Olaf Kuhlmann

A single-center, open-label, one-sequence study of dalcetrapib coadministered with ketoconazole, and an in vitro study of the S-methyl metabolite of dalcetrapib

BACKGROUND Dalcetrapib (RO4607381/JTT-705) is currently under clinical investigation for the prevention of cardiovascular events. It inhibits the activity of cholesteryl ester transfer protein and has been reported to increase levels of high-density lipoprotein cholesterol. OBJECTIVE Because dalcetrapib is likely to be coadministered with agents that inhibit the cytochrome P450 (CYP) 3A4 isozyme, this study aimed to determine the effect of ketoconazole, a strong CYP3A4 inhibitor, on the pharmacokinetics of dalcetrapib. METHODS An open-label, 1-sequence study was conducted in 2 cohorts of healthy, nonsmoking male volunteers aged 18 through 65 years, with a body mass index of 18 to 32 kg/m(2). The first cohort received dalcetrapib 600 mg on days 1 and 7 and ketoconazole 400 mg on days 2 through 7, and, based on the results of a planned interim analysis, the second cohort received dalcetrapib 900 mg alone on days 1 and 7 and ketoconazole on days 2 through 7. Pharmacokinetic and safety parameters were assessed at specific times throughout the study. To confirm CYP involvement in the metabolism of the inactive metabolite dalcetrapib-S-methyl, in vitro studies were performed using human liver microsomes and recombinantly expressed CYP isoforms. RESULTS Of the 26 participants, 96% were white, with a mean age of 38.1 years and a mean weight of 78.6 kg. In the in vivo portion of the study, coadministration of ketoconazole with dalcetrapib 600 mg had no significant effect on any pharmacokinetic parameter of dalcetrapib. Coadministration of ketoconazole with dalcetrapib 900 mg was associated with significant decreases in the dalcetrapib C(max) (-23%; P = 0.002) and AUC(0-infinity) (-18%; P = 0.001) and a significant increase in oral clearance (22%; P = 0.001). Significant increases in the C(max) (P = 0.001) and AUC(0-infinity) (P < 0.001) of dalcetrapib-S-methyl were observed with coadministration of ketoconazole. The combination was generally well tolerated, with 32 of 35 adverse events (91.4%) being mild in intensity. The most frequent adverse events were headache (6/26 [23.1%] in the ketoconazole group; 4/18 [22.2%] in the group receiving dalcetrapib 900 mg plus ketoconazole) and diarrhea (4/26 [15.4%] in the ketoconazole group; 2/18 [11.1%] in the group receiving dalcetrapib 900 mg plus ketoconazole). The in vitro studies confirmed the involvement of CYP3A in the metabolism of dalcetrapib-S-methyl. CONCLUSIONS In this clinical study in healthy male volunteers, coadministration of dalcetrapib 600 mg with the CYP3A4 inhibitor ketoconazole was not associated with any significant changes in the pharmacokinetic parameters of the parent compound. Coadministration of dalcetrapib 900 mg with ketoconazole was associated with significant decreases in the dalcetrapib C(max) and AUC, contrary to the increases that would be expected if dalcetrapib were a substrate for CYP3A4. The combination of dalcetrapib and ketoconazole was generally well tolerated.

In vitro and in vivo assessment of the effect of dalcetrapib on a panel of CYP substrates

