Population PKPD of voclosporin in renal allograft patients.

The aims of this population-pharmacokinetic/pharmacodynamic (POP-PKPD) analysis of voclosporin in renal allograft patients were to build a POP-PKPD model for voclosporin and calcineurin activity (CNa) and identify clinically relevant covariates that could assist dosing of the drug. POP-PKPD modeling was performed using a stochastic approximation of the standard expectation maximization (SAEM) algorithm for nonlinear mixed-effects as implemented in Monolix™ 3.2. Voclosporin whole blood concentrations were obtained from de novo renal allograft patients and assayed using a validated LC/MS/MS assay. CNa was measured using a (32)P-radiolabeled assay. A two-compartment model with simultaneous sigmoid inhibitory Emax model was used to describe the PKPD relationship between voclosporin concentration and CNa. The POP-PKPD model was then utilized to simulate an optimal initial dosing strategy. Eighty-seven patients were included in the POP-PKPD study. Population mean estimates (relative standard error, rse) for oral clearance (CL/F) and first compartment volume of distribution (V1), were 717 mL min(-1) (35%) and 2010 mL (17%), respectively. Maximum CNa Inhibition (Imax), effective concentration (C50), and baseline immunosuppression (S0) were 0.87 pmol/min/mg (8.0%), 123 ng/mL (10%), and 1.15 pmol/min/mg (4.0%), respectively. Covariate analyses demonstrated that age and body surface area significantly influenced CL/F: CLi=717(Agei/48.8)-0.57(BSAi/1.99)1.1, while serum triglycerides significantly altered S0: S0i=1.15(TRIGi/1.97)0.15.

Pharmacokinetics of voclosporin in renal impairment and hepatic impairment.

Voclosporin is a novel calcineurin inhibitor intended for prevention of organ graft rejection and treatment of lupus nephritis. These studies evaluated the effect of renal or hepatic impairment on pharmacokinetics of voclosporin. Thirty-three subjects were enrolled into 1 of 4 groups based on renal function as defined by creatinine clearance and 18 subjects were enrolled into 1 of 3 groups based on hepatic function defined by Child-Pugh classes. Voclosporin 0.4 mg/kg was administered orally. Geometric mean ratios (renal/hepatic impairment-to-normal) and 90% confidence intervals for Cmax and AUC were calculated. A default no-effect interval of 80-125% was set. Although 90% confidence intervals exceeded the no-effect intervals for both parameters, individual Cmax and AUC plots indicate almost complete overlapping range of values for mild and moderate renal impairment and normal subjects. Severe renal impairment resulted in a 1.5-fold increase in AUC without an increase in Cmax . Mild to moderate hepatic impairment resulted in a 1.5- to 2-fold increase in voclosporin exposure. Voclosporin can be administered safely to patients with mild to moderate renal impairment without dose modification. Appropriate safety monitoring with concentration-based adjustments in transplantation are recommended for patients with severe renal impairment, and for patients with hepatic impairment.

Anticoagulant characteristics of HD1-22, a bivalent aptamer that specifically inhibits thrombin and prothrombinase.

BACKGROUND: HD1-22 is a bivalent aptamer that binds to thrombin with high affinity (K(d) = 0.65 nm) and occupies both anion binding exosites without blocking the active centre of the enzyme. HD1-22 has been developed by connecting the exosite 1 binding aptamer HD1 and the exosite 2 binding aptamer HD22 through a poly-dA linker. OBJECTIVES: To characterize the anticoagulant profile of HD1-22 in comparison to the clinically established direct acting thrombin inhibitors bivalirudin and argatroban, and to test the efficacy of antidote-oligodeoxynucleotides. METHODS AND RESULTS: HD1-22 prolongs clotting times of the thrombin time, activated partial thromboplastin time, ecarin clotting time, and lag-time of the tissue factor triggered thrombin generation assay in a dose-dependent manner. On a molar basis, its anticoagulant activity was nearly identical to bivalirudin and superior to argatroban. Thrombin-induced platelet aggregation was more effectively inhibited by HD1-22 than by bivalirudin. The HD1-22 aptamer retains the ability of the HD1-moiety to bind to (pro)exosite 1 of prothrombin and inhibits the prothrombinase activity nearly 2-fold better than HD1. The anticoagulant activities of HD1-22 are fully reversed by addition of antidote-oligodeoxynucleotides. CONCLUSIONS: The strong thrombin-inhibiting activity, together with the availability of a rapid acting antidote strategy, makes HD1-22 an interesting anticoagulant candidate, especially for use in clinical situations where effective anticoagulation and rapid reversal of the anticoagulant effect are required. The data obtained warrant further clinical studies.

