APIs that are non-ionizable or demonstrate poor salt forming ability traditionally present few opportunities for creating crystalline solid forms with desired physical properties. Cocrystals are an additional class of crystalline solid that can provide options for improved properties. In this case, a crystalline molecular complex of glutaric acid and an API was identified and used to demonstrate an improvement in the oral bioavailability of the API in dogs.

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Human cytochrome P450 3A4is recognized as the catalyst for the oxygen-dependent metaboism of a diverse group of medicafly important chemicals, inWuding the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids, such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymaticafly active fusion protein (rF450[mHum3A4/mRatOR]Ll) has been expressed at high levels in Escherichia coli and purified to homogeneity.Enzymatic studies show a requirement for lipid, detergent, and cytochrome bs for the 63-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometricafly detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolim of both testosterone and erthromycin. These resuits relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.One ofthe most versatile ofthe cytochrome P450s is the form present in human liver called CYP3A4, which catalyzes the oxidative metabolism of a wide array of different chemicals with markedly different structural characteristics (1). Interest has centered on this P450 since it is reported to be one of the more abundant P450s in human liver (2); it is inducible by a variety of agents including glucocorticoids as well as phenobarbital (3); it appears to play a central role in the metabolism of the immunosuppressive cyclic peptide cyclosporin A as well as macrolide antibiotics, such as erythromycin (4); it also catalyzes the 63-hydroxylation of a number of steroids including testosterone, progesterone, and cortisol (5). Clinical interest relates to the measurement of erythromycin metabolism by a breath test (6) and the presence of6f-hydroxylated steroids in urine (7) as indicators of CYP3A4 function for evaluation of transplant recipients.P450s ofthe 3A family were first characterized by Guzelian and colleagues (8) based on the ability ofthe catabolic steroid pregnenolone-16a-carbonitrile to induce a unique form of P450 (which they called P-450p). They also recognized the ability of the macrolide antibiotic triacetyloleandomycin (TAO) to serve as a powerful inducer of this type of P450-a result of interest because of the known ability of TAO and other compounds to form stable, metabolite-inhibitor complexes with P450 (9). Reconstitution studies using purified P450s of the 3A family have shown the need to include phospholipids, detergents, and cytochrome b5 when testing enzymatic activities (10, 11).

The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein. In general these electrons are transferred from NADPH to the P450 via an FMN-and FAD-containing flavoprotein (NADPH-P450 reductase). The present paper shows that NADPH can be replaced by an electrochemically generated reductant [cobalt(H) 4A1 and the flavin domains of rat NADPH-P450 reductase was expressed and purified according to methods described previously (2-4). A carboxyl-terminal histidine-tagged P450, rP4504A1(His)6, was expressed and purified according to methods described by Jenkins and Waterman (5). Concentrations of the P450 enzymes were determined spectrophotometrically (reduced-CO bound minus oxidized enzyme) (6). The P450 was reduced under anaerobic conditions with sodium dithionite in the presence of a trace of added methyl viologen. Recombinant rat cytochrome b5 (rHMw(His)4b5), here termed b5, was purified from E. coli membrane fractions as described (7). 7705The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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