Lane J. Brunner

PEGylation of a Vesicular Stomatitis Virus G Pseudotyped Lentivirus Vector Prevents Inactivation in Serum

ABSTRACT One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.

Differences in Lipoprotein Lipid Concentration and Composition Modify the Plasma Distribution of Cyclosporine

AbstractPurpose. The purpose of this study was to define the relationship between lipoprotein (LP) lipid concentration and composition and the distribution of cyclosporine (CSA) in human plasma. Methods. 3H-CSA LP distribution was determined in normolipidemic human plasma that had been separated into different LP and lipoprotein-deficient plasma (LPDP) fractions by either affinity chromatography coupled with ultracentrifugation, density gradient ultracentrifugation or fast protein liquid chromatography. 3H-CSA LP distribution (at a concentration of 1000 ng/ml) was also determined in patient plasma samples with defined dyslipidemias. Furthermore, 3H-CSA LP distribution was determined in patient plasma samples of varying LP lipid concentrations. Following incubation, the plasma samples were separated into their LP and LPDP fractions by sequential phosphotungistic acid precipitation in the dyslipidemia studies and by density gradient ultracentrifugation in the specific lipid profile studies and assayed for CSA by radioactivity. Total plasma and lipoprotein cholesterol (TC), triglyceride (TG) and protein (TP) concentrations in each sample were determined by enzymatic assays. Results. When the LP distribution of CSA was determined using three different LP separation techniques, the percent of CSA recovered in the LP-rich fraction was greater than 90% and the LP binding profiles were similar with most of the drug bound to plasma high-density (HDL) and low-density (LDL) lipoproteins. When 3H-CSA was incubated in dyslipidemic human plasma or specific patient plasma of varying LP lipid concentrations the following relationships were observed. As the very low-density (VLDL) and LDL cholesterol and triglyceride concentrations increased, the percent of CSA recovered within the VLDL and LDL fractions increased. The percent of CSA recovered within the HDL fraction significantly decreased as HDL triglyceride concentrations increased. The percent of CSA recovered in the LPDP fraction remained constant except in hypercholesterolemic/hypertriglyceridemic plasma where the percent of CSA recovered decreased. Furthermore, increases in VLDL and HDL TG/TC ratio resulted in a greater percentage of CSA recovered in VLDL but less in HDL. Conclusions. These findings suggest that changes in the total and plasma LP lipid concentration and composition influence the LP binding of CSA and may explain differences in the pharmacological activity and toxicity of CSA when administered to patients with different lipid profiles.

Simple and rapid assay for acetaminophen and conjugated metabolites in low-volume serum samples.

The use of marker compounds for estimating drug metabolic capacity or pharmacokinetic parameters is common in the biological sciences. Often small laboratory animals are used and thus sample size is a limiting concern. In this report, we describe an assay we developed for measuring the concentration of acetaminophen and its conjugated metabolites in low-volume serum samples. Acetaminophen and metabolites were removed from 10 microl serum samples by a single-step 6% (v/v) perchloric acid deproteination using theophylline as internal standard. Samples were separated in a pH 2.2 sodium sulfate-acetonitrile mobile phase at a flow-rate of 1.5 ml/min on a 15 cm octadecylsilyl column at room temperature. Analytes were detected at a wavelength of 254 nm. The resulting chromatograms showed no interfering peaks from endogenous serum components. The concentration ranges measured were 1.56-200 microg/ml for acetaminophen and acetaminophen sulfate and 3.91-500 microg/ml for acetaminophen glucuronide. The assay was linear in the range of concentrations analyzed. The intra-day and inter-day coefficient of variation ranged from 0.4 to 8.2% and 0.2 to 12.3% for acetaminophen, 0.5 to 12.9% and 0.3 to 16.1% for acetaminophen glucuronide, and 0.4 to 8.1% and 0.2 to 14.3% for acetaminophen sulfate, respectively. Results from the experiments show that acetaminophen and its conjugated metabolites can easily and reproducibly be measured in low-volume serum samples and thus may offer an additional method to measure these compounds when the volume of biological samples may be limited.

