Fatemeh Akhlaghi

Anti-Influenza Prodrug Oseltamivir Is Activated by Carboxylesterase Human Carboxylesterase 1, and the Activation Is Inhibited by Antiplatelet Agent Clopidogrel

Oseltamivir is the main medicine recommended by the World Health Organization in anticipation of next influenza pandemic. This anti-influenza viral agent is an ester prodrug, and the antiviral activity is achieved by its hydrolytic metabolite: oseltamivir carboxylate. In this study, we report that the hydrolytic activation is catalyzed by carboxylesterase human carboxylesterase (HCE) 1. Liver microsomes rapidly hydrolyzed oseltamivir, but no hydrolysis was detected with intestinal microsomes or plasma. The overall rate of the hydrolysis varied among individual liver samples and was correlated well with the level of HCE1. Recombinant HCE1 but not HCE2 hydrolyzed this prodrug and produced similar kinetic parameters as the liver microsomes. Several HCE1 natural variants differed from the wild-type enzyme on the hydrolysis of oseltamivir. In the presence of antiplatelet agent clopidogrel, the hydrolysis of oseltamivir was inhibited by as much as 90% when the equal concentration was assayed. Given the fact that hydrolysis of oseltamivir is required for its therapeutic activity, concurrent use of both drugs would inhibit the activation of oseltamivir, thus making this antiviral agent therapeutically inactive. This is epidemiologically of significance because people who receive oseltamivir and clopidogrel simultaneously may maintain susceptibility to influenza infection or a source of spreading influenza virus if already infected.

Secondary Metabolites Produced by the Marine Bacterium Halobacillus salinus That Inhibit Quorum Sensing-Controlled Phenotypes in Gram-Negative Bacteria

ABSTRACT Certain bacteria use cell-to-cell chemical communication to coordinate community-wide phenotypic expression, including swarming motility, antibiotic biosynthesis, and biofilm production. Here we present a marine gram-positive bacterium that secretes secondary metabolites capable of quenching quorum sensing-controlled behaviors in several gram-negative reporter strains. Isolate C42, a Halobacillus salinus strain obtained from a sea grass sample, inhibits bioluminescence production by Vibrio harveyi in cocultivation experiments. With the use of bioassay-guided fractionation, two phenethylamide metabolites were identified as the active agents. The compounds additionally inhibit quorum sensing-regulated violacein biosynthesis by Chromobacterium violaceum CV026 and green fluorescent protein production by Escherichia coli JB525. Bacterial growth was unaffected at concentrations below 200 μg/ml. Evidence is presented that these nontoxic metabolites may act as antagonists of bacterial quorum sensing by competing with N-acyl homoserine lactones for receptor binding.

Distribution of cyclosporin in organ transplant recipients.

AbstractCyclosporin is an immunosuppressive agent with a narrow therapeutic index. The total concentration of cyclosporin in blood is usually monitored to guide dosage adjustment and to compensate for substantial interindividual and intraindividual variability in cyclosporin pharmacokinetics. Cyclosporin is a highly lipophilic molecule and widely distributes into blood, plasma and tissue components. It mainly accumulates in fat-rich organs, including adipose tissue and liver. In blood, it binds to erythrocytes in a saturable fashion that is dependent on haematocrit, temperature and the concentration of plasma proteins. In plasma, it binds primarily to lipoproteins, including high-density, low-density and very-low-density lipoprotein, and, to a lesser extent, albumin. The unbound fraction of cyclosporin in plasma (CsAfu) expressed as a percentage is approximately 2%.It has been shown that both the pharmacokinetic and pharmacodynamic properties of cyclosporin are related to its binding characteristics in plasma. Furthermore, there is some evidence to indicate that the unbound concentration of cyclosporin (CsAU) has a closer association with both kidney and heart allograft rejection than the total (bound + unbound) concentration. However, the measurement of CsAfu is inherently complex and cannot easily be performed in a clinical setting. Mathematical models that calculate CsAfu, and hence CsAU, from the concentration of plasma lipoproteins may be a more practical option, and should provide a more accurate correlate of effectiveness and toxicity of this drug in transplant recipients than do conventional monitoring procedures.In conclusion, the distribution characteristics of cyclosporin in blood, plasma and various tissues are clinically important. Further investigations are needed to verify whether determination of CsAU improves the clinical management of transplant recipients.

