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D W Holt

Publications and source records attributed to D W Holt.

At least 19 recordsLinked to original sources
PubMed2006

Paradoxical response to tacrolimus assessed by interleukin-2 gene expression.

BACKGROUND: The calcineurin inhibitors cyclosporine and tacrolimus are potent immunosuppressive drugs, used mainly after organ transplantation. Methods to monitor their pharmacodynamic effects are not well established. METHODS: Whole blood samples from healthy volunteers (n = 16) were incubated for 24 hours in culture medium; each sample was preincubated for 2 hours with or without tacrolimus. An identical procedure was performed for 7 samples using blood from renal transplant patients before transplantation. Following the culture period, total RNA was isolated and quantitative reverse transcriptase polymerase chain reaction, using TaqMan probes, was employed to quantify interleukin-2 (IL-2) mRNA expression, and IL-2 mRNA copy number was reported by reference to a standard curve. RESULTS: IL-2 mRNA synthesis was suppressed by the presence of tacrolimus in most cases, compared with a control sample. However, some samples demonstrated up-regulation of mRNA expression. In the patient samples, there was up-regulation of IL-2 mRNA in two samples and, after transplantation, these patients developed acute rejection. CONCLUSION: Quantitative measurement of cytokine IL-2 regulated gene expression may represent a method to assess the efficacy of calcineurin inhibitor drugs.

Adult↗
PubMed2004

Therapeutic drug monitoring of cyclosporine.

The introduction of cyclosporine into clinical practice improved transplant outcome. However, the use of cyclosporine is not without problems. A narrow therapeutic index (the drug causes irreversible kidney damage when given in too high a dose) coupled with variable absorption and unpredictable pharmacokinetics has resulted in the need to measure cyclosporine blood concentrations to enable the dose of the drug to be individualised to the patient. When this is done correctly therapeutic efficacy can be maximised while toxicity is kept to a minimum. The evolution of cyclosporine dose optimisation started with the adjustment of empirical fixed doses by clinical "judgement;" progressed to therapeutic drug monitoring of trough, predose, C0 concentration with non specific assays that measured parent drug and metabolite; then on to "specific" cyclosporine C0 measurements; through area under curve monitoring using full profile measurements and limited sampling scheme procedures; and finally ending up with absorption profiling that targets AUC in the first 4 hours or the 2 hour blood cyclosporine concentration, C2. At the same time the formulation of cyclosporine has changed from Sandimmune to Neoral and now generic forms of the latter are available. The evidence base supporting C2 monitoring continues to grow and the technique will need to be customised as new combination therapies emerge. Therapeutic drug monitoring of cyclosporine may also need to be tailored to avoid the potential negative impact of switching patients to generic forms of the drug.

Area Under Curve↗
PubMed2004

A survey to determine the blood concentration of cyclosporine 2 hours postdose in stable renal transplant patients.

The current method for monitoring cyclosporine measures predose concentrations (C0). A better method has been developed, namely, measurement of the blood cyclosporine concentration at 2 hours postdose (C2). The aim of this survey was to determine the variability of C0 and C2 concentrations among stable renal transplant patients. One hundred two stable renal transplant patients who were at least 6 months posttransplant were recruited from the renal transplant outpatient clinic. The cyclosporine dose was between 100 and 500 mg daily; all patients had been monitored using C0 concentrations. Blood samples for cyclosporine concentration measurements were taken at both C0 and C2 at two consecutive clinic visits. The within- and between-patient variabilities were calculated using nested analysis of variance. The mean age was 50 years (21 to 81); the mean weight was 75 kg. The mean cyclosporine dose was 3.18 mg/kg/d (1.2 to 8.8). The average serum creatinine was 174 micromol/L (77 to 626) and the average cholesterol was 5 micromol/L (3 to 9). The mean (+/-SD) C0 concentration was 150 (47.31) microg/L and C2=895 (239) microg/L. The C0 concentration varied over 16-fold between patients compared to a sevenfold variation in C2. The between-subject coefficient of variation (CV) was 35% for C0 and 30% for C2 and the within subject CV was 23% for C0 and 20% for C2. The results suggest that cyclosporine concentrations at C0 are slightly more variable than those at C2. Whether this modest reduction in variability results in better patient outcomes is the subject of the next phase of this study.

