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A macrocyclic peptide scaffold with well-established structure-property relationship is desirable for tackling undruggable targets. Here, we adopted a natural macrocycle, cyclosporin O ( CsO ) and its derivatives ( CP1 - 3 ), and evaluated the impact of conformation on membrane permeability, cyclophilin A (CypA) binding, and the pharmacokinetic (PK) profile. In nonpolar media, CsO showed a similar conformation to cyclosporin A ( CsA ), a well-known chameleonic macrocycle, but less chameleonic behavior in a polar environment. The weak chameleonicity of CsO resulted in decreased membrane permeability; however, the more rigid conformation of CsO was not detrimental to its PK profile. CsO exhibited a higher plasma concentration than CsA , which resulted from minimal CypA binding and lower accumulation in red blood cells and moderate oral bioavailability ( F = 12%). Our study aids understanding of CsO , a macrocyclic peptide that is less explored than CsA but with greater potential for diversity generation and rational design.

Transporters can play a key role in the absorption, distribution, metabolism, and excretion of drugs. Understanding these contributions early in drug discovery allows for more accurate projection of the clinical pharmacokinetics. One method to assess the impact of transporters in vivo involves co-dosing specific inhibitors. The objective of the present study was to optimize the dose and route of administration of a P-glycoprotein (P-gp) inhibitor, valspodar (PSC833), and a dual P-gp/breast cancer resistance protein (BCRP) inhibitor, elacridar (GF120918), by assessing the transporters' impact on brain penetration and absorption. A dual-infusion strategy was implemented to allow for flexibility with dose formulation. The chemical inhibitor was dosed intravenously via the femoral artery, and a cassette of known substrates was infused via the jugular vein. Valspodar or elacridar was administered as 4.5-hour constant infusions over a range of doses. To assess the degree of inhibition, the resulting ratios of brain and plasma concentrations, Kp's, of the known substrates were compared to the vehicle control. These data demonstrated that doses greater than 0.9 mg/hr/kg valspodar and 8.9 mg/hr/kg elacridar were sufficient to inhibit P-gp- and BCRP-mediated efflux at the blood-brain barrier in rats without any tolerability issues. Confirmation of BBB restriction by efflux transporters in preclinical species allows for subsequent prediction in humans based upon the proteomic expression at rodent and human BBB. Overall, the approach can also be applied to inhibition of efflux at other tissues (gut absorption, liver clearance) or can be extended to other transporters of interest using alternate inhibitors., (© 2021 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

To develop a novel intestinal drug absorption system using intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells, the cells must possess sufficient pharmacokinetic functions. However, the CYP3A4/5 activities of human iPS cell-derived small intestinal epithelial cells prepared using conventional differentiation methods is low. Further, studies of the CYP3A4/5 activities of human iPS-derived and primary small intestinal cells are not available. To fill this gap in our knowledge, here we used forskolin to develop a new differentiation protocol that activates adenosine monophosphate signaling. mRNA expressions of human iPS cell-derived small intestinal epithelial cells, such as small intestine markers, drug-metabolizing enzymes, and drug transporters, were comparable to or greater than those of the adult small intestine. The activities of CYP3A4/5 in the differentiated cells were equal to those of human primary small intestinal cells. The differentiated cells had P-glycoprotein and PEPT1 activities equivalent to those of Caco-2 cells. Differentiated cells were superior to Caco-2 cells for predicting the membrane permeability of drugs that were absorbed through a paracellular pathway and via drug transporters. In summary, here we produced human iPS cell-derived small intestinal epithelial cells with CYP3A4/5 activities equivalent to those of human primary small intestinal cells., Competing Interests: Declaration of competing interest The authors have declared no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

As drug discovery moves increasingly toward previously "undruggable" targets such as protein-protein interactions, lead compounds are becoming larger and more lipophilic. Although increasing lipophilicity can improve membrane permeability, it can also incur serious liabilities, including poor water solubility, increased toxicity, and faster metabolic clearance. Here we introduce a new efficiency metric, especially relevant to "beyond rule of 5" molecules, that captures, in a simple, unitless value, these opposing effects of lipophilicity on molecular properties. Lipophilic permeability efficiency (LPE) is defined as log D 7.4 dec/w - m lipo cLogP + b scaffold , where log D 7.4 dec/w is the experimental decadiene-water distribution coefficient (pH 7.4), cLogP is the calculated octanol-water partition coefficient, and m lipo and b scaffold are scaling factors to standardize LPE values across different cLogP metrics and scaffolds. Using a variety of peptidic and nonpeptidic macrocycle drugs, we show that LPE provides a functional assessment of the efficiency with which a compound achieves passive membrane permeability at a given lipophilicity.

Purpose: The objective of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) formulation for the oral delivery of CRV431, a non-immunosuppressive analogue of cyclosporine A. Relative to cyclosporine A, CRV431 is poorly soluble in lipid solvents and thusly presents a challenge for the development of a formulation of sufficient oral bioavailability for clinical use., Methods: The solubility of CRV431, a cyclosporine derivative, was determined in a range of commonly used surfactants, oils and co-solvents. A pseudo-ternary phase diagram was constructed from the most soluble excipients and prototype formulations, SERIES 1 and SERIES 2 were developed. The pharmacokinetics, following single oral doses of 1 and 3 mg/kg of CRV431 SMEDDS, was studied in healthy human volunteers using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS)., Results: The maximum drug load for the SERIES 1 formulations was less than 40 mg/ml. Manipulation of the excipient ratios allowed for the development of SERIES 2 formulations, which had higher drug loading capacity and stability for CRV431 compared to SERIES 1. Further improvements allowed for the development of an optimized SMEDDS formulation containing up to 90 mg/ml CRV431 and which generated a microemulsion mean particle size of 25 nm when dispersed into aqueous media. The pharmacokinetics of the optimized CRV431 SMEDDS displayed excellent total body exposure and dose-proportional effects in humans, and high drug levels in the liver of rats., Conclusions: The developed SMEDDS formulation should allow for effective clinical development of CRV431, targeted to the treatment of liver diseases including hepatitis B (HBV), fibrosis, and hepatocellular carcinoma.

Cremophor EL is a nonionic surfactant widely used in pharmaceutical formulations. Nonetheless, there are several reports on the influence of this excipient on the protein binding, pharmacokinetics, and pharmacodynamics of drugs. Valspodar is an investigational non-immunosuppressive derivative of cyclosporine A, used in clinical trials for treatment of multidrug resistant tumors. The formulation of valspodar (Amdray ® ) contains cremophor EL and ethanol as solubilizing agents. The main aim of the current study was to assess the plasma protein binding (in vitro) and the pharmacokinetic profile of valspodar in the cremophor EL-based formulation in comparison to a cremophor EL-free formulation following intravenous (i. v.) administration to rats. Valspodar dissolved in PEG 400/ethanol (diluted in Dextrose 5%) was used as the cremophor EL-free formulation. The in vitro plasma unbound fraction (f u ) of valspodar in the cremophor EL formulation was 2.3-fold higher than the PEG 400/ethanol formulation. Following a single i. v. dose of 5 mg/kg, valspodar in the cremophor EL-based formulation had around 50% lower plasma AUC compared to the PEG 400/ethanol formulation. Moreover, the cremophor EL formulation had significantly higher volume of distribution and clearance in comparison to the PEG 400-based formulation. The results highlight the significance of excipient-drug interaction that should not be overlooked during the early stages of drug development., Competing Interests: Conflicts of interest: No conflicts of interest to declare., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background and Objectives: An open-label, multicenter, randomized phase II trial was conducted from July 1, 2005 to March 29, 2011 to compare two protocols for treating children with frequently relapsing nephrotic syndrome using microemulsified cyclosporine., Design, Setting, Participants, & Measurements: Ninety-three children with frequently relapsing nephrotic syndrome were randomly assigned to group A (n=46) or group B (n=47). In both groups, the 2-hour postdose cyclosporine level was monitored. For group A, the cyclosporine target was set to 600-700 ng/ml for the first 6 months and 450-550 ng/ml for the next 18 months; for group B, it was set to 450-550 ng/ml for the first 6 months and 300-400 ng/ml for the next 18 months. The primary end point was the sustained remission rate. At the end of the study, if there was no difference in safety profile between the two groups and the sustained remission rate in group A was superior to group B with a decision threshold of 8%, then the regimen for group A would be determined the better treatment., Results: Eight children from an ineligible institution, where cyclosporine levels were not measured, were excluded from all analyses. At 24 months, the sustained remission rate was nonsignificantly higher in group A (n=43) than group B (n=42; 64.4% versus 50.0%; hazard ratio, 0.57; 95% confidence interval, 0.29 to 1.11; P=0.09), and the progression-free survival rate was significantly higher (88.1% versus 68.4%; hazard ratio, 0.33; 95% confidence interval, 0.12 to 0.94; P=0.03). The relapse rate was significantly lower in group A than group B (0.41 versus 0.95 times/person-year; hazard ratio, 0.43; 95% confidence interval, 0.19 to 0.84; P=0.02). The rate and severity of adverse events were similar in both treatment groups., Conclusion: The sustained remission rate was not significantly different between the two treatment groups, but the regimen with the higher 2-hour postdose cyclosporine level target improved progression-free survival and reduced the relapse rate.

