Inhibitor In Human Plasma Research Articles

High-performance liquid chromatography assay for the quantification of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in human plasma

An accurate, sensitive, and specific reverse-phase high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of HIV-protease inhibitors (PIs) (indinavir, IDV; amprenavir, APV; saquinavir, SQV; nelfinavir, NFV; ritonavir, RTV; and lopinavir, LPV) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) (nevirapine, NVP; delavirdine, DLV; and efavirenz, EFV) in human blood plasma is described. The method provides excellent resolution and peak shape for nine analytes through a linear gradient (36–86%) of 25% phosphate buffer (pH 4.5), 60% acetonitrile, 15% methanol, and 0.75 ml TFA, with a gradient mobile phase flow rate (0.9–1.1 ml) over 30 min run time. The optimized solid phase extraction (SPE) extraction method using (1.0 ml, 100 mg BOND ELUT-C18 Varian) column provides a clean base line and high extraction efficiency using a 550 μl plasma sample. The method was validated over the range of 10–10,000 ng/ml for NVP, IDV, and SQV; 10–5000 ng/ml for EFV; 25–10000 ng/ml for APV; and 25–5000 ng/ml for DLV, NFV, RTV, and LPV. This method is accurate (average accuracies of three different concentrations ranged from 91 to 112%), and precise (within- and between-day precision measures ranged from 0.2 to 5.7% and 0.1 to 5.4%, respectively). This method is suitable for use in clinical pharmacokinetic studies as well as in therapeutic drug monitoring (TDM).

Determination of a cyclooxygenase II inhibitor in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5′-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1–20 and 5–1000 ng/ml, are described. Following liquid–liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 μl) and part of the extract (1 μl) was analyzed by GC/MS/SIM. The drug ( I) and internal standard ( II) were separated on a 25 m×0.2 mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 μm) phase and analyzed by MS/SIM monitoring ions at m/ z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1–20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5–1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91–112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.

Simultaneous Determination of Six HIV Protease Inhibitors, One Metabolite, and Two Non-nucleoside Reverse Transcriptase Inhibitors in Human Plasma by Isocratic Reversed-Phase Liquid Chromatography After Solid-Phase Extraction

A selective and sensitive high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of six human immunodeficiency virus (HIV)-protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine) in a single run. This involve a simple liquid-solid extraction on OASIS® HLB column in the presence of an internal standard (prazepam). Separation is achieved on a Xterra®, C18 (150 × 3.9 mm I. D.) column with a mobile phase consisting of acetonitrile and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer pH 10.5, (37 : 63, v/v) delivered isocratically. A sequential ultraviolet detection (5 minutes sequence set at 320 nm for nevirapine acquisition and 55 minutes at 210 nm for other durgs and internal standard) is performed. The method is linear over the specific ranges investigated. Precision and accuracy at four concentrations are respectively less than 13.6% and 9.1% for intraday assays and less than 4.2% and 5.9% for interday assays. This method is suitable for therapeutic drug monitoring purpose and routinely used in our laboratory.

Rapid quantification of HIV protease inhibitors in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

HIV protease inhibitors are important antiretroviral drugs which have substantially reduced the morbidity and mortality associated with HIV-1 infection. Recent data have shown relationships between plasma concentrations of the protease inhibitors and clinical response, which makes therapeutic drug monitoring valuable. We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of the six licensed protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) and the pharmacologically active nelfinavir metabolite M8 in plasma. The sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using only 100 microl of plasma. Chromatographic separation was performed on an Inertsil ODS3 column (50 x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml min(-1). The analytical run time was 5.5 min. The use of a 96-well plate autosampler allowed batch sizes up to 150 patient samples. The triple-quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 0.01-10 microg ml(-1) for indinavir and saquinavir, 0.1-10 microg ml(-1) for amprenavir, 0.05-10 microg ml(-1) for nelfinavir and ritonavir, 0.1-20 microg ml(-1) for lopinavir and 0.01-5 microg ml(-1) for M8. Saquinavir-d(5) and indinavir-d(6) were used as internal standards. The coefficients of variation were always <10% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/-15%). The validated concentration ranges proved to be adequate in daily practice. This robust and fast LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring and pharmacokinetic studies in our hospital.