Creatinine↗
PubMed2004

The relative proportions of sirolimus metabolites in blood using HPLC with mass-spectrometric detection.

Measurement of sirolimus in blood as a guide to dose adjustment is an accepted practice. To date, most data have resulted from the use of a chromatographic technique. With the imminent introduction of an immunoassay into this field, there is a need to know whether metabolites that could interfere with the performance of this assay, causing a bias compared with measurements made by a chromatographic assay, vary over a period of time or with changes in concomitant immunosuppressive therapy. This preliminary study measured several sirolimus metabolites in blood samples from a variety of clinical settings, using high-performance liquid chromatography with tandem mass-spectrometric detection. Two metabolites known to cross-react in one immunoassay system, single hydroxylation products and 41-O-demethyl rapamycin, were found to constitute the bulk of the metabolic products. They were also found to form a remarkably stable proportion of all metabolites measured, both with respect to the time since transplantation and the concomitant use of cyclosporine or tacrolimus. It is concluded that the analytical bias due to cross-reactivity with metabolites, inherent in this immunoassay, should be consistent across a wide spectrum of patients receiving the drug.

Biotransformation↗
PubMed2003

Therapeutic monitoring of sirolimus: its contribution to optimal prescription.

It is now common practice to measure immunosuppressive drugs in blood as a guide to therapy. The immunosuppressive drug sirolimus, recently approved for use following kidney transplantation, was developed in the context of this clinical approach. Throughout the early clinical studies, validated analytical techniques based on chromatographic techniques were used to measure the drug. After a brief period in which an immunoassay was available, routine measurements are again being performed by chromatographic assays. In this article the use of blood concentration measurements in the assessment of the early and pivotal clinical trials of the drug is documented. Then, the rationale for the routine monitoring of the drug in clinical practice, a regulatory requirement in some countries, is set out. It is concluded that the development of this compound has benefited from experience gained during the pharmacokinetic assessment of other immunosuppressive drugs. The pharmacokinetic data accumulated on sirolimus have been a key element in formulating guidelines on dosing with this drug, both when used in combination with cyclosporine and when used after cyclosporine withdrawal.

Binding Sites↗
PubMed2001

Sirolimus for rescue and primary immunosuppression in transplanted children receiving tacrolimus.

AIMS: The role of sirolimus (SRL) as a rescue agent (n=42) and as a component of primary immunosuppression (n=8) was evaluated in a mixed population of 50 transplanted children receiving tacrolimus (liver: 26, heart: 5, intestinal: 5, liver-intestine: 9, lung: 1, bone marrow: 1, liver-kidney: 1, multivisceral: 1). Rescue indications for tacrolimus (TAC) failure were recurrent acute rejection and acute rejection complicating withdrawal of immunosuppression in posttransplant lymphoproliferative disorder (PTLD). Rescue indications for TAC toxicity were nephrotoxicity, pancreatitis, seizures, hypertrophic cardiomyopathy, and graft-versus-host disease. RESULTS: Mean age at rescue was 11.5 years and mean follow-up was 204 (range 18-800) days. As primary immunosuppression, SRL+TAC prevented early acute rejection in 7/8 children. The indication for rescue resolved in 33/42 children. In children with TAC toxicity, this was associated with decrease in TAC doses by 50%, significant improvements in renal function, and continuing decline in Epstein-Barr virus (EBV) viral load in PTLD patients. Serious adverse events led to discontinuation of SRL in 9/42 rescue patients, 3 of them also experienced acute rejection. Three additional children also experienced acute rejection on SRL therapy (overall incidence 6/50, 12%). Pharmacokinetic analysis in the first week of SRL administration suggested a short half-life (11.8+/-5.5 hr, n=21). CONCLUSIONS: SRL and reduced-dose TAC may achieve adequate immunosuppression without compromising renal function or enhancing EBV viremia significantly.

