Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN. / CHAIRMAN: GORDON L. AMIDON.

Thesis (Ph. D.)--University OF MICHIGAN. / CO-CHAIRMEN: GORDON L. AMIDON; GORDON L. FLYNN.

Tea consumption has been suggested to have beneficial effects for human health, especially in cancer prevention. At present, epidemiological evidence of the protective effect of tea consumption against the development of human cancer is not conclusive. Interpretation of epidemiological data and extrapolation of rodent data to humans are generally hampered by inadequate information on the bioavailability and pharmacokinetics of tea constituents. We have performed studies to determine the pharmacokinetics of green tea in humans after single and multiple oral dose administration of tea polyphenols and the contribution of hepatic first-pass elimination to the low oral bioavailability of green tea catechins in animals. EGCG was present in the systemic blood in the unchanged form in humans after oral administration of two green tea polyphenol products, EGCG and Polyphenon E (a mixture of major green tea polyphenols). Oral administration of EGCG and Polyphenon E resulted in similar systemic exposure of EGCG. EGC and EC were present in glucuronic acid/sulfate conjugates in blood and urine samples after the Polyphenon E administration. Large inter-subject variations in the systemic levels of green tea catechins were observed following oral administration of green tea polyphenols. We found that it is safe for healthy human subjects to take green tea polyphenols for four weeks in amounts equivalent to those contained in 8 to 16 cups of green tea once a day or in divided doses twice a day. Systemic availability of EGCG increased more than 60% after chronic green tea polyphenol administration at high doses once a day. Oral administration of green tea polyphenols at the selected doses and dosing schedules did not elicit overall changes in the selected pharmacodynamic measurements. Oral bioavailability of green tea catechins was demonstrated to be low in animals and possibly in humans. Based on our pre-clinical study, we found that first-pass hepatic elimination of green tea catechins didn't play a significant role in the presystemic elimination of orally administered catechins. Factors within the gastrointestinal tract such as limited membrane permeability, transporter mediated intestinal secretion, or gut wall metabolism may contribute more significantly to the low oral bioavailability of green tea catechins.

Epigallocatechin gallate (EGCG) is a potent polyphenoclic antioxidant extracted from green tea. Due to its antimutagenic and antitumor activities it is a promising candidate for use in topical formulations for skin cancer prevention. The overall objective of this dissertation was to develop a formulation suitable for the topical delivery of (EGCG). A high-performance liquid chromatography (HPLC) assay was developed to evaluate the possible degradation of EGCG from the various solvents used to incorporate it into topical formulations in the preformulation studies. This method was validated to establish linearity, precision, and accuracy and also was successfully applied to determine the stability of the finished formulations. First, the stability of EGCG in 0.05 M aqueous buffers in the pH range 3-9 was evaluated. The results indicated that the degradation of EGCG in aqueous media is strongly dependent on its storage conditions. EGCG exhibited its highest stability at low pH and 4°C. Stability studies of EGCG in organic solvents indicated that the degradation of EGCG can be reduced significantly. Furthermore, it was found that the addition of EDTA and butylated hydroxytoluene (BHT) resulted in an even greater stability of EGCG. Next, different types of polymers were investigated for their ability to form non-aqueous gels with glycerin, the solvent found most suitable for enhancing EGCG's stability. Methacrylic acid derivatives (Carbopols RTM) were found to be the most efficacious gelling agents. Carbopol RTM 974, best known for its mucoadhesive properties, was found to produce the highest viscosity. In the last phase of the study EGCG was incorporated into the anhydrous formulations. The in vitro release characteristics were then evaluated using a Franz-type diffusion cell system. The results indicated that the release of EGCG obeys the Higuchi equation. Furthermore, it was found that a stable EGCG formulation in glycerin can be developed.

