Comparison of an ELISA and an LC/MS/MS method for measuring tacrolimus concentrations and making dosage decisions in transplant recipients

This study compared an enzyme-linked immunosorbent assay (ELISA) to a liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique for measurement of tacrolimus concentrations in adult kidney and liver transplant recipients, and investigated how assay choice influenced pharmacokinetic parameter estimates and drug dosage decisions. Tacrolimus concentrations measured by both ELISA and LC/MS/MS from 29 kidney (n = 98 samples) and 27 liver (n = 97 samples) transplant recipients were used to evaluate the performance of these methods in the clinical setting. Tacrolimus concentrations measured by the two techniques were compared via regression analysis. Population pharmacokinetic models were developed independently using ELISA and LC/MS/MS data from 76 kidney recipients. Derived kinetic parameters were used to formulate typical dosing regimens for concentration targeting. Dosage recommendations for the two assays were compared. The relation between LC/MS/MS and ELISA measurements was best described by the regression equation ELISA = 1.02 . (LC/MS/MS) + 0.14 in kidney recipients, and ELISA = 1.12 . (LC/MS/MS) - 0.87 in liver recipients. ELISA displayed less accuracy than LC/MS/MS at lower tacrolimus concentrations. Population pharmacokinetic models based on ELISA and LC/MS/MS data were similar with residual random errors of 4.1 ng/mL and 3.7 ng/mL, respectively. Assay choice gave rise to dosage prediction differences ranging from 0% to 30%. ELISA measurements of tacrolimus are not automatically interchangeable with LC/MS/MS values. Assay differences were greatest in adult liver recipients, probably reflecting periods of liver dysfunction and impaired biliary secretion of metabolites. While the majority of data collected in this study suggested assay differences in adult kidney recipients were minimal, findings of ELISA dosage underpredictions of up to 25% in the long term must be investigated further.

Isolation and characterisation of Indian Ocean ciguatoxin

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3 H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean. (C) 2002 Elsevier Science Ltd. All rights reserved.

Associations between human liver and kidney cadmium content and immunochemically detected CYP4A11 apoprotein

This present study was undertaken to assess potential effects of cadmium on CYP4A11 apoprotein in human liver and kidney as detected by Western blotting using a highly specific anti-peptide antibody. Liver and kidney cortex samples were autopsy specimens of 37 individuals (26 mates and I I females) whose ages ranged from 3 to 89 years. All were Caucasians who had not been exposed to cadmium in the workplace. Reduced CYP4A11 apoprotein levels were found in chronic hepatitis samples and in liver samples showing fatty changes. In contrast, increased CYP4A11 apoprotein levels were found in liver samples having higher cadmium content compared to the lower cadmium content samples. Increased CYP4A11 levels were also found in liver samples from female donors, compared to male donors; the difference being attributable to higher female liver cadmium burden. In distinction to liver, lowered CYP4A11 levels were seen in the kidney cortex samples which have high cadmium content, It is proposed here that the difference between the absolute cadmium burden of the liver and kidney samples may be responsible for the different patterns of expression of CYP4A11 in these two tissues. Further, since cadmium exposure may be associated with derangement in blood pressure control, it is interesting to note the possible relationship between altered CYP4A11-dependent production of arachidonic acid hydroxy and epoxy metabolites in kidney cortex and altered control of blood pressure. Our findings provide a possible link between these observations. (C) 2002 Elsevier Science Inc. All rights reserved.

Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

Distribution kinetics of solutes in the isolated in-situ perfused rat head using the multiple indicator dilution technique and a physiological two-barrier model

The purpose of this study was to determine the pharmacokinetics of [C-14]diclofenac, [C-14]salicylate and [H-3]clonidine using a single pass rat head perfusion preparation. The head was perfused with 3-[N-morpholino] propane-sulfonic acid-buffered Ringer's solution. Tc-99m-red blood cells and a drug were injected in a bolus into the internal carotid artery and collected from the posterior facial vein over 28 min. A two-barrier stochastic organ model was used to estimate the statistical moments of the solutes. Plasma, interstitial and cellular distribution volumes for the solutes ranged from 1.0 mL (diclofenac) to 1.6 mL (salicylate), 2.0 mL (diclofenac) to 4.2 mL (water) and 3.9 mL (salicylate) to 20.9 mL (diclofenac), respectively. A comparison of these volumes to water indicated some exclusion of the drugs from the interstitial space and salicylate from the cellular space. Permeability-surface area (PS) products calculated from plasma to interstitial fluid permeation clearances (CLPI) (range 0.02-0.40 mL s(-1)) and fractions of solute unbound in the perfusate were in the order: diclofenac>salicylate >clonidine>sucrose (from 41.8 to 0.10 mL s(-1)). The slow efflux of diclofenac, compared with clonidine and salicylate, may be related to its low average unbound fraction in the cells. This work accounts for the tail of disposition curves in describing pharmacokinetics in the head.

Keratinocyte Growth factor (KGF) in hematology and oncology

Keratinocyte Growth factor (KGF) is an epithelial cell growth factor of the fibroblast growth factor family and is produced by fibroblasts and microvascular endothelium in response to proinflammatory cytokines and steroid hormones. KGF is a heparin binding growth factor that exerts effects on epithelial cells in a paracrine fashion through interaction with KGF receptors. Preclinical data has demonstrated that KGF can prevent lung and gastrointestinal toxicity following chemotherapy and radiation and preliminary clinical data in the later setting supports these findings. In the experimental allogeneic bone marrow transplant scenario KGF has shown significant ability to prevent graft-versus-host disease by maintaining gastrointestinal tract integrity and acting as a cytokine shield to prevent subsequent proinflammatory cytokine generation. Within this setting KGF has also shown an ability to prevent experimental idiopathic pneumonia syndrome by stimulating production of surfactant protein A, promoting alveolar epithelialization and attenuating immune-mediated injury. Perhaps most unexpectantly, KGF appears able to maintain thymic function during allogeneic stern cell transplantation and so promote T cell engraftment and reconstitution. These data suggest that KGF will find a therapeutic role in the prevention of epithelial toxicity following intensive chemotherapy and radiotherapy protocols and in allogeneic stem cell transplantation.

Characterisation of multiple Caribbean ciguatoxins and congeners in individual specimens of horse-eye jack (Caranx latus) by high-performance liquid chromatography/mass spectrometry

We studied the variation in toxin profiles of purified extracts of 10 individual specimens and two pools of ciguateric Caranx latus. High-performance liquid chromatography/mass spectrometry (HPLC/MS) identified in all individual samples at least seven Caribbean ciguatoxins (C-CTXs) comprising C-CTX-1 and its epimer C-CTX-2 ([M + H](+) m/z 1141.58), and five new C-CTX congeners with pseudo-molecular ions at m/z 1141.58, 1143.60, 1157.57, 1159.58, and 1127.57. In some samples, additional C-CTX isomers were detected with [M + H](+) ions at m/z 1141.58 (two), 1143.60 (one) and 1157.57 (two). The two low-toxic pools contained only four to six ciguatoxins. The comparison in relative proportions of four different mass classes ([M + H](+) at m/z 1141, 1143, 1157 and 1127) showed that the group at m/z 1157 increased (2-20%) with flesh toxicity. More than 80% of group m/z 1141 comprised C-CTX-1, C-CTX-2 and their isomer C-CTX-1 a whose level in this group correlated with fish toxicity. Contrary to low-toxic fishes, high-risk specimens had C-CTX-1 levels

Attenuation of cardiac fibrosis by pirfenidone and amiloride in DOCA-salt hypertensive rats