ABSTRACT Objective: The primary objective of this study was to investigate the drug–drug interaction potential of dalcetrapib on drugs metabolized via major cytochrome P450 (CYP) isoforms using both in vitro and clinical approaches. A secondary objective was to investigate the safety and tolerability of dalcetrapib alone or co-administered either with a combination of five probe drugs or with rosiglitazone. Research design and methods: Human liver microsomes and a panel of substrates for CYP enzymes were used to determine IC50 for inhibition of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. In addition, two drug–drug interaction studies were conducted in healthy males: dalcetrapib 900 mg plus the Cooperstown 5 + 1 drug cocktail, which includes substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and dalcetrapib 900 mg plus rosiglitazone, a substrate for CYP2C8. Pharmacokinetic and safety parameters were assessed. Results: In vitro, dalcetrapib was inhibitory to all CYP enzymes tested. IC50 values ranged from 1.5 ± 0.1 μM for CYP2C8 to 82 ± 4 μM for CYP2D6. Co-administration of dalcetrapib plus drug cocktail showed no clinically relevant effect of 900 mg dalcetrapib on activity of CYP1A2, CYP2C19, CYP2D6, CYP2C9, or CYP3A4 following repeated administration. Co-administration of dalcetrapib plus rosiglitazone showed no clinically relevant effect of dalcetrapib 900 mg on activity of CYP2C8. Dalcetrapib was generally well tolerated. Conclusions: Although in vitro studies indicated that dalcetrapib inhibits CYP activity, two clinical studies showed no clinically relevant effect on any of the major CYP isoforms at a 900 mg dose, which is higher than the 600 mg dose being explored in phase III studies. Dalcetrapib was generally well tolerated in these studies. However, these studies were limited to a small number of healthy males; additional, larger studies are necessary to study its safety.

Dalcetrapib pharmacokinetics and metabolism in the cynomolgus monkey

The thioester dalcetrapib is undergoing Phase III clinical evaluation for the prevention and regression of atherosclerosis and the prevention of cardiovascular events through targeting cholesteryl ester transfer protein and increasing high-density lipoprotein cholesterol levels. Dalcetrapib undergoes rapid hydrolysis to generate the pharmacologically active form (dalcetrapib-thiol), which undergoes extensive metabolism via glucuronic acid conjugation, methylation, and hydroxylation, predominately forming the pharmacologically inactive S-methyl (dalcetrapib-S-Me) and S-glucuronide (dalcetrapib-S-Glu) metabolites. The purpose of this study was to characterize the absorption and disposition of dalcetrapib-thiol and its primary metabolites in cynomolgus monkeys following first pass through the intestines and liver using an in vivo dual portal and peripheral vein cannulation. Results showed the high influence of glucuronidation of dalcetrapib-thiol on the first-pass effect. Following passage through the primate intestinal wall, area under the plasma concentration–time curve indicated a marked loss (by ~85%) of active compound and formation of dalcetrapib-S-Glu and dalcetrapib-S-Me. Based on time to maximum drug concentrations (Tmax) values in the portal vein, metabolism of dalcetrapib-thiol to dalcetrapib-S-Glu appears to occur almost instantly (median Tmax 6.0 and 5.5 h, respectively), whereas methylation to dalcetrapib-S-Me occurs much more slowly (median Tmax, 24 h). A relatively modest impact on systemic exposure followed hepatic first pass, with a further decrease in dalcetrapib-thiol exposure of 58% (AUC), a 3-fold reduction in exposure levels of dalcetrapib-S-Me and near-complete decrease in exposure of dalcetrapib-S-Glu. Passage of drug-related material through the intestinal wall and the liver results in an overall decrease of exposure to dalcetrapib-thiol of >90%.

Application of a Statistical Method to the Absorption of a New Model Drug from Micellar and Lipid Formulations—Evaluation of Qualitative Excipient Effects

The scope of the present article is to study formulation parameters of micellar and of lipid delivery systems on the exposure of a new drug compound A in Wistar rats. A statistical analysis is to be performed a posteriori from a data set of all rat studies that were conducted during the preclinical development of the drug. Several formulations were evaluated mainly in view of sufficient exposure in toxicological studies. Because of the low solubility and high lipophilicity of compound A, the preclinical formulation development focused on micellar solutions and different kinds of lipid drug delivery systems. Candidate formulations were first tested for their dilution in artificial intestinal fluids before they were evaluated in the rat. A partial least square model was applied to the entire pharmacokinetic data set, and the type of delivery system, as well as excipients, were investigated in view of effects on the area under the plasma level curve. The results showed that self-emulsifying systems and in particular self-microemulsifying drug delivery systems were most effective in pushing the exposure of compound A. Another significant factor was the dose. A data subset showed nonlinearity in the pharmacokinetics with respect to the dose. However, the most important findings of the multivariate data analysis were overall effects of excipients on the exposure. These effects are considered as a sum of several influences so that the underlying mechanism is essentially complex and is not fully understood. Cremophor and lecithin exhibited a positive effect, whereas TPGS containing systems reached only below average exposure. No significant effect was observed with polysorbate 80 or Solutol HS. The model indicated the favorable use of a cosurfactant, in particular Capmul MCM. Similarly the use of a cosolvent showed a positive coefficient and ethanol was here best in class. No marked effects were observed for the oil selection, but a tendency toward below average exposure was displayed when long-chain triglycerides were in the formulation. The a posteriori analysis of the pharmacokinetic data using multivariate statistical models was very helpful to clarify effects of drug delivery systems as well as of general effects of excipients. Guidance was provided for the formulator, but further studies are needed to better understand the complex effects on a mechanistic level.

