Toshiro Niwa

Preparations of biodegradable nanospheres of water-soluble and insoluble drugs with D,L-lactide/glycolide copolymer by a novel spontaneous emulsification solvent diffusion method, and the drug release behavior

Abstract Nanospheres with d , l -lactide/glycolide copolymer (PLGA) were prepared as a biodegradable polymeric carrier for both water-soluble and insoluble drugs by a novel spontaneous emulsification solvent diffusion method. Indomethacin and 5-fluorouracil (5-FU) were employed as poorly water-soluble and water-soluble model drugs, respectively, to investigate the encapsulation efficiency. The drug and PLGA, dissolved in an acetone-dichloromethane (or acetone-chloroform) mixture, were poured into an aqueous solution of polyvinyl alcohol with stirring using a high-speed homogenizer when necessary. The dispersed droplets were finely emulsified into nanometer-sized spheres. The marked decrease of the interfacial tension between organic and aqueous phases and the spontaneous mixing caused by a rapid diffusion of acetone from the organic to aqueous phase resulted in the formation of submicron-sized PLGA spheres. The recovery of indomethacin entrapped in the nanospheres (mean diameter: 400–600 nm) increased to 75% at maximum. The rapid deposition of polymeric film on the droplet was required for improving the encapsulation of 5-FU to prevent leakage from the droplet. The mean diameter of nanospheres formulated with 5-FU were successfully decreased to 200–300 nm even without high-speed homogenizing. The drug release behavior from nanospheres suspended in buffered solution exhibited a biphasic pattern. The initial burst of release might be due to the rapid release of drugs deposited on the surface and in the water channels of nanospheres. At a later stage, the drug release rate was reduced. During the release test, PLGA was not degradated for 100 h irrespective of the molecular weight. The molecular weight of polymer was a main factor in controlling the drug release rate from the nanospheres.

Improvements in flowability and compressibility of pharmaceutical crystals for direct tabletting by spherical crystallization with a two-solvent system

Abstract Pharmaceutical crystals for direct tabletting were agglomerated by the spherical crystallization technique with a two-solvent system, i.e. good and poor solvents for dissolving the drug, to improve their micromeritic properties, such as flowability, packability and compressibility. Flowability and packability of the agglomerates were investigated by measuring tapped densities and interparticle frictions when sheared. Compressibility was investigated by analyzing the compaction process by the Heckel equation and the mechanical strength of the resultant tablet, including tensile strength and capping index. Such micromeritic properties of the agglomerates were much improved due to their reduced interparticle friction and increased interparticle bonding on tabletting.

Comparison of Kinetic Parameters for Drug Oxidation Rates and Substrate Inhibition Potential Mediated by Cytochrome P450 3A4 and 3A5

Cytochrome P450 (P450 or CYP) 3A is one of the most important P450 subfamilies in terms of its broad substrate specificity and relatively high abundance in humans. The substrate specificities of CYP3A4 and CYP3A5 are generally overlapped, but sometimes could differ from each other. It is still important to understand drug interactions more precisely in individual subjects. However, there are few review articles regarding comparative drug oxidation rates catalyzed by CYP3A4 and CYP3A5 and/or substrate inhibition potential towards CYP3A4 and CYP3A5. In this article, we summarize 1) Michaelis-Menten constants (Km), maximal velocities (Vmax), and intrinsic clearance (Vmax/Km) values for 63 substrates (94 reactions) mediated by CYP3A4 and/or CYP3A5, 2) inhibition constants (Ki) and 50% inhibitory concentrations (IC50) of 18 substrates, and 3) maximum inactivation rate constants (kinact) of 14 inhibitors from the literature. The relative contribution of polymorphic CYP3A5 compared with inducible CYP3A4 varies with the substrates and the reaction positions of the substrates. Inhibitory effects of azole antifungal agents and macrolide antibiotics, with low Ki and/or IC50 values for CYP3A4, are likely to be determinant factors for predominant drug interactions in humans, although Asian subjects with relatively high frequency of genetic CYP3A5 expressers should be carefully treated with CYP3A substrates. The collective findings in our present survey provide fundamental and useful information for drug oxidations catalyzed by CYP3A4 and CYP3A5, in spite of some contradictive kinetic parameters for the same reactions reported from many laboratories in different conditions. To understand causal factor(s) and mechanism(s) for such different reports summarized here is still one of the hot research topics to be solved in current drug metabolism.