A comparative pH-dissolution profile study of selected commercial levothyroxine products using inductively coupled plasma mass spectrometry

Levothyroxine (T4) is a narrow therapeutic index drug with classic bioequivalence problem between various available products. Dissolution of a drug is a crucial step in its oral absorption and bioavailability. The dissolution of T4 from three commercial solid oral dosage forms: Synthroid (SYN), generic levothyroxine sodium by Sandoz Inc. (GEN) and Tirosint (TIR) was studied using a sensitive ICP-MS assay. All the three products showed variable and pH-dependent dissolution behaviors. The absence of surfactant from the dissolution media decreased the percent T4 dissolved for all the three products by 26-95% (at 30 min). SYN dissolution showed the most pH dependency, whereas GEN and TIR showed the fastest and highest dissolution, respectively. TIR was the most consistent one, and was minimally affected by pH and/or by the presence of surfactant. Furthermore, dissolution of T4 decreased considerably with increase in the pH, which suggests a possible physical interaction in patients concurrently on T4 and gastric pH altering drugs, such as proton pump inhibitors. Variable dissolution of T4 products can, therefore, impact the oral absorption and bioavailability of T4 and may result in bioequivalence problems between various available products.

Declining intracellular T-lymphocyte concentration of cyclosporine a precedes acute rejection in kidney transplant recipients.

Background. We investigated cyclosporine A (CsA) concentrations at the site of action, inside T-lymphocytes, to evaluate its applicability as a new supplementary therapeutic drug monitoring method after renal transplantation. Method. In this prospective single-center study, 20 kidney transplant recipients, mean age 54 (range 21–74) years, on CsA-based immunosuppression were included within 2 weeks posttransplant and followed for 3 months. Nine patients also had one full 12-hour pharmacokinetic profile performed. T-lymphocytes were isolated from 7 ml whole blood using Prepacyte and intracellular CsA concentrations were determined using a validated liquid chromatography double mass spectrometry method. Results. Seven patients (35%) experienced acute rejections (all biopsy verified) during the first three months posttransplantation. Intracellular CsA concentrations tended to decline 1 week prior to acute rejection and the decrease was significant (−27.1±14.6%, P=0.014) three days before the rejection episodes were recognized clinically. In addition, the intracellular CsA area under the curve 0–12 measured during stable phase was 182% higher in the rejection-free patients (P=0.004). There was no difference between patients experiencing rejection and the rejection-free patients with respect to CsA C2-levels, dose (mg/kg), human leukocyte antigen mismatch, donor age, recipient age, or ABCB1 genotyping. Conclusion. Intracellular CsA T-lymphocyte concentrations declined significantly 3 days prior to a rejection episode and there was a general lower intracellular exposure of CsA in recipients experiencing rejection. Intracellular measurement of CsA therefore seems to have a potential to further improve individualization of therapeutic drug monitoring. Larger studies are needed to elucidate the role for intracellular T-lymphocyte measurements in ordinary clinical care, for both CsA and other immunosuppressive drugs.

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.

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.

Development of a sensitive and selective method for the quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma

A highly selective, sensitive and robust LC-MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M+2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 microm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within +/-15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.

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.

High-performance liquid chromatography method for the determination of mycophenolic acid and its acyl and phenol glucuronide metabolites in human plasma

Measuring the concentration of the pharmacologically active metabolite of mycophenolic acid (MPA), acyl-MPAG (AcMPAG), in addition to the pharmacologically inactive phenol glucuronide metabolite (MPAG) may prove useful in the therapeutic drug monitoring of MPA. A simple high-performance liquid chromatography method with ultraviolet detection (HPLC-UV) was established for simultaneous determination of MPA, AcMPAG, and MPAG in human plasma. The method utilizes 2 internal standards (IS), phenolphthalein glucuronic acid (PGA) for MPAG and a carboxy butoxy derivative of MPA (MPAC) for AcMPAG and MPA. The method consists of solid-phase extraction of the analytes followed by analysis over a Zorbax Rx C8 column (150 × 4.6 mm, 5 μm) at 254 nm. The analytes were separated with a gradient mixture of methanol and 0.1% phosphoric acid over a run time of 14 minutes at a flow rate of 1 mL/min. The assay was linear in the concentration range from 0.2 to 50 mg/L for MPA, 0.5 to 25 mg/L for AcMPAG, and 2 to 500 mg/L for MPAG. The mean ± SD interday accuracy and %CV for MPA were 100.3 ± 5.7 and 5.7%, for AcMPAG, 102.6 ± 5.7 and 5.6%, and for MPAG 100.5 ± 5.3 and 5.3%, respectively. The average ± SD of MPA, MPAG, and AcMPAG maximum concentrations (Cmax) in 23 kidney transplant recipients on 500 or 1000 mg twice daily mycophenolate mofetil were 11.77 ± 9.43, 88.15 ± 46.4, and 3.01 ± 1.73 mg/L, respectively, and the predose trough (Cmin morning) concentrations were 2.24 ± 3.11, 55.44 ± 29.55, and 1.42 ± 0.74 mg/L, respectively. The method described is robust, sensitive, reproducible, and will be useful in therapeutic drug monitoring or pharmacokinetic studies of MPA.