Metabolism and mass balance of SGLT2 inhibitor tofogliflozin following oral administration to humans

Abstract 1. Tofogliflozin is a novel and selective SGLT2 inhibitor increasing glucosuria by inhibition of glucose re-absorption in the kidney for the treatment of type 2 diabetes mellitus. 2. In this study, the metabolism and the mass balance of tofogliflozin was evaluated following administration of a single oral dose of 20 mg [14C]-tofogliflozin to six healthy subjects. 3. Tofogliflozin underwent mainly oxidative metabolism in the ethylphenyl moiety, but also minor glucuronide conjugates of metabolites and the parent drug were formed. 4. In plasma, the parent drug and its major phenyl acetic acid metabolite M1 accounted for 42% and 52% of the total drug-related material, respectively. The hydroxyl metabolites and their successor ketone metabolite showed an exposure well below 5%, along with an acyl glucuronide of M1. 5. Tofogliflozin was completely absorbed with subsequent predominate metabolic clearance and a small contribution of direct urinary elimination. Approximately, 76% of the dose was excreted in urine and 20% in faeces within 72 h. The high absorption of tofogliflozin was exemplified by the small trace of parent drug in faeces. The phenyl acetic acid metabolite M1 was the major component excreted in urine and faeces accounting for more than half of the dose. Tofogliflozin demonstrated a high metabolic turnover.

Determination of dalcetrapib by liquid chromatography-tandem mass spectrometry.

The cholesteryl ester transfer protein modulator dalcetrapib is currently under development for the prevention of dyslipidemia and cardiovascular disease. Dalcetrapib, a thioester, is rapidly hydrolyzed in vivo to the corresponding thiophenol which in turn is further oxidized to the dimer and mixed disulfides (where the thiophenol binds to peptides, proteins and other endogenous thiols). These forms co-exist in an oxidation-reduction equilibrium via the thiol and cannot be stabilized without influencing the equilibrium, hence specific determination of individual components, i.e., in order to distinguish between the free thiol, the disulfide dimer and mixed disulfide adducts, was not pursued for routine analysis. The individual forms were quantified collectively as dalcetrapib-thiol (dal-thiol) after reduction under basic conditions with dithiothreitol to break disulfide bonds and derivatization with N-ethylmaleimide to stabilize the free thiol. The S-methyl and S-glucuronide metabolites were determined simultaneously with dal-thiol with no effect from the derivatization procedure. Column-switching liquid chromatography-tandem mass spectrometry provided a simple, fast and robust method for analysis of human and animal plasma and human urine samples. Addition of the surfactant Tween 80 to urine prevented adsorptive compound loss. The lower limits of quantitation (LLOQ) were 5 ng/mL for dal-thiol, and 5 ng/mL for the S-methyl and 50 ng/mL for the S-glucuronide metabolites. Using stable isotope-labeled internal standards, inter- and intra-assay precisions were each <15% (<20% at LLOQ) and accuracy was between 85 and 115%. Recovery was close to 100%, and no significant matrix effect was observed.

In vitro profiling of the metabolism and drug–drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP

Abstract 1. The metabolism and drug–drug interaction (DDI) risk of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs. 2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1. 3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1. 4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency.

