Development and validation of an LC-MS/MS assay for the quantification of allopregnanolone and its progesterone-derived isomers, precursors, and cortisol/cortisone in pregnancy.

Neuroactive steroids are potent neuromodulators that play a critical role in both maternal and fetal health during pregnancy. These stress-responsive compounds are reportedly low in women with perinatal depression and may be associated with poor pregnancy outcomes in animal models. Chronic stress is a risk factor for adverse birth outcomes. Simultaneous quantification of neuroactive steroids, in combination with stress hormones cortisol/cortisone, provides an opportunity to investigate the synergistic relationship of these analytes within the convenience of one assay. A simple, reliable, and sensitive method for quantifying these endogenous compounds is necessary for further research with the potential to advance clinical diagnostic tools during pregnancy. Analytes were extracted from serum with a simple protein precipitation using methanol and then separated and quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After online extraction, analytes were separated using an Agilent Poroschell 120, 50 × 4.6 mm, 2.7 µm particle size, EC-C18 analytical column. The reliable quantification range was from 0.78 to 1000 ng/mL. QC sample inter- and intraday trueness was between 90 and 110% while inter- and intraday imprecision was less than 10%. Extracted samples were stable up to 7 days at 4 °C and extraction recovery was above 95%. Serum samples from 54 women in pregnancy were analyzed using this method. Here, we provide a validated, fast, and specific assay with sufficient sensitivity that allows for simultaneous quantification of blood serum concentrations of allopregnanolone (3α-hydroxy-5α-pregnan-20-one), pregnanolone (3α-hydroxy-5ß-pregnan-20-one), epipregnanolone (3ß-hydroxy-5ß-pregnan-20-one), pregnenolone, progesterone, cortisol, and cortisone in pregnancy for clinical study samples and clinical diagnostics.

Development of a Physiologically-Based Pharmacokinetic Model for Sirolimus: Predicting Bioavailability Based on Intestinal CYP3A Content.

Sirolimus is an inhibitor of mammalian target of rapamycin (mTOR) and is increasingly being used in transplantation and cancer therapies. Sirolimus has low oral bioavailability and exhibits large pharmacokinetic variability. The underlying mechanisms for this variability have not been explored to a large extent. Sirolimus metabolism was characterized by in vitro intrinsic clearance estimation. Pathway contribution ranked from CYP3A4 > CYP3A5 > CYP2C8. With the well stirred and Qgut models sirolimus bioavailability was predicted at 15%. Interindividual differences in bioavailability could be attributed to variable intestinal CYP3A expression. The physiologically-based pharmacokinetics (PBPK) model developed in Simcyp predicted a high distribution of sirolimus into adipose tissue and another elimination pathway in addition to CYP-mediated metabolism. PBPK model predictive performance was acceptable with Cmax and area under the curve (AUC) estimates within 20% of observed data in a dose escalation study. The model also showed potential to assess the impact of hepatic impairment and drug-drug interaction (DDI) on sirolimus pharmacokinetics.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e59; doi:10.1038/psp.2013.33; published online 24 July 2013.

Time-dependent effects of imatinib in human leukaemia cells: a kinetic NMR-profiling study.

