First-in-class MKK4 inhibitors enhance liver regeneration and prevent liver failure.

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.

Molybdenum Cofactor Catabolism Unravels the Physiological Role of the Drug Metabolizing Enzyme Thiopurine S-Methyltransferase.

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt-/- ) mice as well as human liver cytosol. Urothione levels were quantified by liquid-chromatography tandem mass spectrometry in the kidneys and urine of mice. TPMT-genotype/phenotype and excretion levels of urothione were investigated in human samples and validated in an independent population-based study. As Moco provides a physiological substrate (thiopterin) of TPMT, thiopterin-methylating activity was associated with TPMT activity determined with its drug substrate (6-thioguanin) in mice and humans. Urothione concentration was extremely low in the kidneys and urine of Tpmt-/- mice. Urinary urothione concentration in TPMT-deficient patients depends on common TPMT polymorphisms, with extremely low levels in homozygous variant carriers (TPMT*3A/*3A) but normal levels in compound heterozygous carriers (TPMT*3A/*3C) as validated in the population-based study. Our work newly identified an endogenous substrate for TPMT and shows an unprecedented link between Moco catabolism and drug metabolism. Moreover, the TPMT example indicates that phenotypic consequences of genetic polymorphisms may differ between drug- and endogenous substrates.

Pharmacogenetics of UGT1A4, UGT2B7 and UGT2B15 and Their Influence on Tamoxifen Disposition in Asian Breast Cancer Patients.

Tamoxifen (TAM) is an established endocrine treatment for all stages of oestrogen receptor (ER)-positive breast cancer. Its complex metabolism leads to the formation of multiple active and inactive metabolites. One of the main detoxification and elimination pathways of tamoxifen and its active metabolites, 4-hydroxytamoxifen (4-OHT) and endoxifen, is via glucuronidation catalysed by uridine 5'-diphospho-glucuronosyltransferases (UGTs). However, few studies have comprehensively examined the impact of variations in the genes encoding the major hepatic UGTs on the disposition of tamoxifen and its metabolites. In the present study, we systematically sequenced exons, exon/intron boundaries, and flanking regions of UGT1A4, UGT2B7 and UGT2B15 in 240 healthy subjects of different Asian ethnicities (Chinese, Malays and Indians) to identify haplotype tagging single nucleotide polymorphisms. Subsequently, 202 Asian breast cancer patients receiving tamoxifen were genotyped for 50 selected variants in the three UGT genes to comprehensively investigate their associations with steady-state plasma levels of tamoxifen, its active metabolites and their conjugated counterparts. The UGT1A4 haplotype (containing variant 142T>G, L48 V defining the *3 allele) was strongly associated with higher plasma levels of TAM-N-glucuronide, with a twofold higher metabolic ratio of TAM-N-glucuronide/TAM observed in carriers of this haplotype upon covariate adjustment (P < 0.0001). Variants in UGT2B7 were not associated with altered O-glucuronidation of both 4-OHT and endoxifen, while UGT2B15 haplotypes had a modest effect on (E)-endoxifen plasma levels after adjustment for CYP2D6 genotypes. Our findings highlight the influence of UGT1A4 haplotypes on tamoxifen disposition in Asian breast cancer patients, while genetic variants in UGT2B7 and UGT2B15 appear to be of minor importance.

Association of CYP2C19*2 and associated haplotypes with lower norendoxifen concentrations in tamoxifen-treated Asian breast cancer patients.

AIM: The aim was to examine the influence of CYP2C19 variants and associated haplotypes on the disposition of tamoxifen and its metabolites, particularly norendoxifen (NorEND), in Asian patients with breast cancer. METHODS: Sixty-six CYP2C19 polymorphisms were identified in healthy Asians (n = 240), of which 14 were found to be tightly linked with CYP2C19*2, CYP2C19*3 and CYP2C19*17. These 17 SNPs were further genotyped in Asian breast cancer patients receiving tamoxifen (n = 201). Steady-state concentrations of tamoxifen and its metabolites were quantified using liquid chromatography­mass spectrometry. Non-parametric tests and regression methods were implemented to evaluate genotypic­phenotypic associations and haplotypic effects of the SNPs. RESULTS: CYP2C19 functional polymorphisms and their linked SNPs were not significantly associated with plasma concentrations of tamoxifen and its main metabolites N-desmethyltamoxifen, (Z)-4-hydroxytamoxifen and (Z)-Endoxifen. However, CYP2C19*2 and its seven linked SNPs were significantly associated with lower NorEND concentrations, MRNorEND/NDDM and MRNorEND/(Z)-END. Specifically, patients carrying the CYP2C19*2 variant allele A had significantly lower NorEND concentrations [median (range), GG vs. GA vs. AA: 1.51 (0.38­3.28) vs. 1.28 (0.30­3.36) vs. 1.15 ng ml−1 (0.26­2.45, P = 0.010)] as well as significantly lower MRNorEND/(Z)-END [GG vs. GA vs. AA: 9.40 (3.27­28.35) vs. 8.15 (2.67­18.9) vs. 6.06 (4.47­14.6), P < 0.0001] and MRNorEND/NDDM [GG vs. GA vs. AA: 2.75 (0.62­6.26) vs. 2.43 (0.96­4.18) vs. 1.75 (1.10­2.49), P < 0.00001]. CYP2C19 H2 haplotype, which included CYP2C19*2, was also significantly associated with lower NorEND concentrations (P = 0.0020), MRNorEND/NDDM (P < 0.0001) and MRNorEND/(Z)-END (P < 0.0001), indicating significantly lower formation rates of NorEND. CONCLUSION: These data highlight the potential relevance of CYP2C19 pharmacogenetics in influencing NorEND concentrations in tamoxifen-treated patients, which may influence treatment outcomes.

