Thomas F. Kalhorn

Maintenance of Intratumoral Androgens in Metastatic Prostate Cancer: A Mechanism for Castration-Resistant Tumor Growth

Therapy for advanced prostate cancer centers on suppressing systemic androgens and blocking activation of the androgen receptor (AR). Despite anorchid serum androgen levels, nearly all patients develop castration-resistant disease. We hypothesized that ongoing steroidogenesis within prostate tumors and the maintenance of intratumoral androgens may contribute to castration-resistant growth. Using mass spectrometry and quantitative reverse transcription-PCR, we evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. Testosterone levels within metastases from anorchid men [0.74 ng/g; 95% confidence interval (95% CI), 0.59-0.89] were significantly higher than levels within primary prostate cancers from untreated eugonadal men (0.23 ng/g; 95% CI, 0.03-0.44; P < 0.0001). Compared with primary prostate tumors, castration-resistant metastases displayed alterations in genes encoding steroidogenic enzymes, including up-regulated expression of FASN, CYP17A1, HSD3B1, HSD17B3, CYP19A1, and UGT2B17 and down-regulated expression of SRD5A2 (P < 0.001 for all). Prostate cancer xenografts derived from castration-resistant tumors maintained similar intratumoral androgen levels when passaged in castrate compared with eugonadal animals. Metastatic prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival. We conclude that intracrine steroidogenesis may permit tumors to circumvent low levels of circulating androgens. Maximal therapeutic efficacy in the treatment of castration-resistant prostate cancer will require novel agents capable of inhibiting intracrine steroidogenic pathways within the prostate tumor microenvironment.

Contribution of CYP2E1 and CYP3A to acetaminophen reactive metabolite formation

CYP2E1, 1A2, and 3A4 have all been implicated in the formation of N‐acetyl‐p‐benzoquinone imine (NAPQI), the reactive intermediate of acetaminophen (INN, paracetamol), in studies in human liver microsomes and complementary deoxyribonucleic acid–expressed enzymes. However, recent pharmacokinetic evidence in humans has shown that the involvement of CYP1A2 is negligible in vivo. The purpose of this study was to evaluate the respective roles of CYP2E1 and 3A4 in vivo.

Intestinal and Hepatic CYP3A4 Catalyze Hydroxylation of 1α,25-Dihydroxyvitamin D3: Implications for Drug-Induced Osteomalacia

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D3 metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23- and 24-hydroxylation of 1,25(OH)2D3. No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH)2D3 hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH)3D3, 1,24S,25(OH)3D3, and 1,23S,25(OH)3D3. Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH)2D3 23- and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH)2D3 metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.

Dose‐dependent pharmacokinetics of acetaminophen: Evidence of glutathione depletion in humans

The time course of excretion of acetaminophen and its metabolites in urine was determined in eight healthy adults (seven men and one woman) who ingested 1 gm of the drug and collected timed urine samples for 24 hours. The mean time of peak excretion rate was 1.3 to 3.7 hours for acetaminophen, its glucuronide, sulfate, cysteine, mercapturate, and methoxy metabolites but 13.5 hours for methylthioacetaminophen. The mean half‐life of acetaminophen was 3.1 hours and the mean half‐life of the metabolites other than methylthioacetaminophen ranged from 4.1 to 5.7 hours. The half‐life of methylthiometabolite could not be determined because of its very late peak time. In a second study the effect of dose on the clearance of acetaminophen was determined in nine healthy adult subjects (eight men and one woman) who received doses of 0.5 and 3 gm acetaminophen on separate occasions, separated by 4 to 10 days. The renal clearance of acetaminophen and the formation clearances of the sulfate, glutathione, and catechol metabolites were lower (by 38%, 41%, 35%, and 46%, respectively) at the higher dose. The renal clearance of acetaminophen sulfate and glucuronide conjugates were not different between doses. In a third study (10 men), 10 gm N‐acetylcysteine was found to increase the formation clearance of the sulfate conjugate by 27% and that of the glutathione conjugate by 10%. The data suggest that the hepatic supply of reduced glutathione and 3′‐phosphoadenosine 5′‐phosphosulfate begins to be depleted over the range of 0.5 to 3 gm acetaminophen and that the depletion is overcome by the administration of N‐acetylcysteine.

Inhibition and induction of cytochrome P4502E1‐catalyzed oxidation by isoniazid in humans

We studied the effect of isoniazid administration on the cytochrome P4502E1—catalyzed elimination of chlorzoxazone and acetaminophen. Isoniazid, 300 mg daily, was administered for 7 days to a group of 10 volunteer slow acetylators. Acetaminophen, 500 mg, and chlorzoxazone, 750 mg, were administered on separate occasions before isoniazid, during the period of isoniazid administration, and after the discontinuation of isoniazid. Isoniazid inhibited the clearance of chlorzoxazone by 58%, as assessed from plasma data, and inhibited the formation of acetaminophen thioether metabolites (a measure of the formation of the hepatotoxin N‐acetyl‐p‐benzoquinone imine and catechol oxidative metabolites of acetaminophen, as determined from their recovery in urine, by 63% and 49%, respectively. Two days after the discontinuation of isoniazid, the clearance of chlorzoxazone was increased over the value before isoniazid by 56%. Acetaminophen thioether but not catechol metabolites were increased by 56% 1 day after the discontinuation of isoniazid and had returned to the pre‐isoniazid value 3 days after the discontinuation of isoniazid. We conclude that the time course of the interaction with regard to chlorzoxazone elimination and formation is compatible with an inhibition‐induction effect of isoniazid on cytochrome P4502E1. The mechanism of this biphasic effect is probably induction by protein stabilization, which results in inhibition of catalytic activity while isoniazid is present.

