Formulation and Evaluation of Drug Delivery Systems for the Administration of Ciprofloxacin Hydrochloride to the Female Genital Tract
FORMULATION AND EVALUATION OF DRUG DELIVERY SYSTEMS FOR THE ADMINISTRATION OF CIPROFLOXACIN HYDROCHLORIDE TO THE FEMALE GENITAL TRACT
BY
AKPABIO, EKAETE IBANGA PG/M.PHARM/Ph.D/08/50260
DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY AND INDUSTRIAL PHARMACY
UNIVERSITY OF NIGERIA NSUKKA
JULY, 2012 i
FORMULATION AND EVALUATION OF DRUG DELIVERY SYSTEMS FOR THE ADMINISTRATION OF CIPROFLOXACIN HYDROCHLORIDE TO THE FEMALE GENITAL TRACT
BY
AKPABIO, EKAETE IBANGA PG/M.PHARM/Ph.D/08/50260
A DISSERTATION SUBMITTED TO THE DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY AND INDUSTRIAL PHARMACY FOR THE AWARD OF THE MASTER OF PHARMACY DEGREE OF THE UNIVERSITY OF NIGERIA, NSUKKA
SUPERVISOR: PROF. SABINUS I. OFOEFULE
DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY AND INDUSTRIAL PHARMACY
UNIVERSITY OF NIGERIA NSUKKA
JULY, 2012 ii
CERTIFICATION
We certify that Ekaete Ibanga Akpabio, a postgraduate student in the Department of
Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria, Nsukka, has completed the requirement for the award of the Degree of Master of Pharmacy in
Pharmaceutical Technology and Industrial Pharmacy.
The research work reported in this dissertation is original and has not been submitted in support of an application for another degree or qualification of this or any other university.
…………………………………….. ……………………………… Prof. Sabinus I. Ofoefule Prof. G. C. Onunkwo (Supervisor) (Head of Department) iii
DEDICATION
To my children Rose, Imo-owo and Emmanuel iv
ACKNOWLEDGEMENT
It is my wish to express my gratitude to my Supervisor; Professor Sabinus I. Ofoefule, whose inspiration and contributions to the success of this work can not be over emphasised. I am most grateful to you for your guidance, support and encouragement which made it possible for the timely completion of this work.
I am also grateful to Prof. G. C. Onunkwo for his unreserved support and encouragement. My heartfelt gratitude goes to Professor A. A. Attama for the gift of the artificial membrane and some other materials for my research work and also for his excellent pieces of academic advice.
I wish to express a very special gratitude to Dr. M. A Momoh who was always there for me, and for his technical assistance, Dr. C. N. Obite for the gift of sodium alginate, and Dr. K. C. Ofokansi for the gift of PEG 8000.
My special thanks go to Dr. J. O. Onyechi for developing the method in Kings
College London, which we adopted and modified in the drainage test.
I must not forget to mention the contributions of my colleagues Mrs. P. M. E.
Ubulom, Mr. U. S. Ekong, all the staff of Pharmaceutics/Pharmaceutical Technology
Laboratory and most especially Mr. Offiong E. Umoh, and Mrs. Umoh, a technologist in the Pharmaceutical Chemistry Laboratory, University of Uyo, Uyo, and also Mr.
AKpan of the Pharmaceutical Microbiology laboratory.
I, am most grateful to my siblings for their encouragement and also to Mrs. Ofoefule who assisted me immensely in typing and correcting my synopsis.
Above all, I give thanks to the Almighty God for sustenance throughout this period.
v
TABLE OF CONTENT
Pages
Title Page ------i
Certification ------ii
Dedication ------iii
Acknowledgement ------iv
Table of Content ------v
List of Tables ------xii
List of Figures ------xiii
Abstract ------xiv
CHAPTER ONE
1.0 Introduction ------1
1.1 Vaginal Drug Delivery Systems - - - - 2
1.1.1 Anatomy and Physiology of the Vagina - - - 4
1.1.2 Drug Absorption from the Vagina - - - - 5
1.1.3 Factors Influencing Vaginal Drug - - - - 5
1.1.4 Physicochemical Factors Affecting Vaginal Drug Delivery ------6
1.1.5 Methods of Improving Vaginal Absorption - - - 8
1.1.6 Pharmacokinetics of Vaginally Administered Agents ------9
1.1.7 Permeation of Drug Molecule through Vaginal Mucosa ------10
1.1.8 Classification of Intra-Vaginal Drug Delivery Systems ------11
vi
1.1.9 Ideal Properties of Intra-Vaginal Drug Delivery Systems ------11
1.1.9.1 Advantages of Intra-Vaginal Drug Delivery Systems ------12
1.1.9.2 Disadvantages of the Intra-vaginal Drug Delivery Systems - - - - - 12
1.2 Bacterial Vaginosis ------13
1.2.1 Symptoms ------13
1.2.2 Diagnosis ------13
1.2.3 Treatment ------14
1.2.4 Bacterial Vaginosis and Pregnancy - - - - 15
1.3 Ciprofloxacin Hydrochloride - - - - - 16
1.3.1 Mechanism of Action ------17
1.3.2 Microbiological Assay - - - - - 18
1.3.3 Susceptibility Tests ------18
1.3.3.1 Dilution Techniques ------18
1.3.3.2 Diffusion Techniques ------20
1.4 Pharmacokinetics ------21
1.4.1 Administration and Absorption Profile - - - 21
1.4.2 Distribution ------23
1.4.3 Elimination ------23
1.4.4 Drug Interactions ------24
1.4.5 Adverse Effects ------25
1.5 Pessary ------26
1.5.1 Classification of Pessaries - - - - - 26
1.5.1.1 Pharmaceutical Pessary - - - - - 26
1.5.1.2 Occlusive Pessaries ------27 vii
1.5.1.3 Therapeutic Pessary ------27
1.5.2 Preparation of Pessary - - - - - 28
1.5.2.1 Pessary Base ------28
1.5.2.2 Applications of Polyethylene Glycol in Pharmaceutical formulation - - - - - 30
1.5.2.3 Stability and Storage Condition - - - - 30
1.5.2.4 Incompatibilities ------31
1.5.2.5 Safety of Polyethylene Glycols - - - - 31
1.5.3 Quality Control of Pessaries - - - - - 32
1.53.1 Appearance ------33
1.5.3.2 Uniformity of Weight ------33
1.5.3.3 Uniformity of Content------33
1.5.3.4 Disintegration ------33
1.6 Pharmaceutical Gels ------33
1.6.1 Classification of Gels ------34
1.6.1.1 Inorganic Gels ------34
1.6.1.2 Hydrogels ------34
1.6.1.3 Organogels ------34
1.6.2. Gelling Agents ------35
1.6.2.1 Pectin ------35
1.6.2.2 Sodium Alginate ------36
1.6.2.3 Tragacanth ------36
1.6.2.4 Carbomer ------36
1.6.3 Vaginal Gels ------37
1.6.3.1 Uses of Vaginal Gels ------38
1.6.3.2 Physico-Chemical Characterisation of Vaginal Gels - - 39 viii
1.6.4 Vaginal Tablets ------39
1.6.4.1 Evaluation of Vaginal Tablets - - - - - 40
1.6.4.1.1 Pharmcopoeial Tests - - - - - 40
1.6.4.1.2 Uniformity of Weight Test - - - - - 40
1.6.4.1.3 Uniformity of Weight Test, Permitted Variations - - 40
1.6.4.1.4 Uniformity of Content - - - - - 40
1.6.4.1.5 Disintegration Test ------42
1.6.4.1.6 Dissolution Test ------42
1.6.4.2 Non-Compendial Tests - - - - - 44
1.6.4.2.1 Tablet Thickness ------44
1.6.4.2.2 Tablet Diameter ------45
1.6.4.2.3 Tablet Hardness or crushing strength - - - - 46
1.6.4.2.4 Friability ------46
1.7 Ac-Di-Sol® ------47
1.8 Primojel® ------49
1.9 Sterotex® ------49
1.10 Bioadhesion ------49
1.10.1 Factors Affecting Mucoadhesion - - - - 50
1.10.1.1 Molecular Weight ------50
1.10.1.2 Concentration of Active Polymer - - - - 51
1.10.1.3 Flexibility of Polymer Chains - - - - 51
1.10.1.4 Spatial Conformation - - - - - 51
1.10.1.5 Environmental Factors - - - - - 52
1.10.1.6 Initial Contact Time ------52
1.101.7 Physiological Factor ------52 ix
1.10.1.8 Disease State ------53
1.11 Mechanism of Drug Release - - - - - 53
1.11.1 Immediate Release Dosage Form - - - - 54
1.11.2 Non-Immediate Release Dosage Forms - - - 54
1.11.3 Drug Release Kinetics ------55
1.11.4 Zero Order Kinetics ------55
1.11.5 First order Kinetics - - - - - 56
1.11.6 Hixon – Crowell Model - - - - - 56
1.11.7 Higuchi Model ------57
1.11.8 Korsemeyer – Peppas Model - - - - - 57
CHAPTER TWO
2.0 MATERIALS AND METHODS
2.1 Materials ------59
2.2 Methods ------59
2.2.1 Preparation of Ciprofloxacin Pessaries - - - 59
2.2.2 Evaluation of Ciprofloxacin Pessaries - - - 63
2.2.2.1 Weight Variation ------63
2.2.2.2 Adhesion/Erosion Experiment - - - - 63
2.2.2.3 Dissolution Profiles of Ciprofloxacin Pessaries - - 65
2.2.2.4 Drainage Experiment ------65
2.2.2.5 Melting Point Determination - - - - - 69
2.2.3 Formulation of Ciprofloxacin Hydrochloride Vaginal Tablet ------69
2.2.4 Evaluation of Vaginal Tablets - - - - 69
2.2.4.1 Weight Variation Test - - - - - 69 x
2.2.4.2 Hardness/Crushing Strength Test - - - - 71
2.2.4.3 Friability Test ------71
2.2.4.4 Diameter and Thickness Test - - - - - 71
2.2.4.5 Dissolution Profile of Ciprofloxacin Vaginal Tablet ------71
2.2.4.6 Adhesion/Erosion Test - - - - - 72
2.2.4.7 Bioadhesive Strength Determination - - - - 75
2.2.4.8 Formulation of Ciprofloxacin Vaginal Gel - - - 78
2.2.4.9 Evaluation of Ciprofloxacin Hydrochloride Vaginal Gel ------78
2.2.5.0 Determination of the Organoleptic Properties of the Ciprofloxacin Hydrochloride Vaginal Gel ------78
2.2.5.1 Determination of pH of Ciprofloxacin Hydrochloride Vaginal Gel - - - - - 78
2.2.5.2 Measurement of the Consistency of Ciprofloxacin Vaginal Gel- - - - - 78
2.2.5.3 Microbiological Assay of Ciprofloxacin Hydrochloride Vaginal Gel - - - - - 80
2.2.5.4 Preliminary Sensitivity Test - - - - - 80
2.2.5.5 Antibacterial Sensitivity Evaluation of Ciprofloxacin Hydrochloride Vaginal Gel - - - 81
2.2.5.6 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Ciprofloxacin Hydrochloride Vaginal Gel - - - - - 81
CHAPTER THREE
3.0 Result and Discussion ------83
3.1 Physical Properties of Ciprofloxacin Hydrochloride Pessary and Vaginal Tablet - - - - - 83
xi
3.2 Erosion/Bioadhesion Test of Ciprofloxacin Pessaries and Vaginal Tablet - - - - - 83
3.3 Drainage Experiment with Ciprofloxacin Hydrochloride Pessaries - - - - - 87
3.4 Melting Point Determination - - - - - 87
3.5 Some Release Parameters of Ciprofloxacin Pessaries ------88
3.5.1 Kinetics and Mechanism of Release for Ciprofloxacin Hydrochloride Pessary - - - - 88
3.5.2 Kinetics and Mechanism of Release for Ciprofloxacin Hydrochloride Vaginal Tablet - - - 89
3.5.3 Some Release Parameters of Ciprofloxacin Hydrochloride Vaginal Tablets - - - - 89
3.6 Physicochemical Properties of Ciprofloxacin Vaginal Gel ------97
3.6.1 Organoleptic Properties of Ciprofloxacin Vaginal Gel ------97
3.7 Microbiological Assay of Ciprofloxacin Hydrochloride Gel (Tube Dilution Test) - - - 99
3.7.1 Minimum Inhibitory Test (24 Hours) and Minimum Bactericidal Concentration (MBC) (48 Hours) ------99
3.7.1.1 Minimum Bactericidal concentration (48 Hours)- - - 99
3.7.1.2 Agar – Well Diffusion - - - - - 100
4.0 CHAPTER FOUR
4.1 Summary and Conclusion - - - - - 102
REFERENCES ------103
APPENDIX xii
LIST OF TABLES
TABLE TITLE PAGE
1 Composition of Ciprofloxacin Hydrochloride Pessary ------60
2 Composition of Ciprofloxacin Hydrochloride Vaginal Tablet ------70
3 Composition of Ciprofloxacin Hydrochloride Vaginal Gel ------79
4a Physical Properties of Ciprofloxacin Hydrochloride Pessary ------85
4b Physical Properties of Ciprofloxacin Hydrochloride Vaginal Tablet ------85
5a Erosion/Bioadhesion Test of Ciprofloxacin Pessaries ------86
5b Erosion/Bioadhesion Test of Ciprofloxacin Hydrcohloride Vaginal Tablet - - - - 86
6 Result of Drainage Experiment with Ciprofloxacin Hydrochloride Pessary - - - - - 90
7 Melting Point Determination - - - - - 91
8 Some release parameters of Ciprofloxacin Pessaries ------92
9 Kinetics and Mechanism of Release for Ciprofloxacin Hydrochloride Pessary - - - 93
10 Physico-Chemical Properties of Ciprofloxacin Hydrochloride Vaginal Gel - - - - 98
11a Minimum inhibitory test (24 Hours) - - - - 98
11b Minimum Bactericidal Concentration (48 Hours) - - 101
11c Agar – Well Diffusion - - - - - 101 xiii
LIST OF FIGURES
Fig 1: Pharmacokinetics of Drugs Administered to the Vagina - 9
Fig 2: Structure of Ciprofloxacin Hydrochloride - - - 16
Fig 3: Structure of Polyethylene Glycol - - - - 29
Fig 4: Ciprofloxacin Hydrochloride Pessaries - - - 62
Fig 5: Set up for the Erosion/Bioadhesion Test for Ciprofloxacin Hydrochloride Pessary - - - - - 64
Fig 6: Set up for the Dissolution Test for Ciprofloxacin Hydrochloride Pessary - - - - - 67
Fig 7: Set up for the Drainage Test for Ciprofloxacin Hydrochloride Pessary in Artificial Membrane - - 68
Fig 8: Ciprofloxacin Hydrochloride Vaginal Tablet - - - 73
Fig 9: Dissolution of Ciprofloxacin Vaginal Tablet in Dialysis Membrane 74
Fig 10: Erosion/Bioadhesion Test for Ciprofloxacin Vaginal Table - 77
Fig 11: Release Profile of Ciprofloxacin Hydrochloride from Pessaries Batches 1-4 in phosphate buffer (pH 4.0) - - 94
Fig 12: Release Profile of Ciprofloxacin Hydrochloride from Pessaries Batches 5-8 in phosphate buffer (pH 4.0) - - 95
Fig 13: Release Profile of Ciprofloxacin Hydrochloride from Vaginal Tablet in Phosphate Buffer (pH 4.0) - - - 96
Fig 14: Spectrum of Ciprofloxacin Hydrochloride at λmax (278) - 131
Fig 15: Beer’s plot for ciprofloxacin Hydrochloride at 270nm - 132
xiv
ABSTRACT
Three different dosage forms of Ciprofloxacin hydrochloride (CH), namely pessaries, tablets and gels were formulated and evaluated. Pessaries were evaluated for weight uniformity, bioadhesion on Pig rectum, drainage through pig rectum and dissolution profile through an artificial membrane .as well as determination of their melting points range. Ciprofloxacin vaginal tablets were evaluated for weight uniformity, dissolution through an artificial membrane, and bioadhesion on Pig vaginal epithelium, while CH vaginal gel was evaluated for pH, consistency and its activity against Escherichia coli and Staphylococcus aureus.
The Ciprofloxacin pessaries were torpedo shaped with no visible cracks or depressions. The coefficient of weight variations ranged from 0.006 to 0.183 %.
Pessaries that contained 0.2 % Primogel®, 0.01 % Ac-Di-Sol® and 0.1 % Sterotex® had percentage erosion of 29.73±15.3, 36.46±8.1 and 35.75±1.8 % respectively while those that contained no additive, 0.1% Primogel®, 0.2% Sterotex®, 0.01% Carbopol
971 and the pessary containing a mixture of PEG4000 and 8000 (ratio1:1) had percentage erosions of 22.15±3.60, 22.92±7.30, 22.15±3.60, 21.00±3.90 and
18.51±3.40 % respectively. Results of bioadhesion of the pessaries to pig rectum showed 100 % bioadhesion by the pessaries except the control batch which had bioadhesion of 66.7 %. Pessaries containing no additive exhibited the highest drainage (66.9 %) and the least percentage of pessary retained on the pig rectum, while the pessaries containing 0.01 % Ac-Di-Sol® exhibited the least drainage (11.5
%) and the highest percentage of the pessary (88.5%) retained on the pig rectum. All the pessaries melted within the temperature range of 38 to 42°C. Fastest release through an artificial membrane occurred from pessaries containing 0.2 % Primogel® xv and slowest from pessaries that contained no additive. The weight, thickness, diameter and hardness of CH vaginal tablets were 0.614±0.20 g, 5.53±0.01 mm, 12.54±0.02 mm, and 1.92±0.20 kg/f respectively. There was gradual release of CH from the vaginal tablets through an artificial membrane over ninety min. The CH vaginal gel had a pH of 6.67 and consistency of 115.40 ±0.77 centipoise. Ciprofloxacin vaginal gel was more active against Escherichia coli (MIC = 0.03125 mg/mL) than
Staphyloccocus aureus (MIC = 0.0625mg/ml). The mechanisms and kinetics of drug release were also evaluated. Results indicated that formulations containing 80 %
PEG 4000,0.1 % Primogel® as well as 0.1 %Sterotex followed zero order kinetics via non-Fickian mechanism. Formulations containing 0.01 % Ac-Di-Sol® followed
Higuchi and zero order kinetics(r2 =0.978, 0.9961) with diffusion controlled mechanism (n = 0.50) The formulation containing 0.1% Carbopol971® exhibited
Higuchi kinetics with Fickian diffusion mechanism(n = 0.52).The release kinetics exhibited by the ciprofloxacin hydrochloride vaginal gel was zero order (r2 = 0.9032) via non-Fickian mechanism.