The goal of this study was to evaluate the time course of metabolic changes in leukaemia cells treated with the Bcr-Abl tyrosine kinase inhibitor imatinib. Human Bcr-Abl(+) K562 cells were incubated with imatinib in a dose-escalating manner (starting at 0.1 microM with a weekly increase of 0.1 microM imatinib) for up to 5 weeks. Nuclear magnetic resonance spectroscopy and liquid-chromatography mass spectrometry were performed to assess a global metabolic profile, including glucose metabolism, energy state, lipid metabolism and drug uptake, after incubation with imatinib. Initially, imatinib treatment completely inhibited the activity of Bcr-Abl tyrosine kinase, followed by the inhibition of cell glycolytic activity and glucose uptake. This was accompanied by the increased mitochondrial activity and energy production. With escalating imatinib doses, the process of cell death rapidly progressed. Phosphocreatine and NAD(+) concentrations began to decrease, and mitochondrial activity, as well as the glycolysis rate, was further reduced. Subsequently, the synthesis of lipids as necessary membrane precursors for apoptotic bodies was accelerated. The concentrations of the Kennedy pathway intermediates, phosphocholine and phosphatidylcholine, were reduced. After 4 weeks of exposure to imatinib, the secondary necrosis associated with decrease in the mitochondrial and glycolytic activity occurred and was followed by a shutdown of energy production and cell death. In conclusion, monitoring of metabolic changes in cells exposed to novel signal transduction modulators supplements molecular findings and provides further mechanistic insights into longitudinal changes of the mitochondrial and glycolytic pathways of oncogenesis.

Worsening of long-term myocardial function after successful pharmacological pretreatment with cyclosporine.

Pretreatment with cyclosporine (CsA) decreases infarct size 24h after myocardial ischemia/reperfusion (I/R). The goal of this study was to determine effects of CsA pretreatment on long-term cardiac function after I/R-injury. Rats were randomly assigned to group-1: vehicle-only, group-2: CsA-5mg/kg/day, and group-3: CsA-12.5mg/kg/day given orally for three days prior to I/R-injury (30 min of left anterior descending coronary artery occlusion). Post-I/R survival and cardiac function were evaluated 14 days after I/R-injury by echocardiography and invasive hemodynamic measurements. Rats with I/R-injury showed increased left ventricular pressure (LVEDP) compared to rats without I/R-injury (p<0.005). Although CsA initially decreased infarct size, no differences of LVEDP were seen 14 days after I/R-injury (vehicle: 21.2+/-8.9 mmHg, CsA-5mg/kg/day: 21.5+/-0.7 mmHg, CsA-12.5mg/kg/day: 20.5+/-9.4 mmHg). Ejection fraction and fractional shortening were decreased compared to baseline, but showed no differences between groups. On day 14, a dose-dependent increase in left ventricular end diastolic diameter was seen (p<0.001). CsA pretreatment was associated with a dose-dependent decrease in post-I/R-survival (vehicle: 56%, CsA-5mg/kg/day: 32%, CsA-12.5mg/kg/day: 16%; p=0.017). CsA pretreatment did not improve long-term cardiac function despite decreased infarct size 24h after I/R-injury, but increased post-I/R mortality significantly. Poor cardiac function after CsA pretreatment might be caused by left ventricular dilation.

Biotransformation of cyclosporin in primary rat, porcine and human liver cell co-cultures.

The purpose of this study was to investigate the species-specific cyclosporin biotransformation in primary rat, human, and porcine liver cell cultures, and to investigate the suitability of a modified sandwich culture technique with non-purified liver cell co-cultures for drug metabolism studies. A sandwich culture was found to enhance hepatocellular metabolic activity and improve cellular morphology and ultrastructure. The cyclosporin metabolites AM9 and AM1 were formed in porcine and human liver cell sandwich co-cultures at levels corresponding to the respective in vivo situations. In contrast, metabolite profiles in rat hepatocytes were at variance with the in vivo situation. However, for all cell types, the overall metabolic activity was positively influenced by sandwich co-culture. The initial levels of albumin synthesis were higher in sandwich cultures than in those without matrix overlay. It is hypothesized that the sandwich culture system provides an improved microenvironment and is, therefore, an advantageous tool for in vitro studies of drug metabolism.

Effect of hepatitis C infection on tacrolimus doses and blood levels in liver transplantation recipients.

