Human Pharmacokinetic and CYP3A Drug-Drug Interaction Prediction of GDC-2394 Using Physiologically Based Pharmacokinetic Modeling and Biomarker Assessment.

Physiologically based pharmacokinetic (PBPK) modeling was used to predict the human pharmacokinetics and drug-drug interaction (DDI) of GDC-2394. PBPK models were developed using in vitro and in vivo data to reflect the oral and intravenous PK profiles of mouse, rat, dog, and monkey. The learnings from preclinical PBPK models were applied to a human PBPK model for prospective human PK predictions. The prospective human PK predictions were within 3-fold of the clinical data from the first-in-human study, which was used to optimize and validate the PBPK model and subsequently used for DDI prediction. Based on the majority of PBPK modeling scenarios using the in vitro CYP3A induction data (mRNA and activity), GDC-2394 was predicted to have no-to-weak induction potential at 900 mg twice daily (BID). Calibration of the induction mRNA and activity data allowed for the convergence of DDI predictions to a narrower range. The plasma concentrations of the 4ß-hydroxycholesterol (4ß-HC) were measured in the multiple ascending dose study to assess the hepatic CYP3A induction risk. There was no change in plasma 4ß-HC concentrations after 7 days of GDC-2394 at 900 mg BID. A dedicated DDI study found that GDC-2394 has no induction effect on midazolam in humans, which was reflected by the totality of predicted DDI scenarios. This work demonstrates the prospective utilization of PBPK for human PK and DDI prediction in early drug development of GDC-2394. PBPK modeling accompanied with CYP3A biomarkers can serve as a strategy to support clinical pharmacology development plans. SIGNIFICANCE STATEMENT: This work presents the application of physiologically based pharmacokinetic modeling for prospective human pharmacokinetic (PK) and drug-drug interaction (DDI) prediction in early drug development. The strategy taken in this report represents a framework to incorporate various approaches including calibration of in vitro induction data and consideration of CYP3A biomarkers to inform on the overall CYP3A-related DDI risk of GDC-2394.

Perspective: 4ß-hydroxycholesterol as an emerging endogenous biomarker of hepatic CYP3A.

A key goal in the clinical development of a new molecular entity is to quickly identify whether it has the potential for drug-drug interactions. In particular, confirmation of in vitro data in the early stage of clinical development would facilitate the decision making and inform future clinical pharmacology study designs. Plasma 4ß-hydroxycholesterol (4ß-HC) is considered as an emerging endogenous biomarker for cytochrome P450 3A (CYP3A), one of the major drug metabolizing enzymes. Although there are increasing reports of the use of 4ß-HC in academic- and industry-sponsored clinical studies, a thorough review, summary and consideration of the advantages and challenges of using 4ß-HC to evaluate changes in CYP3A activity has not been attempted. Herein, we review the biology of 4ß-HC, its response to treatment with CYP3A inducers, inhibitors and mixed inducer/inhibitors in healthy volunteers and patients, the association of 4ß-HC with other probes of CYP3A activity (e.g. midazolam, urinary cortisol ratios), and present predictive pharmacokinetic models. We provide recommendations for studying hepatic CYP3A activity in clinical pharmacology studies utilizing 4ß-HC at different stages of drug development.

27-Hydroxycholesterol upregulates the production of heat shock protein 60 of monocytic cells.

Investigating differentially expressed proteins in a milieu rich in cholesterol oxidation products, we found via mass spectrometry-based proteomics that surface levels of heat shock protein 60 (HSP60) were upregulated on monocytic cells in the presence of 27-hydroxycholesterol (27OHChol). The elevated levels of cytoplasmic membrane HSP60 were verified via Western blot analysis and visualized by confocal microscopy. Treatment with 27OHChol also resulted in increased levels of cellular HSP60 without altering its transcription. Cholesterol, however, did not affect cell-surface levels and cellular amount of HSP60. GSK 2033, an LXR antagonist, inhibited expression of live X receptor α, but not of HSP60, induced by 27OHChol. Treatment with 27OHChol also resulted in increased release of HSP60 from monocytic cells, but the release was significantly reduced by inhibitors of endoplasmic reticulum-Golgi protein trafficking, brefeldin A and monensin. Results of the current study indicate that 27OHChol upregulates not only cell-surface and cellular levels of HSP60 but also its release from monocytic cells, thereby contributing to activation of the immune system.

Ceramide generation occurring during 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis is caspase independent and is not required to trigger cell death.

Biological activities of oxysterols seem tightly regulated. Therefore, the ability to induce cell death of structurally related oxysterols, such as those oxidized at C7(7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5-80 microg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7beta-hydroxycholesterol and 7-ketocholesterol, which trigger an apoptotic process characterized by the occurrence of cells with fragmented and/or condensed nuclei, and by various cellular dysfunctions: loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADP-ribose) polymerase, and increased accumulation of cellular C16 : 0 and C24 : 1 ceramide species. This ceramide generation is not attributed to caspase activation since inhibition of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 microM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7beta-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 microM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7alpha-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the alpha or beta hydroxyl radical position of oxysterols oxidized at C7 plays a key role in the induction of the apoptotic process. In addition, our findings demonstrate that 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis involve the mitochondrial signal transduction pathway and they suggest that C16 : 0 and C24 : 1 ceramide species generated through ceramide synthase play a minor role in the commitment of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death.

Metabolism of new anticancer oxysterol derivatives in rats.

