Influence of Artificially Generated Interocular Blur Difference on Fusion Stability Under Vergence Stress.

The stability of fusion was evaluated by its breakage when interocular blur differences were presented under vergence demand to healthy subjects. We presumed that these blur differences cause suppression of the more blurred image (interocular blur suppression, IOBS), disrupt binocular fusion and suppressed eye leaves its forced vergent position. During dichoptic presentation of static grayscale images of natural scenes, the luminance contrast (mode B) or higher-spatial frequency content (mode C) or luminance contrast plus higher-spatial frequency content (mode A) were stepwise reduced in the image presented to the non-dominant eye. We studied the effect of these types of blur on fusion stability at various levels of the vergence demand. During the divergence demand, the fusion was disrupted with approximately half blur than during convergence. Various modes of blur influenced fusion differently. The mode C (isolated reduction of higher-spatial frequency content) violated fusion under the lowest vergence demand significantly more than either isolated or combined reduction of luminance contrast (mode B and A). According to our results, the image´s details (i.e. higher-spatial frequency content) protects binocular fusion from disruption by the lowest vergence demand.

Pharmacokinetic de-risking tools for selection of monoclonal antibody lead candidates.

Pharmacokinetic studies play an important role in all stages of drug discovery and development. Recent advancements in the tools for discovery and optimization of therapeutic proteins have created an abundance of candidates that may fulfill target product profile criteria. Implementing a set of in silico, small scale in vitro and in vivo tools can help to identify a clinical lead molecule with promising properties at the early stages of drug discovery, thus reducing the labor and cost in advancing multiple candidates toward clinical development. In this review, we describe tools that should be considered during drug discovery, and discuss approaches that could be included in the pharmacokinetic screening part of the lead candidate generation process to de-risk unexpected pharmacokinetic behaviors of Fc-based therapeutic proteins, with an emphasis on monoclonal antibodies.

Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases.

Anti-angiogenic therapies using biological molecules that neutralize vascular endothelial growth factor-A (VEGF-A) have revolutionized treatment of retinal vascular diseases including age-related macular degeneration (AMD). This study reports preclinical assessment of a strategy to enhance anti-VEGF-A monotherapy efficacy by targeting both VEGF-A and angiopoietin-2 (ANG-2), a factor strongly upregulated in vitreous fluids of patients with retinal vascular disease and exerting some of its activities in concert with VEGF-A. Simultaneous VEGF-A and ANG-2 inhibition was found to reduce vessel lesion number, permeability, retinal edema, and neuron loss more effectively than either agent alone in a spontaneous choroidal neovascularization (CNV) model. We describe the generation of a bispecific domain-exchanged (crossed) monoclonal antibody (CrossMAb; RG7716) capable of binding, neutralizing, and depleting VEGF-A and ANG-2. RG7716 showed greater efficacy than anti-VEGF-A alone in a non-human primate laser-induced CNV model after intravitreal delivery. Modification of RG7716's FcRn and FcγR binding sites disabled the antibodies' Fc-mediated effector functions. This resulted in increased systemic, but not ocular, clearance. These properties make RG7716 a potential next-generation therapy for neovascular indications of the eye.

Simulation of monoclonal antibody pharmacokinetics in humans using a minimal physiologically based model.

Compared to small chemical molecules, monoclonal antibodies and Fc-containing derivatives (mAbs) have unique pharmacokinetic behaviour characterised by relatively poor cellular permeability, minimal renal filtration, binding to FcRn, target-mediated drug disposition, and disposition via lymph. A minimal physiologically based pharmacokinetic (PBPK) model to describe the pharmacokinetics of mAbs in humans was developed. Within the model, the body is divided into three physiological compartments; plasma, a single tissue compartment and lymph. The tissue compartment is further sub-divided into vascular, endothelial and interstitial spaces. The model simultaneously describes the levels of endogenous IgG and exogenous mAbs in each compartment and sub-compartment and, in particular, considers the competition of these two species for FcRn binding in the endothelial space. A Monte-Carlo sampling approach is used to simulate the concentrations of endogenous IgG and mAb in a human population. Existing targeted-mediated drug disposition (TMDD) models are coupled with the minimal PBPK model to provide a general platform for simulating the pharmacokinetics of therapeutic antibodies using primarily pre-clinical data inputs. The feasibility of utilising pre-clinical data to parameterise the model and to simulate the pharmacokinetics of adalimumab and an anti-ALK1 antibody (PF-03446962) in a population of individuals was investigated and results were compared to published clinical data.