1 This study has administered pirfenidone (5-methyl-l-phenyl-2-[1H]-pyridone) or amiloride to attenuate the remodelling and associated functional changes, especially an increased cardiac stiffness, in DOCA-salt hypertensive rats. 2 In control rats, the elimination half-life of pirfenidone following a single intravenous dose of 200 mg kg(-1) was 37 min while oral bioavailability at this dose was 25.7%. Plasma pirfenidone concentrations in control rats averaged 1.9 +/- 0.1 mug ml(-1) over 24 It after 14 days' administration as a 0.4% mixture in food. 3 Pirfenidone (approximately 250-300 mg kg(-1) day(-1) as 0.4% in food) and amiloride (I mg kg-1 day(-1) sc) were administered for 2 weeks starting 2 weeks post-surgery. Pirfenidone but not amiloride attenuated ventricular hypertrophy (2.69 +/- 0.09, UNX 2.01 +/- 0.05. DOCA-salt 3.11 +/- 0.09 mg kg(-1) body wt) without lowering systolic blood pressure. 4 Collagen deposition was significantly increased in the interstitium after 2 weeks and further increased with scarring of the left ventricle after 4 weeks; pirfenidone and amiloride reversed the increases and prevented further increases. This accumulation of collagen was accompanied by an increase in diastolic stiffness constant; both amiloride and pirfenidone, reversed this increase. 5 Noradrenaline potency (positive chronotropy) was decreased in right atria (neg log EC50: control 6.92 +/- 0.06; DOCA-salt 6.64 +/- 0.08); pirfenidone but not amiloride reversed this change. Noradrenaline was a more potent vasoconstrictor in thoracic aortic rings (neg log EC50: control 6.91 +/- 0.10; DOCA-salt 7.90 +/- 0.07); pirfenidone treatment did not change noradrenaline potency. 6 Thus, pirfenidone and amiloride reverse and prevent cardiac remodelling and the increased cardiac stiffness without reversing the increased vascular responses to noradrenaline.

Present and future pharmacotherapy for benign prostatic hyperplasia

Around 50% of men 51-60 years of age have pathological benign prostatic hyperplasia (BPH). Pharmacotherapy for BPH includes the 5alpha-reductase inhibitor finasteride, and alpha(1)-adrenoceptor antagonists. Finasteride reduces prostate volume and symptom scores, while increasing peak urinary flow rates. The main problem with finasteride treatment is that it increases the incidence of ejaculation disorders. All of the alpha(1)-adrenoceptor antagonists have been shown to reduce symptom scores and increase peak urinary flow rates in BPH. The nonselective alpha(1)-adrenoceptor antagonists (prazosin, terazosin and doxazosin) were developed as antihypertensives, and hypotensive-related side effects are the main problem with these agents in BPH. These side effects can be diminished by reducing peak concentrations of the drugs, as with once-daily alfuzosin, or by using the uroselective antagonist tamsulosin. Phytopharmaceuticals are commonly used in the treatment of BPH, such as saw palmetto berry which has been shown to improve the symptoms and peak urinary flow rate. Androgen receptor antagonists are not used in BPH because of their adverse effects. Newer drugs under development for the treatment of BPH include alpha(1)-adrenoceptor antagonists that show more selectivity for alpha(1A)-adrenoceptors than tamsulosin, combined 5alpha-reductase/alpha(1)-adrenoceptor inhibitors and combined type 1/type 2 5alpha-reductase inhibitors. New targets for the drug treatment of BPH include indothelin, growth factors, estrogens and the phosphodiesterase isoenzymes.

Responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on aortae from normo-, pre and hypertensive rats