This dissertation deals with two major topics: allometric (or interspecies) scaling of pharmacokinetic parameters, and the interaction of cocaine and ethanol. These topics are tied together by the inclusion of allometric analyses of both cocaine and ethanol. Currently, to make initial predictions of human pharmacokinetic parameters using allometry, allometric slopes of 0.75, 1.0, or 0.25 (for clearance, volume, and half-life, respectively) are applied to the appropriate parameter value measured in a single species. Chapter 1 demonstrates the validity of this practice, and, using literature data on many diverse compounds, suggests that the monkey is the best species for predicting human data. Chapter 2 uses allometric principles to show that hepatic extraction ratio (ER) is independent of body weight, and thus constant across species. This is due to the fact that ER is the ratio of two parameters with identical allometric slopes, hepatic clearance and blood flow. Chapter 3 demonstrates that cocaine can be scaled using allometric relationships, leading to reasonably accurate predictions of the parameters in humans. Chapter 4 shows that the pharmacokinetic parameters of ethanol can be scaled allometrically. This was previously thought to be impossible, since ethanol undergoes saturable elimination, and as a result, the pharmacokinetic parameters (clearance and half-life) are dose-dependent. By scaling other parameters that are dose-independent, such as the Michaelis-Menten parameters, predictions of human concentration-time plots can be simulated. The co-administration of cocaine and ethanol is shown to cause a superadditive response. This is a result of two mechanisms: ethanol inhibits cocaine clearance, thus increasing cocaine concentrations, and, an active metabolite, cocaethylene, whose effects are similar to that of cocaine, is formed in vivo after co-administration of the two drugs. Because this drug combination is so common, and because of the resultant increased risk of toxicity and death, it is important to understand how much cocaethylene is formed after co-administration of cocaine and ethanol. Chapter 5 outlines a procedure used to calculate the fraction of cocaethylene formed after co-administration of the two drugs. Chapter 6 then summarizes the pharmacokinetic-pharmacodynamic models of cocaine given alone, compared to the combination.

The current work focuses on the development and evaluation of techniques and models that can facilitate the development of solution based metered dose inhalers. These include an online reverse phase hplc method for analyte quantitation from propellant based pressurized metered dose inhalers. The technique ( direct injection method) finds applications in determining solubility of compounds in aerosol propellants and can possibly be used for stability analysis. With the development of this technique it would be feasible to generate a solubility database in order to understand the physico-chemical factors affecting the solubility and also possibly predict the solubility of compounds in HFA 134a propellant. The regular solution theory based on solubility parameter approach was evaluated for this purpose by utilizing a set of 35 diverse compounds in HFA 134a. A new product performance evaluation tool; the Model 3320 series Aerodynamic Particle Sizer (APS) used in conjunction with Model 3320 Impactor Inlet was evaluated for analysis of solution metered dose inhalers. The Model 3320 APS series provides rapid aerodynamic size distribution data and coupled with Model 3306 Impactor Inlet allows for the chemical analysis of the Inlet port, 'respirable' mass and 'non-respirable' mass. It was shown that in order to obtain comparable results between the Model 3306 Impactor Inlet and the Andersen Cascade Impactor (ACI), an extension to the USP throat may be necessary. The solubility data generated by the direct injection method coupled with the 'respirable deposition' data generated using the APS 3320 series can be used to optimize the product performance of cosolvent based solution metered dose inhalers.