Prevention of Cyclosporine-Induced Nephrotoxicity with Pentoxifylline

Although cyclosporine (CSA) is established in the prevention of allograft rejection, its use has been associated with dose-limiting toxicities, most notably to the kidney and liver. To date, the pathogenesis of the acute form of nephrotoxicity is unclear but may be related to inhibition of vasodilatory prostaglandins resulting in vasoconstriction and ischemia. The present study investigated the coadministration of CSA with a unique hemorheologic agent, pentoxifylline (PTX), in the murine model. A total of 48 rats were orally dosed with CSA 25 mg/kg for 10 days with either PTX 45 mg/kg i.p. or saline every 12 hr. Posttreatment renal function, assessed by creatinine (CCR) and inulin (CIN) clearances and renal electrolyte handling, was compared with baseline data and between groups. In an attempt to assess prostaglandin-mediated changes in enteral absorption, oral CSA pharmacokinetics with and without PTX were compared to the pharmacokinetics of similar groups (N = 8) administered i.v. CSA. Mean CIN of rats coadministered CSA and PTX (942 +/- 214 microliters/min/g KW) was similar to control rats 884 +/- 185 microliters/min/g KW); both were significantly greater than CSA alone (537 +/- 211 microliters/min/g KW; p less than .01). Likewise, percent of baseline CCR was significantly reduced in rats treated CSA (61 +/- 24%) compared to controls 113 +/- 41%) and rats coadministered PTX (117 +/- 75%; p less than .05). No differences in percent change from baseline electrolyte handling were observed among groups. Further, no differences in CSA pharmacokinetics with or without PTX were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of ethanol concentrations in the extracellular fluid of the rat brain: In vivo calibration of microdialysis probes

Abstract: Traditional microdialysis techniques provide qualitative data, although quantitative data are often required for pharmacodynamic analyses. This study evaluated a potentially usefulin vivo delivery technique to calibrate microdialysis probes for ethanol. We measuredin vivo delivery extraction fractions within subjects across 2 days and found no change over time. We tested the effect of diffusion direction on extraction fraction and found that it was higher for ethanol diffusion out of the probe than for diffusion into the probe, bothin vitro andin vivo. Thein vivo extraction fraction ratio of diffusionIN versus diffusionOUT was 0.65 ± 0.03. Finally, we predicted extracellular brain ethanol concentrations after 1 g/kg ethanol administration usingin vivo delivery, “no net flux” dialysis, orin vivo delivery corrected for diffusion direction with thein vivo extraction fraction ratio. Bothin vivo delivery and “no net flux” dialysis predicted brain concentrations that were approximately one‐third lower than blood concentrations, whereas the correctedin vivo delivery predicted extracellular concentrations very similar to blood concentrations. We conclude that microdialysis calibration methods for ethanol require a measure of extraction fraction for diffusion into the probe. Further studies are needed to establish whether this effect is common to other alcohols.

Interaction Between Cyclosporine and Grapefruit Juice Requires Long‐Term Ingestion in Stable Renal Transplant Recipients

Study Objective. To examine the effect of the concurrent administration of increasing amounts of grapefruit juice, an inhibitor of drug metabolism, on the steady‐state pharmacokinetics of cyclosporine.

Metabolic interaction between cyclosporine and sirolimus

Background. When the immunosuppressants cyclosporine (CsA) and sirolimus (SRL) are co-administered to transplant patients, lower doses are used than when either drug is given alone. Since both drugs share similar transport and metabolic pathways, there is the potential for an interaction leading to unpredictable effects. Furthermore, both drugs affect the activity of cytochrome P450 3A1/2 (CYP3A1/2), the rat parallel to human CYP3A4, and the multidrug transporter P-glycoprotein (Pgp). Methods. To clarify the role of metabolic enzymes and membrane transporters involved in the disposition of both drugs, we examined hepatic CYP3A1/2, Pgp, and multidrug resistance gene (mdr) mRNA during chronic therapy with CsA and SRL in salt-depleted rats. Specifically, rats were given intravenous doses of CsA 2.5 mg/kg and SRL 1 mg/kg, alone or in combination, for two weeks via constant rate intravenous infusion. Results. CsA treatment inhibited hepatic CYP3A1/2 protein expression, catalytic activity, and mRNA levels. SRL dosing suppressed CYP3A1/2 protein expression and catalytic activity, without affecting mRNA. With combined dosing, however, there was a much greater reduction. Hepatic Pgp protein levels were elevated after treatment with either drug alone, as well as with combined dosing. Compared to controls, there were significant increases in mdr1a and mdr1b mRNA levels in all treatment groups, with the combined drugs causing the greatest increase. Conclusions. Both CYP3A1/2 and Pgp participate in the disposition of CsA and SRL in rats. Changes in the individual activities of CYP3A1/2 and Pgp may contribute to an interaction between CsA and SRL resulting in unanticipated effects during chronic therapy.

