Solid-state stability of human insulin. II. Effect of water on reactive intermediate partitioning in lyophiles from pH 2-5 solutions: stabilization against covalent dimer formation.

Previous studies have established that at low pH human insulin decomposition proceeds through a two-step mechanism involving rate-limiting intramolecular formation of a cyclic anhydride intermediate at the C-terminal AsnA21 followed by intermediate partitioning to various products, most notably desamido insulin and covalent dimers, in both aqueous solution and in the amorphous (lyophilized) solid state. This study examines the product distribution resulting from insulin degradation in lyophilized powders as a function of water content and the phase behavior of the solid (glassy versus rubbery) between pH 3 and 5. In amorphous solids at low water content (glassy state), the cyclic anhydride intermediate of insulin reacts predominantly with water to form deamidated insulin, whereas the intermolecular reaction with another insulin molecule to form a covalent dimer accounts for < or = 15% of the total degradation. Increasing water content reduces the glass transition temperature of insulin to < 35 degrees C, and covalent dimer formation becomes increasingly favored relative to deamidation. An increase in solid-state pH also favors dimerization as deprotonation of the terminal amino groups of insulin renders them more nucleophilic. Covalent dimerization was almost totally suppressed by incorporation into a glassy matrix of trehalose, which both minimizes molecular mobility and physically separates the insulin molecules. The kinetics and product distribution of human insulin in lyophilized powders between pH 3 and 5 illustrate the differential sensitivities of various solid-state reaction types to the effects of water activity and solid-phase behavior. The intramolecular cyclization at the AsnA21 position requires only short-range conformational flexibility and thus is only modestly restricted even in the glassy state. On the other hand, the competing bimolecular reactions involving either water or another molecule of insulin combining with the intermediate anhydride are dependent on molecular mobility of the reactants, in accord with predictions of free volume theory. In the glassy state, deamidation (reaction with water) is favored because of the restricted molecular mobility of proteins in rigid matrices. Increasing plasticization with increasing water content favors covalent aggregate formation because of the higher dependence of protein mobility on free volume within the solid matrix.

Solid-state stability of human insulin. I. Mechanism and the effect of water on the kinetics of degradation in lyophiles from pH 2-5 solutions.

PURPOSE: Previous studies have established that in aqueous solution at low pH human insulin decomposition proceeds through a cyclic anhydride intermediate leading to the formation of both deamidated and covalent dimer products. This study examines the mechanism and kinetics of insulin degradation in the amorphous solid state (lyophilized powders) as a function of water content over a similar pH range. METHODS: Solutions of 1.0 mg/mL insulin were adjusted to pH 2-5 using HCl, freeze-dried, then exposed to various relative humidities at 35 degrees C. The water content within the powders was determined by Karl Fischer titration, and the concentrations of insulin and its degradation products were determined by HPLC. Degradation kinetics were determined by both the initial rates of product formation and insulin disappearance. RESULTS: Semi-logarithmic plots of insulin remaining in lyophilized powders versus time were non-linear, asymptotically approaching non-zero apparent plateau values, mathematically describable by a reversible, first-order kinetic model. The rate of degradation of insulin in the solid state was observed to increase with decreasing apparent pH ('pH') yielding, at any given water content, solid-state 'pH'-rate profiles parallel to the solution pH-rate profile. This 'pH' dependence could be accounted for in terms of the fraction of the insulin A21 carboxyl in its neutral form, with an apparent pKa of approximately 4, independent of water content. Aniline trapping studies established that the mechanism of degradation of human insulin in lyophilized powders between pH 3-5 and at 35 degrees C involves rate-limiting intramolecular nucleophilic attack of the AsnA21 C-terminal carboxylic acid onto the side-chain amide carbonyl to form a reactive cyclic anhydride intermediate, which further reacts with either water or an N-terminal primary amino group (e.g., PheB1 and GlyA1) of another insulin molecule to generate either deamidated insulin (AspA21) or an amide-linked covalent dimer (e.g., [AspA21-PheB1] or [AspA21-GlyA1]), respectively. The rate of insulin degradation in lyophilized powders at 35 degrees C increases with water content at levels of hydration well below the suspected glass transition and approaches the rate in solution at or near the water content (20-50%) required to induce a glass transition. CONCLUSIONS: The decomposition of human insulin in lyophilized powders between pH 3-5 is a water induced solid-state reaction accelerated by the plasticization effect of sorbed water. The formation of the cyclic anhydride intermediate at A21 occurs readily even in the glassy state, presumably due to the conformational flexibility of the A21 segment even under conditions in which the insulin molecules as a whole are largely immobile.