LC-MS/MS determination of a farnesyl transferase inhibitor in human plasma and urine

To support clinical pharmacokinetic studies in cancer patients, sensitive and specific methods for measuring 4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-1-(3-chlorophenyl) piperazinone (I), a farnesyl transferase inhibitor (FTI), in human plasma and urine were developed and validated. The methods are based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization (APCI) and tandem mass spectrometric (MS/MS) detection in the positive ion mode using a heated nebulizer interface. Drug and internal standard were isolated from plasma or basified urine using automated solid-phase extraction on cyano cartridges. The organic extracts were dried, reconstituted in aqueous acetonitrile and injected into the system. Chromatographic separation of I and internal standard ( IS) was achieved using a BDS Hypersil C8 analytical column, with a mobile phase consisting of acetonitrile:methanol:water (50:4:46) and trifluoroacetic acid (0.05%) at a flow rate of 0.6 ml/min. MS/MS detection was performed on a PE-Sciex API 300 tandem mass spectrometer operated in selected reaction monitoring mode. The parent→product ions monitored were m/ z 406→195 for analyte I and m/ z 448→195 for the internal standard. Unusual in this method is that quantitation is accomplished using a secondary product ion, m/ z 195, of drug I and IS. The assays were validated over the concentration range of 0.5–1000 ng/ml (1.2 nM to 2.5 μM, respectively) in plasma, and 2.5–500 ng/ml (6.2 nM to 1.23 μM) in urine. Accuracy was within ±10% of nominal concentration at all levels in urine, and all but the lowest standard in plasma (±14% at 0.5 ng/ml). Intraday precision (expressed as coefficients of variation, CVs) for standard replicates and interday precision for quality control (QC) samples were less than 8% at all concentrations in both matrices. Detailed descriptions of the extraction procedure and analytical methodology used in the assay of I in plasma and urine are presented. This procedure may have utility in the quantitation of other imidazole-based FTIs with cyanobenzyl substructures.

Simultaneous quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography.

A rapid (less than 30 min), sensitive, and specific liquid chromatography method for simultaneous assay of nine antiretroviral drugs in human plasma is described. This technique allows therapeutic drug monitoring of six approved protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) and two approved non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine). Assays were performed after diethyl ether liquid-liquid extraction from 250-microL plasma samples. Chromatographic separation was achieved on an X-TERRA (Waters; Saint Quentin, France) column using a 58% water (with 3 mmol/L pyrrolidine) and 42% acetonitrile mobile phase. Three ultraviolet wavelengths were used for detection with a diode array detector. This method allowed quantitative assay of all nine antiretroviral drugs within a concentration range of 25 ng/mL to 9000 ng/mL. The method has been validated extensively and has been in routine use in our laboratory for several months for drug monitoring in plasma samples from patients treated with antiretroviral drugs.

Monolithic silica liquid chromatography columns for the determination of cyclooxygenase II inhibitors in human plasma

Methods employing monolithic HPLC columns for the determination of the cyclooxygenase II inhibitors rofecoxib ( I) and 3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5′-dimethyl-5H-furan-2-one (DFP, III) in human plasma are described. Each analyte, together with an internal standard was extracted from the plasma matrix using solid-phase extraction in the 96-well format. The analytes were chromatographed on a Chromolith Speed Rod monolithic HPLC column (4.6×50 mm). Analyte detection for rofecoxib was via fluorescence following post-column photochemical derivatization. Detection for III was based on the native fluorescence of the compound. The precision, accuracy, and linearity of the methods were found to be comparable to those obtained using methods employing conventional packed HPLC columns. Use of the monolithic column permitted mobile phase flow-rates of up to 6.5 ml/min to be employed in the assays. The use of elevated flow-rates enabled the per sample analysis time to be reduced by up to a factor of 5 compared with assays based on packed HPLC columns. The results of experiments aimed at evaluating the ruggedness and reproducibility of monolithic columns employed in bioanalytical methods are presented.

