Comparison of two population pharmacokinetic programs, NONMEM and P-PHARM, for tacrolimus

Objectives: To compare the population modelling programs NONMEM and P-PHARM during investigation of the pharmacokinetics of tacrolimus in paediatric liver-transplant recipients. Methods: Population pharmacokinetic analysis was performed using NONMEM and P-PHARM on retrospective data from 35 paediatric liver-transplant patients receiving tacrolimus therapy. The same data were presented to both programs. Maximum likelihood estimates were sought for apparent clearance (CL/F) and apparent volume of distribution (V/F). Covariates screened for influence on these parameters were weight, age, gender, post-operative day, days of tacrolimus therapy, transplant type, biliary reconstructive procedure, liver function tests, creatinine clearance, haematocrit, corticosteroid dose, and potential interacting drugs. Results: A satisfactory model was developed in both programs with a single categorical covariate - transplant type - providing stable parameter estimates and small, normally distributed (weighted) residuals. In NONMEM, the continuous covariates - age and liver function tests - improved modelling further. Mean parameter estimates were CL/F (whole liver) = 16.3 1/h, CL/F (cut-down liver) = 8.5 1/h and V/F = 565 1 in NONMEM, and CL/F = 8.3 1/h and V/F = 155 1 in P-PHARM. Individual Bayesian parameter estimates were CL/F (whole liver) = 17.9 +/- 8.8 1/h, CL/F (cutdown liver) = 11.6 +/- 18.8 1/h and V/F = 712 792 1 in NONMEM, and CL/F (whole liver) = 12.8 +/- 3.5 1/h, CL/F (cut-down liver) = 8.2 +/- 3.4 1/h and V/F = 221 1641 in P-PHARM. Marked interindividual kinetic variability (38-108%) and residual random error (approximately 3 ng/ml) were observed. P-PHARM was more user friendly and readily provided informative graphical presentation of results. NONMEM allowed a wider choice of errors for statistical modelling and coped better with complex covariate data sets. Conclusion: Results from parametric modelling programs can vary due to different algorithms employed to estimate parameters, alternative methods of covariate analysis and variations and limitations in the software itself.

Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2 mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2 mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24 h, The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity, Conversely, pro-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action. (C) 2002 Elsevier Science Ltd. All rights reserved.

Antagonism by NKP608 of substance P-induced plasma protein exudation in a novel preparation of perfused trachea in rats and guinea-pigs in vivo

Objective: To examine whether NKP608, a novel 1-benzoyl-2-benzyl-4-aminopiperidine NK1 receptor antagonist, inhibits substance P (SP)-induced airway plasma protein exudation in vivo. Material: Anaesthetised English shorthair guinea-pigs and Wistar rats. Treatment: Tachykinin peptides were applied topically onto the trachea and antagonists administered intravenously. Methods: Tracheal segments isolated in situ were perfused with saline and plasma-derived protein assayed in the perfusate. Results: SP (1 muM) caused plasma protein exudation, which was abolished by an NK1 antagonist (RP 67580, 1.75 mumol/kg) but unaffected by an NK2 antagonist (SR 48968, 1.75 mumol/kg) indicating the response is NK1-receptor-mediated. This was confirmed with a response to an NK1 agonist ([Sar(9), Met(O-2)(11)]-SP, 1 muM) but none to an NK2 agonist ([betaAla(8)]-neurokinin A(4-10), 1 muM). NKP608 inhibited SP responses with estimated ID50 values (mumol/kg) of 0.0044 (guinea-pigs) and 0.19 (rats). Conclusions: NKP608 is an antagonist in vivo of NK1 receptor-induced tracheal plasma protein exudation and is more potent in guinea-pigs than rats.