Are endosomal trafficking parameters better targets for improving mAb pharmacokinetics than FcRn binding affinity?

F.W.R. Brambell deduced the existence of a protective receptor for IgG, the neonatal Fc receptor (FcRn), long before its discovery in the early to mid-1990s. With the coincident, explosive development of IgG-based drugs, FcRn became a popular target for tuning the pharmacokinetics of monoclonal antibodies (mAbs). One aspect of Brambell's initial observation, however, that is seldom discussed since the discovery of the receptor, is the compliance in the mechanism that Brambell observed (saturating at 10s-100s of µM concentration), vs. the comparative stiffness of the receptor kinetics (saturating in the nM range for most species). Although some studies reported that increasing the already very high Fc-FcRn affinity at pH 6.0 further improved mAb half-life, in fact the results were mixed, with later studies increasingly implicating non-FcRn-dependent mechanisms as determinants of mAb pharmacokinetics. Mathematical modelling of the FcRn system has also indicated that the processes determining the pharmacokinetics of mAbs have more nuances than had at first been hypothesised. We propose, in keeping with the latest modelling and experimental evidence reviewed here, that the dynamics of endosomal sorting and trafficking have important roles in the compliant salvage mechanism that Brambell first observed nearly 50 years ago, and therefore also in the pharmacokinetics of mAbs. These ideas lead to many open questions regarding the endosomal trafficking of both FcRn and mAbs and also to what properties of a mAb can be altered to achieve an improvement in pharmacokinetics.

Inosine monophosphate dehydrogenase expression and activity are significantly lower in kidney transplant recipients with diabetes mellitus.

BACKGROUND: Inosine 5'-monophosphate dehydrogenase (IMPDH) is a target of the immunosuppressive drug, mycophenolic acid (MPA). A 12-hour clinical pharmacokinetic and pharmacodynamic study was conducted to compare IMPDH1 and IMPDH2 gene expression, IMPDHI and IMPDHII protein levels, and enzyme activity between kidney transplant recipients with respect to diabetes status. METHODS: Nondiabetic (ND, n = 11) and diabetic (D, n = 9) kidney transplant recipients and on nontransplant nondiabetic (n = 10) and diabetic (n = 10) volunteers were included in the study. RESULTS: Area under the effect curve values for gene expression: IMPDH1 [ND: 22.1 (13.8-31.3) versus D: 4.5 (2.3-6.5), P < 0.001] and IMPDH2 [ND: 15.3 (11.0-21.7) versus D: 6.1 (4.6-8.6), P < 0.001], protein level: IMPDHI [ND: 1.0 (0.5-1.3) versus 0.5 (0.4-0.7), P = 0.002] and IMPDHII [ND: 1.0 (0.6-1.6) versus D: 0.7 (0.6-0.8) P < 0.001] and enzyme activity [ND: 180 (105-245) versus D: 29.9 (15.3-35.6) µmole·s(-1)·mole(-1) adenosine monophosphate, P < 0.001] was significantly lower in transplant recipients with diabetes. Similar results were observed in nontransplanted volunteers. Kinetic studies of MPA-mediated suppression of IMPDH activity in nontransplanted individuals revealed an approximately 2.5-fold lower half-maximum effective concentration (EC50) for diabetic as compared with nondiabetic [ND: 50.2 (49.8-50.7) versus D: 15.8 (15.6-16.3) nmole/L, P = 0.004] volunteers. This difference was not related to several IMPDH gene variants. CONCLUSIONS: This study indicates a significantly lower IMPDH gene expression, protein level, and enzyme activity in diabetic patients. Further clinical studies in a larger number of patients are warranted to verify whether MPA dosing must be optimized for kidney transplant recipients with diabetes mellitus.

Pharmacokinetics, pharmacodynamics and physiologically-based pharmacokinetic modelling of monoclonal antibodies.