1
CHAPTER ONE
1.0: INTRODUCTION
Vaginal drug delivery has been of interest to scientist to varying degrees during the last century (1). The vagina as a route of drug delivery, offers a means to administer drugs for local benefit or systemic distribution. For years, antifungal and antibacterial agents have been administered vaginally to treat yeast and bacterial infections, respectively. Administration of local spermicides and cleansing products is also a current practice (2). The introduction of intravaginal rings (IVRS) in the 1990s to administer steroid hormones for hormone replacement therapy represented a major advance in both vaginal drug delivery and the drug delivery field as a whole. These devices were designed to sustain the release of steroid hormones for up to one month in duration. A few years later, a contraceptive IVR product NuvaRing® was introduced. This product has been widely accepted as a once monthly vaginal contraceptive product (3).
Traditionally, available dosage forms include liquids (solutions, emulsions and suspension, pessaries, tablets, creams and ointments). Gels and vaginal rings have gained increased popularity due to their unique advantages and women’s acceptance.
Others such as capsules, foams, vaginal films medicated tampons, sponges or diaphragms are also of interest.
Bacterial vaginosis is a non specific infection associated with positive cultures for
Gardnerella vaginalis, characterised by increased malodourous vaginal discharge. It is the most common vaginal infection of reproductive aged. This condition is treated by using medicine taken orally or intravaginally. Antibiotics such as metronidazole, clindamycin both as tablets and gels as well as tinidazole has been the drug of choice 2 for the treatment of bacterial vaginosis (4). Ciprofloxacin, a fluoroquinolone antibacterial agent has been found to be active against Gardnerella vaginalis.
Treatment of bacterial vaginosis has largely been restricted to use of orally or vaginally administered metronidazole, clindamycin and tinidazole. The susceptibility of Gardnerella vaginosis to ciprofloxacin hydrochloride has open a new window of opportunity in the management and treatment of bacterial vaginosis.
The objectives of the study, therefore, are:
1. to design and formulate three different vaginal dosage forms (pessary, tablet
and gel) for the delivery of ciprofloxacin hydrochloride to the vaginal tract for
the treatment of bacterial vaginosis and other susceptible organism.
2. to evaluate some physical properties of the dosage forms which may affect the
performance of the dosage forms.
3. to evaluate effects of some additives on the release of ciprofloxacin
hydrochloride under environment and conditions which mimic the
environment of the vagina.
1.1: Vaginal Drug Delivery Systems
Vaginal canal is an important route of drug administration for both local and systemic diseases. The administration of drugs in the Vagina is believed to be as old as pharmacotherapy, first written documents dating from 19th century BC. Although traditionally regarded for the treatment of local conditions. e.g vaginitis, in the early
20th century first exponents evidenced the significant systemic drug absorption of some drugs through the vagina, leading to the development of drug products for the management of non- vaginal conditions. Nowadays the acceptability of vaginal drug administration by women and doctors is increasing, augmenting interest for this drug delivery routes. Vaginal drug delivery systems are traditionally used to deliver 3 contraceptive and drugs to treat vaginal infections (5-6). However, vaginal drug delivery is not limited to these drugs as the vagina has promise as a site to topically deliver drugs which will be absorbed systemically because of the dense network of blood vessel in the vaginal wall (7)
Formulations such as pessaries, vaginal tablets, inserts, cream, douches and gel are administered by this route (8).
The first truly controlled drug delivery systems for use in the vagina were developed in 1970, when the first vaginal ring was used for delivery of medroxy-progesterone acetate as a contraceptive. Tablets, creams and pessaries are the usual formulations in over- the-counter (OTC) vaginal medications while vaginal rings are the most common long-term drug delivery systems currently used (5).
In recent years vaginal bioadhesive preparations have been developed as a new type of controlled release dosage form for the treatment of both topical and systemic diseases. The greatest advantage to such dosage forms is the possibility of maintaining them in the vagina for extended period of time thereby enabling lower dosing frequencies. The introduction of intravaginal rings (IVRs) in the 1990s to administer steroid hormones for hormone replacement therapy represented a major advance in both vaginal drug delivery and the drug delivery field as a whole (9).
These devices were designed to sustain the release of steroid hormones for up to one month in duration. Nuva Ring (R), which is a contraceptive IVR product, was introduced a few years later and was widely accepted (9).
4
1.1.1: Anatomy and Physiology of the Vagina:
The vagina is a fibro-muscular tube lined with stratified epithelium, connecting the external and internal organs of reproduction. It runs obliquely upwards and backwards at an angle of 450 between the bladder (in front) of the rectum and anus (behind). The vagina in an adult premenopausal female is approximately 7-8cm long and 2cm wide, shrinking in the postmenopausal female to approximately 4.5-6cm in length and 1-
1.5cm in width. Normal pH of the vagina in premenopausal women ranges from 4 to
5, and rises to almost 7 in post menopausal female. The vagina is characterized by an exceptional elasticity and the surface area of the vagina is increased by numerous folds by microridges covering the epithelium cell surface (10). A layer of relatively thick connective tissue is located between the anterior vaginal wall and the urinary canal as well as the posterior vaginal wall and the intestinal tract. The vaginal wall itself consists of three layers:- the epithelial layer, the tunica adventitia, and the muscular coat. The epithelial layer is made up of an epithelial lamina and a lamina propria. It consists of non cornified, stratified squamous epithelial cells which changes with age. In the sub epithelial layer rests a network of elastic fibres around the lamina propria and collagenous fibres around the tunica adventitia, creating a connection to the muscular coat. The muscular coat of the vagina is composed of smooth muscle and elastic fibers. A spiral arrangement of these fibres provides support to withstand stretching without rupturing the vagina.
The tunica adventitia is made up of loose connective tissue that is attached to the muscular coat. The vagina is encompassed by a vascular supply of arteries, veins and lymph capillaries, as well as sensory and autonomous nerves. Cyclic variation occurs, since the vaginal epithelium is affected by ovarian hormones, and also age related changes associated with menopause, cause reduction in vaginal size, loss of elasticity, 5 decrease in vascularity and a thinning of the mucosa. This thinning of the vaginal epithelium leads to a substantial increase in permeability of this tissue. The structural changes would therefore influence vaginal absorption, and must be taken into consideration as a significant factor in vaginal application of drugs (10).
1.1.2: Drug Absorption from the Vagina
Absorption of a wide range of drugs from the vagina has been studied. A detailed review describes studies on the vaginal absorption of steroids, prostaglandins, antimicrobials, antivirals, proteins and nonoxynol-9 (6). Similar to other mucosal drug delivery routes, drug transport across the vaginal membrane may occur by a number of different mechanisms: i. diffusion through the cell due to a concentration gradient (transcellular route). ii. vesicular or receptor mediated transport mechanism iii. diffusion between cells through the tight junctions (intercellular route) (11).
In most cases, a higher bioavailability is obtained when drugs are given by the intra- vaginal route than the oral route (12).
1.1.3: Factors Influencing Vaginal Drug Absorption
A good understanding of the various factors that can influence drug absorption from the vaginal cavity is necessary in order to design the formulation and device used for intra- vaginal administration. Several factors ranging from physiological conditions, physicochemical properties of the drug and factors related to the administration device must be studied.
(a) Physiological Factors:
These are factors that are related to the physiology of the vagina and they
include pH, effect of the estrous cycle on the permeability of the vaginal 6
mucosa, thickness of the vaginal epithelium, vaginal fluid volume, chemical
composition of fluid, pH, viscosity and surface tension, the pressure exerted
on the dosage form by the rectal wall, (this play a vital role in vaginal drug
absorption) and sexual arousal, mucociliary clearance (MCC), vaginal
obstruction, etc. which affect either the mucus or ciliary heating and vaginal
blood flow.
(b) Formulation factors affecting vaginal drug delivery:
These include factors related to the dosage forms such as the characteristics of
the active ingredients, pH, and mucosal irritancy; osmolarity viscosity
(solutions, gels) and density (powder, tablets) of the formulation;
concentration and volume administrated, type of dosage form; particle size of
drug molecule, chemical nature, and ionization surface charge.
1.1.4: Physicochemical Factors affecting Vaginal Drug Delivery
Peptides and proteins are known to aggregate in the medium due to changes in pH and ionic strength of the medium or concentration of the substance. The aggregated complex have a different diffusion and permeation coefficient as against the monomer. Poorly soluble drugs like metronidazole have rate limiting dissolution in small volume of vaginal fluid. The vagina permeability of straight chain aliphatic alcohols increases in a chain length dependent manner (13). Lipophillic steroids such as progesterone and estrone permeates the vaginal wall to a greater extent than hydrophilic steroids like hydrocortisone and testosterones. Generally, absorption of low molecular weight lipophilic drugs is much more than large molecular weight lipophilic or hydrophilic drugs.
7
(a) Drug release:
The small volume of vaginal fluid makes dissolution the rate limiting step for
systemic absorption of drugs from vaginal formulations. The type of dosage
form affects the rate of dissolution, for instance a drug which is already
dissolved in an aqueous vaginal gel, will be more rapidly absorbed than a drug
which is in solid form within a vaginal tablet preparation.
(b) The effective area of Contact:
The vaginal cavity has an area of approximately 60 cm2. The total area which
a medicament could get deposited will depend to a larger extent on the size of
the dosage form. Also the type of formulation influences the size of area over
which the drug is deposited (12) The hydrophilicity of the drug influences the
extent of its spreading and distribution throughout the vagina.
(c) Contact time:
The extent of flow and retention of the medicament within the vaginal cavity
depends on the type of formulation (13). Lower vaginal residence time can be
improved by a bioadhesive dosage form that results in prolonged contact with
the surface, and hence a better drug absorption.
(d) Concentration: The rate of absorption via passive diffusion can be increased by
increasing drug concentration in vaginal fluid. Long term vaginal drug
delivery system are high concentration drug loaded formulations that
gradually release the drug in the limited vaginal fluid, making the fluid highly
saturated, ensuring better absorption and sustained drug delivery throughout
the intended time of application.
8
1.1.5: Methods of Improving Vaginal Absorption
The low vaginal absorption can be attributed to the poor membrane permeability due to molecular size, lack of lipophilicity, fluid volume, estrous cycle and pH of the vagina. In order to overcome this problem, it is pertinent to utilise penetration enhancers to facilitate the transport of these molecules and hence improve their bioavailability. Generally, enhancers improve the absorption of these molecules by one or several combined mechanisms viz: i) by increasing intracellular transport or utilizing penetration agents e.g.
polyethylene glycol. ii) by increasing the contact time between the dosage form and the vaginal
membrane by making use of muco-adhesive polymer such as carbopol 934,
940 etc in gel formulation to increase their viscosity. iii) by increasing vaginal flow, thereby raising the concentration gradient across
the vaginal mucosa. iv) by the use of bio-adhesive preparations for the treatment of both topical and
systemic disease. This enhances the retention of these dosage forms in the
vagina for extended periods of time, thereby enabling lower dosing
frequencies. Among the polymers, polyacrylic acid, and hydroxypropyl
methyl cellulose are the ideal excipients in bio-adhesive vaginal preparations
due to their high bioadhesive strength (14) v) by the use of chelating agents as penetration enhancers in vaginal
formulations. It was reported that vaginal administration of the protein
leuprolide was much more effective when enhancers such as carboxylic acids
with chelating ability was co-administered (6) 9 vi) the use of pro-drug enhances drug permeability through modification of the
hydrophilicity or lipophilicity of the drug. This can be achieved by
modification of chemical structure of the drug molecule, thus making it
selective, site specific and a safe vaginal drug delivery system. vii) the use of gel formulation, is an extreme case of viscosity enhancement.
Mostly hydrogels are used for the intra vaginal gel drug delivery system.so
that the dosing frequency can be decreased to once or twice a day.
1.1.6: Pharmacokinetics of Vaginally Administered Agents
The absorption, distribution and elimination of a drug molecule after its release from an intra-vaginal drug delivery device in the vaginal lumen follow the pharmacokinetic sequences below:
Drug in Tissue compartment C t
Dosage form in vagina Kct Ktc Release Drug in vaginal Ka fluid Cv Drug in central compartments (Cc)
Ke
Elimination
Figure I: Pharmacokinetics of Drugs Administered to the Vagina.
10
The rate of change in drug concentration in the central compartment can be expressed by: dcc/dt = ka.Cv + Ktc.Ct – Kct. Cc – Ke.Cc------Eqn (1) dcc/dt = Ka.Cv+ Ktc. Ct – (Kct + Ke) Cc ------Eqn (2)
Where
Cv = Concentration of drug in Vagina.
Ka = Absorption rate constant
Cc = Concentration of drug in central compartment.
Kct = Rate constant for the transfer of drug concentration from central compartment to tissue compartment.
Ktc = Rate constant for the transfer of drug concentration from tissue compartment to central compartment.
Ct = Concentration of drug in tissue compartment.
Cc = Concentration of drug in central compartment.
Ke = Elimination rate constant.
1.1.7: Permeation of Drug Molecule Through Vaginal Mucosa
The permeability of drug through vaginal mucosa is affected by the estrous cycle, low lipophilicity of drug and on the drug concentration in the vaginal fluid. It therefore means that the permeability follows a first order rate process (15) The apparent permeability coefficient Papp for vaginal membrane permeability is defined by.
Papp = 1/(1(Paq + 1/Pv)------Eqn (4)
Where
Paq = permeability coefficient of the aqueous diffusion layer
Pv = permeability coefficient of the vaginal membrane. 11
Since the permeability coefficient of the vaginal membrane depends on the permeability coefficient of aqueous pore pathway (Pp) and lipoid pathway (PL),
Therefore Pv = Pp + PL
So eqn. (4) will become
Papp = 1/(1/Paq + 1/(Pp + PL] ------Eqn (5)
1.1.8: Classification of Intra-Vaginal Drug Delivery System
Different systems have been developed to effect drug delivery to the vaginal mucosa, these include; a) vaginal rings b) vaginal gel and creams c) pessaries and tablets d) bioadhesive micro-particulated drug delivery devices e) vaginal foams
1.1.9: Ideal Properties of Intra-Vaginal Drug Delivery Systems
Component of vaginal drug delivery dosage form should melt at vaginal temperature i.e. at 370C. Intra-vaginal drug delivery device should be non-toxic and non-irritating.
It should not have any meta-stable form. The preparation should have high water number, and also good wetting and emulsifying properties. It should be non-sensitized on vaginal pH (3.5-4.9) (16)
It should be stable on storage. The preparation should have proper viscosity, to avoid the leakage of the preparation from the vagina, the preparation should have proper bioadhesive/mucoadhesive properties, in order to increase the contact time between the membrane and preparation (14)
12
1.1.9.1: Advantages of Intra-vaginal drug delivery system
This route is the most preffered and targeted goal of new drugs and dosage forms
(17), vaginal administration can be used as an alternative route in certain cases of therapeutic importance.
1. In cases of nausea and vomiting, the act of taking medication orally may
induce emesis so that drug is vomited before it is absorbed.
2. Irritation to the stomach and small intestine associated with certain drugs can
be avoided.
3. Hepatic first pass metabolism can be avoided.
4. Rapid drug absorption and quick onset of action can be achieved.
5. Drug delivery can be stopped by removing the dosage forms e.g vaginal rings.
6. The vaginal bioavailability of smaller drug molecules is good, while that of
larger molecules can be improved by means of absorption enhancer.
This route encourages self medication. It is convenient for patients on long term therapy, when compared with parenteral medication. Contact with digestive fluid is avoided, thereby preventing enzymatic degradation of some drugs. Use of less total drug than an oral dose is possible via this route.
1.1.9.2: Disadvantages of the Intra-vaginal Drug Delivery System
Some of the disadvantages of this route of drug administration include: i. non-compliance by patients. ii. variability in drug absorption related with menstrual cycle, menopause and
pregnancy, can also limit the vaginal drug delivery route usage. iii. influence of sexual intercourse. iv. gender specificity. 13 v. personal hygiene. vi. leakage of drugs from vagina and wetting of under garments.
1.2: Bacterial Vaginosis
Bacterial vaginosis is a polymicrobial clinical syndrome resulting from the replacement of the normal hydrogen peroxide producing lactobacillus specie in the vagina with high concentrations of anaerobic bacteria. Example include, Prevotella specie, Mobiluncus specie, Gardneralla vaginalis and Mycoplasma hominis. Bacterial vaginosis is the most prevalent cause of vaginal discharge or malodour. However, more than 50 % of women with bacterial vaginosis are asymptomatic1. The cause of this microbial alteration is not fully understood. Bacterial vaginosis is associated with having multiple sexual partners, douching and lack of vaginal lactobacilli.
1.2.1: Symptoms
About half of women with bacterial vaginosis are asymptomatic (4). The primary symptom of BV is an abnormal odourous vaginal discharge, which may look grayish white or yellow. The fish-like odour is noticeable especially after intercourse. Women with BV may also experience burning during urination or itching around the outside of vagina or both (18).
1.2.2: Diagnosis
Bacterial vaginosis can be diagnosed based on the history of symptoms, a vaginal examination or by examining a sample of the vaginal discharge. Laboratory tests to detect signs of BV may include; i. Wet Mount: A sample of vaginal discharge is mixed with normal saline after
placing it on a microscopic slide. The prepared saline is examined to identify
the bacteria present, to look for white blood cells that point to an infection and 14
to look for unusual cells called clue cells, which is the most reliable sign of
BV (19). ii. The Whiff Test: This involves the addition of several drops of potassium
hydroxide solution to a sample of vaginal discharge to ascertain whether a
strong fishy odour is produced. A fishy odour on the Whiff test suggest BV. iii. Vaginal pH: The normal vaginal pH is between 3.8-4.5. Bacterial vaginosis
usually causes an increase in the vaginal pH above 4.5. iv. Gram Stain: When a gram stain is used, determining the relative
concentration of lactobacilli, gram negative and gram positive rods and cocci
(i.e. G. vaginalis, prevotella, porphyromonas and streptococci) and curved
gram-negative rods is considered the gold standard laboratory method for
diagnosing BV. A culture of G. vaginalis is not recommended as a diagnostic
tool because it is not specific (1).