Disposition and oral bioavailability studies involving non-rodent animal models traditionally rely upon the dog to predict human pharmacokinetics and oral bioavailability values during the preclinical phase of drug development. However, differences in oral absorption parameters suggest that the dog, in general, may fail to reasonably predict human values. The Yucatan micropig is proposed and examined as an alternative animal model for such studies. The presence and distribution of four major cytochrome P450 (CYP) enzymes (1A2, 2C19, 2D6, and 3A4) was characterized along the small intestine of the Yucatan micropig. Distribution patterns of these enzymes were similar to humans, especially for CYP3A4. CYP1A2, 2D6, and 3A4 content were greatest in the duodenum-upper jejunum region while CYP2C19 content was relatively consistent throughout the length of the small intestine. The quantity of CYP3A4 enzymes present was substantially greater than any of the other enzymes examined. To examine the validity of using swine to predict human values, an oral bioavailability study was conducted in Yucatan micropigs using antipyrine as a model compound. Rate and extent of absorption of antipyrine in humans were better predicted using the Yucatan micropigs as an animal model than any other species (rat, monkey, dog). After establishing the utility of the Yucatan micropig for absorption studies to predict human values, the disposition and bioavailability of components in two botanicals, turmeric and ginger, were examined. Curcumin, a major active component of turmeric, was found to have a very short terminal half-life due to, in part, a high blood clearance based on data obtained from several in vitro studies. Bioavailability of curcumin varied depending on the formulation administered. Large amounts of a glucuronide metabolite were detected after oral administration of curcumin indicating substantial pre-systemic metabolism. Unlike curcumin, 6-, 8-, and 10-gingerol, major putative components of ginger, were stable in blood. Terminal half-lives were only slightly longer than that for curcumin (10.5, 6.2, and 8.8 minutes, respectively). Formation of a glucuronide metabolite for each of the gingerols was observed after oral administration of capsules containing a crude ginger extract, but absent in the commercial product administered.

The overall objective of this research was to develop controlled-release formulations of Melanotan-I (MT-I). Two polymers were used, Poloxamer 407 (P407) and Poly(D,L lactide-co-glycolide) (PLGA). First, various aqueous formulations of P407 were evaluated containing MT-I and 25% w/v P407 alone, or with one of the following additives present, i.e., polyvinylpyrrolidone, methylcellulose or hydroxypropyl methylcellulose. The in-vitro release profiles of MT-I from the P407 formulations and the dissolution of the gel were obtained simultaneously using a membraneless in vitro model and a zero-order profile was observed for both. The in-vivo release kinetics of selected formulations were analyzed in guinea pigs following intraperitoneal administration. The plasma concentration-time profiles showed an extended release of the peptide formulated with gel compared to the interperitoneal administration of MT-I in solution and exhibited potential for prolonged release up to 24 hrs only. Biodegradable implants of PLGA copolymer were prepared by a melt-extrusion method. The in vitro release of MT-I from the implants exhibited a triphasic profile with an initial rapid release followed by a secondary phase of slow release, then a tertiary phase of rapid release due to erosion of the polymer. Based on release kinetics of MT-I from different viscosity PLGA implants, the polymer having a viscosity of 0.6 dL/gm was selected for preparing an implant system with a one-month duration of release. Molecular weight reduction, mass loss and lactic acid release from PLGA implants were characterized in order to correlate the in vitro release profile of MT-I with hydrolytic degradation of the polymer. The in vivo release profile of the MT-I depot implanted subcutaneously exhibited a one-month release of the peptide and the reflectance reading obtained from a reflectometer showed a prolonged skin darkening for 3 months. A 2.5 fold increase in the melanin pigment (eumelanin) was observed in guinea pig skin biopsy samples. The results indicate that the PLGA implants are a promising delivery device for controlled-release of MT-I over a one-month period.

The objective of this research project is to develop an ocular insert for the systemic delivery of insulin. Insulin was delivered by a Gelfoam®-based device. All formulations were evaluated for their ability to lower blood glucose concentrations in rabbits. Device removal and flow-through methods were used to investigate the in vivo and in vitro release of the insulin device. Results indicate that the proposed ocular device with an enhancer gives a uniform blood glucose reduction for up to 10 hours. The efficacy of the proposed device can be greatly improved by treating the gelatin sponge with either 5% acetic or 1% of HCl acid solutions. The improvements include: the elimination of enhancer and an up to 5-fold reduction of the dose of insulin with comparable efficacy to that produced by the devices containing an absorption enhancer. Based upon the data from both in vivo and in vitro dissolution studies, the prolonged activity of insulin is due to the slow release of insulin from the device. Overall, the proposed Gelfoam®-based ocular device provides uniform prolonged insulin activity for up to 10 hours. The device can be manufactured in a relatively simple way and the ingredients required are inexpensive. The information provided by this project may lead to a major contribution to the treatment of diabetes as well as to ophthalmic drug delivery in general.