Development of monoclonal antibodies (mAbs) and their functional derivatives represents a growing segment of the development pipeline in the pharmaceutical industry. More than 25 mAbs and derivatives have been approved for a variety of therapeutic applications. In addition, around 500 mAbs and derivatives are currently in different stages of development. mAbs are considered to be large molecule therapeutics (in general, they are 2-3 orders of magnitude larger than small chemical molecule therapeutics), but they are not just big chemicals. These compounds demonstrate much more complex pharmacokinetic and pharmacodynamic behaviour than small molecules. Because of their large size and relatively poor membrane permeability and instability in the conditions of the gastrointestinal tract, parenteral administration is the most usual route of administration. The rate and extent of mAb distribution is very slow and depends on extravasation in tissue, distribution within the particular tissue, and degradation. Elimination primarily happens via catabolism to peptides and amino acids. Although not definitive, work has been published to define the human tissues mainly involved in the elimination of mAbs, and it seems that many cells throughout the body are involved. mAbs can be targeted against many soluble or membrane-bound targets, thus these compounds may act by a variety of mechanisms to achieve their pharmacological effect. mAbs targeting soluble antigen generally exhibit linear elimination, whereas those targeting membrane-bound antigen often exhibit non-linear elimination, mainly due to target-mediated drug disposition (TMDD). The high-affinity interaction of mAbs and their derivatives with the pharmacological target can often result in non-linear pharmacokinetics. Because of species differences (particularly due to differences in target affinity and abundance) in the pharmacokinetics and pharmacodynamics of mAbs, pharmacokinetic/pharmacodynamic modelling of mAbs has been used routinely to expedite the development of mAbs and their derivatives and has been utilized to help in the selection of appropriate dose regimens. Although modelling approaches have helped to explain variability in both pharmacokinetic and pharmacodynamic properties of these drugs, there is a clear need for more complex models to improve understanding of pharmacokinetic processes and pharmacodynamic interactions of mAbs with the immune system. There are different approaches applied to physiologically based pharmacokinetic (PBPK) modelling of mAbs and important differences between the models developed. Some key additional features that need to be accounted for in PBPK models of mAbs are neonatal Fc receptor (FcRn; an important salvage mechanism for antibodies) binding, TMDD and lymph flow. Several models have been described incorporating some or all of these features and the use of PBPK models are expected to expand over the next few years.

Insulin resistance, ceramide accumulation and endoplasmic reticulum stress in experimental chronic alcohol-induced steatohepatitis.

AIMS: Chronic alcohol abuse causes steatohepatitis with insulin resistance, which impairs hepatocellular growth, survival and metabolism. However, growing evidence supports the concept that progressive alcohol-related liver injury may be mediated by concurrent mal-signaling through other networks that promote insulin resistance, e.g. pro-inflammatory, pro-ceramide and endoplasmic reticulum (ER) stress cascades. METHODS: Using the Long Evans rat model of chronic ethanol feeding, we characterized the histopathologic and ultrastructural features of steatohepatitis in relation to biochemical and molecular indices of tissue injury, inflammation, insulin resistance, dysregulated lipid metabolism and ER stress. RESULTS: Chronic steatohepatitis with early chicken-wire fibrosis was associated with enlargement of mitochondria and disruption of ER structure by electron microscopy, elevated indices of lipid storage, lipid peroxidation and DNA damage, increased activation of pro-inflammatory cytokines, impaired signaling through the insulin receptor (InR), InR substrate-1, Akt, ribosomal protein S6 kinase and proline-rich Akt substrate 40 kDa, glycogen synthase kinase 3ß activation and constitutive up-regulation of ceramide and ER stress-related genes. Liquid chromatography coupled with tandem mass spectrometry demonstrated altered ceramide profiles with higher levels of C14 and C18, and reduced C16 species in ethanol-exposed livers. CONCLUSION: The histopathologic and ultrastructural abnormalities in chronic alcohol-related steatohepatitis are associated with persistent hepatic insulin resistance and pro-inflammatory cytokine activation, dysregulated lipid metabolism with altered ceramide profiles and both ER and oxidative stress. Corresponding increases in lipid peroxidation, DNA damage and protein carbonylation may have contributed to the chronicity and progression of disease. The findings herein suggest that multi-pronged therapeutic strategies may be needed for effective treatment of chronic alcoholic liver disease in humans.

Diabetes mellitus reduces the clearance of atorvastatin lactone: results of a population pharmacokinetic analysis in renal transplant recipients and in vitro studies using human liver microsomes.

