table 1 1 : Effect of Cannabis crude resin on acute edema of the rat paw induced by egg albumin ...... 87
Table 12: Effect of Cannabis crude resin syrup on acute edema of the rat paw induced by egg albumin ...... 87
Table 13: Effect of Cannabis syrup on acute edema of the rat paw induced by egg albumin ...... -88 Table 14: Effect of Cannabis syrup on acute edema of the rat paw induced by egg albumin ...... 88 .<...... :. .r...... >> Table: 15: Percentage inhibition of edema in rats treated with Cannabis crude resin formulation ...... 88 Table: 16 Percentage inhibition of edema in rats treated with Cannabis crude resin syrup formulation ...... 89 Table 17 Percentage inhibition of edema ip rats treated with Cannabis crude resin syrup containing sodium m-bisulphite formulation ...... 89 Table 18: Percentage inhibition of edema in rats treated with Cannabis crude resin syrup containing sodium m-bisulphite and EDTA formulation ...... 89 Table 19: The table shows the activity of Cannabis crude extracts of the listed microorganisms ...... 96 Table 20: MIC values of the following crude Cannabis resin extracts in B. subtilis and S . azrrew ...... 97 Table 21: Results of the Physical parameters of the Cannabis Crude Resin Extract Suppositories ...... -127 Table 22: Accelerated stability study of Cannabis syrup formulation for six days .....140
xii Table 23: Rate stability (degradation) studies for 36 weeks of Cannabis syrup formulation...... I40 Table 24: Rf value of Cannabis syrup stored at different temperature...... 141 Table 25: Rf value of Cannabis syrup stored at room temperature...... 142 Table 26: Amount of serum Glutamate-Oxaloacetate Transaminase in rats injected with different concentrations of Cannabis crude resin...... 153 Table 27: Amount of serum Glutamate-pyruvate Transaminase in rats injected with different concentrations of Cannabis crude resin...... 153 Table 28: Amount of serum Urea in rats injected with different concentrations of Cannabis crude resin...... 153
xiii List of Figures Figure 1 : Effects of Cannabis crude resin and formulations on peak edema ...... 90 Figure 2: Effects of Cannabis crude resin syrup on paw in rats ...... 91 Figure 3: Effects of Cannabis crude resin syrup containing sodium meta bisulphate edema on paw in rats ...... 92 Figure 4: Effects of Cannabis crude resin syrup containing sodium meta bisulphate and EDTA edema on paw in rats ...... 93 Figure 5: Effects of Cannabis resin edema on paw in rats ...... 94 Figure 6: A graph of Log Conc . Against inhibitory zone diameter (ED2) of crude Cannabis extract WFC (Bacillus subtilis) ...... 98 Figure 7: A graph of 1ZD2 against Log Conc . of crude Cannabis extract WFCH (Bacillus subtilis) ...... 99 Figure 8: A graph of IZD' against Log Conc. of crude Cannabis extract WFH (Bacillus subtilis) ...... 100 Figure9: A graph of IZD' against Log Conc. of crude Cannabis extract WFH (Staph aureus) ...... 101 Figure 10: A graph of IZD' against Log Conc. of crude Cannabis extract WFH (Salmonella typhii) ...... 102 Figure 11: A graph of lZD2 against Log Conc . of crude Cannabis extract WFMC
(Bacillus subt ilis) ...... #, ...... ,...... ,.. : ...... 1 03 Figure 12: A graph of 1zD2against Log Conc. of crude Cannabis extract WFMC (staph
Figure 13: A graph of IZD' against Log Conc . of crude Cannabis extract WFE (Bacillus subtilis) ...... Ir - .. 105 Figure 14: A graph of IZD' against Log Conc . of crude Cannabis extract WFE (staph aureus) ...... 106 Figure 15: A graph of 1ZD2 against Log Conc . of crude Cannabis extract Gentamycin (Bacillus subtilis) control ...... 107 Figure 16: A graph of 1zD2 against Log Conc. of crude Cannabis extract Gentamycin (Staph azrreus) control ...... 108 Figure 17: Killing kinetics of WFH against B . subtilis ...... 109
xiv Figure 18: Killing kinetics of WFH against S. aureus ...... 1 10 Figure 19: Killing kinetics of WFH against Sal. thyphii ...... 111 Figure 20: Killing kinetics of WFC against B. subtilis ...... 112 Figure 2 1 : Killing kinetics of WFCH against B. subtilis ...... 113 Figure 22: Killing kinetics of WFMC against B. subtilis ...... 114 Figure 23: Killing kinetics of WFMC against St . aureus ...... 115 Figure 24: Killing kinetics of WFE against B. subtilis ...... 116 Figure 25: A graph of total white blood cell (WBC) of cell per pL of blood against Experimental Period (weeks) ...... 118 Figure 26: A graph of Total red blood cell (RBC) per pL of Blood against Experimental period (weeks) ...... 119 Figure 27: A graph of Absolute Lyphocyte Count per p1 of blood against Experimental Period (week) ...... 120 Figure 28: A graph of Absolute Monocyte count per pL of Blood against Experimental Period (weeks) ...... 12 1 Figure 29: A graph of Absolute Neuotrophil Counts per pl of blood against Experimental Period (weeks) ...... 122 Figure 30: graph of Absolute Eosinophil Counts per pL of blood against Experimental Period (weeks) ...... -123 Figure 31: A graph of pack ceJJ.,xdume (PCV) (%) per pL of Blood against Experimental Period (weeks) ...... 124 Figure 32: A graph of the release profile of crude Cannabis from the suppository in O.IN HCI ...... 128 Figure 33: A grabh of the release profile of crude Cannabis from the suppository in O.1N HCI ...... 129 Figure 34: A graph of the release profile of crude Cannabis from the suppository in 0 .IN HCI ...... 130 Figure 35: A graph of the release profile of crude Cannabis from the suppository in O.1N HCI ...... 131 Figure 36: A graph of the release profile of crude Cannabis from the suppository in 0 .IN HCI ...... 132 Figure 37: A graph of the release profile of crude Cannabis from the suppository in
0. IN HCI...... , . . . . , .., . , . . , . .I33 Figure 38: A graph of the release profile of crude Cannabis from the suppository in
0. IN HCI...... , . , , . , ...... , . , . . .I34 Figure 39: A graph of the release profile of crude Cannabis from the suppository in O.1N HCI...... 135 Figure 40: A graph of the release profile of crude Cannabis from the suppository in
0.1N HCI...... , ...... ,. 1 36 Figure 4 1 : Cannabis Beer's plot for suppositories release studies...... ,137 Figure 42: A scan of Cannabis crude resin extract showing maximum peak at 274 hnm...... I43 Figure 43: Figure 85: Spectrophotometric scan of Cannabis crude resin sport A from a TLC plate showing various peaks...... 144 Figure 44:: Spectrophotometric scan of Cannabis crude resin sport B from a TLC plate showing various peaks...... , I45 Figure 45: Spectrophotometric scan of Cannabis crude resin sport C from a TLC plate showing various peaks...... , . . .I46 Figure 46: Spectrophotometric scan of Cannabis crude resin sport D from a TLC plate showing various peaks...... I47 Figure 47: A graph of glutamate o$&.wetate transaminase activity in serum controlled and tested animals after administering Cannabis crude resin extract...... 150. Figure 48: A graph of glutamate pyruvate transaminase activity in serum controlled and tested animals after administering Cannabis crude resin extract...... 1 51 Figure 49: A graph of urea activity in serum--controlled and tested animals after administering Cannabis crude resin extract ...... 152
xvi List of Abbreviations Tetrahydrocannabinol (THC) Cannabidiols (CBD) Cannabinols (CBN) Lysergic acid diethylamide (LSD) Central nervous system (CNS) Structure-Activity Relationships (SAR) Hexahydrocannabinol (HHC) Pentylenetetrazole (PTZ) Diphenylhydantoin (DPH) Prostaglandins (PG) Lutenizing hormone-releasing hormone (LHRH) Luteinizing hormone (LH) Multiple Sclerosis (MS) National Organisation for the Reform of Marijuana Legislation (NORML) Cannabinoid receptors (CB) Serum glutamate pyruvate transaminase (SGPT) Serum glutamate oxaloacetate transaminase (SGOT) -< ,"...!. 4. .~.. -- . ' National Drug Law Enforcement Agency (NDLEA) National Agency Food and Drug Administration and Control (NAFDAC)
xvii List of Plates Plate 1: Plate 1 : Normal Liver cells. The liver Section of the rats given neither C'nnnnhis crude resin nor vehicle. Haematoxylin and Eosin stain. X 100 (Untreated, control rats)...... 155 Plate 2: Week one. The liver from dosed positive control vehicle showing mild necrosis of hepatocytes in the hepatic portal area (arrow head). Haematoxylin and Eosin Stain. (X 100) ...... 155 Plate 3: Week one. The liver section to 20 mg/kg dosed group showings vascular hyalinization, portal congestion and sinusoidal dilitation. Haematoxylin and Eosin technique stain. (X 100)...... I56 Plate 4: the liver section of the rats dosed 40 mglkg displaying severe hepatic necrosis, parenchymal degeneration and hyalinisation of hyperaemia. Haematoxylin and Eosin technique Stain. (X 100)...... 156 Plate 5:Liver section of rat treated with 40 mglkg drug for two weeks. Shows sinusoidal dilation, necrosis and hyalinization. Haematoxylin and Eosin stain. (X 100)...... I57 Plate 6: Week two. The liver section of rats dosed positive control vehicle showing sinusoidal dilation and mild degree hyalinisation of hyperaemia. Haematoxylin and Eosin stain. (X 100)...... 157 Plate 7: Liver section of rats h-&edd.w.i.th 20. mg/kg drug for three weeks. Shows pronounced necrosis and hyalinization. Haematoxylin and Eosin stain. (X 100)...... 158 Plate 8: liver section of rats treated with 40 mglkg drug for week three. Shows evidence of pronounced hyalinization and necrosis. Haematoxylin and Eosin stain. (X 100). ..I58 Plate 9: Week three: Live section of rats dosed positive control vehicle and shows necrosis in the parenchyma. Haematoxylin and Eosin stain. (X 100)...... I59 Plate 10: Week one, the positive control (vehicle without drug) of the brain shows evidence of eosinophilia of cell bodies, chromatolysis and edematous vascular congestion. Haematoxylin and Eosin stain. (X 100)...... 16 1 Plate 1 1 : Normal architecture of the brain showing no pathology in controlled animals (untreated rats). Haematoxylin and Eosin stain. (X 100)...... I61
xviii Plate 12: The brain section of the rats treated with 20 mg/kg drug for one week. Shows evidence of nuclear eosinophilia and chromatolysis. Haematoxylin and Eosin technique stain. (X 100)...... I62 Plate 13: The brain section of the rats treated with 40 mg/kg drug for one week. Showing prominent nuclear eosinophilia of the cell bodies. Haematoxylin and Eosin technique stain. (X 100)...... 162 Plate 14: Week 2. The photomicrograph represents the brain of rats treated with vehicle and shows nuclear eosinophilia suggestive of mild toxicity. Haematoxylin and Eosin stain. (X 100)...... I63 Plate 15: Week two. Photomicrograph of the brain of rats treated with 20 mg/kg showing eosinophilia and chromatolysis of the neurons suggestion of neurotoxicity. Haematoxylin and Eosin stain. (X 100)...... 163 Plate 16: : The brain section of the rats treated with 40 mglkg drug for two weeks. Showing prominent eosinophilia of cell bodies suggestive of neurotoxicity. Haematoxylin and Eosin stain. (X 100)...... 164 Plate 17: Week three. Photomicrograph of the brain of rats treated with vehicle and showing mild nuclear eosinophilia. Haematoxylin and Eosin stain. (X 100)...... I64 Plate 18: The brain section of rats treated with 20 mg/kg drug for three weeks. Shows evidence of nuclear eosinophilia in the cell bodies of neurons suggestive of mild neurotoxicity. Haematoxylin and.Eosin staim (X 100)...... I65 Plate 19: Brain section of the rats treated with 40 mg/kg drug for three weeks. Showing nuclear eosinophilia, arterial attenuation and edema which features are indicative of neurotoxicity. Haematoxylin and Eosin stain. (X 100)...... 165 Plate 20: Normal kidney. The kidney sec'tion-of rats given neither drug nor vehicle showing normal architecture. Haematoxylin and Eosin stain. (X 100)...... I66 Plate 21. The photomicrograph represents kidney or the rats treated with 20 mglkg drug for two weeks. Showing droplets of hyaline in the tubules and also degenerative changes in the glomeruli.. Haematoxylin and Eosin stain. (X 100)...... 166 Plate 22: The kidney section of rats treated with 40 mglkg for two weeks displaying interstitial large vacuolation and edema. Haematoxylin and Eosin stain. (X 100). ....I67
xix Plate 23: Kidney section of the rats treated with vehicle for three weeks. Showing normal architecture. Haematoxylin and Eosin stain. (X 100)...... 167 Plate 24: The kidney section of the rats treated with 20 mg/kg drug for three weeks..168 Plate 25: The spleen section of the rats showing normal architecture in the control animals. Haematoxylin and Eosin stain. (X 100)...... 170 Plate 26: The spleen section of the rats treated with vehicle for week one. Haematoxylin and Eosin stain. (X 100)...... 170 Plate 27: The spleen section of rats treated with 20 mg/kg drug for one week. Shows evidence of necrosis, disappearance of lymphocytes at the germinal centre of the follicles. Lymphocytes are also degenerate and condense in some other areas. Haematoxylin and Eosin stain. (X 100)...... 17 1 Plate 28: The spleen section of rats treated with 40 mg/kg drug for one week. Shows evidence of severe necrosis and cellular degeneration of the follicles. Haematoxylin and Eosin stain. (X 100)...... I7 1 Plate 29: The spleen section of rats treated with 40 mg/kg drug for three weeks showing marked follicular derangement, necrosis and lymphocytic loss. Haematoxylin and Eosin stain. (X 100)...... I72 Abstract This work studied the various delivery systems (oral and parental formulations) of crude Cannabis. The stability, pharmacological and toxicological profiles of Cannabis resin in the formulations was also studied. Dried powdered leaves of Cannabis sativa L. were extracted with chloroform, the extract was concentrated to obtain an oil-based tar (resin) and its phytochemical composition determined. The Cannabis crude resin was formulated as syrup, capsule and suppository dosage forms. The stability of the resin in the formulations was studied by accelerated stability technique. The in vivo release profile of the capsules was also studied. The antimicrobial activity of Cannabis crude resin was investigated using clinically isolated Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) and killing rate constant against the organisms were determined by agar diffusion and viable count methods respectively. The anti- inflammatory effect of Cannabis crude resin and syrup formulations was studied using egg-albumin-induced paw oedema in rats. The acute toxicity (LDSo)of the crude extract was determined by the brine-shrimp method while effects on biochemical parameters (glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase and urea) haematological parameters (eosinophile, monocyte, neutrophiles and lymphocyte counts, packed-cell volume and red blood cells) were determined in Wistar rats after 1, 2 and 3 weeks of treatment. The, internal,acgans~(liver, heart, kidney, spleen, lungs and the brain) from the treated rats were examined histopathologically to ascertain degeneration on chronic administration of Cannabis crude resin. Results were analysed statistically using the Analysis of Variance (SPSS) and Student's t-test. Means that differed significantly were identified by the least significant difference (LSD) Post-hoc test at 95 % confidence interval i.e. (Pc0.05). The result showed that the extraction process yielded 10 % (wlw) of the Cannabis crude resin. The presence of alkaloids, glycosides, saponins, tannin, terpenes and steroids was detected in the crude resin and dried plant. Cannabis crude resin syrup was stable even without the addition of EDTA with a shelf-life of 50 days. The degradation of Cannabis crude resin syrup formulation followed a first-order kinetic with the degradation rate constant of 0.0039 min-I. At a minimum inhibitory
xxi concentration (MIC) of 12.5 mglml, the chloroform, methylene chloride, ethanol, n- hexane: chloroform (1:l) and n-hexane extracts of Cannabis sativa showed significantly (Pc0.05) higher antimicrobial activity against S. aureus and Ps. aeruginosa than Penicillin G (MIC 18.0 mgfml). The crude resin syrup formulation exhibited a significant (Pc0.05) dose-related inhibition of paw edema in rats. The crude resin caused changes in biochemical and haematological parameters in a dose- dependent manner with no significant increase in the first two weeks of treatment, which markedly changed at week 3. The histopathological results of the various organs showed lesions of varied intensity suggesting possible toxicity in chronic use of Cannabis crude resin. These findings suggest that crude Cannabis resin can be used for its antimicrobial and antiinflamatory effect and that its formulation into syrup favours its pharmacological activity and have good stability.
xxii Chapter One 1 .O INTRODUCTION For centuries, man has used various preparations of Cannabis saliva L. (Cannabinaceae) for their psychotomimetic and supposed medicinal effects. Since the pioneering studies of Emperor Shan Nung of China 2737 BC, ' this natural product has been investigated in various parts of the world for its potentially therapeutic uses. The world literature is replete with folkloric accounts of its use in medicine. The ancient world of the Middle East and Europe comprising Assyria, ancient Egypt, the Scythians, Judea and ancient Greece and Rome have reported its various medicinal uses. The ancient Assyria of the sixth century B.C cultivated Cannabis for medical purpose. It was known as "qaunnabu" and externally it served as a constituent of various ointments for bruises and swellings, and as a bandage; its fumes were used for managing arthritis and it was prescribed for impotence.3 The ancient Egyptians also used Cannabis (called "3MSM.T') for prevention of/controlling haemorrhages in women during childbirth, in enemas, eye medication and bandages." In ancient Greece and Rome, though its psychotropic properties were not known but Cannabis was commonly used as a laxative, painkiller and an ointment for ear treatment. It was often linked to male impotence.677 In ancient India, Cannabis also referred to as "bhang" was prescribed for the treatment of fever, earache, lepyosy,,,g~n~r;rhoeaasthma, hysteria and sciatica. It was also believed to be an aphrodisiac.*' The leaves were recommended as antiphlegmatic and also as a remedy for catarrh accompanied by diarrhoea. Marijuana - derived drugs were also used in the treatment of cramps, convulsion in children, headache, hysteria and tetanus. " Tetrahydrocannabinol (THC), in particular reduces intestinal mobility ''-I3. In ancient China, Cannabis also known as "Ma" was reported to have psychotropic action. It was used in the treatment of rheumatism, haemorrhages during childbirth and vomiting. It was also used in combination with wine as an anaesthetic in major ~~erationsl~''~.The ancient Scythians, the medieval Arabs, the Syrians were all familiar with Cannabis even though they were more interested in the plant for religious rite17-19. Cannabis use in western Europe and America was only reported as late as lgth century by the following researchers: O'Shaughnessy, et a1 who all agreed on the fact that Cannabis has been in use for a very long time as a medicinal treatment for various maladies. 20-22 The 2oth century use of Cannabis is restricted to the treatment of vomiting in cancer chemotherapy as
well as an appetite stimulant.23,24 It is also used as a painkiller for excruciating pains in multiple sclerosis 25.26 .
1.2 Historical Background of Marijuana (Cannabis) Interest in drugs from Cannabis (marijuana) has had a long history of medical and therapeutic use in India and the middle ~ast~~where it has been variously used as analgesic, anti-convulsant, antispasmodic, anti-emetic and hypnotic. Marijuana was introduced to British medicine in the mid-nineteenth century by O'Shanghnessy who had gained clinical experience with the drug as an Army Surgeon in ~ndia~~.He recommended its use for the relief of skin, muscle spasm, and convulsions occurring in tetanus, rabies, rheumatism and epilepsy29. Partly as a result of his advocacy Cannabis came to be widely used as an analgesic, anti-convulsant and anti-spasmodic throughout the middle part of the 191h century in Britain and the USA. However, the medical use of Cannabis declined around the turn of the 20"' century. Because the active constituents of Cannabis were not isolated until the second half of the 20Ih century, Cannabis continued to be used in the form of natural preparations which varied in purity and hence in effectiveness. The use of Cannabis was largely supplanted by other pharmaceutically purer drugs, which could be given in standardised doses to produce more dependable effects. These include the opiates, aspirin, chloral hydrate and thq.barbitluates30. The promulgations of laws in the early part of the 20Ih century also further discouraged the medicinal use of such Cannabis preparations, since Cannabis was treated as a "narcotic" drug. It finally disappeared from the American Pharmacopoeia in the 1940s after the passage of the Marijuana Tax AC~~'. Tetrahydrocannabinol (THC), the major psychoactive ingredient of Cannabis was not isolated until 1964~',shortly before Cannabis achieved widespread popularity as a recreational drug among American youths. Its widespread recreational use, and its symbolic association with a rejection of traditional social values, undoubtedly hindered pharmaceutical research into its therapeutic uses. Consequently, the rediscovery of some of its traditional therapeutic uses was largely serendipitous, as was the discovery of some newer uses. For example, its value as an anti-emetic agent in the treatment of nausea caused by cancer chemotherapy seems to have been rediscovered by young adults who had used Cannabis recreationally prior to undergoing chemotherapy for Leukaemia 32. From the mid-1970s some clinical research on the therapeutic value of Cannabis and cannabinoids was undertaken. On the whole, however, this research has been very thin and uneven, consequently, many of the claims for the therapeutic efficacy of cannabinoids rely heavily and, in the case of rare medical conditions, solely upon anecdotal evidence; that is, the testimonies of individuals who claim to have derived medicinal benefit from its use33 and small number of cases reported by physicians34. Evidence will be reviewed for the best - supported therapeutic uses of cannabinoids. The review begins with the evidence of the effectiveness of cannabinoids as anti-emetic drugs for nausea caused by cancer chemotherapy, and as an agent to control intra-ocular pressure in glaucoma. Brief reviews are also provided of the evidence in favour of other putative therapeutic uses of cannabinoids, which are less well supported by clinical evidence, chief among which are its uses as an anti-convulsant, an anti-spasmodic and as an analgesic agent.
