Launa J. Aspeslet

Simultaneous determination of fluoxetine and norfluoxetine enantiomers in biological samples by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the simultaneous analysis of the enantiomers of fluoxetine and norfluoxetine. The assay involves basic extraction of these enantiomers from the biological samples, followed by their conversion to diastereoisomers using the chiral derivatizing reagent (S)-(-)-N-trifluoroacetylprolyl chloride. The method was utilized to detect and measure the quantity of these enantiomers in plasma and urine of patients and in liver and brain tissue of rats treated with (R,S)-fluoxetine.

The monitoring of immunosuppressive drugs : A pharmacodynamic approach

Pharmacodynamic monitoring measures biologic response to a drug, which, alone or coupled with pharmacokinetics, provides a novel method for the optimization of drug dosing. Pharmacodynamic monitoring has been investigated by us and other investigators on primarily five immunosuppressive drugs: cyclosporine (CsA), mycophenolate mofetil (MMF), rapamycin (RAPA), azathioprine (AZA), and methylprednisolone (MP). The pharmacodynamic monitoring of CsA and MMF involves measurement of the activity of the enzymes calcineurin and inosine monophosphate dehydrogenase, respectively. The pharmacodynamics of AZA are assessed by measurement of the activity of thiopurine methyl transferase (TPMT), which is induced by a metabolite of AZA, 6-mercaptopurine. The pharmacodyamics for RAPA involve the measurement of a P70 S6 kinase activity within lymphocytes, whereas that for MP involves the measurement of the endogenous synthesis of cortisol by the suppression of the hypothalamic pituitary axis. To date, the most detailed studies have been performed involving pharmacodynamic monitoring of CsA and MMF. Similarities exist in the pharmacodynamic response to CsA and MMF in patients who undergo renal transplantation. At trough concentrations in blood, both drugs result in only a 50% reduction in activity of their target enzymes; however, there is considerable interpatient variability. Throughout the dosing interval, enzyme activity parallels that of drug concentrations. Renal transplant recipients who are treated with AZA and who exhibit an increase in TPMT activity from the time of transplantation experience fewer episodes of active rejection. Renal transplant recipients who are administered MP and in whom suppression of endogenous synthesis of cortisol is greatest exhibit the least incidence of steroid-induced side effects. Additional clinical trials relating pharmacokinetics and pharmacodynamic parameters to clinical response are under way to ascertain which provides the best guide for dosing. Pharmacodynamic monitoring may provide an alternative approach to traditional drug level measurement.

Requirements for therapeutic Drug monitoring of sirolimus, an immunosuppressive agent used in renal transplantation

BACKGROUND On September 15, 1999, sirolimus received approval from the US Food and Drug Administration (FDA) for marketing as an immunosuppressive agent. As with any chronically administered medication, the question arises whether therapeutic drug monitoring (TDM) is required for optimal therapy. In the case of sirolimus, there are data to suggest that TDM may be beneficial in some patients. OBJECTIVE To assess the need for monitoring sirolimus concentrations, this paper reviews the following factors influencing the usefulness of TDM: wide pharmacokinetic variability; toxicity; suspected noncompliance; suspected drug interactions; and specific demographic characteristics. Data supporting the correlation between sirolimus concentration and immunosuppressive efficacy are also discussed. RESULTS The available literature on sirolimus suggests that TDM may be required in some cases. Studies have shown that there is wide interindividual variability in the pharmacokinetic behavior of drugs in transplant patients; that there is a relationship between blood concentrations of sirolimus and adverse events; and that coadministration of cyclosporine alters the pharmacokinetics of sirolimus. Additionally, the correlation between sirolimus concentration and immunosuppressive efficacy in phase III trials suggests a benefit in transplant patients when sirolimus concentrations reach appropriate levels. Finally, noncompliance is a common occurrence in the transplant population, and monitoring is often necessary in suspected noncompliers. CONCLUSION Although additional clinical studies are needed, it appears that TDM is an important aspect of treatment with sirolimus.

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.

