Safety and Pharmacokinetics of Bendamustine Rapid-Infusion Formulation.

Bendamustine hydrochloride (BDM) is approved in the United States to treat chronic lymphocytic leukemia and relapsed indolent B-cell non-Hodgkin lymphoma. The first formulation marketed in the United States (original BDM) was a lyophilized product requiring reconstitution prior to dilution to the final admixture. A liquid formulation of BDM was subsequently introduced that did not require reconstitution before dilution. Both formulations are administered as a 500 mL admixture with a recommended infusion time of 30 or 60 minutes for chronic lymphocytic leukemia and indolent B-cell non-Hodgkin lymphoma, respectively. A newer liquid BDM formulation (rapid BDM) is a ready-to-dilute solution not requiring reconstitution that dilutes into an admixture of only 50 mL and can be safely administered in a shorter infusion time (10 minutes). Rapid BDM admixture also has longer stability at room temperature than both lyophilized and liquid BDM formulations (6 vs 2 to 3 hours). This phase 1, open-label, randomized, crossover (3-period, partially replicated) study, conducted in "end-of-life" cancer patients at 10 oncology centers in the United States, demonstrates that rapid BDM is bioequivalent to original BDM as determined by area under the curve. Expected differences in maximum plasma concentration and time to maximum plasma concentration were observed between study treatments, given the substantially shorter infusion time of rapid BDM. No clinically relevant differences in other evaluated pharmacokinetic parameters were found. Rapid BDM infusions were safe and tolerable for cancer patients in this study. The overall safety profiles of the 2 BDM formulations were comparable, with no new safety signals identified and no differences in infusion-related adverse events.

Discovery of new potentially defective alleles of human CYP2C9.

CYP2C9 is a clinically important enzyme, responsible for the metabolism of numerous clinically important therapeutic drugs. In the present study, we discovered 38 single nucleotide polymorphisms in CYP2C9 by resequencing of genomic DNA from 92 individuals from three different racial groups. Haplotype analysis predicted that there are at least 21 alleles of CYP2C9 in this group of individuals. Six new alleles were identified that contained coding changes: L19I (CYP2C9*7), R150H (CYP2C9*8), H251R (CYP2C9*9), E272G (CYP2C9*10), R335W(CYP2C9*11) and P489S (CYP2C9*12). When expressed in a bacterial cDNA expression system, several alleles exhibited altered catalytic activity. CYP2C9*11 appeared to be a putative poor metabolizer allele, exhibiting a three-fold increase in the Km and more than a two-fold decrease in the intrinsic clearance for tolbutamide. Examination of the crystal structure of human CYP2C9 reveals that R335 is located in the turn between the J and J' helices and forms a hydrogen-bonding ion pair with D341 from the J' helix. Abolishing this interaction in CYP2C9*11 individuals could destabilize the secondary structure and alter the substrate affinity. This new putative poor metabolizer (PM) allele was found in Africans. A second potentially PM allele CYP2C9*12 found in a racially unidentified sample also exhibited a modest decrease in the Vmax and the intrinsic clearance for tolbutamide in a recombinant system. Further clinical studies are needed to determine the effect of these new polymorphisms on the metabolism of CYP2C9 substrates.

Differential metabolism of acrylonitrile to cyanide is responsible for the greater sensitivity of male vs female mice: role of CYP2E1 and epoxide hydrolases.

Acrylonitrile (AN) is a potent toxicant and a known rodent carcinogen. AN epoxidation to cyanoethylene oxide (CEO) via CYP2E1 and its subsequent metabolism via epoxide hydrolases (EH) to yield cyanide is thought to be responsible for the acute toxicity and mortality of AN. Recent reports showed that male mice are more sensitive than females to the acute toxicity/mortality of AN. The present work was undertaken to assess the metabolic and enzymatic basis for the greater sensitivity of male vs female mice to AN toxicity. Male and female wild-type and CYP2E1-null mice received AN at 0, 2.5, 10, 20, or 40 mg/kg by gavage. Cyanide concentrations were measured at 1 or 3 h after dosing. Current data demonstrated that cyanide levels in blood and tissues of AN-treated wild-type mice of both sexes were significantly greater than in vehicle-treated controls and increased in a dose-dependent manner. In contrast, cyanide levels in AN-treated CYP2E1-null mice were not statistically different from those measured in vehicle-treated controls. Furthermore, higher levels of cyanide were detected in male wild-type mice vs females in association with greater sensitivity of males to the acute toxicity/mortality of this chemical. Using Western blot analysis, negligible difference in CYP2E1 expression with higher levels of soluble and microsomal EH (sEH and mEH) was detected in the liver of male vs female mice. In kidneys, male mice exhibited higher expression of both renal CYP2E1 and sEH than did female mice. In conclusion, higher blood and tissue cyanide levels are responsible for the greater sensitivity of male vs female mice to AN. Further, higher expression of CYP2E1 and EH in male mice may contribute to greater formation of CEO and its subsequent metabolism to yield cyanide, respectively.