Acute Disease↗
PubMed2001

Circulating cardiac troponin-T in patients before and after renal transplantation.

BACKGROUND: The diagnostic and prognostic use of cardiac troponin T (cTnT) in patients with renal failure has been questioned. Raised serum concentrations of cTnT, with no apparent signs of cardiac damage using conventional methods of detection, have been reported. We aimed to relate circulating concentrations of cTnT to improved renal function following renal transplantation over a one-year period. METHOD: Plasma cTnT was analysed from patients with end stage renal disease before and after transplantation and subsequently at 1, 3, 6 and 12 months. Eight patients had diabetes, 14 had hyperlipidaemia, 8 were smokers and 4 were ex-smokers; all were hypersensitive. RESULTS: At the time of transplantation, 3 of the 32 patients (9.4%) had plasma cTnT concentrations above 0.1 microg/l. In addition to these three patients, five others showed raised cTnT over the one-year period. CONCLUSIONS: The overall trend in circulating cTnT concentrations did not seem to be affected by improved renal function. However, all of the patients that had raised cTnT concentrations at any stage of the one-year period had explainable pathologies or were exposed to multiple cardiac risk factors.

Adult↗
PubMed2001

Cardiac troponins and creatine kinase content of striated muscle in common laboratory animals.

Animal models are important for the investigation of human heart pathology, novel treatments, and medical or surgical interventions for disease. Serum markers of myocardial damage may also be important tools within this field of research. In order to assess the cardiac specificity of widely utilised serum markers, we measured the cardiac troponins and creatine kinase (CK) isoenzymes in cardiac and skeletal muscle samples taken from dog, monkey, pig and rat. These samples were also analysed by immunoblotting for cardiac troponin I (cTnI) and cardiac troponin T (cTnT). The content of cTnI and cTnT in skeletal muscle was below 0.6% of that found in heart for all animal species studied. This low immunoreactivity in skeletal muscle was confirmed by immunoblot analysis. The content of CK was higher in skeletal muscle than in heart muscle for all species. The CK-MB/total CK ratio was lower in skeletal muscle than in cardiac muscle for all species. The differences in CK-MB content of skeletal muscle and heart muscle were much less pronounced than the tissue differences in the amounts of the cardiac troponins. The cardiac troponins are potentially useful serum markers of myocardial damage, with high specificity for myocardial muscle in these common laboratory animals. Creatine kinase-MB is much less cardiac-specific.

Animals↗
PubMed2001

Elevated serum levels of soluble interleukin-2 receptor, neopterin and beta-2-microglobulin in idiopathic dilated cardiomyopathy: relation to disease severity and autoimmune pathogenesis.

BACKGROUND: It has not been assessed whether high levels of soluble interleukin 2 receptor (sIL-2R), neopterin and beta-2 microglobulin in idiopathic dilated cardiomyopathy reflect heart failure severity and/or an active autoimmune process. The aim of this study was to relate serum levels of these markers to clinical and autoimmune features. METHODS: We studied 60 patients with idiopathic dilated cardiomyopathy, 67 controls with ischemic heart failure and 34 normals. RESULTS: Abnormal levels of sIL-2R, but not of neopterin and beta-2 microglobulin, were more frequent in idiopathic dilated cardiomyopathy than in ischemic patients (35% vs. 16%; P=0.02) or in normals (35% vs. 12%, P=0.01); mean sIL-2R levels were, however, similar in idiopathic dilated cardiomyopathy and ischemic heart failure (842+/-75 vs. 762+/-93 U/ml, P=NS). In idiopathic dilated cardiomyopathy abnormal levels of sIL-2R were associated with lower peak oxygen consumption (P=0.008), higher neopterin and HLA class II expression in the myocardium (P=0.02), but were unrelated to cardiac autoantibody status or titer. In addition, abnormal levels of neopterin were associated with adverse prognosis and higher beta-2 microglobulin; abnormal levels of beta-2 microglobulin with lower echocardiographic percent fractional shortening, higher sIL-2R and higher neopterin. CONCLUSIONS: There is no convincing evidence that abnormal sIL-2R, neopterin and/or beta-2 microglobulin are disease-specific markers of idiopathic dilated cardiomyopathy. The lack of association with cardiac autoantibodies suggests that these abnormalities are mainly related to heart failure severity rather than autoimmune pathogenesis. In keeping with this view, high levels of sIL-2R, neopterin and/or beta-2 microglobulin identified a subset of idiopathic dilated cardiomyopathy patients with advanced disease and poor prognosis.