Regulation of alpha1-proteinase inhibitor release by proinflammatory cytokines in human intestinal epithelial cells.

alpha1-Proteinase inhibitor (alpha1-PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti-inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells. Antiinflammatory actions of alpha1-PI are thought to be of relevance in the pathogenesis of inflammatory bowel disease. To investigate the role of macrophage-derived cytokines on alpha1-PI secretion from intestinal epithelial cells, we cultured Caco-2 cells until differentiation (14 days in culture) on permeable filter supports. Monolayers of differentiated Caco-2 cells were then co-cultured with human peritoneal macrophages, grown on plastic in the basolateral chamber. Under these conditions, alpha1-PI secretion from Caco-2 cells was enhanced by 45%, probably by a direct action of macrophage-derived cytokines on Caco-2 cells. To extend this observation further, we treated differentiated Caco-2 cells with macrophage-derived proinflammatory cytokines (IL-1beta, IL-8, TNF-alpha), as well as with lymphocyte-derived cytokines IL-2, IL-6 and IFN-gamma. As early as after 24h treatment, IL-2 and IL-8 induced a significant and dose-dependent increase of alpha-1-PI secretion into cell culture medium; this effect was completely reversed after immunoneutralization by the antibodies against IL-2 and IL-8 alpha1-PI secretion was only slightly decreased after treatment with IFN-gamma, while IL-1beta, IL-6 and TNF-alpha had no effect. alpha1-PI secretion correlated well with the expression of this protein in differentiated Caco-2 cells after cytokine treatment, as confirmed by Western blot. Our data imply that, in vitro, alpha1-PI secretion in enterocyte-like Caco-2 cells is up-regulated by IL-2 and IL-8. Our results suggest that both lymphocyte- and macrophage-derived cytokines regulate secretion of the anti-inflammatory protein alpha1-PI in intestinal epithelial cells.

Robotic inhibition assay for determination of HMG-CoA reductase inhibitors in human plasma.

The cholesterol-lowering drug simvastatin (SIMV, Zocor reduced heart attacks by 42% in patients who had high cholesterol levels and suffered from heart disease. Upon oral administration, SIMV is quickly hydrolyzed to its beta-hydroxyacid and other acid metabolites, which are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase. A Tecan-based enzyme inhibition assay has been developed to improve the existing Zymark-based assay for the determination of both active and total concentrations of HMG-CoA reductase inhibitors in human plasma. A Tecan Genesis 200 robotic workstation equipped with eight probes and customized hardware was utilized to achieve higher sample throughput and improve assay reproducibility and mechanical stability. The developed enzyme inhibition assay was validated over two concentration ranges of 0.4-20 ng equivalent/mL, and 2-50 ng equivalent/mL. Intra- and interday precision data (coefficient of variation (CV)) for both concentration ranges were less than 9%, with an accuracy of 93-107%. The interday precision for the determination of quality control (QC) samples was less than 2% and 8%, respectively. The respective interday QC accuracy values were 93-103% and 97-104%. Good linearity across the two concentration ranges was observed, with acceptable reproducibility. This improved enzyme inhibition assay has been utilized to analyze human plasma samples from several clinical studies.

Alpha-1 antitrypsin phenotypes by isoelectric focusing in a metropolitan southern Chinese population.

alpha-1 antitrypsin (alpha1AT) is an abundant protease inhibitor in human plasma. Its phenotypic variability has been reported to be associated with pulmonary emphysema and chronic liver diseases. However, alpha1AT deficiency is an uncommon condition in the Chinese population. The aim of this study was to describe the phenotypic distribution of alpha1AT in a southern Chinese population. A total of 1085 healthy blood donors underwent alpha1AT phenotyping by isoelectric focusing. Two thirds (66.1%) were homozygous for either M1 or M2, whereas 32.6% were heterozygous for two different M phenotypes. The frequency of allelic variants was only 0.007, and deficiency variants were absent. Compared with earlier studies on southern Chinese populations, this study found a lower frequency of M2, and a higher number of allelic variants, including E, L, N, P, and S. This phenomenon can be attributed to population migration and mixing. An understanding of the alpha1AT pattern is important for evaluating the predisposition of the population to selected clinical diseases.

Antiretrovirals: simultaneous determination of five protease inhibitors and three nonnucleoside transcriptase inhibitors in human plasma by a rapid high-performance liquid chromatography--mass spectrometry assay.

