An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

Discovery of BLU-945, a Reversible, Potent, and Wild-Type-Sparing Next-Generation EGFR Mutant Inhibitor for Treatment-Resistant Non-Small-Cell Lung Cancer.

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment landscape for EGFR mutant (L858R and ex19del)-driven non-small-cell lung cancer (NSCLC), most patients will eventually develop resistance to TKIs. In the case of first- and second-generation TKIs, up to 60% of patients will develop an EGFR T790M mutation, while third-generation irreversible TKIs, like osimertinib, lead to C797S as the primary on-target resistance mutation. The development of reversible inhibitors of these resistance mutants is often hampered by poor selectivity against wild-type EGFR, resulting in potentially dose-limiting toxicities and a sub-optimal profile for use in combinations. BLU-945 (compound 30) is a potent, reversible, wild-type-sparing inhibitor of EGFR+/T790M and EGFR+/T790M/C797S resistance mutants that maintains activity against the sensitizing mutations, especially L858R. Pre-clinical efficacy and safety studies supported progression of BLU-945 into clinical studies, and it is currently in phase 1/2 clinical trials for treatment-resistant EGFR-driven NSCLC.

Isotretinoin and its Metabolites Alter mRNA of Multiple Enzyme and Transporter Genes In Vitro, but Downregulation of Organic Anion Transporting Polypeptide Does Not Translate to the Clinic.

Isotretinoin [13-cis-retinoic acid (13cisRA)] is widely used for the treatment of neuroblastoma and acne. It acts via regulating gene transcription through binding to retinoic acid receptors. Yet, the potential for isotretinoin to cause transcriptionally mediated drug-drug interactions (DDIs) has not been fully explored. We hypothesized that isotretinoin and its active metabolites all-trans-retinoic acid (atRA) and 4-oxo-13cisRA would alter the transcription of enzymes and transporters in the human liver via binding to nuclear receptors. The goal of this study was to define the DDI potential of isotretinoin and its metabolites resulting from transcriptional regulation of cytochrome P450 and transporter mRNAs. In human hepatocytes (n = 3), 13cisRA, atRA, and 4-oxo-13cisRA decreased OATP1B1, CYP1A2, CYP2C9, and CYP2D6 mRNA and increased CYP2B6 and CYP3A4 mRNA in a concentration-dependent manner. The EC50 values for OATP1B1 mRNA downregulation ranged from 2 to 110 nM, with maximum effect (Emax ) ranging from 0.17- to 0.54-fold. Based on the EC50 and Emax values and the known circulating concentrations of 13cisRA and its metabolites after isotretinoin dosing, a 55% decrease in OATP1B1 activity was predicted in vivo. In vivo DDI potential was evaluated clinically in participants dosed with isotretinoin for up to 32 weeks using coproporphyrin-I (CP-I) as an OATP1B1 biomarker. CP-I steady-state serum concentrations were unaltered following 2, 8, or 16 weeks of isotretinoin treatment. These data show that isotretinoin and its metabolites alter transcription of multiple enzymes and transporters in vitro, but translation of these changes to in vivo drug-drug interactions requires clinical evaluation for each enzyme. SIGNIFICANCE STATEMENT: Isotretinoin and its metabolites alter the mRNA expression of multiple cytochrome P450s (CYPs) and transporters in human hepatocytes, suggesting that isotretinoin may cause clinically significant drug-drug interactions (DDIs). Despite the observed changes in organic anion transporting polypeptide 1B1 (OATP1B1) mRNA in human hepatocytes, no clinical DDI was observed when measuring a biomarker, coproporphyrin-I. Further work is needed to determine whether these findings can be extrapolated to a lack of a DDI with CYP1A2, CYP2B6, and CYP2C9 substrates.

Determinants of Cytochrome P450 2D6 mRNA Levels in Healthy Human Liver Tissue.

Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme that exhibits large interindividual variability. Recent studies suggest that differential transcriptional regulation of CYP2D6 in part may be responsible for the variability. In this study, we characterized potential determinants of CYP 2D6  transcript levels in healthy human liver tissue samples (n = 115), including genetic polymorphisms in CYP2D6 and the genes encoding transcription regulators for CYP2D6 expression; mRNA expression of the transcription factors and their known target genes; and hepatic levels of bile acids and retinoids, agents that modulate the expression/activity of the transcription factors. Their associations with CYP2D6 mRNA levels in the tissues were examined. Results from multivariable linear regression analysis revealed CYP8B1 mRNA level and rs3892097, the single- nucleotide polymorphism defining the nonfunctional CYP2D6*4 allele, as the two most significant predictors of CYP2D6 mRNA levels in the liver tissue samples, explaining 30% of the variability.

Does In Vitro Cytochrome P450 Downregulation Translate to In Vivo Drug-Drug Interactions? Preclinical and Clinical Studies With 13-cis-Retinoic Acid.

All-trans-retinoic acid (atRA) downregulates cytochrome P450 (CYP)2D6 in several model systems. The aim of this study was to determine whether all active retinoids downregulate CYP2D6 and whether in vitro downregulation translates to in vivo drug-drug interactions (DDIs). The retinoids atRA, 13cisRA, and 4-oxo-13cisRA all decreased CYP2D6 mRNA in human hepatocytes in a concentration-dependent manner. The in vitro data predicted ~ 50% decrease in CYP2D6 activity in humans after dosing with 13cisRA. However, the geometric mean area under plasma concentration-time curve (AUC) ratio for dextromethorphan between treatment and control was 0.822, indicating a weak induction of dextromethorphan clearance following 13cisRA treatment. Similarly, in mice treatment with 4-oxo-13cisRA-induced mRNA expression of multiple mouse Cyp2d genes. In comparison, a weak induction of CYP3A4 in human hepatocytes translated to a weak in vivo induction of CYP3A4. These data suggest that in vitro CYP downregulation may not translate to in vivo DDIs, and better understanding of the mechanisms of CYP downregulation is needed.

Sources of all-trans retinal oxidation independent of the aldehyde dehydrogenase 1A isozymes exist in the postnatal testis†.

Despite the essential role of the active metabolite of vitamin A, all-trans retinoic acid (atRA) in spermatogenesis, the enzymes, and cellular populations responsible for its synthesis in the postnatal testis remain largely unknown. The aldehyde dehydrogenase 1A (ALDH1A) family of enzymes residing within Sertoli cells is responsible for the synthesis of atRA, driving the first round of spermatogenesis. Those studies also revealed that the atRA required to drive subsequent rounds of spermatogenesis is possibly derived from the ALDH1A enzymes residing within the meiotic and post-meiotic germ cells. Three ALDH1A isozymes (ALDH1A1, ALDH1A2, and ALDH1A3) are present in the testis. Although, ALDH1A1 is expressed in adult Sertoli cells and is suggested to contribute to the atRA required for the pre-meiotic transitions, ALDH1A2 is proposed to be the essential isomer involved in testicular atRA biosynthesis. In this report, we first examine the requirement for ALDH1A2 via the generation and analysis of a conditional Aldh1a2 germ cell knockout and a tamoxifen-induced Aldh1a2 knockout model. We then utilized the pan-ALDH1A inhibitor (WIN 18446) to test the collective contribution of the ALDH1A enzymes to atRA biosynthesis following the first round of spermatogenesis. Collectively, our data provide the first in vivo evidence demonstrating that animals severely deficient in ALDH1A2 postnatally proceed normally through spermatogenesis. Our studies with a pan-ALDH1A inhibitor (WIN 18446) also suggest that an alternative source of atRA biosynthesis independent of the ALDH1A enzymes becomes available to maintain atRA levels for several spermatogenic cycles following an initial atRA injection.

Inhibition of the all-trans Retinoic Acid (atRA) Hydroxylases CYP26A1 and CYP26B1 Results in Dynamic, Tissue-Specific Changes in Endogenous atRA Signaling.

