Drazen Ostovic

A Quantitative Kinetic Study of Polysorbate Autoxidation: The Role of Unsaturated Fatty Acid Ester Substituents

PurposeTo study the role of unsaturated fatty acid ester substituents in the autoxidation of polysorbate 80 using quantitative kinetics.MethodsOxidation kinetics were monitored at 40°C in aqueous solution by tracking head space oxygen consumption using a fiber optic oxygen sensor with phase shift fluorescence detection. Radical chain initiation was controlled using an azo-initiator and assessed by Hammond’s inhibitor approach, allowing oxidizability constants (kp/(2kt)1/2) to be isolated. Reaction orders were determined using modified van’t Hoff plots and mixed polysorbate micelles.ResultsThe oxidizability constant of polysorbate 80 ((1.07u2009±u20090.19)u2009×u200910−2xa0M−1/2xa0s−1/2) was found to be 2.65 times greater than polysorbate 20 ((0.404u2009±u20090.080)u2009×u200910−2xa0M−1/2xa0s−1/2). The additional reactivity of polysorbate 80 was isolated and was first-order in the unsaturated fatty acid ester substituents, indicating that the bulk of the autoxidative chain propagation is due to these groups. This data, and the observation of a half-order dependence on the azo-initiator, is consistent with the classical autoxidation rate law (-d[O2]/dt = kp[RH](Ri/2kt)1/2).ConclusionsPolysorbate 80 autoxidation follows the classical rate law and is largely dependent on the unsaturated fatty acid ester substituents. Clarification of the substituents’ roles will aid formulators in the selection of appropriate polysorbates to minimize oxidative liabilities.

Regiospecific Intestinal Absorption of the HIV Protease Inhibitor L-735,524 in Beagle Dogs

AbstractPurpose. To evaluate regional intestinal absorption and the feasibility of sustained release dosage form development for an HIV protease inhibitor, L-735,524.nMethods. L-735,524 free base or sulfate salt was administered orally as suspension, solution or in solid dosage forms to fasted or fed Beagle dogs. Delayed-release dosage forms with “slow” or “fast” in vitro dissolution rates were evaluated in vivo to assess plasma concentration profiles. In addition, drug was administered directly into the jejunum or colon of animals, and drug concentrations determined in portal circulation to characterize absorption from these sites.nResults. L-735,524 sulfate was well absorbed orally from a solution or capsule formulation if fasted animals stomachs were preacidified with citric acid solution. A free base suspension, delivered in divided doses to fed animals, was also well absorbed. Prototype extended release dosage forms of L-735,524 produced a reduction in peak plasma levels but failed to prolong absorption and extend plasma concentrations compared to an immediate release capsule. Administration of L-735,524 sulfate solution (pH<3) as bolus solution or by infusion into the jejunum resulted in rapid but incomplete absorption compared to oral gavage. The free base suspension (pH 6.5) delivered into jejunal or colonic regions did not produce measurable systemic plasma concentrations.nConclusions. Extended release formulations did not prolong absorption of L-735,524 in dogs. Optimal L-735,524 absorption was dependent on solubility in an acidic environment in the duodenum.

Identification of oxidative degradates of the thrombin inhibitor, 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylenecarboxamidomethylpyridinyl)pyrazinone, using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

Liquid chromatography/mass spectrometry was used to identify reaction products from a solution of 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methyleneca rboxamidomethylpyridinyl)pyrazinone (L-375,378) and hydrogen peroxide, a system that generates high levels of the oxidative degradates which form in the tablets and intravenous (i.v.) solutions of L-375,378. Two major hydrogen peroxide reaction products of L-375,378 (m/z 407) with m/z values of 369 and 370 were separated and identified. Both compounds were products of ring opening with elimination of three carbon atoms from the center pyrazinone ring. The structural assignments for these two products were alpha-amidinoamide and alpha-diamide compounds, respectively. In addition, five products (m/z 423) with a molecular weight 16 Da greater than that for L-375,378 were separated. Further liquid chromatography/tandem mass spectrometry experiments indicated that three of these M + 16 products were phenolic derivatives of L-375,378. Among them, the para-hydroxy compound has been verified using an authentic standard. The other two phenolic compounds were believed to be the meta- and ortho-hydroxy derivatives of L-375,378. The fourth M + 16 product was derived from hydroxylation of the methyl group on the center pyrazinone ring. The fifth M + 16 product was derived from oxidation on the aminopyridine moiety, most likely N-oxide of the pyridine ring. Other minor hydrogen peroxide reaction products were not studied in detail because they did not appear in tablets or i.v. formulations.

Development of subcutaneous and intramuscular formulations of calcium alendronate salts

AbstractPoorly soluble calcium-alendronate salts were prepared and investigated as potential candidates for subcutaneous or intramuscular formulations. Three such formulations containing calcium-alendronate salts with different stoichiometries were developed for testing in safety, disposition and efficacy studies in animals. All formulations demonstrated a drastic reduction in pain on injection and tissue damaging propensity compared to the soluble salts of ABP. All three were efficacious and showed prolonged absorption from the injection site with the deposition of a large percentage of the dose into the bone. Complex formation between alendronate and calcium was also studied.

A new hydroxyethylamine class of HIV-1 protease inhibitors with high antiviral potency and oral bioavailability

Abstract A new hydroxyethylamine class of inhibitors was designed combining features from our clinical candidate, L-735,524, along with small heterocyclic P 2 -ligands developed in these laboratories. Highly potent inhibitors possessing subnanomolar IC 50 s have been identified, which exhibit good antiviral potency in cell culture. L-738,872, a representative inhibitor in this class, showed 34% oral bioavailability in dogs.

A rapid bioassay for the determination of non-nucleoside HIV-1 reverse transcriptase inhibitor plasma levels

A rapid method for measuring pyridinone HIV-1 reverse transcriptase inhibitor plasma levels was necessary for identifying potential clinical candidates and for choosing a clinical formulation. Due to its sensitivity to the pyridinones, ability to tolerate extraneous material, and its capability for rapid, high through-put screening, the HIV-1 RT assay was developed into a bioassay for determining plasma levels of the pyridinones in Rhesus monkey plasma. With this assay, dose proportionality of L-697, 639 was established. Formulation studies using L-697, 639 indicated that the plasma levels achieved in Rhesus monkeys with the clinical formulation (peak levels = 3.9 microM at 30 min) fall between the levels achieved with polyethylene glycol 300 and hydroxypropyl methyl cellulose formulations (peak levels = 8.9 microM and 0.4 microM respectively, at 60 min). Two other pyridinones, L-696, 229 and L-697, 661, administered as the clinical formulation, had peak plasma levels of 1.6 microM (30 min) and 0.3 microM (60 min), respectively. In Rhesus monkeys, the bioavailabilities of these compounds (administered as the clinical formulation) ranged from 11 to 24% and their half-life values ranged from 24 to 120 min. The results of oral studies in Rhesus monkeys with these compounds were very similar to initial results of studies in humans.