The objective of this study was to determine the responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on the isolated aorta in the maturation of normotensive and hypertensive rats. The effects of a very slowly reversible antagonist, bromoacetylalprenololmenthane (BAAM), on the relaxant responses of the aortae of 5- and 14-week-old Wistar Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHRs) to isoprenaline were determined. Five-week-old SHRs are pre-hypertensive and the aortic rings are less responsive to isoprenaline than age-matched WKY (pD(2) values: WKY, 8.40; SHRs, 8.03). Similar relaxant responses to forskolin were obtained on the aortae of 5- and 14-week-old WKY and SHRs. The K-A value for isoprenaline at the aortic beta(2)-adrenoceptors of the 5-week-old WKY was 2.1 x 10(-7) M, and similar values were obtained on the aortae of 5-week-old SHR and 14-week-old WKY and SHRs. In the maturation of the WKY aortae from 5 to 14 weeks, there was a reduction in the maximum response, a major loss of sensitivity and a loss of 2-adrenoceptor reserve for isoprenaline. On 5-week-old SHR aorta, the sensitivity to isoprenaline was 2.5-fold lower, and the beta(2)-adrenoceptor reserve was less than on age-matched WKY. In the development of hypertension on the SHR aorta from 5 to 14 weeks, there was a reduction in the maximum response to isoprenaline. At 14 weeks, the sensitivity and the 2-adrenoceptor reserve to isoprenaline were similar, but the maximum responses were lower on the SHR than WKY. As there are differences in pre-hypertensive SHR and age-matched WKY aortic responses to isoprenaline, it is no longer valid to consider that the loss of responsiveness to isoprenaline in hypertension is solely owing to the hypertension. There are no changes in affinity, but major changes in the sensitivity, maximum responses and aortic beta(2)-adrenoceptor reserves to isoprenaline in the maturation of normotensive and pre-hypertensive aortae.

Effects of dofetilide on cardiovascular tissues from normo- and hypertensive rats

The aim was to test whether dofetilide has some potential for use in the treatment of heart failure. Dofetilide at less than or equal to 3 x 10(-5) m had no effect on the quiescent Wistar Kyoto (WKY) rat aorta, mesenteric and intralobar arteries, or the spontaneous contractions of the WKY rat portal vein. Dofetilide at 10(-6) to 3 x 10(-5) m relaxed the KCl-contracted aorta. Dofetilide at 10(-9)-10(-7) m augmented the force of contraction of left ventricle strips from 12- and 18-month-old WKY rats at 2 Hz. Spontaneously hypertensive rats (SHRs) at 12 and 17-21 months of age are models of cardiac hypertrophy and failure, respectively. The augmentation of force at 2 Hz with dofetilide was similar on 12- and 18-month-old WKY rats and 12-month-old SHRs but reduced on the 18-month-old SHR left ventricle. At a higher more physiological frequency, 4 Hz, the threshold concentration of dofetilide required to augment the force responses of 21-month-old SHR left ventricles was markedly increased and the maximum augmenting effect was decreased. Dofetilide at 10(-7)-10(-5) m reduced the rate of the 17-month-old WKY rat right atrium, and had a similar effect on age-matched SHR right atrium. In summary, dofetilide is a positive inotrope and negative chronotrope in the rat. However, as the positive inotropic effect is not observed with clinically relevant concentrations at a physiological rate in heart failure, dofetilide is unlikely to be useful as a positive inotrope in the treatment of heart failure.

The therapeutic potential of dopamine modulators on the cardiovascular and renal systems

In the periphery, physiological dopamine increases renal blood flow, decreases renal resistance and acts on the kidney tubule to enhance natriuresis and diuresis. The loss of dopamine function may be involoved in the deterioration in kidney function associated with ageing and may have a role in the pathogenesis of hypertension and diabetes. Intravenous dopamine is used as a positive inotrope in the treatment of acute heart failure and cardiogenic shock and as a diuretic in renal failure. The clinical uses of dopamine are limited, as it must be given intravenously, and also has widespread effects. The levels of peripheral dopamine can be increased by the administration of L-dopa to increase synthesis, prodrugs to release dopamine (docarpamine, glu-dopa) or by inhibiting the breakdown of dopamine (nitecapone). Preliminary clinical trials suggest that docarpamine may be useful in patients with low cardiac output syndrome after cardiac surgery and in refractory cirrhotic ascites. Ibopamine is an agonist at dopamine D1 and D2 receptors, which may retard the progression of chronic renal failure. Gludopa is selective for the kidney thus avoiding widespread side effects. The early clinical studies with ibopamine as a diuretic in heart failure were favourable but the subsequent large mortality study showed that ibopamine increased mortality. Fenoldopam is a selective dopamine D1 receptor agonist. Intravenous fenoldopam may be useful in the treatment of hypertension associated with coronary artery bypass surgery or in hypertensive emergencies. Although our understanding of physiological and pathological roles of peripheral dopamine has been increasing rapidly in recent times, we still need more information to allow the design of clinically useful drugs that modify these roles. One priority is an orally-active selective dopamine D1 receptor agonist.