1.3 Cultivation Cannabis sutiva is usually planted in the months of May, June and July. The seed germinates in less than one week if the soil is moist. Weed control however, is important to the young plant, since their growth would be greatly inhibited by the weed, which is competing for light, water and soil nutrients. During arid conditions Cannabis plant are watered at least once a week. The plants mature in August, September and October after. 3;to. 5 months of growth. Most Cannabis products are obtained from cutting the stem of plants beneath the lowest branches, uir-drying and hand-stripping seed, bracts, flowers, leaves and small stems. Cannabis grows easily in temperate climates; it also requires good soil fertilizer and water. I) ..... There are two basic types of Cannabis based on genetic characteristics: (i) Drug type which originates in hot climates such as India and is high in tetrahydrocannabinol (THC) but low in cannabidiols (CBD) and (ii) Fiber type, which originates in temperate climate and is low in THC but high in CBD and it is used industrially for fiber and food. No social implications arise from cultivation of fiber types. 35,36
1.4 Chemistry Marijuana is a plant product, which has numerous chemical constituents. That is, it contains about 426 chemical entities of which 60 are cannabinoids, cannabidiols (CBD) and cannabinols (CBN), but it is the (-)-~~-6a,10a trans-tetrahydrocannabinol (A~-THC),which is responsible for the main pharmacological effect37. Many cannabinoids have been isolated and their structures determined. Mechoulam et alJ7 first proposed the term, cannabinoids for C21 groups of compound present in Cunnabis sativa and these includes their analogue and transformation products. Though the name in a wide sense is a general term for compounds structurally related to THC, cannabinoids can be categorized in terms of the alkyl side chain as shown in structures 1,2 and 3
Pentyl Cannabinoids (1) l7
Propyl Cannabinoids (2)
Methyl Cannabinoids (3) The following three numbering systems: (i) Monoterpenes (often used in chemical structure research) (4) (ii) Dibenzopyran used in literature in chemical synthesis (5) and (iii) Diphenyl, which is only, used for (CBDs) cannabidiols, generally used for naming the cannabinoids (6). Based on the monoterpenoid numbering system, A9-THC is also known as A'-THC. The other physiological active isomers, A'-THC (alternate name A6-THC) are found only in a few varieties of the plant. The other two isomers with 6a, 10a - cis ring junction are cis-A9-THC and cis-A8-THC, both of which have been synthesized and are physiologically inactive and have not been found in the plant3! The trans compounds of cannabinoids are more thermodynamically stable than the cis compounds. However, the A'-THC is more stable than A~-THCin the trans series. This is due to the isomerization of A9-THC to As-THC on treatment with acids. The main pharmacological interest centers on the thermodynamically less stable A9-THC and its various derivatives and metabolites. This has posed many synthetic problems, because during chemical reactions the more stable derivatives of transd8-THC are mostly formed. In addition, the chemistry of cannabinoids is much more complex than the structure of A9-THC would indicate. The reactions are capricious and sensitive to reaction conditions, and they form complex mixtures, which are difficult to separate. A9-THC is an optically active resinous material, which is very lipid soluble and water insoluble. It is important to note that most cannabinoids display similar properties, which makes their pharmacological testing difficult. The cannabinoid compounds have to be administered in various solvents such as polyethylene glycol, Tween 80, Triton, Emulphor, egg - albumin, or alcohol, which are not without pharmacological a~tivit~~~-~'. .' ,.<'..!. *. .. .4.. . . The clinical effects produced by investigating marijuana or A9-THC are unique and it cannot be classified pharmacologically as being due to a stimulant, sedative tranquilizer or a hallucinogen, although they share some properties with each of these. In general, the effects produced by the drug are dose dependent. Thus, in small doses, it produces subjective effects more typical of a hallucinogen and somewhat resembling those of lysergic acid diethylamide (LSD). It also produces sedative effects with no significant mimetic action and no cross-tolerance. When compared to drugs such as opiates and barbiturates, it dose not produce physical dependence; thus it is not a narcotic42. Interestingly, the user may be in a high state of intoxication, but to an observer he may appear to be near normal state. Mild states of intoxication are generally undetected, and the mood may vary from being happy and gregarious to quiet and introspective. M onoterpeniod (4) Dibenzopyran (5)
CBN (8) CBD (9) With high doses, notable signs are minimal but the most reproducible signs include an
.' ,, .,'.-7. ,:- , ,.Wi , . increase in pulse rate and bloodshot eyes. Dryness of the mouth and throat and increase in appetite are common. In some cases, there may be slurring of the speech. With moderate dosage, the user can perform simple physical and metal task, but the performance of-more complicated physical. , and_ .- psychological tasks may be impaired. The most common subjective effect appears to be time distortion with other effects varying from pleasant relaxation to acute anxiety, loss of contact with reality, hallucination and panic. These latter effects are generally associated with large doses. As with other psychoactive drugs, the effects vary greatly and are mostly dictated by the psychological makeup of the individual and the setting under which the drug is used. However, in various animals, the cannabinoids show predominantly central nervous system (CNS) depression and ataxia, which last from several hours to days, depending on the dose administered. The characteristic effect of cannabinoids, which distinguishes them from all other psychoactive drugs, is a postural arrest phenomenon with relaxed staring and is accompanied by hyper sensibility to external stimuli.39,43
1.5 Structure-Activity Relationships (SAR) The SAR of cannabinoids is presently based on their psychoactive component of Cannabis - like effect.
1.5.1 SAR in Man The various cannabinoids, which have been evaluated clinically, are summarized in Table 1. In this table the dose corresponding to total doses (mg) in man, and in some instances, where the values are in pglkg, these have been converted to mgl75 kg man. The relative potencies of various compounds are at best very rough estimates.
Basically, these are estimates showing the potency of A9 - and A* - THCs, their metabolites and some other homologs and analogs44. For other Cannabis in this table, the relative potencies have been estimated on the basis of subjective effects described in the literature using total dose of 20 mgpost oral (p.0.) or 2 mg intra venous (i.v.) of ALTHCas ~tandard.~' On the basis of comparative data, A9-THC by smoking is considered to be 2 -3 times more potent and i.v administered A9-THC is about 10 times more potent than p.0. ingested -THC?~.47. By either smoking or i.v, the effects of A9 - THC appears within seconds or minutes, whereas'Wit'h.p.%.doses, the onset of symptoms is delayed from 30 minutes to 2h. The clinical syndrome, however, is very similar in all cases. ~ollister~'reported definite marijuana -like effects, although the potency was much
less with A6" 'Oa ,- THC by smoking, but some.~orkers~~failed to find any activity I, _ with this compound at a dose of 400 pglkg (30 - mg total dose) by the same route of administration. This discrepancy might be due to the fact that the source of material used by these workers was different, and in addition, the technique of smoking used may have resulted in more than the usual amount of pyrolysis of the THC. Similarly, difference in activity was reported with 8a -hydroxy -A9- THC (10) when given i.~.~~ and Perez-Rayes et a149found it to be inactive. This inactivity in man is surprising in view of its reported activity, albeit slight, in various animals.49 An examination of table 1 suggest the following SAR in man Benzopyran ring system(l0)
THC ring system (1 1) Table 1 : Cannabinoids in manss Compound Structure Dose Route of Estimated Comments (ms> administ relative ration potency 12. - THC 4-15* Smoking 1 oo* Marihuana 'high'; 2 0 p.0. 100 2.6-3 times more 1-6 i.v. 100 potent by smoking than p.0.
p. 0. Marijuana "high"; the potency ration i. v. was the same by both routes of administration
14. A6"'Oa - Smoking Marijuana, "high" THC but much less potent
15. Synhexyl p. 0. Marijuana "high" but much less potent; Smoking Slow onset but longer duration Marijuana "high" questionable; mainly tachycardia, drowsiness, etc
17. Cannabinol No effect
Very mild "high'
18. Inactive; no "high" Cannabidiol Shows anticonvulsant properties
19.Cannabidiol Inactive dimethyl ether
20. 11- Marijuana "high" hydroxy-~' - but less intensive THC and shorter duration of action than - THC (i.v in ethanol) 21. 8a- Marijuana "high' hydroxy-A~ - but mild (i, v, in THC ethanol) 22. o Inactive \ qH Cannabichrom ene
23. Nabilone At 2.5 mg, 113 felt "high"; at 5 mg, 313 felt high
Effective antinauseant at 2 mg p. o. dose
25. Nabitan Lightheaded at 10 mg; has analgesic properties
26. Nabocate Very mild "high" at 5 mg dose; reduces intraocular pressure
1. It appears that an intact benzopyran ring is a definite requirement for activity; since the compound, cannabidiol, having an open ring, is inactive. However, the benzopyran by itself does not confer activity, since cannabichromene is inactive. Moreover, the oxygen in the benzopyran ring can be substituted by nitrogen without loss of activity as in Levonantradol(22). 2. Attachment of a nonplanar alicyclic ring (i.e., ring C) to the benzopyran ring in the 3, 4 - position is important for activity. However, planar ring attachment as in cannabinol reduces activity (nearly inactive). 3. In placement of a nonpolar ring attachment, a bulky substituent in the 4- position of the benzopyran ring can also confer activity to the molecule as in (nonabine) BRL-4664 (Structure) (25) 4. A variety of substituents can be introduced in the alicyclic C without loss of activity. Thus, the methyl in 9 - position as present in A9 - THC is not essential and can be replaced by a hydroxyl (levonantradol [24]) a hydroxymethyl (1 1-hydroxy -A9 - THC[4]), or a ketone (Nabilonet231) without loss of activity. Even the presence of two different substituents in the alicyclic ring, such as a methyl group at 9 - position and a double bond in the ring (e.g. A' - A*, A6 - THC), or a hydroxyl in a 8- position
(8- hydroxy - THC's) retains activity. In the double bond isomers, the position of the double bond at 9 - position appears optimum for activity (A9 - > A~ -> A6 - THC). 5. The alicyclic ring attachment to the benzopyran ring can be substituted by a heterocyclic ring (i.e., tetrahydropyridine ring as in SPI, Nabitan and Abbott - 41988) without loss of activity (25). 6. In the aromatic ring, esterification of the phenolic group retains activity (Nabocate[26] and levonantradol[24]) 7. The length of the aromatic side chain can be varied without loss of activity, but a three-carbon chain seernsarhifiim81 for activity, and branching of the chain increases potency. Attachment of the side chain to the aromatic ring can also be via an oxygen atom (i.e. ether as in levonantradol) without loss of activity. It is note worthy that, within the structural constrains as present in the THC ring system, changing the position of double bond inthe alkyclic ring, the length and branching of the side chain, or producing hydroxyl metabolites at the 11 - or 8 - position dose not alter qualitatively the THC effects but may strongly affect potency. Changing the double bond from A9 - to A8 - or A~ reduces potency. Decreasing the length of the side chain by two carbons reduces potency by 75%. Increasing the length of the side chain with branching enhances potency several fold. Substitution of hydroxyl group in the 11 - position retains potency, while hydroxylation in the 8 - position reduces potency by 80%. From the examination of table 1, it is also apparent that, in various structurally modified THCs, there is an indication of separation of pharmacological activities, which could be taken advantage of in therapeutics. Apparently this is the primary basis for the use of A9 - THC and Nabilone as antinauseants for cancer chemotherapy treatment, the development of levonantradol and Nabitan as analgesics, and the development of Nabocate as an antiglaucoma agent. In summary, of all the 15 compounds evaluated for clinical use only nine have been tested for therapeutic utility. Others simple have been tested only for their marijuana - like "high" effect.
1.5.2 SAR in Animals Some tetrahydrocannabinols have been tabulated in Table 2 with A~-THC,its derivatives, A' - THC, CBD, cannabinol (CBN), cannabichromene, other related natural constituents of the plant, and miscellaneous compounds including hexahydrocannabinols (HHCs) and their derivatives; The behavioural effects observed in monkeys and ataxia tests on dogs and mouse activity cage EDsOshave also been summarized in Table 2. It is important to note that in this test, the dosages are in mglkg given i.v. and the generated data could provide comparative studies in both man and animals. Table 3 shows some numbers of THCs with double bond in the Aha' Ioa - position and this is important because the initial SAR in THCs was developed on the basis of these ebmpounds; using mainly dog ataxia test as used by ~oewe'~and rabbit corneal areflexia tests3. Similarly, the figures in the dog ataxia test indicated the relative potencies of various THCs compared to (f) -A 6a IOa _ THC. However, some significant differences were observed between the SAR in the dog , - .- and the rat. 54,55 Table 4 show list of heterocyclic analogs of THCs. These are compounds, which have a heteroatom in one of the rings of THCs. On the basis of some of the data available from animal studies in Tables 2 - 4, the following conclusions are drawn regarding the SAR in cannabinoids. (1) Essentially a benzopyran structure, with an aromatic hydroxyl group at C - 1 position and an alkyl or alkoxyl group at C - 3 positions, is a requirement for activity. If the above substitution pattern is altered it leads to major loss of potency, 56, 57 the exception is the dimethylheptyl analog (compound 56 in Table 3), which was found to be 10 times more potent than synhexyl (13) in the rat. Although a benzopyran ring is a definite requirement for activity the benzopyran by itself dose not confer activity. This is based on the observation that the ring-opened compound CBD (compound 41 in Table 2) is inactive in both the monkey and the dog and did not generalize to - THC in the drug discrimination test in the rat. Similarly, cannabichromene CBC
(Compound 48 in Table 2) and (Compound 69 - 71 in Table 3) which have an intact benzopyran ring are inactive. However, it is interesting to note that although, Cannabis - like effects may be absent, and other CNS effects may be retained. This is exemplified by several ring-opened compounds, which have shown potent analgesic effects (Compound 80 - 87 in Table 4) and several pyran depressant properties (Compounds 55 -58 in Tables 3 and Compound 73 in Table 4)
TABLE 2: Natural and synthetic THC and their pharmacological effects
I Compound Man (mglkg i. v) Mouse (mg/kg i. v) Comments N A
I A' - THC methyl ether (27) Much more toxic than A9 - THC in mice
I Diethylaminoethyl ether of - THC (28)
1 2 - Methyl - - THC (29) 8.0 reduced activity Active; by37 % at 10 equipotent with mglkg i.p. - THC compared to 42 % by A9 - THC
1 12(1Hydroxy - A9 - THC (30) -111 0' potency of A8 - THC for producing catalepsy but equipotent for producing hypothermia in mice A' - THC phosphate ester 9disodium salt) 1 (31) Inactive; dose not produce catalepsy in mice. High acute toxicity
I A' - THC sulphate (potassium salt) (32) Inactive; dose not produce catalepsy in mice
I Ax - THC glucuronide (sodium salt) (33) Inactive
1 A' - THC methyl ether (34) Mice: no analgesic activity
/ 1 1 - Methyl - A' - THC (35)
1 9 Nor- 9 - phenyl - A" THC (36)
1 9- Nor - 9n - butyl - A' - THC (37
I& .< ,I * 9 - Nor - 9 - isopropyl - A' - THC (38)
1 9 - Nor - 9 - benzyl - A' - THC (39)
1 9 - Nor - 9 - phenylethyl - A' - THC (40) As active as A' - THC; similar pharmacological I& profile in mice 9 - Nor - A' - THC (41) Inactive up to 30 Man; show mg i.v. and 100 anticonvulsant mg p.0. properties at dose of 200-300 mg and hypnotic properties at 160
(-) - Cannabidiol (CBD) (42) mg. C- I 1 methyl group is axial
1 1 a- Hexahydrocannabinol (HHC) (43) C-1 1 methyl group is equatorial more portent than axial isomer 1 1 P- Hexahydrocannabinol HHC (44) .<,, CH20Ac Acetoxymethyl I group is axial
1 I j3 - Acetoxy- Hexahydrocannabinol HHC (45) CH20Ac- Acetoxymethyl - group is equatorial more portent than axial isomer 11 u - Acetoxy- Hexahydrocannabinol HHC (46) 10a-Hyd roxy group is equatorial; more active
Hexahydrocannabinol HHC (47) Mice produces catalepsy, hypothermia etc but more potent than A' - THC 8 p,9 P - Epoxy - Hexahydrocannabinol HHC (48) Decrease activity at Potent analgesic in mice
Canbisol (49) --
Decrease activity 5- Drug discrimination in rats: EDSo0.36 mglkg; effective antinauseant in man at 2-mg p.0. dose.
Nabilone (50) Table 3: Synthetic 'Oa - THC and their pharmacological effects
1 Compound Biological test Comments Rat: relative potency Relative potency (synhexyl), 0.5 compared to synhexyl.
(R=C5H1,)
(+)-A~~,'Oa - THC (51) Synthetic Rat; relative potency (synhexyl), <0.4
(52) (-) I", 2"- dimethylhepthyl acetate homolog of THC (DMPH Synthetic) General CNS depression; analgesic activity
(53) DMPH water soluble derivative Potent to moderate CNS activity in rodent . screens p.0. - .-
Inactive at 20 mgkg Relative potency in dogs, 0.04
Rat: relative potency (synhexyl), 10
Rat: relative potency (synhexyl), 1
Inactive
R'=H or CH, IN _ R = CH,, C5H11,OH
Inactive at 10-20 mglkg p.0. in mice and rats Inactive
NHCOCH,
R
Active in mouse activity test only
NHCH,
CH(Me)CH(Me)C,H,,
Inactive
Active in mouse activity test only Active in mouse activity test only
Active in muricidal and mouse activity test only
Active in muricidal, septal-lesioned rat test and mouse activity test only
Inactive
Inactive at 10 mgkg Steroidal analog of i.v. THC inactive Cyclic analog of DMHP
CH,
Cyclic analcyclic Inactive analog of DMHP
(2) The position and the environment around the aromatic hydroxyl group are very important for activity, viz., (a) The OH at position C - 1 is in itself necessary for activity. Thus, the abnormal compounds where the hydroxyl and the side chain have been interchanged (i.e. side chain at C - 1 and OH at C - 3) are all inactive (Compounds 74 -79 in Table 4).
-, ,r . -1. *l' , . .'+ . Similarly, removal of OH at C - 1 eliminates'a~tivit~(Compound 57 -58 in Table 3). (b) Etherification of the OH at C -1 retains activity and, in some carbocyclic and heterocyclic analogs, can lead to greater selectivity of action e.g. in carbocyclic series (Compound 53 in Table 3); the morphine butyrate is a potent analgesic, whereas the 81 _ ." homopiperidin butyrate series shows potent anticonvulsant properties. Etherification of the OH at C -1 eliminates activity, as is clear from the methyl ethers of both A9 - and A8 - THCS (Compound 26 and 33 in Table 2 and Compound 52 in Table 3). The glucoronide and the sulfate of A8 - THC are also inactive (Table 2: Compound 31 and 32) and the phosphate (Compound 33 in Table 2) has only one-tenth activity of A8 -
THC. Replacement of the OH by NH2 retains, activity (Compound. 60 - 64 in Table 3); replacement by SH eliminates activity (Compound 59 in Table 3). (c) Since C -10 in the alicyclic ring C is in close proximity to the OH group, a methyl substituent at C - 10 significantly alters the activity in the case of a planar five-membered ring.57
(3) Substitution in the aromatic ring by electronegative group like carboxyl, carbomethoxyl, and acetyl groups eliminates activity, whereas alkyl or hydroxyl groups at C-2 position retain and in C-4 position reduce activity. The exception is compound 27 and 28 (Table 2), which are reported to produce general CNS depression.58
Table 4: Miscellaneous and their pharmacological effects Compound Comment Mice: decreased activity, Ed50 4.2 mgtkg i.v. Dog; ataxia; inactive Active in mice but at 0.5 mglkg i.v inactive in dog
Mice ; inactive at 100 inactive mgkg i.p.
(74) (-) - Abnormal -A~- THC Mice ; inactive at 100 inactive mglkg i.p.
(75) (-)-Abnormal -A' - THC 1 Mice: inactive at 100 mglkg i.p. ( (76) (-) - Abnormal -cis-A' - THC Mice: inactive
antihypertensiveDog; Antihypersensive in dogs but no behavioral effects koHOH (77) Rat ; antiflamatory activity
OH
(78) Rat: antiflamatory activity
(79) nonabine (BRL-4664) OH Mice: potent Potent analgesic analgesic
..,-?..I..,' ....I> . .
(80) fl-hydroxy isomer Mice; analgesic but Analgesic less potent than the 1' - ... P-hydroxy isomer
1 (81) a-Hydrory isomer I Mice; analgesic but Analgesic less potent than the 0-hydroxy isomer Mice; some analgesic 1 activity
Mice; inactive inactive
Potential analgesic in 3-10-fold potency animal tests of morphine
Potential analgesic in 8-25-fold potency animal tests of morphine
Potential analgesic in 60-190 -fold animal tests potency of morphine Rat; ptosis and Active Potential catalepsy, etc.; analgesic in decrease motor animal tests activity at 10-200 mgk3 P.0.
R = C5Hll
R' = HI CH, R" = HI CH,
(88) Rat; ptosis and catalepsy, etc.; decrease motor activity at 1- 10 mdkg P.0.
Rat; ptosis and Active catalepsy, etc.; decrease motor activity at 1-50 mglkg p.0.
R" = H, CH,
(90) Rat; ptosis and Active catalepsy, etc.; decrease motor activity at 50 mglkg p.0. (1) A minimum length of the aromatic side chain is necessary to elicit activity, and the branching of the alkyl side chain increases potency. Thus l', 2- dimethylheptyl or l', 1'-dimethylheptyl gives the most potent compounds. Similarly,
P-fluorophenyl alkyl and side chain good activity. - 0- CH (CH3) (CH2) 3 Ph, - CH2 -
CH = CH - CH C- CsHl I and - CH2CH2CHC5Hl1 (2) On ring B, the gem dimethyl group at C - 6 is essential for activity (Compounds number. 73, 77, and 78 in Table 3). Replacement of one of the geminal methyl groups at C-6 by a hydroxyl-methyl group retains activity (Compound 29 in Table 2 is as active - THC). Replacement of pyran 0 by N and ring expansion of ring B by one carbon (Compound 88 in Table 3):) leads to retention of activity. loa- (3) In the alicyclic ring C, compounds with the double bond in -,As, or position are active. It is noteworthy that compounds with a double bond in the A10, 10a
- position are also active. The 6a, 10a - cis isomer decreases inactivity when compared to the trans isomer. Tetrahydrocannabinols (natural occurring) are active in the 10a R and 6a R series only. A methyl at C - 9 increases and is essential for activity (Compound 85, 73 -78 in Table 2), but metabolism to the 11-hydroxymethy is not as prerequisite for THC activity, since 9 -nor-A8 THC - is active. Compounds are active even when no double bound are present in the ring e.g. HHCs. In this compounds, an equatorial group at C-9 increase activity and axial group decrease activity (Compound 42 and 44,..vs.;.A3..and45 in Table 2). Similarly, the activity is retained even when diverse groups are present such as epoxide, a ketone, or an alcohol (Compound 47,49,48 & 46 in Table 2). (4) The C-ring can be substituted by a variety of nitrogen and sulfur -containing rings without loss of activity. The most' active CNS agents are obtained when the heteroatom (Nitrogen and sulphur) is in a phenethyl orientation e.g. inserted in place of C-7 or C-9. (5) Planarity of the C - ring is not a necessary criterion for activity, can be seen in the quinuclidine analog and benzoxocine compounds. (Compound 88-91 in Table 4). (6) In both carbocyclic and heterocyclic analogs opening the pyran ring generally decreases activity. 1.6 Pharmacology (Therapeutic potential in man and laboratory animals) 1.6.1 Health Aspect of Cannabis The effects of Cannabis in man and animal models have their inherent health hazards, as they are with all drugs. Many adverse reactions to drugs are not recognized until after much exposure had occurred. Often these are idiosyncratic or allergic reactions. On the other hand, adverse reactions due to expansions of the pharmacological action of a drug may be recognized both early and late. A similar pattern holds for Cannabis: The ambiguity currently surrounding the health hazards of Cannabis may be attributed to a number of factors, which include: (1) It has been difficult either to prove or to disprove health hazards in man from animal studies, since such studies reveal possible harmful effects, the dose used are often large and treatment is generally short. (2) Cannabis is still used mainly by young persons in the best of health between the ages of 18 and 25 years and declines sharply after the age of about 34. 59,60 (3) Cannabis is often used in combination with tobacco and alcohol, as well as a variety of other illicit drugs. Thus potential health hazard from Cannabis may be difficult to distinguish from those of concomitantly used drugs. (4) The whole issue of Cannabis use is so often laden with emotion that serious investigations of the health hazards of the drug have been coloured by the prejudices of the experimenter, either for or against the drug as a potential hazard to health.
Similarly, the assessment of the. ,...... therapeutic, , ..?, potentials of marijuana is also clouded by prejudices, either for or against the drug. This is because virtually every claim of therapeutic benefit made of Cannabis is for conditions for which there are already many effective treatments. Under the health aspect of Cannabis main areas of interest are: (i) Acute and chronic effects of Cannabis in human, and (ii) Issues regarding its adverse effect on health, including its effect on driving ability.
1.6.2 Acute and Chronic Effects of Cannabis in Humans/Animals 1.6.2.1 Acute studies A~-THC is the major component of Cannabis and when smoked, a variable fraction of it is lost in the smoke, escaping into the air or exhaled from respiratory dead space. Relatively little is lost by pyrolysis, since it is likely that cannabinoid is volatilized in advance of the burning segment of the cigarette. The efficiency of the delivery of a dose by smoking has been estimated to be about 18%, but frequent smokers obtain
23%, while infrequent users obtain only 10%. 6' THC and marijuana extracts are also active by mouth; the systemic bioavailability of oral administration is only about 6%, one-third of that obtained from smoking.62 The effects and absorption of THC appears within minutes when smoked and if Cannabis is of how potency the effects may be subtle or brief. Seldom does the effect last longer than 2 to 3h after a single cigarette, although these can be prolonged by repeating smoking. Oral dose delays unset of symptoms for about 30 minutes to over 2 h, as well as prolonging the span action of the drug. Smoking is similar to intra venous (i.v.) administration in producing maximum plasma concentrations early, while post oral (p.0.) administration produces slower rises of maximum plasma concentrations, which are also lower than those for smoking63.The effects of rate of administration on the time course and intensity of Cannabis effects in man and animals have been reported. 64,65 Constant increase in pulse rate, slightly falling or unchanged blood pressure at higher doses, orthostatic hypotension and conjunctiva reddening has been reported. 66 This symptoms and the increasing pulse rate have been reported to correlate quite well in time with the appearance and duration of psychic effects of the drug as well as the plasma concentration of , ,the,, . . ,$rug.67.... '.. Muscle strength decreases, appetite is inconsistently augmented, and along with an increased food intake68 has been reported. Observed physiological effects have not included changes in pupil size, respiratory rate, or deep tendon reflexes. Perceptual and psychic changes are biphasic. An initial period of euphoria or "high" ii followed by drowsiness. Time sense is altered, hearing is less discriminate, and vision is apparently sharper with many visual distortions. Depersonalization, difficulty in concentration and thinking and dream - like state is prominent. However, the effects that users derive from Cannabis are extremely variable. Some of the variability depends on individual variation in degree of tolerance to the drug, based on prior use. 1.6.2.2 Chronic Studies The effects of chronic use of Cannabis are more to the point when considering the issues of its status as a possible social drug. In a study of Cannabis use in Jamaica with about sixty adult workers, all men, 30 were Cannabis smokers, and 30 were not, although the later might have used Cannabis as tea. No significant abnormalities were revealed from an intensive hospital study. The smokers claimed to use Cannabis so as to work better, but evidence in a selected subgroup supported slightly decreased performance.68 Similar study carried out in Costa Rica and Greece confirmed the finding above69, 70 . 8. Though, the finding concluded that the Cannabis users had a higher prevalence of personality disorders, probably unrelated to their use of Cannabis but possibly contributing to it.
