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Biomedical subjects

V W Armstrong

Publications and source records attributed to V W Armstrong.

At least 37 records · Page 2Linked to original sources
PubMed2000

Mycophenolate mofetil in stem cell transplant patients in relation to plasma level of active metabolite.

OBJECTIVES: To investigate mycophenolate mofetil (MMF) plasma levels and impact on acute graft versus host disease (aGvHD) after stem cell transplantation (SCT). METHODS: SCT patients (n = 14) with aGvHD (>/= II) receiving MMF (1-3 g/d) in addition to cyclosporine, prednisolone, and methotrexate for aGvHD prophylaxis were investigated. Plasma levels of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) were determined by high-performance liquid chromatography. RESULTS: Overall median steady state pre-dose plasma MPA concentration was 0.47 mg/L and increased within 75 min after administration to 1.64 mg/L. In comparison to patients with skin aGvHD, patients with gut aGvHD had lower MPA concentrations, both pre-dose (p = 0.16) and after 75 min, (p = 0.02). All 7 patients with skin aGvHD but only 2 patients with gut aGvHD responded to MMF. Overall, the pre-dose plasma MPA concentration was significantly (p = 0.007) greater in responders (n = 9) than in non-responders (n = 5). CONCLUSION: MMF seems to be an effective treatment for aGvHD in SCT patients particular in those patients without gut involvement.

Drug Therapy, Combination↗
PubMed2000

Induction of cytokine release by the acyl glucuronide of mycophenolic acid: a link to side effects?

OBJECTIVES: We have identified an acyl glucuronide (M-2) of the immunosuppressant mycophenolic acid (MPA). Acyl glucuronides have toxic potential and may contribute to drug toxicity. Whether acyl glucuronides are able to induce release of proinflammatory cytokines is unknown. Gastrointestinal disturbances have been observed during MPA therapy and may involve an inflammatory reaction. This study investigated whether M-2 can induce IL-6 and TNF-alpha release as well as gene expression of these cytokines in leukocytes. DESIGN AND METHODS: M-2 was produced by incubation of MPA with human liver microsomes. Human mononuclear leukocytes were incubated in the presence of M-2. Concentrations of IL-6 and TNF-alpha were measured by ELISA. Expression of mRNA was determined by quantitative RT-PCR. RESULTS: Incubation of 3 x 10(6) cells with M-2 resulted in a time and dose dependent release of cytokines, whereas MPA or its phenolic glucuronide MPAG were without effect. Cytokine liberation depended on mRNA induction. Response to M-2 showed much inter individual variability (30-fold for IL-6, 3-fold for TNF-alpha). CONCLUSIONS: If M-2 promotes release of cytokines in vivo, these may mediate some of the toxic actions of MPA.

Adult↗
PubMed2000

Pharmacokinetic and metabolic investigations of mycophenolic acid in pediatric patients after renal transplantation: implications for therapeutic drug monitoring. German Study Group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients.

The need for mycophenolic acid (MPA) monitoring is still under discussion. Key issues for the PK/PD relationships of this drug are: the role of metabolites, the usefulness of AUC versus predose levels, and the need to monitor the free concentration of MPA (f-MPA). Recent advances have revealed that, in addition to 7-O-MPAG, three additional MPA metabolites are present in the plasma of transplant recipients. One of these metabolites (M-2), identified as an acyl glucuronide of MPA, was found to inhibit IMPDH-II in vitro. This active metabolite was also found to cross-react in the Emit assay for MPA. In an ongoing multicenter study, the authors are evaluating the relevance of monitoring total (t-MPA) and free mycophenolic acid (f-MPA) in pediatric renal transplant recipients. As in adults, a time-dependent increase of t-MPA-AUC(0-12h) within the first 3 months posttransplant (35 versus 64 mg x h/L, [corrected] 3 weeks versus 3 months respectively; daily dosage: 0.6 g/m2 bid) was seen. Receiver operating characteristics curve analyses were used to test the ability of predose levels or AUC(0-12h) to discriminate between cases with no complications and those with acute rejection, adverse events (severe infections, leukopenia), or gastrointestinal disorders observed during the early posttransplant course. In agreement with observations in adults, a significant (p = 0.001) association was observed between AUC(0-12h) and acute rejection. A t-MPA-AUC(0-12h) of approximately 30-60 mg x h/L [corrected], as determined by HPLC, seems to be a reasonable target for the early posttransplant period. It remains to be elucidated whether regular predose level monitoring may be of more practical value. A higher incidence of rejection was observed at predose MPA concentrations < or = 1 mg/L, as measured by HPLC. In contrast to t-MPA, f-MPA-AUC(0-12h) was significantly related to severe infections and leukopenia. The risk for severe adverse events was increased at f-MPA- AUC(0-12h) values > or =600 microg x h/L [corrected]. On the basis of these data and the observed variability in the pharmacokinetics of MPA, the development of monitoring strategies for this drug appears to be promising.