The presence of clue cells, an increased vaginal pH and a positive Whiff test are enough evidence to make a diagnosis (4).
1.2.3: Treatment
Two treatment options are employed in the treatment of BV. They are; i. Watchful waiting: This line of treatment involves allowing the symptoms of
BV to go away on their own. This happens when the vaginal lactobacilli
organisms increase to their normal levels and while the level of other bacteria
drop. ii. Antibiotics: Antibiotics such as metronidazole, clindamycin and tinidazole
have been used to treat bacterial vaginosis either by mouth or vaginally (4).
The recommended regimens include; a. Metronidazole , 500 mg, orally twice a day for 7 days. 15 b. Metronidazole, 0.75 % one full applicator (5 g) intravaginally, once a day for
5 days. c. Clindamycin cream, 2 %, one full applicator (5 g) intravaginally at bed time
for 7 days (19).
Alternative regimens include the use of clindamycin 300 mg orally twice a day for 7 days or clindmycin ovules 100 mg intravaginally once at bedtime for 3 days.
Metronidazole 2 g single dose therapy has been found to possess the lowest efficacy for BV and is no longer a recommended or alternative regimen
The Food Drug Act has cleared metronidazole 750 mg extended release tablets once daily for 7 days and a single dose of clindamycin intravaginal cream (20).
Limited data have been published that compares the clinical or microbiological equivalences of these regimens with other regimens. From the results, cure rates do not differ between intravaginal clindimycin cream and ovules (20).
Recently, ciprofloxacin hydrochloride has been employed both as pessary at a dose of
0.2 g and effervescent ciprofloxacin lactate vaginal tablets at a dose of 0.1 g respectively (21,22) .
1.2.4: Bacterial Vaginosis and Pregnancy
Pregnant women with BV are at increased risk of pre-term birth. There is no benefit to testing and/or treating all pregnant women for BV unless the woman has symptoms of infection (23). Some experts recommend testing all pregnant women who have a history of a pre-term delivery. Pregnant women with symptoms of BV infections are usually treated with metronidazole tablets for seven days (24).
16
1.3: Ciprofloxacin Hydrochloride
Ciprofloxacin hydrochloride, USP, is a synthetic chemotherapeutic antibiotic of the fluoroquinolone drug class (25). It is a second generation fluoroquinolone antibacterial agent. The fluoroquinolones are significantly more potent than the quinolones and have much broader spectra of activity. The fluoroquinolones possess both a 6-fluoro substituent and a 7- piperzinyl group on the quinolone pharmacophore. The introduction of these two groups expanded the spectrum, increased potency and appears to have prevented the development of plasma-mediated resistance (26). Ciprofloxacin hydrochloride is the monohydrochloride monohydrate salt of 1-cycloproply-6-fluoro-1, 4 dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid. It is fairly yellowish to light yellow crystalline substance with a molecular weight of 385.8. Its empirical formula is C17 H18FN3O3.HCl.H2O, and its chemical structure is as follows: O
F COOH .HCl.H2O
HN N N
Figure 2: Structure of Ciprofloxacin Hydrochloride
Ciprofloxacin is marketed worldwide with over three hundred different brand names.
In the United States, Canada and the UK, it is marketed as Bayap, Ciloxan, Ciflox,
Cipro, Cipro XR, Cipro XL, Ciproxin, Prociflor and most recently Proquin. It is also marketed as Ciprex in India and Russia and, “Cetraxal” in Spain. In addition it is marketed in Nigeria as Cefroden, Ciprox, Cipronol, Cipro care etc. Ciprofloxacin film-coated tablets are available in 250 mg, 500 mg and 500 mg of extended release oral tablets. It is also available as the oral suspension and as an ophthalmic lotion. 17
1.3.1: Mechanism of Action
They inhibit DNA synthesis, initially by inhibiting ATP-dependent DNA super coiling by binding to subunit A of DNA-gyrase, secondarily they also inhibit the relaxation of supercoiled DNA, a reaction not dependent on ATP. Finally they also block the DNA nicking – closing enzyme that in the absence of drug interference is ultimately responsible for DNA elongation (26), thereby inhibiting cell division. This mechanism can also affect mammalian cell replication. In particular, some congeners of this drug family display high activity not only against bacteria topoisomerases but also against eukaryotic topoisomerases and are toxic to cultured mammalian cells and in vivo tumor models (27) Although quinolones are highly toxic to mammalian cells in culture, its mechanism of cytotoxic action is not known. However, quinolone- induced DNA damage was first reported in 1986 (28).Recent studies have demonstrated a correlation between mammalian cell cytotoxicity of the quinolones and the induction of micronuclei (29-32). As such, some fluoroqiunolones may cause injury to the chromosome of eukaryotic cells(33,34). Presently there is an ongoing debate as to whether or not this DNA damage is to be considered one of the mechanisms of action concerning the severe adverse reactions experienced by some patients following fluoroquinolone therapy (35,36).
The fluoroquinolones exhibit bactericidal activity against a broad spectrum of aerobic gram-positive and gram-negative bacteria, including many gram-positive cocci, gram- negative bacilli and acid-fast bacilli. The drugs are highly effective in eradicating organisms that cause urinary tract infections and prostatitis (36). The fluoroquinolones are highly bactericidal in vitro and are considerably more potent against E. coli and various species of Salmonella, Shigella, Enterobacter,
Campylobacter and Weisseria. Minimal inhibitory concentrations of ciprofloxacin for 18
90% of these strains are less than 0.2 g/mL (37). The drug is less active against
Pseudomonas aeruginosa, Enterococci, and Pneumococci. Values of MIC90 range from 0.5 g/mL - 2 g/mL. Ciprofloxacin also has good activity against staphylococci including methicilin-resistant strains (MIC90 = 1 g/mL). Several intracellular bacteria are inhibited by ciprofloxacin at concentrations that can be achieved in plasma. These include; Mycoplasma, Legionella, Brucella and
Mycobacterium including Mycobacterium tuberculosis (38) most anaerobic microorganisms are resistant to the fluoroquinolones. Resistant to these drugs develop during therapy, especially with Pseudomonas aeruginosa. Ciprofloxacin is used in the treatment of a number of infections including infections of bones and joints, endocarditis, gastroenteritis, malignant otitis externa, respiratory tract infections prostatitis, anthrax, chancroid etc. Ciprofloxacin as well as other fluoroquinolones are increasingly being relied upon for the empirical therapy of urinary tract infections due to increasing resistance to sulphamethoxazole-trimetoprim combination. However, they should not be used for routine treatment of upper and lower respiratory tract, skin and soft tissue infection (38).
1.3.2: Microbiological Assay
Ciprofloxacin is less active when tested at acidic pH. The inoculums size has little effect when tested in vitro. The minimal bactericidal concentration (MBC) generally does not exceed the MIC by more than a factor of 2.
1.3.3: Susceptibility Tests
1.3.3.1: Dilution Techniques: Quantitative methods are used to determine antimicrobial minimum inhibitory concentrations (MICs). These MICs provide estimates of the susceptibility of bacteria to antimicrobial compounds. Standardised 19 methods are based on a dilution method (39) (broth or agar) or equivalent with standardised inoculums concentrations and standardised concentrations of ciprofloxacin powder. The MIC values should be interpreted according to the following criteria:
For testing Enterobacteriaceae;
MIC (mcg/mL) Interpretation
1 Susceptible (S)
2 Intermediate (I)
4 Resistant (R)
A report of ‘susceptible’ shows that the pathogen is likely to be inhibited if the antimicrobial compound in the blood reaches a concentration usually achievable. A report of ‘intermediate’ indicate that the result should be considered equivocal, and if the microorganism is not fully susceptible to alternative, clinically feasible drugs, the test should be repeated (39). A report of ‘Resistant’ indicates that the pathogen is not likely to be inhibited if the antimicrobial compound in the blood reaches the concentration usually achievable. Standardised susceptibility test procedures require the use of laboratory control microorganisms to control the technical aspects of the laboratory procedures.
Standard ciprofloxacin powder should provide the following MIC values;
Microorganism MIC Range (mcg/ml)
Escherichia coli ATCC 25922 0.004 – 0.015
Staphylococcus aureus ATCC 29213 0.12 – 0.5
20
1.3.3.2: Diffusion Techniques
Quantitative methods that require measurement of zone diameters also provide reproducible estimates of the susceptibility of bacteria to antimicrobial compounds.
One such standardised procedure (40) requires the use of standardised inoculum concentrations. This procedure use paper disks impregnated with 5-mcg ciprofloxacin to test the susceptibility of microorganisms to ciprofloxacin.
Reports from the laboratory providing susceptibility test with a 5mcg ciprofloxacin disk should be interpreted according to the following criteria:
For testing Enterobacteriaceae:
Zone Diameter (mm) Interpretation
21 Susceptible (S)
16 – 20 Intermediate (I)
15 Resistant (R)
Interpretation involves correlation of the diameter obtained in the disk test with the
MIC for ciprofloxacin. As with standardised dilution techniques, diffusion methods require the use of laboratory control microorganisms that are used to control the technical aspects of the laboratory procedures. For the diffusion technique, the 5mcg ciprofloxacin disk should provide the following zone diameters in these laboratory quality control strains;
Microorganism Zone Diameter (mm)
Escherichia coli ATCC 25922 30 – 40
Staphylococcus aureus ATCC 25923 22 – 30
21
1.4: Pharmacokinetics
1.4.1 Administration and Absorption Profile
Ciprofloxacin hydrochloride is rapidly well absorbed from the (GIT) following oral administration and undergoes minimal first pass metabolism (39). The absolute bioavailability is approximately 70%. The rate of absorption of ciprofloxacin administered as conventional tablets is decreased by the presence of food in the GIT.
The extent of absorption is however, not affected. Also, food does not affect the pharmacokinetics of ciprofloxacin administered as the oral suspension (39)..
Concomitant administration with dairy products alone may substantially reduce GIT absorption of ciprofloxacin. Absorption is not affected to a larger extent by dietary calcium that is part of a meal. Magnesium, aluminium and/or calcium containing antacids or products containing calcium, iron or zinc decrease the oral bioavailability of ciprofloxacin hydrochloride.
The oral bioavailability of ciprofloxacin administered as conventional tablets is 50 –
85% in healthy, fasting adults, and peak serum concentrations of the drug generally are attained within 2–3 hours. Peak serum concentrations and the area under the serum-concentration time curve (AUC) increase in proportion to the dose over the oral dosage range of 250-1000 mg and are affected by gender. Following oral administration of a single 250, 500, 750 or 1000 mg dose of ciprofloxacin hydrochloride as conventional tablets or oral suspension in healthy, fasting adults, peak serum concentrations average 0.76-1.5, 1.6-2.5, 2.5-4.3 or 3.4-5.4 mcg/mL respectively (40). Peak plasma concentrations are attained within 1-4 hours following oral administration of ciprofloxacin extended release tablets. Oral administration of ciprofloxacin 500 mg as extended release tablets or 250mg twice daily as conventional tablets results in steady-state mean peak plasma concentrations of 1.59 22 or 1.14 mcg/mL respectively. The AUC is similar with both regimens (40). Peak serum concentrations of ciprofloxacin and AUCs of the drug are slightly higher in geriatric patients than in younger adults. This may result from increased bioavailability, reduced volume of distribution and/or reduced renal clearance in these patients. Single dose oral studies using ciprofloxacin conventional tablets and single and multiple-dose intravenous studies indicated that, peak plasma concentrations are
16 – 40 % higher in younger adults than older ones and elimination half-life is prolonged approximately to 20 % in individuals older than 65 years of age (41). The manufacturer states that these differences can be partially attributed to decreased renal clearance in this age group and are reported to be not clinically important.
Following IV infusion over 60 minutes of a single 200 or 400 mg dose of ciprofloxacin in healthy adults, peak serum concentrations average 2.1 and 4.6mcg/ml respectively immediately following the infusion. Serum concentrations 6 hours after the start of infusion average 0.3 and 0.7 mcg/mL respectively. Adults receiving
400mg ciprofloxacin IV every 12 hours, mean peak concentrations at steady state are
4.56 or 0.2 mcg/mL respectively.
In a limited number of pediatric patients within the ages of 6 - 16 years, who received two doses of ciprofloxacin of 10 mg/kg by IV infusion over 30 minutes, 12 hours apart, mean peak plasma concentrations were 8.3mcg/ml and trough concentrations ranged from 0-0.09 mcg/mL. After the second IV infusion, these pediatric patients were switched to oral ciprofloxacin given in a dosage of 15 mg/kg every 12 hours and achieved a mean peak concentration of 3.6 mcg/mL after the initial oral dose.
23
1.4.2: Distribution
Ciprofloxacin is widely distributed into body tissue and fluids following oral or IV administration. Highest concentration of the drug generally are attained in bile, lungs, kidney, liver, gall bladder, uterus, seminal fluid, prostatic fluid, tonsils, endometrium, fallopian tubes and ovaries (42).Concentrations of the drug achieved in most of these tissues and fluids substantially exceed those in serum. Ciprofloxacin is also distributed into bone, aqueous humor, sputum, saliva, nasal secretions, skin, muscle, adipose tissue, cartilage etc. In healthy adults, the apparent volume of distribution is
2-3.5 L/kg and the apparent volume of distribution at steady state is 1.7-2.7 L/kg. The apparent volume of distribution of ciprofloxacin in geriatric patients averages 3.5 - 3.6
L/kg (42).
Low concentrations of ciprofloxacin are distributed into cerebrospinal fluid and the peak CSF concentrations may be 6-10 % of peak serum concentrations. After oral administration, ciprofloxacin is widely distributed throughout the body. Tissue concentrations often exceed serum concentrations in both men and women, particularly in genital tissue including the prostate(42).
1.4.3: Elimination
The serum elimination half-life of ciprofloxacin in adults with normal renal function is 3-7 hours (42). Following IV administration in healthy adults, the distribution half- life of ciprofloxacin averages 0.18-0.37 hours and the elimination half-life averages 3-
4 hours. In patients with impaired renal function ciprofloxacin elimination are higher and the half-life prolonged.
Serum concentrations of ciprofloxacin are higher in patients with impaired renal function. The half-life of the drug ranges from 4.4-12 hours on adults with creatinine clearances of 30 mL/minute or even less (41). 24
It is eliminated by both renal and non-renal mechanisms, and is partially metabolized in the liver by modification of the piperazinyl group to at least four metabolites, which have been identified as desethylene ciprofloxacin, (M1), Sulfociprofloxacin
(M2), Oxociprofloxacin (M3), and IV formyl ciprofloxacin (M4). All these metabolites have been found to have microbiological activity less than that of the parent drug, but may be similar or greater than that of some other quinolones (39).
Ciprofloxacin and its metabolites are excreted unchanged in urine by both glomenular filtration and tubular secretion. Most of the drug appears unchanged in feaces as a result of biliary excretion. Small amounts of ciprofloxacin are removed by hemodialysis, and the amount depends on several factors such as type of coil used and dialysis flow rate.
1.4.4: Drug Interactions
The toxicity of drugs that are metabolized by the cytochrome P450 system is enhanced by concomitant use of some quinolones. Concurrent administration of ciprofloxacin with magnesium or aluminium antacids, sucralfate or products containing calcium, iron or zinc (including multivitamins or other dietary supplements) may substantially decrease the absorption of ciprofloxacin, resulting in serum and urine levels considerably lower than desired. Current or past treatment with oral corticosteroids is associated with an increased risk of Achilles tendon rapture, especially in elderly patients who are also taking the fluoroquinolones (45).
Ciprofloxacin’s renal clearance may affect other drugs subject to renal clearance or otherwise affecting the kidney. The concomitant use of ciprofloxacin with cyclosporins has also been associated with transient elevations in serum creatinine.
Renal tabular transport of methotrexate may be inhibited by concomitant 25 administration of ciprofloxacin potentially leading to increased plasma level of methotrexate and risk of methotrexate toxicity.
Probenecid interferes with renal tubular secretion of ciprofloxacin and produces an increase in the level of ciprofloxacin in serum(44). Ciprofloxacin can reduce effects of the co-administered drug; including adverse effects, for example, it has been shown to interact with thyroid medications resulting in unexplained hypothyroidism (45).
Altered serum levels of phenytoin have been reported in patients receiving ciprofloxacin concomitantly (44) because ciprofloxacin is an inhibitor of the hepatic
CYP1A2 enzyme pathway. Coadministration of ciprofloxacin and other drugs primarily metabolized by CYPIA2, for example, theophylline, methylxanthines and tizanidine results in increase in plasma concentrations of the coadministered drugs and could result in clinically significant pharmacodynamic side effects of the co- administered drug (43). Serious and fatal reactions have been reported in patients receiving concurrent administration of ciprofloxacin with tizanidine and theophylline, these include cardiac arrest, seizure, status epilepticus and respiratory failure.
1.4.5: Adverse effects
Some adverse effects that may occur as a result of ciprofloxacin therapy include irreversible peripheral neuropathy, (46,47). Spontaneous tendon rapture and tendonitis
(48-51), acute liver failure or hepatitis (52,53), toxic epidermal syndrome, severe central nervous disorders as well as photosensitivity/photoxicity reactions (55).
Psychotic reactions and confusional states, acute pancreatitis, bone marrow depression, interstitial nephritis and hemolytic anaemia may also occur during
Ciprofloxacin therapy (56,57).
Additional serious adverse reactions include temporary as well as permanent loss of vision (58,59), irreversible double vision and drug induced psychosis (60,61) and 26 chorea (62) impaired colour vision, exanthema, abdominal pain, malaise, drug fever, dysesthesia and eosinophilia (63,64). Pseudotumour cerebri, commonly known as idiopathic intracranial hypertension also referred to as increased intracranial pressure, has been reported to occur as a serious adverse reaction to ciprofloxacin (65).