An analytical technique using liquid chromatography (LC) coupled with electrospray-mass spectrometry (ESI--MS) has been developed for the simultaneous determination of five protease inhibitors (PIs): saquinavir, indinavir, ritonavir, nelfinavir, and amprenavir; and three non-nucleoside reverse transcriptase inhibitors (NNRTIs): nevirapine, delavirdine, and efavirenz, in human plasma. This assay allows the elution and identification of these drugs in a single run (10 minutes) using a linear gradient with water and acetonitrile. The procedure involves liquid--liquid extraction. High-performance liquid chromatography (HPLC) separation was achieved on a C18 reversed-phase column, with a linear gradient elution followed by mass spectrometry detection. The calibration curves, obtained by automatic process peak area integration, show a good linearity in a range of concentrations between 20 and 10,000 ng/mL (40--10,000 ng/mL for efavirenz). The limit of detection was approximately 10 ng/mL for seven drugs (25 ng/mL for efavirenz). The coefficients of variation (CV) were always less than 15% for both intraday and interday precision for each compound. The recovery of the eight drugs ranged from 88.5% to 100%. This novel LC/ESI--MS assay provides an excellent method for simultaneous quantitative monitoring of different components of the highly active antiretroviral treatments (HAARTs) in patients treated simultaneously with PIs and NNRTIs, and it has been successfully applied to therapeutic drug monitoring and pharmacokinetic studies.

The effect of the Glu342Lys mutation in alpha1-antitrypsin on its structure, studied by molecular modelling methods.

The structure of native alpha1-antitrypsin, the most abundant protease inhibitor in human plasma, is characterised primarily by a reactive loop containing the centre of proteinase inhibition, and a beta-sheet composed of five strands. Mobility of the reactive loop is confined as a result of electrostatic interactions between side chains of Glu342 and Lys290, both located at the junction of the reactive loop and the beta structure. The most common mutation in the protein, resulting in its inactivation, is Glu342-->Lys, named the Z mutation. The main goal of this work was to investigate the influence of the Z mutation on the structure of alpha1-antitrypsin. Commonly used molecular modelling methods have been applied in a comparative study of two protein models: the wild type and the Z mutant. The results indicate that the Z mutation introduces local instabilities in the region of the reactive loop. Moreover, even parts of the protein located far apart from the mutation region are affected. The Z mutation causes a relative change in the total energy of about 3%. Relatively small root mean square differences between the optimised structures of the wild type and the Z mutant, together with detailed analysis of 'conformational searching' process, lead to the hypothesis that the Z mutation principally induces a change in the dynamics of alpha1-antitrypsin.

High-throughput, semi-automated determination of a cyclooxygenase II inhibitor in human plasma and urine using solid-phase extraction in the 96-well format and high-performance liquid chromatography with post-column photochemical derivatization-fluorescence detection

Compound I, 5-chloro-3-(4-methanesulfonylphenyl)-6′-methyl-[2,3′]bipyridinyl, has been found to be a specific inhibitor of the enzyme cyclooxygenase II (COX II). The anti-inflammatory properties of this compound are currently being investigated. HPLC assays for the determination of this analyte in human plasma and human urine have been developed. Isolation of I and the internal standard ( II) was achieved by solid-phase extraction (SPE) in the 96-well format. A C 8 SPE plate was used for the extraction of the drug from human plasma (recovery >90%) while a mixed-mode (C 8/Cation) SPE plate was used to isolate the analytes from human urine (recovery approximately 71%). The analyte and internal standard were chromatographed on a Keystone Scientific Prism-RP ® guard column (20×4.6 mm) connected to a Prism-RP ® analytical column (150×4.6 mm), using a mobile phase consisting of 45% acetonitrile in 10 m M acetate buffer (pH=4); the analytes eluted at retention times of 5.2 and 6.9 min for I and II, respectively. Compounds I and II were found to form highly fluorescent products after exposure to UV light (254 nm). Thus, the analytes were detected by fluorescence ( λ ex=260 nm, λ em=375 nm) following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 5–500 ng/ml for human plasma and human urine yielded a linear response ( R 2>0.99) when a 1/ y weighted linear regression model was employed. Based on the replicate analyses ( n=5) of spiked standards, the within-day precision for both assays was better than 7% C.V. at all points on the calibration curve; within-day accuracy was within 5% of nominal at all standard concentrations. The between-run precision and accuracy of the assays, as calculated from the results of the analysis of quality control samples, was better than 8% C.V. and within 8% of nominal. I was found to be stable in human plasma and urine for at least 8 and 2 months, respectively. In addition, the human plasma assay was semi-automated in order to improve sample throughput by utilizing a Packard liquid handling system and a Tom-Tec Quadra 96 SPE system. The precision and accuracy of the semi-automated procedure were comparable to the manual procedure. Over 5000 clinical samples have been analyzed successfully using these methods.