Genetic findings and functional studies of human CYP3A5 single nucleotide polymorphisms in different ethnic groups.

OBJECTIVES: Genetic polymorphisms of cytochromes P450 (CYPs) are a principal reason for inter-individual variations in the metabolism of therapeutic drugs and environmental chemicals in humans. The present study identifies 34 single nucleotide polymorphisms (SNPs) of CYP3A5 including 27 previously unidentified SNPs by direct sequencing of the exons, intron-exon junctions and 5'-upstream region of CYP3A5 from 92 racially diverse individuals (24 Caucasians, 24 Africans, 24 Asians, and 20 individuals of unknown racial origin). RESULTS: Four new CYP3A5 SNPs produced coding changes: R28C, L82R, A337T, and F446S. CYP3A5 R28C occurred in African populations (allelic frequency of 4%). CYP3A5 A337T occurred in Asians (2% allelic frequency), CYP3A5 L82R (occurred in the racially unidentified group) and CYP3A5 F446S (identified in Caucasians with a 2% allelic frequency) were on an allele containing the splice change g.6986A>G known as CYP3A5*3. The newly identified allelic proteins were constructed by site-directed mutagenesis, expressed in Escherichia coli and purified. CYP3A5 L82R was expressed only as denatured CYP420, suggesting it may be unstable. CYP3A5*1 exhibited the highest maximal clearance for testosterone followed by CYP3A5 A337T > CYP3A5 R28C >> CYP3A5 F446S. CYP3A5*1 exhibited a higher V(max) for nifedipine oxidation than CYP3A5 A337T > CYP3A5 R28C >> CYP3A5 F446S. CYP3A5 A337T and CYP3A5 R28C exhibited a 42-64% lower V(max) for nifedipine oxidation than CYP3A5*1. CYP3A5 F446S exhibited a > 95% decrease in the intrinsic clearance for both 6beta-hydroxytestosterone and nifedipine oxidation. CONCLUSION: This study identifies four new potentially defective coding alleles. CYP3A5 F446S is predicted to be more catalytically defective than the splice change alone.

Identification and functional characterization of new potentially defective alleles of human CYP2C19.

CYP2C19 is a clinically important enzyme responsible for the metabolism of a number of therapeutic drugs, such as mephenytoin, omeprazole, diazepam, proguanil, propranolol and certain antidepressants. Genetic polymorphisms in this enzyme result in poor metabolizers of these drugs. There are racial differences in the incidence of the poor metabolizer trait, which represents 13-23% of Asians but only 3-5% of Caucasians. In this study, single nucleotide polymorphisms (SNPs) in CYP2C19 were identified by direct sequencing of genomic DNA from 92 individuals from three different racial groups of varied ethnic background, including Caucasians, Asians and blacks. Several new alleles were identified containing the coding changes Arg114 His (CYP2C19*9), Pro227 Leu (CYP2C19*10), Arg150 His (CYP2C19*11), stop491 Cys (CYP2C19*12), Arg410 Cys (CYP2C19*13), Leu17 Pro (CYP2C19*14) and Ile19 Leu (CYP2C19*15). When expressed in a bacterial cDNA expression system, CYP2C19*9 exhibited a modest decrease in the V(max) for 4'-hydroxylation of -mephenytoin, and no alteration in its affinity for reductase. CYP2C19*10 exhibited a dramatically higher K(m) and lower V(max) for mephenytoin. CYP2C19*12was unstable and expressed poorly in a bacterial cDNA expression system. Clinical studies will be required to confirm whether this allele is defective in vivo. CYP2C19*9, CYP2C19*10 and CYP2C19*12 all occurred in African-Americans, or individuals of African descent, and represent new potentially defective alleles of CYP2C19 which are predicted to alter risk of these populations to clinically important drugs.

Cytochrome P450 2E1 (CYP2E1) is essential for acrylonitrile metabolism to cyanide: comparative studies using CYP2E1-null and wild-type mice.