BACKGROUND AND OBJECTIVE: Patients with diabetes mellitus might be at a higher risk of HMG-CoA reductase inhibitor (statin)-induced myotoxicity, possibly because of reduced clearance of the statin lactone. The present study was designed to investigate the effect of diabetes on the biotransformation of atorvastatin acid, both in vivo in nondiabetic and diabetic renal transplant recipients, and in vitro in human liver samples from nondiabetic and diabetic donors. SUBJECTS AND METHODS: A total of 312 plasma concentrations of atorvastatin acid and atorvastatin lactone, from 20 nondiabetic and 32 diabetic renal transplant recipients, were included in the analysis. Nonlinear mixed-effects modelling was employed to determine the population pharmacokinetic estimates for atorvastatin acid and atorvastatin lactone. In addition, the biotransformation of these compounds was studied using human liver microsomal fractions obtained from 12 nondiabetic and 12 diabetic donors. RESULTS: In diabetic patients, the plasma concentration of atorvastatin lactone was significantly higher than that of atorvastatin acid throughout the 24-hour sampling period. The optimal population pharmacokinetic model for atorvastatin acid and atorvastatin lactone consisted of a two- and one-compartment model, respectively, with interconversion between atorvastatin acid and atorvastatin lactone. Parent drug was absorbed orally with a population estimate first-order absorption rate constant of 0.457 h(-1). The population estimates of apparent oral clearance (CL/F) of atorvastatin acid to atorvastatin lactone, intercompartmental clearance (Q/F), apparent central compartment volume of distribution after oral administration (V(1)/F) and apparent peripheral compartment volume of distribution after oral administration (V(2)/F) for atorvastatin acid were 231 L/h, 315 L/h, 325 L and 4910 L, respectively. The population estimates of apparent total clearance of atorvastatin lactone (CL(M)/F), apparent intercompartmental clearance of atorvastatin lactone (Q(M)/F) and apparent volume of distribution of atorvastatin lactone after oral administration (V(M)/F) were 85.4 L/h, 166 L/h and 249 L, respectively. The final covariate model indicated that the liver enzyme lactate dehydrogenase was related to CL/F and alanine aminotransferase (ALT) was related to Q/F. Importantly, diabetic patients have 3.56 times lower CL(M)/F than nondiabetic patients, indicating significantly lower clearance of atorvastatin lactone in these patients. Moreover, in a multivariate population pharmacokinetics model, diabetes status was the only significant covariate predicting the values of the CL(M)/F. Correspondingly, the concentration of atorvastatin acid remaining in the microsomal incubation was not significantly different between nondiabetic and diabetic liver samples, whereas the concentration of atorvastatin lactone was significantly higher in the samples from diabetic donors. In vitro studies, using recombinant enzymes, revealed that cytochrome P450 (CYP) 3A4 is the major CYP enzyme responsible for the biotransformation of atorvastatin lactone. CONCLUSIONS: These studies provide compelling evidence that the clearance of atorvastatin lactone is significantly reduced by diabetes, which leads to an increased concentration of this metabolite. This finding can be clinically valuable for diabetic transplant recipients who have additional co-morbidities and are on multiple medications.

Effect of diabetes mellitus on pharmacokinetic and pharmacodynamic properties of drugs.

The effects of diabetes mellitus on the pharmacokinetics and pharmacodynamics of drugs have been well described in experimental animal models; however, only minimal data exist for humans and the current knowledge regarding the effects of diabetes on these properties remains unclear. Nevertheless, it has been observed that the pharmacokinetics and pharmacodynamics of drugs are changed in subjects with diabetes. It has been reported that diabetes may affect the pharmacokinetics of various drugs by affecting (i) absorption, due to changes in subcutaneous adipose blood flow, muscle blood flow and gastric emptying; (ii) distribution, due to non-enzymatic glycation of albumin; (iii) biotransformation, due to regulation of enzymes/transporters involved in drug biotransformation; and (iv) excretion, due to nephropathy. Previously published data also suggest that diabetes-mediated changes in the pharmacokinetics of a particular drug cannot be translated to others. Although clinical studies exploring the effect of diabetes on pharmacodynamics are still very limited, there is evidence that disease-mediated effects are not limited only to pharmacokinetics but also alter pharmacodynamics. However, for many drugs it remains unclear whether these influences reflect diabetes-mediated changes in pharmacokinetics rather than pharmacodynamics. In addition, even though diabetes-mediated pharmacokinetics and pharmacodynamics might be anticipated, it is important to study the effect on each drug and not generalize from observed data. The available data indicate that there is a significant variability in drug response in diabetic subjects. The discrepancies between individual clinical studies as well as between ex vivo and clinical studies are probably due to (i) the restricted and focused population of subjects in clinical studies; (ii) failure to consider type, severity and duration of the disease; (iii) histopathological characteristics generally being missing; and (iv) other factors such as varying medication use, dietary protein intake, age, sex and obesity. The obesity epidemic in the developed world has also inadvertently influenced the directions of pharmacological research. This review attempts to map new information gained since Gwilt published his paper in Clinical Pharmacokinetics in 1991. Although a large body of research has been conducted and significant progress has been made, we still have to conclude that the available information regarding the effect of diabetes on pharmacokinetics and pharmacodynamics remains unclear and further clinical studies are required before we can understand the clinical significance of the effect. An understanding of diabetes-mediated changes as well as of the source of the variability should lead to the improvement of the medical management and clinical outcomes in patients with this widespread disease.