Molecular analysis of the Pi*Z allele in patients with liver disease.

Alpha1-antitrypsin (AAT) is the main protease inhibitor in human plasma. There are more than 75 variants of this protein that differ from each other by their isoelectric point. Most of these alleles cause a reduction in AAT levels; the most common allele is Pi*Z. The main complications related to the Pi*Z allele are obstructive pulmonary disease and liver disease. Some Pi*Z allele carriers present cholestatic jaundice and cirrhosis. The Z type is associated with a secretion defect, which leads to deficiency of AAT and to the formation of intrahepatocytic inclusions in affected subjects. The diagnosis of AAT deficiency can be made by different techniques, including molecular analysis, although the final diagnosis should be done in conjunction with demonstration of the periodic acid-Schiff-positive globules on liver biopsy. In this study, specimens of 29 patients with cryptogenic cirrhosis between age 1 month and 18 years, and of 100 controls were submitted to polymerase chain reaction followed by digestion with TaqI enzyme. Five of the 29 patients had undergone liver transplantation. Three patients were heterozygous for the Pi*Z allele, and two were homozygous (allele frequency = 12.07%; 7/58). Among the controls, who represented the population of Porto Alegre, 1 in 100 individuals was heterozygous for the Pi*Z allele, resulting in an allele frequency of 0.5% (1/200). The high frequency of Pi*Z alleles among the patients indicates the usefulness of AAT molecular testing in children with cholestatic jaundice and cirrhosis.

1-Oxacephem-based human chymase inhibitors: discovery of stable inhibitors in human plasma

1-Oxacephem derivatives were evaluated as a novel series of chymase inhibitors. The structure–activity relationship studies of 1-oxacephems led to compounds 15, which exhibited 27 nM inhibition of human chymase and improvement of stability in human plasma ( t 1/2 1.5 h).

Serpins Identified as Cell Growth Inhibitors in Human Plasma

We have identified a new factor, CFX, in human serum and plasma that inhibits the growth of cultured human and mouse cell lines. CFX was determined to be a negatively charged, hydrophobic glycoprotein, with a native molecular weight of 110–120 kDa and a minimal active subunit of 55 kDa. It is precipitated by 60% ammonium sulfate and is resistant to heat treatment at 100°C for 30 min. CFX was purified from human plasma to a single band on a gel which retained the cell growth inhibitory activity. Amino acid sequence analysis of the CFX band revealed sequences from four human glycoproteins, α1-antichymotrypsin, C1-esterase inhibitor, α1-antitrypsin, and α2-antiplasmin, all members of the superfamily of serpins. Of the four, C1-esterase inhibitor was shown to be the most potent cell growth inhibitor. These results suggest that serpins may play a cell growth inhibitory role in vivo, in addition to their role as protease inhibitors.