A 30 - day high - dose Cannabis study in which 2 mg of THC per day were administered post oral (p.0.) to volunteers reveal that the subjects tolerated such large doses remarkably.70 A mild withdrawal reaction due to discontinuation of the drug, bradycardia and psychotomimetic effects were absent. A larger experimental study in which Cannabis was smoked rather than taken post oral (p.0.) exposed subjects from 35 to 198mg of THC daily for 78 days. It was found that Cannabis lowered intraocular pressure, lowered serum testosterone levels, narrowing of airways, lack of chromosomal alteration and unchanged response?'
, ., ,* -...... v , . *..-a 1.6.3.1 Possible Adverse Effects of Cannabis on Health 1.6.3.1.1 Immunity The human body protects itself form invaders, such as bacteria and viruses through the elaborate and dynamic network of organs.a;ld cells referred to as the immune system. Cannabinoids, especially THC, can modulate the function of immune cells in various ways (in some cases enhancing and in others diminishing the immune response). In vitro studies, using either human or animal material, suggest that cell mediated immunity may be impaired after exposure to Cannabis. However clinical evidence has been discovered suggesting that sustained impairment of cell mediated immunity might lead to an increase prevalence of opportunistic infections or malignancy. Despite some degree of impairment of immune response, however, the remaining immune function may be adequate, especially in young persons who are the major users of Cannabis. An impairment of cellular immunity in 51 chronic users of Cannabis was shown by inhibition of lymphocyte blastogenesis from the mitogen, phytohemagglutin.72 A decrease in T-lymphocytes was found in 9 of 23 chronic users, employing rosette formation as a way of quantifying; T-lymphocytes the number of lymphocytes was different from non users.73 Immunosuppression has also been shown in animals by prolonged allogenic skin graft survival, inhibited primary antibody induction to sheep erythrocytes and a diminished blastogenic response.74 Further studies have tend to also confirm an immunosuppressant action of Cannabis in animals given either post oral (p.0.) or injected inter peritoneal (i.p). 74p 75 Mice treated with THC and challenged with gram-negative bacteria showed enhanced susceptibility76 and other studies using in vitro techniques for studying lymphocytes have found no alteration in nucleic acid synthesis in the presence of as much as 10.6 x 1o-~ M concentration of an nab is.^^ However, other studies cast doubt on some of the earlier positive observations of impaired cellular immunity. Dinitrochlorobenzene is used as a skin test for intact delayed hypersensitivity, mediated by cellular immunity. No differences were observed in 34 chronic Cannabis smokers as compared to 279 non-smokers7'. Similarly, the response of cultured lymphocytes from 12 long-term smokers of Cannabis to two mitogens was not impaired as contrasted with lymphocytes from .',. '..c 71. %,.Z , , non-smokers79. Even the ingestion of Cannabis in amounts of 210 mg daily of THC failed to alter the response of the subject's lymphocytes to mitogen stim~lation.'~ In summary, it is still not known how much impairment is necessary to make someone vulnerable. Is . .:..
1.6.3.2 Chromosomal Damage Adverse effects on chromosomes of somatic cells have been especially controversial. Since the technique of human cytogenetic studies still leave much to be desired. As assessing damage to chromosomes is more of an art than a science. Early reports of Cannabis in the above were positive, but a more recent report was negative. A significant increase (3.4 versus 1.2 %) of chromosomal abnormalities was reported in Cannabis users as compared with non-users." Changes were larger breaks or translocation of chromosomes with more of the later found in chronic Cannabis users than in non-users. When these breaks where included in the counts the differences ~anished.'~No increase in chromosomal breaks was found in cells from subjects taking post oral (p.0.) Cannabis extract (containing only THC) or synthetic THC.') Finally both retrospective and prospective studies have flaws and as such one simply cannot conclude the issue as settled.
1.6.3.3 Pregnancy and Foetal Development 1.6.3.3.1 Animal Studies Virtually every drug that has been studied for dysmorphogenic effects has been found to have them if the doses are high enough, if enough species are tested or if treatment is prolonged. Since the placenta is not a barrier to the passage of most drugs, it should be assumed that the drugs would reach the foetus if taken during pregnancy.84 This assumption is well validated for THC, based on autocardiographic studies." A high incidence of stunting of foetuses was seen in mice tested on the 6th day of pregnancy with a single inter peritoneal (i.p) dose of 16 mg Cannabis resin per kg. No changes in litter sizes or malformations were reported and when the experiment was repeated from day 1 to 6 of pregnancy, foetal resorption was complete.86. Exposure of pregnant rats to either Cannabis smoke or smoke from extracted Cannabis throughout gestation period produced less fertile offspring with small reproductive organs in Cannabised-treated animals87p*'. Similarly exposure to THC in uterus can result in long-term ~hang,es..~..Many uterus effects interfere with embryo implantations9 it has been reported that exposure of THC shortly before or after birth can result in impaired reproductive behaviour in mice when they reach adulthood;
female are slower to show sexual receptivity and males are slower to mount 90. However, it has been reported that pregnant rabbits treated post oral (p.0.) with daily doses of THC at 15 mglkg on days 6 to 18 of gestation delivered infants without visible abnormalities and injecting subcutaneous (s.c.) doses of THC up to 100 mglkg daily in days 6 to 15 gestation had no tetratogenic effect. 91,92 Although THC can act directly on endocrine tissues, such as the testes and ovaries, it appears to affect reproductive physiology through its actions on the brain, some where other than the pituitary. Some of the effects of THC are exerted through its action on stress hormones, such as cortisol.93 1.6.3.3.2' Human Studies Few human studies are consistent with the acute animal studies and THC inhibits reproductive functions. An epidemiological study found more meconium staining of the foetus and more disturbances of the duration of labour (either short or long) among 35 users as compared to 36 non-users of ann nab is^^. However, no significant differences were reported for moderate and heavy users, as regard to neonatal outcomes." A large study involving 12,424 women of whom, 1246 (1 1%) were Cannabis users indicated lower birth weight, a shorter gestation period and more major malformations were reported against offspring of users.9s No change in serum human chorionic gonadotrophin, placental lactogen, progesterone, estradiol, and estriol were found in 13 women who smoked Cannabis during pregnancy, compared with a matched control number who did not.96
1.6.3.4 Cell Metabolism There is limited information concerning the effects of Cannabis in cell physiology and metabolism. Smoke from both Cannabis and tobacco increased the size of the cytoplasm, nuclei, and nucleoli along with an increase in DNA content of human lung cell e~~lants.~~Mitotic abnormalities were also noted with an increase of 10 to 25% over those of controls.89 A combination of both smokes (tobacco and Cannabis) produced greater abnormalities than either one alone. Malignant cell transformation of hamster lung culture was ..o)x~ed..,.after.the administration of both types of smoke97. These findings suggest that Cannabis smoke are. harmful to lung cells in cultures and contributes to the development of premalignant and malignant lesions. Cannabinoids have also been reported to interfere with the normal cell cycle by a reduction in growth rate during log phase and zilengthening of the mean division time upon exposure of THC to protozoan, ~etrah~mena~~and are dose dependent. The' decrease in the synthesis of DNA, RNA, and protein on addition of THC to various human and animal cultures has been reported.99
1.6.3.5 Psychopathology Cannabis may produce directly an acute panic reaction, a toxic delirium, an acute paranoid state or acute mania. Whether it can directly evoke depression or schizophrenic states, or whether it can lead to sociopathy or even "amotivational syndrome" is much less certain. The existence of specific Cannabis-related psychosis, postulated for many years, is still not established. The fact that users of Cannabis may have higher levels of various types of psychopathology does not infer a causal relationship. Rather, the evidence suggests that virtually every diagnosable psychiatric illness among Cannabis users began before the first use of the drug. Use of alcohol and tobacco, as well as sexual experience and "acting-out" behaviour, usually antedate the use of Cannabis.loo When the contributions of childhood misbehaviour, school behavioural problems, and associated use of other illicit drugs were taken into account, it was difficult to make a case for a deleterious effect of . ., ,, regular Cannabis use.lO' Thus, it seems likely that psychopathology may predispose individual to Cannabis use rather than the other way round.
1.6.3.5.1 Acute Panic Reaction This adverse psychological consequence of Cannabis use is probably the most frequent. It has been reported that about one in five users of Cannabis in schools experienced anxiety, confusion or other unpleasant effects. These unpleasant experiences were not always associated with unfamiliarity with the drug. Some subjects experienced these adverse reactions after repeated use.lo2 the conventional wisdom, however, is that such acute panic reactions occurs more commonly in relatively inexperienced users of Cannabis, more commonly when the dose is larger than that to which prior users may have become accustomed, and more commonly in older users who may enter the dsg .st& ~itha higher level of initial apprehension.lo3 the acute panic reactions associated with Cannabis are similar to those previously reported to be caused by hallucinogens. The subject is most concerned about losing control, or even of losing his or her mind. Reactions are usually self-limited and may respond to reassurance or "talking down'"; in the case of Cannabis use, sedatives are rarely required as the inherent sedative effect of the drug, following the initial stimulation, often is adequate. Occasionally one may see a dissociative reaction, but this complication is readily reversible.
1.6.3.5.2 Toxic delirium Very high doses of Cannabis may evoke toxic delirium, manifested by marked memory impairment, confusion and disorientationlo4. This non-specific adverse psychological effect is seen with many drugs, but the exact mechanism is not clear in the case of Cannabis smokers. 1.6.3.5.3 Psychoses A variety of psychotic reactions have been ascribed to Cannabis use. Many are difficult to fit into the usual diagnostic classifications. Two cases of manic reactions have been reported105 in children who were exposed to Cannabis by elders. In both cases the manic reaction was treated with antipsychotic drugs and both ultimately showed full recovery.lo5 Similarly, there are clinical reports of Cannabis-induced psychosis-like states (schizophrenia-like, depression and /or mania) lasting for a week or more.Io6 ~ollister'~~su~~estedthat because of the varied nature of the psychotic states induced by Cannabis, there is no specific "Cannabis psychosis". Rather, the marijuana experience might trigger latent psychopathology of many types. It has also been reported that there is reasonable evidence that heavy use, and perhaps acute use in sensitive individuals can produce an acute psychosis in which confusion, amnesia, delusion, hallucinations, anxiety, agitation and hypo-manic symptoms predominate108. The association between Cannabis and schizophrenia is not well understood. The scientific literature indicates general agreement that heavy Cannabis use can precipitate episodes but not that Cannabis can cause the underlying psychotic disorder ' I. Estimates of the prevalence of Cannabis use among schizophrenics vary considerably but there is general agreement that it is at least as great as that among the general population'12. Schizophrenics prefer the effects of Cannabis to those of alcohol and cocaine .~hicb.,theyseem to use less often than in the general population. 113, 114 The reason for this are unknown, but it is possible that schizophrenics might obtain some symptomatic relief from moderate Cannabis use. But when compared with by the general population, people with schizophrenia or with family history of schizophrenia" are- likely to be at great risk for adverse psychiatric effects from the use of cannabinoids.
1.6.3.5.4 Flash backs This curious phenomenon, in which events associated with drug use are suddenly thrust into consciousness in the no drugged state, has never been satisfactorily explained. It is most common with LSD and other similar hallucinogens but has been reported fairly often with Cannabis use. It was initially thought to be associated with the use of LSD as well as Cannabis, but more recent experiences indicate that it occurs in those whose sole drug use is Cannabis115. One possibility is that flashback represents a kind of de'jd vu phenomenon. Another is that they are associated with recurrent paroxysmal seizure-like activity in the brain. The most unlikely possibility is that they are related to a persistent drug effect. They may occur many months removed from the use of either LSD or Cannabis, so that it is highly unlikely that any active drug could still be present in the body. Further, the interval between last drug use and flashback is one in which the subject is perfectly lucid. For the most part, the reactions are mild and require no specific treatment.
1.6.3.5.5 Violence The myth dies hard that Cannabis makes otherwise docile subjects violent. Virtually every experimental study of Cannabis that has tried to measure violent or aggressive behaviour or thought during Cannabis intoxication has come to the same conclusion; that they are decreased rather than increased. It has been reported that physical aggression was related to the quantity of alcohol taken, but not to any dose of THC when Cannabis was compared with alcohol in forty students 1 16,117 . Similarly, it has been reported that Cannabis does not precipitate violence in the vast majority of users. 'I8 The possibility was entertained that a rare individual with some special predisposition to aggressive or violent behaviour may be triggered into expressing such behaviour under the influence of the drug.'
1.6.3.5.6 Amotivational S,yadrome,> One of the more controversial effects claimed for Cannabis is the production of an "amotivational syndrome". This syndrome is not a clinical situation, but it has been used to describe young people who drop out of social activities and show little interest in school, work or other goal-directed' activity. It has been postulated that the apparent loss of motivation seen in some Cannabis users is really a manifestation of a concurrent depression, for which Cannabis may have been self-prescribed treatment 'I9. When moderately heavy Cannabis use accompanies these symptoms, the drug is often sited as the cause, but no convincing data have demonstrated a causal relationship between smoking Cannabis and these behavioural characteristics 120 - 125 Laboratory studies have provided only scanty evidence for this concept. It has been reported in a study in Canada that decrease in productivity following smoking Cannabis was due to less time put into the work than lessened efficiency at work.126 There is not enough evidence to conclude that a chronic Cannabis user lacks motivation, instead, relevant personality traits and behaviour of subjects must be assessed before and after the subject becomes a heavy Cannabis user.
1.6.3.5.7 Residual Psychomotor Impairment Cannabis administration has been reported to affect psychomotor performance on a number of tasks. The types of psychomotor function that have been shown to be disrupted by acute administration of Cannabis include body sway, hand steadiness, rotary pursuit, driving and flying simulation, divided attention, sustained attention, and the digit-symbol substitution test. A study of experienced airplane pilots showed that after a single Cannabis cigarette their performance in flight simulator tests was impaired'27. Similarly, experimental studies carried out in rats following the chronic administration of Cannabis indicates psychomotor impairment but, such studies also are always difficult to extrapolate to man.12* A comparison of 23 chronic users of Cannabis with 11 non-users revealed some evidence of impairment in the users with the latter having lower intelligence and memory quotients with lower scores on psychomotor test.'29
1.6.3.6 Tolerance and Dependence The rate at which tolerance to the various effects of any drug develops is an important consideration for its safety and efficacy. For medical use, tolerance to some effects of cannabinoids might be desirable..<,.D$Eerence.in the rates at which tolerance to the multiple effects of a drug develops can be dangerous. For example, tolerance to the various effects of cannabinoids might develop at different rates; it is important to evaluate independently their effects on mood, motor performance, memory, and attention, as well as any therapeutic use udder investigation. Tolerance to most of the effects of Cannabis can develop rapidly after only a few doses, and it also disappears rapidly. Tolerance to large doses has been found to persist in experimental animals for long periods after cessation of drug use. Performance impairment is less among people who use Cannabis heavily than it is among those who use Cannabis only oc~asionall~'~~-'~~possibly because of tolerance. Definite evidence of tolerance to the effects of THC in man was adduced only when it became permissible to use comparably large doses over longer period of time. Subjects in one 30-day study were given high doses (70 to 210 mglkg) of THC post oral (p.0.) around the clock. Tachycardia actually reverts to bradycardia, and a progressive loss of "high" was noted.'15 Similarly tolerance to Cannabis smoking was observed in a 64-day study in which at least one cigarette daily had to be smoked with smoking as desired later in the same day. Additionally, on this study tolerance developed to the respiratory depressant effect of THC.'~~.Heavy users tend to reach higher plasma concentration of THC than light users after similar doses of THC, going against the arguing that heavy users show less performance impairment because they somehow absorb less THC (perhaps due to differences in smoking behaviour) 13'. The different effects of Cannabis smoking and oral THC in the development of tolerance have been reported in a daily Cannabis residential study. 138p139 Onegroup was given Cannabis cigarettes to smoke four times per day for four consecutive days; another group was given THC pills in the same schedule. During the four-day period, both groups became tolerant to feeling "high" and what they reported as "good drug affect". In contrast, neither group became tolerant to stimulatory effects of Cannabis or THC on appetite.
1.6.3.6.1 Physical dependence /Withdrawal Evidence from both animals and man indicates that physical dependence can be induced by abuse of THC. All monkeys given automatic injections of doses of THC of 0.1 to 0.4 mglkg showing abstinence signs when withdrawn. When monkeys were allowed to self-administration of the drug for 3 to 8 weeks, majority has an abstinence
syndrome when the drug was .,abypfly,,,discontinued. ,I . The syndrome appeared approximately 12 h after the last administration and lasted 5 days. It was characterized by irritability, aggressiveness, tremors, yawning, photophobia, piloerection and penile erection.l4' In man similar, though mild, withdrawal reactions was observed after abrupt cessation of doses of 30 mg of THC given every 4 hours post oral (po) for 10 to 20 days. Subjects became irritable, had sleep disturbances and had increased appetite. Nausea, vomiting and occasional diarrhoea were encountered. Sweating, salivation and tremors were autonomic signs of abstinence.14' A distinctive Cannabis and THC withdrawal syndrome has been identified, but it is mild and subtle compared to the profound physical syndrome of alcohol or heroin withdrawal. 142, 143 THC symptoms of Cannabis withdrawal include restlessness, irritability, mild agitation, insomnia, sleep disturbance, nausea and crimping; fatigue and illusion or hall~cination'~~.In a residential study of daily Cannabis users, withdrawal symptoms included sweating and runny nose, in addition to those mentioned above.'45
1.6.3.7 Lung Problems Tobacco is the predominant cause of such lung diseases as cancer and emphysema, and Cannabis smoke contains many of the components of tobacco smoke146. However, smoking is the most efficient method administering Cannabis, due to the enormously high lipid solubility of THC. The pulmonary surfactant is a perfect trap for THC, which is then rapidly absorbed into the blood. The phannacokinetics of the THC administered by smoking is similar to those of its intra venous (i.v) administration.
1.6.3.7.1 Bronchial and Pulmonary Damage A number of animal studies have revealed respiratory tract changes and disease associated with Cannabis smoking. Extensive damage to the smaller airways, which are the major site of chronic obstructive pulmonary disease (COPD) 147 and acute and chronic pneumonia have been reported in various species exposed to different doses of Cannabis smoke. Is', 152p153Similarly, results of human studies suggest that there is a greater chance of respiratory illness in people who smoke Cannabis. Studies carried out in Cannabis smokers, compared with tobacco smokers and non-smokers revealed that out of a group of 446 volunteers, IS -20.% of the Cannabis smokers showed or reported symptoms of chronic bronchitis, including cough, and phlegm production'48. Similar, findings were reported on subjects who had an additive effect of smoking both Cannabis and tobacco. 149
1.6.3.7.2 Bronchial Tissue changes Habitual Cannabis smoking is associated with changes in the lining of the human respiratory tract. It has been reported that many Cannabis and tobacco users have increased redness (erythema) and swelling (edema) of the airway tissue and increased mucous secretion.150, 151 A similar report involving both Cannabis and tobacco smokers revealed that the number and size of small blood vessels in the bronchial wall are increased, tissue edema is present and the normal ciliated cells lining the inner surface of the bronchial wall are largely replaced by mucous-secreting goblet cells. '52, A 1998 study has shown that both Cannabis and tobacco smokers have significantly more cellular and molecular abnormalities in bronchial epithelium cells than non- smokers; these changes are associated with increased risk of cancer.'54Earlier studies, similarly, have shown that such abnormalities are greatest in people who smoke both Cannabis and tobacco; hence these agents might have additive effects in airway tissue.15j' 150p I5l Similar results in U.S servicemen who suffered from respiratory symptoms and where heavy hashish smokers (hashish as the resin from the Cannabis plant) have been reported. ' 55
1.6.3.7.3 Macrophages Alveolar macrophages are the principal immune effector cells in the lung and are primarily responsible for protecting the lung against infections by microorganisms, inhaled foreign substances and tumour cells. They are increased during tissue inflammation. It has been reported that in a large sample of volunteers, habitual marijuana smokers had twice as many alveolar macrophages as non-smokers and smokers of both tobacco and marijuana had twice as many again.156 Cannabis smoking has been reported to reduce the ability of alveolar macrophages to kill fungi, such as Candida albicans, 157 pathogenic bacteria such as Staphylococcus aureus and tumour cells. The reduction in ability to destroy fungal organisms was similar to that seen in tobacco smokers. Furthermore, it has been reported that Cannabis smoking depresses production of proinflampy$~ry..~ytokines,., ,, such as INF-I and IL-6, but not of immunosuppressive cytokine.15'
1.6.3.7.4 Cardiovascular Problems Cannabis smoke and oral THC can causeltachycardia (rapid heart beat) in humans.'59 In some cases, blood pressure increases while the person is standing resulting in postural hypotension (deceased blood pressure due to a changing posture from a lying or sitting position to a standing position, which can cause dizziness and faintness) has also been reported. 160, 161 In animals, decreases in heart rate and blood pressure due to THC have been reported1607162. However, most of animal studies have been conducted in anaesthetized animals, and anaesthesia causes hypotension. Thus, these studies should be interpreted as reports on the effect of cannabinoids in hypertensive subjects. The results of the animal and human studies are consistent with the conclusion that cannabinoids cause hypotension at high doses in animals, as well as man.'63 A direct test of the effects of Cannabis smoking compared with Cannabis placebo cigarette in exercise-induced angina have been reported to be harmfbl with the Cannabis smoking deceasing exercise time until angina by 48 % and Cannabis placebo cigarette smoking exercise time until angina by only 9 % has been re~0rted.l~~Premature ventricular contractions have been reported following marihuana smoking '". However, when subjects were continually monitored electrocardiographically while smoking cigarettes containing approximately 20 mg of THC, no increase in such premature beats was formed.'66
1.6.4 Therapeutic uses
Throughout its history, Cannabis has been reported to have clinical utility. 167 The literature is replete with anecdotal reports of the therapeutic usefulness in various disorders but few of these are backed by sound scientific research. It was not until pure active principles and synthetic analogues became available, that folklore stories could be tested clinically in both laboratory animals and man for predicting possible 168, 169 psychic side effects as well as defining their therapeutic potential , There have been reports to indicate that the cannabinoids may be effective in the treatment of pain, convulsion, glaucoma, muscle plasticity, bronchial asthma, nausea and vomiting'70. These disorders .are.ounently. treated with drugs that are structurally distinct from cannabinoids.
1.6.4.1 Multiple Sclerosis/Neurology Multiple sclerosis is an autoimmune disease-'in which the victim's immune system attacks the myelin sheath of the nerves. It is an extremely variable disease, but often manifests itself with symptoms of plasticity, tremour, pain bladder and bowel dysfunction, and consequently sleep problems.171 Cannabis has been found to reduce muscle spasm and tremors in people who suffer from cerebral palsy or multiple sclerosis 172. It has, however, also been found to impair posture and balance in patients with spastic multiple sclerosis 170 A double-blind study reported in 1984 of four patients with extension spasticity found that THC significantly reduced spastically by clinical mea~urement'~~.A study of single MS patient found that his chronic motor handicaps acutely improved while he smoked a Cannabis cigarette; there spasticity and ataxia were quantitatively assessed.174 Dose related improvement in dystonia, ranging from 20 -50 %, was- observed in a patient administered with cannabidiol ((CBD) a non-psychoactive cannabinoid of marijuana) orally but worsened the coexisting Parkinsonian features in two persons when the dose was higher.I7' Similarly, accounts of Cannabis being used successfully to treat motics in Tourette's syndrome, torsion dystonia and Huntington's chorea have been reported. 176, 177 It has been argued that the effect of Cannabis in such cases may be related more to its sedative, anxiolytic or analgesic effects than to any specific central effect. This was the conclusion drawn from the study of Parkinsonian tremor.'78 This trial, though prompted by anecdotal reports of benefit, failed to find any improvement in patients' tremor. The benefit of the use of Cannabis by patients suffering from MS has been reported and Cannabis found to ease the muscle spasm in the bladder that makes it difficult for them to urinate179. Some doctors have similarly, prescribed the synthetic cannabinoids, nabilone, to MS patients with good result, although some patients apparently found nabilone less effective than Cannabis. A general improvement of the well being of MS patient with muscle spasm has been reported when nabilone was administered as compared to placebo180.