All-trans retinoic acid (atRA), the active metabolite of vitamin A, is a ligand for several nuclear receptors and acts as a critical regulator of many physiologic processes. The cytochrome P450 family 26 (CYP26) enzymes are responsible for atRA clearance, and are potential drug targets to increase concentrations of endogenous atRA in a tissue-specific manner. Talarozole is a potent inhibitor of CYP26A1 and CYP26B1, and has shown some success in clinical trials. However, it is not known what magnitude of change is needed in tissue atRA concentrations to promote atRA signaling changes. The aim of this study was to quantify the increase in endogenous atRA concentrations necessary to alter atRA signaling in target organs, and to establish the relationship between CYP26 inhibition and altered atRA concentrations in tissues. Following a single 2.5-mg/kg dose of talarozole to mice, atRA concentrations increased up to 5.7-, 2.7-, and 2.5-fold in serum, liver, and testis, respectively, resulting in induction of Cyp26a1 in the liver and testis and Rar ß and Pgc 1ß in liver. The increase in atRA concentrations was well predicted from talarozole pharmacokinetics and in vitro data of CYP26 inhibition. After multiple doses of talarozole, a significant increase in atRA concentrations was observed in serum but not in liver or testis. This lack of increase in atRA concentrations correlated with an increase in CYP26A1 expression in the liver. The increased atRA concentrations in serum without a change in liver suggest that CYP26B1 in extrahepatic sites plays a key role in regulating systemic atRA exposure.

Impact of Sample Matrix on Accuracy of Peptide Quantification: Assessment of Calibrator and Internal Standard Selection and Method Validation.

Protein quantification based on peptides using LC-MS/MS has emerged as a promising method to measure biomarkers, protein drugs, and endogenous proteins. However, the best practices for selection, optimization, and validation of the quantification peptides are not well established, and the influence of different matrices on protein digestion, peptide stability, and MS detection has not been systematically addressed. The aim of this study was to determine how biological matrices affect digestion, detection, and stability of peptides. The microsomal retinol dehydrogenase (RDH11) and cytosolic soluble aldehyde dehydrogenases (ALDH1As) involved in the synthesis of retinoic acid (RA) were chosen as model proteins. Considerable differences in the digestion efficiency, sensitivity, and matrix effects between peptides were observed regardless of the target protein's subcellular localization. The precision and accuracy of the quantification of RDH11 and ALDH1A were affected by the choice of calibration and internal standards. The final method using recombinant protein calibrators and stable isotope labeled (SIL) peptide internal standards was validated for human liver. The results demonstrate that different sample matrices have peptide, time, and matrix specific effects on protein digestion and absolute quantification.

Role of Retinoic Acid-Metabolizing Cytochrome P450s, CYP26, in Inflammation and Cancer.

Vitamin A (retinol) and its active metabolite, all-trans-retinoic acid (atRA), play critical roles in regulating the differentiation, growth, and migration of immune cells. Similarly, as critical signaling molecules in the regulation of the cell cycle, retinoids are important in cancers. Concentrations of atRA are tightly regulated in tissues, predominantly by the availability of retinol, synthesis of atRA by ALDH1A enzymes and metabolism and clearance of atRA by CYP26 enzymes. The ALDH1A and CYP26 enzymes are expressed in several cell types in the immune system and in cancer cells. In the immune system, the ALDH1A and CYP26 enzymes appear to modulate RA concentrations. Consequently, alterations in the activity of ALDH1A and CYP26 enzymes are expected to change disease outcomes in inflammation. There is increasing evidence from various disease models of intestinal and skin inflammation that treatment with atRA has a positive effect on disease markers. However, whether aberrant atRA concentrations or atRA synthesis and metabolism play a role in inflammatory disease development and progression is not well understood. In cancers, especially in acute promyelocytic leukemia and neuroblastoma, increasing intracellular concentrations of atRA appears to provide clinical benefit. Inhibition of the CYP26 enzymes to increase atRA concentrations and combat therapy resistance has been pursued as a drug target in these cancers. This chapter covers the current knowledge of how atRA and retinol regulate the immune system and inflammation, how retinol and atRA metabolism is altered in inflammation and cancer, and what roles atRA-metabolizing enzymes have in immune responses and cancers.