Daniel J. Trepanier

RAPAMYCIN : DISTRIBUTION, PHARMACOKINETICS AND THERAPEUTIC RANGE INVESTIGATIONS : AN UPDATE

Based on the findings above, a number of conclusions can be made regarding the distribution, pharmacokinetics, and therapeutic range investigations with RAPA: (a) the majority of the drug is sequestered in erythrocytes, resulting in whole blood concentrations being considerably higher than plasma concentrations; (b) the drug is metabolized by the same cytochrome P450 3A enzyme involved in the metabolism of CsA and FK506. Metabolites are primarily simple demethylations and hydroxylations with 41-O-demethyl RAPA being the major metabolite both in vivo and in vitro; (c) the drug has a relatively long half-life in both humans and animals with 24-h trough concentrations being within the analytical range of HPLC when immunosuppressive doses are administered; (d) the drug exhibits a degree of proportionality between trough concentrations and dose; (e) a strong correlation exists between area under the concentration-time curve and trough blood concentration at steady state; (f) trough concentrations of the drug appear to be related to immunosuppressive efficacy and drug-related side effects; (g) the nephro- and neurotoxic properties of CsA are not augmented by concurrent treatment with RAPA; and (h) phase IIB trial results have shown a decrease of acute rejection episodes from 40% to < 10% among patients treated with full-dose CsA plus RAPA. The studies described here should provide a basis for the establishment of therapeutic monitoring protocols for RAPA. In addition, new derivatives of RAPA, such as SDZ RAD, designed to overcome formulation problems associated with RAPA, while maintaining similar pharmacokinetics and in vivo activity, show promise as alternatives to RAPA.

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.

Carbamylation of erythrocyte membrane proteins: an in vitro and in vivo study

OBJECTIVES To establish the degree of erythrocyte membrane protein carbamylation in uremic and nonuremic patients, and to characterize the in vitro binding of cyanate to the individual proteins of the cytoskeletal matrix. DESIGN AND METHODS For in vivo studies, erythrocyte ghosts were digested with proteinase K and the released peptides colorimetrically assayed for carbamylation, using the diacetyl monoxime reagent, and quantitated using homocitrulline. For in vitro studies, erythrocyte ghosts were incubated with [14C] cyanate, and the membrane proteins separated by SDS-PAGE. Cyanate incorporation was quantitated by liquid scintillation counting and imaging densitometry of the excised bands. RESULTS Erythrocytes from uremic patients were found to have a greater level of carbamylation than those from nonuremic patients (47.09 +/- 7.80 and 25.89 +/- 6.92 nmol homocitrulline/mg proteolyzed protein released, respectively). In vitro incorporation of [14C] cyanate into membrane protein followed the sequence: spectrin > ankyrin > Band 4.1 > Band 3 > actin > Band 7. CONCLUSIONS The increased level of erythrocyte membrane protein carbamylation in uremic compared to nonuremic patients may lead to membrane destabilization and contribute to the decreased erythrocyte survival time observed in uremia.

Carbamylation of erythrocyte membrane aminophospholipids: an in vitro and in vivo study

OBJECTIVES To study the binding of cyanate to erythrocyte membrane aminophospholipids in vitro, and to investigate whether carbamylated aminophospholipids can be detected in the plasma membrane of native erythrocytes. DESIGN AND METHODS For in vitro studies, the lipid components of 14C-carbamylated erythrocyte membranes were resolved by thin-layer chromatography (TLC). The covalent incorporation of cyanate was visualized by autoradiography and quantitated by phosphorus analysis. For the in vivo studies, phospholipid headgroups were enzymatically hydrolyzed by phospholipase D and subsequently reacted with diacetyl monoxime. RESULTS Both phosphatidylethanolamine (PE) and phosphatidylserine (PS) were covalently modified by [14C] cyanate; incorporating 15.76 +/- 0.09 and 13.34 +/- 0.81 mol%, respectively, following a 15-h incubation. Carbamylated PE (carb-PE) was resolved with PE by TLC in a solvent system consisting of chloroform/methanol/ammonia (65/35/5, v/v/v). Treatment of native erythrocyte membrane lipid micelles with phospholipase D, followed by reaction with diacetyl monoxime, suggests the presence of intrinsic carb-PE (2.85 +/- 0.65 percent of the total PE). CONCLUSIONS Carbamylation of erythrocyte aminophospholipid may be involved in some of the hematological consequences of uremia on the erythrocyte.

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.

In Vitro Phase I Metabolism of CRV431, a Novel Oral Drug Candidate for Chronic Hepatitis B

The cytochrome P450-mediated Phase I in vitro metabolism of CRV431 was studied using selective chemical inhibition and recombinant human enzymes. Additionally, the metabolic profile of CRV431 in human, rat, and monkey liver microsomes was investigated. Liver microsomes were incubated for 0–80 min with CRV431, and the metabolite profile was assessed by electrospray ionization liquid chromatography mass spectrometry (ESI-LCMS). CRV431 was extensively metabolized through oxidation to produce various hydroxylated and demethylated species. Species identified included monohydroxylated CRV431 (two distinct products), dihydroxylated CRV431, demethylated CRV431 (two distinct products), demethylated and hydroxylated CRV431 (two distinct products), didemethylated and hydroxylated CRV431, and didemethylated and dihydroxylated CRV431. The magnitude of metabolism was greatest in monkey, followed by human, followed by rat. Importantly, all of the species identified in human microsomes were correspondingly identified in monkey and/or rat microsomes. Human liver microsome studies using selective chemical inhibition, as well as studies using recombinant human cytochrome P450 enzymes, revealed that the major enzymes involved are cytochromes P450 3A4 and 3A5. Enzymes 1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 are not involved in the in vitro metabolism of CRV431. This information will be useful for the further development of CRV431 both preclinically and clinically.