1.6.4.2 Antiemetic Effects Antiemetic agents are drugs ~hat+,~pn~~l/prevent/amestnausea and vomiting."' Cancer chemotherapy, especially with the agent cisplatin, produces sever nausea and vomiting, which is extremely difficult to treat with conventional antiemetic drugs such as prochlorperazine. This complication is so severe that many patients forgo the effective cancer 'chemotherapy. I*' Thisuphas-led to the use of some cannabinoid analogs or synthetic materials like nabilone and dronabinol as an antiemetic for treatment of nausea and vomiting associated with cancer chemotherapy (has become popular) Cannabinoids are as effective in this area as phenothizaines, with which they may be combined, but incidence of adverse effect is high. A third of patients in some studies experienced dysphoria and up to 80 % had somno~ence.'~~ The suggestion that Cannabis (THC) might be useful as an antiemetic arose as early as 1972 when it was used along side placebo in 20 patients under cancer chemotherapy in a controlled trial, definite antiemetic effect was reported by 14 of the patients and none from placebo. Is6 THC was compared with two other antiemetics, effect. Cannabis does not cure the disease but can slow the progressive loss of sight when conventional medicines fail and surgery is too dangerous'97p "8. Many experiments done in rabbits using various routes of administration, including instillation of cannabinoids to the eye, have confirmed the ability of Cannabis to reduce intraocular pressure. Topical administration would be especially desirable for treating glaucoma as with the other drugs used for this purpose. Smoking Cannabis or taking THC iv would be totally unusable for patients with glaucoma. Rather the eye-drops containing THC is preferable since the THC, being lipid soluble, is stabilized using mineral oil as the vehicle for instillation into the eye.lg9The degree of lowering of intraocular pressure is at least as great as that with conventional eye drop, such as pilocarpine, and the duration of effect is often longer. Studies have continued on certain synthetic and natural cannabinoids such as cannabinol or A3 and p8 - 1 1-dihydroxy -A9-THC. They produce some minimal systemic absorption when applied to the conjunctivae, but it is of no consequence in producing mental effects.200 Similarly, an extract of a nonpsychoactive component of Cannabis whose composition is still uncertain has been tested both alone and in combination with timolol eye drops in patients with increased intraocular pressure. The effects of the two agents are additive and are said to be effective when other measures have failed.201 Intraocular pressure, though reduced acutely, have not been shown to be reduced following long-term treatment, nor has there been any demonstr~t~n,~thatzvisualfunction is preserved by the use of cannabinoids in glaucoma. In summary, there has been considerable interest in the use of Cannabis as anti- glaucoma agent despite their side effects (psychiatric, hypertension etc) and the lack of topical application.202 Understanding'carfnabinoids mechanism of action could provide valuable insight into the etiology of glaucoma.
1.6.4.5 Prostaglandin synthesis It has been suggested203 that cannabinoids alter intraocular pressure by altering prostaglandin synthesis in part because prostaglandin has been reported to increase intraocular pressure204. In addition, it has been shown that A9 -THC attenuated increase in intraocular pressure produced by arachidonic acid in rabbit, an effect which was interpreted as being due to inhibition to prostaglandin synthesis.205While there is an interesting relationship between cannabinoids and prostaglandin, definitive evidence linking prostaglandin to cannabinoids effect on intraocular pressure is lacking.
1.6.4.6 Anticonvulsant One of the first therapeutic uses suggested for Cannabis was as an anticonvulsant and its effect was documented experimentally many years ago.206 Subsequent studies in various animals species have validated that action. THC in cats temporarily reduced clinical and electrographic seizure activity induced by electric stimulation of subcortical Mice were protected by cannabidiol (CBD) against maximal electroshock seizures but not against those caused by pentylenetetrazole. Its profile of activity resembled that of phenytoin than that of THC208-2 10 . THC and cannabidiol both potentiated the anticonvulsant effects of phenytoin against electrically induced seizures in mice. The two cannabinoids in combination produced the most effect?'
2'2 THC given chronically to rats prevented the kindling effect l . Using the hind limb extension effect of a larger dose of pentylenetetrazole (PTZ) it was shown that CBD, like diphenylhydantoin (DPH), was effective in blocking the actions, whereas
THC was only partially active.215'2'6 Thus cannabinoids appear to possess a real potential for anticonvulsant activity in human; with cannabidiol being of particular interest due to its lack of other CNS effects.
.,,.-. + .r. .v .. ... Y.? , . 1.6.4.7 Analgesic There are many folkloric/anecdotal reports of Cannabis smokers using the drug to reduce migraine, dysmenorrhoea and other types of pain. However, its disadvantage is its inconsistent effect; (i.e. sometlines it heightens sensitivity to pain). The synthetic cannabinoid, levonantradol, is an effective analgesic for postoperative and pain caused by cancer218but at the cost of adverse effects. A clinical trial is now underway to investigate whether Cannabis can provide analgesia at doses that do not produce central effecta219 Similarly they have been studies involving the use of Cannabis as analgesia in neurotransmitter receptors, opioid receptors and prostaglandin receptors.220 1.6.4.7.1 Neurotransmitter receptors It has been suggested that a catecholaminergic link between morphine and A~-THCin that yohimbine, an alpha2 antagonist, will block the effect of both in the tail flick procedure.221However, it is quite possible that opiates and cannabinoids can exert similar effects on catecholamines through different mechanisms. It has also been reported that cannabinoids exert their effects through a pathway that is parallel to that of morphine and this may account for cannabinoid insensitivity to opiate antagonists.222
1.6.4.7.2 Opioid receptors The interrelationships between cannabinoids and opioids have been examined and the findings were inconsistent, particularly with regard to opioid antagonist blockage by cannabinoid antinociception with respect to its interaction with opioid receptors. At higher concentrations that far exceed those of opioid analgesics, cannabinoids alter the in-vitro binding of ~~ioids.~~)
1.6.4.7.3 Prostaglandins It is generally recognized that prostaglandins are involved in some aspects of the expression of pain e.g. amplification of pain during the inflammatory process.224It is therefore reasonable to speculatehai .prostaglandins can be involved in cannabinoid induced antinociception in light of their association with pain and the fact that cannabinoids alter prostaglandin synthesis. A striking observational similarities between PGE, levonantradol, a cannabinoid with potent antinociceptive properties, have been reported.225 It was postiilated.".that levonantradol interacts with a prostaglandin receptor coupled to adenylate cyclase to inhibit AMP formation the same way that has been proposed for morphine.226In this manner, cannabinoids and morphine would be capable of sharing certain pharmacological properties, such as antinociception and antidiarrhoeal activity, without the necessity of cannabinoids binding to an opioid receptor. The ability of PGE2 to produce diarrhoea and emesis in
228 was noted as a further support for this hypothesis in addition to the observations that levonantradol can block prostaglandin-induced diarrhoea225 and numerous emetic stimuli in cats229and humans. 230.23 1 Similarly, early work in animals using Cannabis extracts indicated that there was
some analgesic activity in ann nab is.^^^. 233. Since these studies, it has been demonstrated that A'-THC is active in a wide variety of procedures used to test 236,237 analgesics. It is achieved in the writhing test2341 235 the hot plate procedure, and
in the tail flick test234, 235. In these studies both rats and mice were Furthermore, it was shown that the antinoceptive effects of A'-THC were not antagonized by nalorphine, indicating that A'-THC differs from morphine in these procedures.237 The only non-rodent reports of analgesic activity for Cannabis constituent are the activity in cats"' and A'-THC effects on dog against electric stimulation.240
1.6.4.8 Anti inflammatory Activity The traditional use of Cannabis for medicinal purposes of certain inflammatory disorders has been reported and CBD which is one of the components was found to be more effective than aspirin as an anti-inflammatory agent.241However, considerable discrepancy has been reported as to the activity of cannabinoids in a number of animal tests for anti-inflammatory, mild analgesic and anti-pyretic activity. It has been reported that A'-THC was 20 times as potent as aspirin and approximately twice as potent as hydrocortisone in carrageenan edema test in rats.242These results were not confirmed when A'-THC was orally administered and it was inactive in blocking the carrageenan-induced edema in We 'fht''pit6 The investigators of the above reports disagreed as to the activity of A'-THC as an antipyretic and as a mild analgesic. The former reported analgesic but not antipyretic and the later reported antipyretic and not analgesic. These discrepancies, however, were attributed to the ,' . ... vehicles used to suspend A'-THC. The former suspended the cannabinoid in undiluted propylene glycol whereas the later used fatty acid poor serum albumin. In addition, studies have shown that cannabinoids can exhibit anti-inflammatory . activity. For example, in rats with autoimmune encephalomyelitis (an experimental model used to study multiple sclerosis), cannabinoids were shown to, attenuate the
signs and symptoms of central nervous damage."'? 245 Cannabichromene, a cannabinoid found in marijuana, has also been reported to have anti-inflammatory properties.2461t is important to note that no mechanism of action for possible anti- inflammatory effects of cannabinoids has been identified, and the effects of these atypical cannabinoids and effects of marijuana are not yet established.
1.6.4.9 Anti-asthmatic Cannabis causes bronchodilation and Cannabis derivatives have therefore been tested as anti-asthma Smoking Cannabis results in chronic irritation of the bronchi by tars and other substances, so recent research has recommended other methods of administration. THC (cannabinoids) administered orally or in the form of an inhalerlaerosol spray has been investigated and used for treating early phase of bronchial asthma.248-25 1 However, little is known regarding the mechanism by which cannabinoids produce this effect. When compared to other more familiar anti-asthma drugs, it does not appear that the bronchodilation action is due to antimuscarinic or P - adrenergic agonistic activity.252
1.6.4.10 Antibiotic Cannabis has shown some promises as an antibacterial and anti-fungal agent.253 Cannabidiol has been found to have antibiotic activity against Gram-positive bacteria in ~itro.~~~Both THC and cannabidiol have been reported to inhibit and kill staphylococci and streptococci in ~itro.~~~
1.6.4.11 Appetite Stimulant. ... .,. %., . There is considerable information in the anecdotal literature about the effect of marijuana on food intake. Cannabis and THC may help to increase food intake and slow down weight loss in cancer patients.256Dornabinol is under trial in the US as an appetite stimulant for AIDS patients.25' - ." A double-blind study of smokers of Cannabis or placebo cigarettes showed increased caloric consumption in those using the drug. 257 It has been reported that inter peritoneal (i.p) administration of A~-THCcause a dose-related decrease in food intake if the food was presented 2 h after the drug was given in animals. It was also reported that, if the A~-THCis given 16 h before eating instead of immediately before eating, there was little effect.258A decrease in food intake and body weight has been reported in rats given behaviourally effective doses.259Similarly, A~-THCgiven intravenously twice a day causes reduction in food intake and body weight; though, if administered for more days the magnitude of the decrease was less as a result of tolerance to THC.~~O However, most antipsychotic agents will stimulate appetite. THC as compared with ethanol and dextroamphetamine produces a variable response on appetite, both in fed and fasted subjects. The majority had increased appetite and food consumption as compared with placebo.257
1.6.4.12 Miscellaneous Uses .. , 1.6.4.12.1 Antineoplastic and Immunosuppressant Activity Both the d8-and A9-THC isomers, as well as cannabinol, have some antineoplastic effect on transplanted lung tumours in animals, as well as on tumours in ~itro.~~'THC may have a general ability to reduce the synthesis of nucleic acids, which may account for the reported immunosuppressant effects as well. d9-THChas been reported to inhibit some marrow leukopoesis and 11- hydroxy -d9-THCwas found to inhibit myelopoesis. 262,263
1.6.4.12.2 Anorexia A dose-related anorexia in rats has been reported following the administration of d9- THC. The potency was approximately half that of de~tro-am~hetamine.~~~
1.6.4.12.3 Antifertility Actiw "' -,.'..;' ' . It is now well established that d9-THCand a number of THC analogs have a pronounced effect throughout the male and female reproductive systems. In essence, it suppresses the secretion of luteinizing hormone and ovulation.264 It _ ... A~-THChas been reported to decrease biosynthesis of lutenizing hormone-releasing hormone (LHRH) 265 rather than increasing its release. Similarly, the administration of testosterone in the male reproductive system of rat was not blocked when A9-THC was given.266In another study; d9-THCproduced a decrease in testosterone levels as well as a decrease in LH in rats. Both of these effects appeared to be temporary since the levels of hormones returned to control values during chronic treatment with the ~annabinoid.~~~~functional disruption of gonad functions has been reported to be caused by cannabinoid~.~~~Similarly, some reports have shown that d9-THCand other cannabinoids are estrogenic, while others are anti-e~tro~enic.~~~Thepotential use of A9-THC as an antifertility agent is low but it is possible that some of the newer derivations and analogs may possess activity in this area.
1.6.4.12.4 Gastrointestinal effects Detailed gastroenterological data have not been published on cannabinoids. The charcoal-meal test has been used to demonstrate that A9-THC and cannabidiol cause parallel dose-dependent inhibition of intestinal mobility in mice. 237p 270 The effects of, A~-THCand cannabidiol were synergistic though, when compared to morphine these agents were less potent and not antagonized by nalorphine. A9-THC does not leave any anti-secretory activity, neither does it increase gastric secretions.
1.7 The Legal - Illegality of Clinical Cannabis use in Modern Therapy Drugs, says psychiatrist Thomas Szasz, have taken the lead role from sex in "the grand morality play of human existence". "No longer", says Szasz "are men, women, and children tempted, corrupted and ruined by the irresistibly sweet pleasures of sex; instead, they are tempted, corrupted and ruined by the irresistibly sweet pleasures of drugs". 27' Because dealing with drugs is viewed as a moral problem, politicians tend to compete in their zeal to banish the evil from the society. Those who talk of legalisation are dismissed as mavericks, and whipped back to line2721 273. some form of legalization would help since an exception for legal drugs like alcohol, nicotine and caffeine has been approved. Yet-apworld devoid.of drugs seems as unlikely as a world devoid of poverty and sin. Thomas Syndenham observed 300 years ago that "among the remedies which it has pleased Almighty God to give to man to relieve his sufferings, none is so universal and so efficacious as opium" 274 and Aldons Huxley wrote, "that humanity at large will ever bk able-in dispense artificial paradises seems very unlikely. Most men and women lead lives at the large to escape longing to transcend themselves if only for a few moments, is and has always been one of the principal appetites of the If we accept that a world without drugs is unachievable (and probably intolerable) then the important question, becomes "what are the best means to regulate the production, distribution and consumption of the great variety of psychoactive substances available today and in the foreseeable future?" 276 TO reduce the debate to arguments between "prohibitionists" and "legalizers" is to oversimplify it, but it is a useful device for beginning to understand the issues. Thus, the case for legalising drugs begins with the failure of current prohibitionist policies. The smoking of Cannabis, even long term, is not harmful to health. Yet this widely used substance is illegal just about everywhere. There have been numerous calls over the years for the legalisation, or at least decriminalisation, of soft drugs, among which Cannabis remains the most popular with all social groups. However, the demand for the legalization of Cannabis is due to its medicinal or therapeutic efficacy and not for its recreational abilities. It may also be useful to outline the direction in which other jurisdictions are going. In particular we refer to the situation that presently prevails in all of the major western countries like Netherlands, Germany, Spain, Italy, the United States and Australia. Of all of the major western countries outside of North America, only France and New Zealand have taken no measures to ease the impact of Cannabis laws. The national government of Canada and the US appear to be somewhat out of step with most of the rest of the western world and, of course, our country, Nigeria.
1.7.1 In The Netherlands The Netherlands, in 1976, effectively decriminalized penal possession of drugs by passing the "Opium Act" in the country. It was amended to draw a clear distinction between so called hard drug on one hand and Cannabis product on the other hand.
Since then, there has been .a ,, go)ii~y..Qf. . non-enforcement of the law as it relates to marijuana, and hashish can be openly purchased in licensed "coffee shops" throughout the country. Studies have shown that since 1976 changes in the "Opium Act" in Netherlands there has been a fall in the use and consumption ok Cannabis: from 13 % of those aged 17 - 18 in 1976 to 6 % in 1985. The consumption in the Netherlands is substantially lower than that in the US. Current use among Dutch high school students is around 5.4 % compared with 29 % in the United States. 272,273,277
1.7.2 In Germany In Germany, public prosecutors have been given discretion to dismiss minor cases of drug possession unconditionally or on condition that a fine is paid or that community service is completed. Prosecutors have used discretionary powers to dismiss minor drug cases in which the offender purchased or was in possession of drugs for personal use. Each of the German states has developed its own guidelines as to when it would be permissible to dismiss a drug case.278
1.7.3 In Spain In Spain, a 1995 amendment to the penal code stipulates that a criminal offence for drug possession is only established upon proof of a subjective intent to traffic or facilitate drug use by others. Possession of any illicit drug for personal use is no longer subjective to any criminal or administrative sanction.279
1.7.4 In Italy In Italy, there has been movement towards replacing the criminal sections for the drug possession and use with an administrative sanction. Essentially, the Italian drug laws put the drug user beyond the reach of the criminal law by creating drug law exemption for possession, purchase and import of drugs for personal use while still keeping the drug user under administrative controls.280
1.7.5 In Australia In 1987, in South Australia and in 1992, in the Australian capital territory, "expiation" schemes were introduced which effectively de-facto decriminalized the use and possession of Cannabis. Under these schemes, the police have the option of issuing an expiation notice to anyone caught with a specific amount of Cannabis instead of .,,, . .-5 .,. . . ..E charging the individual with a criminal offence. The expiation notice allows the offender to pay a small fine and avoid being saddled with criminal record. Small scale Cannubis possession, cultivation or use remains criminal offences. But they are no longer penalized as though they were. .In South Australia, the designated amount allowing for the assurance of an expiation notice in lieu of a criminal charge is 100 g of Cannabis or 20 g of Cannabis resin. In addition an expiation notice can be used for someone cultivating up to 10 Cannabis plants. In the Australian capital territory, an expiation notice can be issued for 25 g of Cannabis or up to 5 plants being ~ultivated.~~'Finally the Australian National Task Force on Cannabis has identified five options for Cannabis legislation: total prohibition; prohibition with civil penalties; partial prohibition, regulation of the product, distribution and sale of Cannabis and free availability.282 The task force opted for keeping possession, cultivation and sale in any quantity illegal but decriminalising "simple personal use or possession .. . without compromising activities aimed at deterring Cannabis use".
1.7.6 In The USA In the 2oth century, Cannabis has been used more for its euphoric effects than as a medicine. Its psychological and behavioural effects have concerned public officials since the drug first appeared in the South-western and Southern states during the first two decades of the century. By 193 1, at least 29 states had prohibited use of the drug for non medical purpose.283It was first regulated at the federal level by the Marijuana Tax Act of 1937, which required anyone producing, distributing or using marijuana for medical purpose to register and pay a tax and which effectively prohibited non medical use of the drug. Although the act did not make medical use of marijuana illegal, it did make it expensive and inconvenient. In 1942 Cannabis was removed from the U.S. Pharmacopoeia because it was believed to be harmful and addictive drug that causes psychoses, mental deterioration and violent behaviour. The current legal status of marijuana was established in 1970 with the passage of Controlled Substances Act, which divides drugs into five schedules and placed marijuana in Schedule I, the category for drugs with high potential for abuse and accepted medical use. In 1972, the National Organisation for the Reform of Marijuana Legislation (NORML), an organization that supports decriminalisation of marijuana unsuccessfully petitioned the Bureau of Narcotics and Dangerous Drugs to move . ,, ..!. .I. .%' . + ' marijuana from Schedule I to Schedule 11, NORML argued that marijuana is therapeutic in numerous ailments, less toxic and in many cases more effective than conventional medicines.284 Thus, for 25 years in the U.S. the medical marijuana movement has'been closely linked with the marijuana decriminalisation which has coloured the debate. However, since NORML's petition in 1972, there have been a variety of legal decisions concerning Cannabis. From 1973 to 1978, 11 states adopted statutes that decriminalized use of marijuana, although some of their decriminalized marijuana use in the 80s and 1990s. For instance, in Alaska it is not against the 'law to possess marijuana in the privacy of ones residence but it is still illegal to possess marijuana anywhere else in the state and Alaska appears to be moving towards overturning de~riminalisation.~~~In Alaska, Miami, Minnesota, Mississippi, Nebraska, and Oregon, possession of small amounts of marijuana is treated as a "civil violation" rather than a crime, much like minor traffic offences. In New York, and North Carolina, possession of small amount is deemed a misdemeanour. In Ohio, it is a "minor misdemeanour" and in Colorado it is a "petty ~ffence'"'~. Against this backdrop, voters in California and Arizona in 1996 passed two referenda that attempted to legalize the medical use of marijuana under particular conditions. The conditions are: under the law, physicians cannot be punished or denied any right or privilege for recommending marijuana and controlled substances, including those of Schedule I to treat the diseases or relieve the suffering of terminally ill patients.2877 288. Thus, it can be seen that nowhere in the USA has the simple possession of marijuana been legalized, although as noted above in many states the consequences of simple possession have been eased to a greater or lesser extent.
1.7.7 In Nigeria In Nigeria there is not much of literature reporting the use of Cannabis or marihuana for medicinal use or research. However, this may be as a result of the laws surrounding the use and procurement of marihuana in the country. The law is given by the Dangerous drug Act (Cap 91 and it states as follows: - An Act to regulate the importation, exportation, manufacture, sale and use of opium and other dangerous drugs. This law is also enforced by National Drug Law Enforcement Agency (NDLEA) and the National Agency Food and Drug Administration and Control (NAFDAC). The punishment for the use, sale of the substance is 25 years or life .. ,, -. ..-r. .I' ...... i-> imprisonment. 289
1.7.8 In United kingdom The British Medical Association published a major report on the therapeutical uses of Cannabis and drew a clear distinction with its recreational use and presented a great deal of anecdotal evidence in favour of change in government With the above reason the House of Lords selected committee on Science and Technology which is currently carrying out inquiry into the science behind call for medicinal use of Cannabis, by taking evidence on the physiological and psychological effects and how they vary with different modes, methods of preparation and administration, extent of addiction and tolerance, the strength of the evidence permitting medical use and for maintaining prohibition of recreational use. This were done by taking oral evidence from the following: (A) Roger Petwee of the University of Aberdeen's Institute of Biomedical Sciences. (B) President of the International Cannabinoid Research Society. (C) Multiple Sclerosis (MS) Society.
Finally, in U.K under its current legislation, (Misuse Drug Act of 1971) 29' Schedule 1 Drugs: drugs with a high potential of abuse and no therapeutic value, such as those that head to ecstasy are banned. Schedule 2 Drugs: - use of Cannabis and / or its constituents could be restricted in the same way as morphine, without being banned.
1.8 AIM AND SCOPE OF THE STUDY Documented evidences abound on the use of the leaves, stem, seeds and some chemically active constituents of Cannabis sativa (marijuana) as therapeutic agents in the treatment of diverse diseases. These evidences have. propelled interest in developing new drugs based on cannabis that can treat more effectively or more safely the constellation of symptoms for which cannabinoids might have therapeutic benefit. Through the process of rational drug design scientists manipulate the chemical structure of known cannabinoids to design better therapeutic agents. Thus, the basis for pursuit of evaluation of phyto-chemical and medicinal properties of Cannabis sativa (preparation or formulation) from cannabis has risen as a result of the inherent health hazards associated with the tar, from smoking cannabis as compared to tobacco as a long term health risk. Thus this research work was set out to .,,,.....,I. ..> ' . investigate the phyto-chemical and medicinal properties of Cannabis sativa. In line with these, this work covered such areas as:- Formulation of crude cannabis resin into syrups, suppositories and capsules. Evaluation of the crude resin extracts:..of various solvents for antimicrobial activities. Accelerated and stability studies of syrup formulation. Effects of cannabis sativa on biochemical assay of enzyme markers like serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and urea. In addition to these, the histopathological determination of its effects on the liver, brain, spleen, and the kidney will also be presented. CHAPTER TWO 2.0 Experimental 2.1 Materials 2.1.1 Chemicals and Reagents All chemical solvents and reagents listed and used in this study were of analytical grade and products of May and Baker, England; BDH, England; Merck, Darmstadt, Germany; Sigma-Aldrich, USA; Riedel-Dehaen, Germany.