Child↗
PubMed2000

The monoethylglycinexylidide (MEGX) test as a marker of hepatic dysfunction in septic patients with pneumonia.

The aim of this study was to monitor hepatic function in patients with pneumonia meeting the sepsis criteria of the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) and to determine if hepatic dysfunction is related to the systemic inflammatory response. Twenty patients were recruited. The monoethylglycinexylidide (MEGX) test was carried out on days 1-10 after admittance to the intensive care unit. Blood samples for determination of serum concentrations of hyaluronic acid, C-reactive protein (CRP), interleukin (IL)-6, IL-8, IL-10 and conventional liver function tests (aspartate aminotransferase, alanine aminotransferase, bilirubin, albumin) were also drawn. Patients were classified into two groups according to illness severity estimated by the simplified acute physiology score (SAPS II) on the day of admission. Patients in group I (n=10) had a SAPS II probability of mortality >3% while those in group II (n=10) had a SAPS II < 3%. The MEGX level over the first five days was significantly lower in group I than in group II (p<0.0001). Significant inverse correlations during the first 5 days were observed between the MEGX 30 min test results and IL-6, CRP and SAPS II and more modest correlations with hyaluronic acid (p=0.0025) and IL-10 (p=0.021). The conventional liver function tests did not differ between the two groups and were mostly within the respective reference ranges. We conclude that the MEGX test is a sensitive marker of liver dysfunction early in sepsis and that low MEGX values are associated with an enhanced inflammatory response.

Adult↗
PubMed2000

Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit.

BACKGROUND: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated. METHODS: The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents. RESULTS: The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2-25 mg/L), <4.4% for 7-O-MPAG (10-250 mg/L), and < or =14% for AcMPAG (0.1-5 mg/L). The between-day imprecision was <6.2%, <4.5%, and < or =14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at -20 degrees C. CONCLUSIONS: The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.

Chromatography, High Pressure Liquid↗
PubMed1999

Assessment of liver function by the MEGX test in patients with schistosomiasis and cirrhosis.

OBJECTIVES: The aim of this study was to determine the effect of schistosomiasis infection on hepatic function in Egyptian patients with posthepatitic cirrhosis. DESIGN AND METHODS: Hepatic function, was assessed in 66 Egyptian patients, with (n = 30) and without (n = 36) schistosomal liver fibrosis due to Schistosoma mansoni and in 20 healthy controls, using the monoethylglycinexylidide (MEGX) test. Serum MEGX concentrations were measured before and 5, 15, 30, 60, 120, and 180 min after a lidocaine bolus. The sero-prevalence of antibodies to hepatitis C was also determined in the patients. RESULTS: MEGX test results were significantly lower in patients than in controls at all time points. MEGX test results declined with advancing Child Class. Receiver operating characteristic (ROC) curve analysis revealed the following areas under the ROC curves for discrimination of Child Class C from Child Classes A/B: 30 min, 0.762; 60 min, 0.743; 120 min, 0.731; 15 min, 0.728; 180 min, 0.728; 5 min, 0.602. Schistosomiasis infection had no influence on MEGX test results when cirrhotic patients with (Schisto+) and without (Schisto-) schistosomiasis were compared. While the prevalence of the hepatitis B surface antigen was only 16.7% (Schisto-) and 26.7% (Schisto+), there was an extremely high sero-prevalence of antibodies to hepatitis C (HCV) in both groups: 88.9% (Schisto-) and 73.3% (Schisto+). CONCLUSIONS: The association of schistosomal liver fibrosis with cirrhosis does not additionally influence MEGX formation. In addition, HCV rather than schistosomiasis infection must be considered as a major cause for the progressive liver disease in these patients.