Partial-filling micellar electrokinetic chromatography and non-aqueous capillary electrophoresis for the analysis of selected agrochemicals.

Selected agrochemicals (s-triazines and phenoxy acids) have been investigated with partial-filling micellar electrokinetic chromatography (PFMEKC) and non-aqueous capillary electrophoresis (NACE). Because these two techniques are compatible for coupling of capillary electrophoresis with mass spectrometry, different conditions affecting the separation efficiency (reproducibility, method linearity) were systematically tested, and the results were compared with those from classical MEKC. The conditions tested included buffer molarity, pH, the concentrations of the organic modifier and surfactant, the applied voltage, the injection time of the sample, and the length of the partial-filling plug. The respective limits of detection (LOD) using UV-detection were determined. Reduction of the electrophoretic raw data using the mobility scale transformation (micro-scale) improved qualitative comparison of the electropherograms and the reproducibility of quantitative data (integrated peak area) thus extending this data treatment from CZE to other endoosmotic flow-driven CE-techniques such as PFMEKC and NACE.

[Anaplastic astrocytomas of the medulla oblongata. A rare differential diagnosis in orthostatic hypotension]. / Anaplastisches Astrozytom der Medulla oblongata. Eine seltene Differentialdiagnose bei orthostatisch bedingter Hypotonie.

HISTORY AND PHYSICAL EXAMINATION: A 39-year-old women was admitted for evaluation of dizziness and hypotension. During standing the blood pressure dropped from 130/80 to 80/40 mmHg. Ten weeks before admission she had recurrent cerebral convulsions. Neurological evaluation showed a slight left hemiparesis. Computed tomography of the brain revealed a 1.5 +/- 1 cm cyst in the left tempral region. It was thought that the cause of the recurrent convulsions was alcohol abuse. During the next few weeks orthostatic hypotension increased and she was not able to work. INVESTIGATIONS: At admission abnormal findings included hypotension, horizontal nystagmus, and deviation of the soft palate to the right. After the patient was brought to an upright position during standardized passive tilt testing she showed a defect in the sympathetic limb of the baroreceptor reflex arc. Head magnetic resonance tomography showed a signal-enhancing tumour in the cervicomedullary region. TREATMENT AND CLINICAL COURSE: Before a planned biopsy could be performed the patient died of respiratory arrest. Postmortem examination revealed an anaplastic grade III astrocytoma extending form the pons to the medulla oblongata. CONCLUSION: Upright tilting leads to pooling of blood in the legs. One of the normal compensatory responses is a reflex tachycardia which our patient did not show as a sign of an afferent defect. Patients with orthostatic hypotension as a prominent symptom should be investigated with a standardized tilt test. In special patients, additional neurological investigations are necessary.

Quantitative and qualitative precision improvements by effective mobility-scale data transformation in capillary electrophoresis analysis.

By transforming the time-based x-axis of electropherograms in capillary zone electrophoresis (CZE) into the corresponding effective mobility-scale, we propose a simple and robust data representation for a better qualitative and quantitative capillary electrophoresis (CE) analysis. The time scale of the raw electrophoretic data (detection signal versus time) is transformed into an effective electrophoretic mobility scale (mu eff-scale) with account of the electroosmotic flow (EOF) peak or of an internal standard of known effective mobility. With the new scaling (detection signals versus effective mobility), the obtained electropherograms are more representative of the velocity-based electrophoretic separation and the comparison of complete electropherograms is directly possible. This is of importance when tracking peaks in real samples where alteration in EOF stability can occur or when comparing electrophoretic runs from different experimental setups (independence in column length and voltage). Beside the qualitative possibilities, a quantitative improvement is achieved in the mu eff-scale with significant better peak area reproducibility and equal to more precision in quantitative analysis than with the primary time-scale integration.