Solubilization and stabilization of an anti-HIV thiocarbamate, NSC 629243, for parenteral delivery, using extemporaneous emulsions.

The O-alkyl-N-aryl thiocarbamate, I, (2-chloro-5-[[(1-methyl-ethoxy)thioxomethyl]amino]benzoic acid, 1-methylethylester, NSC 629243, also known as Uniroyal Jr.) is an experimental anti-HIV drug with very low water solubility (1.5 micrograms/mL). Early clinical studies required an injectable solution at approximately 15 mg/mL, representing a solubility increase of approximately 10(4)-fold. Adequate solubilization of this hydrophobic drug was achieved in 20% lipid emulsions. Extemporaneous emulsions were prepared by adding a concentrated drug solution to a commercially available parenteral emulsion. Various methods of preparation to minimize drug precipitation during its addition and enhance redissolution of precipitated drug were evaluated. The stability and mechanism(s) of decomposition of NSC 629243 in both 20% lipid emulsions and in natural oil vehicles were examined. In lipid emulsions, the shelf life at 25 degrees C varied from 1 to > 10 weeks, depending on the extent to which air was excluded from the preparation. The shelf life of 50 mg/mL solutions in natural oils at 25 degrees C varied from < 1 to > 100 days depending on the oil and its supplier. A qualitative correlation was found between the initial rate of oxidation and the peroxide concentration in the oil. The primary degradation product in both systems was shown to be a disulfide dimer, II, formed via oxidation. Oxidation was inhibited by vacuum-sealing of emulsion formulations or incorporation of an oil-soluble thiol, thioglycolic acid (TGA), into oil formulations. TGA may inhibit oxidation by consuming free radicals or peroxide initiators or by reacting with the disulfide, II, to regenerate the starting drug.

High-performance liquid chromatographic (HPLC) and HPLC-mass spectrometric (MS) analysis of the degradation of the luteinizing hormone-releasing hormone (LH-RH) antagonist RS-26306 in aqueous solution.

The kinetics of the degradation of an LH-RH antagonist, RS-26306,1, in aqueous solution from pH 1 to pH 11 were studied by reverse-phase HPLC. The pH-rate profiles at 50, 60, and 80 degrees C were U-shaped with the rate law of kobs = kHaH + kw + kOHaOH. The predicted 25 degrees C shelf life at the pH of maximum stability, pH approximately 5, is greater than 10 years. The products from the degradation were analyzed by HPLC-MS using thermospray ionization. Below pH 3, the primary product, 2, forms from the acid-catalyzed deamidation of the C-terminal amide. Above pH 7, epimerization of the individual amino acids is the principal reaction. Between pH 4 and pH 6, intramolecular serine-catalyzed peptide hydrolysis becomes important, yielding a tripeptide, 3, and a heptapeptide, 4. At the pH of maximum stability all three pathways for degradation are observed.

Drug-excipient incompatibility studies of the dipeptide angiotensin-converting enzyme inhibitor, moexipril hydrochloride: dry powder vs wet granulation.

The drug-excipient incompatibility screen for moexipril hydrochloride (1) using various isothermal stress methods is reported herein. It was found that most of the commonly used filters, disintegrants, lubricants, glidants, and coating agents were incompatible with 1 in dry powder mixtures; moisture and basic (or alkalizing) agents were determined to be the dominant destabilizing factors. In wet granulations, basic agents, however, were found to suppress drug degradation even in the presence of moisture. Supported by the product distribution studies, the stabilization is proposed to involve the neutralization of the acidic drug by the basic excipients.