The therapeutic potential of endothelin-1 receptor antagonists and endothelin-converting enzyme inhibitors on the cardiovascular system

Clinical trials have established bosentan, an orally active non-selective endothelin (ET) receptor antagonist, as a beneficial treatment in pulmonary hypertension. Trials have also shown short-term benefits of bosentan in systemic hypertension and congestive heart failure. However, bosentan also increased plasma levels of ET-1, probably by inhibiting the clearance of ET-1 by endothelin type B (ET.) receptors, and this may mean its effectiveness is reduced with long-term clinical use. Preliminary data suggests that selective endothelin type A (ETA) receptor antagonists (BQ-123, sitaxsentan) may be more beneficial than the non-selective ET receptor antagonists in heart failure, especially when the failure is associated with pulmonary hypertension. Experimental evidence in animal disease models suggests that non-selective ET or selective ETA receptor antagonism may have a role in the treatment of athero-sclerosis, restenosis, myocarditis, shock and portal hypertension. In animal models of myocardial infarction and/or reperfusion injury, non-selective ET or selective ETA receptor antagonists have beneficial or detrimental effects depending on the conditions and agents used. Thus clinical trials of the nonselective ET or selective ETA receptor antagonists in these conditions are not presently warranted. Several selective endothelin-converting enzyme inhibitors tors have been synthesised recently, and these are only beginning to be tested in animal models of cardiovascular disease, and thus the clinical potential of these inhibitors is still to be defined.

Functional beta(1)- and beta(2)-adrenoceptors in the left and right atrium of pre-hypertensive rats

There is a small increase in the functional beta(2)-adrenoceptor response on the spontaneously hypertensive rat (SHR) left atrium in the early stages of hypertension. In the present study, the functional beta(1)- and beta(2)-adrenoceptors of the left and right atrium in SHR pre-hypertension and age-matched (5-week-old) Wistar Kyoto (WKY) rats were characterized. Contractility methods with isoprenaline, T-0509 (a selective beta(1)-adrenoceptor agonist) and procaterol (a selective beta(2)-adrenoceptor agonist) were used. At 5 weeks, the SHRs were pre-hypertensive. Isoprenaline was more potent on the left atrium of 5-week-old SHRs than WKY rats. Bisoprolol, a selective beta(1)-adrenoceptor antagonist, was more potent against isoprenaline and T-0509 on the SHR than WKY rat left atrium. ICI 118,551, a selective beta(2)-adrenoceptor antagonist, was more potent against procaterol and T-0509 on the SHR than WKY rat left atrium. The results with bisoprolol and ICI 118,551 suggest that there are more functional beta(1)- and beta(2)-adrenoceptors on the left atrium of 5-week-old SHRs than WKY rats. Isoprenaline, T-0509 and procaterol were equipotent on the right atrium of 5-week-old WKY rats and SHRs. Bisoprolol was more potent against isoprenaline, T-0509 and procaterol on the SHR than WKY rat right atrium. ICI 118,551 was more potent against T-0509, but not isoprenaline and procaterol, on the SHR than WKY rat left atrium. This suggests there are more functional beta(1)-adrenoceptors, and probably more functional beta(2)-adrenoceptors, on the right atrium of 5-week-old SHRs than WKY rats. These functional differences in beta(1)-and beta(2)-adrenoceptor-mediated responses of the left and right atria of pre-hypertensive SHRs cannot be caused by hypertension, and may be associated with the onset of hypertension.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.