Detection of volatile organic peroxides in indoor air.

A supercritical fluid extraction cell filled with adsorbent (Carbotrap and Carbotrap C) was used directly as a sampling tube to enrich volatile organic compounds in air. After sampling, the analytes were extracted by supercritical fluid CO2 with methanol as modifier. Collected organic peroxides were then determined by a RP-HPLC method developed and validated previously using post-column derivatization and fluorescence detection. Some volatile organic peroxides were found in indoor air in a new car and a newly decorated kitchen in the lower microg m(-3) range. tert-Butyl perbenzoate, di-tert-butyl peroxide, and tert-butylcumyl peroxide could be identified.

Analysis of agrochemicals by capillary electrophoresis.

An increasing amount of articles using capillary electrophoresis as an investigation tool for pesticides and environmental pollutants were found over the last few years in analytical chemistry oriented journals. This review covers a wide literature range of the 1990s and concentrates on the analysis of organic agrochemicals (herbicides, fungicides, insecticides, acaricides, etc.) with capillary electrophoresis (capillary zone electrophoresis, micellar electrokinetic chromatography with CE-UV-visible or laser-induced fluorescence detection) as well as with the on-coming hyphenated techniques like capillary electrophoresis-electrospray ionization mass spectrometry. The principal preconcentration methods that allowed real sample analysis with CE are also briefly discussed. The pesticides, the separation methods, the used electrolytes, the detection types, the detection limits and the preconcentration methods were classified and presented in tabulated form as a rapid information tool.

Analysis of cyanogenic glycosides by micellar capillary electrophoresis.

The separation of amygdalin, prunasin and their isomers neoamygdalin and sambunigrin could be achieved with micellar capillary electrophoresis (MEKC). The two isomers were obtained in alkaline conditions and were produced in less than 15 min at pH 11.0. The developed methods showed a good selectivity in the separation of the isomers only in the presence of SDS micelles. The working pH was optimized to allow best resolution and quantitative analysis of these compounds. With a linear calibration over an injection time from 1 to 20 s, the detection limit was found to be in the range of 5 microM (S/N=3; 20 s injection time). Two pH buffer systems (pH 5.2 and pH 9.1) were chosen to confirm the peak attributions of the compounds in the apple and peach seeds samples. Sambunigrin was found in both apple and peach seeds but could not be quantified because of missing standards. Prunasin and amygdalin were not found in the apple sample, while they were quantified in the peach seeds in concentrations of 50 microg/g and 90 microg/g (dry weight), respectively.

Pharmacokinetics and metabolism of sirolimus.

Sirolimus (rapamycin, Rapamune) is a potent immunosuppressive drug that received marketing approval from the US Food and Drug Administration on September 15, 1999. Research into defining its pharmacokinetic (PK) behavior, interaction with other agents, and metabolism is ongoing. It has been established that oral doses of both liquid and solid formulation are rapidly, though incompletely and variably, absorbed. Metabolism by the intestinal and hepatic CYP3A family of enzymes likely contributes to variability in absorption and low bioavailability. Sirolimus has a long terminal half-life, the AUC correlates well with trough and peak concentrations, and it exhibits a moderate degree of dose proportionality. There is significant interpatient variability in PK parameters of sirolimus, though it exhibits predictable PK behavior when used with prednisone and cyclosporine neoral. There is a decreased rejection risk with higher doses and target level attainment. Several species of sirolimus metabolites have been characterized, and are measurable in whole blood and tissue specimens. Many more species of sirolimus metabolites are detectable, but they are not quantifiable at this time. The total concentration of metabolites appears to be less than that of the parent drug when examined through the PK profile. A reference method for the quantitation of metabolites remains elusive because of a lack of proper standardization. The clinical significance of sirolimus metabolites remains to be proven.

Donor/Acceptor Metallocenes: A New Structure Principle in Catalyst Design.

Suitable as highly effective polymerization catalysts is the new class of donor/acceptor metallocenes in which the rotation of the two pi ligands is restricted through the formation of a dative D(+)-->A(-) bond (see picture). Specifically optimized substitution patterns yield excellent properties for the synthesis of high-melting, highly crystalline thermoplastic materials, amorphous thermoplastic materials with high glass transition temperatures as well as polyolefin elastomers with low glass T(g).

