S u r v iv a l o f B a c t e r ia in P e l l e t s , T a b l e t s a n d C a p s u l e s By M a r ia K o u im t z i T h e s is p r e s e n t e d f o r t h e d e g r e e o f D o c t o r o f P h il o s o p h y IN THE F a c u l t y o f M e d i c i n e o f THE U n iv e r s it y o f L o n d o n S eptem ber 1999 T he School of Pharm acy U niversity of London ProQuest Number: 10104248 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10104248 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 A b s t r a c t The survival of probiotic and model organisms in pellets, tablets and capsules was investigated in an attempt to formulate stable solid oral dosage forms, containing probiotic bacteria. Two new incorporation methods were investigated: direct incorporation of bacterial suspensions into pellet formulations and the freeze drying of mixtures of bacterial suspensions with tableting excipients. The mixing of freeze dried bacteria with tableting excipients was also investigated. Gram-negative aerobic {Escherichia coli), Gram-positive aerobic {Staphylococcus saprophyticus and Bacillus subtilis) and Gram-positive anaerobic {Bifidobacterium longum and Lactobacillus acidophilus) vegetative bacteria, together with spores of Bacillus subtilis, were introduced separately into a formulation, which was extruded, spheronised and dried to produce pellets. Spores survived all stages of the process. Survival levels of the Gram-positive organisms after extrusion, spheronisation and drying were significantly higher than the Gram-negative E. coli. The effects of extrusion speed, extrusion die length to radius ratio, and extrusion pressure on the viability of the more sensitive E. coli were investigated. The level of killing was not affected by extrusion speed or die length to radius ratio. However, survival was inversely proportional to extrusion pressure over the range 1-8000 kPa. A range of compaction forces was employed to investigate the susceptibility ofL acidophilus, incorporated into a lactose and a microcrystalline formulation mix, to the forces produced by tableting. Samples from both mixtures were compressed at pressure ranging from 1-300 MPa. A strong negative correlation between bacterial survival and compaction pressure was observed, suggesting that survival decreased with increase in tablet compaction pressure. Mechanical strength and friability tests showed that all tablets produced were of acceptable strength. Results show that L. acidophilus can be successfully inoculated into a tablet formulation providing the compaction pressure is monitored carefully. Stability testing of the L. acidophilus formulations showed that freeze dried L. acidophilus does not remain viable after eight and nine days in the mixtures with microcrystalline cellulose and lactose respectively. Capsule filling with the L acidophilus/ldictose mixture was proved to be the most successful approach, since the lethal effects of drying and pressure were kept to a minimum. Furthermore, these capsules were successfully coated at room temperature with an ethylcellulose/amylose colon-specific coat, without loss of bacteria viability. Five percent coat thickness was shown to withstand simulated gastric and intestinal conditions, whilst remaining susceptible to degradation in the colon by bacterial enzymes. A cknowledgments This thesis is dedicated to my parents. Without them I would not have been! They taught me that I can achieve anything I set my mind to and encouraged me to set my targets high. They have supported me in every possible way, both emotionally and financially. Everything I have achieved I owe it to them. This dedication is a small token of my appreciation. Thank you Mum. Thank you Dad. George, you have kept me sane when the going got tough and most importantly talked me out of quitting when it all got too much. Friends are there for the hard times and you have proved this to me on countless occasions. Thank you for all your support and confidence in me. Prof. J. M. Newton and Dr. R. J. Finney showed incredible trust in me. They continuously encouraged me and gave me advice and ideas when I got stuck. No matter how busy, they always made themselves available to me and proofread my drafts tirelessly. Thank you. My PhD studies were financed by a scholarship from the Greek State Scholarships Foundation (I.K.Y.), after the endorsement of Prof. P. Mahairas from the University of Athens, Greece. I wish to thank them both. Finally, a big thank you to all my friends and colleagues