ABSTRACT
The solubility of gentamicin in various formulation vehicles (oils, surfactants and co-surfactants) was determined using the shake flask method. Emulsifying ability of the surfactants for the selected oil was screened. Different SNEF prototypes were developed following the construction of ternary phase diagrams using water titration method. The effect of drug and other additives on the ternary phase diagrams were studied and the selected formulations were optimized. Optimized formulations were characterized by weight uniformity of their capsule shells and visual assessment of their self nanoemulsification. The optical clarity and robustness to dilution of the SNEFs were evaluated. Emulsification time, droplet size, zeta potential, polydispersity index and Fourier transform-infrared spectroscopy were measured. SNEFs were also characterized by scanning electron microscopy (SEM). Apparent viscosities, absolute drug content and drug content efficiencies were determined. Stability studies and octanol/water partition coefficient were evaluated, while in vitro antibacterial studies of the SNEFs and permeation studies were carried out. In vitro anti-pneumococcal study of the SNEFs against S. pneumoniae was done followed by extrapolation of their minimum inhibitory concentrations (MICs). In vivo anti-pneumococcal studies of gentamicin released in the sera and CSF of white albino rats were evaluated. Haematological studies were carried out to measure packed cell volume (PCV), total white blood cells (WBC), red blood cells (RBC) and haemoglobin (Hb) concentrations. Biochemical studies were performed to evaluate alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin and creatinine concentrations. This was followed by histopathological examinations of the brain, kidney and liver of the animals. Results were expressed as mean ± SD. ANOVA and student t-tests were performed on the data sets. Ternary phase plots were analyzed using SigmaPlot® 11.0. Permeation calculations were performed with a special Microsoft excel programme. Differences were considered significant for p values < 0.05. Gentamicin produced maximum solubility in soyabean oil, Kolliphor® EL, Kolliphor® P188, and in Transcutol® HP. PEG 4000 and gentamicin reduced the area of nanoemulsion formation in the ternary phase diagrams for the selected systems. The emulsion droplet size was in the nanometer scale. The SNEF capsules had uniform average weight of 300 mg ± 0.7. The SNEFs had good optical clarity with percentage transmittances above 50 % and showed low propensity to drug precipitation, and exhibited rapid emulsification rate (17 – 40 s). FTIR revealed that the structure of gentamicin remained completely intact in all the formulations. SEM micrographs showed smooth and spherical globules. Rheological studies showed decrease in apparent viscosities with increase in shearing speed. Drug content ranged from 36.3 – 99.8 %. Stability studies suggest that the SNEFs were relatively stable over 4 months. Octanol/water partition coefficient ranged from 0.38 – 1.46. In vitro antibacterial studies showed susceptibility in the order: K. pneumonia > E. coli > S. aureus > B. subtilis. In vitro permeation studies of showed overall extended permeation of gentamicin. In vitro anti-pneumococcal study showed MICs of 2.5 – 5 mg/ml. In vivo anti-pneumococcal study of gentamicin in sera suggests that sera from animals administered with batch C (3:1 w/w) gentamicin SNEFs at 7 mg/kg produced good inhibition of the bacteria. The in vivo anti-pneumococcal activity of gentamicin in CSF showed rapid establishment of a biocidal concentration after 30 min. Haematological studies showed increase in PCV, RBC and Hb counts while WBC count and its differentials decreased. Biochemical studies showed decreased ALP and varying AST, ALT, bilirubin and creatinine concentrations. Histopathological findings showed dominant astrocytosis of the brain for gentamicin-loaded SNEFs indicating a breach of the integrity of the BBB.
CHAPTER ONE
INTRODUCTION
Over the past years, the fraction of new drug products that are new chemical entities has steadily decreased, reflecting the tremendous cost required to bring new drugs to the marketplace. Increased understanding of drug metabolic and toxicological factors, such as the effect of the patient age on drug distribution, the genetic factors that may result in dramatic intersubject variability in metabolism, short-term versus long-term exposure toxicities, and the potential for teratogenic, mutagenic and embryotoxic effects, has increased the scrutiny under which governmental agencies view the chemical entity. This careful inspection is intended to minimize the possibility of toxic reaction(s) and to demonstrate the safety and efficacy of new drug products. The regulatory process has also resulted in significantly more costly and time-consuming testing prior to commercialization. This increased emphasis on safety has placed an additional burden on those who are involved in the development of new drugs, while increasing financial pressures have led to the need for decreased development time. The investigation of approved drugs has resulted in enhanced patient safety and therapeutic efficacy by directing research efforts toward the more efficacious delivery of known pharmacologically active agents to the appropriate physiologic site. This trend has caused pharmaceutical researchers to seek the most suitable methods to deliver both new and existing compounds in the most pharmacologically appropriate manner. The methods may be designed to optimize bioavailability, minimize toxicity and side effects, and improve stability.
1.1 Drug delivery systems
A drug delivery system (DDS) is defined as a formulation or a device that enables the introduction of a therapeutic substance in the body and improves its efficacy and safety by controlling the rate, time, and place of release of drugs in the body [1]. Therefore, DDS, which include particulate carriers, composed primarily of lipids and/or polymers, and their associated therapeutics, can be used to improve the pharmacological properties of many conventional drugs. DDS are designed to alter the pharmacokinetics (PK) and biodistribution (BD) of the associated drugs, or to function as drug reservoirs (i.e. as sustained release systems) or both [2]. The concerned pharmacokinetic parameters are absorption, distribution, metabolism and excretion. The nature of conventional therapeutics, especially their low molecular weight, confers on them the capacity to cross various body compartments and access numerous cell types and subcellular organelles. Thus, these drugs are suitable for the treatment of diseases. However, this form of indiscriminate distribution leads to the occurrence of side effects and to the need for higher doses of the drug to elicit a satisfactory pharmacological or therapeutic response. Rapid renal clearance as a result of the low molecular weight of these compounds, among other factors such as protein binding, lipophilicity, ionizability, etc, implies frequent administration and/or a high dose to achieve a therapeutic effect. Formulation scientists are involved in serious researches into new formulations that ensure a greater pharmacological response, which in turn, would lead to lower doses and therefore the minimization of side effects. Thus, it is necessary to improve the bioavailability of drugs. Bioavailability of drugs is affected by several factors including the physical and chemical characteristics of the drug, the dose and concentration, the frequency of dosing, and the administration route.
Active drugs are not administered alone but in dosage forms that generally include other substances called excipients. The latter are added to formulations in order to improve the bioavailability and the acceptance of the drug by patients. Excipients used include emulsifiers, dyes, lubricants, diluents, chemical stabilizers, etc. These substances were initially considered inert because they do not exert therapeutic action nor do they modify the biological action of a drug. However, it is currently upheld that excipients influence the speed and extent of drug absorption, and therefore, the pharmaceutical form of these substances affects drug bioavailability. In the light of this reality, there has been an intense increase in research on the modification of drug release and absorption. The development of new drug delivery systems (NDDS) will offer additional advantages to drugs currently in use. They may also allow an extension of patent protection for an active pharmaceutical ingredient (API). They will facilitate more patient-friendly administration, thus resulting in patient increased compliance and satisfaction. Since human health is continuously threatened by autoimmune, neurodegenerative, metabolic and cancer diseases, etc, which are very difficult to treat with systemically delivered drugs, NDDS seek to improve the pharmaceutical profile and stability of a drug, ensure its correct concentration, achieve maximum biocompatibility, minimize side effects, stabilize the drug in vivo and in vitro, facilitate the accumulation of the drug at a specific site of action, and increase exposure time in the target cell [3].