Adolescent↗
PubMed2001

Increased plasma endothelin levels in angina patients with rapid coronary artery disease progression.

AIMS: To investigate the association between plasma endothelin levels and rapid coronary artery disease progression, as assessed by quantitative angiography. METHODS AND RESULTS: Changes in diameter were assessed in 224 coronary stenoses of 92 consecutive patients (62 men) with chronic stable angina pectoris who were on a waiting list for routine coronary angioplasty and underwent coronary angiography on two occasions: the first (diagnostic) angiogram was carried out at study entry and the second 5.5+/-3.0 months later, immediately prior to coronary angioplasty. A digital quantitative angiographic analysis system was used to assess differences in stenosis diameter between the first and second angiogram. Plasma immunoreactive endothelin levels were estimated by radioimmunoassay at study entry. Rapid coronary artery disease progression occurred in 29 (31.5%) patients according to pre-established criteria: 12 (41%) had a > or =10% diameter reduction of at least one pre-existing stenosis > or =50%, 10 (34%) had a > or =30% diameter reduction of a pre-existing stenosis <50%, 5 (17%) patients developed a new stenosis and 2 (7%) had progression of a lesion to total occlusion by the second angiogram. Baseline demographic, clinical and angiographic data were similar in patients with and without stenosis progression. Plasma endothelin levels were significantly higher in patients with rapid disease progression than in those without (5.7+/-2.0 pg. ml(-1)vs 3.9+/-1.6 pg. ml(-1), P<0.001). Multiple logistic regression analysis revealed that endothelin was an independent predictor of disease progression (P=0.001). Moreover, endothelin levels above 4.26 pg. ml(-1)(the median of the total endothelin concentrations) were associated with a sixfold increase in the risk of developing rapid stenosis progression. CONCLUSIONS: Plasma endothelin is raised in patients with coronary artery disease progression and may be a marker of risk of rapid stenosis progression. Endothelin may also play a pathogenic role in this process.

Aged↗
PubMed2001

Simultaneous determination of plasma prednisolone, prednisone, and cortisol levels by high-performance liquid chromatography.

Recipients of organ transplants remain particularly dependent on prednisolone as part of their maintenance immunosuppression. Despite this, the pharmacokinetics of prednisolone have never been fully characterized in these patients, and consequently dosing remains empirical. Accurate monitoring of prednisolone, its primary metabolite prednisone, and endogenous cortisol suppression in such patients may provide a means of improving the clinical outcome by adjusting for variability in prednisolone pharmacokinetics and pharmacodynamics. Measurement of endogenous cortisol may provide an independent marker of prednisolone pharmacodynamics. A simple isocratic reverse-phase high-performance liquid chromatography procedure, using betamethasone as an internal standard, was developed to quantify plasma prednisolone, prednisone, and cortisol simultaneously. The steroids were extracted from 0.5 mL plasma with 3 mL (1:1 v/v) ethyl acetate/tert-methyl butyl ether and 0.1 mL phosphoric acid, washed in 0.1 mol/L NaOH before a final drying step and reconstitution in mobile phase for injection. Separation was achieved using a Supelcosil LC-18-DB, 150 x 4.6-mm, 5-microm particle size, reverse-phase column attached to a Newguard 15 x 3-mm, RP8 guard column maintained at 25 degreesC, with ultraviolet detector set at 254 nm. The mobile phase consisted of 16% isopropanol in water containing 0.1% trifluoroacetic acid, set at a flow rate of 1.2 mL/min. The assay was linear up to 1,002 microg/L for prednisolone, 982 microg/L for prednisone, and 545 microg/L for cortisol. Mean intra-assay and interassay imprecision levels were 6.0% and 7.2%, respectively, for prednisolone, 5.8% and 7.2% for prednisone, and 5.6% and 7.9% for cortisol. Intra-assay inaccuracy was <7% of nominal values for prednisolone, prednisone, and cortisol. The lower limit of quantification was 7 microg/L for prednisolone and prednisone and 10 microg/L for cortisol. Corticosteroid recoveries were 73%, 74%, and 90% for prednisolone, prednisone, and cortisol, respectively. The authors describe a robust, inexpensive, and simple method suitable for therapeutic drug monitoring or pharmacokinetic studies of prednisolone; it may also be used to measure the suppression of endogenous cortisol production.