New water soluble derivatives of oxysterols--the phosphodiesters of oxysterols and of nucleosides--have been synthesized. In vitro, these compounds share the biological properties of their parent oxysterols. Furthermore, they display anticancer activity when injected i.p. in mice bearing experimental tumors. The pharmacokinetic study described here proved that the water-soluble derivatives of oxysterols act as prodrugs releasing free oxysterol in the blood, the liver and the kidney after i.p. or i.v. injection in rats. The hydro-solubility of such compounds as well as their slow metabolism into the active principle could account for their biological activity and make them suitable as new therapeutic agents.

[Metabolism of 3beta-(2-hydroxyethoxy)cholest-5-ene after intravenous administration to rats]. / Metabolizm 3beta-(2-gidroksiétoksi)kholest-5-ena pri vnutrivennom vvedenii krysam.

The intravenous injection of 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest- 5-ene into rats with the cannulated common bile duct resulted in the primary accumulation of radioactivity in liver (24%) and spleen (12%) tissues, bile (7%), and blood serum (15%) after 3 hours. The distribution of 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest-5-ene throughout various tissues was close to that of [14C]cholesterol being administrated under the same conditions. The analysis of radioactive products from blood serum showed that 40-60% of 3 beta-(2-hydroxy-2-[3H]ethoxy)- cholest-5-ene was converted to the acyl derivative under experimental conditions.

Cholesterol metabolite, 5-cholesten-3ß-25-diol-3-sulfate, promotes hepatic proliferation in mice.

UNLABELLED: Oxysterols are well known as physiological ligands of liver X receptors (LXRs). Oxysterols, 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol as endogenous ligands of LXRs, suppress cell proliferation via LXRs signaling pathway. Recent reports have shown that sulfated oxysterol, 5-cholesten-3ß-25-diol-3-sulfate (25HC3S) as LXRs antagonist, plays an opposite direction to oxysterols in lipid biosynthesis. The present report was to explore the effect and mechanism of 25HC3S on hepatic proliferation in vivo. Following administration, 25HC3S had a 48 h half life in the circulation and widely distributed in mouse tissues. Profiler™ PCR array and RTqPCR analysis showed that either exogenous or endogenous 25HC3S generated by overexpression of oxysterol sulfotransferase (SULT2B1b) plus administration of 25HC significantly up-regulated the proliferation gene expression of Wt1, Pcna, cMyc, cyclin A, FoxM1b, and CDC25b in a dose-dependent manner in liver while substantially down-regulating the expression of cell cycle arrest gene Chek2 and apoptotic gene Apaf1. Either exogenous or endogenous administration of 25HC3S significantly induced hepatic DNA replication as measured by immunostaining of the PCNA labeling index and was associated with reduction in expression of LXR response genes, such as ABCA1 and SREBP-1c. Synthetic LXR agonist T0901317 effectively blocked 25HC3S-induced hepatic proliferation. CONCLUSIONS: 25HC3S may be a potent regulator of hepatocyte proliferation and oxysterol sulfation may represent a novel regulatory pathway in liver proliferation via inactivating LXR signaling.

Absorption of cholesterol oxidation products from ordinary foodstuff in humans.

BACKGROUND: Information on the absorption of cholesterol oxidation products (COP) from ordinary foodstuff in humans is scarce. METHODS: Five healthy young men were offered a salami- and Parmesan-containing meal naturally rich in COP. Plasma and lipoprotein COP concentrations were measured over the following 9 h. RESULTS: The mean plasma free (nonesterified) COP concentration showed its maximal increase 3 and 5 h after meal consumption. In contrast, the raise in plasma total COP concentration began 6 h after the meal with a maximum at 8 h and was statistically significant for 7alpha- and 7beta-hydroxycholesterol and 7-ketocholesterol. The increase in plasma total cholesterol concentration was comparable to that of total COP. Comparing the COP composition of the chylomicrons and the test meal, cholestanetriol, 7-ketocholesterol, and to a lesser extent cholesterol-alpha-epoxide were underrepresented in the chylomicrons as was the opposite for 7beta-hydroxycholesterol. In very-low-density lipoprotein, a steady increase in the COP:cholesterol ratio was observed from 6 h on. CONCLUSION: COP from ordinary foodstuff were absorbed in the human intestinal tract but differences in the bioavailability of the single COP compounds were found.

7 Alpha-hydroxylation of 27-hydroxycholesterol in rat brain microsomes.

27-hydroxycholesterol is shown to be 7 alpha-hydroxylated by microsomal preparations of rat brain. The apparent Km was about 2 microM and Vmax about 15 pmol/min x mg protein. The reaction might modulate biological functions of 27-hydroxycholesterol, such as its suppressive effect on the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and supports the possibility of bile acid formation in the brain.

Metabolism of 4 beta -hydroxycholesterol in humans.

One of the major oxysterols in the human circulation is 4 beta-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4 beta-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7 alpha-, 27-, and 24-hydroxycholesterol to be approximately 0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4 beta-hydroxycholesterol. The apparent half-life of deuterium-labeled 4 beta-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4 beta-Hydroxycholesterol was converted into acidic products at a much slower rate than 7 alpha-hydroxycholesterol in primary human hepatocytes, and 4 beta-hydroxycholesterol was 7 alpha-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4 beta-hydroxycholesterol. These results suggest that the high plasma concentration of 4 beta-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7 alpha-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4 beta-hydroxycholesterol as a ligand for the nuclear receptor LXR.