1.5: Pessary
Pessaries are solid preparations suitably shaped for vaginal administration and contain one or more medicaments that are usually intended to act locally. These preparations are employed principally to combat infections in the female genito-urinary tract, to restore the vaginal mucosa to normal state, and for contraception (66). The usual pathogenic organisms are Trichomonas vaginalis, Candida albicans or other species, and Heamophilus vaginalis. Among the anti-infective agents available as vaginal preparations are nystatin, clotrimazole, miconazole, sulfanilamide, povidone iodine, clindamycin phosphate, metronidazole and oxytetracycline. Nonoxynol-9, a spermicide is employed for vaginal contraception. An estrogenic substance such as dienestrol is available as are found in vaginal preparation and it serves to restore the vaginal mucosa to its normal state (66).
1.5.1: Classification of Pessaries
Pessaries can be classified into the following groups viz: pharmaceutical pessary, occlusive pessaries and therapeutic pessaries.
1.5.1.1: Pharmaceutical Pessary
Pharmaceutical pessaries are used as a very effective means of delivery of easily absorbed pharmaceutical substances through the skin of the vagina or rectum and they are intended to have a local action. The active ingredient is usually mixed with a suitable base such as cocoa butter, glycero-gelatin or macrogol which are solid at 27 room temperature to assist insertion. It melts or disperses at body temperature. Oil- based pessaries undergo leakage with the potential to spoil clothing.In addition, the oil may cause deterioration of rubber thereby causing problems with contraceptives made from these materials (67).
Hormones can also be administered into the body in the form of pessaries.
Advantages of such pessaries include: i. some drugs can only achieve adequate concentrations in the body when
administered through this route. ii. optimal drug concentration can be achieved in the desired area without
causing systemic side effects which occur with oral, intramuscular or
intravenous drug administration. iii. they can be used to treat irregular periods and amenoerrhia in women
example, is progesterone pessary.
1.5.1.2: Occlusive Pessaries
An occlusive pessary is generally used in combination with spercimide as a contraceptive.
1.5.1.3: Therapeutic Pessary
A therapeutic pessary is used to support the uterus, vagina, bladder or rectum.
Therapeutic pessaries are most commonly used to treat prolapse of the uterus, urinary incontinence, a retroverted uterus, cystocele and rectocele. Side effects and complications often associated with therapeutic pessaries include; increased vaginal discharge, vaginal irritation, ulceration, bleeding and dyspareunia. Cervical cancer has, however,been reported to be more likely in older women with a prolonged history of pessary use.(67). 28
1.5.2: Preparation of Pessary
Pessaries may be prepared by moulding or by compression and by methods designed to minimise microbial contamination. They usually weigh between 3-5g. Moulded pessaries are either globular or ovoid in shape, while compressed pessaries are usually conical in shape. The most commonly used base for pessaries consists of combinations of the various molecular weight PEGs, surfactants and preservatives such as the parabens. Many pessaries and other types of vaginal products are buffered to an acid pH, usually about pH 4.5, which resembles that of the normal vagina.This acidity futher discourages pathogenic organism and provides a favourable environment for eventual colonization by the acid-producing bacilli normally found in the vagina. The PEG based vaginal suppository are water miscible and are generally sufficiently firm for the patient to handle and insert without great difficulty.
1.5.2.1: Pessary Base
The most commonly used base for vaginal suppositories consists of combinations of the various molecular weights of PEG. The various polyethylene glycols polymers are marketed in United States as carbowax and polyglycols.
Polyethylene glycol polymers have received much attention as suppository bases in recent years because they possess many desirable properties. They are chemically stable, non irritating, miscible with water and mucous secretions and can be formulated either by moulding or compression in a wide range of hardness and melting point. They do not melt at body temperature but dissolve to provide a more prolonged release than theobroma oil. Some polyethylene glycols polymers may be used singly as suppository bases but more commonly, formulas call for compounds of two or more molecular weights mixed in various proportions, as required to yield products of satisfactory hardness and dissolution time. Since the water miscible 29 suppositories dissolve in body fluids and need not be formulated to melt at body temperature, they can be formulated with much higher melting points and thus may be safely stored at room temperature.
The empirical formula and molecular weight of PEG is given as HOCH2
(CH2OCH2)mCH2OH where ‘m’ represents the average number of oxyethylene groups. Alternatively, the general formula H (OCH2CH2) nOH may be used to represent PEG, where ‘n’ is a number ‘m’ in the previous formula +1.
O H H O n
Figure 3: Structure of Polyethylene glycol.
The USP-NF (67) describes polyethylene glycol as being an addition polymer of ethylene oxide and water. Polyethylene glycol grades with molecular weight of 200 to 600 are liquids, grades 1000 and above are wax-like solids at ambient temperatures, while PEG grade with molecular weight above 1000 are white or off-white in colour, and range in consistency from pastes to waxy flakes. They have a faint, sweet odour.
Grades of PEG 6000 and above are available as free flowing milled powder, with melting ranges of 50-58oC for PEG 4000, 55-63 oC for PEG 6000 and 60-63 oC for
PEG 8000.
All grades of PEG are soluble in water and miscible in all proportions with other polyethylene glycols. Aqueous solutions of higher molecular weight grades may form gels. Liquid PEGs are soluble in acetone, alcohols, benzene, glycerin and glycols, whereas solid PEGs are soluble in acetone, dichloromethane, ethanol and methanol; 30 they are slightly soluble in aliphatic hydrocarbons and ether but insoluble in fats, fixed oils and mineral oil.
1.5.2.2: Applications of PEGs in Pharmaceutical Formulation
PEGs are widely used in a variety of pharmaceutical formulations including parenteral, topical ophthalmic, oral and rectal preparations. Mixtures of PEG can be used as suppository base (69). They are chemically reactive than fats. Greater care is needed in processing them into pessaries to avoid inelegant, contraction holes in the pessaries or suppositories. The rate of release of water soluble medications decreases with the increasing molecular weight of the PEG.
In solid dosage formulations higher molecular weight PEGs, can enhance the effectiveness of tablet binders and impart plasticity to granules (70). However they have only limited binding action when used alone, and can prolong disintegration if present in concentrations greater than 5%w/w. When used for thermoplastic granulations, (71-74) a mixture of the powdered constituents with 10-15%w/w PEG
6000 is heated to 70-75oC. The mass then becomes paste like and forms granules if stirred while cooling. This technique is useful for the preparation of dosage forms such as lozenges when prolonged disintegration is required.
1.5.2.3: Stability and Storage Conditions
Polyethylene glycols are chemically stable in air and in solution, although grades with molecular weight less than 2,000 are hygroscopic. Polyethylene glycols do not support microbial growth, and do not become rancid.
Polyethylene glycols and aqueous PEG solutions can be sterilized by autoclaving, filtration or gamma irradiation (75). Sterilization of solid grades by dry heat at 150oC for I hour may induce oxidation, darkening and the formation of acidic degradation 31 products. Ideally, sterilization should be carried out in an inert atmosphere. Oxidation of PEGs may also be inhibited by the inclusion of a suitable antioxidant.Polyethylene glycols should be stored in well-closed containers in a cool, dry place. Stainless steel, aluminum, glass or lined steel containers are prefered for the storage of liquid grades.
1.5.2.4: Incompatibilities.
The chemical reactivity of PEGs is mainly confined to the two terminal hydroxyl groups, which can be esterified. All grades, however, can exhibit some oxidizing activity owing to the presence of peroxide impurities and secondary products formed by autoxidation. The antibacterial activity of certain antibiotics is reduced in polyethylene glycol bases, particularly that of penicillin and bacitracin. The preservative efficacy of the parabens may also be impaired owing to binding with polyethylene glycols.
Physical effects caused by PEG bases include softening and liquefaction in mixtures with phenol, tannic acid and salicylic acid. Discoloration of sulphonamides and ethanol can also occur and sorbitol may be precipitated from mixtures (68). Plastics made of materials such as polyethylene, phenol formaldehyde, polyvinyl chloride and cellulose - ester membranes (in filters) may be softened or dissolved by PEGs.
Migration of PEG can occur from tablet film coatings, leading to interaction with core components.
1.5.2.5: Safety of Polyethylene Glycols
Polyethylene glycols are widely used in a variety of Pharmaceutical formulations.
They are generally regarded as non toxic and non irritant material (76-78). Adverse reactions to PEGs have been reported, the greatest toxicity is common with PEGs of low molecular weight. Their toxicities however, are very low. PEGs administered 32 topically may cause stinging, especially when applied to mucous membranes.
Hypersensitivity reactions to PEGs applied topically has also been reported. This include urticaria and delayed allergic reaction.(79) The most serious adverse effects associated with PEGs are hyperosmolarity, metabolic acidosis, and renal failure following the topical use of PEGs in burn patients (80). Topical preparations containing PEGs should therefore be used cautiously in patients with renal failure, extensive or open wounds. Oral administration of PEGs in large quantity can elicit a laxative effect. Therapeutically, up to 4 litres of an aqueous mixture of electrolytes and high molecular weight PEGs is consumed by patients undergoing bowel cleansing. (81).
Liquid PEGs may be absorbed when taken orally, but the higher molecular weight
PEGs are not significantly absorbed from the gastro-intestinal tract. Absorbed polyethylene glycol is excreted largely unchanged in the urine, although PEGs of low molecular weight may be partially metabolized. The (WHO) World Health
Organization, has set an estimated acceptable daily intake of PEGs at up to 10mg/kg body weight (82). In parenteral products, the maximum recommended concentration of PEG 300 is approximately 30%v/v. Hemolytic effects have been observed at concentrations greater than about 40%v/v.
1.5.3: Quality Control of Pessaries
Quality control procedures listed in the (BP) British Pharmacopoiea for manufactured pessaries include:appearance, disintegration, dissolution, uniformity of content and uniformity of weight.
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1.5.3.1: Appearance
The BP states that when cut longitudinally and examined with the naked eye; the internal and external surface of the pessary should be uniform in appearance.
Compliance with this standard indicates satisfactory subdivision and dispersion of suspended material.
1.5.3.2: Uniformity of Weight
Twenty pessaries are weighed singly and the average weight determined. No pessary must deviate from the average weight by more than 5 % except that two may deviate by not more than 7.5 %.
1.5.3.3: Uniformity of Content
The preparation complies with the test if not more than one individual content is outside the limits of 85 % - 115 % of the average content and none is outside the limits of 75 % - 125 % of the average content. If 2 or 3 individual contents are outside the limits of 85 % - 115 %, but within the limits of 75 %-125 %, the individual contents of another 20 dosage units taken at random has to be determined. The preparation complies with the test if not more than one individual content of the 30 units are outside the limits of 85-115 % of the average content and none is outside the limits of 75-125 % of the average content.
1.5.3.4: Disintegration
For vaginal product, when a dissolution test is prescribed a disintegration test may not be required (83).
1.6: Pharmaceutical Gels
Gels are defined as semi-solid systems in which the movement of the dispersing medium is restricted by an interlacing three dimensional network of particles or 34 solvated macromolecules of the dispersed phase (84). A high degree of physical and chemical cross-linking may be involved. The semi-solid nature of gels may result from an increased viscosity caused by the interlacing and consequential internal friction. A gel may consist of twisted matted strands often wound together by stronger types of van-der -Waals forces to form crystalline and amorphous regions throughout the system such as tragacanth and carboxymethylcellulose (84). Some gel systems have clear appearance as water, while some are turbid, since the ingredients may be completely molecularly dispersed, or they may form aggregates which disperse light.
The concentration of the gelling agents is mostly less than 10%, usually in range of
0.5-2 %.
1.6.1: Classification of Gels
Generally gels are classified into four major groups viz: inorganic, organic, hydrogels and organo gels.
1.6.1.1: Inorganic gels: are two-phase systems such as aluminum hydroxide gel and bentonite magma. Organic gels are single phase systems and may include such gelling agents as carbomer, tragacanth and those that contain an organic liquid, such as plastibase.
1.6.1.2: Hydrogels: these ingredients are dispersable colloidals or soluble in water and include organic hydrogel, natural and synthetic gums and inorganic hydrogels.
Other examples include hydrophilic colloids such as silica, bentonite, tragacanth, pectin, sodium alginate, methyl cellulose and alumina which in high concentration, form semi solid gels.
1.6.1.3: Organogels: these include the hydrocarbons, animal and vegetable fat, soap base greases and the hydrophilic organogels. Included in the hydrocarbon type is
Jelene or plasti-base, which is a combination of mineral oils and heavy hydrocarbon 35 waxes with a molecular weight of about 1300. The hydrophilic organogels or polar organogels include the polyethylene glycols of higher molecular weight, the carbowaxes. They are soluble to about 75 % in water and are completely washable.
The gels look and feel like petrolatum; they are non-ionic and stable. Jellies are a class of gels in which the structural coherent matrix contains a high proportion of liquid usually water. They are usually formed by adding a thickening agent such as tragacanth or carboxymethlycellulose to an aqueous solution of drug substance. The resultant product is usually clear and uniformly semi solids. They are highly susceptible to bacterial contamination and growth and as such are mostly preserved with antimicrobial. They should be stored with tight closures since water evaporates, drying out the product.
1.6.2: Gelling Agents
The consistency of gels can vary widely depending on the gelling agent used in their preparation. Common gelling agents include tragacanth, pectin, sodium alginate, gelatin, cellulose derivatives, carbomer, polyvinyl alcohols and clays.
1.6.2.1: Pectin
Pectin is obtained from the inner rind of citrus fruit or from apple pulp remaining after cider making. It is extracted with dilute acid and purified (85). It dissolves in about 20 parts of water, producing an opalescent acid solution. It is a valuable gelling agent for acid products and has been used often with glycerol as a dispersing agent and humectants in dermatological jellies. It is highly prone to microbial degradation and as such a preservative is very essential. Jellies made with pectin should be packed in tightly closed containers since they lose water rapidly by evaporation and this is increased by the susceptibility of pectin gel to syneresis (86). 36
1.6.2.2: Sodium Alginate
Sodium alginate jellies may be used both as lubricant (1.5 to 2%) and dermatological vehicles (5-10 %). A trace of calcium salt may be added to increase viscosity and most formulations contain glycerol as a dispersing agent. It consists mainly of the sodium salt of alginic acid, a polyuronic acid from sea weed, Laminaria. It is a white or buff powder (85). Maximum viscosity is at about pH 7 and at pH 4 and 10 it is only
10 % lower. At pH 3, however alginic acid is precipitated.
1.6.2.3: Tragacanth
Tragacanth jellies are sometimes called bassorin pastes, because the hydrophilic component of tragacanth that gels in water has been named bassorin (85). The gum has been used in the preparation of gels that are most stable at pH 4 to 8. They are preserved with either 0.1 % benzoic acid or sodium benzoate or a combination of 0.17
% methylparaben and 0.03 % propyl paraben. These gels may be sterilized by autoclaving. Aqueous dispersions are prepared by adding the powder to vigorously stirred water, since the powdered gum tends to form lumps when added to water.
Ethanol, glycerin or propylene glycol is used as wetting agents to wet the tragacanth before mixing with water.
1.6.2.4: Carbomer
Carbomer resins are high molecular weight allyl penaterythritol-cross linked acrylic acid based polymers modified with C10 to C30 alkyl acrylates. They are fluffy white dry powders with large bulk density. There are many carbomer resins with viscosity ranges from 0-80,00 cps (85), these include carbomer 910, 934, 934P, 940 and 1342 which are official in the USP-NF. carbomer 934 is highly effective in thick formulations such as viscous gels, while carbomer 934P is used for the preparation of 37 oral gels or gels meant for mucosal contact application and is the most widely used in the pharmaceutical industry. In addition to thickening, suspending and emulsifying in both oral and topical formulations, Carbopol 934 is used in commercial products to provide sustained release properties in the stomach and intestinal tract (85). Carbopol
940 forms sparkly clear water or hydroalcoholic gels, it is the most efficient of all the carbopol resins and has very good non drip properties.
The addition of alcohol to prepared carbomer gels may decrease their viscosity and clarity. An increase in the concentration of carbomer can be used to overcome the loss of viscosity. The viscosity of the gel also depends on the presence of electrolytes and on the pH. Generally, a maximum of 3% electrolytes can be added before a rubbery mass is formed. An excess of the electrolyte will result in decrease viscosity that cannot be reversed by the addition of an acid. Maximum viscosity and clarity occur at pH 7, but acceptable viscosity and clarity begin at pH 4.5 to 5.0 and extend to a pH of
11.
A neutralizer such as sodium hydroxide or potassium hydroxide is added to thicken the gel after the carbomer has been dispersed. Other neutralizing agents include sodium carbonate, ammonia and borax.
1.6.3: Vaginal Gels
The vagina has been used as a mucosal drug delivery route for a long time. Its single characteristics can be limitative or advantageous when drug delivery is considered.
Vaginal gels have been used and are receiving a great deal of interest as drug delivery system (9). Gels are versatile and have been used as delivery systems for microbicides, contraceptives, labour inducers, and other substances. Vaginal gels are known to possess a higher bio-compatibility and bioadhesivity and can be rapidly eliminated through normal catabolic pathways; thus decreasing the risk for irritative 38 or allergic host reaction at the application site (86). It was reported that, 0.75 % intra vaginal metronidazole gel was proved to have a clinical cure rate similar to that for oral metronidazole for the treatment of bacterial vaginosis (86). Typical examples of vaginal gels currently in used include; metronidazole vaginal gel (Metrogel) and
Clindamycin vaginal gel (Cleocin) gel, both utilized in the treatment of bacterial vaginosis.
1.6.3.1: Uses of Vaginal Gels
Vaginal gels may contain microbicide, which are agents used in the treatment of sexually transmitted infection. Sexually transmitted infection has remained a significant problem, both in developed and developing world settings (87).
According to the latest statistics from UNAIDS/WHO, 33.3 million people are living with HIV-1 infection, with 2.6 million new infections in 2009.
In addition, the WHO estimated that 340 million new cases of gonorrhea, Chlamydia, syphilis and trichomoniasis occurred throughout the world in 1999 in both men and women aged 15-49 years (88). Among STIs, the prevention of HIV-1 transmission has been a high funding priority for the National Institutes of Allergy and Infectious diseases, the United States Agency for International Development, and the Bill and
MelindaGate’s Foundation (89). Early attempts to prevent transmission of HIV-1 from men to women were based on the use of surfactants polyanions and acid buffers.