Background & Aims: SCY-635 is a non-immunosuppressive analog of cyclosporin A that inhibits cyclophilins A and B and hepatitis C virus (HCV) replication in vitro. In a phase 1b multi-dose escalation study, we evaluated the safety, plasma pharmacokinetics, and antiviral activity of 15 days of monotherapy with SCY-635 in adults with chronic genotype 1 HCV infection., Methods: Twenty adults with chronic HCV genotype 1 were randomized to SCY-635 oral doses of 100, 200, or 300 mg three times daily for 15 days., Results: No dose-limiting clinical or laboratory toxicities were identified. On day 15, the mean decline in plasma viremia was 2.24±1.74 log(10) IU/ml with SCY-635 900 mg/d. Individual antiviral responses correlated with host IL28B genotype. Post hoc analyses indicated treatment with SCY-635 increased plasma protein concentrations of interferon α (IFNα), IFNs λ(1) and λ(3), and 2'5' oligoadenylate synthetase 1 (2'5'OAS-1), with the greatest increases in IL28B CC and CT subjects. Changes in plasma concentrations for all markers were coincident with changes in the plasma concentration of SCY-635. Peaks of IFNs α, λ(1), and λ(3) and 2'5'OAS-1 were observed within 2 h after drug administration. In replicon cells, SCY-635 enhanced secretion of type I and type III IFNs and increased the expression of IFN-stimulated genes (ISG)., Conclusions: These studies establish clinical proof of concept for SCY-635 as a novel antiviral agent and suggest that restoration of the host innate immune response to chronic hepatitis C infection may represent a major mechanism through which cyclophilin inhibitors exert clinical antiviral activity., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to assess the potential of polymeric micelles to solubilize valspodar and modify its pharmacokinetics following intravenous and oral administration in rat. Drug-loaded methoxy poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) micellar solutions were prepared and administered either intravenously or orally to healthy Sprague-Dawley rats. Plasma pharmacokinetic parameters of valspodar in its polymeric micellar formulation were compared to its clinical formulation, which uses Cremophor EL and ethanol as solubilizing agents. High loading level was achieved for valspodar in PEO-b-PCL leading to an aqueous solubility of 2.8 mg/mL. Following i.v. administration (5 mg/kg), valspodar in the PEO-b-PCL micelles provided significantly higher (approximately 77%) plasma AUC compared to the Cremophor EL formulation. The PEO-b-PCL micelles also significantly decreased the volume of distribution (Vd(ss)) and clearance (CL) of valspodar by nearly 49% and 34%, respectively. After oral administration (10 mg/kg), the average C(max) were similar for both formulations and were both reached at approximately 2 h. The plasma unbound fraction of valspodar in the polymeric micellar formulation was significantly lower than control (8.27% versus 14.85%). Our results show that PEO-b-PCL micelles can efficiently solubilize valspodar and favorably modify its pharmacokinetic profile in rat after i.v. administration by decreasing the CL and Vd., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Valspodar, a non-immunosuppressive analog of cylosporine, is a potent P-glycoprotein (MDR1) inhibitor. As MDR1-mediated efflux of chemotherapeutic agents from leukemic blasts may contribute to drug resistance, a phase 1 study of valspodar combined with mitoxantrone and etoposide in pediatric patients with relapsed or refractory leukemias was performed., Procedure: Patients received a valspodar-loading dose (2 mg/kg) followed by a 5-day continuous valspodar infusion (8, 10, 12.5, or 15 mg/kg/day) combined with lower than standard doses of mitoxantrone and etoposide. The valspodar dose was escalated using a standard 3 + 3 phase I design., Results: Twenty-one patients were evaluable for toxicity and 20 for response. The maximum tolerated dose (MTD) of valspodar was 12.5 mg/kg/day, combined with 50% dose-reduced mitoxantrone and etoposide. The clearance of mitoxantrone and etoposide was decreased by 64% and 60%, respectively, when combined with valspodar. Dose-limiting toxicities included stomatitis, ataxia, and bone marrow aplasia. Three of 11 patients with acute lymphoblastic leukemia (ALL) had complete responses while no patient with acute myeloid leukemia (AML) had an objective response. In vitro studies demonstrated P-glycoprotein expression on the blasts of 5 of 14 patients, although only 1 had inhibition of rhodamine efflux by valspodar., Conclusions: While this regimen was tolerable, responses in this heavily pretreated population were limited to a subset of patients with ALL.

SCY-635 is a novel nonimmunosuppressive cyclosporine-based analog that exhibits potent suppression of hepatitis C virus (HCV) replication in vitro. SCY-635 inhibited the peptidyl prolyl isomerase activity of cyclophilin A at nanomolar concentrations but showed no detectable inhibition of calcineurin phosphatase activity at concentrations up to 2 microM. Metabolic studies indicated that SCY-635 did not induce the major cytochrome P450 enzymes 1A2, 2B6, and 3A4. SCY-635 was a weak inhibitor and a poor substrate for P-glycoprotein. Functional assays with stimulated Jurkat cells and stimulated human peripheral blood mononuclear cells indicated that SCY-635 is a weaker inhibitor of interleukin-2 secretion than cyclosporine. A series of two-drug combination studies was performed in vitro. SCY-635 exhibited synergistic antiviral activity with alpha interferon 2b and additive antiviral activity with ribavirin. SCY-635 was shown to be orally bioavailable in multiple animal species and produced blood and liver concentrations of parent drug that exceeded the 50% effective dose determined in the bicistronic con1b-derived replicon assay. These results suggest that SCY-635 warrants further investigation as a novel therapeutic agent for the treatment of individuals who are chronically infected with HCV.

Valspodar is a P-glycoprotein inhibitor widely used in preclinical and clinical studies for overcoming multidrug resistance. Despite this, the pharmacokinetics of valspodar in rat, a commonly used animal model, have not been reported. Here, we report on the pharmacokinetics of valspodar in Sprague-Dawley rats following intravenous and oral administration of its Cremophor EL formulation, which has been used for humans in clinical trials. After intravenous doses, valspodar displayed properties of slow clearance and a large volume of distribution. Its plasma unbound fraction was around 15% in the Cremophor EL formulation used in the study. After 10 mg kg(-1) orally it was rapidly absorbed with an average maximal plasma concentration of 1.48 mg l(-1) within approximately 2 h. The mean bioavailability of valspodar was 42.8%. In rat, valspodar showed properties of low hepatic extraction and wide distribution, similar to that of its structural analogue cyclosporine A.

UNIL088 is a water-soluble prodrug of cyclosporine A (CsA) designed for topical ocular delivery. The pro-moiety is grafted via an ester function to CsA and the solubilizing group is a phosphate ion. The aim of this study was to elucidate the conversion mechanisms by which UNIL088 generates CsA. UNIL088 was incubated in rabbit tears at physiological temperature to study its enzymatic and chemical conversion, respectively. Metabolites and intermediates were identified using a quadrupole-time of flight (QqTOF) mass spectrometer, which allowed biotransformation pathways to be deduced. Conversion is activated by the chemical or enzymatic hydrolysis of the terminal ester function of the pro-moiety, leading to the phospho-serine-sarcosine-cyclosporine A that spontaneously converts into CsA. In addition to the main biotransformation pathway, a secondary reaction involved hydrolysis of the phosphate ester group of the pro-moiety, probably by phosphatases present in tears.

Topical cyclosporin A (CsA, 1) is not effective in the treatment of skin diseases, due to its low skin penetration. Following a prodrug strategy, a series of novel derivatives of 1 and of 2-[O-(2-hydroxyethyl)-d-Ser(8)]-CsA (SDZ IMM 125, 5) with potentially enhanced skin penetration properties were synthesized, in order to achieve higher levels of the active parent drugs in the skin. Permeation through skin and prodrug/drug levels in the skin were measured in vitro using rat and human skin. Introduction of a polar side chain, either in the form of a positively charged quaternary amine, a negatively charged phosphate or sulfate, or an amphiphilic phosphocholine moiety, generally increased permeability. Maximal increase in permeability through skin relative to CsA was up to 300-fold with rat skin, and up to 16-fold with human skin. Penetration into skin, as evaluated by measurement of prodrug/drug concentrations in the skin after 48 h, could be enhanced up to 14-fold (rat and human skin). Increases of permeation rates and skin concentrations showed no strict correlation. Using the phosphate 10 as prodrug, a 2.5-fold higher concentration of the active parent compound (5) could be achieved in rat skin as when administering 5 itself. The results demonstrate that in contrast with the '500 Dalton rule', which postulates poor skin penetration of molecules larger than 500 Da, high skin permeation can be achieved also with compounds of a molecular weight in the range between 1200 and 1600 Da. Results also indicate that in principle higher skin levels of active drug can be attained with a prodrug strategy in this class of compounds.