Insulin resistance, ceramide accumulation, and endoplasmic reticulum stress in human chronic alcohol-related liver disease.

BACKGROUND: Chronic alcohol-related liver disease (ALD) is mediated by insulin resistance, mitochondrial dysfunction, inflammation, oxidative stress, and DNA damage. Recent studies suggest that dysregulated lipid metabolism with accumulation of ceramides, together with ER stress potentiate hepatic insulin resistance and may cause steatohepatitis to progress. OBJECTIVE: We examined the degree to which hepatic insulin resistance in advanced human ALD is correlated with ER stress, dysregulated lipid metabolism, and ceramide accumulation. METHODS: We assessed the integrity of insulin signaling through the Akt pathway and measured proceramide and ER stress gene expression, ER stress signaling proteins, and ceramide profiles in liver tissue. RESULTS: Chronic ALD was associated with increased expression of insulin, IGF-1, and IGF-2 receptors, impaired signaling through IGF-1R and IRS1, increased expression of multiple proceramide and ER stress genes and proteins, and higher levels of the C14, C16, C18, and C20 ceramide species relative to control. CONCLUSIONS: In human chronic ALD, persistent hepatic insulin resistance is associated with dysregulated lipid metabolism, ceramide accumulation, and striking upregulation of multiple ER stress signaling molecules. Given the role of ceramides as mediators of ER stress and insulin resistance, treatment with ceramide enzyme inhibitors may help reverse or halt progression of chronic ALD.

The concentration of cyclosporine metabolites is significantly lower in kidney transplant recipients with diabetes mellitus.

BACKGROUND: Diabetes mellitus is prevalent among kidney transplant recipients. The activity of drug metabolizing enzymes or transporters may be altered by diabetes leading to changes in the concentration of parent drug or metabolites. This study was aimed to characterize the effect of diabetes on the concentration of cyclosporine (CsA) and metabolites. METHODS: Concentration-time profiles of CsA and metabolites (AM1, AM9, AM4N, AM1c, AM19, and AM1c9) were characterized over a 12-hour dosing interval in 10 nondiabetic and 7 diabetic stable kidney transplant recipients. All patients were male, had nonfunctional CYP3A5*3 genotype, and were on combination therapy with ketoconazole. RESULTS: The average daily dose (±SD) of CsA was 65 ± 21 and 68 ± 35 mg in nondiabetic and diabetic subjects, respectively (P = 0.550). Cyclosporine metabolites that involved amino acid 1 (AM1, AM19, AM1c) exhibited significantly lower dose-normalized values of area under the concentration-time curve in patients with diabetes. Moreover, during the postabsorption phase (≥3 hours after dose), metabolite-parent concentration ratios for all metabolites, except AM4N, was significantly lower in diabetic patients. The pharmacokinetic parameters of ketoconazole were similar between the 2 groups thus excluding inconsistent ketoconazole exposure as a source of altered CsA metabolism. CONCLUSIONS: This study indicates that diabetes mellitus significantly affects the concentration of CsA metabolites. Because CsA is eliminated as metabolites via the biliary route, the decrease in the blood concentration of CsA metabolites during postabsorption phase would probably reflect lower hepatic cytochrome P450 3A4 enzyme activity. However, other mechanisms including altered expression of transporters may also play a role. Results of cyclosporine therapeutic drug monitoring in diabetic patients must be interpreted with caution when nonspecific assays are used.

