Derrick G. Freitag

Pharmacokinetics and metabolism of sirolimus.

Sirolimus (rapamycin, Rapamune) is a potent immunosuppressive drug that received marketing approval from the US Food and Drug Administration on September 15, 1999. Research into defining its pharmacokinetic (PK) behavior, interaction with other agents, and metabolism is ongoing. It has been established that oral doses of both liquid and solid formulation are rapidly, though incompletely and variably, absorbed. Metabolism by the intestinal and hepatic CYP3A family of enzymes likely contributes to variability in absorption and low bioavailability. Sirolimus has a long terminal half-life, the AUC correlates well with trough and peak concentrations, and it exhibits a moderate degree of dose proportionality. There is significant interpatient variability in PK parameters of sirolimus, though it exhibits predictable PK behavior when used with prednisone and cyclosporine neoral. There is a decreased rejection risk with higher doses and target level attainment. Several species of sirolimus metabolites have been characterized, and are measurable in whole blood and tissue specimens. Many more species of sirolimus metabolites are detectable, but they are not quantifiable at this time. The total concentration of metabolites appears to be less than that of the parent drug when examined through the PK profile. A reference method for the quantitation of metabolites remains elusive because of a lack of proper standardization. The clinical significance of sirolimus metabolites remains to be proven.

Structural basis for the cyclophilin A binding affinity and immunosuppressive potency of E-ISA247 (voclosporin)

X-ray crystal structures of the cyclosporin A analogue E-ISA247 (voclosporin) and its stereoisomer Z-ISA247 bound to cyclophilin A suggest the molecular basis for the differences in their binding affinities and immunosuppressive efficacies.

Development and validation of a LC/MS/MS method for quantifying the next generation calcineurin inhibitor, voclosporin, in human whole blood

A rapid, accurate, and reproducible liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for the therapeutic drug monitoring of voclosporin in human whole blood. Sample aliquots of 100muL were processed utilizing a protein precipitation procedure that contained a mixture of methanol, 0.2M ZnSO(4), and deuterated voclosporin internal standard. Supernatant was injected onto a Zorbax SB-C8, 2.1x12.5mm column (at 60 degrees C), and washed with water-acetonitrile, supplemented with 0.02% glacial acetic acid and 0.02mM sodium acetate, to remove poorly retained components. After washing, water-MeOH (with 0.02% glacial acetic acid and 0.02mM sodium acetate) was used to elute the voclosporin and internal standard to the Applied Biosystems/MDS-Sciex API3000 mass spectrometer for detection in multiple reaction monitoring. Analytical performance was assessed in the range of 1-200ng/ml in whole blood. This method has been used to quantify concentrations of voclosporin in whole blood from healthy volunteers participating in a pharmacokinetic study.

Cytochrome P450 3A and P‐glycoprotein drug–drug interactions with voclosporin

AIMS Voclosporin is a novel calcineurin inhibitor intended for prevention of organ graft rejection and treatment of lupus nephritis. Pharmacokinetic drug interactions between voclosporin and a CYP3A inhibitor, inducer and substrate and a P-glycoprotein inhibitor and substrate were evaluated. METHODS Voclosporin 0.4 mg kg(-1) was administered to 24 subjects in each of five studies, as follows: every 12 h (Q12H) alone and concomitantly with ketoconazole 400 mg once daily (QD); single dose before and single dose after rifampin 600 mg QD; Q12H where midazolam 7.5 mg was administered as a single dose alone before voclosporin and with last the dose of voclosporin; Q12H alone and concomitantly with verapamil 80 mg every 8 h; and Q12H with digoxin 0.25 mg QD. The noncompartmental pharmacokinetic parameters maximal concentration (Cmax ) and area under the concentration-time curve (AUC) were obtained, and geometric least squares mean ratios and 90% confidence intervals were evaluated. RESULTS Ketoconazole increased voclosporin Cmax (6.4-fold) and AUC (18-fold); rifampin reduced voclosporin AUC (0.9-fold); voclosporin did not change exposure of midazolam or α-hydroxy-midazolam; verapamil increased voclosporin Cmax (2.1-fold) and AUC (2.7-fold); and voclosporin increased digoxin Cmax (0.5-fold), AUC (0.25-fold) and urinary excretion (0.2-fold). CONCLUSIONS Administration of voclosporin concomitantly with strong inhibitors and inducers of CYP3A resulted in increased and decreased exposures, respectively, and should be considered contraindicated. Drug-drug interactions involving voclosporin and CYP3A substrates are not expected. Administration of voclosporin concomitantly with inhibitors and substrates of P-glycoprotein resulted in increased voclosporin and substrate exposures, respectively. Appropriate concentration and safety monitoring is recommended with co-administration of voclosporin and P-glycoprotein substrates and inhibitors.