Adult↗
PubMed1999

Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil.

1. Mycophenolic acid (MPA), is primarily metabolized in the liver to 7-O-MPA-beta-glucuronide (MPAG). Using RP-h.p.l.c. we observed three further MPA metabolites, M-1, M-2, M-3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP-glucuronic acid, UDP-glucose or NADPH; (C) hydrolysis of metabolites was investigated using beta-glucosidase, beta-glucuronidase or NaOH; (D) cross-reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). 2. Mass spectrometry of M-1, M-2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. 3. Incubation of microsomes with MPA and UDP-glucose produced M-1, with MPA and UDP-glucuronic acid MPAG and M-2 were formed, while with MPA and NADPH, M-3 was observed. 4. Beta-Glucosidase hydrolysed M-1 completely. Beta-Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M-2 was reduced by approximately 30%. Only M-2 was labile to alkaline treatment. 5. M-2 and MPA but not M-1 and MPAG cross-reacted in the EMIT assay. 6. These results suggest that: (i) M-1 is the 7-OH glucose conjugate of MPA; (ii) M-2 is the acyl glucuronide conjugate of MPA; (iii) M-3 is derived from the hepatic CYP450 system.

Chromatography, High Pressure Liquid↗
PubMed1999

Area under the plasma concentration-time curve for total, but not for free, mycophenolic acid increases in the stable phase after renal transplantation: a longitudinal study in pediatric patients. German Study Group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients.

Mycophenolate mofetil, an ester prodrug of the immunosuppressant mycophenolic acid (MPA), is widely used for maintenance immunosuppressive therapy in pediatric renal transplant recipients. However, little is known about the pharmacokinetics of MPA in this patient population in the stable transplant phase, and dosage guidelines are preliminary. The authors therefore compared the pharmacokinetics of MPA, free MPA, and the renal metabolite MPA glucuronide (MPAG) in the initial (sampling at 1 and 3 weeks) and stable phases (sampling at 3 and 6 months) posttransplant in 17 children (age, 12.0 +/- 0.77 years; range, 5.9 to 15.8 years), receiving the currently recommended dose of 600 mg MMF/m2 body surface area (BSA) twice a day. Plasma concentrations of MPA and MPAG were measured by reverse phase HPLC. Because MPA is extensively bound to serum albumin and only the free drug is presumed to be pharmacologically active, the authors also analyzed the MPA free fraction by HPLC after separation by ultrafiltration. The intraindividual variability of the area under the concentration-time curves (AUC0-12) of MPA throughout the 12-hour dosing interval was high in the immediate posttransplant period, but declined in the stable phase, whereas the interindividual variability remained unchanged. The median MPA-AUC0-12 values increased 2-fold from 32.4 (range, 13.9 to 57.0) mg x h/L at 3 weeks to 65.1 (range, 32.6 to 114) mg x h/L at 3 months after transplantation, whereas the median AUC0-12 values of free MPA did not significantly change over time. This discrepancy can be attributed to a 35% decline of the MPA free fraction from 1.4% in the initial phase posttransplant to 0.9% (p < 0.01) in the stable phase. In conclusion, pediatric renal transplant recipients given a fixed MMF dose exhibit a 2-fold increase of the AUC0-12 of total MPA in the stable phase posttransplant and a 35% decrease of the MPA free fraction, whereas the AUC0-12 of free MPA remains unchanged over time. Because the latter pharmacokinetic variable is theoretically best predictive of the clinical immunosuppressive efficacy of MMF, these findings may have consequences for the dosing recommendations of MMF in renal transplant recipients.

Adolescent↗
PubMed1999

Application of a thermodynamic nearest-neighbor model to estimate nucleic acid stability and optimize probe design: prediction of melting points of multiple mutations of apolipoprotein B-3500 and factor V with a hybridization probe genotyping assay on the LightCycler.