Acrylonitrile (AN) is a rodent carcinogen and suspected human carcinogen. Metabolism of AN proceeds via conjugation with glutathione or epoxidation via cytochrome P4502E1 (CYP2E1) to cyanoethylene oxide (CEO). It was hypothesized that CEO metabolism via epoxide hydrolase (EH) is the primary pathway for cyanide formation. The objective of this work is to assess the enzymatic basis of metabolism to cyanide. Male wild-type and CYP2E1-null mice received 0, 2.5, 10, 20, or 40 mg of AN/kg by gavage, and cyanide was measured in blood and tissues. CYP2E1 and EH expression were assessed using Western blot analyses. Present results demonstrated that cyanide concentrations in blood and tissues of AN-treated wild-type mice were higher at 1 versus 3 h, increased in a dose-dependent manner, and were significantly higher in AN-treated versus vehicle-treated mice. In contrast, cyanide concentrations in the blood and tissues of AN-treated CYP2E1-null mice were not statistically different from those of vehicle-treated mice. Furthermore, this work showed that EH is expressed in CYP2E1-null and wild-type mice. In conclusion, under the current experimental conditions using CYP2E1-null mice, current work demonstrated for the first time that CYP2E1-mediated oxidation is a prerequisite for AN metabolism to cyanide. Since earlier studies showed that CYP2E1 is the only enzyme responsible for AN epoxidation, it is concluded that AN metabolism to CEO is a prerequisite for cyanide formation, and this pathway is exclusively catalyzed by CYP2E1. Finally, this work confirmed that cyanide plays an essential role in the causation of the acute toxicity/mortality of AN.

Effect of methacrylonitrile on cytochrome P-450 2E1 (CYP2E1) expression in male F344 rats.

Tissue-specific induction of cytochrome P-450s (CYP) followed by increased in situ bioactivation may contribute to chemical-induced site-specific toxicity. In rats, methacrylonitrile (MAN) is metabolized by cytochrome P-450 2E1 (CYP2E1) to acetone, which is eliminated along with parent MAN in breath. Gavage administration of MAN to rats causes olfactory epithelial damage and liver enlargement. It was hypothesized that treatment of rats with MAN may result in differential expression of CYP2E1 in tissues leading to tissue-specific toxicity via increased in situ formation of cytotoxic MAN metabolites. In this study, male F344 rats received 60 mg MAN/kg and were sacrificed 6, 12, or 24 h after a single dose, or 24 h after 7 consecutive daily doses. Liver, lung, and nasal tissues were collected. Reverse-transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining were used to assess CYP2E1 expression and localization, and chlorzoxazone hydroxylation was used as a measure of CYP2E1 catalytic activity. Present results showed that CYP2E1 mRNA was increased in lung and nasal tissues with minimal effects in liver of MAN-treated rats. Induction of CYP2E1 protein expression was detected in lung. CYP2E1 activity was higher in liver and lung microsomes from MAN-treated rats when compared to control animals. To compare the effects of MAN and acetone, male F344 rats received a single acetone dose (5 ml/kg) by gavage. After 12 h, acetone treatment resulted in a significant increase in the levels of CYP2E1 mRNA and protein in lung and nasal tissues, with no obvious change noted in the liver. Overall, these data suggest that administration of MAN to rats causes increased expression of CYP2E1 in lung, liver, and nasal tissues. These results also show that acetone induces the expression of CYP2E1 at both the mRNA and protein levels in rat nasal and lung tissues. In conclusion, MAN increased the expression of CYP2E1, and this effect varied as a function of time, length of exposure, and tissue examined. While the damage in the olfactory mucosa due to MAN treatment may not be explained by the observed induction of CYP2E1, it is possible that other CYPs may play a role in the in situ bioactivation of MAN.

Does the skeleton of a sponge provide a defense against predatory reef fish?

Sponge tissue often contains two structural components in high concentrations: spicules of silica, and refractory fibers of protein (spongin). Some terrestrial plants contain analogous structures, siliceous inclusions and refractory lignins, that have been demonstrated to deter herbivory. We performed feeding experiments with predatory reef fish to assess the deterrent properties of the structural components of three common Caribbean demosponges, Agelas clathrodes, Ectyoplasia ferox, and Xestospongia muta. The concentrations of spicules and spongin in the tissues varied widely between the three species, but when assayed at their natural volumetric concentrations, neither spicules (all three species assayed) nor the intact spiculated spongin skeleton (A. clathrodes and X. muta assayed) deterred feeding by reef fish in aquarium or field assays using prepared foods of a nutritional quality similar to, or higher than, that of sponge tissue. Spicules deterred feeding in aquarium assays when incorporated into prepared foods of a nutritional quality lower than that of sponge tissue (15-19 times less protein), but spiculated spongin skeleton was still palatable, even in prepared foods devoid of measurable protein, and even though spicules embedded in spongin were oriented in their natural conformation. Based on comparisons of the nutritional qualities of the tissues of the three sponge species and of the prepared foods, sponge tissue would have to be much lower in food value (5 times less protein or lower) for spicules to provide an effective defense, and spicules in combination with the spongin skeleton would be unlikely to provide an effective defense regardless of the nutritional quality of the tissue. Unlike terrestrial plants, marine sponges may use silica and refractory fibers solely for structural purposes.