This study was conducted to define the maximum tolerated dose (MTD), dose limiting toxicity (DLT), and pharmacokinetics of idarubicin when administered with and without the P-glycoprotein inhibitor PSC-833 in combination with cytarabine, and etoposide. Fifteen patients with relapsed and refractory acute leukemia were enrolled and received cytarabine as a 7-day continuous infusion, with etoposide and idarubicin administered for any three consecutive days during the cytarabine infusion. Two hours prior to the second dose of idarubicin, PSC-833 administration was initiated. The pharmacokinetics of idarubicin alone and with PSC-833 was assessed at three idarubicin dose levels (6, 8 and 10 mg/m(2)). The MTD of idarubicin in this combination was 8 mg/(m(2) day) with a DLT of oral mucositis. The complete remission rate (on an intent-to-treat basis) for this regimen was 33%, with a median duration of 6 months. The clearance of idarubicin was 140 +/- 200 and 181 +/- 94.3 l/h for idarubicin alone and with PSC-833, respectively. The volume of distribution of the central compartment was 423 +/- 443 and 337 +/- 394 l for idarubicin alone and in combination with PSC-833, respectively. This combination including PSC-833 was well tolerated. Although a pharmacokinetic interaction might have been expected, PSC-833 did not significantly alter the disposition of idarubicin.

The mean time to peak absorption of cyclosporine (CsA) in liver transplant patients is approximately 2 hours, but in some patients the peak occurs later. The goal of this study was, therefore, to investigate the incidence of delayed absorption in 27 de novo liver transplant recipients receiving CsA > or =10 mg/kg/day (C(2) monitoring) and in 15 maintenance patients. Patients were categorized as 'normal' absorbers (C(2) exceeding C(4) and C(6)) or 'delayed' absorbers (C(4) or C(6) exceeding C(2)), and as 'good' (>800 ng/mL at C(0), C(2), C(4), or C(6)) or 'poor' absorbers (C(0), C(2), C(4) and C(6) <800 ng/mL) on the day of study. Among de novo patients, 15 (56%) had 'normal' CsA absorption and 12 (44%) 'delayed' absorption. Good CsA absorption occurred in 16 patients (59%) and poor absorption in 11 (41%). The proportion of poor absorbers was similar in patients with normal (6/15, 40%) or delayed (5/12, 42%) absorption. Among the 12 delayed absorbers, 11 had peak CsA concentration at C(4). Mean C(0) level was significantly higher in delayed absorbers (282 +/- 96 ng/mL) than in normal absorbers (185 +/- 88 ng/mL; P = .01). Delayed absorbers reverted to normal absorption (C(2) > C(4)) after a median of 6 days from the day of study, and no cases of delayed absorption were found among maintenance patients. In conclusion, almost 50% of the patients had delayed CsA absorption early posttransplant; around half of these exhibited normal CsA exposure. Measurement of C(4) in addition to C(2) differentiates effectively between delayed and poor absorbers of CsA such that over- or underimmunosuppression can be avoided.

The hydrophobic cyclic undecapeptide cyclosporin A (CyA) used in the prevention of graft rejection and in the treatment of autoimmune diseases was encapsulated by nanoprecipitation within non-biodegradable polymeric nanoparticles. The effect of polymers (Eudragit RS or RL) and additives within the alcoholic phase (fatty acid esters and polyoxyethylated castor oil) on the size, zeta potential and the encapsulation efficiency of the nanoparticles was investigated. The mean diameter of the various CyA nanoparticles ranged from 170 to 310 nm. The size as well as the zeta potential increased by adding fatty acid ester and polyoxyethylated castor oil within the organic phase. No significant differences in surface potential were observed for all formulations tested. Probably due to the very low water solubility of the drug, high encapsulation efficiencies were observed in a range from 70 to 85%. The oral absorption of CyA from these polymeric nanoparticles was studied in rabbits and compared to that of Neoral capsule. Based on comparison of the area under the blood concentration-time curve values, the relative bioavailability of CyA from each nanoparticulate formulation ranged from 20 to 35%.

Purpose: P-glycoprotein (Pgp) inhibitors have been under clinical evaluation for drug resistance reversal for over a decade. Valspodar (PSC 833) inhibits Pgp-mediated efflux but delays drug clearance, requiring reduction of anticancer drug dosage. We designed an infusional schedule for valspodar and vinblastine to mimic infusional vinblastine alone. The study was designed to determine the maximally tolerated dose of vinblastine, while attempting to understand the pharmacokinetic interactions between vinblastine and valspodar and to determine the response rate in patients with metastatic renal cell cancer., Patients and Methods: Thirty-nine patients received continuous infusion valspodar and vinblastine. Vinblastine was administered for 3 days to compensate for the expected delay in clearance and the required dose reduction. Valspodar was administered initially at a dose of 10 mg/kg/d; the dose of vinblastine varied., Results: The maximum-tolerated dose of vinblastine was 1.3 mg/m(2)/d. As suggested previously, serum valspodar concentrations exceeded those needed for Pgp inhibition. Consequently, the dose of valspodar was reduced to 5 mg/kg, allowing a vinblastine dose of 2.1 mg/m(2)/d to be administered. Pharmacodynamic studies demonstrated continued inhibition of Pgp at lower valspodar doses by functional assay in Pgp-expressing CD56+ cells and by (99m)Tc-sestamibi imaging. A 15-fold range in cytochrome p450 activity was observed, as measured by midazolam clearance. No major responses were observed., Conclusions: These results suggest that the pharmacokinetic impact of cytochrome P450 inhibition by valspodar can be reduced although not eliminated, while preserving Pgp inhibition, thus separating the pharmacokinetic and pharmacodynamic activities of valspodar.

Methadone is an opiate drug that has been identified as an in-vitro substrate of the efflux pump P-glycoprotein (P-gp), active in the intestinal epithelium and in the blood-brain barrier (BBB), among other sites. The objective of this study was to test in vivo, in the rat model, the role of P-gp modulation on the analgesic effect and brain uptake of methadone, as well as identify the most relevant site via dual oral and intravenous (i.v.) experiments. The P-gp specific inhibitor (valspodar or PSC833) was preadministered (10 mg kg(-1) i.v.) to test groups. Analgesia was measured using the tailflick test. The ED50 for oral methadone (2, 3, 6 and 8 mg kg(-1)) decreased three-fold in valspodar groups compared with controls (2.23 +/- 0.002 mg kg(-1) and 6.07 +/- 0.07 mg kg(-1); P < 0.0001). The overall analgesic effect (% antinociception) was elevated 3.1 times in pretreated compared with control rats (90.65% +/- 0.22 vs 29.23% +/- 14.0; P < 0.01) after 6 mg kg(-1) oral methadone and 2.8 times after i.v. (0.35 mg kg(-1)) administration (91.75% +/- 4.27 vs 32.45% +/- 9.0; P < 0.01). The brain:plasma distribution ratio was higher in pretreated animals and AUCbrain (overall brain concentration) was 6 times higher after oral methadone and 4 times higher after i.v. compared with controls, disproportionally increased relative to plasma, implying an active process at the BBB. P-gp, and hence substrate comedication, plays a critical role in the evolution of the methadone analgesic effect and in its brain uptake, independent of the administration route.

The main cause of illness and death after the first year of heart transplantation is vasculopathy of the cardiac allograft, probably initiated by early immunological and non-immunological endothelial damage. The incidence of multiple episodes of grade 3A rejection 6 months after primary heart transplantation was lower with everolimus (1.5 mg, 8.1% and 3 mg, 6.6%) than in the azathioprine group (14%). Allograft vasculopathy was less frequent with everolimus than azathioprine. A follow-up study is necessary to determine whether these effects translate into the important end points of reduced incidences of death, graft loss or a second transplantation.

Study Objective: To evaluate P-glycoprotein (P-gp)-mediated renal drug interactions in an in vitro model of tubular secretion., Design: In vitro experiment., Setting: University-affiliated pharmacokinetics laboratory. CELL LINES: Madin-Darby canine kidney (MDCK), multidrug-resistant-1 (MDR1)-MDCK, and human colon carcinoma (Caco-2) cells., Intervention: Transepithelial transport (basolateral-to-apical and apical-to-basolateral) of cimetidine was assessed in the absence and presence of various concentrations of the P-gp inhibitors itraconazole and PSC-833 in a renal P-gp cell culture model (MDR1-MDCK)., Measurements and Main Results: Apparent permeability of cimetidine was characterized, and level of P-gp expression was determined by Western blot analysis, in MDCK (wild type), MDR1-MDCK, and Caco-2 cells (for relative comparison). In the presence of PSC-833, cimetidine's apparent permeability value for basolateral-to-apical transport decreased from 2.96 to 1.15 x 10(-6) cm/second, coupled with a decrease in efflux ratio from 2.36 to 1.80. The effect of itraconazole was concentration dependent, with cimetidine's apparent permeability value for basolateral-to-apical transport decreasing from 3.96 to 1.92 x 10(-6) cm/second (p < 0.05), resulting in a 50% decrease in efflux ratio. Expression of P-gp was negligible in MDCK (wild-type) cells, but high-level expression was confirmed in both MDR1-MDCK and Caco-2 cells., Conclusion: P-glycoprotein plays a significant role in the renal tubular secretion of organic cations such as cimetidine, and the high level of P-gp expression in MDR1-MDCK cells makes this a well-suited model for evaluating mechanisms of renal drug interactions.