Phytochemical test Reagents Fehling's solution 1: 20 g of Rochelles salts (Potassium Sodium Tartarate), 15 g of NaHO were dissolved in water and made up to 100 ml with water. Fehling's solution 11 : 4 g of Copper (1 1) Sulphate (May and Baker, England) was dissolved in water and made up to 100 ml with distilled water. Wagner's reagent: 2.0 g of iodine crystals, potassium iodide (Merck, Darmstadt, Germany) were dissolved in water and made up to 100 ml of distilled water Mayer's reagent: 1.53 g of mercuric chloride (Merck, Germany) was dissolved in 60 ml of distilled water, 0.5 g of patassuim iodide was dissolved in 20 ml of distilled water and the solutions were then mixed and the volume made up to 100 ml solution a-naphthol (Riedel-Dehaen, Gmaay) Tetraoxosulphate (vi) acid (BDH, England) Ammonium hydroxide (BDH, England) Chloroform (May and Baker, England) Sodium hydroxide (May and Baker, England) Carbon tetrachloride (May and Baker, England) Ferric chloride (Sigma-Aldrich, USA) Ethanol (70%, 90%) (BDH, England) Lead subacetate (Sigma-Aldrich, USA) 2% 3,5-dinitrobenzoic acid (BDH, England), in 90% ethanol (BDH, England) Glacial acetic acid (May and Baker, England) Ethyl acetate (BDH, England) Aluminum chloride (Sigma-Aldrich, USA) Arachis oil.
Extraction and Chromatography Reagents Chloroform (May and Baker, England) . Dioxane (May and Baker, England) N-hexane (BDH, England) Ethanol (BDH, England) Silica Gel GF254(Merck, Darmstadt, Germany)
Microbiological studies Methanol (BDH, England) Chloroform (May and Baker, England) N-hexane (BDH, England) Ethanol (BDH, England), Distilled Water Nutrient agar (Oxoid, England) Saboraud dextrose agar (Oxoid, England)
Pharmacological studies Acetylsalicylic acid (Sigma-Aldrich, USA) <,,,., '+.?. .v.,:...w ' Fresh egg albumin
Biochemical Assay Reagents used for the entire assay wFre imprted commercial kits and product of RANDOX, USA; QCA, Spain; Teco (TC), USA; Biosystems Reagents and Instruments, Spain
Haematology/blood Chemisty Laboratory Reagents: (a) Leukocyte diluting fluid containing 10% glacial acetic acid tinged with gentian violent. (b) Erythrocyte diluting fluid containing sodium citrate, formaldehyde (May and Baker) and distilled water. (c) Leishman's stain containing Leishman stain powder in pure methyl alcohol. (d) Ethylene-diamine tetraaacetic acid (EDTA) (BDH, England) anticoagulant
Histopathology Normal saline Ethanol (BDH, England) Xylene (BDH, England) Paraffin wax (BDH, England) Formaldehyde (May and Baker, England)
Stability Studies Syrup (Sucrose, (BDH, England)) Ethanol (BDH, England) Sodium meta bisulphate (Sigma-Aldrich, USA) EDTA (BDH, England) Distilled water Chloroform (May and Baker, England) Propylene glycol (BDH, England) Dioxane (May and Baker, England) N-hexane (BDH, England) Diethyl ether (May and ~&i<& Instruments/Equipment Water bath Gallenkamp, England C hemical balance Gallenkamp, England Refrigerator Kelvinator, Germany Rat cages Swctro~hotometer(UV-2 102) Unico. USA Haematocytometer set containing improved Neubauer counting chamber and diluting pipettes (Hawksley, England) Microhaematocrit centrifuge and Reader (Hawksley and Sons ltd., England) Laboratory counters - total and differential counters (Clay Adams, New Jersey). Sample bottles treated with EDTA. 2.2 Source and Identity of plant materials The fresh leaves of Cannabis sativa L. were collected in July 1999, from the Crude Drug and Research Unit of National Drug Law Enforcement Agency (NDLEA) in Enugu, Enugu State of Nigeria. Common Name:- Marihuana Nigerian Names:- Igbo, wee-wee, bana, hashish Botanical Names:- Cannabis sativa Linn Geographical distribution: - Found in both tropics and tropical regions of the world. Morphological part used: - Leaves, seed, stem, roots, flower and bract. Time of Collection: - August, September and October or three to five month after growth. Condition of collection before use: - fresh parts of plant before drying. -, ,. -:.... .I. .$ . .') . . 2.3 Preparation of plant extracts Whole leaves of the plant (Cannabis sativa) were rinsed thoroughly in running tap water sun-dried in open air for 48 hours and pulverized to coarse powder using If _ mortar. One thousand grams (1000 g) of the powdered leaves of Cannabis sativa L. was extracted with 1 litre of chloroform for 8 h using a soxhlet extractor. The crude chloroform extract was evaporated to dryness under reduced pressure, using a rotary evaporator at an optimum temperature of between 40 and 45OC, to yield 173.25 g of crude resin tar. 2.3.1 Preparation of n-Hexane: Chloroform extract (1:l) Whole leaves of the plant (Cannabis sativa) were rinsed thoroughly in running tap water, sun-dried in open air for 48 hours and pulverized to coarse powder using mortar. Two hundred grams (200 g) of the powdered leaves of Cannabis sativa L. was extracted with n-hexane: chloroform (1 :1) for 8 h using a soxhlet extractor. The crude n-hexane: chloroform extract was evaporated to dryness under reduced pressure, using a rotary evaporator at an optimum temperature of between 40 and 45"C, to yield 12.03 g of crude resin tar. Same procedure was adopted for the extraction of Cannabis sativa from n-hexane, chloroform, methylene chloride and ethanol to yield 7.13, 35.16,5.23 g and 11.98 g of crude resin tar respectively. 2.4 Ultraviolet (UV) Absorption Spectra The UV absorption spectra of a 250 mg % of crude Cannabis resin extract in absolute alcohol (ethanol) was determined using W-visible spectrophotometer fitted with a scanning device. Figure 42 shows the absorption spectra maxima obtained to be 274 nrn. 2.5 Phytochemcal Tests A small quantity of the Cannabis saliva L. leaves was sun-dried, powdered and subjected to the following phytochemical analyses using standard phytochemical 2.5.1 Test for the presence of reducing sugar. One gram (0.1 g) of the ground plant material was shaken vigorously with 5.0 ml of distilled water, heated in water bath f&. 5 minutes and filtered. A 1.0 ml. volume of the filtrate was added to equal volumes of Fehling's solution I1 and I and shaken vigorously. A brick red precipitate was observed which indicated the presence of reducing sugar. 2.5.2 Test for the Presence of Plavonoids A quantity of 0.2 g of the macerated sample was heated with 10.0ml of ethyl acetate in boiling water for 3 minutes. The mixture was filtered. About 4.0 ml of the filtrate was shaken with 1.0 ml of 1 % aluminum chloride solution and observed for light yellow colouration in the ethyl acetate layer. A yellow colouration was observed in the ethyl acetate layer, which indicated the presence of flavonoids. 2.53 Test for the Presence of Glycosides About 2.0 g of the macerated Cannabis saliva L. material was mixed with 30.0 ml of water. The mixture was heated on a water bath for 5 minutes filtered and used for the following test. A 5.0 ml volume of the filtrate was added to 0.2 ml of Fehling's solution A and Fehling's solution B until it turned alkaline (when tested with litmus paper). The mixture was then heated on a water bath for 2 minutes. A brick red precipitate was observed which indicates the presence of glycoside. Using 15.0 ml of dilute sulphuric acid instead of water the above process was repeated and the quantity of precipitate formed was the same as in the former experiment. 2.5.3.1 Tests for 0 - and C - glycosides The powder of 1.O g quantity of the powdered Cannabis was heated with 5.0 ml of water in a water bath for 10 minutes and later cooled and filtered. The filtrate was treated with 5.0 ml of 25 % wlv hydrochloric acid solution and the solution heated for 15.0 minutes, cooled and filtered. The filtrate was extracted thrice with diethyl acetate was shaken with 5.0, ml of 3.5 % dilute ammonia solution and ,.-...,..1 ..'i observed. The aqueous layer was further treated with 0.5 ml of ferric chloride solution, heated for 30 minutes and cooled. This solution was extracted with 10.0 ml of chloroform using a separating funnel. The chloroform layer was washed with 5.0 ml of water; then 3.0 ml of 3.5 % dilute arnmo&a solution was added and the mixture was observed. 2.5.3.2 Borntagger's test for Anthraeene glyeosides A quantity of 0.1 g quantity of the Cannabis placed in a conical flask was mixed with 5.0 ml of dilute tetraoxosulphate (VI) acid and 5.0 ml of ferric chloride solution. The resultant mixture was boiled actively for 5.0 minutes, cooled and filtered into a separating fhnel. The filtrate was shaken with equal volume of carbon tetrachloride and the lower organic layer drawn into a test tube. Finally 5.0 ml of dilute ammonia was added to it with gentle shaking and observed for colour change. 2.5.3.3 Test for cyanogenic glycosides Five milliliter (5.0 ml) of distilled water was added to 0.1 g of the powdered Cannabis in a conical flask. A piece of sodium picrate paper was suspended in the flask with the aid of a cork. The flask was then placed in an oven at 45'~for one hour and the colour change of the paper observed. 2.5.4 Test for the presence of alkaloids The powder of 2.0 g of the Cannabis saliva L. was pounded on a motar. A quantity of about 0.2 g was boiled with 5.0 rnl of 2 % hydrochloric acid on a steam bath. It was filtered and 1.0 ml portion of the filtrate treated with 2 drops of the following reagents. Dragendorff s reagent (Bismuth potassium iodide solution) Red precipitates indicates the presence of alkaloid. Mayer's reagent (Potassium mercuric iodide solution). A creamy white precipitate indicates the presence of alkaloid. Wagner's reagent (Iodine in potassium iodide solution). A reddish brown precipitate indicates the presence of alkaloid. .<,,..,?.I:.I",...... r ' . 2.5.5 Test for Starch A dispersion of the powdered Cannabis was boiled, cooled and filtered. To the filtrate, was added two drops of 5.0 % iodine solution and the mixture observed for any colour change. I, 2.5.6 Test for the Presence of Proteins A 5.0 ml volume of distilled water solution was added to O.lg of the ground plant material and left for 3 hours and filtered. Two drops of Millon's reagent were added unto a 2.0 ml portion of the aqueous extract of the Cannabis. A yellow precipitate (was observed which) indicates the presence of protein. 2.5.7 Test for Tannin Two grams of the ground plant material was boiled with 5.0 ml of 45 % ethanol for 5 minutes. The mixture was cooled and filtered. The filtrate was used for the following tests. Lead Subacetate To 1.0ml of the filtrate was added 3 drops of lead subacetate solution. A white precipitate (was observed which) indicates the presence of Tannins. Bromine Water To 1.0 ml of the filtrate was added 0.5 ml bromine water and then observed for a pale brown precipitate. Ferric Chloride A quantity 1.0 ml of filtrate was diluted with distilled water and 2 drops of ferric chloride solution added. A transient greenish to black colour indicates the presence of tannins. 2.5.8 Test for Saponins The powder of 0.1 g of the ground plant material was boiled with 5.0 ml of distilled water for 5 minutes. The mixture was filtered while still hot. The filtrate then was used for the following test.s. .. ,.'..r..d...... -* ' . 2.5.8.1 Frothing Test A quantity (1.0 ml) of the filtrate was diluted with 4.0 ml of distilled water in a test tube and shaken vigorously and then ,obserGed on standing for stable froth which indicates the presence of saponins. 2.5.8.2 Emulsion Test Two drops of olive oil was added to 1.0 ml of an aqueous filtrate of the powdered Cannabis and shaken vigorously and observed for the formation of emulsion. The formation of emulsion indicates the presence of saponins. 2.5.9 Test for Flavonoids A quantity of 0.1 g the powdered Cannabis material was heated with 10 ml of ethyl acetate in a boiling water bath for 3 minutes. The mixture was cooled and filtered, and lml of dilute ammonia solution was added to a 4.0 ml quantity of the filtrate. The flask was shaken gently and the lower alkaline layer observed. A yellow colour indicates the presence of flavonoids. 2.5.10 Test for Carbohydrates One gram (0.1 g) of the Cannabis material was shaken vigorously with distilled water and then filtered. Few drops of Molisch's reagent were added to an aqueous solution of the Cannabis followed by vigorous shaking. Thereafter, 1.0 ml of conc. H2S04was added carefully thro the sides of the test tube to form two layers. A brown ring observed at the interface indicates the presence of carbohydrate. 2.6 Microbiological Evaluations 2.6.1 Isolation and characterisation of test microorganisms. Clinical isolates of Staphylococcus aureus, E. coli, Salmonella Typhi, B subtilis were collected from the Department of Pharmaceutics, University of Nigeria, Nsukka. Each test organism was purified by sub-lattaring three times in Mueller Hinton agar, MHA (Oxoid) and identity confirmed after characterization by standard bacteriological methods.296p297 Stock.,,.. qu1,ture.s ., were maintained in nutrient agar at 4'~. 2.6.2 Maintenance and standardisation of stock cultures A stock culture of each clinical isolate was maintained on MHA slants (for bacteria) and SDA slants (for fungi) at1.4~~..-.Prior to use successive daily sub- culturing into fresh agar slants for a period of 3 days activated the cultures. The overnight (20h) cultures were standardized by diluting the Gram-negative organisms 1:5000 and Gram-positive organisms 1: 1000 to obtain population density of approximately lo6 cfdml before use298. For C. albicans and Aspergillus niger, subcultures in Saboraud's dextrose broth were adjusted to 90 % transmittance at 530 nm using distilled ate?^ 2.6.3 Preliminary Sensitivity Tests 2.6.3.1 Sensitivity test of Cannabis Crude Resin Extracts The sensitivity of selected bacteria and fungi to the crude ethanol, chloroform, n-hexane, aqueous, methylene chloride and n-hexane: chloroform extracts of the Cannabis resin was evaluated by the cup-plate agar diffusion method. 299 A small portion of each of the above extracts (except the aqueous extract) was evaporated to dryness by heating on a water bath and the weight of the extract determined. The extracts were then dissolved in 2.0 ml methanol and the resulting solution diluted to a concentration of 100 mglml using distilled water. For the aqueous extract, the equivalent amount to form 100 mglml was dissolved directly in distilled water. Molten nutrient agar and SDA (20.0 ml each) were seeded with 0.1 ml of standardised broth cultures of bacteria and fungi respectively. A total of 5 wells, each 8 mm in diameter were made in the agar using a sterile cork borer. Two drops (0.02 ml per drop) of each of the extracts were carefully placed into each of the wells. Two drops of diluted gentamicin was put in the centre well as control. The plates were left for 1 h at room temperature for diffusion, after which they were incubated at 37'~for 24 h for bacteria and at 25'~for 48 h for fungi. Inhibition zones Diameters IZD were measured at the end of incubation period. The mean of duplicated determinations was taken. 2.6.4 Minimum Inhibitory Concentration,(MIC) of Extracts .',,...,. 4 .** The MIC of the chloroform, n-hexane, and n-hexane: chloroform, methylene chloride (C12CH2)and aqueous extracts of the Cannabis against the selected bacteria and fimgi were determined by a modified cup-plate agar diffusion method3''. Two fold serial dilutions of a methanol solution .of the extracts were made in distilled water. Two drops (0.02 ml per drop) of different dilutions of extracts were placed in wells (8.0 rnrn diameter) bored on either nutrient or SDA plates already seeded with 0.1 ml of a standardized culture (10~cfdml)of the test microorganism. The plates were left for 1 hr. at room temperature for diffusion. They were then incubated at 37'~for 24h for bacteria and at 25'~for 48 h for fungi. The inhibition zone diameters (IZD~)of the different concentrations of each extract were measured and the MIC obtained from the intercepts on the log concentration axis of the graphs of logarithm of concentration (log conc.) against the squares of the inhibition zone diameter (IZD~). 2.6.5 Determination of Rate of the Kill of the Crude Extracts The killing rate was carried out using viable counting of bacteria (the surface-viable method). A total of three isolates were selected, that showed sensitivity to five extracts of Cannabis crude resin. The test organisms were grown in 3.0 ml of nutrient broth in the incubator at 37OC for 14 h in order to achieve an innoculum size (turbidity) of approximately 0.5 McFarland standards. Thereafter, the organisms were sub-cultured in 3.0 ml of fresh nutrient broth in eight different test tubes and incubated at 37OC for 1 h to activate the organism. Samples (0.1 ml) were withdrawn from all the test tubes and diluted in sterile normal saline before estimating the viable bacteria count in oven-dried nutrient agar plates. The Cannabis crude extracts were then added into three test tubes and one drop of samples taken from these tubes at different time intervals of 20,40,60 and 90 minutes. After appropriate dilution in sterile normal saline, samples were then spread plated in oven-dried nutrient agar plates. The plates were incubated for 24 h and the viable cell count was estimated. The viable count of the 8" tube (control), which contained only test bacteria (without Cannabis crude extract) was similarly estimated. Calculation of the original viable count: Mean count I drop x DF Original number of cells I ml = 0.01 7 Note: 0.01 7 = volume of one drop. .<,,...I..)' '.Z 2.7 Toxicity Tests Tests to determine the acute toxicity and lethality of the crude Cannabis resin were performed. The leaves were used and. the route of administration was I* _ ..- intraperitoneal. The toxicological studies helped to determine the doses of crude resin employed in subsequent whole animal experiments. 2.7.2 Cytotoxicity Assay (CDSo) The cytotoxicity (and biological activity) assay (CD50) was by the Brine Shrimp Lethality Bioassay technique. 301p 302 About 5.0 mg of the eggs of brine shrimp, Artemia salina (Interpet Ltd., UK), obtained from Department of Pharmacology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, were hatched in 200.0 ml of natural sea water obtained from the Atlantic Ocean at Bar Beach, Lagos Nigeria, after incubation for 48 h at about 30°C. The emergent larvae (nauplii) were separated from the eggs and held for further 48 h at 30°C before use for the following experiments. Procedures: The Cannabis crude resin extracts were reconstituted and dissolved serially in ethanol: propylene glycol (9: l), to give three concentrations; namely, 1000.0 parts per million (ppm), 100.0 and 10.0 ppm, respectively. The controls were seawater + ethanol: propylene glycol or seawater for the crude extract. The assays were done in triplicate (5.0 ml) in appropriately labeled clean, transparent vials. Ten (10) brine shrimp larvae (nauplii) were added to each vial containing a concentration of the extract in seawater or ethanol: propylene glycol- in- seawater 5.0 ml respectively. Incubation was at room temperature for 24 hours after which the surviving shrimps in each vial were counted and the median lethal concentration (LC5())calculated using the Finney Probit Analysis Computer Programme. 2.8 Administration of Cannabis Suspension Twenty rats were divided into 4 groups of five per group (each group containing 3 males and 2 females). The rats were weighed before they were fed. Crude Cannabis resin suspended in 9:l ethanol: propylene glycol corresponding to 20 and 40 mg to lkg body weight of the rats was administered i.p. to the groups. The administration of the drug suspension..,,,...l..r in..%+ the above order was repeated at the same hour everyday for 14 days. The animals were behavioral changes. 2.9 Determination of Haematological Parameters 2.9.2 Sample Collection IS . .:.. Blood for the total leukocyte, differential leukocyte, erythrocyte counts and packed cell volume (PCV) determination was collected before commencement of treatment on (day 0) and on the day 8 and day 14 post treatment from the retro-bulbar plexus of the medial canthus of the eye by carefully inserting a micro capillary tube into the medial canthus of the eye to puncture the plexus and enable outflow of blood into the sample bottle treated with EDTA. This was shaken gently to prevent clotting. A 0.02 ml volume of the blood sample was added to 0.38 ml of the leukocyte diluting fluid in a test tube for the total leukocyte count. Another 0.2 ml of blood was added to 4.0 ml of erythrocyte diluting fluid for the red blood cell counts. A micro capillary tube was newly filled with the blood sample for PCV determination. 2.9.3 Procedure for the Haematological Determination For the total leukocyte counts, a drop of the diluted sample was used to load the Neubauer chamber, and all cells within the chamber were enumerated. The dilution was 1 :20; the number of enumerated cells for each sample was multiplied by 50 to obtain the absolute leukocyte count per micro litre of blood 303. For the erythrocyte counts too, a drop of the blood sample diluted with erythrocyte diluting fluid was used to load the Neubauer chamber, and all red blood cells in the five groups of sixteen small squares in the central area of the Neubauer chamber were enumerated. The dilution was 1 :200; the number of cells enumerated for each animal was multiplied by 10,000 to obtain the erythrocyte counts per micro litre of blood 304. The filled micro capillary tube for PCV determination was sealed at one end and centrifuged for five minutes using a microhaematocrit centrifuge. After the packing of the blood cells, the PCV was read as a percentage using a microhaematocrit reader. For the differential leukocyte counts, a drop of the blood sample was placed at one end of a clean grease free slide. The blood was carefully smeared to make a thin blood film, which was dried in air and thereafter stained by the Leishrnan technique.303 The slides were later examined under oil immersion with light microscope, two hundred cells were enumerated by the longitudinal counting method .....+ and each cell type was scored using differential cell counter. Results for each type of leukocyte were expressed as a percentage of the total count, which was thereafter converted to absolute values per micro litre of blood. Let N = No. of cells counted in 4 square (mmi .v Then - = No of cells in 1 square (mm) 4 Note: The volume of each square (mm)= 1 x KOmm3 :. No. of cells in 1 mm3 = /i x I0 (the blood was diluted 1 in 10) N x 200 :. No. of cells per mm3 of undiluted blood = N/ x 10 x 20 (i.e =Nx50) 4 2.9.4 Data Analysis Data on the total leukocyte counts; absolute values of the different leukocytes, erythrocytes counts and PCV were expressed as group means with standard deviations for each determination. Means of the four groups were compared for significant difference of each day of the determination-using single factor analysis of variance (ANOVA). 2.10 Preparation of Cannabis Formulations 2.10.2 Materials 0.1 N HC1 solution, Theobroma oil, and Cannabis crude resin. 2.10.2.1 Preparation of Suppositories Using the displacement value of 1.5 for theobroma oil, the correct quantity of the base in each batch was calculated. The suppositories were prepared to contain 300 mg (2 %, 4 % and 6 % Tween 85), 300 mg, 600 mg and 900 mg respectively of crude Cannabis resin per suppository. Enough quantities to yield 12 suppositories were calculated. The correct quantity of the drug was added to the base (after melting it), with continuous stirring until it was cool but pourable. The preparation was poured into the 1.0 g moulds (previously lubricated with glycerine) until there was an overflow and then cooled at O'C for 30 minutes. After cooling, the suppositories were removed from the mould and stored,.in.themfigerator for further experiments. The same procedure was carried out for all the batches. 