Interactions of hydroxy-s-triazines with sodium dodecyl sulfate-micelles investigated by micellar capillary electrophoresis.

The electrophoretic behavior of fourteen 4,6-diamino-s-triazines was investigated in the presence of an anionic surfactant (sodium dodecyl sulfate, SDS) using micellar capillary electrophoresis (MCE). The measurements were performed at the pH of zero charge of the hydroxytriazines and the existence of strong ionic and H-bond interactions of hydroxy-s-triazine species with the anionic micelles could be shown. Their migration behavior was compared to the n-octanol-water partition coefficients (log Kow) measured with reverse-phase HPLC and calculated with different fragment contribution methods. A partition model was proposed to understand the interactions of the three major hydroxy-s-triazine species: cationic, anionic and neutral (presenting enol and keto forms) with the charged SDS micelles taken as model for charged natural polyelectrolytes like humic substances. These results strongly indicate that hydroxy-striazines are polarized in the presence of the charged micelles and that they are essentially present in their keto form in the micellar phase (and enol form in the water phase), confirming previous studies suggesting the presence of zwitterionic resonance structures at a neutral pH around their isoelectric point.

Studies of the behaviour and fate of the polymer-additives octadecyl-3-(3.5-di-t-butyl-4-hydroxyphenyl)propionate and tri-(2.4-di-t-butylphenyl)phosphite in the environment.

In order to evaluate the environmental risk potential of the polymer-additives octadecyl-3-(3.5-di-t-butyl-4-hydroxyphenyl)propionate (additive 1) and tri-(2.4-di-t-butylphenyl)phosphite (additive 2) we have studied the degradation of the 14C-labelled single components and their release from a polypropylene matrix in abiotic and biotic test systems as well as their transport behaviour in soil. The biotic degradation in waste compost, activated sludge and in soil was studied. In order to conduct release tests, waste disposal material were used as well. The abiotic degradation was effectuated by photooxidation at a catalytic surface. The highest mineralisation rates were obtained by photooxidative degradation of the two test substances. The maximum content of bounded residues was found for additive 1 in soil and for additive 2 in waste compost. In the extracts of soil and compost, the principal metabolites could be identified in the case of additive 1 as 7.9-di-t-butyl-1-oxaspiro[4.5]deca-6.9-dien-2.8-dion and in activated sludge as the methyl ester of 3-(3.5-di-t-butyl-4-hydroxyphenyl) propionic acid. Additive 2 metabolised in all degradation tests nearly completely under formation of two products. The main component was identified as tri-(2.4-di-t-butylphenyl)phosphate. Both polymer-additives were eluated from the chosen soil types. The transport behaviour of additive 1 was independent from the soil type. In all test matrices additive 1 as well as additive 2 were released from polypropylene. Because the amounts of additive 1 and 2 absorbed onto soil particles were taken into account, the highest liberation rates were found in the soil test.

Study of FK-binding protein: FK506-metabolite complexes by electrospray mass spectrometry: correlation to immunosuppressive activity.

Electrospray ionization mass spectrometry was used to study several non-covalent FK-binding protein (FKBP) immunosuppressant complexes in the gas phase. Relative FKBP binding affinities were determined from the signal ratio for the 7+ charge states of bound and unbound complexes as a function of capillary exit voltage. All complexes displayed a 1:1 binding stoichiometry. The relative gas-phase binding affinities were found to be well correlated with in vitro FKBP binding and in vitro immunosuppression (rapamycin > FK506 > or = 31-demethyl FK506 > 13-demethyl FK506 >> Cyclosporin A; CsA). The method demonstrates potential as a simple, rapid, and automatable technique for prediction of the immunosuppressive activity of FKBP:drug complexes.

Second generation assay for thyrotropin receptor antibodies has superior diagnostic sensitivity for Graves' disease.