Determination of a novel thrombin inhibitor in human plasma and urine utilizing liquid chromatography with tandem mass spectrometric and ultraviolet detection

An LC–MS–MS method and an HPLC–UV method have been developed for the assay of a novel thrombin inhibitor in human fluids. The LC–MS–MS method is developed for plasma, which usually requires maximum sensitivity. The HPLC–UV method is for urine. In both methods, analytes are extracted using liquid–liquid extraction, and analyzed by reversed-phase high-performance liquid chromatography. A tandem mass spectrometer in the multiple reaction monitoring (MRM) mode is used for detection of the analytes in the plasma method. UV is the detector for the urine method. The plasma method has a lower limit of quantitation (LOQ) of 0.1 ng/ml with a linearity range of 0.1–100 ng/ml. The urine method has an LOQ of 8.12 ng/ml (20 nM) and the linear dynamic range is 8.12–8120 ng/ml (20–20 000 nM). Both methods are fast, specific and sensitive. Various validation procedures have proven that both methods are rugged, robust and reproducible. The research also suggested that, while LC–MS–MS provides superior sensitivity and selectivity for the determination of drugs and their metabolites at very low concentrations, HPLC with a conventional detector such as UV is still useful in the analysis when the sensitivity requirement is not crucial.

Generation of Catalytically Active Granzyme K from Escherichia coli Inclusion Bodies and Identification of Efficient Granzyme K Inhibitors in Human Plasma

Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions.

Regulation of the activities of thrombin and plasmin by cholesterol sulfate as a physiological inhibitor in human plasma.

Thrombin and plasmin, both of which are serine proteases in the plasma of vertebrates, play essential roles in blood clotting and fibrinolysis, respectively, and regulation of their activities is important to suppress the excessive reactions within the vascular network and to prevent tissue injury. Along with the peptidic inhibitors belonging to the serpin family, we found that cholesterol sulfate (CS), which is present at the concentration of 2.0+/-1.2 nmol/ml in human plasma, was a potent inhibitor of both plasma thrombin and plasmin. Thrombin, as determined both using a chromogenic substrate and the natural substrate, fibrinogen, was inactivated upon reaction with CS in a dose-dependent manner, but not in the presence of the structurally related steroid sulfates, I3SO3-GalCer and II3NAalpha-LacCer, suggesting that both the sulfate group and the hydrophobic side chain of CS are necessary for the inhibitory activity of CS. Preincubation of thrombin with CS at 37 degrees C for 10 min was required to achieve maximum inhibition, and virtually complete inhibition was achieved at a molar ratio of CS to thrombin of 18:1. CS-treated thrombin had the same Km and a lower Vmax than the original enzyme, and a higher molecular weight. The molecular weight and activity of the original enzyme were not observed on the attempted separation of the CS-treated enzyme by gel permeation chromatography and native PAGE, indicating that the inactivation of thrombin by CS is irreversible. In contrast, CS was readily liberated from the enzyme by SDS-PAGE, suggesting that hydrophobic interactions are involved in the CS-mediated inactivation of thrombin. When acidic lipids were reacted with thrombin after dissolving them in DMSO, I3SO3-GalCer, steroid sulfates and II3NAalpha-LacCer, as well as CS, but not SDS and sodium taurocholate, exhibited inhibitory activity, probably due to micellar formation facilitating interaction between thrombin and negatively charged lipids. On the other hand, plasmin, as determined using a chromogenic substrate, was more susceptible to acidic lipids than thrombin. CS, I3SO3-GalCer and II3NAalpha-LacCer, all of which are present in serum, inhibited the activity of plasmin in aqueous media, as well as in DMSO-mediated lipid solutions. Thus, acidic lipids in plasma were demonstrated to possess regulatory activity as endogenous detergents toward both enzymes for blood clotting and fibrinolysis.

Determination of an in vivo metabolite of a human immunodeficiency virus protease inhibitor in human plasma by high-performance liquid chromatography with tandem mass spectrometry

A method for the determination of a metabolite of the human immunodeficiency virus protease inhibitor indinavir, in human plasma is described. Isolation of the analyte and the internal standard from plasma was achieved via liquid-liquid extraction with a mixture of isopropanol-chloroform (5:95, v/v). The analytes were chromatographed under reversed-phase conditions on a Waters Symmetry C 8 column. A Sciex API III + tandem mass spectrometer equipped with a heated nebulizer was used as a detector and was operated in the positive ion mode. Multiple reaction monitoring using the precursor→product ion combinations of m/ z 523.4→273.4 and 512.4→345.2 was used to quantify analyte and internal standard, respectively. The method was validated in the concentration range of 5–500 ng/ml plasma with adequate assay precision and accuracy. The assay was used to analyze samples collected during drug interaction studies of indinavir.