Development and validation of a sensitive, simple, and rapid method for simultaneous quantitation of atorvastatin and its acid and lactone metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The aim of the proposed work was to develop and validate a simple and sensitive assay for the analysis of atorvastatin (ATV) acid, ortho- and para-hydroxy-ATV, ATV lactone, and ortho- and para-hydroxy-ATV lactone in human plasma using liquid chromatography-tandem mass spectrometry. All six analytes and corresponding deuterium (d5)-labeled internal standards were extracted from 50 µL of human plasma by protein precipitation. The chromatographic separation of analytes was achieved using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 µm). The mobile phase consisted of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). All analytes including ortho- and para-hydroxy metabolites were baseline-separated within 7.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode, with multiple-reaction monitoring scan. The calibration curves for all analytes were linear (R(2) ≥ 0.9975, n = 3) over the concentration range of 0.05-100 ng/mL and with lower limit of quantitation of 0.05 ng/mL. Mean extraction recoveries ranged between 88.6-111%. Intra- and inter-run mean percent accuracy were between 85-115% and percent imprecision was ≤ 15%. Stability studies revealed that ATV acid and lactone forms were stable in plasma during bench top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at -80 °C for 3 months. The method was successfully applied in a clinical study to determine concentrations of ATV and its metabolites over 12 h post-dose in patients receiving atorvastatin.

Significantly reduced cytochrome P450 3A4 expression and activity in liver from humans with diabetes mellitus.

BACKGROUND AND PURPOSE: Patients with diabetes mellitus require pharmacotherapy with numerous medications. However, the effect of diabetes on drug biotransformation is not well understood. Our goal was to investigate the effect of diabetes on liver cytochrome P450 3As, the most abundant phase I drug-metabolizing enzymes in humans. EXPERIMENTAL APPROACH: Human liver microsomal fractions (HLMs) were prepared from diabetic (n= 12) and demographically matched nondiabetic (n= 12) donors, genotyped for CYP3A4*1B and CYP3A5*3 polymorphisms. Cytochrome P450 3A4, 3A5 and 2E1 mRNA expression, protein level and enzymatic activity were compared between the two groups. KEY RESULTS: Midazolam 1'- or 4-hydroxylation and testosterone 6ß-hydroxylation, catalyzed by P450 3A, were markedly reduced in diabetic HLMs, irrespective of genotype. Significantly lower P450 3A4 protein and comparable mRNA levels were observed in diabetic HLMs. In contrast, neither P450 3A5 protein level nor mRNA expression differed significantly between the two groups. Concurrently, we have observed increased P450 2E1 protein level and higher chlorzoxazone 6-hydroxylation activity in diabetic HLMs. CONCLUSIONS AND IMPLICATIONS: These studies indicate that diabetes is associated with a significant decrease in hepatic P450 3A4 enzymatic activity and protein level. This finding could be clinically relevant for diabetic patients who have additional comorbidities and are receiving multiple medications. To further characterize the effect of diabetes on P450 3A4 activity, a well-controlled clinical study in diabetic patients is warranted.

Diabetes mellitus reduces activity of human UDP-glucuronosyltransferase 2B7 in liver and kidney leading to decreased formation of mycophenolic acid acyl-glucuronide metabolite.

Mycophenolic acid (MPA) is an immunosuppressive agent commonly used after organ transplantation. Altered concentrations of MPA metabolites have been reported in diabetic kidney transplant recipients, although the reason for this difference is unknown. We aimed to compare MPA biotransformation and UDP-glucuronosyltransferase (UGT) expression and activity between liver (n = 16) and kidney (n = 8) from diabetic and nondiabetic donors. Glucuronidation of MPA, as well as the expression and probe substrate activity of UGTs primarily responsible for MPA phenol glucuronide (MPAG) formation (UGT1A1 and UGT1A9), and MPA acyl glucuronide (AcMPAG) formation (UGT2B7), was characterized. We have found that both diabetic and nondiabetic human liver microsomes and kidney microsomes formed MPAG with similar efficiency; however, AcMPAG formation was significantly lower in diabetic samples. This finding is supported by markedly lower glucuronidation of the UGT2B7 probe zidovudine, UGT2B7 protein, and UGT2B7 mRNA in diabetic tissues. UGT genetic polymorphism did not explain this difference because UGT2B7*2 or *1c genotype were not associated with altered microsomal UGT2B7 protein levels or AcMPAG formation. Furthermore, mRNA expression and probe activities for UGT1A1 or UGT1A9, both forming MPAG but not AcMPAG, were comparable between diabetic and nondiabetic tissues, suggesting the effect may be specific to UGT2B7-mediated AcMPAG formation. These findings suggest that diabetes mellitus is associated with significantly reduced UGT2B7 mRNA expression, protein level, and enzymatic activity of human liver and kidney, explaining in part the relatively low circulating concentrations of AcMPAG in diabetic patients.