Solubility and stability of dalcetrapib in vehicles and biological media

Dalcetrapib solubility was determined in aqueous and in non-aqueous vehicles and in biorelevant media. In a pure aqueous environment the solubility was low but could be increased by addition of surfactants or complexing agents. This was also reflected in the solubility seen in simulated gastrointestinal (GI) fluids, with almost no solubility in simulated gastric fluid, but reasonable solubilisation in simulated intestinal fluids containing lecithin and bile salt. Additionally, the stability of dalcetrapib was determined in simulated GI fluids with and without pancreatic lipase. In solutions without lipase, dalcetrapib was slowly hydrolysed, but in the presence of lipase the hydrolysis rate was significantly faster depending on pH and enzyme activity. In biological fluids, dissolved dalcetrapib appeared to behave similarly being rapidly hydrolysed in human intestinal fluids with a half-life below 20s with no degradation observed in human gastric fluids at low pH. The results provide supportive evidence that absorption is higher under fed conditions and indicate lipase inhibitors might interfere with oral absorption of dalcetrapib.

Pharmacokinetics and metabolism of the dipeptidyl peptidase IV inhibitor carmegliptin in rats, dogs, and monkeys.

The pharmacokinetics and excretion of carmegliptin, a novel dipeptidyl peptidase IV inhibitor, were examined in rats, dogs, and cynomolgus monkeys. Carmegliptin exhibited a moderate clearance, extensive tissue distribution, and a variable oral bioavailability of 28–174%. Due to saturation of intestinal active secretion, the area under the plasma concentration–time curve (AUC) in dogs and monkeys increased in a more than dose-proportional manner over an oral dose range of 2.5–10 mg/kg. Following oral administration of [14C]carmegliptin at 3 mg/kg, > 94% of the radioactive dose was recovered in 72-h post-dose from Wistar rats and Beagle dogs. Virtually, the entire administered radioactive dose was excreted unchanged in urine, intestinal lumen, and bile. Approximately 36%, 29%, and 19% of the dose were excreted by respective routes. Consistently, in vitro, carmegliptin was highly resistant to hepatic metabolism in all species tested. Based on in vitro studies, carmegliptin is a good substrate for Mdr1/MDR1. Breast cancer resistance protein (Bcrp) is not expected to be involved in the transport of carmegliptin since in vitro carmegliptin was not significantly transported by this transporter. The very high extravascular distribution of carmegliptin in the intestinal tissues, as demonstrated in Wistar rats and Beagle dogs, could play a significant role in its therapeutic effect.

Profiling of dalcetrapib metabolites in human plasma by accelerator mass spectrometry and investigation of the free phenothiol by derivatisation with methylacrylate

&NA; Dalcetrapib, a thioester prodrug, undergoes rapid and complete conversion in vivo to its phenothiol metabolite M1 which exerts the targeted pharmacological response in human. In clinical studies, M1 has been quantified together with its dimer and mixed disulfide species that represent the ‘dalcetrapib active form’ in plasma. In this article, we describe the determination of the free phenothiol M1 by derivatisation with methylacrylate as a percentage of ‘dalcetrapib active form’. Pharmacokinetic profiles of M1 after oral administration of dalcetrapib to humans could be established, underscoring the validity to use a composite measure of ‘dalcetrapib active form’ as a surrogate marker for pharmacodynamic evaluations. ‘Dalcetrapib active form’ and M1 made up 8.9% and 3.6% of total drug‐related material, respectively. In addition, complete metabolite profiling of 14C‐labeled dalcetrapib was conducted after two‐dimensional HPLC using fast fractionation into 384‐well plates and ultrasensitive determination of the 14C‐content by accelerator mass spectrometry. M1 underwent further biotransformation to its S‐methyl metabolite M3, which was further oxidized to its sulfoxide and sulfone. Another metabolic pathway was the formation of the S‐glucuronide. All of these species underwent further oxidation in the ethylbutyl cyclohexyl moiety leading to a multitude of hydroxyl and keto metabolites undergoing further conjugation to O‐glucuronides. More than 80 metabolites were identified, demonstrating extensive metabolism. However, it was unambiguously demonstrated that none of these metabolites were major according to the MIST guideline (exceeding 10% of drug related material in circulation). The combination of accelerator mass spectrometry with HPLC together with high resolution mass spectrometry allowed for structural characterization of the most relevant human metabolites.