Pharmacokinetics of voclosporin in renal impairment and hepatic impairment

Voclosporin is a novel calcineurin inhibitor intended for prevention of organ graft rejection and treatment of lupus nephritis. These studies evaluated the effect of renal or hepatic impairment on pharmacokinetics of voclosporin. Thirty‐three subjects were enrolled into 1 of 4 groups based on renal function as defined by creatinine clearance and 18 subjects were enrolled into 1 of 3 groups based on hepatic function defined by Child‐Pugh classes. Voclosporin 0.4 mg/kg was administered orally. Geometric mean ratios (renal/hepatic impairment‐to‐normal) and 90% confidence intervals for Cmax and AUC were calculated. A default no‐effect interval of 80–125% was set. Although 90% confidence intervals exceeded the no‐effect intervals for both parameters, individual Cmax and AUC plots indicate almost complete overlapping range of values for mild and moderate renal impairment and normal subjects. Severe renal impairment resulted in a 1.5‐fold increase in AUC without an increase in Cmax. Mild to moderate hepatic impairment resulted in a 1.5‐ to 2‐fold increase in voclosporin exposure. Voclosporin can be administered safely to patients with mild to moderate renal impairment without dose modification. Appropriate safety monitoring with concentration‐based adjustments in transplantation are recommended for patients with severe renal impairment, and for patients with hepatic impairment.

Population PKPD of voclosporin in renal allograft patients

The aims of this population‐pharmacokinetic/pharmacodynamic (POP‐PKPD) analysis of voclosporin in renal allograft patients were to build a POP‐PKPD model for voclosporin and calcineurin activity (CNa) and identify clinically relevant covariates that could assist dosing of the drug. POP‐PKPD modeling was performed using a stochastic approximation of the standard expectation maximization (SAEM) algorithm for nonlinear mixed‐effects as implemented in Monolix™ 3.2. Voclosporin whole blood concentrations were obtained from de novo renal allograft patients and assayed using a validated LC/MS/MS assay. CNa was measured using a 32P‐radiolabeled assay. A two‐compartment model with simultaneous sigmoid inhibitory Emax model was used to describe the PKPD relationship between voclosporin concentration and CNa. The POP‐PKPD model was then utilized to simulate an optimal initial dosing strategy. Eighty‐seven patients were included in the POP‐PKPD study. Population mean estimates (relative standard error, rse) for oral clearance (CL/F) and first compartment volume of distribution (V1), were 717 mL min−1 (35%) and 2010 mL (17%), respectively. Maximum CNa Inhibition (Imax), effective concentration (C50), and baseline immunosuppression (S0) were 0.87 pmol/min/mg (8.0%), 123 ng/mL (10%), and 1.15 pmol/min/mg (4.0%), respectively. Covariate analyses demonstrated that age and body surface area significantly influenced CL/F: CLi=717(Agei/48.8)−0.57(BSAi/1.99)1.1 , while serum triglycerides significantly altered S0: S0i=1.15(TRIGi/1.97)0.15 .

Voclosporin Food Effect and Single Oral Ascending Dose Pharmacokinetic and Pharmacodynamic Studies in Healthy Human Subjects

Voclosporin (VCS) is a novel calcineurin (CN) inhibitor intended for prevention of organ graft rejection and treatment of lupus nephritis. These studies evaluated the single ascending dose pharmacokinetics (PK) and pharmacodynamics (PD, CN activity) of VCS and the effect of food. VCS was administered orally in single doses of 0.25 through 4.5 mg/kg in 62 subjects in the single ascending dose study and as a single oral 1.5 mg/kg dose to 18 subjects after fasting, consumption of a low‐fat and high‐fat meal. Non‐compartmental PK, PD, and PKPD correlation were evaluated. Following single oral doses, systemic exposure increased in a linear manner and demonstrated 1:1 dose‐proportional, first‐order linear PK above 1.5 mg/kg. VCS inhibited CN activity in a dose‐related fashion with maximal inhibition peaking at 3.0 mg/kg. PKPD correlation indicated an EC50 of 78.3 ± 6.8 ng/mL. Administration of VCS with a low‐fat and high‐fat meal decreased Cmax by 29% and 53%, respectively, and AUCinf by 15% and 25%, respectively. Following ascending single doses of VCS, exposure increased in a linear fashion. A food effect on exposure was demonstrated, with a more pronounced effect following a high‐fat meal. VCS concentrations were also found to correlate with CN activity.

Study of FK-binding protein: FK506-metabolite complexes by electrospray mass spectrometry: correlation to immunosuppressive activity.

Electrospray ionization mass spectrometry was used to study several non-covalent FK-binding protein (FKBP) immunosuppressant complexes in the gas phase. Relative FKBP binding affinities were determined from the signal ratio for the 7+ charge states of bound and unbound complexes as a function of capillary exit voltage. All complexes displayed a 1:1 binding stoichiometry. The relative gas-phase binding affinities were found to be well correlated with in vitro FKBP binding and in vitro immunosuppression (rapamycin > FK506 > or = 31-demethyl FK506 > 13-demethyl FK506 >> Cyclosporin A; CsA). The method demonstrates potential as a simple, rapid, and automatable technique for prediction of the immunosuppressive activity of FKBP:drug complexes.