Detection of autoantibodies to the TSH receptor (TSH-R) in Graves' disease has found widespread use in clinical routine and is performed mostly by commercial RRAs measuring TSH binding inhibitory activity. We report in this study on a second generation TSH binding inhibitory assay using the human recombinant TSH-R with two major improvements: 1) superior diagnostic sensitivity for Graves' disease, and 2) for the first time, nonradioactive and radioactive coated tube (CT) technology. Full-length human recombinant TSH-R was expressed in the K562 leukemia cell line and grown in suspension at a high density. A murine monoclonal antibody was selected for binding to the native TSH-R without interfering with autoantibodies or TSH and was coated to polystyrene tubes. After detergent extraction, TSH-R was affinity immobilized on antibody-coated tubes. The binding of TSH to the TSH-R could be demonstrated by the addition of 125I- or acridinium ester-labeled bovine TSH, and this binding could be inhibited by sera from patients with Graves' disease up to 95%. Subsequently, these novel assays, a CT RRA and a CT luminescence receptor assay, were compared to the conventional RRA based on porcine antigen in a blinded clinical multicenter trial. Sera from 328 patients with Graves' disease (86 untreated, 116 treated, and 126 in remission) and 520 controls (comprised of healthy blood donors and patients with autoimmune diseases or goiter) were tested in all 3 assays. Receiver-operating characteristic plot analysis resulted in a specificity of 99.6% with a sensitivity of 98.8% for both CT assays, compared to 99.6% specificity and 80.2% sensitivity for the conventional RRA (P < 0.001). In all 3 groups of patients with Graves' disease, the 2 CT assays were significantly more sensitive for the disease than the conventional assay, without loss of specificity in the control groups. This increase in sensitivity and the nonradioactive or radioactive CT format constitute a significant improvement over the currently available assays.

Heat shock protein 80 of Neurospora crassa, a cytosolic molecular chaperone of the eukaryotic stress 90 family, interacts directly with heat shock protein 70.

The subunit structure of Hsp80, the most abundant heat-shock protein of Neurospora crassa, was examined by chemical cross-linking of the purified protein in vitro. Resolution of glutaraldehyde-treated Hsp80 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE suggests that the native state of this protein is a tetramer; the relative proportion of cross-linked species, estimated by the fraction of protein recovered in each category, is consistent with a dimer-of-dimer structure. Upon interaction with nucleotides, higher order cross-linked oligomers were detected, indicating ligand-induced conformational changes. The effect of nucleotides was also monitored by following tryptophan fluorescence: CTP, UTP, and NAD led to fluorescence quenching, the effect of CTP being the most pronounced. As individual molecular chaperones often act in concert with cochaperones, interaction between the two major cytosolic stress proteins--Hsp80 and Hsp70--was examined. Purified Hsp70 was immobilized on ATP-agarose and purified Hsp80 was applied to the Hsp70-saturated matrix; while Hsp80 did not bind to ATP-agarose by itself, it was bound strongly by immobilized Hsp70. The [Hsp70-Hsp80] complex was eluted with ATP and coelution of both proteins was confirmed by Western blot analysis, using specific polyclonal antibodies raised against each protein. The physical association of stress-inducible Hsp70 and Hsp80 was verified by interprotein cross-linking in vitro followed by immunoblot analysis and by immunoprecipitation.

Stereoselective metabolism of rac-mexiletine by the fungus Cunninghamella echinulata yields the major human metabolites hydroxymethylmexiletine and p-hydroxymexiletine.

rac-Mexiletine is an orally effective class 1b antiarrhythmic agent used to treat ventricular arrhythmias. In vivo experiments have demonstrated. It is predominantly metabolized by the liver with < 10% excreted as unchanged drug. The major mammalian metabolites have been identified as p-hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM). The purpose of our study was to determine whether the fungus Cunninghamella echinulata, which possesses a cytochrome P450 system analogous to that found in humans, could be used as a suitable in vitro model for studying the oxidative metabolism of rac-mexiletine. To accomplish this, a high performance liquid chromatographic assay was used that was capable of simultaneously quantifying the enantiomers of mexiletine, HMM, and PHM. Utilizing this procedure, it was demonstrated that C. echinulata stereoselectively converted rac-mexiletine into HMM (4% of added drug) and PHM (32% of added drug) after an incubation period of 50 hr. In addition, metabolite biosynthesis could be optimized by altering fermentation media components. Seven media values and seven pH values were evaluated. It was determined that a medium at pH 7.0 containing yeast extract and sucrose yielded optimal amounts of metabolites.