The cyclosporine analog Valspodar (PSC 833, Novartis Pharma) is a strong inhibitor of the mdr1 gene product p-glycoprotein (pgp). A phase I/II study was conducted in order to evaluate if addition of Valspodar to treatment with daunorubicin and cytarabine, given to patients with primary refractory or relapsed acute myeloid leukemia, could increase the complete remission rate.Fifty-three patients were treated in cohorts of three to six patients. Twelve patients reached a complete remission in bone marrow, five of whom also normalized their peripheral blood values. Three patients experienced treatment-related deaths from pneumonia, liver failure and cerebral hemorrhage, respectively. It is concluded that Valspodar 10 mg/kg per 24 h in combination with daunorubicin 45 mg/m(2) for 3 days and cytarabine 1 g/m(2) twice daily for 4 days is tolerable in this heavily pre-treated group of patients. Due to the moderate treatment results, the phase II part of the study was ended prematurely. The modulation of only pgp did not give an obvious improvement of the treatment results in this group of patients.

The pharmacokinetics of valspodar (PSC 833), a selective second-generation P-glycoprotein modulator, was evaluated as part of a Phase I study to modulate paclitaxel therapy in 15 patients with refractory malignancies. Valspodar was given intravenously at 1.42 mg/kg/h for 2 hours, followed by a 27-hour continuous infusion at 0.42 mg/kg/h. Serial blood samples were obtained after intravenous infusion of valspodar and paclitaxel. Valspodar disposition was best described by a linear two-compartment model. The median (range) valspodar clearance was 0.40 ml/min/kg (0.07-1.40 ml/min/kg). The 20-fold interpatient variability in valspodar clearance was not correlated with age, body weight, orgender but might be associated with coadministered medications that were metabolized via cytochrome P450 3A-mediated elimination. Valspodar whole-blood concentrations were maintained above the target threshold of 1000 ng/ml for a median of 32 hours. The pharmacokinetic model generated from this study allows for application in future studies to optimize the use of valspodar.

Information about the pharmacokinetics, safety, and efficacy of target of rapamycin (TOR) inhibitors, such as sirolimus and everolimus, in pediatric renal transplant recipients is limited. In an ascending single-dose pharmacokinetic study of sirolimus in pediatric dialysis patients, no clinically significant association was observed between patient age and absorption of sirolimus from the gastrointestinal tract. However, young pediatric patients (5 to 11 years of age) exhibited significantly greater apparent oral clearances, suggesting that pediatric patients require slightly higher doses than do adults when adjusted for body weight or surface area. Similarly, in studies performed in pediatric renal transplant recipients, the half-life of sirolimus was shorter and the clearance was greater in younger patients. On the other hand, in single-dose pharmacokinetic studies of everolimus, the apparent clearance was reduced in pediatric renal transplant recipients compared with clearance in adults. This reduced clearance was attributed to a smaller apparent volume of distribution in pediatric patients, rather than to a difference in terminal half-life. This suggested that, although the adult 12-hour dosing interval was appropriate for pediatric patients, they would require reduced dosing based on body size compared with adults. In a large trial (N = 719) of sirolimus versus azathioprine in combination with cyclosporine microemulsion and prednisone, 6 pediatric patients (13 to 18 years of age) received sirolimus at 2 mg/d, 3 received sirolimus at 5 mg/d, and 3 received azathioprine. Seven of the nine patients who received sirolimus experienced no rejection episodes. Six infectious episodes occurred in the 6 patients receiving sirolimus at 2 mg/d, 10 episodes occurred in the 3 patients receiving sirolimus at 5 mg/d, and 8 episodes occurred in the 3 patients receiving azathioprine. At 6 months after transplantation, renal function was similar in all 3 groups, although there was a statistically nonsignificant increase in the group receiving sirolimus at 5 mg/d. The mean cholesterol and triglyceride levels were generally comparable in all 3 groups. TOR inhibitors are promising agents for the prevention of graft rejection in pediatric renal transplant recipients, but more pharmacokinetic data are required to assess the optimal dosing regimens in this population. In addition, further data are needed on the efficacy and safety of TOR inhibitors in combination with other agents in pediatric transplantation recipients to best assess the role of TOR inhibition in corticosteroid and/or calcineurin inhibitor-sparing regimens.

The transfer kinetics of cyclosporine across the dually perfused rat placenta in the maternal to fetal direction and a possible involvement of P-glycoprotein were investigated. The transplacental clearance of cyclosporine in the materno-fetal direction was found to be dependent on the maternal inflow concentration of cyclosporine. Coadministration of cyclosporine with an excess of quinidine or chlorpromazine into the maternal compartment revealed 1.7- and 1.9-fold increase in cyclosporine concentration in the fetal compartment. In the experiments where quinidine was present both in the maternal and fetal compartments, cyclosporine appeared in the fetal compartment significantly faster, and its amount was three times higher when compared with controls. Conversely, quinidine or chlorpromazine did not affect the transplacental passage of L-[(3)H]-glucose. The interference of quinidine with the metabolism of cyclosporine in the placenta was excluded because only traces of M-1 and M-17 metabolites were found in the fetal solutions. Sodium azide, a mitochondrial respiratory inhibitor, was found to double the rate of cyclosporine, but not L-[(3)H]-glucose, passage across the placenta. Our findings indicate that P-glycoprotein pumps cyclosporine out of the trophoblast cells of the rat placenta in the ATP-dependent manner and restricts the passage of cyclosporine across the placental barrier., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1583-1592, 2001)

Background: PSC 833 is a second-generation P-glycoprotein (Pgp) antagonist developed to reverse multidrug resistance (MDR). The authors conducted a Phase I study of orally administered PSC 833 in combination with vinblastine administered as a 5-day continuous infusion., Methods: Seventy-nine patients with advanced malignant disease were enrolled in the trial and treated with escalating doses of PSC 833. Pharmacokinetic interactions between PSC 833 and vinblastine were anticipated. Accordingly, when dose limiting toxicities were observed, the dose of vinblastine was reduced as PSC 833 was escalated. Three schedules and two formulations of PSC 833 were used in the study., Results: The maximum tolerated doses of PSC 833 were 12.5 mg/kg orally every 12 hours for 8 days for the liquid formulation in combination with 0.9 mg/m(2) per day vinblastine as a continuous intravenous infusion (CIV) for 5 days; and 4 mg/kg orally every 6 hours for 8 days for the microemulsion formulation in combination with 0.6 mg/m(2) per day vinblastine CIV for 5 days. The principal toxicities for PSC 833 were ataxia and paresthesias and for the combination, constipation, fever. and neutropenia. Increased oral bioavailability and increased peak and trough concentrations were observed with the microemulsion formulation. Significant interpatient variability in pharmacokinetic parameters was observed. Ten patients studied at the MTD for PSC 833 (4 mg/kg orally every 6 hours for 8 days) had inhibition of rhodamine efflux from CD56 positive peripheral lymphocytes as a surrogate for Pgp antagonism. Among 43 evaluable patients with clear cell carcinoma of the kidney, 3 patients had complete responses, and 1 patient had a partial response., Conclusions: PSC 833 in combination with vinblastine can be administered safely to patients provided the vinblastine dose is adjusted for pharmacokinetic interactions. The high interpatient variability is a significant confounding factor. Surrogate studies with CD56 positive cells suggest that Pgp inhibition in the clinical setting is achievable. Improved methods for predicting pharmacokinetic interactions should improve future studies., (Copyright 2001 American Cancer Society.)