Natural human antibody-mediated destruction of porcine neonatal islet cell grafts

Abstract: Porcine pancreata may be considered a potential source of islets for transplantation into diabetic recipients; however, whether porcine islet grafts will be susceptible to damage by natural antibody‐mediated hyperacute rejection remains unknown. In this study, we performed Western blots to determine whether membrane proteins present on porcine neonatal islet cells (NIC) are recognized by xenoreactive antibodies present in human sera. Western blots of freshly isolated porcine NICs with AB sera detected the presence of 14 antigens (MW 24–164 kDa) and 4 antigens (MW 101–150 kDa) to which antiserum against human IgM and IgG bound, respectively. The most prominent antigens with IgM reactivity had MWs of 36, 63, and 120 kDa, whereas for IgG, the most intensely reactive antigen had a MW of 120 kDa. When membrane fractions prepared from purified porcine aortic endothelial cells and LLC‐PK1 cells were analyzed, the major antigens had molecular weights comparable to those seen for NICs. After culturing the NICs for 5 days, the number of detected xenoreactive antigens binding IgM or IgG decreased and the antigens present at 36, 63, and 120 kDa with IgM reactivity were shown to have a decreased intensity of binding. Incubation of cultured porcine NICs for 18 hr in the presence of human AB serum containing complement resulted in a 55% loss of cellular insulin content (P < 0.0001), a 45% reduction in recoverable DNA (P < 0.0001), and a marked reduction in insulin secretory response to an in vitro glucose challenge. Recovery and viability of porcine NICs was not affected when incubated with AB serum depleted of anti‐Gal antibodies with Synsorb 90. These results demonstrate that natural human antibodies of both IgM and IgG subtypes bind to antigens present on Department of Laboratory Medicine and complement reduces islet cell survival and functional viability. Adsorbing serum with the αGal(1–3)βGal(1–4)βGlc carbohydrate removes natural human antibody‐mediated destruction of porcine neonatal islet cell grafts.

Determination of p-trifluoromethylphenol, a metabolite of fluoxetine, in tissues and body fluids using an electron-capture gas chromatographic procedure

An electron-capture gas chromatographic procedure was developed for the analysis of p-trifluoromethylphenol, an O-dealkylated metabolite of fluoxetine, in biological samples. A basic extraction of the biological sample was employed, followed by derivatization with pentafluorobenzenesulfonyl chloride. The internal standard, 2,4-dichlorophenol, was added to all samples used in the procedure to aid in quantitation. The practical limit of detection (signal-to-noise ratio>3) for p-trifluoromethylphenol was <5 ng/ml in human plasma samples, <10 ng/g of rat brain tissue, <25 ng/g of rat liver tissue and <25 ng/ml in human and rat urine samples. In the rat, the levels of free p-trifluoromethylphenol in the liver were 10-fold higher than those in the brain, and a substantial amount was excreted in the urine. Human urine samples contained levels of free p-trifluoromethylphenol approximately 30-fold higher than those found in human plasma samples. The procedure described is useful for the detection and quantitation of free p-trifluoromethylphenol in humans and rats treated with fluoxetine.

ISA247: quality of life results from a phase II, randomized, placebo-controlled study.

Background: Psoriasis is a chronic skin condition that can negatively affect a patients quality of life (QoL), often hindering social functioning. ISA247, a novel psoriatic agent, has shown clinical efficacy in moderate to severe psoriasis sufferers, but its effect on QoL is currently not reported. Objective: The objective of this study was to assess the effect of ISA247 on the QoL in patients with stable, plaque-type psoriasis. Methods: A phase II, randomized, double-blind, placebo-controlled, parallel-group, multicenter study assessed the effects of ISA247 doses of 0.5 mg/kg/d (n = 77) or 1.5 mg/kg/d (n = 83) compared with placebo (n = 41) for 12 weeks. QoL was assessed using the Dermatology Life Quality Index (DLQI) and Psoriasis Disability Index (PDI) scales. Results: ISA247 treatment (pooled groups) significantly improved QoL scores as assessed by both the DLQI and the PDI compared with those receiving placebo (p < .05). Treatment with the higher dose of 1.5 mg/kg/d demonstrated a significantly greater response to many of the QoL scales compared with the 0.5 mg/kg/d group (p < .05). Conclusions: ISA247 appears to improve the QoL while also providing effective treatment for chronic, moderate to severe, plaque-type psoriasis.

A gas chromatographic procedure for separation and quantitation of the enantiomers of the antidepressant tranylcypromine

A novel assay procedure has been developed that allows for the separation and quantification of the enantiomers of the monoamine oxidase inhibitor tranylcypromine (TCP) in brain and liver of rats. The analytical method involves extraction of the drug from rat tissue with an organic solvent. TCP is then derivatized with S-(-)-N-(trifluoroacetyl)-prolyl chloride to allow gas chromatographic analysis of the resulting diastereoisomers. Conditions for analysis by a gas chromatograph equipped with a nitrogen-phosphorus detector and a capillary column are described. The method has been applied to the separation and quantification of the enantiomers of TCP in samples of brain and liver of rats that had been injected with this drug alone and after pretreatment with iprindole, a drug known to block aromatic ring hydroxylation.

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 .