The effect of St John's wort (hypericum perforatum) on cytochrome p450 1a2 activity in perfused rat liver.

AIMS: We previously reported the effect of St John's wort (Hypericum perforatum) standardised extract LI 160 on the activities of cytochrome P450 2C6, 2D2 and 3As (Dostalek et al., Life Sciences 2005;78(3):239-44). In this study, we aimed to assess the effect of St John's wort on the activity of cytochrome P450 1A2. METHODS: The isolated perfused rat liver model was used in our experiments with phenacetin as a marker substrate for cytochrome P450 1A2. Male Wistar rats were treated with St John's wort extract (100 mg/kg, once daily for 10 days); comparative inhibitor (alpha-naphthoflavone, 100 mg/kg) or comparative inducer (omeprazole, 30 mg/kg). RESULTS: The rate of formation of acetaminophen was significantly inhibited by the use of St John's wort (P<0.001). In addition, St John's wort extract inhibited cytochrome P450 1A2 activity significantly more than the control inhibitor (P<0.001). CONCLUSIONS: St John's wort significantly inhibited cytochrome P450 1A2 enzyme activity. It remains to be determined whether the co-administration of St John's wort extract and other medications or substrates for cytochrome P450 1A2 could result in significant drug interactions.

Single-dose and steady state pharmacokinetics of CSA and two main primary metabolites, AM1 and AM4n in patients with rheumatic/autoimmune diseases.

BACKGROUND: Cyclosporine A (CsA) is an immunomodulatory agent used in standard immunosuppressive regimens in solid organ transplantations as well as in the treatment of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus and psoriasis. Its immunosuppressive activity is primarily due to parent drug. However, following oral administration, absorption is incomplete and varies between individuals. Further, there is a dearth of pharmacokinetic data for CsA in autoimmune patients compared to transplant recipients. AIM: The goal of this study was to investigate the single-dose and steady state pharmacokinetics of CsA and two main primary metabolites, AM1 and AM4N, in patients with rheumatic/autoimmune diseases. METHODS: Thirty-eight subjects, average age (years± SD) 46.8 (±11.6) years with rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis, ankylosing spondylitis and undifferentiated SpA were included in an observational open study. The single dose pharmacokinetics (area under the concentration-time curve of CsA and its metabolites (AUC) and other PK parameters) were determined over a 24 h period following oral administration of 1.3 mg/kg oral CsA. Two CsA formulations-Neoral and the Czech generic substitute Consupren®, were used. Pharmacokinetic analysis was performed on all 38 patients after administration of a single dose of CsA (1.34 mg/ kg/day). In 12 patients only, a second series of blood samples was taken to calculate monitored PK parameters under steady state conditions. RESULTS: Pharmacokinetic assessment showed AUC(0-24) 3009.66 ± 1449.78 ng/ml.h and C(max) 827.84 ± 425.84 after administration of a single dose of CSA, AUC(0-24) 3698.50 ± 2147 ng/ml.h and C(max) 741 ± 493 ng/ml after repeated dose. The proportion of the AM1 metabolite (AUC(0-24)) after a single dose of CsA corresponded to 40% of the parent compound and to approximately 35% of the parent compound in steady state conditions. The proportion of AM4N metabolite was low in both conditions and represented only 3 and 4.5% after a single dose and at steady state, respectively. CONCLUSION: The pharmacokinetic data (AUC(CsA), C(max)) for the whole 24 h interval were similar to the published findings, mainly under steady state conditions. The AM1 (AUC(0-24)) after a single dose of CsA and in steady state conditions represented about 40% of the parent drug. The ratio of AM4N metabolite was low in both conditions.

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1'-hydroxymidazolam, and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry.

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC-MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1'-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D5) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 microL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 microL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm x 100 mm, 3.5 microm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100-250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85-115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.