PSC 833 (Valspodar) can reverse multidrug resistance (MDR) in patients with hematologic malignancies, but alters the pharmacokinetics of concomitant anticancer agents. A phase I, dose-finding study was initiated to define a safe and effective regimen of mitoxantrone, etoposide, and cytarabine (MEC) when administered with PSC 833 to patients with early relapsed or refractory acute myeloid leukemia (AML). Poor-prognosis AML patients refractory to first-line induction therapy or relapsing within 9 months of attaining complete remission (CR) were treated with cytarabine (1.0 g/m2/day), etoposide (30 mg/m2/day), and mitoxantrone at a dose of either 3.0 mg/m2/day (cohort 1) or 4.5 mg/m2/day (cohorts 2 and 3) for 6 days plus continuous-infusion PSC 833 (10 mg/kg/24 h with a 2.0 mg/kg loading dose) for 6 or 7 days each 21-day cycle. Patients achieving CR were given a 4-day MEC plus PSC 833 consolidation cycle. Twenty-three patients were enrolled (eight with primary refractory AML and 15 in relapse). Dose-limiting toxicity occurred in one of six patients in cohort 2 (grade 4 mucositis) and one of seven patients in cohort 3 (grade 4 hyperbilirubinemia). The maximum tolerated dose of mitoxantrone was defined as 4.5 mg/m2/day. Clinically significant grade 4 hyperbilirubinemia, possibly related to PSC 833, occurred in four patients. Hematologic toxicities were as expected in this patient population, but were not dose limiting. Mild to moderate cerebellar ataxia and paresthesia occurred in six (26%) and five (22%) patients, respectively, but were not dose limiting. Overall, six of 23 (26%) patients achieved CR, including five patients with demonstrated P-glycoprotein expression and/or function. The median overall survival was 4 months. All six patients with a CR were alive and four (17%) patients were disease free at 12 months. Blood levels of PSC 833 were well above the target level of 1000 ng/ml, a concentration that is known to reverse MDR in vitro. PSC 833 reduced the clearance of etoposide by approximately two-fold. No correlation was observed between the mitoxantrone or etoposide area under the curve and response. In conclusion, the MEC plus PSC 833 tested regimen was well tolerated and the 26% CR rate warrants further testing of this regimen in a randomized, phase III trial.

Purpose: To determine the maximum-tolerated dose, pharmacokinetic interaction, and activity of PSC 833 compared with daunorubicin (DNR) and cytarabine in patients with poor-risk acute myeloid leukemia., Patients and Methods: Patients received ara-C 3 g/m(2)/d on 5 consecutive days, followed by an IV loading dose of PSC 833 (1.5 mg/kg) and an 84-hour continuous infusion escalating from 6, 9, or 10 mg/kg/d. Daunorubicin was administered as a 72-hour continuous infusion at 34 or 45 mg/m2/d [corrected]. Responding patients received consolidation chemotherapy with DNR pharmacokinetics performed without PSC-833 on day 1, and with PSC-833 on day 4. Response was correlated with expression of P-glycoprotein and lung resistance protein (LRP), and in vitro sensitization of leukemia progenitors to DNR cytotoxicity by PSC 833., Results: All 43 patients are assessable for toxicity and response. Grade 3 or greater hyperbilirubinemia (70%) was the only dose-dependent toxicity. Four patients (9%) succumbed to treatment-related complications. Twenty-one patients (49%) achieved a complete remission or restored chronic phase, including 10 of 20 patients treated at the maximum-tolerated dose of 10 mg/kg/d of PSC-833 and 45 mg/m(2) of DNR. The 95% confidence interval for complete response was 33.9% to 63.7%. Administration of PSC 833 did not alter the mean area under the curve for DNR, although clearance decreased approximately two-fold (P =.04). Daunorubicinol clearance decreased 3.3-fold (P =.016). Remission rates were not effected by mdr-1 expression, but LRP overexpression was associated with chemotherapy resistance., Conclusion: Combined treatment with infused PSC 833 and DNR is well tolerated and has activity in patients with poor risk acute myeloid leukemia. Administration of PSC 833 delays elimination of daunorubicinol, but yields variable changes in DNR systemic exposure.

PSC-833 reverses multidrug resistance by P-glycoprotein at concentrations < or = 1000 ng / ml. A phase I study of PSC-833 and doxorubicin was conducted to determine the maximum tolerated dose and to investigate pharmacokinetics. PSC-833 was intravenously infused as a 2-h loading dose (LD) and a subsequent 24-h continuous dose (CD). Doxorubicin was infused over 5 min, 1 h after the LD. The starting dose was 1 mg / kg for both LD and CD with 30 mg / m(2) doxorubicin; these dosages were increased to 2 and 10 mg / kg and 50 mg / m(2), respectively. Thirty-one patients were treated. Nausea / vomiting was controllable with granisetron and dexamethasone. Neutropenia and ataxia were dose limiting. Steady-state concentrations of PSC-833 > 1000 ng / ml were achieved at a 2 mg / kg LD and a 10 mg / kg CD. Ex-vivo bioassay revealed that activity in serum for reversing multidrug resistance was achieved in all patients; IC(50) of P-glycoprotein expressing 8226 / Dox(6) in patients' serum was decreased from 5.9 to 1.3 microg / ml (P < 0.0001) by PSC-833 administration. Doxorubicin clearance was 24.3 +/- 13.7 (mean +/- SD) liter / h/m(2), which was lower than the 49.0 +/- 16.9 liter / h/m(2) without PSC-833 (P < 0.0001). The relationship between doxorubicin exposure and neutropenia did not differ between patients treated and not treated with PSC-833. The recommended phase II dose of PSC-833 was 2 and 10 mg / kg for LD and CD, respectively, which achieved a sufficient concentration in serum to reverse drug resistance, as confirmed by bioassay. The dose of doxorubicin should be reduced to 40 mg / m(2), not because of the pharmacodynamic interaction between PSC-833 and doxorubicin affecting hematopoiesis, but because of pharmacokinetic interaction.

Purpose: To evaluate the maximum-tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetic (PK) profile of paclitaxel and carboplatin when administered every 3 weeks with the oral semisynthetic cyclosporine analog valspodar (PSC 833), an inhibitor of P-glycoprotein function., Patients and Methods: Fifty-eight patients were treated with escalating doses of paclitaxel ranging from 54 to 94.5 mg/m(2) and carboplatin area under the plasma concentration versus time curve (AUC) ranging from 6 to 9 mg.min/mL, every 21 days. The dose of valspodar was fixed at 5 mg/kg every 6 hours for a total of 12 doses from day 0 to day 3. The MTD was determined for the following two groups: (1) previously treated patients, where paclitaxel and carboplatin doses were escalated; and (2) chemotherapy-naïve patients, where paclitaxel dose was escalated and carboplatin AUC was fixed at 6 mg.min/mL. PK studies of paclitaxel and carboplatin were performed on day 1 of course 1., Results: Fifty-eight patients were treated with 186 courses of paclitaxel, carboplatin, and valspodar. Neutropenia, thrombocytopenia, and hepatic transaminase elevations were DLTs. In previously treated patients, no DLTs occurred at the first dose level (paclitaxel 54 mg/m(2) and carboplatin AUC 6 mg.min/mL). However, one of 12, two of six, two of four, four of 11, and two of five patients experienced DLTs at doses of paclitaxel (mg/m(2))/carboplatin AUC (mg.min/mL) of 67.5/6, 81/6, 94.5/6, 67. 5/7.5, and 67.5/9, respectively. In chemotherapy-naïve patients, one of 17 developed DLT at paclitaxel 81 mg/m(2) and carboplatin AUC 6 mg/mL.min. There was prolongation of the terminal phase of paclitaxel elimination as evidenced by an increased time that plasma paclitaxel concentration was >/= 0.05 micromol/L, ranging from 16.6 +/- 6.7 hours to 41.5 +/- 9.8 hours for paclitaxel doses of 54.5 mg/m(2) to 94.5 mg/m(2), respectively., Conclusion: The recommended phase II dose in chemotherapy-naïve patients is paclitaxel 81 mg/m(2), carboplatin AUC 6 mg.min/mL, and valspodar 5 mg/kg every 6 hours. In previously treated patients, the recommended phase II dose is paclitaxel 67.5 mg/m(2), carboplatin AUC 6 mg.min/mL, and valspodar 5 mg/kg every 6 hours. The acceptable toxicity profile supports the rationale for performing disease-directed evaluations of paclitaxel, carboplatin and valspodar on the schedule evaluated in this study.

Many systemically effective drugs such as cyclosporin A are ineffective topically because of their poor penetration into skin. To surmount this problem, we conjugated a heptamer of arginine to cyclosporin A through a pH-sensitive linker to produce R7-CsA. In contrast to unmodified cyclosporin A, which fails to penetrate skin, topically applied R7-CsA was efficiently transported into cells in mouse and human skin. R7-CsA reached dermal T lymphocytes and inhibited cutaneous inflammation. These data establish a general strategy for enhancing delivery of poorly absorbed drugs across tissue barriers and provide a new topical approach to the treatment of inflammatory skin disorders.