at The School of Pharmacy for their help during the last four years. Index In d e x A b st r a c t 2 A cknowledgments 3 In d e x 4 In d e x of fig u r e s 8 In d e x OF TABLES 11 C hapter 1: INTRODUCTION 12 1.1 Intestinal flora 12 1.2 Infection control by intestinal flora 16 1.2.1 Antibiotic treatment and antibiotic associated diarrhoea (AAD) 16 1.2.2 Pseudomembranous colitis 18 1.2.3 Necrotising enterocolitis 19 1.2.4 Traveller’s diarrhoea 20 1.2.5 Radiation therapy 21 1.3 Probiotics 21 1.3.1 Pathogenicity of probiotics 23 1.3.2 Probiotic survival during passage through the gastrointestinal tract 24 1.3.3 Mode of action of probiotics 24 1.3.4 Efficacy of probiotics 26 1.3.5 Delivery of probiotics 30 1.4 Solid dosage forms 32 1.4.1 Tablets 32 1.4.2 Capsules 39 1.4.3 Pellets 40 1.5 Colonic Delivery 50 Index 1.5.1 Colonie delivery systems 52 1.6 Aim 63 er 2: Materials and Methods 64 2.1 Materials 65 2.1.1 Excipients 65 2.1.2 Solvents 69 2.1.3 Water 69 2.1.4 Media and nutrients 69 2.1.5 Bacterial strains 73 2.2 Apparatus 75 2.3 Methods 77 2.3.1 Pellet production 77 2.3.1.1 Formulation 77 2.3.1.2 Mixing 77 2.3.1.3 Extrusion 77 2.3.1.4 Spheronisation 84 2.3.1.5 Air drying 84 2.3.1.6 Freeze drying 84 2.3.2 Tablet production 84 2.3.2.1 Breaking load 84 2.3.2.2 Friability testing 84 2.3.3 Capsule production 84 2.3.3.1 Capsule filling 84 2.3.3.2 Capsule coating 88 2.3.3.3 Disintegration testing for coated capsules 90 2.3.4 Preparation of media 90 2.3.5 Preparation of bacterial suspensions 92 Index 2.3.6 Viable counting 95 2.3.6 Statistical analysis 95 2.3.6.1 Significance tests 96 2.3.6.2 Correlation analysis 97 Chapter 3: B a c ter ia l Su r v iv a l D u r in g E x tr u sio n - S pheronisation 100 3.1 Introduction 101 3.2 Survival of model bacteria during pellet production 103 3.2.1 Survival of Bacillus subtilis spores 103 3.2.2 Survival of vegetative cells of 104 3.2.3 Survival of vegetative cells of Staphylococcus saprophyticus 105 3.2.4 Survival of vegetative cells of Escherichia coli 106 3.3 Effect of extrusion parameters on the viability ofEscherichia coli 107 3.3.1 Ram speed 108 3.3.2 Die dimensions 109 3.3.3 Variation in viable count with water content of extrudate 109 3.3.4 Extrusion pressure 112 3.3.5 Shear stress 115 3.4 Survival of probiotic bacteria during pellet production 119 3.4.1 Effect of freeze drying on the viability of probiotic bacteria at various stages of the extrusion-spheronisation process 121 3.4.2 Bacerial loss after inoculum preparation 124 3.4.3 Alternative pellet formulations for /ongwm 126 3.4.4 Bifidobacterium longum survival in different formulations during extrusion 132 Index C hapter 4: SURVIVAL OF L actobacillus acidophilus in s o l i d s i n g l e u n it DOSAGE FORMS 134 4.1 Introduction 135 4.2 Survival of Lactobacillus acidophilus in tablets 135 4.2.1 Formulation mix homogeneity 136 4.2.2 Stability of formulation mixes 136 4.2.3 Susceptibility of Lactobacillus acidophilus to compaction 138 4.2.4 Breaking load 140 4.2.5 Friability testing 142 4.3 Survival of Lactobacillus acidophilus in capsules 142 4.3.1 Capsule filling 143 4.3.2 Ethylcellulose coat 144 4.3.3 Ethylcellulose/amylose coat applied and hot air dried 147 4.3.4 Ethylcellulose/amylose coat applied without heat 150 Co n c l u d in g Re m a r k s 152 Re f e r e n c e s 157 Index In d e x o f fig u r e s C hapter 1; INTRODUCTION 1.1 The human gastrointestinal tract 14 1.2 Movements involved in tablet compression 35 1.3 Force and displacement data obtained form a tablet press 36 1.4 Spheroniser featuring cross-hatch geometry plate 46 1.5 Shape changes during the spheronisation process 47 1.6 Pellet forming mechanisms 48 1.7 Colonic delivery system by Quadros et al (1995) 55 1.8 Bacterial degradation of sulphasalazine 58 1.9 Newer azo-prodrugs 59 Chapter 2: MATERIALS AND METHODS 2.1 Ram extruder 79 2.2 Lloyds MX50 extruder 80 2.3 Force displacement profile 81 2.4 Rheometric Scientific Acer Series 2000 Capillary Rheometer 83 2.5 Hofliger Karg device 87 2.6 Capsule coating 89 2.7 Perfect positive correlation between variables x and y 99 2.8 Perfect negative correlation between variables x and y 99 2.9 Zero correlation between variables x and y 99 Chapter 3: B a c t e r ia l Su r v iv a l D u r in g E xtrusio n - Spheronisation 3.1 S ampling of batch A 102 3.2 Survival of Bacillus subtilis spores 103 3.3 Survival of vegetative cells of Bacillus subtilis 104 8 Index 3.4 Survival of vegetative cells of Staphylococcus saprophyticus 105 3.5 Survival of vegetative cells