BACKGROUND: In our cohort of patients with hepatitis-C virus, which is the most common indication for liver transplantation, we have noted higher relative blood levels of tacrolimus compared to patients without hepatitis-C virus. AIM: To verify this observation and determine its clinical significance, we performed a comparison of doses and blood levels of tacrolimus in hepatitis-C virus and non- hepatitis-C virus liver transplantation recipients. METHODS: Tacrolimus dose and trough level, as well as mean alanine aminotransferase, for all patients transplanted at our center with a deceased donor between 1/1995 and 12/1999 with hepatitis-C virus were recorded at monthly intervals during the first 24 months following transplantation and compared to patients without hepatitis-C virus. RESULTS: The tacrolimus levels for hepatitis-C virus and non-hepatitis-C virus patients were not significantly different at any of the monthly intervals, except month 9. In addition, the overall mean tacrolimus levels for hepatitis-C virus and non-hepatitis-C virus patients were not significantly different (P = ns). However, the mean tacrolimus dose (mg/kg) was significantly higher for hepatitis-C virus patients at 12, 15, 18, 21 and 24 months, P < 0.01. The total mean tacrolimus dose in hepatitis-C virus patients was lower during year one by 39% (P = 0.018) and by 73% (P = 0.001) during year two. The total difference in cost of tacrolimus (for year one and two) administered to hepatitis-C virus patients was $4920, P = 0.03. The serum alanine aminotransferase was significantly higher in hepatitis-C virus patients at each monthly interval except month 1, P < or = 0.01. CONCLUSIONS: Liver transplant recipients with hepatitis-C virus require significantly lower oral doses of tacrolimus to achieve the same blood levels compared to non-hepatitis-C virus patients. This difference may result in a significant reduction in the cost of tacrolimus in hepatitis-C virus patients. The most likely explanation for these findings is decreased hepatic clearance of tacrolimus caused by mild hepatic injury from recurrent hepatitis-C virus.

Overcoming MDR by ultrasound-induced hyperthermia and P-glycoprotein modulation.

We assessed the effects of combining ultrasound-induced hyperthermia (USHT) with the P-glycoprotein modulator PSC 833 on cellular retention and cytotoxicity of rhodamine 123 (R123) and doxorubicin in the parent and multidrug resistance (MDR) variants of two human cancer lines. USHT significantly increased cellular uptake of R123 and doxorubicin. Without PSC 833, release of R123 and doxorubicin from both USHT-treated and untreated cells was rapid. As expected, PSC 833 (0.5 microM) only slowed their release into the MDR lines. Interestingly, despite the differences in their starting amounts, PSC 833 was effective in prolonging R123 and doxorubicin release from both USHT-treated and untreated MDR cells. PSC 833 did not augment the cytotoxicity of doxorubicin in parent lines but did cause a significant increase in cytotoxicity of doxorubicin in the MDR lines. However, the combined effect of USHT and PSC 833 on cytotoxicity of doxorubicin far exceeded that produced by USHT or PSC 833 alone.

Red wine decreases cyclosporine bioavailability.

BACKGROUND: Many commonly ingested substances such as grapefruit juice and Hypericum perforatum (St John's wort) have been found to interact with important therapeutic agents such as cyclosporine (INN, ciclosporin). The mechanism for these interactions is thought to involve modulation of the activity of the drug-metabolizing enzyme cytochrome P4503A4 (CYP3A4) and/or the drug transport protein Pglycoprotein. In vitro data suggest that red wine may interact with CYP3A4 substrates such as cyclosporine. METHODS: We conducted a randomized, 2-way crossover study of 12 healthy individuals. Subjects received a single 8-mg/kg dose of oral cyclosporine with water (control) and with 12 oz of red wine (Blackstone Merlot, 1996; Blackstone Winery, Graton, Calif). Whole blood was analyzed for cyclosporine and 6 metabolites by specific fluorescence polarization immunoassay and tandem liquid chromatography-mass spectrometry. Blood levels of cyclosporine were compared between the 2 arms. RESULTS: Red wine caused a 50% increase in the oral clearance of cyclosporine. Systemic exposure as measured by the area under the concentration-versus-time curve (AUC) and peak concentration (C(max)) were significantly decreased by red wine. However, half-life was not affected, suggesting that red wine decreased cyclosporine absorption. In vitro, the solubility of cyclosporine in red wine appeared to be lower than in water. CONCLUSIONS: Administration of cyclosporine with red wine causes a significant decrease in cyclosporine exposure. Because cyclosporine is a narrow therapeutic range compound, caution may be warranted with concomitant intake of red wine and cyclosporine.