Aims: To investigate the effect of acute P-glycoprotein inhibition by the multidrug-resistance (MDR) modulator valspodar (SDZ PSC 833; PSC) on the pharmacokinetics, and potentially adverse pharmacodynamic effects of morphine, and its principal pharmacologically active metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G)., Methods: In a double-blind, three-way crossover study, the pharmacokinetic and potentially adverse pharmacodynamic effects (reaction time, transcutaneous PCO2, blood pressure) of morphine were compared with and without acute inhibition of P-glycoprotein by PSC. The effects of PSC alone were also evaluated. The study was performed in 18 healthy male volunteers and pharmacodynamic effects analysed by measuring the area under the effect (AUE) curve. 150 mg PSC (or its placebo) was given as an i.v. infusion over 2 h. With the expected inhibition of Pgp 1 h after starting PSC infusion, 7.5 morphine HCl (or its placebo) was infused over 2 h., Results: The infusion of PSC resulted in blood concentrations expected to inhibit Pgp mediated transport. While the pharmacokinetics of plasma morphine and M6G. were unaffected there was a small but statistically significant increase in the AUC and Cmax of M3G (11.8 and 8.3%, respectively). The t(1/2) and tmax were unaffected. The pharmacokinetic parameters of PSC were not affected by coadministration with morphine. PSC did not significantly affect the adverse events of morphine, as assessed by spontaneous reporting. Compared with PSC alone, morphine elicited an increase in reaction time (Emax 48 ms, compared with the predose absolute reaction time of 644 ms), which was not detected by the alertness-drowsiness score, indicating only slight sedation. There was a significant decrease in systolic blood pressure (Emin -9 mm Hg), and a trend for a fall in diastolic blood pressure (Emin -14.5 mm Hg) and respiratory rate (Emin -1.8 breath x min(-1)). For all these parameters, the effects of PSC/morphine were similar to that of PSC alone, suggesting some attenuation of morphine's effect. In contrast, morphine caused a significant increase in PCO2 (Emax 0.69 kPa) compared to PSC alone, indicating slight respiratory depression. This increase was similar to that of the PSC/morphine combination., Conclusions: Acute inhibition of P-glycoprotein by PSC in this setting does not affect the pharmacokinetic or safety-related pharmacodynamic profile of morphine in a clinically significant manner.

Older patients with acute myelogenous leukemia (AML) have overexpression of P-glycoprotein (Pgp+), and this has been shown to correlate quantitatively with therapeutic outcome. Since Pgp-mediated efflux of cytotoxic drugs can be inhibited by the cyclosporine analogue, PSC 833, we investigated the use of this agent with a 5-day mitoxantrone/etoposide regimen in patients over age 55 with newly diagnosed AML. Previous studies suggested a 33% incidence of grade IV/V non-hematologic toxicity with the use of mitoxantrone 10 mg/M(2) and etoposide 100 mg/M(2), each for 5 days, in this patient population. Since PSC 833 alters the pharmacokinetic excretion of MDR-related cytotoxins, this phase I dose-finding study was performed to identify doses of mitoxantrone/etoposide associated with a similar 33% incidence of grade IV/V non-hematologic toxicity, when given with PSC 833. Mitoxantrone/etoposide (M/E) doses were escalated in fixed ratio from a starting dose of M: 4 mg/M(2) and E: 40 mg/M(2), to M: 7 mg/M(2) and E: 70 mg/M(2), in successive cohorts of eight patients each. PSC 833 was well tolerated and the MTD of this M/E regimen with PSC 833 in this population was M: 6 mg/M(2) and E: 60 mg/M(2). The complete response (CR) rate for all patients was 50% (15/30) and was considerably higher for de novo than for secondary AML. These data suggest that the addition of PSC 833 to an M/E regimen for older patients with untreated AML is well tolerated but requires a reduction in M/E dosing to avoid increased toxicity.

The gut contains drug metabolizing enzymes and drug export proteins, of which the most important are CYP3A4 and P-glycoprotein. P-glycoprotein is localised to the apical membrane and CYP3A4 in the subapical cytoplasmic membranes in the enterocytes. The substrate and modulator specificity overlap between the two systems. The function of the two systems may be integrated. The intestinal first pass effect is the fraction of a drug which is metabolised or excreted during the absorption from the lumen. The significance of the intestinal first pass effect for the bioavailability of the three model drugs, midazolam, cyclosporin and digoxin, has been reviewed. The impact of different disease states in the gastrointestinal tract on the function of intestinal drug metabolising enzymes and P-glycoprotein is far from elucidated. Further insight into the function of these systems may lead to optimisation of drug therapy.

Purpose: To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacokinetics of paclitaxel when given with PSC 833 (valspodar) to patients with refractory solid tumors., Patients and Methods: Patients were initially treated with paclitaxel 175 mg/m(2) continuous intravenous infusion (CIVI) over 3 hours. Subsequently, 29 hours of treatment with CIVI PSC 833 was started 2 hours before paclitaxel treatment was initiated. In this combination, the starting dose of paclitaxel was 52.5 mg/m(2). Paclitaxel doses were escalated by 17.5 mg/m(2) increments for four subsequent cohorts. Each cohort consisted of three patients with the exception of the last cohort, which consisted of six patients. Data for the pharmacokinetics of paclitaxel with and without concurrent PSC 833 administration were obtained., Results: All 18 patients completed at least one course of concurrent treatment (median, two courses; range, one to six) and were evaluable for toxicity. The MTD for paclitaxel with PSC 833 was 122.5 mg/m(2). Neutropenia was the DLT. All patients had PSC 833 blood concentrations greater than 1, 000 ng/mL before, during, and 24 hours after the paclitaxel infusion. PSC 833 produced small increases in the paclitaxel peak plasma concentrations and areas under the concentration-time curve. However, PSC 833 greatly prolonged the terminal phase of paclitaxel, resulting in plasma paclitaxel concentrations of more than 0.05 micromol/L for much longer than expected. As a result, myelosuppression was comparable to that produced by full-dose paclitaxel given without PSC 833. Of the 16 patients who were assessable for response, one patient experienced a partial response and an additional nine patients experienced disease stabilization after paclitaxel treatment alone., Conclusion: Treatment with paclitaxel 122.5 mg/m(2) as a 3-hour CIVI concurrent with a 29-hour CIVI of PSC 833 results in acceptable toxicity. The addition of PSC 833 alters the pharmacokinetics of paclitaxel, which explains the enhanced neutropenia experienced by patients treated with this drug combination.

We have previously demonstrated that liposome encapsulation of doxorubicin (DOX) can alleviate adverse interactions with non-encapsulated DOX and the cyclosporine multidrug-resistant (MDR) modulator Valspodar. We have now investigated the behavior of different liposomal DOX formulations in MDA435LCC6/MDR-1 human breast cancer solid tumor xenograft models to identify liposome characteristics associated with enhanced therapeutic activity and the mechanism whereby increased chemosensitization is achieved. Toxicity studies incorporating conventional phosphatidylcholine (PC)/cholesterol (chol) and sterically stabilized (polyethylene glycol 2000 [PEG]-containing) formulations of DOX indicated that whereas PC/Chol DOX was approximately 3-fold more toxic in the presence of Valspodar, PEG containing distearoylglycerophosphocholine (DSPC)/Chol DOX was minimally affected. In mice bearing MDR tumors, co-administration of Valspodar and egg phosphocholine (EPC)/Chol DOX resulted in modest MDR modulation and efficacy, whereas the sterically stabilized formulation induced reductions in tumor growth equivalent to that achieved for drug-sensitive tumors treated with non-encapsulated DOX. Pharmacokinetic studies revealed a 2.5-fold increase in plasma DOX area under the curve (AUC) upon co-administration of Valspodar with EPC/Chol DOX whereas no such alterations were observed with the sterically stabilized liposomes. Compared to non-encapsulated DOX combined with Valspodar, improvements in efficacy and toxicity correlated with the extent to which liposomal DOX formulations were able to circumvent pharmacokinetic interactions. Confocal microscopy demonstrated that Valspodar increased cell-associated DOX which correlated with the level of anti-tumor efficacy., (Copyright 2000 Wiley-Liss, Inc.)

Resistance of cancer cells is the major limitation to the success of chemotherapy. Although many mechanisms of cellular resistance to anticancer drugs have been defined, the best understood of these is multidrug resistance (MDR), caused by the multidrug transporter, P-glycoprotein (P-gp), the product of the MDR1 gene. New drugs developed specifically to inhibit P-gp and modulate MDR, such as valspodar (PSC 833 [Amdray]), are currently undergoing clinical testing. Moreover, agents designed to inhibit other mechanisms of drug resistance are currently in development, and concurrent blockade of multiple mechanisms of resistance appears to be a promising approach. Coadministration of MDR1-related chemotherapeutic drugs with an MDR modulator may enhance the bioavailability of these agents sufficiently to enable oral dosing, which would potentially be more convenient and less toxic.

Background: Our aim was to determine the value of 99mTc-Sestamibi scanning as functional detection method of P-glycoprotein (Pgp) blockade by PSC 833 in solid tumour patients., Patients and Methods: Day 1 and day 4 after 2,200 mg orally administered PSC 833 the tumour area was scanned after intravenous (i.v.) administration of 400 MBq 99mTc-Sestamibi. In tumours with net 99mTc-Sestamibi uptake and in the hepatic region K-efflux was determined. Whole blood was analyzed for 99mTc-Sestamibi, and PSC 833 levels., Results: Fourteen patients were included. In the only Pgp-positive tumour with positive 99mTc-Sestamibi scanning K-efflux of 99mTc-Sestamibi decreased significantly after PSC 833 intake. A net inhibition of liver efflux of Sestamibi after PSC 833 intake was observed in all evaluable patients. PSC 833 blood levels were all above 2 mg/L during scanning; 99mTc-Sestamibi blood levels post versus pre PSC 833 were unchanged., Conclusions: PSC 833 induced modulation of K-efflux of 99mTc-Sestamibi in a Pgp positive tumour and in all patients in the liver.

