New applications of electron diffraction in the pharmaceutical industry: polymorph determination by using a combination of electron diffraction and synchrotron X-ray powder diffraction techniques.

Electron diffraction has been recently used in the pharmaceutical industry to study the polymorphism in crystalline drug substances. While conventional X-ray diffraction patterns could not be used to determine the cell parameters of two forms of the microcrystalline GP IIb/IIIa receptor antagonist roxifiban, a combination of electron single-crystal and synchrotron powder diffraction techniques were able to clearly distinguish the two polymorphs. The unit-cell parameters of the two polymorphs were ultimately determined using new software routines designed to take advantage of each technique's unique capabilities. The combined use of transmission electron microscopy (TEM) and synchrotron patterns appears to be a good general approach for characterizing complex (low-symmetry, large-unit-cell, micron-sized) polymorphic pharmaceutical compounds.

Physical chemical stability of warfarin sodium.

Crystalline warfarin sodium is an isopropanol clathrate containing 8.3% isopropyl alcohol (IPA) and 0.57% water upon receipt. The hygroscopicity and impact of moisture on IPA status as well as on the stability of the clathrate was studied at different relative humidities. The IPA loss and water uptake were simultaneous but they did not exchange at 1:1 molar ratio. At 58% relative humidity (RH) or below, the exchange process was insignificant. At 68% RH or above, the clathrate tended to lose IPA while absorbing water and reverting to the amorphous state. The rate of IPA loss and moisture uptake was a function of RH. The thermal stability of the crystalline warfarin sodium was also examined. Physical change occurred after isothermal storage for 24 hours at 80 degrees C and 11 hours at 120 degrees C. The rate of IPA loss was temperature dependent.

Electronic and resonance effects on the ionization of structural analogues of efavirenz.

The solubility of 4 analogues of efavirenz was studied as a function of pH. The study evaluated the ionization behavior and determined the relative contribution of electronegative substituents versus resonance effects on the pK(a) value of the cyclic carbamate. The most profound lowering effect on the pK(a) was due to the presence of multiple electronegative substituents and in particular the trifluoromethyl and acetylene groups. The presence of chlorine on the benzoxazinone ring was found to have a slight impact on the pK(a), although to a lesser extent. In the absence of any functional groups on the benzoxazinone ring system, the pKa shifted to a value of 13.2, which is 3 pH units above that of efavirenz and more closely correlates with typical literature values for cyclic carbamates.

Variable heating rate thermogravimetric analysis as a mechanism to improve efficiency and resolution of the weight loss profiles of three model pharmaceuticals.

The effect of variable heating rates on the efficiency or resolution of the derivative of the thermogravimetry profiles of three model pharmaceutical compounds was investigated. The variable heating system utilized computer controlled algorithms for the evaluation of crystalline sodium warfarin, DuP 532 and hydrated DuP 925 as model compounds with one, two and three step weight loss profiles, respectively. As the heating modes were increased through each of the eight settings, the minimum heating rate decreased while the efficiency and resolution and the analysis times increased. The observed weight loss remained relatively constant for each of the model compounds as the heating modes were increased. The efficiency of the derivative of the weight loss profile of crystalline sodium warfarin increased from 121 to 621 as the heating mode increased. The resolution between the two steps of the derivative of the weight loss profile of DuP 532 increased from 1.73 to 3.88 as the heating mode increased. For hydrated DuP 925, the resolution increased from 1.49 to 5.46 between steps 1 and 2 of the derivative of the weight loss profile and from 1.87 to 3.24 between steps 2 and 3 of the derivative of the weight loss profile. Variable heating rates provided a valuable aid in obtaining high efficiency/resolution thermograms. The enhanced efficiency/resolution permitted greater separation of the volatilization process, especially for samples with multi-step weight loss profiles. Increasing the heating mode afforded higher efficiencies/resolutions that typically reached a maximum value at mode 6.

Crystalline versus amorphous content of lumaxistrade mark analog XP280 using X-ray and electron diffraction methods.

In the course of the development of Lumaxistrade mark (roxifiban), the physical state of XP280 (the besylate salt of the active metabolites of roxifiban) and SC887 (the mesylate salt of the free base of roxifiban) were characterized. Powder X-ray diffraction patterns of XP280 were ambiguous in that a high degree of background signal was present and potentially indicative of the existence of an amorphous phase. Herein the results of combined synchrotron X-ray diffraction and electron microscopy (diffraction and imaging) studies on XP280 and SC887 are reported. The combination of these two techniques allowed an unambiguous assessment of the crystallinity, as well as determination of four of the unit cell parameters of XP280 and complete determination of the unit cell parameters for SC887.

Polymorph determination for the GP IIb/IIIa antagonist, roxifiban, using a combination of electron diffraction and synchrotron X-ray powder diffraction techniques.