BACKGROUND: PCR-based mutation detection is prone to methodological errors, e.g., in restriction length fragment polymorphism (RFLP) and allele-specific amplification (ASA), false PCR results may occur because of technical faults or atypical new mutations. METHODS: We investigated the ability of a genotyping assay based on hybridization of labeled oligonucleotides to detect and discriminate known and as yet unknown mutations in the factor V and apolipoprotein B-100 genes. Expected melting points were calculated using a nearest-neighbor model for nucleic acid duplex stability and compared with experimental findings derived from LightCycler melting curves. A method for genotyping the apolipoprotein B-100 G10699A and C10698T mutations is presented. RESULTS: All mismatches tested for in the probed sequence could be detected with a single probe. The measured melting points were in good agreement with their values predicted using the nearest-neighbor model (r = 0.96; y = 0.98x + 1.18; S(y|x) = 0.96; n = 24). CONCLUSIONS: This procedure not only allows the identification of the mutation of interest but also enables the discrimination from other potential mutations in the vicinity of the former. The nearest-neighbor model is valid for hybridization probe assays on the LightCycler and should be of general value in setting up such assays. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detectable, but they will not necessarily be discriminated from each other in all cases.

Apolipoproteins B↗
PubMed1998

[Age related decrease of high density lipoproteins (HDL) in women after menopause. Quantification of HDL with genetically determined HDL arylesterase in women with healthy coronary vessels and in women with angiographically verified coronary heart disease].

BACKGROUND: The decline in the concentration of high density lipoproteins (HDL) observed in postmenopausal women is thought to contribute to the increasing incidence of coronary artery disease (CAD) after menopause. Human serum arylesterase (EC 3.1.1.2) is exclusively associated with HDL. We therefore investigated possible differences in the decline of HDL-levels and of HDL-subfractions HDL2 and HDL3 between postmenopausal women without and with angiographically documented CAD. PATIENTS AND METHODS: HDL-, HDL2-and-HDL3- concentrations were studied in postmenopausal women with angiographically documented CAD (n = 24; 51 to 72 years mean: 62 years) and compared to HDL-parameters of women without CAD (n = 22; 51 to 81 years, mean: 58 years). Arylesterase activities of HDL2-and HDL3-subfractions and HDL2-cholesterol concentrations were determined after differential precipitation with polyethylene glycol (4.7 mM PEG). Phenotyping of HDL-arylesterase was achieved in CAD patients and in women without CAD after determining hydrolysis of arylesterase substrates paraoxon (PO) and phenylacetate (PA) by calculating paraoxonase/arylesterase activity ratios R (R = [PO]/[PA] x 1000): phenotype A (n = 26) with R < 2.5, phenotype AB (n = 16) with 5.0 < R < 10.7, and phenotype B (n = 4) with R > 13.5. RESULTS: In postmenopausal women with documented CAD, as compared to women without CAD, HDL-cholesterol (55 +/- 3 mg/dl vs. 69 +/- 3 mg/dl HDL2-arylesterase (25 +/- 1 kU/l vs. 33 +/- 2 kU/l), and HDL3-arylesterase (89 +/- 4 kU/l vs. 106 +/- 5 kU/I) were found to be significantly reduced. Analysis of the correlation of lipid parameters and age revealed in CAD patients, but not in postmenopausal women without CAD, a significant increase of total cholesterol (r = 0.42), and significant reductions of both HDL2-arylesterase (r = -0.47) and HDL3-arylesterase (r = 0.74) with increasing age. In contrast, HDL-cholesterol (r = -0.14) and HDL2-cholesterol (r = -0.06) of CAD patients showed only slight and non-significant reductions with age. Since HDL3-arylesterase was found to be age-dependently reduced in women without CAD (r = 0.17), HDL2-arylesterase of postmenopausal women, among all lipid parameters showed the most pronounced differences between women without CAD and CAD patients. The age-dependent decrease of HDL2-arylesterase in postmenopausal women with CAD does not result from an increased frequency of B-allele carriers in the subgroup of CAD patients with an age above the median (64 years). CONCLUSION: Genetically determined serum HDL-arylesterase is well suited to quantify HDL in postmenopausal women without and with CAD. HDL2-arylesterase of postmenopausal women should be evaluated as a screening parameter for both primary and secondary CAD prevention.

Aged↗