Unfortunately, these compounds failed to protect women against HIV-1 infection. A recently completed study of women in South Africa using the nucleotide reverse transcriptase inhibitor Tenofovir® in a vaginal gel showed a significant reduction in
HIV – 1 transmission (89). This success and the desire to reduce transmission of other sexually transmitted diseases have spurred the development of novel delivery systems for a wide range of drugs. Vaginal gels containing nonoxynol-9-(N-9) has been used 39 as contraceptive (9). Vaginal gel containing 0.75 % metronidazole and 2 % clindamycin has been used in the treatment of bacterial vaginosis
(86).Bioadhesive gels containing (200mg) miconazole nitrate has been used in the treatment of vulvo vaginitis associated with Candida and/or Gram positive bacteria. In addition, vaginal gel has been utilized for the induction of labour and abortion (84).
1.6.3.2: Physico-Chemical Characterization of Vaginal Gels
Pharmaceutical characterization of vaginal gel is an important step in order to optimize safety, efficacy and acceptability. The physicochemical properties of vaginal gel of importance include pH, viscosity, colour and odour.
1.6.4: Vaginal Tablets
Vaginal tablets also reffered to as inserts, are uncoated bullet-shaped or ovoid tablets inserted into the vagina for local effects. They are prepared by compression and shaped to fit snugly on plastic inserter devices that accompany the product. They contain antibacterials for the treatment of vaginitis caused by Haemophilus vaginalis or antifungals for the treatment of vulvo vaginitis candidiasis caused by Candida albicans and related species (90). They are prepared by compression and are commonly formulated to contain lactose as a filler, a disintegrant and binder such as polyvinyl pyrrolidone as well as lubricants such as magnesium stearate. These tablets are intended to disintegrate within the vagina, releasing their medication (90). Vaginal tablets currently sold in the market include Mycelex-G vaginal tablets® Estradeol vaginal tablets, Copnesten vaginal tablet, Nystatin vaginal tablet, Effervescent
Ciprofloxacin lactate vaginal tablets etc.
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1.6.4.1: Evaluation of Vaginal Tablets
1.6.4.1.1: Pharmcopoeial Tests
The British pharmacopoeia 1993, contains standards and test methods for the following tablet properties: -uniformity of weight, content of active ingredient, uniformity of content, disintegration and dissolution.
The first three tests are designed to control the amount of active material in the tablet, while the last two control the ability of the drug to be released from the tablet.
1.6.4.1.2: Uniformity of Weight Test
This test involves the weighing of 20 tablets from a batch individually and the calculation of the mean. Not more than two tablets are permitted to deviate from the mean by more than the percentage given below, and none by more than double that percentage.
1.6.4.1.3: Uniformity of weight test, permitted variations
Average weight of tablet percentage deviation
80mg or less 10
More than 80mg but less than
250mg 7.5
250mg or more 5
1.6.4.1.4 Uniformity of Content
The content of active ingredient is determined from a sample of 20 tablets, by crushing the tablets and subjecting an aliquot of the resultant power to the stipulated assay procedure (91). The result of the assay gives the average drug content of the 20 tablets, but gives no indication of the variation of drug content among the individual tablets. Cross variation would be excluded by the uniformity of weight test only if the 41 drug comprises the bulk of the tablet; this only holds for relatively non potent drugs which do not require dilution before tableting. However content variation among tablets of high potency drugs in which most of the tablet will be diluents, cannot be picked up by a combination of these two tests. In such cases, the uniformity of content must be established by individual tablet assays. Ten tablets are assayed by the specified method. The preparation being examined fails to comply if more than one tablet is outside the range of 85 %-115 % of the average value or if any tablet is outside the range of 75 %-125 % of the average. If one tablet is outside the 85 %-115
% range, then a further 20 tablets are assayed and no more tablets outside this range should be found (91). The USP XX11 adopts a somewhat different approach. It permits uniformity to be demonstrated by either weight variation or content uniformity, but accepts that weight variation is not an adequate test when the active ingredient is a minor component of the tablet formulation (92). Hence weight variation can only be used when there is 50 mg or more of active ingredient present which comprises at least 50 % of the weight of the dosage form. A sample of 30 tablets is randomly selected. Ten out of the thirty tablets are weighed and the mean calculated. From the result of the assay, the content of active ingredient is calculated, assuming homogenous distribution of the drug among the tablets. Content uniformity is also established using a random sample of 30 tablets. Ten are assayed and the mean content and relative standard deviation calculated. If one tablet is outside the range 85 %-115 % of the claimed content but within the range 75 %-125 % or the relative standard deviation (RSD) is greater than 6% or both, the remaining 20 tablets are assayed. No further tablets outside the 85 %-115 % range maybe present.
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1.6.4.1.5: Disintegration Test
For a drug to be absorbed from a solid dosage form after oral administration, it must first be in solution, and the first important step toward this is usually the break-down of the tablet, a process known as disintegration (93). The disintegration test is a measure of the time required under a given set of conditions for a group of tablets to disintegrate into particles which will pass through a 10-mesh screen. Generally, the test is useful as a quality assurance tool for conventional dosage forms. The disintegration is carried out using the disintegration tester which consists of a basket rack holding 6 plastic tubes; open at the top and bottom. The bottom of the tube is covered by a 10-mesh screen. The basket is immersed in a bath of suitable liquid held at 37oC, preferably in 1 L beaker. For compressed uncoated tablets, the testing fluid is usually water at 37oC but some monographs direct that simulated gastric fluid be used. If one or two tablets fail to disintegrate, the test is repeated using 12 tablets. For most uncoated tablets, the BP requires that the tablets disintegrate in 15 minute while for coated tablets, up to 2 hours may be required (94). The individual drug monographs specify the time disintegration must occur to meet the pharmacopoeial standards. The test described above is applied to tablets which are designed to disintegrate in the gastro-intestinal tract. If this is not the case, the test is modified.
For example dispersable tablets must disintegrate in water at 19-21oC within 3 minutes, while effervescent tablets must disintegrate in 200ml of non-agitated cold water at 15-20oC within 5 minutes.
1.6.4.1.6: Dissolution Test
The British pharmacopoeia of 1993 permits three types of apparatus, the rotating basket, the paddle and the flow through cell methods. In vitro dissolution testing of solid dosage forms is important for a number of reason; 43 i. The dissolution test guides formulation and product development toward
product optimization. When carried out in the early stages of a product’s
development, it allows differentiation between formulations and correlations
identified with in vivo bioavailability data. ii. manufacturing may be monitored by dissolution testing as a component ,from
early product development through approval and commercial production
ensures control of any variables of materials and processes that could affect
dissolution and quality standards. iii. consistent in vitro dissolution testing ensures bioequivalence from batch to
batch. The United States Food and Drug administration (FDA) allow
manufacturers to examine scale up batches up to 10 % of the proposed size of
100,000 dosage units, whichever is grater (90). iv. tablet dissolution is a requirement for regulatory approval of marketing for
products registered with the FDA and the regulatory agencies of other
countries.
Dissolution testing and interpretation can be continued through three stages if necessary (95). In stage 1 (S1), six tablets are tested and are acceptable if all of the tablets are not less than the monograph tolerance limit (Q) plus 5 % (96). If the tablet fails in stage 1 (S1) then an additional six tablets are tested in stage 2 (S2). The tablets are accepted if the average of the twelve tablets is greater than or equal to Q and no unit is less than Q minus 15 %. If the tablets fail the test, an additional twelve tablets are tested., and the tablets are acceptable if the average of all 24 tablets is greater than or equal to (Q ) and if not more than two tablets are less than Q minus 15 %.
Industrial pharmacist routinely tests their formulations for dissolution. The results are plotted as concentration versus time and the time taken for 50 %(t50),90 %(t90)and the 44
percentage dissolved in 30 minutes. A value for t90% of 30 minutes is often considered satisfactory and is an excellent goal since a common dissolution tolerance in the USP/NF is not less than 75 % dissolved in 45 minutes.
1.6.4.2: Non-Compendial Tests
There are many tests that are frequently applied to tablets for which there are non- pharmacopoeial requirements but will form a part of the manufacturer’s product specification. These include;
1) Tablet Thickness
2) Tablet Hardness
3) Tablet Friability
1.6.4.2.1: Tablet Thickness
The thickness of a tablet is the only dimensional variable related to the compression process (96). Thus, at a constant compressive load it varies with changes in die fill with particle size distribution and packing of the particle mix being compressed, and with tablet weight, while with a constant die fill, thickness varies with variations in compressive load (96). Tablet is consistent batch to batch or within the batch when the tablet granulation or powder blend is adequately consistent in particle size and size distribution, if the punch tooling is of consistent length, and if the tablet press is clean and also in good working condition (96).
The thickness of individual tablets is usually measured with a micrometer, which permits accurate measurements and provides information on the variation between tablets. Other techniques utilized in production control involved placing 5-10 tablets in a holding tray, where their total crown thickness may be measured with a sliding caliper. This method is much more rapid than measurement with a micrometer in 45 providing an overall estimate of tablet thickness in the production operations.
However, it does not readily provide information on variability between tablets. This method is more suitable for production work.
Tablet thickness should be controlled within a ± 5 % variation of a standard value.
Any variation in tablet thickness within a particular lot of tablets or between the manufacturer’s lots should not be apparent to the unaided eye for consumer acceptance of the product. It is also necessary to control tablet thickness for the ease of packaging, because of some difficulties which may result in the use of unit dose and other types of packaging equipment if the volume of the material being packaged is not consistent.
Tablet thickness is important for tablet packaging; very thick tablets affect packaging either in blisters or plastic containers. The tablet thickness is determined by the diameter of the die, the amount of fill permitted to enter the die and the force of pressure applied during compression (96). As part of Good manufacturing practice
(GMP), the production run is monitored under control chart at regular intervals usually 10-15min, during the course of manufacturing, the operator must sample specified number of tablets for testing (in-process control) e.g weight of tablet, tablet thickness, friability, disintegration time, and dissolution studies.
1.6.4.2.2: Tablet Diameter
Although the Pharmacopoeia does not insist on a standard weight for official tablets, it nevertheless specifies their diameter. The thickness is not directly controlled, but in practice most manufacturers make uncoated tablets of thickness equal to half the diameter since these results in an elegant and weight of tablet are obviously fixed and there is now very little variation in the dimensions of official tablets produced by different manufacturers. 46
The Pharmacopoeia permits some slight deviation, usually ± 5 % from the nominal diameter.
1.6.4.2.3: Tablet Hardness or Crushing Strength
The resistance of tablets to capping, abrasion or breakage under conditions of storage, transportation and handling before usage depends on its hardness. A hardness tester in common use, is that designed by the Monsanto Chemical Company limited. In this design, the tablet to be tested is placed between the spindle and the anvil, and pressure is applied by turning the knurled knob just sufficiently to hold the tablet in position.
The reading of the pointer on the scale is then adjusted to zero mark; pressure is then increased as uniformly as possible until the tablet breaks, when the pointer will read the pressure required to break the tablet. The ‘hardness factor’ is taken as the average of several determinations. The force required to break the tablet is measured in kg/cm2 and a crushing strength of 4kgf is usually considered to be the minimum for satisfactory tablets (94). Oral tablets normally have a hardness of 4-10 kgf, however, hypodermic and chewable tablets are usually much often (3 kgf) and some sustained release tablets are much harder (10-20 kgf). Tablet hardness has been associated with other tablet properties such as density and porosity. Hardness generally increases with normal storage of tablets and depends on the shape, chemical properties, binding agent and pressure applied during compression.
1.6..4.2.4: Friability
Friction and shock are the forces that most often cause tablets to chip, cap or break.
The friability test is closely related to tablet hardness and is designed to evaluate, the ability of the tablet to withstand abrasion in packaging, handling and shipping. It is usually measured by the use of the Roche friabilator. Twenty (20) tablets are weighed 47 and placed in the apparatus when they are exposed to rolling and repeated shocks as they fall 6 inches in each turn within the apparatus.
After 4 minutes of this treatment or 100 revolutions, the tablets are weighed and the weight compared with the initial weight. The loss due to abrasion is a measure of the tablet friability. The value is expressed as a percentage. A maximum weight loss of not more than 1 % of the weight of the tablets being tested during the friability test is considered generally acceptable and any broken or smashed tablets are not picked.
Normally, when capping occurs friability values are not calculated. A thick tablet may have less tendency to cap, whereas thin tablets of large diameter often show extensive capping, thus indicating that tablets with greater thickness have reduced internal stress
(94)
1.7: Ac-Di-Sol®
Ac-Di-Sol® is an internally cross-linked sodium carboxymethyl cellulose that aids in the disintegration and dissolution of pharmaceutical tablets, capsules and granules.
Ac-Di-Sol® was originally created to solve formulators’ problems with long-term stability, effectiveness at high tablet hardness levels, and the need for high use levels.
Ac-Di-Sol® is now the standard by which super disintegrants are judged. It is made from internally cross-linked form of sodium carboxymethyl cellulose. Ac-Di-Sol® exhibits consistent disintegrant functionality as a result of several distinctive properties which include good water uptake due to high capillary action, rapid swelling properties and efficient fluid channeling, resulting from long fiber length.
The superior disintegration properties of Ac-Di-Sol® makes it effective even at low use levels in very hard tablets or granules. It has also demonstrated excellent long- term stability in a wide range of formulations. The cross linking greatly reduces water solubility while still permitting the material to swell and absorb many times its weight 48 of water without losing individual fibre integrity. It is utilized at concentrations of 1-3
%, (2 % for direct compression and 3 %, for wet granulation (97).
Tablet dissolution can be greatly increased by the use of a “super disintegrant”.
Wicking and swelling represent two means commonly used in determining disintegrant performance. The fibrous nature of Ac-Di-Sol® gives it outstanding water wicking capabilities and its cross-linked chemical structure creates an insoluble hydrophilic highly absorbent material which results in good swelling properties. This dual functionality translates into superior disintegration characteristics at very low use levels compared to other super disintegrants. (98). In order to increase drug dissolution in direct compression formulations, Ac-Di-Sol® is usually incorporated to other formulation components and dry blended to a homogenous mixture prior to the addition of lubricants. This uniform distribution of Ac-Di-Sol® in the tablet matrix enables it to wick water and swell, thereby breaking the tablet apart and exposing more surface area of the drug to gastric fluid (98). Ac-Di-Sol® should be added during the wet granulation process and to the dry granules prior to compression. This improves drug dissolution by taking full advantage of its wicking and swelling properties. The addition of Ac-Di-Sol® during the wet mixing phase results in an intimate mixture of Ac-Di-Sol®, drug binder and filler within the granular matrix.
This intragranular addition of Ac-Di-Sol® allows it to break down the granules by swelling, and exposes greater surface area of the drug to gastric fluids. The addition of
Ac-di-sol® during the dry blending phase integrates Ac-Di-Sol® into the extragranular structure of the tablet, where its fibrous nature, wicking ability and swelling properties carry fluid throughout the tablet to cause rapid disintegration. This combined intragranular and extragranular distribution of Ac-Di-Sol® optimizes its functionality. 49
1.8: Primojel®
This is one of the modifications of starch aimed at improving its disintegrant properties. Primojel® is chemically cross-linked and carboxymethylated potato starch. It is manufactured by cross-linking starch with sodium trimetaphosphate followed by carboxymethylation with sodium monochloroacetate. Approximately one glucose monomer unit in every four is substituted. The benefits of potato starch and the optimization of the degrees of cross-linking/substitution have been reported (99).
The presence of carboxymethyl groups makes the starch more hydrophilic, but not completely water soluble. The process also causes a two to three fold increase in the size of the starch grains. It is used at a relatively low concentration of 2-4 % (100).
1.9: Sterotex®
Sterotex® powder is prepared from fully refined vegetable oil. The oil is hydrogenated to near saturation, bleached and deodorized before being spraychilled to fine powders. Sterotex is used at a level of 1-5 %. It is insoluble in water and is suitable for drugs that degrade in the presence of magnesium stearate or metallic stearates.
1.10: Bioadhesion
Bioadhesion is a state in which two materials at least, one of which is of a biological nature, are held together for extended period of time by interfacial forces (103). For drug delivery purposes, the term bioadhesion implies attachment of a drug carrier system to a specific biological location, which can be the epithelial tissue or the mucous coat on a tissue surface (103), thus increasing drug absorption and overall bioavailability (104-106). In recent years many bioadhesive drug delivery systems have been developed for oral, buccal, nasal, rectal, vaginal, cervical, gastro intestinal 50 tract, occular, sublingual regions, for both systemic and local effects. This reduces toxic side effects and increase the therapeutic efficacy of drugs (107).
Mechanism of Mucoadhesion: For bioadhesion to occur, a succession of phenomenon whose role depends on the nature of the bioadhesive is required. The first stage involves an intimate contact between a bioadhesive and membrane, either from a good wetting of the bioadhesive surfaces or from the swelling of the bioadhesive (108). In the second stage, after contact is established, penetration of the bioadhesive into the crevices of the tissue surface or interpenetration of the chains of the bioadhesive with those of the mucus take place. Low chemical bonds can then settle. On a molecule level, mucoadhesion can be experienced based on molecular interactions. The interaction between two molecules is composed of attraction and repulsion. Attractive interactions arise from van der Waals forces, electrostatic and steric repulsion. For mucoadhesion to occur, the attractive interaction should be larger than the non-specific repulsion (109).
1.10.1: Factors Affecting Mucoadhesion
1.10.1.1: Molecular Weight
The optimum molecular weight for maximum bioadhesion depends largely on the type of mucoadhesive polymer used. It is generally understood that the threshold required for successful bioadhesion is at least 100,000 molecular weight (107). For example, PEG 20,000 has little adhesive character, whereas PEG 200,000 improved adhesive character and PEG 400,000 has superior adhesive properties. The fact that mucoadhesiveness improves with increasing molecular weight for linear polymers implied that interpenetration is more critical for a low molecular weight polymer to be a good mucoadhesive and entanglement is important for high molecular weight 51 polymers. Adhesiveness of a non linear structure by comparison, follows a quite different trend. The adhesive strength of dextran, with a high molecular weight of
19,500,000 is similar to that of PEG, with a molecular weight of 200,000 (107) The reason for this similarity may be that the helical conformation of dextran may shield many of the adhesive groups, which are primarily responsible for adhesion unlike the conformation of polyethylene glycol.