Unit cell parameters of two polymorphs of roxifiban have been determined by a combination of transmission electron microscopy (TEM) single-crystal and synchrotron X-ray powder diffraction techniques. While it was difficult to differentiate the two forms by their standard X-ray diffraction patterns, the high-resolution synchrotron patterns clearly showed striking differences. Unit cells for the two forms required the use of cell parameters derived from TEM diffraction patterns. The two unit cells are, not surprisingly, very similar except for a doubling of one of the axes for form II. The combined use of TEM and synchrotron patterns appears to be a good general approach for characterizing complex (low-symmetry, large unit cell) polymorphs.

Applications of modulated differential scanning calorimetry in preformulation studies.

Characterization of the thermal properties of active pharmaceutical ingredients is critical in the selection of appropriate physical forms for development and defining proper manufacturing, handling and storage conditions of those chemical entities. Modulated differential scanning calorimetry (MDSC) has proven to be an effective tool in the thorough characterization of thermal behavior of compounds in preformulation studies. Selected applications of MDSC for various preclinical compounds are presented, thereby demonstrating the utility of this analytical method in the determination of glass transitions, characterization of desolvation and degradation processes as well as in the study of polymorphic transformations and crystallizations.

Utility of microcalorimetry in the characterization of the browning reaction.

An isothermal microcalorimeter was utilized to characterize a model solid-state interaction. The degradation of the HIV protease inhibitor, DMP 450, in a binary mixture with hydrous lactose was followed in the presence of 5% additional water. Heat produced in the microcalorimeter sample vessel from either chemical or physical change is channeled through extremely sensitive thermopile blankets and is measured as it flows into infinite heat sinks. Solid-state 1:1 mixtures of DMP 450 and hydrous lactose each with 5% water added were analyzed in the microcalorimeter at 50, 60 and 65 degrees C. The resulting heat flow profiles were consistent with an autocatalytic rate law. An activation energy of 26.12 kcal mol(-1) for the DMP 450:lactose mixture was determined from the slope of the Arrhenius plot of the microcalorimetry heat flow maximum value versus the reciprocal of the absolute temperature. The activation energy determined by the traditional method with HPLC analysis was found to be in excellent agreement with the microcalorimetry value at 26.38 kcal mol(-1).

Solubility behavior, phase transition, and structure-based nucleation inhibition of etanidazole in aqueous solutions.

PURPOSE: The solubility behavior, phase transition and inhibition of the nucleation process of etanidazole were characterized. METHODS: Solubility measurements as a function of time permitted characterization of the solubility behavior and phase transition. The precipitate from saturated solutions was isolated and characterized by differential scanning calorimetry, polarized light microscopy, x-ray powder diffraction and coulometric analysis. The physical stability of metastable systems was examined in the presence of various structure-based nucleation inhibitors. RESULTS: Etanidazole is soluble in water with an equilibrium solubility of 68.1 mg/mL, pH 6.5 with changes in pH having virtually no effect on the solubility. Etanidazole reaches concentrations in excess of 150 mg/mL within one hour. Etanidazole solutions prepared at 150 mg/ mL contained crystals after rotating for 24 hours. The crystals were isolated and characterized as etanidazole monohydrate. the solubility of etanidazole monohydrate in water increased with time reaching an equilibrium solubility of 68 mg/mL after 24 hours. Therefore, the solubility studies were actually determining the solubility of the more stable monohydrate from of etanidazole. Etanidazole solutions at concentrations of 50, 100 and 150 mg/mL were stabilized to varying degrees with structure-based nucleation inhibitors (imidazole, ethanolamine or diethanolamine). CONCLUSIONS: Anhydrous etanidazole undergoes a transition in aqueous solutions to the more stable monohydrate when the solubility of the monohydrate is exceeded. The physical stability of etanidazole solutions at 4 degrees C is improved following autoclaving. The addition of structure-based nucleation inhibitors effectively stabilized the metastable systems.

Improved cyclic urea inhibitors of the HIV-1 protease: synthesis, potency, resistance profile, human pharmacokinetics and X-ray crystal structure of DMP 450.

BACKGROUND: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds. RESULTS: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg-1 DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC90 suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response. CONCLUSIONS: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.

Determination of the pKa and pH-solubility behavior of an ionizable cyclic carbamate, (S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4- (trifluoromethyl)-2H-3,1-benzoxazin-2-one (DMP 266).

The solubility of a nonnucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl )- 2H-3,1-benzoxazin-2-one (DMP 266), was investigated as a function of pH. A dramatic increase in the aqueous solubility was observed at pH > or = 10, which was consistent with going from a neutral to a charged species. The ionization of the proton positioned on the carbamate functionality was confirmed spectrophotometrically (pKa = 10.1). The spectrophotometric result was in excellent agreement with that obtained from the solubility studies (pKa = 10.2). The ionization behavior of DMP 266 represents a unique case in which the pKa for a carbamate functional group is quite low. The anomalous pKa value may be attributed to stabilization of the negatively charged species through inductive effects, which originate from the surrounding substituents and delocalization of the negative charge via resonance effects.

Solubilization of nicardipine hydrochloride via complexation and salt formation.