Testing the applicability of molecular genetic markers to population analyses of scleratinian corals

The abundance of coral reefs worldwide is in decline, and despite the ecological importance of reefs, only a limited number of DNA markers have been identified for scleractinian coral genetic studies. This paper addresses the search for new coral molecular markers and investigates the applicability of the cytochrome c oxidase subunit I (COI), the internal transcribed spacer region 1 (ITS1), and the pocilloporin gene to the question of intraspecific variation in the scleractinian coral Pocillopora verrucosa along the southeast African coastline. The COI fragment was 710 bp long and was identical for P. verrucosa (n = 10) and P. damicornis (n = 3). Only two different ITS1 sequences were found (differing by 13 bp insertion), but more importantly, 24% of the sequences were heterogenous indicating that different multiple copies of the sequence exist. Pocilloporin is an intronless gene that was absolutely conserved throughout all P. verrucosa populations (n = 50). Thus, the three DNA regions studied appear unsuitable for the population genetic analyses of P. verrucosa.

Binder treatment and lubricant system for aluminium P/M

Organic binders are used in premixes for powder metallurgy applications to prevent dusting and segregation. This is a particular problem for aluminium powder metallurgy because the dust is a potential safety hazard. The binder must also burn out completely at low temperatures in an inert environment and not react with the metal powders. It is demonstrated that cellulose acetate, polyvinyl acetate and polyvinyl alcohol are effective dedusting agents but they react with the metal powders during sintering and decrease the sintered density. Paraffin wan is an effect dedusting agent that provided die wall lubricity, does not interfere with sintering and increases tensile strength and ductility.

Epidemiology and strain characteristics of Echinococcus granulosus in the Benghazi area of eastern Libya

The incidence of surgically confirmed cystic echinococcosis in eastern Libya was estimated to be at least 4.2 cases/100,000, with significantly more female cases than male. The prevalences of infection with Echinococcus granulosus among 1087 sheep, 881 goats, 428 camels and 614 cattle from the same region, determined postmortem in abattoirs, were 20%, 3.4%, 13.6% and 11%, respectively. Infection in the livestock was age-dependent and, generally, the female animals were more often infected than the male. The measurements of rostellar hooks on protoscoleces collected from sheep and cattle were similar but significantly different from the corresponding measurements of parasites of human or camel origin. However, when a portion of the cytochrome c-oxidase subunit I (coxl) gene from each of 30 protoscolex samples from Libya (12 from cattle, three from humans, five from camels and 10 from sheep) was sequenced, the sequences were all found to be identical to that published for the common sheep strain of E. granulosus.

Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6

Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K-l of 0.9 muM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity.

Bioactivation of tamoxifen by recombinant human cytochrome P450 enzymes

Tamoxifen is a major drug used for adjuvant chemotherapy of breast cancer; however, its use has been associated with a small but significant increase in risk of endometrial cancer. In rats, tamoxifen is a hepatocarcinogen, and DNA adducts have been observed in both rat and human tissues. Tamoxifen has been shown previously to be metabolized to reactive products that have the potential to form protein and DNA adducts. Previous studies have suggested a role for P450 3A4 in protein adduct formation in human liver microsomes, via a catechol intermediate; however, no clear correlation was seen between P450 3A4 content of human liver microsomes and adduct formation. In the present study, we investigated the P450 forms responsible for covalent drug-protein adduct formation and the possibility that covalent adduct formation might occur via alternative pathways to catechol formation. Recombinant P450 3A4 catalyzed adduct formation, and this correlated with the level of uncoupling in the P450 incubation, consistent with a role of reactive oxygen species in potentiating adduct formation after enzymatic formation of the catechol metabolite. Whereas P450s 1AI, 2D6, and 3A5 generated catechol metabolite, no covalent adduct formation was observed with these forms. By contrast, P450 2136, 2C19, and rat liver microsomes catalyzed drug-protein adduct formation but not catechol formation. Drug protein adducts formed specifically with P450 3A4 in incubations using membranes isolated from bacteria expressing P450 3A4 and reductase, as well as in reconstitutions of purified 3A4, suggesting that the electrophilic species reacted preferentially with the P450 enzymes concerned.

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.