2.10.3 Evaluation of Suppositories ,I _ .- 2.10.3.1 Appearance Two suppositories were selected from each batch and the external and internal surfaces when cut longitudinally examined with the naked eye and also with a hand lens. The suppositories were examined for the presence or absence of air bubbles, brittle fracture, uniformity of mixing and for presence or absence of contraction holes. 2.10.3.2 Uniformity of Weight Six suppositories were picked at random and weighed together using a torsion balance. They were also weighed individually and the mean, variance, standard derivation and coefficient of variation were calculated using this formula: (see appendix B) 2.10.3.3 Liquefaction time Liquefaction time apparatus, which can also be used to determine the melting point of fatty base suppositories proposed by Setnikar and ~antelli~'~was modified and used in this study. Each suppository was placed in a heat-resistance and inelastic polyethylene material and tied directly on the bulb of a thermometer using an in- extensible thread. The thermometer with suppository were inserted into a 0.1 N HCl solution maintained at 37' + 0.1 OC by means of a thennostated heating mantle (Jargon & Co.). The time taken for the suppository to melt at that temperature was recorded. Average of four determinations was taken as the liquefaction time. 2.103.4Construction of Calibration Curve (Beer's plot) Cannabis crude resin (100 mg) was weighed out and dissolved in 100 ml solution of ethanol to obtain a stock solution. From the stock solution, 0.2 ml was diluted to 100 ml with ethanol (concentrationO.1 mg %). Similarly 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 to 2.0 ml of the stock were diluted to 100 ml to obtain the corresponding strengths in mg %. The absorbance for each diluted sample was determined at 274 nm using a spectrophotometer. The absorbance values were plotted against the concentration terms to yield Beer's .,,, ..> plot. The slope of the graph was determined. AaC...... (1) A=KC ...... (2) Where A= Absorbance (nm) I, _ ..- C = concentration of Cannabis crude resin K = constant (slope) 2.103.5 Release studies The Erweka dissolution test apparatus was used for the determination of the release rate of the suppositories. Each suppository was placed in the appropriate compartment of the dissolution apparatus containing 400.0 ml of the buffer solution. The paddle was rotated at 120 revlmin and the dissolution medium was maintained at 37OC + 1°C. At predetermined time intervals, 5.0 ml samples were withdrawn and appropriately diluted. A 5.0 ml quantity of the buffer solution was added to the dissolution medium at each time interval to compensate for the sampling. The absorbances of the diluted solutions were measured at 274 nm with a spectrophotometer, and the concentrations were determined from the calibration curve. Average of two-absorbance readings at each time internal was used for all batches. 2.1 1 Anti-inflammatory test The effect of the Cannabis crude resin on acute inflammation was evaluated using the rat paw edema test of Winter et al., 307. Increase in the rat hind paw linear circumference induced by sub plantar injection of undiluted fresh egg albumin (phlogistic agent) was used as model of acute inflammation '08. '09. The Cannabis crude resin was administered as syrup at doses 10 and 25 mg/kg. Animals were allowed free access to food and water before and during test. Procedure Adult albino rats (100 - 150 g) of either sex were used. The animals were divided into 16 groups of 6 animals each. Each group received dose level (1 0 or 25 mglkg) of Cannabis crude resin administered orally. Control animals received an equivalent volume of the syrup (vehicle)- ,, pr 1,90rnglkg acetylsalicylic acid. One hour after administration of the extract or acetylsalicylic acid, inflammation was induced by injecting O.lml of undiluted fresh egg albumin into the sub plantar region of the right hind paw of the rats. The circumference of the paw l.was .:.measured using a tape before administration and at 0.5, 1,2, 3 and 4 h intervals after egg albumin injection. Edema formation was assessed in terms of the difference between the zero time linear circumference of the injected paw and its circumference at the various time intervals after egg albumin injection. For each dose of the Cannabis crude resin, edema rate was calculated using the relation; 309 Where CT= paw circumference at a time T Co = Paw circumference at time To The percent inflammation was calculated using the formula '0 x 100 .....( 2) While percent inhibition of edema was calculated using the formula {(c,.-c,)/c,) XI00 ...... (3) Where Co = the average inflammation (paw circumference) of the control at a given time and CT= the average inflammation of the treated group at the same time. 2.12 Determination of Biochemical Parameters: Blood samples were collected after 1, 2 and 3 weeks of treatment and the biochemical parameters: serum glutamate-oxaloacetate-transaminase (SGOT), serum glutamate-pyruvate-transaminase (SGPT) and urea levels were determined using the colorimetric assay 2.12.1 Determination of the serum glutamateoxaloacetate-transaminase (SGOT) The following solutions were prepared: (1) BufferISubstrate (0.1 M of Phosphate buffer pH 7.4, 0.1 M of aspartate and 2 rnmol/L 2-oxoglutarate): 1.50 g K2HP04,0.20 g KH2P04,30.0 mg 2-oxoglutaric acid, 1.57 g L-aspartate, mono sodium salt (or 1.32 g L-aspartic acid) were dissolved in ., ,, 4 ... 4. . , .>> 70.0 ml of distilled water. The pH was adjusted to 7.4 with 0.1 M NaOH and diluted to 100.0 ml with distilled water. (2) Chromogen (l,2,4-dinitophenylhydrazine): 20.0 mg of 2,4- dinitrophenylhdrazinewas dissolved in 0.1 HCLand made up to 100.0 ml with 0.1 M HC1. (3) Sodium hydroxide (0.4 M): 16 g of NaOH was dissolved in distilled water and made up to 100.0 ml with distilled water. (4) Sodium pyruvate (2.0 M): 22 mg of sodium pyruvate was dissolved in distilled water and made up to 100.0 ml with distilled water. Procedure A 1.0 ml volume of the buffer substrate solution and 0.20 ml of serum were mixed in test tubes and incubated for 60 minutes at 37OC in water bath. At the end of the incubation period 1.0 ml of Chromogen solution was added. They were mixed and allowed to stand for 20 minutes at room temperature. Ten millilitres (10 ml) of NaOH was added, mixed and allowed to stand for 5.0 minutes. The absorbance readings were then taken at 505 nrn against water as blank. The unit activity was read off from the standard curve. 2.12.1.1 Preparation of standard curve for SGOT Different quantities of sodium pyruvate (ml) and bufferlsubstrate solution (ml) were mixed in 6 different test tubes numbered 1-6 as shown below. Table 5 Test tube no 1 2 3 4 5 6 Sodium pyruvate 0.00 0.05 0.10 0.15 0.20 0.25 solution (ml) Buffertsubstrate 1.00 0.95 0.90 0.85 0.80 0.75 solution (ml) Into each test tube 0.2 ml of distilled water and 1.0 ml of chromogen solution (2, 4- dinitrophenylhydrazine) was added and mixed thoroughly. Ten millilitres (10 ml) of NaOH solution were added after 20.0 minutes to all the test tubes. They were mixed and allowed to stand for another .5.0< ,, . .,.minutes s9 , after which the absorbance reading of test tubes 2- 6 were taken against test tube 1 as blank at 505 rim. 2.12.2 Determination of the serum glutamate-pyruvate-transaminase (SGPT) The following solutions were prepared: 1- - -:. Bufferlsubstrate (0.1 M of Phosphate buffer pH 7.4, 0.2 M of DL - alanine and 2 mmoVL 2-oxoglutarate): 1SO g K2HP04,0.20 g KH2P04,30.0 mg 2-oxoglutaric acid, 1.78 g DL-alanine. They were dissolved in distilled water. The pH was adjusted to 7.4 with NaOH. Procedure A 1.0 ml volume of the bufferlsubstrate solution and 0.20 ml of serum were mixed in test tubes and incubated for 30 minutes at 37OC in water bath. At the end of the incubation period 1.0 ml of Chromogen solution was added. They were mixed and allowed to stand for 20 minutes at room temperature. Ten millilitres (10 ml) of NaOH was added, mixed and allowed to stand for 5.0 minutes. The absorbance readings were then taken at 520 nm against water as blank. The i.r unit activity was read off from the standard curve. 2.12.2.1 Preparation of standard curve for SGPT Into six test tubes (1-6) different quantities (ml) of sodium pyruvate solution and bufferlsubstrate solution were mixed as shown below. Table 6 Test tube no 1 2 3 4 5 6 Sodium pyruvate 0.00 0.10 0.20 0.30 0.40 0.50 solution (ml) Bufferlsubstrate 1.00 0.90 0.80 0.70 0.60 0.50 solution (ml) Into each test tube 0.2 ml of distilled water and 1.0 ml of chromogen solution (2, 4-dinitrophenylhydrazine) were added and mixed thoroughly. Ten millilitres (10 ml) of NaOH solution was added after 20.0 minutes to all the test tubes. They were mixed and allowed to stand for another 5.0 minutes after which the absorbance reading of test tubes 2- 6 were ta&en.ggimt, test tube 1 as blank at 520 nm. 2.12.3 Determination of urea levels The following solutions were prepared: (1) Mixed colour reagent: 1.34 g of ldiacetyl monoxirne (DAM) and 6.335 g of thiosemicarbazide were dissolved in1 .O litre of distilled water. (2) Mixed acid reagent: 0.01 g of FeC13.6H20 and 0.1 ml of 85 % orthophosphoric acid were dissolved in 600.0 ml of distilled water. 200.0 ml of H2S04 (concentrated) was added and made up to 1.0 litre with distilled water. Water was added first before gradual addition of conc. tetraoxosulphate (VI) acid to prevent explosion. (3) Urea working standard: 200.0 mg of urea in 100.0 ml of benzoic acid solution (0.5 g of benzoic acid in 500.0 ml of distilled water). Procedure To 3 different test tubes containing 0.02ml of test sample (T), 0.02 ml of standard (S) and 0.02 ml of blank (B), 2.0 ml of mixed colour reagent and 2.0 rnl of A of test sample Concentration Concentrall'on of urea (mgl ml) = x A of standard 1 A of test sample 200 - X - A of standard 1 mixed acid reagent were added to each test tube. They were mixed and placed in a boiling bath for 10 minutes. Afier which the tube were allowed to cool at room temperature in a bowl of water for about 5 minutes. Absorbance readings were taken at 520 nm. 2.13 Histopathological determination: 2.13.1 Isolation of some vital organs of rats The rats were dissected and some vital organs (liver, kidney, spleen, lung, heart and brain) were isolated and examined. The organs were fixed in formalin for preservation. Isolation of these organs was done after 3 weeks of treatment. A. Fixing the Tissue Ten percent (10 % vlv) formalin was used as the fixative agent and for the purpose of preservation. A thin section of the tissue (about 1 to 2 cm in diameter) was trimmed with a sharp razor blade. The small pieces of the tissue were placed in the 10 % formalin for 24 h. After placi~~t~e"'tis~ein'the solution, the container was shaken gently several times to make sure that the fluid had reached all surfaces and that pieces were not sticking to the bottom. B. Washing the Tissues I' - .-. After fixation was accomplished, the excess of the many fixatives was washed out of the tissue to prevent interference with subsequent processes. The tissue was washed with running water for a period of 24 h in 12 jars divided into four sets of three each. C. Dehydration All water was removed fiom the tissue before embedding the tissue in paraffin. The dehydration was achieved by immersing the thin section of the tissue in automatic tissue processor. The automatic tissue contained 12 jars. The first three (3) jars contained 70, 90, and 90 % absolute alcohol respectively. This was done to remove the water content in the tissues. The absolute alcohol reduced the shrinking that occurred in the tissue. The time for each step was 30 minutes. A second change of absolute alcohol was included to ensure complete removal of water. This was achieved in the second three (3) jars of automatic tissue processor. This is called a well-refining step. D. Clearing Solutions of xylene were used for this purpose. The step was achieved in the third three (3) jars of automatic tissue processor. This is because the alcohol (ethanol) used for dehydration would not dissolve or mix with molten paraffin; the tissue was immersed in xylene solution, which was miscible with both alcohol and paraffin before infiltration could take place. Clearing removes opacity from dehydrated tissue making them transparent. A period of 15 minutes was allowed to elapse before the tissue was removed from the solution for infiltration with paraffin. E. Infiltration with Paraffin Paraffin wax with melting point 50 to 52OC range was used to infiltrate the tissue. The tissue was transferred directly from the clearer to a bath containing melted paraffin. After 30 minutes to 1 h in the first bath, the tissue was then removed to a fresh dish of paraffin for a similar length of time. This was achieved in the forth (3) jars of the automatic tissue processor which contained paraffin wax. ., ,, ..T. .I . '. .. F. Embedding (Blocking) with Paraffin As soon as the tissue was thoroughly infiltrated with paraffin, it was ready to be embedded. Paraffin was allowed to soliditjl around and within the tissue. The tissue was placed in a smaller container already filled with melted paraffin and the whole was cooled rapidly with water. G. Paraffin Sectioning The embedded blocks were trimmed into squares and fixed in the microtome knives for sectioning and were floated on a water bath. H. Mounting Glass slides were thoroughly cleaned and thin smear of albumen fixative was made on the slides. The albumenised slide was used to collect the required section from the rest of the ribbon in the water. The section on the glass slide was kept moist before staining. I. Staining with Hematoxylin The slide was passed through a series of jars containing alcohols of decreasing strength and various staining solutions in the following order: 1 Xylene: 3 minutes 2 Absolute Alcohol: 2-3 minutes 3 95 % Alcohol: 2 minutes 4 70 % Alcohol: 2 minutes 5 Lug01 Solution: 3 minutes 6 Running water: 3 minutes 7 5 % sodium thiosulphate: 3 minutes 8 Running water: 5 minutes 9 Delafield hematoxxylin: 5 minutes 10 Running water: 3 minutes 1 I Scott solution: 9 minutes 12 Running water: 3 minutes J. Counterstaining with Eosin Counterstaining of the tissue with eosin was followed in the order as below: 1 70 % Alcohol 1 dip 2 95 % Alcohol 2 dips 3 Absolute Alcohol 3 minutes 4 Absolute Alcohol-xylene (I :1) , 3 minutes . ,, .,v,,r' ..I* 5 Xylene 3 minutes 6 Mounting Medium: the section was kept with xylene while cover glass was added on the slide. 2.14 Stability studies of Cannabis crude resin formulation 2.14.1 Preparation of stock solutions (A) Cannabis syrup stock solution: The powder of 1.0 g of Cannabis crude resin was dissolved in 50.0 ml of absolute ethanol to obtain a stock solution of 20.0 mglml. (B) Stock solution of sucrose: Sixty seven grams (67.0 g) of sucrose was dissolved in sterilized distilled water and made up to 100.0 ml thereby giving 67.0 % sucrose solution. (C) Chloroform water stock solution: A 0.5 ml volume of chloroform was dissolved in 99.5 ml of sterilized distilled water to obtain the chloroform water solution. (D) Sodium m-bisulphite stock solution: Zero point one gram (0.1 g) of sodium metabisulphite was added to 100.0 ml of distilled water to prepare a stock solution of 0.1 % sodium metabisulphite. (E) Ethylenediaminetetraacetic acid (EDTA) stock solution: A quantity of 1.5 g EDTA was added to 50.0 ml of distilled water to prepare a stock solution of 3.0 % EDTA. (F) Propylene glycol. Three batches of Cannabis crude syrup formulation were prepare as listed below:- (1) Cannabis crude resin syrup (CS) (2) Cannabis crude resifi Smp'(CS) '+ Sodium metabisulphite (CSN) (4) Cannabis crude resin syrup (CS) + Sodium metabisulphite (CSN) + EDTA (CSNE) Procedure: (I _ .- To 90 bottles, 2.0 ml and 5.0 ml of 20 mglml Cannabis stock solution were added respectively and made up to 30.0 ml with other stock solution of sucrose propylene glycol, chloroform water, sodium m-bisulphite and EDTA respectively in the following order as shown in the Table 7 below. Table 7: A table showing the formulation ratios of Cannabis resin syrup Batch No. of Cannabis Sucrose Propylene Chloroform 0.1 % 3.0 % bottles stock (67.0 YO) glycol water Sodium EDTA solution (ml) (mu solution m- (ml) 20.0 (mu bisulphite (mg/ml) (ml) CSN CSNE Key: -=nil 2.14.2 Exposure of the Samples to various Temperatures Ninety bottles containing 30 ml each of the syrup were stored at five different . ,. .. v. .v. ,, t ., temperatures, room temperature, 37", 40°, 50" and 60°C respectively. The accelerated stability studies of the syrup were determined over a period of six days and stability at room temperature for 11 months. The various syrup preparations on a pre-coated thin layer chromatography (TLC) plates wereI, developed- .. in n-hexane: dioxane 1: 1 solvent system and the various bands present in the form of their Rf values determined. 2.14.3 TLC determination The various batches containing the Cannabis crude resin formulations were applied to the chromatogram (silica gel 60F254) precoated TLC plates (Merck, Darmstradt)). Chromatography solvents: N-hexane: dioxane (1 :1) Reference1 standard solution: Freshly prepared Cannabis leaves extract in Chloroform and Syrup. Detection: b. Without chemical treatment: In UV-254 nm. The cannabinoids show fluorescence quenching. c. Fast blue salt reagent (FSB): Fast blue salt B 0.5 g was dissolved in 100.0 ml of distilled water. The developed TLC plates was sprayed, dried in warm air, and then immediately sprayed with 0.1 M NaOH. Cannabinoids form colour reaction forming violet-red, orange-red or carmine: the intensity of the colour depends strongly on the quantity of applied material and the subsequent NaOH or KOH 0.1 M treatment. After which the various Rfranges for the developed zone and the diameter of the zones dete~nined.~'' 2.14.4 Determination of Percentage Degradation Substances or medicinal products are subjected to a controlled exaggerated stress over a short period of time so that the rate of reaction is enhanced. The results provide information for the stability in the early stage of product development. Isothermal accelerated stability testing was carried out at room temperature, ie 37', 40°, 50' and 60°C for each sample as indicated in Table 7 and the rate constant determined at each temperature. The quantity of Cannabis crude resin remaining at each point in time in each sample under..> investigation was spectrophotometrically ., ,, - ..$ .I' . determined. From the values and with the aid of Arrhenius equation calculated mathematically, the quantity of Cannabis crude resin syrup degraded in each sample was obtained at the corresponding time interval and the rate constant K, determined. The Arrhenius equation: I' _ .:. KT=Ae - ...... Equation (1) where : KT = Specific reaction rate or rateconstant reactionat temperatureT A = Arrhenius, Ea = Energy of activation, R = Gas constant (1.987 caltdeg mol) T = Absolute temperature. 1 Ea A plot of Log KTversus-a straight line with aslopeof - -and an intercept of Log A. T 2.303 2.14.4.1 Determination of Shelf Life The decomposition rate constants of the samples at various temperatures were obtained by plotting log concentration against time as seen in Figures 47-76. The values of the specific rates of decomposition were then plotted against the reciprocals of the absolute temperature as illustrated in Figure 77-82. The K25extrapolated from the graph was used to obtain a measure of the stability of the sample at ordinary shelf conditions. 0.0 15 T =- ,where K = degradation first order constant lo K Data were expressed as the mean * standard error of mean (SEM) by means of SPSS soft ware. The trapezoid rule method was used for the pharmacokinetic data calculations. CHAPTER THREE 3.0 Results and Discussion 3.1 Yield The yield of Cannabis crude resin from whole leave extract in chloroform, n-hexane: chloroforn~,n-hexane, methylene chloride and ethanol respectively was, 17.58 %, 6.02 %, 3.57 %, 2.62 % and 5.99 % (wlw) respectively. 3.2 Phytochemical Analysis of Cannabis sativa leaves. Table 8 shows the phytochemical constituents of Cannabis sativa leaves. It indicates the presence of the following components: alkaloids, glycoside, saponins, flavonoids, resin, steroids, terpenes and carbohydrates, while reducing sugar, fats and oils are absent. Table 9 shows the results of the phytochemical analysis of the various solvent extracts of crude Cannabis resin used during studies. From Table 9 the order of extraction as against the various solvent is chloroform (WFC) > ethanol (WFET) > methylene chloride (WFMC) > n-hexane (WFH) and n-hexane: chloroform (WFHC) respectively. 3.3 Cytotoxicity Assay LC50 Table 10 shows the results of cytotoxicity assay of Cannabis crude resin extract, singly and in combinations attributed R. Table 8: Phytochemical constituents of Cannabis sativa leaves Constituents Relative presence Saponins ++++ Tannins +++ Flavonoids +++ Resin +++ Terpenoids ++ Alkaloids + Glycoside + Steroids + Carbohydrate + Reducing Sugar ND Fats and Oil ND Proteins ND Key: ++++ = Very abundant, +++ = Moderate present, ++ = present, + = Present in trace amount, ND = Not Detected Table 9: Phytochemical constituents of solvent extracts of crude Cannabis resin Constituents CHC13 CHzClz ETOH n-HEX n-HEX:CHC13 Saponins ++++ ..ND1;,...... Jq\JD . ND ND Tannins +++ + + + + Flavonoids +++ +++ +++ ND +++ Resin +++ +++ +++ +++ +++ Terpenoids +++ +++ " +++. + +++ Alkaloids + ND +++ ND ND Glycoside + ND +++ ND ND Steroids ++ ++ + + ++ Carbohydrate + + +++ + + Reducing Sugar + + + + ND Fats and Oil ND ND ND ND ND Proteins ND ND ND ND ND Key: ++++ = Very abundant, +++ = Moderate present, ++ = present, + = Present in trace amount, ND = Not Detected Table 10: Cytotoxicity of Cannabis crude resin extract singly and in combination % Lethality 40.00 26.67 6.27 650.23 3.4 Pharmacological Parameters 3.4.1 Effects of formulation of Cannabis resin on rat paw edema: The crude Cannabis resin (CCR) and its various formulations caused varying degrees of suppression of paw-edema in rats. The Cannabis crude resin (CCR) produced both dose and non-dose related inhibition of paw-edema (Tables 11 and 18). At 15 mg Kg- I , CCR caused an inhibitory effect comparably greater than that of 100 mg Kg-] piroxicam (Table I I). Cannabis syrup formulation (CS) caused a significant (P<0.05) dose-related inhibition of paw edema. However, the positive control, acetylsalicylic acid (100 mg Kg-]) caused a greater suppression of edema than the two doses of CS (Table 12 and Figure 1). The Cannabis syrup containing sodium m-bisulphite (CNS) caused a significant (P<0.05) non-dose related inhibition of paw-edema (Tables 13 and 17). The CNS formulation (25 mg Kg") caused inhibition markedly better than 100 mg kgm' acetylsalicylic acid (Figure 1). The formulation of Cannabis syrup containing sodium m-bisulphite and EDTA (CSNE) significantly (P<0.05) inhibited paw-edema (Tables 14 and 18). However, acetylsalicylic acid (100 mg kg-]) caused a greater inhibition of paw-edema than both doses of CSNE (Figure 1). The potency increased in the order of magnitude CS> CSN > CSNE > CCR. However, acetylsalicylic acid caused a more progressive inhibition of paw edema with time (Table 18). Various preparations .of,.Gmnabis sativa have been employed for their medicinal effect, including antipyretic, antirheumatic, antiallergic, analgesic and anti- inflammatory purposes.46 Extracts of Cannabis have been shown to possess anti- inflammatory activity2421'I2 and A~-THC,the, psychoactive component of Cannabis has been shown to possess anti-inflamikatdri activity in various models. 