Conditioning regimen-dependent disposition of cyclophosphamide and hydroxycyclophosphamide in human marrow transplantation patients.

PURPOSE The pharmacokinetics of cyclophosphamide (CY) and 4-hydroxycyclophosphamide (HCY) were studied in 14 patients being prepared for bone marrow transplantation with either busulfan (BU)/CY (n = 7) or CY/total-body irradiation (TBI) (n = 7) to determine whether exposure to CY and its proximate toxic metabolite HCY is modulated by other agents used in the preparative regimen. PATIENTS AND METHODS HCY was assayed by a new method that stabilized the metabolite at bedside. In BU/CY patients (who also received phenytoin), CY clearance was 112% greater (P = .0014), half-life 54% less (P = .0027), peak HCY concentration in plasma/CY dose 113% greater (P = .0006), and the ratio of area under the plasma concentration-time curves (AUCs) of HCY to CY 166% greater (P = .0116) than in CY/TBI patients. The ratio of the AUC of HCY/CY dose was 48% greater in BU/CY patients than in CY/TBI patients when one CY/TBI patient with an apparent impaired ability to eliminate HCY was excluded from analysis. In CY/TBI patients, there was an inverse correlation between the AUC of HCY and that of CY (R2 = .740, P = .028). Also, the ratio of the AUC of HCY/CY dose was correlated with the average concentration of BU at steady-state (Css, Bu) (R2 = .646, P = 0.29). Variability in CY and HCY pharmacokinetics among the 14 patients overall was pronounced, with the highest variability (15-fold) observed in the ratio of the AUC of HCY to that of CY. CONCLUSION Prior administration of BU and/or phenytoin significantly alters exposure to CY and HCY. Interpatient variability in HCY exposure at a given CY dose is substantial.

Biosynthesis and IroC‐dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium

In response to iron deprivation, Salmonella enterica serovar Typhimurium secretes two catecholate‐type siderophores, enterobactin and its glucosylated derivative salmochelin. Although the systems responsible for enterobactin synthesis and acquisition are well characterized, the mechanisms of salmochelin secretion and acquisition, as well as its role in Salmonella virulence, are incompletely understood. Herein we show by liquid chromatography‐mass spectrometry analysis of culture supernatants from wild type and isogenic mutant bacterial strains that the Major Facilitator Superfamily pump EntS is the major exporter of enterobactin and the ABC transporter IroC exports both salmochelin and enterobactin. Growth promotion experiments demonstrate that IroC is not required for utilization of Fe‐enterobactin or Fe‐salmochelin, as had been previously suggested, but the ABC transporter protein FepD is required for utilization of both siderophores. Salmonella mutants deficient in salmochelin synthesis or secretion exhibit reduced virulence during systemic infection of mice.

The effect of omeprazole pretreatment on acetaminophen metabolism in rapid and slow metabolizers of S‐mephenytoin

This study evaluates the cost‐effectiveness of vancomycin serum concentration monitoring in patients with hematologic malignancies.Omeprazole, a widely used and potent gastric proton pump inhibitor, induces cytochrome P450 (CYP) 1A2 in humans. Induction is most pronounced in slow metabolizers of S-mephenytoin because CYP2C19 (S-mephenytoin hydroxylase) is responsible for the elimination of omeprazole. Acetaminophen (INN, paracetamol), a widely used and effective analgesic and antipyretic agent, causes serious hepatic and renal toxicity at high doses by conversion of acetaminophen to the toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI) through CYP1A2, CYP2E1, and CYP3A4. This study evaluated whether omeprazole pretreatment in five rapid and five slow metabolizers of S-mephenytoin could increase thioether (an estimate of NAPQI production) metabolite formation from acetaminophen. The results of this study show that, despite induction of CYP1A2 activity in slow metabolizers (a 75% increase in plasma clearance of caffeine), the formation of NAPQI from acetaminophen was not increased after 7 days of omeprazole administration (40 mg/day). This suggests that induction of CYP1A2 activity by omeprazole is unlikely to increase the risk of acetaminophen hepatotoxicity.

Metabolism of dapsone to its hydroxylamine by CYP2E1 in vitro and in vivo

Dapsone toxicity is putatively initiated by formation of a hydroxylamine metabolite by cytochromes P450. In human liver microsomes, the kinetics of P450‐catalyzed N‐oxidation of dapsone were biphasic, with the Michaelis‐Menten constants of 0.14 ± 0.05 and 0.004 ± 0.003 mmol/L and the respective maximum velocities of 1.3 ± 0.1 and 0.13 ± 0.04 nmol/mg protein/min (mean ± SEM). Troleandomycin (40 μmol/L) inhibited hydroxylamine formation at 100 μmol/L dapsone by 50%; diethyldithiocarbamate (150 μmol/L) and tolbutamide (400 μmol/L) inhibited at 5 μmol/L dapsone by 50% and 20%, respectively, suggesting that the low‐affinity isozyme is CYP3A4 and the high‐affinity isozymes are 2E1 and 2C. Disulfiram, 500 mg, 18 hours before a 100 mg oral dose of dapsone in healthy volunteers, diminished area under the hydroxylamine plasma concentration‐time curve by 65%, apparent formation clearance of the hydroxylamine by 71%, and clearance of dapsone by 26%. Disulfiram produced a 78% lower concentration of methemoglobin 8 hours after dapsone.

Pharmacokinetics of cyclophosphamide and its metabolites in bone marrow transplantation patients

To characterize the pharmacokinetics of cyclophosphamide and 5 of its metabolites in bone marrow transplant patients and to identify the mechanism of the increase in 4‐hydroxycyclophosphamide area under the plasma concentration‐time curve (AUC) from day 1 to day 2 of cyclophosphamide administration.