Bruton’s tyrosine kinase (Btk) is a nonreceptor cytoplasmic tyrosine kinase involved in B-cell and myeloid cell activation, downstream of B-cell and Fcγ receptors, respectively. Preclinical studies ...have indicated that inhibition of Btk activity might offer a potential therapy in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here we disclose the discovery and preclinical characterization of a potent, selective, and noncovalent Btk inhibitor currently in clinical development. GDC-0853 (29) suppresses B cell- and myeloid cell-mediated components of disease and demonstrates dose-dependent activity in an in vivo rat model of inflammatory arthritis. It demonstrates highly favorable safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles in preclinical and Phase 2 studies ongoing in patients with rheumatoid arthritis, lupus, and chronic spontaneous urticaria. On the basis of its potency, selectivity, long target residence time, and noncovalent mode of inhibition, 29 has the potential to be a best-in-class Btk inhibitor for a wide range of immunological indications.
ABSTRACT
Purpose
To evaluate 26 marketed oncology drugs for time-dependent inhibition (TDI) of cytochrome P450 (CYP) enzymes. Evaluate TDI-positive drugs for potential to generate reactive ...intermediates. Assess clinical drug–drug interaction (DDI) risk using static mechanistic models.
Methods
Human liver microsomes and CYP-specific probes were used to assess TDI in a dilution shift assay followed by generation of K
I
and k
inact
. Reactive metabolite trapping studies were performed with stable label probes. Static mechanistic model was used to predict DDI risk using a 1.25-fold AUC increase as a cut-off for positive DDI.
Results
Negative TDI across CYPs was observed for 13/26 drugs; the rest were time-dependent inhibitors of, predominantly, CYP3A. The k
inact
/K
I
ratios for 11 kinase inhibitors ranged from 0.7 to 42.2 ml/min/μmol. Stable label trapping agent–drug conjugates were observed for ten kinase inhibitors. DDI predictions gave no false negatives, one true negative, four false positives and three true positives. The magnitude of DDI was overestimated irrespective of the inhibitor concentration selected.
Conclusions
13/26 oncology drugs investigated showed TDI potential towards CYP3A, formation of reactive metabolites was also observed. An industry standard static mechanistic model gave no false negative predictions but did not capture the modest clinical DDI potential of kinase inhibitors.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The cytochrome P450 26 family is believed to be responsible for all- trans -retinoic acid (atRA) metabolism and elimination in the human fetus and adults. CYP26A1 and CYP26B1 mRNA is expressed in a
...tissue-specific manner, and mice in which the CPY26 isoform has been knocked out show distinct malformations and lethality.
The aim of this study was to determine differences in CYP26A1 and CYP26B1 regulation and expression. Analysis of CYP26A1 and
CYP26B1 expression in a panel of 57 human livers showed CYP26A1 to be the major CYP26 isoform present in the liver, and its
expression to be subject to large interindividual variability between donors. CYP26A1 and retinoic acid receptor (RAR) β were
found to be greatly inducible by atRA in HepG2 cells, whereas CYP26B1, RARα, and RARγ were induced to a much lesser extent.
Based on treatments with RAR isoform-selective ligands, RARα is the major isoform responsible for CYP26A1 and RARβ induction
in HepG2 cells. Classic cytochrome P450 inducers did not affect CYP26 transcription, whereas the peroxisome proliferator-activated
receptor (PPAR) γ agonists pioglitazone and rosiglitazone up-regulated CYP26B1 transcription by as much as 209- ± 80-fold
and CYP26A1 by 10-fold. RARβ was also up-regulated by pioglitazone and rosiglitazone. CYP26B1 induction by PPARγ agonists
was abolished by the irreversible PPARγ antagonist 2-chloro-5-nitrobenzanilide (GW9662), whereas RARβ and CYP26A1 induction
was unaffected by GW9662. Overall, the results of this study suggest that CYP26B1 and CYP26A1 are regulated by different nuclear
receptors, resulting in tissue-specific expression patterns. The fact that drugs can alter the expression of CYP26 enzymes
may have toxicological and therapeutic importance.