236, 313 1, addition cannabinol (CBN) and not cannabidiol (CBD) was shown to exhibit these activities.314However, constituents of Cannabis are known to stimulate 315,316 and inhibit317-319 prostaglandin (PG) release by influencing enzyme of this pathway. 320,321 Considerable discrepancy exists in the literature as to the activity of cannabinoids in a number of animal assays for anti-inflammatory activity, but no report has documented the assay using syrups. However, reports, suggest that THC, a cannabinoid, is twenty times as potent as aspirin and approximately twice as potent as hydrocortisone in the carrageenan edema test in rats242.This is in contrast to studies by Kosersky et a1243which reported that oral administration of A -9-THC was inactive in blocking carrageenan-induced edema in the rat paw model. These discrepancies may be attributed to the vehicle In the former propylene was while in the latter fatty acid-poor serum albumin was used. 243 Secondly, the anti-inflammatory effects may be due to the presence of both cannabinoids and non-cannabinoid components (for example cannflavin) have been shown to inhibit PG mobilization 318,319 and synthesis.321Cannabinoids, however stimulate and inhibit phospholipase A2 activityl%s well as inducing and inhibition of cyclogenase and lipoxygenase 321. Table 11: Effect of Cannabis crude resin on acute edema of the rat paw induced by egg albumin. Extract Dose Edema (cm) (Mean f SEM) CCR 10 0.550 + 0.09 0.475 f 0.10 0.350 f 0.100 0.275 f 0.1 1 0.300 f 0.057 Piroxicam 100 0.08 f 0.1 1 0.58 f 0.09 0.48 f 0.09 0.37f0.10 0.25f0.10 Control 0.550 f 0.086 0.550 f 0.10 0.450 f 0.086 0.400 f 0.07 0.325 f 0.096 CCR = Cannabis crude resin; N=4; Control 1 = Syrup Table 12: Effect of Carrnabis crude resin syrup on acute edema of the rat paw induced by egg albumin. Extract Dose Edema (cm) ( Mean + SEM ) CCRS 10 0.650 f 0.06* 0.475 f 0.047** 0.400 f 0.04 0.325 f 0.047 0.175 + 0.06* control2 - 0.925 f 0.047 0.850 f 0.095 0.700 f 0.10 0.600 f 0.08 0.675 f 0.125 , .. .. , , . . CCRS = Cannabis crude resin syrup; Note: N=4;Control2 = Syrup + sodium m-bisulphite * - P < 0.05; ** - P < 0.01 Table 13: Effect of Calimbis syrup on acute edema of the rat paw induced by egg albumin. Extract Dose EDEMA (cm) {mean fSEMI CSN 10 0.550 f 0.064** 0.500 f 0.07* 0.450 f 0.05** 0.400 f 0.1 15* 0.250 f 0.05** 25 0.575 f0.062* 0.450 f 0.028* 0.425 + 0.06* 0.400 f 0.08** 0.250 f 0.06** AS A 100 0.600 f 0.00** 0.600 f 0.00 0.500 +0.04** 0.600 f 0.08 0.500 + 0.04 control3 - 0.900 + 0.04 0.800f0.08 0.900f0.04 0.900f0.04 0.700f0.04 - CCR = Cunnabis crude resin Note: N=4; Control 3 = syrup + sodium m-bisulphite + EDTA * - P < 0.05; ** - P < 0.01 Table 14: Effect of Cannabis syrup on acute edema of the rat paw induced by egg albumin Extract Dose EDEMA (cm) {mean +SEMI CSN 10 0.575 f 0.025 0.550 f 0.028* 0.475 + 0.047 0.400 + 0.04* 0.400 f 0.04 2 5 0.625 f 0.085 0.575 f 0.075* 0.450 + 0.064 0.450 + 0.1 19 0.400 :!: 0.04 ASA 100 0.600 f 0.07 0.400 f 0.08** 0.200 f 0.07 0.267 i- 0.06** 0.260 f 0.07** ~ontrol" 0.8325 k 0.865 f 0.047 0.6325 f 0.06 0.550 + 0.028 0.565 f 0.069 ------CCR = Cannabis crude resin; Note: N=4; Control 4 =Propylene Glycol: Ethanol (10: 1) * - P < 0.05; ** - P < 0.01 ., ,,-,'? ..1: d. .,...;i ' . Table 15: Percentage inhibition of edema in rats treated with Crrntznbis crude resin formulation. Extract Dose Percentage inhibition of Edema ----- (mp/kg) 0.5 h ' 1.o-11" 2.0 11 3.0 h 4.0 h CCR 10.00 0.000 13.630 22.220 3 1.250 7.960 Piroxicam 50.00 85.450 5.450 6.670 7.500 23.070 Table: 16 Percentage inhibition of edema in rats treated with Cannabis crude resin syrup formulation. Extract Dose Percentage inhibition of Edema (%) Cannabis 10.00 22.77 0 40.625 42.205 50.757 78.125 Table 17 Percentage inhibition of edema in rats treated with Cannabis crude resin syrup containing sodium m-bisulphite formulation Extract Dose Percentage inhibition of Edema (%) CSN 10.00 38.888 37.500 50.000 61.110 64.285 25.00 25.000 43.750 52.770 55.555 64.285 Table 18: Percentage inhibition of edema in rats treated with Cannabis crude resin syrup containing sodium m-bisulphite and EDTA formulation. .< ,, % +...r. Extract Dose ~e2entageinhibition of Edema (%) (mgf kg) 0.5 h 1.0 h 2.0 h 3.0 h 4.0 h CSNE 10.00 30.970 36.489 24.960 29.320 29.328 ASA 100.00 27.970 53.810 68.404 54.060 64.660 / 0 10 mglkg 0 25 mghg 100 mglkg (ASA) I - - - .- .- .. .. CCR CSN CSNE Cannabis forrnulatlon (CCR= Cannabis crude resin (1.0h); CS = Cannabis Syrup (0.5h); CSN = Cannabis Syrup + Sodium meta bisulphite (2.0h) and CSNE = Cannabis syrup + sodlum meta bisulphite +EDTA (1.0h) InhibRon of Peak edema) Figure 1 : Effect of Cannabis crude resin and formulations on peak edema. +Cannabis syrup 10 mgkg - +- Cannabis syrup 25 mglkg -.-A---ASAlOOmgkg - -X- - -ve control Time (hour) ,I _... Figure 2: Effect of Cannabis crude resin syrup on paw edema in rats Cannabissyrup 10 mgkg - -S-- Cannabis syrup 25 mglkg - - -A-- - MA100 mgkg - - x . - -ve control Time (hour) I, Figure 2: Effect of Cannabis crude resin syrup on paw edema in rats +Can syrup + sodium meta bisdphite 10 m@g - +-Can syrup + sodium meta bisdphite 25 m@g -4 --ve control . ).. m x-. .Re / \b%-O*H I . .: Time (hour) Figure 3: Effect of Cannabis crude resin syrup containing sodium m-biiphite on paw edema in rats -+- Can syrup + sodium meta bisulphite + EIJrA (10 mglkg) - +- Can syrup + sodium meta bisulphite + EDTA (25 rqlkg) ---A---ASA 100 mlkg - .x - - -ve control *. *. *. *. .. -- .,.-----&--*...... "A- -**- ""---A 0.1 I ' -,,-....r..r,...t> ' 0 .. - ---- 7--- 1. -I--, . --,--I- -7 .sf0 0.5 1 1.5 2 2.5 3 3.5 4 4.: Time (hour) I' ? ... -+10 mglkg Cannabis crude resin - +- 15 mg/kg Cannabis crude resin - - -&- - .I00 mg/kg Piroxicam - -K-- -ve control 0 0.5 1 1.5 2 . 2.5 3 3.5 4 4.5 Time (hour) Figure 5: Effect of Cannabis resin on paw edema in rats. 3.5 Antimicrobial Assay Table 19 shows the results of the antimicrobial sensitivity test on the whole n-hexane: chloroform (WFHC), chloroform (WFC), n-hexane (WFH), methylene chloride (WFMC) and ethanol (WFE) extracts of Cannabis crude rein. The MICs of the above mentioned crude Cannabis resin extracts in B. subtilis, S. aureus and Salmonella typhii are shown in Table 20 and Figures 6 to 16. The five extracts were active against B subtilis. WFH, WFMC and WFE extracts were active against S, aureus while only WFH was active against Salmonella typhii. The MIC values of the whole extracts against B. subtilis were in the order of WFH > WFE > WFMC > WFHC while the control (gentamycin) showed a relatively low MIC value (0.0108 mglml) as compared to that of the above whole extracts. Similarly the MIC value of extract WFE was more potent against S. aureus as compared to WFH and WFMC while the whole extracts MIC values where higher than the control. Only WFH had any activity against Salmonella Typhii and its MIC value was 1.021 1 mglml. The total viable count or killing rate of the above mentioned extracts are indicated in Table 20. The results indicate the killing rate or viable rate count at four times the MIC (4 x MIC), two times MIC (2 x MIC), MIC and half MIC (% x MIC) against the stated test organisms. The result reveals that at 4 x MIC the Cannabis crude extract WFH and WFHC killed the test organisms (B. subtilis) as against WFC, WFMC and WFE respectively. Similarly, at twice the MIC all extracts with the exception of WFE ., ,a -'a .l, ,v. ,, ..,a . . killed the test organisms (B. subtilis) in the order of WFCH > WFC > WFMC > WFH. At the determined MIC values from the graphs in Figures 17 to 24 the killing rate of the extracts were in the order of WFHC > WFH > WFMC as against WFC and WFE that had no activity. Table 20 jndicates that the extract WFMC had a better viable count than other extracts. The viable count in Table 20 also indicates that against test organisms S. aureus, the Cannabis crude extract WFH and WFMC had better activity than other extracts in the order of its regressional analysis 0.698 > 0.0755. The result of these study demonstrates that the Cannabis extracts have an unset of bactericidal activity against S. aureus. Table 19: The table shows the activity of Cannabis crude extracts of the listed microorganisms DRUG B.subtilis S. aureus Salmonella E coli P.ariginosa Typhii WFH + + WFCH + + WFH + + WFMC + + + WFE + + + Table 19: The table shows the activity of Cannabis crude extracts of the listed microorganisms DRUG Bmbtilis S. aureus Salmonella E coli P,ariginosa Typhii WFH + + W FCH + + WFH + + WFMC + + + WFE + + + Table 20: MIC VALUES OFTHE FOLLOWING CRUDE CANNABSRESIN EXTRACTSIN B. subtilis, S. aureus and Salmonella Typhi DRUG B.subtilis S. aureus Salmonella Typhii 4xMIC 2xMIC MIC* 112 x 4xMIC 2xMIC MIC* 112 x 4xMIC 2xMIC MIC* 112 x *SEM *SEM SEM MIC * *SEM *SEM SEM MIC* * SEM SEM SEM MIC * SEM SEM SEM WFH 0.763 + 0.821 * 0.208 + 0.643 + 0.725 + 0.833 * 0.643 + 0.164 i WFCH 0.065 + 0.142 * WFH 0.643* 0.939+ 0.132* f' - WFMC - 0.14 1 0.048 + + - 0.014 * - 0.07 0.01 0.3 1 I' - WFE 0.978 * CONTROL 2.206 (GENTAM *0.41 0.33 YCIN) Coi~inc(pglrn~) + 6.subfilis -Linear (6.subtilis) Figure 6: A graph of Log conc against ED' of crude Cannabis extract WFC (Bacillus subtilis ) I B subtilis -Linear (B subtilis) -200 -1 Figure 7: A graph of Log conc against IZD~of crude Cannabis extract WFCH (Bacillus subtilis) I Log conc (pglml) 0 9. subtilis -Linear (9, subtilis) I* ..... Figure 8: A graph of Log conc against IZD~of crude Cannabis extract WFH (Bacillus subtilis ) / 1 Log Conc (pglml) / / / / / ..,, ,'.. v. ...;/;;pi a ' / ureus - - - Linear (staph aureus) Figure 9: A graph of Log conc against IZD' of crude Cannabis extract WFH (Staph aureus ) Salmonella typhi - - - - - Linear (Salmonella typhi) Figure 10: A graph of Log conc against IZD' of crude Cannabis extract WFH (Salmonella typhi) 500 - 300 - .<,,. 4 ,.r, ,I- ., ,.I* . . 7- I 7- 2.5 Log Conc (pglml) + 6. 'subt#is'"- Linear (8.subtilis) Figure11 : A graph of Log conc against IZD~of crude Cannabis extract WFMC (Bacillus subtilis ) Log Conc (~glml) Staph,,aurey<. - - - Linear (Staph aureus) Figure 12: A graph of Log conc against IZD~of crude Cannabis extract WFMC (Staph aureus) 0 0.5 1 1.5 2 2.5 Log Conc (pglml) + 6. subtilis -Linear (6,subtilis) Figure 13: A graph of Log conc against IZD' of crude Cannabis extract WFE (Bacillus subtilis) / 1 Log Conc (pglml) / Staph aureus - - - Linear (Staph aureus) Figure 14: A graph of Log conc against IZD' of crude Cannabis extract WFE (Staph aureus) / Log Conc.( yglml) 0, . .:. 0 B subtilis -Linear (B subtilis) Figure 15 A graph of Log conc against IZD~of Gentamycin (Bacillus subtilis ) control y = 626.18~- 591.85 + R' = 0.9677 I / / / / / / /I+ / 4 / / Ii / 4 / / / - I 3-- \ I I I 0.5 1 1.5 2 2.5 3 / / 1 Log Conc. (pglml) / / / -, *, .. + .q. .v ,....*> ' . / / / / / / / / Staph. aureus - - - Linear (Staph, aureus) Figure 16: A graph of Log conc against ED* of Gentamycin (Staph aureus) control Time (min) B, subtilis 4xMIC B.subtilis 2 x MIC A B. subtilis MIC X Contorl Gentamycin Linear (B, subtilis 4xMIC) --- Linear (B.subtilis 2 x MIC) ----- Linear (B.subtilis MIC) - - - -Linear (Contorl Gentamycin) Figure 17: Killing kinetics of WFH against 6.subtilis Time (min) S.aureus 4 xMlC W S. aureus 2 x MIC A S. aureus MIC X Control (gentamycin) Linear (S.aureus 4 xMIC) ---Linear (S. aureus 2 x IVIIC) - - - - - Linear (S. aureus MIC) I, . ---- Linear (Control (gentamycin)) Figure 18: Killing kinetics of WFH against S.aureus Time (min) Sal. Thyphii 2 x mix W Sal thyphii MIC Linear (Sal. Thyphii 2 x mix) - - - - - Llnear (Sal thyphii ME) Figure 19: Killing kinetics of WFH against Sal. thyphii \ Time (rnin) \ 6, subtilis 4 x MIC I! _ .,..: B subtilis 2 x MIC A Control (gentamycin) Linear (6. subtilis 4 x MIC) --- Linear (B subtilis 2 x MIC) - - - - Linear (Control (gentamycin)) Figure 20: Killing kinetics of WFC against B. subtilis Time (min) 6. subtilis 2 x MIC 6. subtilis MIC A B. subtilis 7/2 MIC I) _.... X Control (Gentarnycin) - - - Linear (6. subtilis 2 x MIC) - - - -.Linear (6. subtilis ME) --- - Linear (6, subtilis 1/2 MIC) - - - - Linear (Control (Gentarnycin)) Figure 21: Killing kinetics of WFCH against 6.subtilis \ Time (min) \ -8 B, subtilis 2 x MIC , . B. subtilis MIC A Control (gentamycin) - - - Linear (8,subtilis 2 x MlC) ----- Linear (6.subtilis MIC) -.. - Linear (Control (gentamycin)) Figure 22: Killing kinetics of WFMC against B. subtilis Time (min) ' control (gentamycin) St. aureus MIC 0 - .------Linear (St. aureus MlC) ---- Linear (control (gentamycin)) Figure 23: Killing kinetics of WFMC against St. aureus Time (min) St. aureus MIC 0 _ : control (gentamycin) - - - - - Linear (St, aureus MIC) - - - - Linear (control (gentamycin)) Figure 23: Killing kinetics of WFMC against St. aureus Time (min) 8,subtilis 1/2 x MlC , . i Control (gentamycin) - - - - Linear (B.subtilis 1/2 x MIC) - - - - Linear (Control (gent amycin)) Figure 24: Killing kinetics of WFE against B. subtilis 3.6 Effects of crude Cannabis resin extract on Haematological Parameters The haematological indices are represented in Figure 25 to 32 and appendix B. These results indicate that the haematological erythrocytic and leucocytic indices were not significantly different in all the treated animal groups for the first two weeks of treatment. However, on the third week, results showed a significant increase for the treated groups WA and WB in terms of the pack cell volume (PCV), red blood count (RBC) and total leucocytes count, absolute lymphocyte counts, monocyte count, neutrophiles and eosinophil counts. Results of the PCV, showed that there was a significant change in week 3 between the dosed groups WB and WC as against the vehicle WA and the untreated group WD (P < 0.05) (Figure 25). The results of WBC total count, total RBC count, differential counts (that is absolute lymphocyte count, monocyte count and neutrophiles count respectively) all followed the same pattern described above (Figure 26 to 30). However, the elevations observed in the treated groups were positively and significantly dose related (P<0.05) at week three. The eosinophil counts, however, did not indicate any significant differences in the first two weeks of treatment. In contrast to other haematological indices, eosinophils disappeared from the blood of the treated groups in the third week (Figure 3 1). It should be noted that eosinophils serve as detoxifiers and the disappearance of eosinophils in the blood of the dosed groups in weeks two and their total . ,, .. ..' . + disappearance in week three signifies that they have been fully consumed in the detoxification process. Therefore there was a toxic effect that used up the eosinophils from the blood. The presence of basophils is usually only incidental and therefore their presence in rats in the different dosed groups did not follow any trend. These general increases in haematological indices may be attributed to haemoconcentration induced by dehydration, bearing in mind that though Cannabis is an appetizer one of its side effects is dehydration. 322 - + - Pos~bvecontrol 4 -20 mgM of Cannab~scrude resin ---t- 40 mgMof crude Cannabis resin x negative control Experimental Period (weeks) , _ .- Figure 25: A graph of total WBC of cell per pL of blood against Experimental Period (weeks) - + - Positive control 4 -20 mg/d of crude Cannabis resin +40 mg/ml of cn~deCannabisresin -. - r-. Negative Control 0 0.5 1 1.5 2 2.5 3 3.5 Experimental Period ( weeks) Figure 26: A graph of Total RBC per pL of blood against Experimental period (weeks). 0 0.5 1 1.5 2 2.5 3 3.5 Experimental Period (weeks). - -0- - Positwe Control - - 20 mg/ml of oude Cannabis resin -40 mg/ml of crude Cannabis resin - X- Negative control Figure 27: A graph of Absolute Lyphocyte Count per pI of blood against Experimental Period (week). Experimenta1:Period (weeks) - C - Positwe Control - - 20 mglml of Crude Cannab~sresin -40 mglml of Crude Cannabis resin x Negat~veControl Figure 28: A graph of Absolute Monocyte count per pL of blood against Experimental Period (weeks). - + Positive Control - .I - 20 mglml of Crude Cannabis resin -40 rnglml of Crude cannabis resin r Negative Control 0 0.5 1 1.5 2 2.5 3 3.5 I' _ Experimental Period (weeks) Figure 29: A graph of Absolute Neuotrophil Counts per pl of blood against Experimental Period (weeks). 1 " -2 3 4 Experimental Period (weeks) Positive Control - a - 20 rnglrnl of Crude Cannabis resin -40 rnglrnl of Crude Cannabis resin - -X - Negative Control Figure 30: A graph of Absolute Eosinophil Counts per pL of blood against Experimental Period (weeks) 0 1 2 3 4 5 Eperimental Period (weeks) - f - Positive Control - a - 20 mghl of Crude Cannabis resin -40 mglrnl of Crude Cannabis Resin X Negative Control Figure 31 : A graph of PVC (%) per yL of blood against Experimental Period (weeks) 3.7 Cannabis Suppositories formulation and analysis 3.7.1 Appearance of the suppositories The suppositories were torpedo-shaped, smooth in texture with absence of entrapped air, contraction holes or brittle fracture. The external and internal surfaces of the suppositories were uniform in appearance when examined with the naked eye and hand lens. The uniformity in appearance was in terms of colour (greenish brown) and texture. This indicates satisfactory subdivision and dispersing of suspended material and all the batches passed the test according to British. Pharmacopoeia specifications. 323 The suppositories had fairly uniform weights as shown in Table 21 with the exception of batch 3, which was out of range. The coefficients of variation (CV) were 2.91, 3.05, 6.54, 2.45, 3.79 and 1.69 for batches 1, 2, 3, 4, 5 and 6 respectively. This indicates that the mixture of ingredients and the suppository base was fairly homogenous before pouring into moulds. However, the overall variation in weight of the suppositories may have resulted from sedimentation of drug during pouring since the drug (that is Cannabis) was only dispersed in the base. The batches, with the exception of batch 3 nevertheless passed the test according to B. P specifications 323. The results of the liquefaction time test carried out on the suppositories are given in table 21. The monograph (USPXX) 324 did not specify any range for the liquefaction time of Cannabis crude resin extract suppositories since the liquefaction time depends to some extent, upon the size and.,,... shape'., '.I* of the rectal suppositories used. Liquefaction time take considerable time (although it can be shortened by the addition of drug, propylene glycol, water etc) and for drugs with local action, the softening or liquefying is not always essential, but it is for those including a drug for a general action example Cannabis release of which depknds in first instance liquefaction of the suppositories. 325 The knowledge of the liquefaction time is essential because a suppository, which takes too long to liquefy, may be expelled before liquefaction together with the drug it contains. Besides liquefaction time is analogous to disintegration time of tablets. A drug formulation that dose not liquefy easily may be expelled before drug release occurs and may also exert a mechanical irritant action on the ampulla even if the base and the drug, per se are not irritant '05. The 900 mg Cannabis crude in 4 % Tween 85 showed highest melting time. This may be attributed to the heat resistance of Tween 85, which modified the liquefaction of theobroma oil. Figure 35 to 41 shows the release profile of Cannabis Crude resin from the suppositories. The release of Cannabis crude was prolonged in batches 1 and 2 containing 2 and 6 % Tween 85 respectively, while batches 4, 5 and 6 percentage releases was very low. This may be as the result of in cooperation of various percentages of Tween 85 into batches 1 to 3 while batches 4 to 6 had no Tween 85 in cooperated in to them. Table 21: Results of the Physical parameters of the Cannabis Crude Resin Extract Suppositories Batch Parameter Weight uniformity Liquefaction Time Absolute Drug Content (X * CV) (X * SD) 1 1.08 k 2.91 11.00 * 3.60 300 in 2 % Tween 85 2 1.04 * 3.05 11.67 * 0.57 300 in 2 % Tween 85 3 0.96 * 6.54 10.00 * 1.OO 300 in 2 % Tween 85 4 1.04 * 2.45 9.33 * 0.57 300 5 1.04 k 3.79 8.67 * 0.57 600 6 1.09 * 1.69 7.43 * 1.25 900 X = Mean, CV = Coefficient of Variation, SD = Standard Deviation Can sup 300 mg (2% Tween - a - Can sup 300 mg ( 4 % Tween 85) 0.07 1 - - t - -Can sup 300 mg (6% Tween 85) +Can sup 300mg - -I - Can sup 600 mg - C - Can supp 900 mg 150 200 Time (min) Figure 32: A graph of the release profile of crude Cannabis from the suppository in 0.1N HCI Time (min) Figure 33: A graph of the release profile of crude Cannabis from the suppository in 0.1N HCI +Cannabis suppository 300 mg (2Ya Tween 85) 0 50 100 150 200 Time (min) Figure 34: A graph of the release profile of crude Cannabis from the suppository in 0.1N HCI +Cannabis suppository (300 mg 4OhTween 85) 0 50 100 150 200 Time (min) Figure 35: A graph of the release profile of crude Cannabis from the suppository in 0.1N HCI +Cannabis suppository300 mg ( 6% Tween 85) 0.002 a V) -ma 2 s 0.0015 . ,, ,. . s. .v. , .. . I-- -- I 0 50 1,oo - ... 150 200 Time (min) Figure 36: A graph of the release profile of crude Cannabis from the suppository in 0.1N HCI +Cannabis suppository 300 mg Time (min) Figure 37: A graph of the release profile of crude Cannabis from the suppository in 0.1N HCI +Cannabis suppository 600mg 0 50 100 150 200 Time (min) Figure 38: A graph of the release profile of crude Cannabis from the suppository in 0.1N HCI +Cannabis suppository 900 mg .,,.; ..-!: .I' . . ..-> ' +Can sup 100 mg(2% Tween 85) . I' - .:- 0 50 100 150 200 Time (min) Figure 39: A graph of the release profile of crude Cannabis from the suppository in 0.1N HCI 0 50 100 150 200 Time (min) Figure 40: A graph of the release profile of crude Cannabis from the suppository in 0.1 N HCI +Can sup 100 mg 4% Tween 85) Conc (mgOh) Figure 41 : Cannabis Beefs plot for suppsitories release studies + Cannabis -Linear (Cannabis ) 3.8 Stability studies Figure 42 shows the UV absorption spectra of crude Cannabis resin extract and it has an absorption maxima at 274 nm. The results of accelerated stability studies of Cannabis crude resin syrup formulations are shown in Table 22. From Table 22 the accelerated stability (degradation) studies indicated that the rate constant &) and shelf-life (tsO) values containing 200 mg % all significantly increased (P < 0.05) in the various batches in the order CRSN > CRS > CRSNE. The formulations containing the 500 mg % show a shelf-life in the increasing order of CRSNE > CRSN > CRS. The result of rate stability studies of Cannabis crude resin syrup formulations is shown in Table 25. Table 25 shows the magnitude of significantly increasing (P < 0.05) in both rate constant and shelf-life of the rate stability studies in the various batches in the order CRSN > CRSNE > CRS for the 200 mg% formulation. Only CRNS had significant increase of (P > 0.0.5) in the 500 mg% formulation for the shelf life. The shelf-life of Cannabis resin syrup formulation in the various batches were estimated from the extrapolation intercepts estimate of Arrhenius plot of log k against the reciprocal of absolute temperature 1/T at 27 O C (room temperature) as shown in Tables 22 and 23 respectively. ., ,, .. .. ?. ,,'. . , , .'.