Heterotropic cooperativity in oxidation mediated by cytochrome p450.

Cytochrome P450s (P450 or CYPs) comprise a superfamily of enzymes that catalyze the oxidation of a wide variety of xenobiotic chemicals. Although most of P450 inhibitors decrease the metabolic activities mediated by the corresponding P450 forms, unexpected phenomena, which are called as activation or heterotropic cooperativity, have been often observed. We summarize Michaelis-Menten constants (K(m)), maximal velocities (V(max)), V(max)/K(m) (intrinsic clearance) values, and/or metabolic activities for 22 activators and 24 substrates (30 reactions) mainly mediated by CYP3A4 among human P450 forms. Although an allosteric mechanism has been invoked to explain the cooperativity, the activation patterns or phenomena are dependent on substrates and selected enzyme sources in vitro. Interestingly, recent studies have been shown that human P450 forms other than CYP3A4, such as CYP1A2, CYP2C8, CYP2C9, CYP2D6, and CYP3A7, are also activated by some compounds, whereas there are few reports on CYP3A5. Several models describing interaction among substrates, effectors, and enzymes have been proposed, however, the detailed mechanism for the activation is still generally unknown even though some crystal structures have been shown. A few cases of the cooperativity of CYP3A in experimental animals have been presented, whereas the clinical significance of P450 cooperativity is still unclear. The collective findings provide fundamental and useful information for the activation of P450s by chemicals despite some contradictive kinetic parameters for the same reactions reported. To understand causal factor(s) and mechanism(s) for such different reports summarized here is still one of the hot research topics to be solved in current activation reactions.

Drug interactions between nine antifungal agents and drugs metabolized by human cytochromes P450.

This article reviews in vitro metabolic and in vivo pharmacokinetic drug-drug interactions of nine antifungal agents: six azoles (fluconazole, itraconazole, ketoconazole, miconazole, posaconazole, and voriconazole) and three echinocandins (anidulafungin, caspofungin, and micafungin). In in vitro interaction studies, itraconazole, ketoconazole, and miconazole were found to have higher inhibitory effects on cytochrome P450 (P450 or CYP) 3A4 and 3A5 activities than the other azoles or echinocandins did. Fluconazole, itraconazole, and voriconazole were relatively less potent inhibitors of CYP3A5 than of CYP3A4. The inhibitory effects of fluconazole, itraconazole, ketoconazole, and voriconazole against CYP3A4 and CYP3A5 seemed to be correlated with their dissociation constants for CYP51 (lanosterol 14α-demethylase) from Candida albicans. In in vivo pharmacokinetic studies, itraconazole was found to be a potent clinically important inhibitor of CYP3A4/5 substrates, and fluconazole and voriconazole increased the blood/plasma concentrations of not only CYP3A4/5 substrates but also CYP2C9 substrates. Miconazole was a potent inhibitor of all P450s investigated in vitro, although there are few detailed studies on the clinical significance of this except for CYP2C9. For the echinocandins, no marked inhibition of P450 activities, except for some inhibition of CYP3A4/5 activity, was observed in vitro. The blood/plasma concentrations of concomitant drugs were not markedly affected by coadministration of echinocandins in vivo, suggesting that echinocandins do not cause clinically significant interactions with drugs that are metabolized by P450s via the inhibition of metabolism. The differential effects of these antifungal agents on P450 activities must be considered when clinicians select antifungal agents for patients also receiving other drugs.