A recent advancement in isolation and cryopreservation has resulted in commercially available primary human enterocytes that express various drug metabolizing enzymes (DMEs) and transporters. The ...main objective of this study was to further evaluate the utility of pooled cryopreserved enterocytes, specifically MetMax™ cryopreserved human enterocytes (In vitro ADMET Laboratories), as an in vitro model for assessing intestinal clearance in comparison to hepatocytes.
It was found that, for CYP3A4/5 substrates such as midazolam, amprenavir and loperamide, in vitro metabolic clearance is generally lower in enterocytes compared to that of hepatocytes, which is consistent with the relative abundance of the enzyme between the intestine and liver. In contrast, raloxifene, a surrogate UGT activity substrate, showed 3-fold greater turnover in enterocytes than hepatocytes, which is likely attributed to the differential expression of individual UGTs in human liver and intestine. For procaine, a known CES2 substrate, the measured apparent clearance was higher in hepatocytes, but formation of 4-aminobenzoic acid, a CE2-specific metabolite, was more pronounced in enterocytes, suggesting that CE2 is more active in enterocytes. Salbutamol, a SULT1A3 substrate, showed little turnover in both enterocytes and hepatocytes, and more abundant sulfate conjugate was detected in enterocytes, indicating higher SULT activity in enterocytes than hepatocytes. As expected, ketoconazole inhibited CYP3A4/5-mediated metabolite formation in enterocytes for midazolam, amprenavir and loperamide, suggesting that cryopreserved enterocytes may be useful in determining intestinal CYP3A inhibition parameters. Interestingly, elacridar, a P-gp inhibitor, suppressed metabolite formation in enterocytes for loperamide, a substrate of CYP3A4 and P-gp, suggesting that enterocytes in suspension do not have active P-gp efflux functions, and the suppression of metabolism in enterocytes is probably caused by inhibition of CYP3A4/5 by elacridar.
Our results suggest that pooled cryopreserved human enterocytes, specifically the MetMax™ cryopreserved human enterocytes, represent a valuable in vitro model for assessing first-pass clearance and potential drug interactions in human intestine.
During human pregnancy, CYP2C9, CYP2C19, and CYP2D6 activities are altered. The aim of the current study was to determine if this phenomenon can be replicated in the rat, and to evaluate the ...mechanisms that contribute to the changes in Cyp2c and Cyp2d activity during pregnancy. The intrinsic clearance of dextromethorphan
O-demethylation, a measure of Cyp2d2 activity, was decreased 80% at both days 9 and 19 of gestation when compared to non-pregnant controls. The decreased intrinsic clearance was a result of both decreased
V
max and increased
K
m-values at both days of gestation. Quantitative RT-PCR revealed that transcripts of Cyp2d2 and Cyp2d4 were significantly decreased at day 19 of pregnancy (
p
<
0.05) when compared to day 9 and non-pregnant controls. The decrease in Cyp2d mRNA levels correlated with a decrease in several nuclear receptor mRNA levels (RARα, RXRα, HNF1 and HNF3β) but not with the mRNA levels of nuclear receptors usually associated with regulation of P450 enzymes (PXR, CAR and HNF4α). In contrast, Cyp2c12 and Cyp2c6 transcription and protein expression were not significantly altered during rat pregnancy although the intrinsic clearance of Cyp2c6 mediated diclofenac 4′-hydroxylation was increased 2-fold on day 19 of gestation when compared to non-pregnant controls. The increase in intrinsic clearance was due to a decrease in the
K
m-value for 4′-hydroxydiclofenac formation. These data show that pregnancy significantly alters the expression and activity of drug metabolizing enzymes in an enzyme and gestational stage specific manner. These changes are likely to have toxicological and therapeutic implications.