> . . Table 24 shows the TLC Rf analyses of the various identified spots in the Cannabis crude resin syrup formulation for both accelerated and rate degradation studies. Table 25 shows the disappearance of some of the developed spots in the rate studies in week 28 of the studies. Figure 43. t~ 46 shows the isolated spots obtained from preparative TLC and which were spectrophotometrically determined by scanning. The results of both accelerated and a rate stability study indicate that the various batches of Cannabis crude resin syrup formulation may have undergone a first order kinetic degradation. This degradation may have been due to the conversion of the constituents of Cannabis, which are thermodynamically unstable to their degradation product as shown in the TLC (Rf values) in week 28 when most of the active constituents have been converted to their acidic constituents. The chemistry of cannabinoids is much more complex than the structure of h9-tetrahydrocannbinol would indicate. The reactions are capricious and sensitive to reaction conditions and they form complex mixtures, which are difficult to separate. Secondly, some authors have reported that Cannabis upon smoking, chemical changes occur within the cigarette including the formation of b9-tetrahydrocannbinol by decarboxylation of b9- tetrahydrocannbinol acid, a component of the plant and isomerization of Cannabis to their thermodynamically more stable components. 336 This is in line with the extraction of the resinous tar with soxhlet extractor at slight elevated temperature. Note that Cannabis is an optically active resinous material which is very lipid soluble and water insoluble, it is also a photolabile and thermolabile compound susceptible to heat, acid and oxidation by oxygen and can be partly oxidized to CBN. 337 Figures 42 to 46 show the absorbance spectra obtained when some of the active constituents from TLC were spectrophotometrically scanned to ascertain their effect on stability of the syrup formulation. From the above figure one may deduce that from the sharpness of the curve obtained there is an overlap of many constituents outside the spot the samples scanned. This accounts for the observed peak at 274 nm used in the main study. Table 22: Accelerated stability study of Cannabis syrup formulation for six days. Batch 2 ml of Cannabis crude resin 5 ml of Cannabis crude resin (20 mglml) * SEM (20 mg/ml) * SEM K27 (days") t go(da~s) K27 (days-')' t 90 (days) CRS 0.0 178 * 0.0022* 5.89 * 0.073* 0.00597 * 0.0182 20.47 * 4.820 CRSN 0.01 65 * 0.0005* 6.38 * 0.213* 0.00386 * 0.0000 27.1 6 * 0.236 CRSNE 0.01 86 * 0.0506* 5.67 * 0.073* 0.00320 * 0.0001 33.00 * 1.790 Table 23: Rate stability (degradation) studies for 36 weeks of Cannabis syrup formulation. Batch 2 ml of Cannabis crude resin 5 ml of Cannabis crude resin (20 mg/m!) * SEM (20 mg/ml) * SEM K27(weeks") t rn(weeks) K27(weeks-')' t 90 (weeks) CRS 0.077 * 0,001 5* 14.596 * 2.95* CRSN 0.017 * 0.0032 63.83 * 12.38 0.00084 * 0.000073 126.81 * 0.000007* CRSNE 0.026 h 0.0080* 40.50 h 1 SO* 0.00076 * 0.0001 3 152.17 h 0.00013 NOTE: CRS = Crude Cannabis resiqgy-up;,CRS,N = Crude Cannabis resin syrup + sodium metabisulphite; CRSNE = Crude Cannabis resin syrup + sodium meta bisulphite + EDTA *P < 0.05 Table 24: Rf values of Cannabis syrup stored at different temperature. Batch Cannabis Rf values for different Temperatures of Cannabis crude Cannabis derivatives extract mot. crude 5000 mg (%) 5 ml 2000 mg (%) 2 ml resin 37' 40' 50' 60' 37' 40' 50' 60' extract CCR CBD 0.58 0.516 0.808 0.78 0.58 0.516 0.808 0.76 0.608 THC 0.525 0.45 0.716 0.69 0.525 0.45 0.725 0.69 0.54 CBN 0.44 ------0.44 CBD 0.33 ------0.35 CBDA 0.275 0.1 16 - - 0.266 0.116 - - 0.275 CBDA 0.21 0.09 0.208 - 0.21 0.09 0.208 - 0.141 CBDA 0.1 16 0.066 - 0.375 0.1 16 0.066 - 0.108 CBDA 0.066 - 0.15 0.19 0.066 - 0.16 0.18 0.055 CBDA - - - 0.15 - - - 0.14 - CCRN CBD 0.566 0.58 0.79 0.73 0.583 0.58 0.79 0.725 0.6 THC 0.5 0.508 0.708 0.65 0.5 0.508 0.708 0.65 0.525 CBN ------0.416 CBDA - - 0.4 - - - 0.38 - 0.308 CBDA 0.26 0.26 - 0.33 - - - 0.33 0.26 CBDA 0.135 - 0.2 0.183 0.114 - - 0.183 0.15 CBDA 0.106 0.135 0.133 0.137 0.118 0.135 0.133 0.14 0.1 CCRNE CBD .583 0.5 0.68 0.675 0.583 0.808 0.71 0.675 0.616 THC 0.475 0.433 0.608 0.59 0.475 0.441 0.64 0.6 0.516 CBN 0.39 - - - 0.375 - - - 0.39 CBDA 0.25 0.225 0.375 - 0.26 0.237 - - 0.28 CBDA - 0.133 - 0.33 - 0.133 - 0.33 0.225 CBDA 0.125 0.091 0.175 0.175 0.15 0.091 0.18 0.175 0.125 CBDA 0.083 - 0.133 0.133 0.1 16 - 0.15 0.14 0.083 CBDA - - 0.09 0.083 - - 0.09 0.090 Key = CCR = Cannabis crude resin foimuhtio~CCRN = Cannabis crude resin formulation with sodium metabisulphite, CCRNE = Cannabis crude resin formulation with sodium meta bisulphite and EDTA. Table 25: Rfvalues of Cannabis syrup stored at room temperature. BATCH Cannabis Rf values for room Temperatures of Cannabis derivatives Cannabis crude extract spot. crude resin Week one Week 28 extract 5000 2000 5000 2000 CCR CBD 0.808 0.825 - - 0.84 THC 0.733 0.74 1 - - 0.758 CBN 0.625 0.625 0.705 0.705 0.64 1 CBD 0.508 - - - 0.525 CBDA 0.433 0.425 - - 0.433 CBDA 0.233 0.425 - - 0.233 CBDA 0.183 0.233 - - 0.183 CBDA - - - - 0.15 CBDA 0.1 0.1 0.12 0.12 0.108 - 0.07 0.07 0.09 CCRN CBD 0.69 0.69 - - -do- THC 0.608 0.616 - - CBN 0.476 - - - 0.4 16 - - - CBDA 0.36 0.36 - - CBDA 0.83 0.176 - - CBDA 0.141 0.16 - - CBDA 0.1 0.09 0.1 0.85 CCRNE CBD - - - - -do- THC 0.558 0.53 - - CBN 0.483 0.46 - - CBDA 0.38 0.375 - - CBDA 0.266 0.258 - - CBDA 0.158 .,. .,,,, p.158 0.12 0.1 1 Key = CCR = Cannabis crude resin'formulation, CCRN = Cannabis crude resin formulation with sodium meta bisulphite, CCRNE = Cannabis crude resin formulation with sodium meta bisulphite and EDTA, Figure 42: A scan of Cannabis crude resin extract showing maximum peak at 274 nm Figure 42: Figure 43: Spectrophotometric scan of Cannabis crude resin sport A from a TLC plate showing various peaks. Fisure 44: Spectrophotometric scan of Cannabis crude resin sport B from a TLC plate ... showing various peaks. Figure 45: Spectrophotometric scan of Cannabis crude resin sport C fiom a TLC plate showing various peaks. Figure 46: Spectrophotometric scan of Cannabis crude resin sport D fiom a TLC plate showing various peaks. 3.9 Biochemical Parameters 3.9.1 Effect of Cannabis Crude Resin on Glutamate-Oxaloacetate Transaminase (SGOT) Activity in Rats Figure 47 and Table 26 shows dosed-related differences in all parameters assayed when the rats were administered varying doses of 20 to 40 mglkg body weight of the extract, Cannabis crude resin. There was marked significant difference (Pc0.05) in the level of SGOT in the rats after one-week treatment (i.p) with the extract (20 mglkg) when compared with the untreated group. A significant difference (p<0.05) was also observed in the SGOT level of rats administered (i.p) high dose of 40 mglkg of the extract after one week of administration when compared with the untreated animal group. There was no significant difference (P>0.05) in the SGOT level when the two doses (20 and 40 mglkg) were administered (i.p) to rats after one week of treatment. However, SGOT activity increased significantly (P<0.05) in the serum of the test animals exposed (i.p) to both the vehicle and the extract when compared with the untreated animal. 3.9.2 Effect of Cannabis Crude Resin on Glutamate-Pyruvate Transaminase Activity in Rats Figure 48 and Table 27 shows dosed-related differences in all parameters determined when rats were administered varying dose of 20 to 40 mg/kg body weight of the ., ,* ;? .-!.r)'...... " > extract. 3.9.3 Effect of Cannabis Crude Resin on Urea Activity in Rats Figure 49 and Table 28 shows the result of .kffects of urea on the rats after the administration of Cannabis crude resin for two weeks. The results indicated a decrease in urea activity after week one. However, after two weeks of treatment with Cannabis crude resin the activities of these enzymes decreased or increased. It has been reported that an increase in the activity of serum enzyme is an indication of damage to the hepatocytes, which causes these enzymes to leak into the serum. However, certain conditions that could lead to elevated level of serum enzymes such as diabetes mellitus hypothyroidism and hyperadrenocorticism especially in SGOT and alkaline phosphatase as they are not 1 c3 Week 0%; I-!!! I-!!! Week Two 40 mglkg pos. cont neg. cont Dosed group Figure 47: A graph of Glutamate Oxaloacetate Transaminase Activity in serum controlled and test animals after administering Cannabis crude resin extract. 20 mglkg 40 mglkg pos. cont neg. cont Dosed Group Figure 48:A graph of Glutamate Pyruvate Transaminase Activity in serum controlled and test animals after administering Cannabis crude resin extract Week One 5l Week Two 20 mglkg 40 mglkg pos. cont neg. cont Dosed Group Figure 49: A graph of Urea Activity in serum controlled and test animals after administering Cannabis crude resin extract Table 26: Amount of serum Glutamate-Oxaloacetate Transaminase in rats injected with different concentrations of Cannabis crude resin - Dose (mg/Kg) Week I * SEM Week 2 * SEM (MmoVliter) (MmoVliter) 20 32.39 * 0.87 2.03 * 0.26 40 35.11 * 2.02 7.72 * 0.94 Positive control (vehicle) 43.26 * 15.92 12.06 * 2.86 Negative control 2 1.48 * 8.65 4.46 * 0.1 1 Table 27: Amount of serum Glutamate-pyruvate Transaminase in rats injected with different concentrations of Cannabis crude resin 20 29.29 * 3.92 35.42 * 3.03 40 3 1.82 * 0.93 43.18 * 9.15 Positive control (vehicle).. ,.-...... ,...... '.-58.,42 * 4.20 59.27 * 0.92 Negative control 34.66 =t6.45 42.27 * 1.19 Table 28: Amount of serum Urea in rats injkted with different concentrations of Cannabis crude resin Dose (mg/Kg) Week 1 * SEM Week 2 * SEM (MmoVliter) (MmoVliter) 20 9.04 * 0.75 4.64* 0.72 Positive control (vehicle) 7.26 * 0.84 2.27 * 0.45 Negative control 12.70 * 1.25 6.27 * 1.55 3.10 Histopathological effects of Cannabis crude resin 3.10.1 Effects of Cannabis Crude Resin on the Liver of Rats No histological change was observed in the liver of the negative control rats administered with neither Cannabis crude extract nor the vehicle after one to three weeks. The negative control rats had liver sections with normal centrilobular and portal area of the hepatic lobule. Also, a normal architecture of the hepatic cords was observed (Plate 1). However, in the first week, sections of the liver treated with 20 and 40 mg/kg (Plate 3 and 4 respectively) of Cannabis crude extract showed some histological changes along the centrilobular and portal area of the hepatic lobule. These sections were compared with sections of the liver administered ethanol: propyleneglycol (vehicle) Plate 2. The liver belonging to the 20 mglkg dosed group showed vascular hyalinization, portal congestion and sinusoidal dilation in the portal area. The sections of the liver of rat dosed 40 mglkg displayed severe hepatic necrosis and parenchymal degeneration while the section of the liver of the rat given the vehicle showed mild necrosis of hepatocyte. At week 2, the liver of the rats treated with 20 and 40 mgkg respectively showed features of sinusoidal dilation, necrosis and hyalinization of the hepatic lobule, while that given the vehicle showed signs of sinusoidal dilation and mild degree of hyalinization. The liver sections given 40 mg/kg and the vehicle are shown in Plate 5 and 6 respectively. During week 3, the rats given 20 and 40 mgkg drug respectively did show evidence of pronounced hepatocellular necrosis and hyalinization indicative of hepatotoxicity, ., ,,. 4.r. .I. . ,.$> which appeared to be dose dependent. Similar, to week 1 and 2, the liver of rats' dosed vehicle in week 3 did show necrosis in the parenchyma. The sections of the liver of rats given 20 and 40 mglkg and the vehicle respectively are shown in Plate 7, 8 and 9 respectively. The degree of inducement of liver lesions by an nab is crude resin was dose/ and time dependent. These lesions observed may be as a result of direct effect of Cannabis on the liver. Indeed all direct hepatotoxins interfere with protein synthesis. Cannabis can have direct hepatotoxic effects in rats. Toxicity is especially liable when protein synthesis is under stress. Plate 1: Normal Liver cells. The liver Section of the rats given neither Cannabis crude resin nor vehicle. Haematoxylin and Eosin stain. X 100 (Untreated, control rats). Plate 2: Week one. The liver from dosed fisitive control vehicle showing mild necrosis of hepatocytes in the hepatic portal area (arrow head). Haematoxylin and Eosin Stain. (X 100) Plate 3: Week one. The liver section to 20 mgkg dosed group showings vascular hyalinization, portal congestion and sinusoidal dilitation. Haematoxylin and Eosin technique stain. (X 100) Plate 6: Week two. The liver section of rats dosed positive control vehicle showing sinusoidal dilation and mild degree hyalinisation of hyperaemia. Haematoxylin and Eosin stain. (X 100 Plate 7: Liver section of rats treated with 20 mglkg drug for three weeks. Shows pronounced necrosis and hyalinization. Haematoxylin and Eosin stain. (X 100) Plate 8: liver section of rats treated with 40 mglkg drug for week three. Shows evidence of pronounced hyalinization and necrosis. Haematoxylin and Eosin stain. (X 100) Plate 9: Week three: Live section of rats dosed positive control vehicle and shows necrosis in the parenchyma. Haematoxylin and Eosin stain. (X 100) 3.10.2 Effects of Cannabis Crude Resin on the Brain of Rats The brain samples of rats treated with 20 and 40 mglkg of Cannabis crude resin showed lesions after week one. The brain cells of rats given 20 and 40 mg/kg are shown in Plates 11 and 12 respectively. The photomicrography (that is Plate 11 and 12) displayed evidence of nuclear eosinophilia of the cell bodies and chromatolysis, which are suggestive of neurotoxicity. Similarly, the vehicle shows evidence of chromatolysis, edematous vascular congestion and eosinophilia of the cell bodies, which are suggestive of mild neurotoxicity. The sections of the brain as shown in Plates 13 and 14. At week two, rats treated with 20 and 40 mglkg showed evidence of eosinophilia of cell bodies and chromatolysis of the neurons suggestive of neurotoxicity. Plates 15 and 16 showed the affected sections of the brain. At week three, rats dosed with 20 and 40 mgkg respectively showed evidence of nuclear eosinophilia in the cell bodies, arterial attenuation and edema which features are indicative of neurotoxicity. The brain cells of rats given 20 and 40 mglkg are shown in Plates 17 and 18 respectively. Similarly, Plate 19 shows the brain cells of rats given the vehicle suggestive of mild nuclear eosinophilia. However, this neurotoxicity in the rat's brain are dose dependent on the Cannabis crude resin concentration. 3.10.3 Effects of Cannabis Crude Resin on the Kidney of Rats The kidney cells of the rats treated .wit.h different concentrations of Cannabis crude resin showed lesions after weeks 1, 2 and 3. However, it is not always that rise in the level of biochemical parameter in the serum may lead to disease condition. Though the rise in the level of serum urea in itself indicates possible malfunction of the kidney as confirmed by sectons of the kidney cells iri'..week 2 dosed with 20 and 40 mglkg of the crude extract in Plates 20 and 21. The lesions observed were interstitial large vacuolation, edema and showing droplets of hyaline in the tubular and degenerative changes in the glomeruli. At week 3, rats dosed with 20 mglkg of the drug showed necrosis and hyalinization as shown in Plate 22. The section of the rats' kidney treated with vehicle and those neither given the drug nor vehicle showed normal architecture in after weeks 1,2 and 3. Plate 10: Week one, the positive control (vehicle without drug) of the brain shows evidence of eosinophilia of cell bodies, chromatolysis and edematous vascular congestion. Haematoxylin and Eosin stain. (X 100) Plate1 1: Normal architecture of the brain showing no pathology in controlled animals (untreated rats). Haematoxylin and Eosin stain. (X 100) Plate 12: The brain section of the rats treated with 20 mg/kg drug for one week. Shows evidence of nuclear eosinophilia and chromatolysis. Haematoxylin and Eosin technique stain. (X 100) Plate 13: The brain section of the rats treated with 40 mg/kg drug for one week. It shows prominent nuclear eosinophilia of the cell bodies. Haematoxylin and Eosin technique stain. (X 100) Plate 14: Week 2. The photomicrograph of the brain of rats treated with vehicle. It shows nuclear eosinophilia suggestive of mild toxicity. Haematoxylin and Eosin stain. (X 100) Plate 15: Week two. Photomicrograp4,of the,brain of rats treated with 20 mglkg showing eosinophilia and chromatolysis of the neurons suggestion of neurotoxicity. Haematoxylin and Eosin stain. (X 100) Plate 16: The brain section of the rats treated with 40 mglkg drug for two week. Showing prominent eosinophilia of cell bodies suggestive of neurotoxicity. Haematoxylin and Eosin stain. (X 100) Platel7: Week three. Photomicrograph of the brain of rats treated with vehicle and showing mild nuclear eosinophilia. Hsiimatoxylin and Eosin stain. (X 100) Platela: The brain section of rats treated with 20 mglkg drug for three weeks. Shows evidence of nuclear eosinophilia in the cell bodies of neurons suggestive of mild neurotoxicity. Haematoxylin and Eosin stain. (X 100) Plate 19: Brain section of the rats treated with' 40 mgtkg drug for three week. Showing nuclear eosinophilia, arterial attenuation and edema which features are indicative of neurotoxicity. Haematoxylin and Eosin stain. (X 100) Plate 20: Normal kidney. The kidney section of rats given neither drug nor vehicle showing normal architecture. Haematoxylin and Eosin stain. (X 100) 22: The kidney section of rats treated with 40 mglkg for two weeks lying interstitial large vacuolation and edema. Haematoxylin and Eosin (X 100) Plate 23: Kidney section of the rats treated with vehicle for three weeks. It shows the normal architecture. Haematoxylin and Eosin stain. (X 100) 3.10.4 Effects of Cannabis Crude Resin on the Spleen of Rat Platc 25 and 26 show the normal architecture and the positive conlrol section of [he spleen of rats treated with Cannabis crude resin cxtract. Similarly Plates 25 to 27 shows ~hcsections of the rats splccn aftcr 1, 2 and 3 weeks of administration of the crude extract. The spleen sections of plate 27 to 29 of both treated rats and vehicle showed lesions. This shows that at these concentrations and within the period that Cannabis crude resin was administered, it produced pathological effects on these organs. The spleen cells of rats treated with 20 and 40 mglkg of Cannabis crude resin are shown in Plates 27 and 28 after week one and Plate 29 after week three. In week one, the section of the rats spleen dosed with 20 and 40 mg/kg respectively displayed evidence of necrosis, lymphocytic loss in the germinal centre of the follicles, degenerate and condensation of the lymphocytes in some areas. At week three, the section of the rat spleen dosed with 40 mglkg showed marked and severe follicular derangement, necrosis and lymphocytic loss as indicated in Plate 27. However, the section of the rat spleen cells given vehicle and the section of the rat spleen not given drug or vehicle both showed a normal architecture as indicated in Plate 25. Plate 25: The spleen section of the rats showing normal architecture in the control animals. Haematoxylin and Ecwin stain. (X 100) Plate 26: The spleen section of the rats treated with vehicle for week one. Haematoxylin and Eosin stain. (X 100) Plate 27: The spleen section of rats treated with 20 mg/kg drug for one week. It shows evidence of necrosis, disappearance of lymphocytes at the germinal centre of the follicles. Lymphocytes are also degenerate and condense in some other areas. Haematoxylin and Eosin stain. (X 100) Plate 28: The spleen section of rats treated with 40 mg/kg drug for one week. Shows evidence of severe necrosis and cellular degeneration of the follicles. Haematoxylin and Eosin stain. (X 100) Plate 29: The spleen section of rats treated with 40 mgkg drug for three weeks. showing marked follicular derangement, necrosis and lymphocytic loss. Haematoxylin and Eosin stain. (X 100) CONCLUSION In conclusion, the result of this study indicated that:- 3 The leaf of Cannabis sativa Linn was found to contain, saponins, tannins, flavonoids, carbohydrates, proteins, glycosides and alkaloids. The crude resin from the leaf is bioactive, as indicated by the cytotoxicity assay. Suffice it to mention that brine shrimp lethality assay is an accepted method for screening plant material for presence of bioactive metabolites to which the pharmacological and antimicrobial activity observed could be attributed. 3 The present study has provided a direct evidence for antimicrobial activity of Cannabis crude resin extract. i; Extracts of Cannabis was shown to possess anti-inflammatory activity and a9- THC, the psychoactive component of Cannabis has been shown to possess anti- inflammatory activity in various models. In addition, cannabinol (CBN) and not cannabidiol (CBD) was shown to exhibit these activities. 3 The results of both accelerated and rate stability study indicate that the various batches of Cannabis crude resin syrup formulation may have undergone a first order kinetic degradation. 3 The toxicological parameters indicated that Cannabis on administration chronically is toxic and dose-dependent. Reference Li, H. I, and Lin, H. (1974). 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Re~tal.Suppository.. , Am1 Ther. lO(6): 1-4 325 Perez-Reyes, M., Wagner, D., Brine, D. R., Christensen, D. H., Davis, K. H. and Wall, M. E. (1976). Tetrahydrocannabinol: Plasma disappearance in man and rate of penetration to mouse brain. In The Pharmacology of Marijuana, ed. by M. C. Braude and S. Szara, Raven Press, New York, pp. 1 17-124. I' . .I. 326 Truitt, E. B. (1971). Biological disposition of tetrahydrocannabinol. Pharmacol. Rev. 23: 273-278. Appendix A Formulas used for calculations in suppositories. - Mean (x)- Cx...... (1) n S - X Variance (var) = U...... n-1 S tan hrd devicr~ions= ...... (3) n - 1 Where n is the number of suppositories sampled; Cx is the total weight of suppositories Appendix B TABLE A: Total PVC Count Week Positive control 20 (mghl) 40 (mg/ml) Negative control Table B: WBC Total Count Week Posiiivc control 20 (mglml) 40 (mglrnl) Negative control 0 7,233.33 *666.19 8,983.33 * 886.91 7,883.33 + 8,893.75 f Table C: RBC Count Wcek Positive control 20 (mg/ml) 40 (mg/ml) Negative control Table D: Lyphocyte Counts Weck Positive control 20 (mg/ml) 40 (mg/ml) Negative control 0 4,676.67 * 357.34 5,332.50 574.20 4,987.00 * 722.16 4,730.44 * 694.50 Table E: Monocyte Count /PI of blood Wcek Positive control 20 (mglml) 40 (mglml) Negative control 0 1303.44 257.02 1913.00 * 292.67 1457.78 * 241.24 132 1.63 * 15 1.85 SD (722.80) SD (825.33) SD (680.30) SD (428.24) 1 1343.50 168.39 1196.1 1 * 200.67 1622.67 263.91 1234.87 * 178.24 SD (474.86) SD (565.91) SD (744.23) SD (502.64) 2 1087.83 150.74 1147.67 * 146.84 1559.83 11 1.84 1253.36 * 119.22 SD (425.1 1) SD (414.11) SD (315.41) SD (336.22) 3 1687.33 232.3 l"455.00 * 3 17.57" 2343.83 118.28"557.67 * 137.12" SD (655.13) SD (895.55) SD (333.56) SD (386.70) Table F: Neutroplul Counts Wecks Positivc control 20 (mg/ml) 40 (mglml) Negative control Table G: Eosinophil Counts Wceks Positive control 20 (mg/ml) ,40 (mglml) Negative control 0 176.83 * 40.74 178.67 *173.'11 - ' 46.33 * 76.29 46.50 * 28.43 SD (1 14.91) SD (488.19) SD (215.15) SD (80.18) 1 92.04 * 33.42 152.89 * 48.93 4 1.33 =t 32.26 43.78 * 34.9 1 SD (94.25) SD (138.13) SD (91.00) SD (98.08) 2 94.72 * 34.96 43.83 f 17.10 40.67 k 3 1.03 42.67 + 28.10 SD (98.61) SD (18.25) SD (87.52) SD (79.27) 3 73.83 * 22.80 SD 0.00 0.00 43.67 * 34.03 (64.30) SD (95.97)