In an in vitro study, the cytochrome P-450 3A (CYP3A)-dependent metabolism and drug interactions of the 3-hydroxy-3-methylglutaryl-Co A reductase inhibitors lovastatin and pravastatin were compared. Lovastatin was metabolized by human liver microsomes to two major metabolites: 6'beta-hydroxy [Michaelis-Menten constant (Km): 7.8 +/- 2.7 microM] and 6'-exomethylene lovastatin (Km,10.3 +/- 2.6 microM). 6'beta-Hydroxylovastatin formation in the liver was inhibited by the specific CYP3A inhibitors cyclosporine (Ki, 7.6 +/- 2.3 microM), ketoconazole (Ki, 0.25 +/- 0.2 microM), and troleandomycin (Ki, 26.6 +/- 18.5 microM). Incubation of pravastatin with human liver microsomes resulted in the generation of 3'alpha,5'beta, 6'beta-trihydroxy pravastatin (Km, 4,887 +/- 2,185 microM) and hydroxy pravastatin (Km, 20,987 +/- 9,389 microM). The formation rates of 3'alpha,5'beta,6'beta-trihydroxy pravastatin by reconstituted CYP3A enzymes were (1,000 microM pravastatin) 1.9 +/- 0.6 pmol.min-1.pmol CYP3A4 and 0.06 +/- 0.04 pmol.min-1.pmol CYP3A5, and the formation rates of hydroxy pravastatin were 0.12 +/- 0.02 pmol.min-1.pmol CYP3A4 and 0.02 +/- 0.004 pmol.min-1.pmol CYP3A5. The specific CYP3A inhibitors cyclosporine, ketoconazole, and troleandomycin significantly inhibited hydroxy pravastatin formation by human liver microsomes, but only ketoconazole inhibited 3'alpha, 5'beta,6'beta-trihydroxy pravastatin formation, suggesting that other CYP enzymes are involved in its formation. It is concluded that, compared with lovastatin [CLint formation 6'beta-hydroxylovastatin (microl.min-1.mg-1): 199 +/- 248, 6'-exomethylene lovastatin: 138 +/- 104)], CYP3A-dependent metabolism of pravastatin [CLint formation 3'alpha,5'beta, 6'beta-trihydroxy pravastatin (microl.min-1.mg-1): 0.03 +/- 0.03 and hydroxy pravastatin: 0.02 +/- 0.02] is a minor elimination pathway. In contrast to lovastatin, drug interactions with pravastatin CYP3A-catalyzed metabolism cannot be expected to have a clinically significant effect on its pharmacokinetics.

The aim of this study was to compare two types of cyclosporin (Cs) particles, SDZ OXL 400 and SDZ IMM 125, the latter being more hydrophilic, to understand their uptake by airway macrophages. Alveolar macrophages (AM), harvested by bronchoalveolar lavage (BAL) of hamster lungs, were cultured with two different doses (0.1 mg and 0.5 mg) for 1 h, 6 h, and 24 h. Control incubations without Cs particles or with latex particles were carried out simultaneously. Cell viability, cell activation (i.e., respiratory burst, interleukin-6 (IL-6) synthesis) and mean volume of particles phagocytosed per macrophage were measured. Both types of Cs particles did not modify the AM viability, and failed to induce IL-6 synthesis during phagocytosis but slightly decreased the cell oxidative respiratory burst. The comparison between SDZ OXL 400 and SDZ IMM 125 showed that for the lower dose the mean volume of both Cs types phagocytosed was similar at 1 h and 6 h. At 24 h an increase of the mean volume phagocytosed was seen for SDZ IMM 125 but not for SDZ OXL 400. For the higher dose the mean volume of SDZ IMM 125 phagocytosed was higher than SDZ OXL 400 at 1 h and 6 h and comparable for both types at 24 h. SDZ IMM 125 particles were phagocytosed more rapidly than SDZ OXL 400. The mean volume of phagocytosed latex particles increased with time and dose and was higher than for both Cs particle types. In conclusion, AM were seen to phagocytose particles of different physical properties (i.e., form, size, and shape), chemical properties (i.e., inert or peptidic) and degrees of hydrophilicity in a different manner.

Study Objective: To assess the potential for a drug-drug interaction between valspodar, a P-glycoprotein (mdrl) modulator used as a chemotherapy adjunct, and dexamethasone, widely included in oncology antiemetic regimens., Design: Randomized, open-label, three-period crossover study., Setting: Clinical pharmacology research center., Subjects: Eighteen healthy men volunteers (age 25.8+/-3.5 yrs, weight 71.6+/-10.3 kg)., Interventions: Subjects received single fasting oral doses of valspodar 400 mg, dexamethasone 8 mg, and both drugs concomitantly with 2- to 3-week washout phases between administrations., Measurements and Main Results: Lack of a pharmacokinetic drug-drug interaction with respect to valspodar was conclusively demonstrated for both Cmax,b (2.3+/-0.4 vs 2.4+/-0.5 microg/ml) and AUCb (19.8+/-4.8 vs 19.6+/-4.9 microg x hr/ml) inasmuch as bioequivalence criteria were satisfied when comparing administration alone with coadministration, respectively. Although no changes in the rate of dexamethasone absorption were noted on coadministration with valspodar (Cmax 88+/-23 vs 91+/-20 ng/ml), overall exposure was significantly increased by 24% on average (AUC 400+/-87 vs 494+/-90 ng x hr/ml). Regression analysis of valspodar Cmax,b and AUCb during coadministration versus the extent of the interaction (percentage increase in dexamethasone AUC) did not reveal a concentration-effect relationship (p=0.7299 and 0.9718, respectively)., Conclusion: Given dexamethasone's wide therapeutic index and the short duration of coadministration foreseen for these drugs in a clinical setting (maximum 1 wk/chemotherapy cycle), the 24% increase in dexamethasone's AUC is unlikely to be relevant. Thus no alterations in valspodar or dexamethasone dosages appear warranted when the two drugs are coadministered. Multiple-dose experience in patients would be desirable to confirm these conclusions.

PSC 833, a nonimmunosuppressive cyclosporin, is a potent inhibitor of the efflux of antitumor drugs mediated by P-glycoprotein and thus has been introduced in clinical trials as an agent to overcome multidrug resistance. The purpose of this study was to evaluate the dose-dependent pharmacokinetics of PSC 833 and its effects on the biliary excretion of endogenous substrates in rats. The major elimination route for PSC 833 is metabolism, followed by excretion into bile. The biliary clearance of PSC 833 was reduced in a dose-dependent manner, whereas no urinary excretion of PSC 833 was detectable. The tissue/blood concentration ratios for PSC 833 in the liver, kidney, intestine, and spleen were reduced in a dose-dependent manner, suggesting the presence of a saturable uptake process and/or saturable binding in these tissues. The dose-dependent increase in the tissue/blood concentration ratio in the brain suggests the presence of efflux transporters on the blood-brain barrier. PSC 833 reduced the bile flow rate by decreasing the biliary excretion of bile acids and reduced and oxidized glutathione, in a dose-dependent manner. The mechanism for the dose-dependent disposition of PSC 833 and its effects on the biliary excretion of endogenous substrates could be related to interactions with transporters.

The isolation from human liver microsomes and identification by electrospray mass spectrometry and tandem mass spectrometry of a new metabolite of IMM-125 resulting from the biotransformation of the amino acid 1 vinylic methyl group to a carboxylic acid, called the IMM-125-COOH metabolite, is described. It was found that the complex of this new metabolite with cyclophilin A is formed less easily than the corresponding cyclophilin A-IMM-125-CH2OH main metabolite and cyclophilin A-IMM-125 complexes. However, when formed, the IMM-125-COOH metabolite-cyclophilin A complex requires more collision-induced dissociation (CID) to dissociate the complex than the complexes formed with the two other ligands. The nanospray tandem mass spectrum of the IMM-125-COOH metabolite-cyclophilin A complex (m/z 1755) gives rise to cyclophilin A-ligand complexes of m/z 1751 by elimination of CO2 and of m/z 1749 by loss of CO2 and H2O or glycerol. Since immunosuppressive activity is known to be dependent on the formation of a binary complex between cyclophilin A and the drug and since the target for the binary complex was found to be the calcium- and calmodulin-dependent protein phosphatase, calcineurin, it could be interesting to measure for structurally related immunosuppressive drugs the CID energy necessary to dissociate the binary complexes in order to evaluate whether a correlation with the phosphatase activity could be derived.