Potential impact of cytochrome P450 3A5 in human liver on drug interactions with triazoles

Cytochrome P450 3A is the main enzyme subfamily involved in the metabolism of a variety of marketed medicines. It is generally believed that the substrate specificity of polymorphic P450 3A5 is similar to that of the predominant P450 3A4 isoform, although some differences in catalytic properties have been found. It has been hypothesized that individuals with CYP3A5 1 (P450 3A5 expresser) might clear the HIV protease inhibitor saquinavir, administered by mouth, more rapidly than subjects lacking functional CYP3A5 alleles. Enhanced midazolam hydroxylation and cyclosporin metabolism occur in an in vitro P450 3A5 system and in liver microsomes expressing P450 3A5 in the presence of thalidomide. However, inhibition constants (K(i)) of three triazole anti-fungal drugs (itraconazole, fluconazole, and voriconazole) for liver microsomal P450 3A5 are higher than for liver microsomal P450 3A4. To predict drug interactions in vivo, we estimated increases of areas under the curves (AUC) dependent on polymorphic P450 3A5 expression, using both 1 +[Inhibitor] / K(i) (recommended in US FDA guidance), and 1 +[Inhibitor](unbound) / K(i) (as recommended by Japanese MHLW Notice). Voriconazole would be expected to cause approximately a three-fold higher increase in AUC in subjects with CYP3A5 3/3 than in those with CYP3A5 1/3, especially when estimated using the FDA guidance. We conclude that drug interactions between marketed drugs may differ substantially between individuals with genetically distinct P450 3A5 catalytic functions.

Oxidation of Endobiotics Mediated by Xenobiotic-Metabolizing Forms of Human Cytochrome P450

Cytochrome P450s (P450 or CYPs) comprise a superfamily of enzymes that catalyze the oxidation of a wide variety of xenobiotic chemicals including drugs and environmental carcinogens. Recent studies have demonstrated that endogenous chemicals are also oxidized by human P450s which mainly metabolize xenobiotics. In this review, we summarize the expected physiological significance of the biotransfornation as well as Michaelis-Menten constants (Km), maximal velocities (Vmax), Vmax/Km (intrinsic clearance) values, and/or metabolic activities for 33 endogenous substrates, including (1) arachidonic acid and fatty acids, (2) steroid hormones, such as testosterone, progesterone, and allopregnanolone, (3) amines, such as tyramine, and (4) lipid-soluble vitamins, such as retinol and vitamin D3 analogues, mediated human P450 isoforms consisting of so-called drug-metabolizing enzymes for the purpose of predicting the key enzyme(s) in vivo. Arachidonic acid is metabolized via the epoxidation and omega-hydroxylation to many biologically active eicosanoids such as epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids by multiple P450 isoforms including CYP2C, CYP2E1 and CYP4A11. CYP2D in the brain may be involved in the metabolism of neuronal amines and steroids and in the regulation of the central nervous system. CYP1A2 and CYP3A4 appear to be the major P450 enzymes catalyzing the oxidation of all-trans-retinol to all-trans-retinoic acid in human liver, and CYP3A4 is one of the vitamin D3 25-hydroxylases. Although the significance of the contribution is still unknown in detail, the collective findings provide fundamental and useful information for the biological contribution of the metabolism of endogenous substances by drug-metabolizing enzymes, P450s. In addition, genetic polymorphism of these drug-metabolizing P450s may affect the metabolism of the endobiotics. Forthermore, these findings imply that xenobiotic oxidations by P450 enzymes are affected by endobiotic molecules and that the endobiotic-xenobiotic interactions as well as drug-drug interactions or drug-food/beverage interactions may be of great importance when understanding the basis for pharmacological and toxicological actions of a number of xenobiotic chemicals.

Role of the solvent-diffusion-rate modifier in a new emulsion solvent diffusion method for preparation of ketoprofen microspheres

A new emulsion solvent diffusion method to prepare the microspheres of ketoprofen with an acrylic polymer was developed by utilizing sugar esters as solvent diffusion modifiers. The microspheres were produced via transient o/w emulsion droplets of the polymer, which was formed by the interaction of drug and water-miscible organic solvent, e.g. ethanol. The solvent consisting in oil droplets diffused into the outer aqueous medium. In the droplets, ethanol interacted with ketoprofen via hydrogen bonding between -OH group of ethanol and both -COOH and = CO groups of ketoprofen. These hydrogen bonds made ethanol solution strongly hydrophobic. The sugar ester added in the ethanol could inhibit such intermolecular interaction between ethanol and the = CO group of ketoprofen. Modulation in the binding force of ketoprofen-ethanol by the sugar ester contributed to achieving a desirable initial ethanol diffusion rate from the oil droplets for the formation of ketoprofen microspheres with high yield (> 85 per cent) and drug entrapment ratio (> 90 per cent).