The solubility behavior of nicardipine (1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic+ ++ acid methyl 2-[methyl(phenyl-methyl)amino]ethyl diester), a calcium channel blocker, used in the treatment of chronic stable angina and mild essential hypertension was investigated. Two techniques that are known to improve solubility, complexation and salt formation, were examined. Concentrations were determined with a specific reversed-phase HPLC assay. The solubility of nicardipine hydrochloride was enhanced exponentially via complexation with aliphatic carboxylic acid buffer systems in a pH dependent fashion. The solubility increased from 5 to 68.6 and 270 mg/mL as the acetate or propionate buffer concentrations, respectively, increased from 0.001 to 5 M, showing a positive deviation from linearity. The conversion of nicardipine hydrochloride to the phosphate salt resulted in a approximately 10-fold solubility improvement. The surface tension of the nicardipine phosphate in water as a function of concentration indicated a critical micelle concentration of 5-6 mg/mL. The critical micelle concentration was greater than the equilibrium solubility of the hydrochloride salt in water, suggesting that a self-association phenomena is responsible for the enhanced solubility of the phosphate salt. Both routes provided potential alternatives for the solubilization of nicardipine.

Complexation of moricizine with nicotinamide and evaluation of the complexation constants by various methods.

The solubility of the antiarrhythmic drug moricizine at physiologic pH is very low. Precipitation after rapid intravenous injection of the hydrochloride salt of moricizine could be a concern. The enhancement of solubility of moricizine near physiologic pH via complexation with nicotinamide was examined as a potential solubilization technique. The studies were performed in pH 6 and pH 7 phosphate buffers at 25 degrees C by the phase solubility method. Moricizine formed 1:1 and 1:2 complexes with nicotinamide at pHs of 6 and 7. The complexation constants K1:1 and K1:2 were estimated by a previously described scheme and equation and compared with those obtained by fitting a line and a parabola to the equations derived from the scheme for both the approximate and exact solutions. The data were best represented by a parabolic regression analysis of the exact solution of the derived equation with values for K1:1 and K1:2 at pH 6 of 16.60 and 0.93 M-1, respectively, and at pH 7 of 7.70 and 5.41 M-1, respectively.

Physicochemical properties of the novel heteropolyanion antiviral hexapotassium-alpha-vanado-11-tungstoborate (DuP 925).

Hexapotassium-alpha-vanado-11-tungstoborate (DuP 925) is a yellow irregular-shaped crystalline powder. The DSC thermogram indicates that decomposition begins to occur above 250 degrees C. The compound exhibits a volatile loss of 3.7% by thermogravimetric analysis. The drug substance adsorbs water reaching a 7.1% volatile loss after 3 weeks at 85% relative humidity. The solubility of DuP 925 in water is high (1.6 g/ml at pH 4.8). Changes in pH have a negligible effect on the solubility with values of 1.3 g/ml in 0.1 N HCl and 1.4 g/ml in 0.1 N NaOH. The solubility is minimally affected by changes in sodium ion concentration. The compound ion pairs with tetrabutyl-ammonium ions at 1:2 and 1:6 ratios, with the 1:6 ion pair having an affinity over six orders of magnitude greater than that of the 1:2 ion pair. The degradation of DuP 925 in solution follows apparent first-order kinetics over the pH range of 0.6 to 12.6 at 80 degrees C. Citrate, EDTA, and phosphate buffers are catalytic at the pH minimum, with citrate and EDTA being stronger catalysts than phosphate. Acetate buffers appear to have negligible catalytic effects at pH 4 to 5. The degradation proceeds through the formation of the symmetrical dodecatungstoborate [BW12O40]-5. In acid, the dodecatungstoborate is stable, while in base it degrades further. Increasing the ionic strength has a catalytic effect on the degradation of DuP 925, while changes in the initial concentration of DuP 925 have a negligible effect on the stability. The pH-rate profile indicates a pH minimum of approximately 3.

Mechanism of diffusion of monosubstituted benzoic acids through ethylene-vinyl acetate copolymers.

Ethylene-vinyl acetate (EVAc), a biocompatible copolymer, has been employed as the rate-controlling membrane in several drug delivery systems. To study the mechanism(s) of diffusion of drugs through EVAc membranes, the diffusion, permeability, and partition coefficients of monosubstituted benzoic acids were studied as a function of vinyl acetate content. The diffusion coefficients were found to occupy a narrow range, but the permeability and partition coefficients were found to increase in a nonlinear fashion as a function of vinyl acetate content, indicating that the diffusion process was partition governed. The partitioning data were analyzed on the basis of the partitioning between the vinyl acetate moiety and the aqueous phase, assuming the formation of 1:1 benzoic acid:vinyl acetate complexes. The effects of ionization and the addition of 2-propanol to the diffusion medium were studied. The results suggest that the un-ionized neutral forms of the benzoic acids are responsible for transport across the copolymer. Altering the composition of the medium by addition of 2-propanol increased the donor phase solubility of the acid, the steady-state rate, and the permeability, suggesting that cosolvent modification provides an excellent chemical means to increase release rates.