Anti-Inflammatory Agents↗
PubMed2001

Cardiac troponin T and creatine kinase MB are not increased in exterior oblique muscle of patients with renal failure.

BACKGROUND: Serum cardiac troponin T (cTnT) concentrations may be increased in patients with renal dysfunction without evidence of cardiac damage, as assessed by conventional methods. It has been suggested that these positive measurements result from the expression in skeletal muscle of fetal isoforms of cTnT, which are detected by the cTnT immunoassay. METHODS: Skeletal muscle (exterior oblique) biopsies were taken from healthy living kidney donors (n = 5) and transplant recipients (n = 19). The amounts of cTnT and creatine kinase (CK) isoenzymes in skeletal muscle of healthy controls were compared with those in patients with renal failure (Wilcoxon-Mann-Whitney test). cTnT was measured quantitatively by a second-generation assay, with a limit of detection of 1 microg/g of protein, and qualitatively by immunohistochemistry and immunoblotting. CK-MB was measured by quantitative electrophoresis. RESULTS: Minute quantities of cTnT were detected in 2 of the 5 (40%) control samples and 9 of the 19 (47%) renal failure samples, respectively, at mean concentrations of <5 microg/g of protein for both subject groups. This was <1/6000th that found in heart muscle. There was no significant difference in cTnT or CK-MB content in skeletal muscle between healthy controls and patients with renal failure. Increased serum cTnT did not predict detectable cTnT in skeletal muscle. cTnT was not detected qualitatively by immunoblotting or immunohistochemistry in any skeletal muscle samples. CONCLUSIONS: Uremia does not affect the content of cTnT or CK-MB in exterior oblique muscle, suggesting that cTnT detected in serum from patients with renal failure does not originate from skeletal muscle.

Adult↗
PubMed2000

A controlled trial of substrate-enhanced, warm reperfusion ("hot shot") versus simple reperfusion.

BACKGROUND: Modified reperfusion after aortic cross-clamping is claimed to reduce myocardial injury, thus improving postoperative myocardial performance. METHODS: We measured perioperative release of creatine kinase-MB and troponin-T in 40 patients undergoing valve replacement (combined with coronary grafts in 12 cases) to determine whether infusion of a modified reperfusate before cross-clamp removal reduced myocardial injury. Patients were randomly allocated to one of two groups with minimization for age, surgeon, operation, and ventricular function. The control group received unmodified reperfusion, while the study group received a normothermic reperfusate, enhanced with glutamate and aspartate, for 5 minutes before removal of the cross-clamp. Serial determinations of troponin-T, creatine kinase-MB isoforms, and total creatine kinase-MB activity were made up to 5 days postoperatively. Requirements for inotropic support and evidence of myocardial infarction were documented. RESULTS: Creatine kinase-MB activity, creatine kinase-MB isoforms, and troponin-T were not significantly different between the two groups. There were no differences in the incidence of postoperative myocardial infarction or in inotrope requirement. CONCLUSIONS: Our study did not demonstrate any advantage in using modified reperfusion in this group of patients.

Adult↗