1.10.1.2: Concentration of Active Polymer
There is an optimum concentration for a mucoadhesive polymer to produce maximum bioadhesion. In highly concentrated system, beyond the optimum level, however, the adhesive strength drops significantly because the coiled molecules become separated from the medium so that the chain available for interpenetration becomes limited
(107)
1.10.1.3: Flexibility of Polymer Chains
Chain flexibility is critical for interpenetration and entanglement. As water soluble polymers become cross linked, the mobility of an individual polymer chain decrease and thus the effective length of the chain that can penetrate into the mucus layer decreases, thus reducing the mucoadhesive strength.
1.10.1.4: Spatial Conformation
Spatial conformation of a molecule plays a very important role in mucoadhesion.
Besides chain length and molecular weight of 19,500,000 for dextrans, they exhibit bioadhesive strength similar to that of PEG, with a molecular weight of 200,000. The helical conformation of dextran with many adhesively active groups is primarily responsible for adhesion, unlike PEG Polymers, which have a linear conformation.
52
1.10.1.5: Environmental Factors pH of polymer – substrate interface. pH can influence the formal charge on the surface of the mucus as well as certain ionizable mucus adhesive polymers. The charge density on the mucus surface varies depending on the pH due to the difference in dissociation of functional groups on the carbohydrate moiety as well as the amino acids of the polypeptide backbone. Some studies had shown that the pH of the medium is important for the degree of hydration of cross linked polycyclic acid.
Consistently increased hydration occur from pH 4 through pH 7,and then a decrease as alkalinity or ionic strength increases(107). For example polycarbophil does not show a strong mucoadhesive property above pH 5 because uncharged carboxyl group reacts with mucin molecule, rather than the ionized group presumably through numerous hydrogen bonds. However, at higher pH, the chain is fully extended due to electrostatic repulsion of the carboxylate anions (110).
1.10.1.6: Initial Contact Time
Contact time between the bioadhesive and mucus layer determines the extent of swelling and interpenetration of the bioadhesive polymer chains. Moreover, bioadhesive strength increases as the contact time increases (111).
1.10.1.7: Physiological Factor
Mucin Turnover Rate
The natural turnover of molecules from the mucus layer is important for at least two reasons. Firstly, the mucin turnover is expected to limit the resident time of the mucoadhesive on the mucus layer. No matter how high the mucoadhesive strength, they are detached from the surface due to mucin turnover. Secondly, mucin turnover results in substantial amount of soluble mucin molecules. These molecules interact 53 with mucoadhesive before they have chance to interact with the mucus layer. Mucin turnover may depend on the presence of food. In this case, the gastric mucosa accumulates secreted mucin on the luminal surface of the tissue during the early stages of fasting. The accumulated mucin is subsequently released by freshly secreted acid or simply by the passage of ingested food. Estimation of mucin turnover varies widely, depending on location and method of measurement (112). Values ranging from a few hours to a day have been reported (112). However, residence times of bioadhesives that are thought to attach to mucin are typically longer than the reported mucin turnover, suggesting that the presence of bioadhesive polymer on mucin may alter the turnover of this biopolymer. Lehr et al (112) calculated the mucin turnover time of 47-270 minutes in rats and 12-24h in humans. The residence time of dosage forms is limited by the mucin turnover time.
1.10.1.8: Disease State
The physicochemical properties of mucus are known to change during disease conditions such as common cold, gastric ulcers, ulcerative colitis, cystic fibrosis, bacterial and fungal infection of female reproductive tract and inflammatory conditions of the eye, (109) thereby changing the degree of mucoadhesion.
1.11: Mechanism of Drug Release
After administration of a solid dosage form, the active principle has to be first released before the effect of the drug can be felt. Dosage forms can thus be classified based on the mechanism of release of the active ingredient from the dosage formulation into the classes viz: immediate and non immediate release dosage forms.
54
1.11.1: Immediate Release Dosage Forms
Conventional dosage forms release their ingredients into an absorption pool immediately after administration (113). Immediate release dosage forms implies that absorption through a biological membrane is the rate limiting step in the delivery of the drug to it target area
1.11.2: Non-Immediate Release Dosage Forms
Sustained release, sustained action, prolonged action, controlled release, extended action, timed release, depot and repository dosage forms are terms used to identify drug delivery systems that are designed to achieve a prolonged therapeutic effect by continuously releasing medication over an extended period of time after administration of a single dose. (114). In the case of injectable dosage forms, this period may vary from days to months whereas in orally administered dosage form, this period is measured in hours and critically depends on the residence time of the dosage form in the gastrointestinal tract. The term “controlled release” has become associated with those systems from which therapeutic agents may be automatically delivered at predefined rates over a long period of time. Products of this type have been formulated for oral, injectable and topical use as well as inserts for placement in body cavities (115). The release of drugs from sustained release dosage form is the rate limiting step. The absorptive phase of the kinetic scheme becomes insignificant compared to the drug release phase. Efforts to develop a non-immediate release drug delivery system must be directed primarily at altering the release rate. The benefits offered by modified release systems include reduced dosing frequency with improved patient compliance, better and more uniform clinical effects with lower incidence of side effects and possible enhanced bioavailability (116).
55
1.11.3: Drug Release Kinetics
Drug release from sustained release dosage forms is usually by diffusion, leaching or erosion (117). In which case dissolution rate is a more important factor than disintegration. Diffusion is the dominant mechanism controlling the dissolution of water soluble drugs, and erosion of matrix is the dominant mechanism controlling the release of water–insoluble drugs. Generally, release of drugs will occur by a mixture of these two mechanisms (118).
Although interactions between water, polymer excipient and drug are the main factors determining drug release, formulation variable such as drug and polymer levels and types, drug polymer/excipient ratios and polymer and drug particle size have been reported to influence the rate of drug release from the hydroxypropyl methyl cellulose matrices (119).
The quantitative analysis of the values obtained in dissolution/release test is easier when mathematical formulae that express the dissolution results as function of some of the dosage forms characteristics are used.
1.11.4: Zero Order Kinetics
Dissolution of drug from dosage forms that do not disintegrate and release the drug slowly where drug release rate is independent of its concentration can be represented as:
CO - Ct = Kt ------Eqn (6)
Where CO is the initial amount of drug in the dosage form; Ct is the amount of drug in dosage form at time‘t’ and ‘K’ is the proportionality constant. Dividing this equation by CO;
1 – (Ct/Co) = Kot ------Eqn (7) 56
Where 1 – (CO - Ct) represents the fraction of drug dissolved in time ‘t’ and KO the zero order release constant. A graphical representation of fraction of drug dissolved versus time will be linear. This relationship can be used to determine the dissolution of various types of modified release dosage forms e.g some transdermal system, matrix tablets with low soluble drugs, coated forms and osmotic system. The dosage form following this profile, release the same amount of drug by unit time and it is the ideal method of drug release in order to achieve a prolonged pharmacological action
(120).
1.11.5: First Order Kinetics
The first order kinetics describes drug release from systems in which the release rate is concentration dependent. The equation is represented mathematically as
Log Ct = Log CO + K1t/2.303 ------Eqn (8)
Where Ct is the amount of drug released in time ‘t’, Co is the initial amount of the drug in the solution and K1 is the first order release constant. Here the graphical representation of the decimal logarithm of drug released versus time will be liner.
Example of a dosage form which follow this release profile is water soluble drug in a porous matrix.
1.11.6: Hixon – Crowell Model
In order to evaluate the drug release with changes in the surface area and the diameter of particles/tablets, Hixon-Crowell in 1931 (121) recognized that the particle regular area is proportional to the cubic root of its volume. This is represented mathematically as follows:
1/3 1/3 CO - Ct = Kst ------Eqn (9) 57
Where CO is the initial amount of drug in the dosage form, Ct is the remaining amount of drug in the dosage form at time ‘t’ and Ks is a constant incorporating the surface volume relation.
1.11.7: Higuchi Model
This model describes the release of drugs from insoluble matrices as a square root of time dependent process based on Fickian diffusion. Higuchi in 1961 (122) and also in
1963 (123) developed models to study the release of water soluble and low soluble drugs incorporated in semisolid and solid matrices.
The Higuchi model is represented as:
1 A = KH-t /2 ------Eqn (10)
Where KH is the Higuchi constant. Higuchi describes drug release as a diffusion process based on the Fick’s law. Examples of this model include drug dissolution from some modified release dosage form such as some transdermal systems and matrix tablets with water soluble drugs.
1.11.8: Korsemeyer – Peppas Model
In 1963 Korsemeyer et al (124) developed a simple, semi empirical model when diffusion is the main drug release mechanism, relating exponentially the drug release to the elapsed time (t).
n Mt/M∞ = Kt ------Eqn (11)
Where Mt/M∞ is the fraction of drug released at time t, K is the proportionality constant, which accounts for the structural and geometrical properties of the matrix and ‘n’ is the diffusional exponent indicative of the mechanism of drug release. The exponent ‘n’ depends on the polymer swelling characteristics and the relaxation rate at the swelling front (125). The values of release parameters, ‘n’ and K, are inversely 58 related. A high value of K may suggest burst drug release from the matrix. The equation is however, valid only for the early stages (< 70 % of drug release (125).
In 1985 Peppas (125) use this n value in order to characterize different release mechanism concluding for values for a slab, of ‘n’ = 0.5 for Fickian diffusion, between 0.5 and 1.0 or n = 1.0, for mass transfer following a non-Fickian model. 0.5 < n > 1.0, for anomalous transport. To determine the exponent ‘n’ the position of the release curve. Where Mt/M∞ < 0.6 should only be used. This model is generally used to analyze the release of polymeric dosage forms, where the release mechanism is not well known or more than one type of release phenomenon could be involved (126).
The release models with major applications and best describing drug release phenomenon are the Higuchi model, zero order kinetics and Korsemeyer Peppas model. The Higuchi and zero order models represent the limit cases in the transport and drug release phenomena and the Korsemeyer-Peppas model can be a decision parameter between these two models. While the Higuchi model had a large application in polymeric matrix systems, the zero order models becomes the ideal to describe coated dosage forms or membrane controlled dosage forms. The criterion to choose the best model to study the drug dissolution/release phenomenon is the use of the coefficient of determination (r2), to assess the fit of a model equation (127).
Usually, this value tends to get greater with the addition of more parameters irrespective of the variable model. 59
CHAPTER TWO
MATERIALS AND METHODS
2.1: Materials
Ciprofloxacin hydrochloride (99.53 % purity, Batch No. CH 1177-11010) was obtained as a gift sample from (Arch Pharma Labs. Ltd, India). Polyethylene glycol
4000 was procured from (Carl Roth GMBH, Karlsruhe Germany), polyethylene glycol 4000, polyethylene glycol 8000 (Sigma-Aldrich, Germany), polyvinyl pyrrolidone (E. Merck Darmstadt) microcrystalline cellulose,glycerol,magnesium stearate,talc (BDH chemicals, Ltd, Poole, England) Sodium alginate (Batch No.
AC177775000) was a free gift from (Janssen Pharmaceuticalaan Geel, Belgium) Ac-
Di-Sol® (FMC, Philadelphia, pa) Sterotex® (Abitech, USA) Primojel® (DMV, international, Vieghel, the Netherlands). Carbopol 971 (BF, Goodrich Co. USA) methyl paraben (Nipa Lab; Hamburg, West Germany) cellulose membrane, M. W.
Cut-off 6000-8000 (Spectrum Labs; Breda, The Netherlands).
2.2: Methods
2.2.I: Preparation of Ciprofloxacin Pessaries
The compositions of the different formulations of ciprofloxacin pessaries are shown in Table 1. Ciprofloxacin pessaries containing 0.2 g of Ciprofloxacin, Polyethylene glycol 4000, 1.6 g and distilled water 0.2 g were prepared by the fusion method, using a metal mould with twelve cavities each with a capacity of 2 g. Polyethylene glycol 4000 was melted over a water bath at a temperature range of 60-63oC. On cooling a solution of 0.2 g Ciprofloxacin hydrochloride in distilled water was added to the base and stirred slowly to avoid the incorporation of air bubbles. The mixture was 60
Table 1: Composition of Ciprofloxacin Hydrochloride Pessary
Ingredient (%) B1 B2 B3 B4 B5 B6 B7 B8 Ciprofloxacin 10 10 10 10 10 10 10 10 Hydrochloride Polyethylene glycol 4000 80 80 80 80 80 80 80 40 Primogel 0.1 0.2 Ac-Di-Sol 0.01
Sterotex 0.1 0.2 Carbopol 0.01 Polyethlene glycol 8000 40
Key: Batch 1 = 80% PEG 4000
Batch 2 = 80% PEG 4000 + 0.1 % Primogel®
Batch 3 = 80% PEG 4000 + 0.2 % Primogel®
Batch 4 = 80% PEG 4000 + 0.01 % Ac-Di-Sol®
Batch 5 = 80% PEG 4000 + 0.1 % Sterotex®
Batch 6 = 80% PEG 4000 + 0.2 % Sterotex®
Batch 7 = 80% PEG 4000 + 0.01 % Carbopol 971®
Batch 8 = 40% PEG 4000 + 40 % PEG 8000
61 poured into the mould and refrigerated for three hours. Batches of pessaries containing varying concentrations of Primojel®, Ac-Di-Sol®, Sterotex®, Cabopol
971® as excipients and Polyethylene glycol 8000, PEG 4000 were similarly prepared. 62
Figure 4: Ciprofloxacin hydrochloride pessaries
63
2.2.2: Evaluation of Ciprofloxacin Pessaries
2.2.2.1: Weight Variation
Eight pessaries were selected at random and weighed individually on an electronic scale (OHAUS, Galaxy). The average weight of the pessaries was determined, and the weight of individual pessaries was compared with the average weight.
2.2.2.2: Adhesion/Erosion Experiment
The apparatus for this experiment is shown in Fig 2.It is made up of a separating funnel clamped on a retort stand with a metal support used to position a plastic support at an angle of 250 (128). Freshly isolated and excised Pig rectum (11.5 cm by
7.5 cm) was pinned on the plastic support. A collecting dish was placed directly under the plastic support to collect detached pessaries. The Pig rectum was flushed with 50 mL of normal saline and the experiment was set at 10 drops of the phosphate buffer
(pH 4) per minute. The pessary was allowed to equilibrate on the intestine for 5 minutes, and the duration of the experiment was 60 minutes. The procedure was repeated for all the batches. The percent bioadhesion and erosion were calculated for each of the batches, from Equation 1and 2 respectively.
Percentage bioadhesion=No of pessary after bioadhesion experiment x100----Eqn (12) Initial number of pessary 1
Percentage erosion = Initial weight –final weight x 100 ------Eqn (13) Initial weight 1 64
Figure 5: Set up for the Erosion/Bloadhesion test for Ciprofloxacin Pessary
65
2.2.2.3: Dissolution Profiles of Ciprofloxacin Pessaries
Dissolution studies of the pessaries were conducted with Erweka dissolution apparatus using USP method. The dissolution medium was 500 mL of phosphate buffer (pH 4) maintained at a temperature of 36± 0.5oC. The agitation rate of the basket was 50rpm.Ten millilitres (10 mL) samples were withdrawn up to 85 min. For each 10 mL withdrawn, 10 mL of phosphate buffer maintained at the same temperature was added to the dissolution medium. The drug content was determined spectrophotometrically at 278 nm using Unico Spectrophotometer model UV 2100 PC
(Shanghai instrument Co. Ltd China). In each of the dissolution profile study a pessary was wrapped in an artificial celloluse membrane as shown in figure 6.
Absorbances of withdrawn samples were converted to concentration from the Beer’s calibration curve of ciprofloxacin hydrochloride.
2.2.2.4: Drainage Experiment
The method of Onyechi et al (125) was adopted, with slight modification. The experiment lasted between 5-21 hours. The result is presented on Table 6.
A metallic stand (Figure 7) was constructed which allowed the Pig rectum to be mounted and maintained vertically in the stand. The intestine was attached to the metal support with cotton thread which was used to prevent the tissue from collapsing. The stand was suspended vertically in a high humidity environment
(Figure 7) and allowed to equilibrate in a hot air oven at a temperature of 39oC. The pessary under test was weighed and inserted at the upper end of the mounted Pig’s rectum and supported by office pins to prevent it from falling off. A Petri dish of known weight was placed beneath the tissue assembly to collect the molten and detached pessary mass. The whole set up was placed in a glass jar containing 300 ml of normal saline and left in the oven for an average time interval of 680 min. After 66 complete drainage the Petri dish and its content were dried and weighed. The percentage of pessary drained out was calculated using the formula below:
% Pessary drained out = (Wi-Wf/Wi)100 ------Eqn(14)
Where Wi is initial weight of pessary, Wf is the final weight of pessary
Percentage of pessary retained =(initial weight of pessary– weight of pessary drained)100 Initial weight of pessary ------Eqn (15)
67
Figure 6: Set up for the dissolution test for Ciprofloxacin Hydrochloride Pessary
68
Figure 7: Set up for the drainage test for Ciprofloxacin Pessary 69
2.2.2.5: Melting Point Determination
The various Batches of pessaries (1-8) were placed in fusion tubes with dimensions 1 cm diameter and 11 cm in length fabricated in the glass blowing section of the
Chemistry Department, University of Uyo, Uyo. The fusion tubes containing the different batches of pessaries were placed inside a thermostated water bath (Grant,
England). The melting point was taken as the temperature range at which the pessary just sintered and passed into solution.
2.2.3: Formulation of Ciprofloxacin Hydrochloride Vaginal Tablet
The composition of ciprofloxacin hydrochloride tablet is shown in Table1b. A modified wet granulation method was adopted. The tablet was formulated with a blend of Ciprofloxacin hydrochloride (10g) microcrystalline cellulose (16.75 g).The blend was granulated with a solution of 7.5 % polyvinyl Pyrrolidone in sufficient isopropyl alcohol. The wet mass was passed through a 2.00 mm stainless steel sieve and dried in a Genlab hot air oven at 60oC for 2 hours. The dried granules were passed through a 1.00 mm sieve. PEG 8000 (11.25 g), talc (0.30 g) and magnesium Stearate
(0.15 g) were added extra granularly to the screened granules and the final mixing was carried out in a powder bottle for 3 minutes. Compression was carried out in a single punch tablet press (Cadmach, Ahmedabad, India), fitted with 12.5 mm flat faced punches at low pressure to a hardness of 2 kg/f.