Coadministration of either cyclosporine or steroids with humanized monoclonal antibodies against CD80 and CD86 successfully prolong allograft survival after life supporting renal transplantation in cynomolgus monkeys.

BACKGROUND: Recent studies have shown some efficacy using monotherapy with monoclonal antibodies (mAb) against CD80 and CD86 receptors after life-supporting renal transplantation in non-human primates. Our study was designed to evaluate the efficacy of combinations of the same mAbs with either microemulsion cyclosporine (CsA) or steroids. METHODS: Unilateral renal transplantation was performed in 16 blood group-matched and MLR-mismatched cynomolgus monkeys that were assigned to four different treatment groups. All monkeys in groups I, II, and IV were treated with the combination of a CD80 (h1F1) and CD86 (h3D1) mAb given at 20 mg/kg each preoperatively, then 5 mg/kg at weekly intervals starting postoperative (po) day 0 until poday 56 (9 doses). In group I the animals (n=4) were treated with mAbs only. In group II (n=4) mAbs were combined with a CsA regimen adjusted daily to maintain target 24 hr trough levels of 150-300 ng/ml CsA for poday 0 to poday 56. In group III (n=4) the animals received CsA monotherapy according to the same regimen as group II. In group IV methylprednisone was administered at 2 mg/kg IV on poday 0-2, then at 0.5 mg/kg/day prednisone per gavage that was and tapered to 0.2 mg/kg/day on which they were maintained until poday 56. All animals were off all immunosuppressive treatment after poday 56 and were then followed until poday 119. RESULTS: The mean survival of groups I-IV was 74 (range 9-119 days), 113 (96-119 days), 39 (22-71 days), and 79 days (6 to 119), respectively. All animals in group I showed clinical evidence of acute severe rejection (fever, creatinine increase, anuria) within the first week posttransplant, including those that retained renal function until poday 119. Only one animal in group II had a moderate clinical rejection during the treatment period and three of four animals survived the intended follow-up period. All animals in group III had multiple biopsy proven or severe clinical rejection episodes within the first 21 days and only one animal survived beyond poday 40. Moderate or severe acute rejection was diagnosed in three of four animals of group IV within the first 28 days post transplant and only one animal survived until poday 119. CONCLUSION: Our data show that combining a calcineurin inhibitor or prednisone with mAbs designed to block costimulatory signals does not antagonize the immunosuppressive efficacy of these mAbs. In addition, combining CsA with mAbs directed against the CD80 and CD86 receptors significantly prolongs graft survival when compared to CsA monotherapy. Therefore clinical trials of humanized mAbs to CD80 and CD86 used in combination with conventional immunosuppression can be considered.

Sirolimus, but not the structurally related RAD (everolimus), enhances the negative effects of cyclosporine on mitochondrial metabolism in the rat brain.

Clinical studies have shown enhancement of cyclosporine toxicity when co-administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the effects of sirolimus with those of the structurally related RAD. Two-week-old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg(-1) d(-1)), III. sirolimus (3 mg kg(-1) d(-1)), IV. RAD (3 mg kg(-1) d(-1)), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS. Cyclosporine significantly inhibited mitochondrial glucose metabolism (glutamate: 78+/-6% of control; GABA: 67+/-12%; NAD(+): 76+/-3%; P<0.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81+/-3% of control, RAD: 69+/-2%; P<0.02). Sirolimus enhanced cyclosporine-induced inhibition of mitochondrial glucose metabolism (glutamate: 60+/-4%; GABA: 59+/-8%; NAD(+): 45+/-5%; P<0.02 versus cyclosporine alone). Lactate production was significantly reduced. In contrast, RAD antagonized the effects of cyclosporine (glutamate, GABA, and NAD(+), not significantly different from controls). The results can partially be explained by pharmacokinetic interactions: co-administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co-administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria. The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their effects on brain high-energy metabolism and tissue distribution in the rat.