Regioselective hydroxylation of steroid hormones by human cytochromes P450

Abstract This article reviews in vitro metabolic activities [including Michaelis constants (Km), maximal velocities (Vmax) and Vmax/Km] and drug–steroid interactions [such as induction and cooperativity (activation)] of cytochromes P450 (P450 or CYP) in human tissues, including liver and adrenal gland, for 14 kinds of endogenous steroid compounds, including allopregnanolone, cholesterol, cortisol, cortisone, dehydroepiandrosterone, estradiol, estrone, pregnenolone, progesterone, testosterone and bile acids (cholic acid). First, we considered the drug-metabolizing P450s. 6β-Hydroxylation of many steroids, including cortisol, cortisone, progesterone and testosterone, was catalyzed primarily by CYP3A4. CYP1A2 and CYP3A4, respectively, are likely the major hepatic enzymes responsible for 2-/4-hydroxylation and 16α-hydroxylation of estradiol and estrone, steroids that can contribute to breast cancer risk. In contrast, CYP1A1 and CYP1B1 predominantly metabolized estrone and estradiol to 2- and 4-catechol estrogens, which are endogenous ultimate carcinogens if formed in the breast. Some metabolic activities of CYP3A4, including dehydroepiandrosterone 7β-/16α-hydroxylation, estrone 2-hydroxylation and testosterone 6β-hydroxylation, were higher than those for polymorphically expressed CYP3A5. Next, we considered typical steroidogenic P450s. CYP17A1, CYP19A1 and CYP27A1 catalyzed steroid synthesis, including hydroxylation at 17α, 19 and 27 positions, respectively. However, it was difficult to predict which hepatic drug-metabolizing P450 or steroidogenic P450 will be mainly responsible for metabolizing each steroid hormone in vivo based on these results. Further research is required on the metabolism of steroid hormones by various P450s and on prediction of their relative contributions to in vivo metabolism. The findings collected here provide fundamental and useful information on the metabolism of steroid compounds.

Comparison of Cytochrome P450 2D6 and Variants in Terms of Drug Oxidation Rates and Substrate Inhibition

This review focuses on identification of the important active site residues of CYP2D6 in terms of CYP2D6 polymorphism. A meta-analysis was performed on the reported literature regarding (1) values of the Michaelis-Menten constant (K(m)), maximal velocity (V(max)), and intrinsic clearance (V(max)/K(m)) for 41 metabolic reactions of 31 substrates mediated by human cytochrome P450 2D6 and its variants and mutants and (2) inhibition constants (K(i)) for 15 inhibitors. The mean ratios of V(max)/K(m) values with respect to the wild type (CYP2D6.1) for CYP2D6.2 (R296C/S486T), CYP2D6.10 (P34S/S486T), CYP2D6.17 (T107I/R296C/S486T), CYP2D6.31 (R296C/R440H/S486T), CYP2D6.34 (R296C), CYP2D6.36 (P34S/S486T and 6 other amino acids substitutions), CYP2D6.49 (P34S /F120I/S486T), and P34S and G42R mutants but not CYP2D6.39 (S486T) were in the range 0.03-0.61, and the median ratios were in the range 0.03-0.57. More than 90% of V(max)/K(m) values for CYP2D6.10, CYP2D6.17, and CYP2D6.36 were less than half of those for CYP2D6.1. In addition, 20-59% of V(max)/K(m) values for these variants were less than one-tenth those of the wild type. These results suggest that the CYP2D6 polymorphism may affect the metabolic activities of many compounds. However, the kinetic behaviors of these variants and mutants depended on the metabolic reaction. The K(i) values of many of the inhibitors of CYP2D6.10 and CYP2D6.17 were comparable with or higher than those for CYP2D6.1. Collectively, these findings provide insights into the contributions of CYP2D6 polymorphisms to drug metabolism and adverse drug interactions.