2.2.4: Evaluation of Vaginal Tablets
2.2.4.1 Weight Variation Test.
Twenty tablets selected at random were weighed using an electronic balance
(OHAUS, Galaxy).
70
Table 2: Composition of Ciprofloxacin Hydrochloride Vaginal Tablet Formulation
Ingredients (mg per tablet) Ciprofloxacin hydrochloride 200 Microcrystalline 375 Polyethylene glycol 8000 225 Polyvinyl pyrolidone (in ethanol) 7.5 Talc 3 Magnesium stearate 6
71
2.2.4.2: Hardness/Crushing Strength Test
Hardness of the vaginal tablet was determined using the Monsanto hardness tester
(Rolex, Chandigarh, India). Five tablets selected at random were tested.The mean and standard deviation were calculated.
2.2.4.3: Friability Test
The friability test was carried out using 10 tablets. A Roche friabilator (UNID
056830 Campbell Electronic, Mumbai, India)was used. The tablets were selected at random, dedusted and subjected to abrasive shock at 100 rpm for 4 minutes. The friability ‘F’ was calculated from Equation 3.
100(Wo-Wi------Eqn (16) Wo
Where, Wo is the initial weight of tablets, and Wi is the weight after abrasive shock.
2.2.4.4: Diameter and Thickness Test
The diameter and thickness of 10 tablets were measured using a micrometer screw gauge. The result was expressed as mean ± standard deviation.
2.2.4.5: Dissolution Profile of Ciprofloxacin Vaginal Tablets
The release profile of ciprofloxacin hydrochloride from the vaginal tablets was carried out in Erweka dissolution apparatus (DA – DD model) using the USP basket method.
The dissolution medium was 500 mL phosphate buffer (pH4) maintained at a temperature of 36± 0.5oC and agitation speed of 50rpm.Asample (10 mL) was withdrawn at 5 minute interval. For each 10 mL of phosphate buffer maintained at the same temperature was added to the dissolution medium. Absorbance of withdrawn samples were measured at 278 nm in a UV/visible spectrophotometer model UV
2100PC (Shanghai Instr. Co. Ltd China). Cumulative percentage drug released was 72 calculated using an equation obtained from the standard plot. The dissolution study was carried out with the tablet placed in an artificial cellulose membrane( figure 3)
Results obtained were fitted into zero order, first order, and the Higuchi square root kinetics and Korsemeyer-Peppas model to ascertain drug release mechanism.
2.2.4.6: Adhesion/Erosion Test
The vaginal tablets were subjected to the same experiment as described for pessaries.
The percentage bioadhesion and erosion were calculated from the equations 17 and 18 below;
Percentage bioadhesion =100 (No of tablet after bioadhesion experiment)-----Eqn (17) Initial number of tablets
Percentage erosion =100 (Initial weight –final weight) ------Eqn (18) Initial weight 73
Figure 8: Ciprofloxacin hydrochloride vaginal tablet 74
Figure 9: Dissolution of Ciprofloxacin Vaginal Tablet in a dialysis membrane 75
2.2.4.7: Bioadhesive Strength Determination
The Lecomte du Nouy tensiometer (model Nr 3124, A. Kruss Hamburg, Germany) was used for this study. A freshly excised pig vaginal epithelium with dimensions
11.5cm by 7.5cm was used for this study. The tissue was pinned unto the polyethylene support of the bioadhesive instrument placed on a metal support. The instrument was zeroed, and the bioadhesion of the clean plate determined in degrees, after the addition of 1µl of phosphate buffer pH 4.0 at the interface and leaving it for
10 minutes (103). A tablet was glued to the plastic plate using a cyanoacrylate adhesive. The tablet was then brought into contact with the everted mucous surface and 1µl of the phosphate buffer pH 4.0 added at the interface to activate the interaction. A contact time of 10 minutes was allowed for bioadhesive interaction to take place. The force required to remove the tablet was recorded in degrees and appropriate conversion to bioadhesive strength was obtained from the modified equation of Harkins and Jordan (130). Average of five determinations on a fresh vaginal epithelium was taken as the bioadhesive strength
T = (Mg/2L).F ------Eqn (19)
Where T is the tension equivalent to bioadhesive strength, M is the mass required to return the lever to zero position after each bioadhesion experiment, g is the acceleration due to gravity, F is the instrument constant and L is the area of the bioadhesive interface. The tablets were circular and flat faced. The bioadhesive interface, L, is the area of the tablet in contact with the mucus and is equivalent to the area (A) of one side of the tablet.
A = πr2 = π (D2/4) ------Eqn (20)
Where A is the area of one side of the tablet,
L from Equation 4 becomes: L = π (D2/4)------Eqn (21) 76
Therefore, T = [ Mg/(2 [ D2/4] )] . F ------Eqn
(22)
T = (2. mg/πD2). F------Eqn (23)
Where D is the mean tablet diameter.
Equation 23 was used to calculate the tension equivalent to the bioadhesive strength of the tablets.
77
Figure 10: Erosion/Bioadhesion test for Ciprofloxacin Hydrochloride Vaginal Tablet 78
2.2.4.8: Formulation of Ciprofloxacin Hydrochloride Vaginal Gel
The composition of Ciprofloxacin hydrochloride vaginal gel is shown in Table 2. The gel was prepared by triturating a mixture of ciprofloxacin hydrochloride (1%), sodium alginate (7 %) and methyl paraben (0.2 %) in a mortar.
Glycerol (7 %) was measured and placed in a beaker, the powder mix was added slowly with stirring to the beaker to form a smooth flowing liquid. The Distilled water
(84 mL) was added all at once and stirred gradually to avoid the incorporation of air bubbles.A portion of the prepared gel was filled into a collapsible tube while the remaining was stored in an airtight specimen bottle for further experiments.
2.2.4.9: Evaluation of Ciprofloxacin Hydrochloride Vaginal Gel.
2.2.5.0: Determination of the Organoleptic properties of the Ciprofloxacin Hydrochloride Vaginal Gel
The ciprofloxacin hydrochloride vaginal gel was examined visually for colour and the odour was perceived by smelling.
2.2.5.1: Determination of pH of Ciprofloxacin Hydrochloride Vaginal Gel
The vaginal gel (1g) was dispersed in 30 mL of distilled water and the pH was measured using a pH meter.
2.2.5.2: Measurement of the Consistency of Ciprofloxacin Vaginal Gel
The viscosity of the vaginal gel was measured at 37o±oC using a penetrometer
(isetamatic). The time lapse between each reading was 10 secs. The result is shown in able 9.
79
Table 3: Composition of Ciprofloxacin Hydrochloride Vaginal Gel
Ingredients Weight (%) Ciprofloxacin Hydrochloride 1.0 Sodium alginate 7 Polyhydric alcohol 7 Preservative 0.2-0.4 Vehicle to 100
80
2.2.5.3: Microbiological Assay of Ciprofloxacin Hydrochloride Vaginal Gel
2.2.5.4: Preliminary Sensitivity Test
The antibacterial activity of the vaginal gel was evaluated using the spread plate method (131) with Staphylococcus aureus (NCTC 6571), and Escherichia coli
(NCTC 10418) as the test organisms. A 0.1mL volume of standardized bacterial suspensions were aseptically spread plated on sterile nutrient agar (NA) plates, incorporated 1 mL of 50, 40, 30, 20 and 10 mg/mL of the gel preparation respectively.
The gel were solubilized with Tween 20, and incubated at 37oC for 24 hours for growth. After incubation the presence of growth denotes non-sensitivity of the organisms to the gel; or no potency; while the absence of growth denotes sensitivity of the test organisms to the gel, hence high potency. 81
2.2.5.5: Antibacterial Sensitivity Evaluation of Ciprofloxacin
Hydrochloride Vaginal Gel
The antibacterial potency of the ciprofloxacin hydrochloride (1%) gel was evaluated using the agar well diffusion method (131). on a 50 mg quantity of the vaginal gel was used.The gel was solubilized with Tween 80. Serially dilutions of 0.5, 0.25,
0.125, 0.0625, 0.03125, and 0.0156 mg/mL were made. A 0.1mL volume of standardized bacterial suspension were aseptically spread on different sterile nutrient agar plates. With a sterile cork-borer (4 mm in diameter) wells were aseptically bored on the previously seeded plates, after which 0.2 mL of the respective gel dilutions were introduced into the wells. The plates were then held at 4oC for 1 hour followed by incubation at 37oC for 24 hours. At the end of incubation, diameters of zones of inhibition were measured
2.2.5.6: Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Ciprofloxacin Hydrochloride Vaginal Gel
The MIC of the vaginal gel for each of the test organism was determined according to the macrodilution broth method (132-133). Serial dilutions of the vaginal gel (0.5,
0.25. 0.125, 0.0625, 0.03125 and 0.0156mg/ml) were made. To each of the test tubes concentrations of the anti bacterial gel, was added 0.1ml standardized bacterial suspensions and incubated at 37oC for 24 hours. Uninoculated nutrient broth served as control. The MICs of the gel for each of the bacteria was taken as the least concentration of the gel that inhibited the growth of the test organisms as measured by turbidity.
The MBC of the gel was determined from the 24 hour macrodilution broth MIC tubes with no visible growth (turbidity). The non-turbid MIC tubes were further incubated 82 for 48 hours. At the end of incubation, the appearance of growth (turbidity) in the non
– turbid MIC tubes, denoted the MBC of the gel against the organism (134). 83
CHAPTER THREE
3.0: Results and Discussion
3.1: Physical Properties of Ciprofloxacin Hydrochloride Pessary and Vaginal Tablet
The data of physical parameters like thickness, hardness, diameter, weight variation, friability are presented on Table 4. All the parameters except hardness and friability lie within acceptable limits (90). The average weight of the pessaries was 2.32 g and the weight variation for every batch was less than ± 7.5 %. The average weight of the tablets was 0.614 g and the weight variation in the batch was less than ± 5 %. The crushing strength of the tablet was between 1.92-2 kgf. The crushing strength was kept at this low level to ensure adequate swelling of the tablet and relatively fast release of the incorporated drug within the vagina epithelium.
The friability of the tablets was 8.05 %. This value though high was not considered to be deleterious to the dosage form. Vaginal preparations are usually enclosed in foils and other primary pages prior to packaging in a secondary container. Such products are not usually exposed to vigorous handling processes.
3.2: Erosion/Bioadhesion Test of Ciprofloxacin Pessaries and Vaginal Tablet
The result of the erosion/bioadhesion test of ciprofloxacin, hydrochloride pessaries and vaginal tablet is show in Table 5. From the result, batch 4 exhibited the highest percentage erosion. This was followed closely by batch 5. Batch 8 showed the least percentage erosion. All the batches except batch I exhibited percentage bioadhesion of
100 %.
The high value of percentage erosion for pessary containing Ac-Di-Sol® could be due to the super disintegrant property of this compound, resulting from its good water 84 uptake, rapid swelling properties and efficient fluid channeling on account of its long fiber length. (99). The least value of percentage erosion exhibited by Batch 8 pessaries could be attributed to the non-inclusion of other excipient that could have resulted in alteration in the hardness and dissolution time of the pessary which would affect the rate of erosion. The vaginal tablet showed 100% bioadhesion as shown on
Table 4b. It swelled when in contact with the buffer solution (pH 4.0). It exhibited a bioadhesive strength of 101.1Nm-1. This indicates that it could adhere to the mucous surface for a period of time long enough to release its medicament (106)
85
Table 4: Physical properties of Ciprofloxacin Hydrochloride Pessary and Vaginal Tablet
(a) Vaginal Pessary
Parameter Batches Mean + 1 2 3 4 5 6 7 8 SD Weight 2.34± 2.42± 2.21± 2.50± 2.26± 2.09± 2.51± 2.25± Variation 0.18 0.17 0.12 0.01 0.10 0.21 0.05 0.16 (g) n = 8
(b) Vaginal Tablets
Parameter (Mean + SD) Weight Thickness (mm) Diameter (mm) Hardness (kgf) Friability (%) Variation (g) (n=10) (n=10) (n=5) (n=10) (n = 20) 0.614 ± 0.20 5.53 ± 0.01 12.54 ± 0.01 1.92 ± 0.20 8.05 ± 0.20.
Key: Batch 1 = 80% PEG 4000
Batch 2 = 80% PEG 4000 + 0.1% Primogel®
Batch 3 = 80% PEG 4000 + 0.2 % Primogel®
Batch 4 = 80% PEG 4000 + 0.01 % Ac-Di-Sol®
Batch 5 = 80% PEG 4000 + 0.1 % Sterotex®
Batch 6 = 80% PEG 4000 + 0.2 % Sterotex®
Batch 7 = 80% PEG 4000 + 0.01 % Carbopol 971®
Batch 8 = 40% PEG 4000 + 40 % PEG 8000 86
Table 5: Erosion/Bioadhesion Test of Ciprofloxacin Pessaries and Vaginal Tablet
(a) Vaginal Pessary
Batch Erosion (%) Mean ± SD Bioadhesion (%) 1 22.15 ± 3.62 66.7 2 22.92 ± 7.31 100 3 29.73 ± 15.30 100 4 36.46 ± 8.10 100 5 35.75 ± 1.81 100
6 22.15 ± 3.62 100 7 21.00 ± 3.91 100 8 18.51 ± 3.40 100 Key: Batch 1 = 80 % PEG 4000
Batch 2 = 80% PEG 4000 + 0.1 % Primogel®
Batch 3 = 80% PEG 4000 + 0.2 % Primogel®
Batch 4 = 80% PEG 4000 + 0.01 % Ac-Di-Sol®
Batch 5 = 80% PEG 4000 + 0.1 % Sterotex®
Batch 6 = 80% PEG 4000 + 0.2 % Sterotex®
Batch 7 = 80% PEG 4000 + 0.01 % Carbopol 971®
Batch 8 = 40% PEG 4000 + 40 % PEG
(b) Vaginal Tablets
Batch Bioadhesion (%) Bioadhesion (Nm-1) 1 100 % 101.10
87
3.3: Drainage Experiment with Ciprofloxacin Hydrochloride Pessaries
The result of the drainage experiment is shown in Table 6. From this result, the pessary containing 0.01 % Ac-Di-Sol® (Batch 4) had the least percentage of pessary drained and the highest percentage of pessary retained on the tissue. This was followed by batch 7 which contain 0.01 % Carbopol 971® with values of 19.4 % as mean percentage drainage and 80.6 % as mean percentage retained. Batch 1 containing 0.1 % Primogel® exhibited the least percentage retained and the highest mean percent age drainage.
According to Onyechi et al (129) drainage technique is used in bioadhesion studies as an aid to develop sustained release bioadhesive formulation. Ac-Di-Sol® is a super disintegrant, and possesses outstanding water wicking capabilities and its cross-linked chemical structure creates an insoluble hydrophilic absorbent material which results in good swelling properties (100). Its super disintegrant properties could have been responsible for the spreading of the pessary on the tissue thus resulting in very low percentage drainage.
Carbopols usually exhibit good bioadhesive properties largely because they contain carboxy groups which ensure good hydration and strong interaction, which may require greater force to detach the polymer film from a mucus surface. This property provides an explanation for the increased percent retention of pessary containing
Carbopol 971 on the tissues (128).
3.4: Melting Point Determination
The result of the melting point determination is show on Table 7. From the result, the melting point of all the pessary batches fell within 38 and 42oC respectively. This 88 makes room for easy handling since the melting point range is above room temperature. And since PEGs pessaries are usually formulated to dissolve rather than melt at body temperature, this provides more prolonged release than other bases like theobroma oil (124).
3.5: Some Release Parameter of Ciprofloxacin Pessaries
The release rate of ciprofloxacin hydrochloride from the pessaries was analysed on the basis of time taken for 50% and 70% of the drug to be released (T50 and T70 as shown on Table 8. Batch 1 exhibited the slowest rate of release this could possibly result from the hydrophobic nature of polyethylene 4000 since there was no additive which must have retarded the rate of release of ciprofloxacin hydrochloride from the pessary. Batch 3 exhibited the fastest release rate, releasing 50 % and 70 % of the drug at 6.6 and 9.5 minutes respectively. This is because, this batch contains primojel which is a super disintegrant (97) and must have conferred fast disintegration time and hence fast dissolution of the pessary to enhance the fast release of the drug.
3.5.1: Kinetics and Mechanism of Release for Ciprofloxacin Hydrochloride Pessary
The data obtained were fitted into different kinetic models viz: zero order, first order
Higuchi and Korsemeyer Equations. The results shown on Table 9 indicated that,
Batch I exhibited zero order release kinetics as indicated by high regression value (r2
=0.9889), as well as Higuchi kinetics regression value (r2 =0.9900). In Batch 3, zero order, first order and Higuchi release models were operational,(r2=0,9226,0.9210 and
0.9657), drug release from Batch 4 followed Higuchi kinetics (r2=0.9878 )which describes the release of drugs from an insoluble matrix as a square of time dependent process based on Fickian diffussion , as well as zero order kinetics (r2 = 0.7625). The zero order rate describes systems where rate of drug release is independent of con 89 centration, in order to confirm the exact mechanism of drug release from these pessaries the data obtained were fitted into Korsemeyer Equation (124).Batch 5 followed zero order release kinetics (r=0.7625) while Batch 6 followed the Higuchi and zero order release kinetics respectively (r2=0.9878,0.9961). The kinetics of drug release from Batch 7 predominantly followed the Higuchi model. Regression analysis was performed and regression values r2, were 0.7489-0.9946 for different formulations. Slope value of 0.5
3.5.2: Some Release Parameters of Ciprofloxacin Hydrochloride Vaginal Tablets
The release rate of ciprofloxacin hydrochloride from the vaginal tablet was analysed on the basis of time taken for 50 % and 70 % of the drug to be released (t50 and t70).
The result obtained showed that 50 % of the drug was release at 1.3 minutes while 70
% was released at 5.0 minutes. Traditional delivery systems are characterized by immediate and uncontrolled drug release kinetics (127). The vaginal tablet release the drug at an uncontrolled rate as depicted by the results.
3.5.3: Kinetics and Mechanism of Release for Ciprofloxacin Hydrochloride Vaginal Tablets
The dissolution data was fitted into zero-order, first order, Higuchi and Korsemeyer models. The regression values for zero order, first models were 0.9032, 0.691, 0.8807 and 0.8164 respectively. From the regression values, the release of CH followed zero order release kinetics. In order to confirm the exact mechanism of drug release from the tablets the data was fitted into the Korsemeyer Equation (126). The ‘n’ value 90 obtained was 1.0 which implies that the predominant mechanism of release is anomalous non-Fickian transport.