Use of heterologously-expressed cytochomre P450 and glutathione transferase enzymes in toxicity assays

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1131, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

A new species of Phyllurus (Lacertilia : Gekkonidae) and a revised phylogeny and key for the Australian leaf-tailed geckos

Phyllurus gulbaru, sp. nov., is a highly distinct species of leaf-tailed gecko restricted to rocky rainforest of Pattersons Gorge, north-west of Townsville. The possession of a cylindrical, non-depressed, tapering original and regenerated tail separates P. gulbaru from all congeners except P. caudiannulatus. From this species P. gulbaru is separated by having a partially divided, as opposed to fully divided, rostral scale. Furthermore, the very small spinose body tubercles of P. gulbaru are in marked contrast to the large spinose body scales of P. caudiannulatus. An analysis of 729 bp of mitochondrial 12S rRNA and cytochrome b genes reveals P. gulbaru to be a deeply divergent lineage with closer affinities to mid-east Queensland congeners than the geographically neighbouring P. amnicola on Mt Elliot. In conservation terms, P. gulbaru is clearly at risk. Field surveys of Pattersons Gorge and the adjacent ranges indicate that this species is restricted to a very small area of highly fragmented habitat, of which only a small proportion receives a degree of protection in State forest. Further, there is ongoing, unchecked destruction of dry rainforest habitat by fire. Under current IUCN criteria, P. gulbaru warrants an Endangered ( B1, 2) listing.

Thermodynamic non-ideality as an alternative source of the effect of sucrose on the thrombin-catalyzed hydrolysis of peptide p-nitroanalide substrates

The inhibitory effect of sucrose on the kinetics of thrombin-catalyzed hydrolysis of the chromogenic substrate S-2238 (D-phenylalanyl-pipecolyl-arginoyl-p-nitroanilide) is re-examined as a possible consequence of thermodynamic non-ideality-an inhibition originally attributed to the increased viscosity of reaction mixtures. However, those published results may also be rationalized in terms of the suppression of a substrate-induced isomerization of thrombin to a slightly more expanded (or more asymmetric) transition state prior to the irreversible kinetic steps that lead to substrate hydrolysis. This reinterpretation of the kinetic results solely in terms of molecular crowding does not signify the lack of an effect of viscosity on any reaction step(s) subject to diffusion control. Instead, it highlights the need for development of analytical procedures that can accommodate the concomitant operation of thermodynamic non-ideality and viscosity effects.

Numerical study of hydrogenic effective mass theory for an impurity P donor in Si in the presence of an electric field and interfaces

In this paper we examine the effects of varying several experimental parameters in the Kane quantum computer architecture: A-gate voltage, the qubit depth below the silicon oxide barrier, and the back gate depth to explore how these variables affect the electron density of the donor electron. In particular, we calculate the resonance frequency of the donor nuclei as a function of these parameters. To do this we calculated the donor electron wave function variationally using an effective-mass Hamiltonian approach, using a basis of deformed hydrogenic orbitals. This approach was then extended to include the electric-field Hamiltonian and the silicon host geometry. We found that the phosphorous donor electron wave function was very sensitive to all the experimental variables studied in our work, and thus to optimize the operation of these devices it is necessary to control all parameters varied in this paper.

Description of Paeon asymboli n. sp. (Copepoda : Sphyriidae), parasitic on Asymbolus spp. (catsharks) and a new host record for P-australis Kabata, 1993.

Paeon asymboli n. sp. ( Copepoda: Sphyriidae) is described and illustrated from two female specimens taken from the gills of a grey spotted catshark Asymbolus analis ( Ogilby) and an orange spotted catshark A. rubiginosus ( Last, Gomon & Gledhill) ( Scyliorhinidae) from off southeastern Queensland, Australia. The key features for identification are: a pear- shaped trunk, longer than it is wide, along with a cephalothorax characterised by two large ventral papillae, projecting laterally and supporting a number of secondary lobes; a single mid- line, subconical papilla located antero- dorsal to the ventral papillae; an anterior surface bearing two prominent stalked papillae; and an absence of posterolateral lobular processes. P. australis Kabata, 1993 is recorded for the first time from the eastern shovelnose ray Aptychotrema rostrata ( Shaw & Nodder) ( Rhinobatidae).