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Using physiologically based pharmacokinetic modeling, we predicted the magnitude of drug-drug interactions (DDIs) for studies with rifampicin and seven CYP3A4 probe substrates administered i.v. (10 ...studies) or orally (19 studies). The results showed a tendency to underpredict the DDI magnitude when the victim drug was administered orally. Possible sources of inaccuracy were investigated systematically to determine the most appropriate model refinement. When the maximal fold induction (Indmax) for rifampicin was increased (from 8 to 16) in both the liver and the gut, or when the Indmax was increased in the gut but not in liver, there was a decrease in bias and increased precision compared with the base model (Indmax = 8) geometric mean fold error (GMFE) 2.12 vs. 1.48 and 1.77, respectively. Induction parameters (mRNA and activity), determined for rifampicin, carbamazepine, phenytoin, and phenobarbital in hepatocytes from four donors, were then used to evaluate use of the refined rifampicin model for calibration. Calibration of mRNA and activity data for other inducers using the refined rifampicin model led to more accurate DDI predictions compared with the initial model (activity GMFE 1.49 vs. 1.68; mRNA GMFE 1.35 vs. 1.46), suggesting that robust in vivo reference values can be used to overcome interdonor and laboratory-to-laboratory variability. Use of uncalibrated data also performed well (GMFE 1.39 and 1.44 for activity and mRNA). As a result of experimental variability (i.e., in donors and protocols), it is prudent to fully characterize in vitro induction with prototypical inducers to give an understanding of how that particular system extrapolates to the in vivo situation when using an uncalibrated approach.
The N-methyl-d-aspartate receptor (NMDAR) is a Na+ and Ca2+ permeable ionotropic glutamate receptor that is activated by the coagonists glycine and glutamate. NMDARs are critical to synaptic ...signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders, including schizophrenia, depression, and Alzheimer’s disease. Herein we describe the discovery of potent GluN2A-selective NMDAR positive allosteric modulators (PAMs) starting from a high-throughput screening hit. Using structure-based design, we sought to increase potency at the GluN2A subtype, while improving selectivity against related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). The structure–activity relationship of channel deactivation kinetics was studied using a combination of electrophysiology and protein crystallography. Effective incorporation of these strategies resulted in the discovery of GNE-0723 (46), a highly potent and brain penetrant GluN2A-selective NMDAR PAM suitable for in vivo characterization.
All-trans-retinoic acid (
atRA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of
atRA via oxidative ...metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are
atRA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from
atRA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from
atRA. Quantitatively, CYP26B1 had a lower
K
m (19
nM) and
V
max (0.8
pmol/min/pmol) than CYP26A1 (
K
m
=
50
nM and
V
max
=
10
pmol/min/pmol) for formation of 4-OH-RA. The major
atRA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2–10-fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of
atRA in tissues that function as a barrier for
atRA exposure.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Purpose
To evaluate an alternative
in vitro
system which can provide more quantitatively accurate drug drug interaction (DDI) prediction for 10 protein kinase inhibitors for which DDI risk was ...over-predicted by inhibition data generated in human liver microsomes (HLM).
Methods
Three cryopreserved human hepatocyte (hHEP) systems: 1) plated hHEPs; 2) hHEPs suspended in Dulbecco’s Modified Eagle Medium (DMEM) and 3) hHEPs suspended in human plasma (plasma hHEPs) were developed to detect CYP3A time dependent inhibition, and the static mechanistic model was used to predict clinical outcomes.
Results
A general trend was observed in the CYP3A inactivation potency (
k
inact
/
K
I, app
) as HLM > plated > DMEM ≥ plasma hHEPs. Using the static mechanistic model, DDIs predicted using parameters estimated from plated, DMEM and plasma hHEPs had 84, 74 and 95% accuracy (out of 19 clinical interaction studies) within 2-fold of the reported interaction, respectively. They demonstrated significant improvement compared to the DDIs predicted using parameters estimated from HLMs where 58% accuracy was obtained.
Conclusions
Based on 19 DDIs, plasma hHEPs demonstrate a more reliable clinical DDI prediction for 10 protein kinase inhibitors and prototypical CYP3A time dependent inhibitors.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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