Breast cancer treatment outcome with adjuvant tamoxifen relative to patient CYP2D6 and CYP2C19 genotypes.

PURPOSE: The clinical outcome of tamoxifen-treated breast cancer patients may be influenced by the activity of cytochrome P450 enzymes that catalyze the formation of antiestrogenic metabolites endoxifen and 4-hydroxytamoxifen. We investigated the predictive value of genetic variants of CYP2D6, CYP2C19, and three other cytochrome P450 enzymes for tamoxifen treatment outcome. PATIENTS AND METHODS: DNA from 206 patients receiving adjuvant tamoxifen monotherapy and from 280 patients not receiving tamoxifen therapy (71 months median follow-up) was isolated from archival material and was genotyped for 16 polymorphisms of CYP2D6, CYP2C19, CYP2B6, CYP2C9, and CYP3A5 by matrix-assisted, laser desorption/ionization, time-of-flight mass spectrometry, and by copy number quantification. Risk and survival estimates were calculated using logistic regression, Kaplan-Meier, and Cox regression analyses. RESULTS: Tamoxifen-treated patients carrying the CYP2D6 alleles *4, *5, *10, *41-all associated with impaired formation of antiestrogenic metabolites-had significantly more recurrences of breast cancer, shorter relapse-free periods (hazard ratio [HR], 2.24; 95% CI, 1.16 to 4.33; P = .02), and worse event-free survival rates (HR, 1.89; 95% CI, 1.10 to 3.25; P = .02) compared with carriers of functional alleles. Patients with the CYP2C19 high enzyme activity promoter variant *17 had a more favorable clinical outcome (HR, 0.45; 95% CI, 0.21 to 0.92; P = .03) than carriers of *1, *2, and *3 alleles. CONCLUSION: Because genetically determined, impaired tamoxifen metabolism results in worse treatment outcomes, genotyping for CYP2D6 alleles *4, *5, *10, and *41 can identify patients who will have little benefit from adjuvant tamoxifen therapy. In addition to functional CYP2D6 alleles, the CYP2C19 *17 variant identifies patients likely to benefit from tamoxifen.

Cytokeratin-18 is a useful serum biomarker for early determination of response of breast carcinomas to chemotherapy.

PURPOSE: With a widening arsenal of cancer therapies available, it is important to develop therapy-specific predictive markers and methods to rapidly assess treatment efficacy. We here evaluated the use of cytokeratin-18 (CK18) as a serum biomarker for monitoring chemotherapy-induced cell death in breast cancer. EXPERIMENTAL DESIGN: Different molecular forms of CK18 (caspase cleaved and total) were assessed by specific ELISA assays. Drug-induced release of CK18 was examined from breast carcinoma cells and tissue. CK18 protein composition was examined in serum. CK18 levels were determined in serum from 61 breast cancer patients during docetaxel or cyclophosphamide/epirubicin/5-fluorouracil (CEF) therapy. RESULTS: Caspase-cleaved CK18 molecules were released from monolayer cultures and tumor organ cultures to the extracellular compartment. CK18 was present in complexes with other cytokeratins in serum. Such CK18 protein complexes are remarkably stable, leading to favorable performance of CK18 biomarker assays for clinical investigations. Docetaxel induced increased levels of caspase-cleaved CK18 in serum from breast cancer patients, indicating apoptosis. CEF therapy led to increases predominantly in uncleaved CK18, indicating induction of necrotic cell death in many tumors. The increase in total CK18 at 24 h of the first treatment cycle correlated to the clinical response to CEF therapy (P < 0.0001). CONCLUSIONS: Induction of necrotic cell death may explain the clinical efficacy of anthracycline-based therapy for breast carcinomas with defective apoptosis pathways. We suggest that CK18 biomarkers are useful for early prediction of the response to CEF therapy in breast cancer and may be useful biomarkers for clinical trials.