Table 6: Result of Drainage Experiment with Ciprofloxacin Hydrochloride Pessary
Batch Mean % Mean % Retained Mean drainage Time Drainage (mm) 1 66.9 33.1 ± 3.81 680 2 25.2 74.8 ± 14.61 680 3 55.0 45.0 ± 22.70 680
4 11.5 88.5 ± 6.65 680 5 32.7 67.3 ± 4.48 680 6 19.8 80.3 ± 20.82 680
7 19.4 80.6 ± 12.62 680 8 26.4 73.6 ± 18.43 680
Key: Batch 1 = 80 % PEG 4000 Batch 2 = PEG 4000 + 0.1 Primogel®
Batch 3 = 80% PEG 4000 + 0.2 % Primogel®
Batch 4 = 80% PEG 4000 + 0.01 % Ac-Di-Sol®
Batch 5 = 80% PEG 4000 + 0.1 % Sterotex®
Batch 6 = 80% PEG 4000 + 0.2 % Sterotex®
Batch 7 = 80% PEG 4000 + 0.01 % Carbopol 971®
Batch 8 = 40% PEG 4000 + 40 % PEG 8000
91
Table 7: Melting Point Determination
Batch Melting Point Range (oC) 1 38-40 2 38-42 3 38-42 4 38-42 5 38-40 6 38-40 7 38-40 8 38-40
Key: Batch 1 = 80 % PEG 4000
Batch 2 = PEG 4000 + 0.1 % Primogel®
Batch 3 = 80 % PEG 4000 + 0.2 % Primogel®
Batch 4 = 80 % PEG 4000 + 0.01% Ac-Di-Sol®
Batch 5 = 80 % PEG 4000 + 0.1 % Sterotex®
Batch 6 = 80 % PEG 4000 + 0.2 % Sterotex®
Batch 7 = 80 % PEG 4000 + 0.01 % Carbopol 971®
Batch 8 = 40 % PEG 4000 + 40 % PEG 8000
92
Table 8: Some Release parameters of Ciprofloxacin Pessaries
Batch T50 (Min) T70 (Min) Cmax (%) 1 27.0 45 100 2 11.2 13 100 3 6.6 9.5 100 4 11.5 20 100 5 7.2 95 100 6 8.0 20 100 7 9.5 22.5 100 8 9.5 19.5 100
Key: Batch 1 = 80% PEG 4000
Batch 2 = 80 % PEG 4000 + 0.1 % Primogel
Batch 3 = 80 % PEG 4000 + 0.2 % Primogel
Batch 4 = PEG 4000 + 0.01 % Ac-Di-Sol
Batch 5 = 80 % PEG 4000 + 0.1 % Sterotex
Batch 6 = 80 % PEG 4000 + 0.2 % Sterotex
Batch 7 = 80 % PEG 4000 + 0.1 % Carbopol 971®
Batch 8 = 40 % PEG 4000 + 40 % PEG 8000 93
Table 9: Kinetics and mechanism of Release for Ciprofloxacin Hydrochloride Pessaries.
Batch Zero- First- Higuchi Korsmeyer N order order 1 0.9889 0.7928 0.9900 0.9946 0.65 2 0.9276 0.8903 0.9127 0.9210 0.98 3 0.9226 0.9210 0.9657 0.9576 0.74 4 0.9961 0.7775 0.9878 0.9825 0.50 5 0.7625 0.7292 0.7750 0.7849 0.58 6 0.9612 0.6877 0.9576 0.9599 0.61 7 0.9097 0.6460 0.9098 0.9219 0.52 8 0.9730 0.8425 0.9796 0.9757 0.64
Key: Batch 1 = 80 % PEG 4000
Batch 2 = 80 % PEG 4000 + 0.1 % Primogel
Batch 3 = 80 % PEG 4000 + 0.2 % Primogel
Batch 4 = PEG 4000 + 0.01 % Ac-Di-Sol
Batch 5 = 80 % PEG 4000 + 0.1 % Sterotex
Batch 6 = 80 % PEG 4000 + 0.2 % Sterotex
Batch 7 = 80 % PEG 4000 + 0.1 % Carbopol 971®
Batch 8 = 40 % PEG 4000 + 40 % PEG 8000 94
d e s a e l e r
%
Time (Min)
Figure 11 Release profile of Ciprofloxacin Hydrochloride Pessaries Batches 1-4 in Phosphate buffer (pH 4.0)
Batch 1 Batch 2 B atch 3 Batch 4
95
d e s a e l e r
%
Ti me (Min)
Figure 12: Release profile of Ciprofloxacin Hydrochloride Pessaries Batches 5-8 in Phosphate buffer (pH 4.0)
Batch 5 Batch 6 Batch 7 Batch 8 96
Figure 13: Release profile of Ciprofloxacin Hydrochloride Vaginal Tablet in Phosphate buffer (pH 4.0)
97
3.6: Physicochemical Properties of Ciprofloxacin Vaginal Gel
3.6.1: Organoleptic Properties of Ciprofloxacin Vaginal Gel
The ciprofloxacin vaginal gel was light yellow in colour with a characteristic odour.
It had a pH of 6.67 and a consistency of 115.4 Cp as shown on Table 10. The pH is slightly acidic and it could be applied successfuly in the female genital tract for the treatment of infections caused by susceptible microorganism. Beyond this pH, CH, a weakly acidic drug precipitates. The consistency which was above 100 Cp will prevent the leakage of the gel from the female genital tract thereby preventing staining of underwears (7).
98
Table 10: Physicochemical Properties of Ciprofloxacin Vaginal Gel
Parameters Colour Light yellow Odour Characteristic pH 6.67 Viscosity 115.40 ± 0.77 Centipoise
Table 11: Microbiology Assay (Tube Dilution Test)
Table 11a: Minimum Inhibitory Test (24 Hours)
Test organisms Concentration (mg/ml) 0.5 0.25 0.125 0.0625 0.03125 0.0156 Escherichia - - - - - + coli Staphylococcus - - - - + + aureus + (Growth); MIC: E. coli = 0.03125mg/ml
- (No growth) Staph. aureus = 0.0625mg/ml
99
3.7: Microbiology Assay of Ciprofloxacin Hydrochloride Gel. (Tube Dilution Test)
3.7.1: Minimum Inhibitory Test (24 Hours) and Minimal Bactericidal Concentration (MBC) (48hrs)
The result of the minimum inhibitory test is presented on Table 11a. From the result the MIC for Escherichia coli was 0.03125 mg/mL, while that of Staphylococcus aureus was 0.0625 mg/mL. This indicates higher activity of the gel against infections caused by E. coli. than those caused by Staph.aureus
3.7.1.1: Minimum Bactericidal Concentration (48 Hours)
The results showing the MBC of ciprofloxacin hydrochloride vaginal gel is presented in Table 11b. From the results the MBC of E. coli was 0.0625 mg/mL, while that of
Staph aureus was 0.125 mg/mL.
Minimum inhibitory concentrations MICs are considered the gold standard for determining the susceptibility (131) of organisms to antimicrobials and are therefore used to judge the performance of all other methods of susceptibility testing. The range of antibiotic concentrations used for determining MICs is universally accepted to be in doubling dilution step up and down from 1 mg/L as required (131). According to literature (44,53) the suggested MIC values for standard ciprofloxacin powder is given as 0.004-0.500 mg/mL, for Staph aureus and E. coli respectively. The observed values fell within the accepted limits, and therefore shows that ciprofloxacin gel has significant activity against E. coli and Staphylococcus aureus. The MIC values gives indication of the susceptibility or the resistance of a bacteria strain to a given antibiotic. A value of < 1 mcg/mL is being interpreted as susceptible, a value of 2 mcg/mL mean intermediate activity, and a value of > 4 is being interpreted as resistant. From the results in Table-10a, Escherichia coli and Staph. aureus are both 100 susceptible to ciprofloxacin hydrochloride. The minimum bacterial concentration
(MBC) generally does not exceed the MIC by more than a factor of 2 (132). The result shown conforms to this standard as the difference between the MIC and MBC in this case is less than 2. (MBC for E. coli and Staph aureus are 0.0625 mg/mL and
0.125 mg/mL respectively).
3.7.2: Agar-Well Diffusion
This result is show in Table – 11c. For the diffusion technique, the expected inhibition zone diameters for both E. coli and Staph. aureus are 30-40 mm and 22-30 mm respectively (133).
From the result, the inhibition zone diameter is seen to increase with increase in concentration of the test drug, and the results falls within the accepted limits, with maximum inhibition (37.0 mm) against E. coli and minimum (12.0 mm) against
Staphylococcus aureus. Ciprofloxacin hydrochlorides showed marked activity against
E. coli based on the zone of inhibition and MIC values (134). This is in accordance to what was stated in the literature (134). 101
Table 11b: Minimum Bactericidal Concentration (48 hours)
Test organisms Concentration (mg/mL) 0.5 0.25 0.125 0.0625 0.03125 0.0156 Escherichia - - - - + + coli Staphylococcus - - - + + + aureus + (Growth); MIC: E. coli = 0.0625 mg/mL
- (No growth) Staph. aureus = 0.125 mg/mL
Table 11c: Agar-Well Diffusion
Organism Staph. aureus Esherichia coli Concentration Inhibition zone Concentration Inhibition zone (mg/ml) diameter (mm) (mg/mL) diameter (mm) 0.5 21.0 0.5 37.0 0.25 15.0 0.25 35.0 0.125 12.0 0.125 33.0 0.0625 - 0.0625 25.0 0.03125 - 0.03125 21.0 0.0156 - 0.0156 19.0
102
CHAPTER FOUR
4.1: Summary and Conclusion
The physical properties of both ciprofloxacin pessaries and vaginal tablets were within the acceptable limits except hardness (1.92-2 kgf) and friability (8.05 %). All batches of the pessaries except Batch1 exhibited 100 % bioadhesion to the Pig’s intestine. Batch 4 containing 0.01 % AC-Di-Sol shown the highest percent erosion, followed by Batch 6 containing 0.01 % Sterotex while Batch 8 containing 40 %
Carbopol 971 exhibited the least percentage erosion. Batch 4 exhibited the least percent drainage, with a resultant highest percentage of pessary retained on the tissue, while Batch1 had the highest value of percentage drainage and hence the lowest value for pessary retained on the tissue.
The melting point range for all the batches of pessaries lied between 38-42oC.
Batch 1 containing no additive showed the fastest rate of release at t50 and t70 while
Batch 3 containing 0.2 % Primojel exhibited the slowest release rate at the same time interval. The Ciprofloxacin vaginal gel was light yellow in colour with a characteristic odour. It was slightly acidic with a consistency of 115.4 Cp. The MIC of the vaginal gel for E. coli and S. aureus were 0.03125 mg/mL and 0.0625 mg/mL respectively, while the MBC value were 0.625 mg/L and 0.125 mg/mL for E. coli and
S. aureus respectively. The zone of inhibition for both E. coli and S. aureus were between 19.0-37.0 mm for E. coli and 12.0-21.0 mm for S. aureus. It is evident from this study that Ciprofloxacin hydrochloride has more activity against E. coli than S. aureus. The results of the study has shown that formulation of ciprofloxacin hydrochloride into pessaries, vaginal tablets and vaginal gel could be utilized to 103 maintain adequate vaginal hygienic in women and for the treatment of vaginal infections caused by susceptible micro-organisms.
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APPENDICES
Appendix 1: Weight Variation of Ciprofloxacin Hydrochloride Pessaries, Batches 1-8
Batch Weight variation 1 2.55 2.15 2.44 2.43 2.52 2.30 2.33 2.02
2 2.52 2.42 2.51 2.52 2.55 2.06 2.25 2.49
3 2.24 2.39 2.20 2.06 2.21 2.31 2.34 2.12 2.02
119
4 2.46 2.50 2.56 2.42 2.62 2.50 2.48 2.52
5 2.39 2.30 2.17 2.39 2.20 2.06 2.33 2.26 2.25
6 2.30 2.30 2.00 2.46 2.48 2.02 1.90 2.42 2.00
7 2.42 2.52 2.54 120
2.48 2.60 2.54 2.54 2.52
8 2.33 2.45 2.17 2.46 2.29 2.14 1.97 2.20
121
Appendix 2: Weight Variation of Ciprofloxacin Vaginal tablets
S/N Weight (g) 1 630 2 607 3 611 4 622 5 600 6 612 7 627 8 629 9 603 10 631 11 608 12 615 13 641 14 580 15 605 16 630 17 595 18 610 19 617 20 607
122
Appendix 3: Friability of Ciprofloxacin Hydrochloride Vaginal Tablet
S/N Friability (%) 1 7.6 2 8.5 3 8.2 4 7.6 5 8.3
Appendix 4: Diameter of Ciprofloxacin Vaginal Tablet
S/N Diameter (mm)
1 12.53
2 12.57
3 12.53
4 12.55
5 12.54
6 12.53
7 12.53
8 12.55
9 12.57
10 12.53
123
Appendix 5: Thickness of Ciprofloxacin Vaginal Tablet
S/N Thickness (mm) 1 5.528 2 5.530 3 5.528 4 5.530 5 5.527 6 5.527 7 5.528 8 5.530 9 5.530 10 5.528
Appendix 6: Crushing strength of Ciprofloxacin Hydrochloride Vaginal Tablet
S/N Hardness (kgf) 1 1.9 2 2 3 2 4 1.8 5 1.9
124
Appendix 7: Release of Ciprofloxacin Hydrochloride from Pessaries Batches 1-8 in Phosphate Buffer (pH 4.0)
Batch Time(min) Cumulative % release 1 2 10.2 5 15.6 10 24.0 15 32.5 20 38.9 25 46.9 30 53.1 35 59.2 40 64.4 45 70.4 50 74.2 55 78.7 60 83.4 65 88.9 70 95.9 75 100
2 5 28.2 10 40.1 15 88.6 20 100
3 5 36.2 10 73.2 15 88.6 20 100
4 2 24.6 5 35.0 125
10 46.9 15 58.1 20 69.8 25 80.2 30 90.4 35 100
5 5 27.0 10 73.4 15 61.3 20 62.5 25 73.7 30 94.3 35 100
6 5 38.9 10 53.6 15 54.9 20 70.5 25 77.6 30 77.7 35 85.7 40 100.0
7 5 32.9 10 51.6 15 65.2 20 59.6 25 79.2 30 77.7 35 100
126
8 5 29.2 10 51.6 15 53.3 20 72.4 25 818 30 93.5 35 100
127
Appendix 8: Release of Ciprofloxacin Hydrochloride from Vaginal Tablet in Phosphate Buffer (pH 4.0)
Time(mn) Cumulative % release 2 66 5 73.7 10 75.8 15 76.2 20 77.4 25 78.2 30 78.4 35 79.5 40 85.0 45 84.9 50 85.3 55 85.3 60 85.1 65 92.8 70 96.1 75 95.6 80 89.4 85 100
128
Appendix 9: Erosion/Bioadhesion Test of Ciprofloxacin Hydrochloride Pessaries. Batches 1-8
Batch Initial Final Bioadhesion (%) Erosion (%) Time (min) Weight (g) Weight (g) 1 2.55 - 66.7 - 2.43 1.98 100 18.5 65 2.44 1.82 100 25.8 65
2 2.53 1.70 100 32.8 66 2.50 1.98 100 20.8 66 2.03 1.72 100 15.3 66
3 2.44 1.19 100 51.2 60 2.22 1.74 100 21.6 60 2.20 1.84 100 16.4 60
4 2.48 1.27 100 51.2 60 2.64 1.65 100 21. 60 2.47 1.90 100 16.4 60 5 2.35 1.45 100 38.3 45 2.15 1.40 100 34.9 45 2.20 1.45 100 34.1 45
6 2.20 2.10 100 45.4 85 2.05 1.92 100 63.4 85 1.82 1.70 100 65.9 85
7 2.52 1.85 100 26.6 50 2.51 2.03 100 19.1 50 2.58 2.13 100 17.4 50
8 2.28 1.76 100 22.8 41 2.16 1.72 100 18.5 41 2.50 2.14 100 44.4 41
129
Appendix 10: Result of Drainage Experiment of Ciprofloxacin Pessaries
Batch Weight Weight % Mean % Drainage Mean of of pessary retained time (min) drainage sample sample retained time (min) (g) drained on tissue out(g) 1 2.55 1.95 23.529 2.6 ± 3.81 1,440 680
2. 48 1.89 23.790 300
2.52 1.72 31.746 300
2 2.52 1.49 40.873 2.6 ± 14.6 1,440 680
2.51 0.67 97.211 300
2.52 0.34 86.507 300
3 2.24 1.95 12.946 45 ± 22.7 1,440 680
2.31 0.90 61.038 300
2.34 0.92 60.683 300
4 2.45 0.48 80.408 88.5 ± 6..65 1,440 680
2.47 0.08 96.761 300
2.47 0.28 88.663 300
5 2.39 0.09 62.343 67.3 ± 4.48 1,440 680
2.39 0.64 73.221 300
2.33 0.78 66.523 300
6. 2.30 1.13 50.869 80.3 ± 20.8 1,440 680
2.30 0.09 96.086 300
2.24 0.14 94.238 300
7 2.54 0.09 96.456 80.6 ± 12.6 1,440 680
2.54 0.88 65.354 300
2.52 0.50 80.158 300
8 2.33 0.09 96.137 73.6 ± 18.4 1,440 680
2.45 1.20 51.020 300 130
2.47 0.65 73.684 300
Appendix 11: Determination of Viscosity of Vaginal Gel
S/N Consistency (Centipoises) 1 115 2 116 3 114 4 116 5 115
Appendix12: Preparation of Dissolution Medium
The sodium hydrogen phosphate (5.04g) and potassium dihydrogen phosphate 3.01g were dissolve in sufficient water to produce 1000ml and the pH was adjusted to 4.0 with glacial acetic acid. 131
Figure 14: Spectrum of Ciprofloxacin Hydrochloride at max 278 nm 132
Figure 15: Beer’s Plot for Ciprofloxacin Hydrochloride at 278 nm.