We prepared a microencapsulated sustained-release formulation of cyclosporine A (CsA) and compared its efficacy to the solution formulation of cyclosporine A (Sandimmune, Sandoz) in an attempt to improve the treatment of rheumatoid arthritis. Microspheres containing cyclosporine were prepared with poly(lactic co-glycolic acid) (PLGA), a polymer in the submicron particle range of 0.22-0.8 micron. Studies were carried out to determine uptake rates and mechanisms of lymphocyte inhibition mediated by macrophages containing CsA microspheres in vitro. The results of these studies were used to establish whether lower doses of the microencapsulated cyclosporine could be used in in vivo studies in the polyarthritic rat model for rheumatoid arthritis. In vitro dissolution testing revealed that CsA was released extremely slowly from microspheres for up to 48 hr (0.002%). Radiolabeled 3H CsA was incorporated into some PLGA microspheres or the microspheres were labeled using a 99mTc radioligand when needed, and radiolabeling efficiency was consistently above 50%. Uptake studies at various microsphere-to-macrophage ratios (1:1, 1:5, 1:10) were carried out using 99mTc radiolabeled microspheres and macrophages obtained from normal and polyarthritic rats. Normal macrophages behaved significantly differently from arthritic macrophages throughout the study. Arthritic macrophages cause increased amounts of CsA to be released (68% of the dose) into the culture medium past 24 hr compared to normal macrophages (48% of the dose). This factor may account for the significantly increased inhibition (68.2%) of mixed lymphocyte culture proliferation in the presence of arthritic macrophages containing CsA-loaded PLGA microspheres over normal macrophages (48.2%) that were pre-exposed to the same microsphere dose. The equivalent quantity of CsA as that contained in the microspheres when placed in solution or the same quantity of blank PLGA microspheres caused decreased levels of lymphocyte inhibition when compared to the effects of CsA microspheres in macrophages of normal cells, but significantly decreased levels of inhibition in arthritic cells. From the in vivo studies, it is evident that CsA microspheres, even at low dose levels, were highly effective in inhibiting polyarthritis in rats.

Background: P-glycoproteins are membrane-associated transporters that can render cells resistant to a variety of chemotherapeutic drugs. Reversal agents are (preferably nontoxic) drugs that can inhibit these P-glycoproteins and thereby overcome multidrug resistance. PSC833, a cyclosporin A analog, is a reversal agent that has shown potential in in vitro experiments and in clinical trials. We tested PSC833 to determine whether it is a transported substrate of human and murine P-glycoproteins associated with multidrug resistance (encoded by the human MDR1 gene and its murine homolog, mdr1a) and whether it can completely inhibit these P-glycoproteins under simulated in vivo conditions., Methods: Monolayers of polarized LLC-PK1 pig kidney cells transfected with complementary DNA containing either MDR1 or mdr1a sequences were used to measure the directional transport of P-glycoprotein substrates under various serum conditions., Results: In contrast to two previous studies, we found that PSC833 is transported by both the MDR1 and the mdr1a P-glycoproteins, albeit at a low rate. PSC833 has a very high affinity for the MDR1 P-glycoprotein, and its Michaelis constant (Km) for transport is 50 nM, fourfold lower than for cyclosporin A. Inhibition of drug transport by PSC833 is approximately eightfold less effective in 100% fetal bovine serum than in tissue culture medium containing 10% serum. The concentration of PSC833 necessary to fully inhibit transport of digoxin and paclitaxel (Taxol) under complete (i.e., 100%) serum conditions is higher than the plasma concentrations achieved in clinical trials., Conclusions: Although PSC833 binds efficiently to the MDR1 P-glycoprotein and is released only sluggishly, the high concentrations of PSC833 necessary to inhibit this P-glycoprotein under complete serum conditions in our in vitro system suggest that it may be difficult for PSC833 alone to produce total inhibition of P-glycoprotein activity in patients.

The metabolism of valspodar (PSC 833; PSC), which is developed as a multidrug resistance-reversing agent, was investigated to assess the potential for drug-drug interactions and the pharmacological activity of major metabolites. The primary metabolites of PSC produced by human liver microsomes were monohydroxylated, as revealed by LC/MS. The major site of hydroxylation was at amino acid 9, resulting in M9, as determined by cochromatography with synthetic M9. Dihydroxylated and N-demethylated metabolites were also detected. PSC metabolism in two human livers exhibited KM values of 1.3-2.8 microM. The intrinsic clearance was 9-36 ml/min/kg of body weight. PSC biotransformation was cytochrome P450 (CYP or P450) 3A dependent, based on chemical inhibition and on metabolism by Chinese hamster ovary cells expressing CYP3A. Ketoconazole was a competitive inhibitor (Ki = 0.01-0.04 microM). The inhibition by 27 compounds, including four antineoplastic agents, corresponded to the inhibitory potentials of these compounds toward CYP3A. For vinblastine, paclitaxel, doxorubicin, and etoposide, the IC50 values were 5, 12, 20, and 150 microM, respectively. M9 was also an inhibitor, with a lower apparent affinity for CYP3A (IC50 = 21 microM), compared with that of PSC. M9 was also less active as a multidrug resistance-reversing agent. M9 demonstrated low potency in sensitizing resistant cells to paclitaxel and was a poor inhibitor of rhodamine-123 efflux from paclitaxel-resistant cells. In addition, compared with PSC, a higher concentration of M9 was needed to compete with the photoaffinity labeling of P-glycoprotein. Conversely, PSC inhibited only reactions catalyzed by CYP3A, including cyclosporine A metabolism (IC50 = 6.5 microM) and p-hydroxyphenyl-C3'-paclitaxel formation (Ki = 1.2 microM). Thus, PSC behaves in a manner very similar to that of other cyclosporines, and a comparable drug-drug interaction profile is expected.

The involvement of mdr1a P-glycoprotein (P-gP) on the tissue distribution of the multidrug resistance-reversing agent SDZ PSC 833 was assessed by use of mdr1a (-/-) mice. The mdr1a (-/-) and wild-type mdr1a (+/+) mice received a 4-h constantrate i.v. infusion (2 micrograms/min) of [14C]SDZ PSC 833. Mice were sacrificed at 0, 0.5, 1, 2 and 4 h during infusion and at 0.5, 1, 3, 8 and 24 h after stopping the infusion. Blood and tissues were analyzed on total (14C) and parental SDZ PSC 833 concentrations. Mdr1a (-/-) mice exhibited increased SDZ PSC 833 accumulation in cerebrum, cerebellum and somewhat in testes and small intestine compared with the wild-type mice. The difference between mdr1a (-/-) and (+/+) brain (cerebrum and cerebellum) penetration depended on SDZ PSC 833 blood concentrations, because this cyclosporin analog apparently governs its own brain penetration by inhibiting the P-glycoprotein pump in mdr1a (+/+) mice. Thus the mdr1a (-/-)/(+/+) ratio of brain concentrations tended to decrease and increase at high and low blood concentrations, respectively. These findings clearly demonstrate the interaction of SDZ PSC 833 with the P-glycoprotein present at the blood-brain barrier. The SDZ PSC 833 distribution in other mdr1a P-glycoprotein-expressed tissues, as well as its metabolism and elimination, was not affected by the mdr1a gene disruption. This suggests that factors other than mdr1a P-gP are involved in the disposition of this multidrug resistance-reversing agent.

A semi-preparative (high-performance liquid chromatography) method for separation of cyclosporin metabolites in a fungal fermentation sample was developed. By using the optimized chromatographic separation, 20 cyclosporin metabolites in a process sample were isolated, and their molecular masses measured by double focusing sector mass spectrometry. The structures of some of the cyclosporin congeners were investigated by tandem sector mass spectrometry using fast atom bombardment ionization. The isobaric cyclosporins D ([Val2] CS) and G ([Nva2] CS) were differentiated by significantly different relative intensities of a fragment ion at m/z 30 [CH4N]+ from a precursor ion at m/z 72 [C4H10N]+. Otherwise the tandem mass spectrometry (MS/MS) spectra of the fragment ions of the two isomers are very similar. Cyclosporin A and iso-cyclosporin A were also analysed by high energy MS/MS. No significant fragment ions were found to provide distinctions between them. Relatively good overviews of process samples containing very similar structures were achieved by combining LC separation, molecular ion recognition and MS/MS characterization.

Objective: To assess the effects of pregnancy on cyclosporine levels in six renal allograft patients., Methods: Maternal demographic, laboratory, clinical, and perinatal outcome data were recorded in six pregnant women with previous renal allografts receiving cyclosporine immunosuppression. The cyclosporine and serum creatinine levels were measured before pregnancy, during each trimester, and postpartum., Results: The mean (standard deviation [SD]) maternal age was 29.1 (3.8) years. Parity ranged from 0 to 3. Mean serum creatinine levels tended to be lower during pregnancy than before or after, as did the mean cyclosporine levels. After adjusting for dose, five of six patients had declines in cyclosporine level during pregnancy. The mean (SD) gestational age at delivery was 37.5 (2.8) weeks with a mean (SD) birth weight of 2837 (538) g., Conclusion: Pregnancy in patients with renal allografts can lead to a substantial decline in cyclosporine levels.