Metabolism of sirolimus and its derivative everolimus by cytochrome P450 3A4: insights from docking, molecular dynamics, and quantum chemical calculations.

A combination of quantum chemical calculations and molecular simulations (DOCKing and molecular dynamics) is used to investigate the metabolism of sirolimus (rapamycin) and its derivative everolimus (SDZ-RAD) by cytochrome P450 3A4. Both molecules are drugs with high immunosuppressive activity. Our calculations yield qualitative predictions of the regiospecificities of the hydroxylations and O-dealkylations occurring in these two substrates which are in good agreement with recent experimental results. An analysis of the modeled enzyme-substrate interactions allows us to rationalize the reduced metabolic activity of the larger substrate everolimus compared to sirolimus. Moreover, our simulations suggest that hydrogen donor functionalities close to the metabolic site are important for anchoring the substrate at the active center of the enzyme. In particular, we predict that replacing one hydroxyl group by a fluorine atom should considerably suppress the major metabolic reaction in sirolimus, 39-O-demethylation.

Comparison of the effects of tacrolimus and cyclosporine on the pharmacokinetics of mycophenolic acid.

Mycophenolate mofetil (MMF) is almost completely absorbed from the gut and is rapidly de-esterified into its active drug, mycophenolic acid (MPA). The main metabolite is glucuronidated MPA (MPAG), which is excreted into bile and undergoes enterohepatic recirculation. Studies in healthy volunteers treated with cholestyramine show that interruption of the enterohepatic recirculation decreases MPA exposure by approximately 40%. Published data show a difference in mycophenolic acid plasma concentrations between kidney transplant recipients treated with MMF plus cyclosporine (CsA) and those treated with MMF plus tacrolimus (TRL). However, the interpretation of these data is complicated by interpatient differences in variables that may influence MMF pharmacokinetics (e.g., underlying disease, co-medication, and time since transplantation). To understand the influence of TRL and CsA on MMF pharmacokinetics (PK) more completely, the authors eliminated confounding variables in clinical studies by performing drug interaction studies in inbred rats. To achieve a steady state, 3 groups of Lewis rats (n = 8 per group) were treated once daily with oral CsA (8 mg/kg), TRL (4 mg/kg), or placebo on days 0-6 before all rats began once-daily oral treatment with MMF (20 mg/kg) on day 7. Combined treatment with either MMF + CsA, MMF + TRL, or MMF + placebo was continued for 1 week (days 8-14). Thereafter, CsA and TRL treatments were stopped but MMF treatment was continued on days 14-21. Blood was sampled during the 24 hours subsequent to dosing on day 7 (after the first MMF dose), on day 14 (after multiple MMF doses) and on day 21 (after CsA/TRL washout). Rats in the MMF + TRL group and in the MMF + placebo group showed a second peak in the MPA-PK profiles consistent with enterohepatic recirculation of MPA. The MPA-PK profiles for the MMF + CsA-treated animals did not show a second MPA peak. On Day 14, the mean plasma MPA-AUC(0-24 hours) for the CsA-treated animals was significantly less than MPA exposures for rats in the MMF + TRL- and the MMF + placebo-treated groups. Furthermore, in contrast to results from other investigators, co-administration of CsA and MMF significantly increased MPAG-AUC(0-24 hours). Serum creatinines did not differ among rats in the three groups. CsA but not TRL decreased MPA plasma levels and increased MPAG-AUC(0-24 hours). These data suggest that CsA inhibits MPAG excretion into bile and offer an explanation for the well-known increased MPA exposure in organ transplant patients caused by conversion from CsA- to TRL-based immunosuppression.