Effect of grapefruit juice on cyclosporin A pharmacokinetics in pediatric renal transplant patients

Abstract: Cyclosporin A (CsA) is an important immunosuppressant that is prone to numerous drug interactions. Grapefruit juice has been investigated, as a possible adjunct to CsA dosing in adult renal transplant recipients, to decrease CsA metabolism and reduce dosages. This study investigated this combination in pediatric renal transplant patients. Six stable pediatric renal transplant patients were entered into an open‐label, four‐period cross‐over study in which patients were given their current CsA dose as either an oral solution (CsA‐Sol) or a microemulsion (CsA‐ME). In addition, drugs were administered concurrently with water or grapefruit juice. Steady‐state pharmacokinetic profiles were taken during each of the four periods. Following the concurrent administration of grapefruit juice, CsA whole‐blood 12‐h trough levels were significantly increased during CsA‐Sol dosing. Furthermore, the CsA elimination rate constant was significantly reduced during the same period. After CsA‐ME dosing, no differences in CsA pharmacokinetics were found between concurrent water or grapefruit ingestion. Grapefruit juice co‐administration reduced the production of CsA metabolites, AM1 and AM9, during CsA‐Sol dosing. No changes in CsA metabolite production were found when patients were given CsA‐ME with grapefruit juice as compared with water. Hence, alterations in CsA absorption and elimination occur with concurrent grapefruit juice ingestion when stable pediatric renal transplant patients are taking the oral CsA solution, but not the microemulsion formulation. These changes may be mediated by alterations in intestinal or hepatic metabolism, or drug absorption. The effect of grapefruit juice on CsA absorption is not readily predictable in these patients.

Considerations for use of recombinant adenoviral vectors: Dose effect on hepatic cytochromes P450

Recombinant adenovirus (Ad) serotype 5 is a vector commonly used for gene delivery. Although this vector has a natural tropism for the liver, there is a limited understanding of how Ad administration affects one of the primary hepatic processes, drug metabolism. The effects of systemic administration of a model recombinant adenoviral vector on two hepatic cytochrome P450 (P450) enzymes, CYP3A2 and 2C11, were investigated. Sprague-Dawley rats were treated with one of six vector doses, ranging from 5.7 × 106 to 5.7 × 1012 virus particles (vp)/kg. Hepatic P450 protein expression, catalytic activity, and mRNA levels were measured over 14 days. Ad administration (5.7 × 1010-5.7 × 1012 vp/kg) reduced CYP3A2 over the duration of the study. Six hours after administration of 5.7 × 1012 vp/kg, CYP3A2 activity and mRNA levels were suppressed by 45 and 65%, respectively (P ≤ 0.01). This continued throughout the study with levels dropping to 36 and 45% of controls by 14 days, respectively (P ≤ 0.01). A similar trend was detected for CYP2C11 within this dosing range. Administration of 5.7 × 106, 5.7 × 108, and 5.7 × 109 vp/kg of Ad significantly increased both CYP2C11 protein expression by 86, 71, and 107% and activity 110, 118, and 53%, respectively, above those of animals treated with saline (P ≤ 0.01). These results clearly indicate that a single dose of adenovirus significantly alters key drug metabolizing enzymes for an extended period of time and should be investigated further in the context of the design and implementation of clinical trial protocols.

Time-dependent pharmacokinetics and toxicity of cyclosporine

Although the circadian pattern of cyclosporine (CSA) pharmacokinetics and toxicity has been described previously in both animal and clinical studies, the mechanism of this action is unknown. The present study compared the pharmacokinetics and experimental nephrotoxicity of chronic CSA in both the genetically-hyperlipidemic rat model and the lean litter-mate. Once daily dosing (25 mg/kg via gavage) was either at the start of the active (1900) or inactive (0700) cycle (Nov 1987 to Jan 1988). Serial serum samples following the final dose were assayed by both polyclonal (nonspecific) and monoclonal (specific for parent CSA) RIA. Renal toxicity was assessed by 24-hr creatinine clearances, fractional clearances of sodium and potassium, and inulin clearances (CIN). Despite a greater than 2-fold increase in serum CSA concentrations, there were no changes in renal function in obese rats dosed at the start of the active period compared to the inactive period. Furthermore, mean CIN of the lean group administered drug at the start of the active period was not significantly different from time-matched placebo-treated lean rats. However, there was an 80% drop in CIN in rats treated with CSA at the start of the inactive period compared to control group. There were no differences in electrolyte handling. Insulin concentrations, independent of time of dosing, were markedly elevated in obese rats dosed CSA compared to placebo-treated obese or both lean groups. Serum triglyceride levels were significantly correlated with pharmacokinetic parameters of total but not parent CSA. In summary, significant differences in toxicity were observed due to time of dosing and lipid profiles. Although the mechanism of this action remains unclear, it appears that increased non-fasting serum triglyceride levels following the active period most likely reduced CSA distribution into kidney tissue preventing the dose-limiting nephrotoxicity.