An unexpected pH effect on the stability of moexipril lyophilized powder.

Because of the limited stability of moexipril (RS-10085; 1) in aqueous solution, lyophilized parenteral formulations were evaluated as a function of pH in this study. In general, the lyophilized powder of 1 showed about two orders of magnitude less reactivity at 50 degree C than in aqueous solution at pH values below 3 or above 6. At pH 5.1, however, the lyophilized powder had maximum reactivity, with the rate actually comparable to that observed in aqueous solution. When the distribution of the two major products, diketopiperazine (DKP) 2 and ester hydrolysis analogue 3, was compared to the observed kinetics as a function of pH, it was clear that removal of water via lyophilization suppressed the spontaneous k1 cyclization process, the spontaneous k3 hydrolysis process, and the specific base-catalyzed k4 hydrolysis process. The overall spontaneous k2 cyclization process, however, was not affected by lyophilization. The latter result is accounted for by the increased equilibrium constant for the formation of the tetrahedral intermediate, To, as a result of lyophilization. This study demonstrates that stability data in solution can not be used for predicting the stability of moexipril in lyophilized powder form.

Preformulation stability studies of the new dipeptide angiotensin-converting enzyme inhibitor RS-10029.

The degradation kinetics, products, and mechanisms of RS-10029 (2), 2-[2-[(1-carboxylic acid)-3-phenylpropyl]amino-1-oxopropyl] 6,7-dimethoxy- 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (S,S,S), in aqueous solutions from pH 1 to pH 13 were studied at 50, 60, and 80 degrees C. Pseudo-first-order kinetics were obtained throughout the entire pH range studied, and the log(rate)-pH profile reflected four kinetic processes (ko, k'o, k"o, and kOH) as well as the three pka's of 2. Excellent mass balance (greater than 96%) was obtained for the four major products 3-6 throughout the entire pH range studied even though four other minor products can be detected by high-performance liquid chromatography (HPLC). At pH 8.0 and below, intramolecular aminolysis leading to diketopiperazine (DKP) 5 accounted for greater than 65% of the neutral or water-catalyzed (ko and k'o) processes. Amide hydrolysis leading to products 3 and 4 and epimerization of DKP 5 to the (R,S,S) diastereomer 6 accounted for the remaining 35% of the neutral or water catalyzed processes. At pH values above 8.0, DKP 5 formation begins to decrease as the amide hydrolysis increases so that both mechanisms account for the neutral or water-catalyzed k"o process. Above pH 11.0 amide hydrolysis dominates and is responsible for the specific base-catalyzed (kOH) process. The four minor products detected by HPLC are two diastereomers (7 and 8) of 2 and the two diastereomers (9 and 10) of the DKP 5. The stability results between 2 and its ester prodrug (1) are compared.

Diketopiperazine formation, hydrolysis, and epimerization of the new dipeptide angiotensin-converting enzyme inhibitor RS-10085.

The degradation kinetics, products, and mechanisms of RS-10085(1), 2-[2-(1-ethoxycarbonyl)-3-phenylpropyl]amino-1-oxopropyl]-6,7- dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(S,S,S), in aqueous solution were investigated at 40, 60, and 80 degrees C from pH 1 to pH 13. Pseudo-first-order kinetics were observed throughout the pH range studied and the log(rate)-pH profiles reflected four kinetic processes (ko, k'o, k'o, and kOH) as well as the two pKa's of 1. Excellent (greater than 98%) mass balance was obtained through products 2-5. At pH 4 or below, intramolecular cyclization leading to diketopiperazine 5 accounted for greater than 93% of the observed neutral- or water-catalyzed processes (ko and k'o). At pH levels greater than 5, hydrolysis giving 2 predominated and was responsible for the observed neutral- or water-catalyzed (k''o) and specific base-catalyzed (kOH) kinetic processes. Some epimerization leading to the S,S,R drug isomer (4) was also observed at pH levels greater than 7. The relative acidity of the protons at the three chiral centers of 1 was qualitatively compared and was used to explain the observed specificity in epimerization.