Charlene Willis

Population pharmacokinetics of tacrolimus in adult kidney transplant recipients

The aims of this study were to investigate the population pharmacokinetics of tacrolimus in adult kidney transplant recipients and to identify factors that explain variability.

A retrospective analysis of mycophenolic acid and cyclosporin concentrations with acute rejection in renal transplant recipients.

OBJECTIVES Although monitoring of cyclosporin (CsA) is standard clinical practice postrenal transplantation, mycophenolic acid (MPA) concentrations are not routinely measured. There is evidence that a relationship exists between MPA area under the concentration-time curve (AUC) and rejection. In this study, a retrospective analysis was undertaken of 27 adult renal transplant recipients. METHODS Patients received CsA and MPA therapy and had a four-point MPA AUC investigation. The relationship between MPA AUC performed in the first week after transplantation, as well as median trough cyclosporin concentrations, and clinical outcomes in the first month posttransplant were evaluated. RESULTS A total of 12 patients experienced biopsy proven rejection (44.4%) and 4 patients had gastrointestinal adverse events (14.8%). A statistically significant relationship was observed between the incidence of biopsy proven rejection and both MPA AUC (p = 0.02) and median trough CsA concentration (p = 0.008). No relationship between trough MPA concentration and rejection was observed (p = 0.21). Only 3 of 11 (27%) patients with an MPA AUC > 30 mg x h/L and a median trough CsA > 175 microg/L experienced acute rejection, compared with a 56% incidence of rejection for the remaining 16 patients. Patients who experienced adverse gastrointestinal events had significantly lower MPA AUC (p = 0.04), but median trough CsA concentrations were not significantly different (p = 0.24). Further, 3 of these 4 patients had rejection episodes. CONCLUSIONS In addition to standard CsA monitoring, we propose further investigation of the use of a 4-point sampling strategy to predict MPA AUC in the first week posttransplant, which may facilitate optimization of mycophenolate mofetil dose at a time when patients are most vulnerable to acute rejection.

Evaluation of Limited Sampling Strategies for Estimation of 12-hour Mycophenolic Acid Area Under the Plasma Concentration–time Curve in Adult Renal Transplant Patients

Mycophenolate mofetil, the oral prodrug of mycophenolic acid, is indicated as immunosuppressive therapy after renal transplantation. To aid in the investigation of pharmacokinetic–pharmacodynamic relationships of mycophenolic acid in the clinical setting, limited blood sampling strategies have been proposed, and models from these developed, for the estimation of mycophenolic acid area under the concentration–time curve (AUC). In the current study, the authors investigated the predictive performance of six published models to estimate AUC. A total of 49 profiles from 25 renal transplant patients were used to test each models performance against a full 14 time-point AUC. A wide range of agreement was found when predicted AUCs were compared with full AUCs using linear regression analysis (range:r2 = 0.499 to 0.836). Model 1, which uses 4 time-points over 6 hours, was found to be superior to all other models. The range of time-points used in this model takes into account patients with variable absorption. This model should be further tested on data sets from other centers. The relatively poor performance of the other models may be caused by their inability to describe the peak concentration in these patients. Caution is warranted when using limited sampling strategies on patients whose absorption of mycophenolic acid is altered, compared with those of the pharmacokinetic profiles from which the model was developed.

A Tractable Experimental Model for Study of Human and Animal Scabies

Background Scabies is a parasitic skin infestation caused by the burrowing mite Sarcoptes scabiei. It is common worldwide and spreads rapidly under crowded conditions, such as those found in socially disadvantaged communities of Indigenous populations and in developing countries. Pruritic scabies lesions facilitate opportunistic bacterial infections, particularly Group A streptococci. Streptococcal infections cause significant sequelae and the increased community streptococcal burden has led to extreme levels of acute rheumatic fever and rheumatic heart disease in Australias Indigenous communities. In addition, emerging resistance to currently available therapeutics emphasizes the need to identify potential targets for novel chemotherapeutic and/or immunological intervention. Scabies research has been severely limited by the availability of parasites, and scabies remains a truly neglected infectious disease. We report development of a tractable model for scabies in the pig, Sus domestica. Methodology/Principal Findings Over five years and involving ten independent cohorts, we have developed a protocol for continuous passage of Sarcoptes scabiei var. suis. To increase intensity and duration of infestation without generating animal welfare issues we have optimised an immunosuppression regimen utilising daily oral treatment with 0.2mg/kg dexamethasone. Only mild, controlled side effects are observed, and mange infection can be maintained indefinitely providing large mite numbers (>6000 mites/g skin) for molecular-based research on scabies. In pilot experiments we explore whether any adaptation of the mite population is reflected in genetic changes. Phylogenetic analysis was performed comparing sets of genetic data obtained from pig mites collected from naturally infected pigs with data from pig mites collected from the most recent cohort. Conclusions/Significance A reliable pig/scabies animal model will facilitate in vivo studies on host immune responses to scabies including the relations to the associated bacterial pathogenesis and more detailed studies of molecular evolution and host adaption. It is a most needed tool for the further investigation of this important and widespread parasitic disease.

Novel scabies mite serpins inhibit the three pathways of the human complement system.

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.

Quantification of free mycophenolic acid by high-performance liquid chromatography–atmospheric pressure chemical ionisation tandem mass spectrometry

To facilitate the investigation of free mycophenolic acid concentrations we developed a high-performance liquid chromatography tandem mass spectrometry method using indomethacin as an internal standard. Free drug was isolated from plasma samples (500 microl) using ultrafiltration. The analytes were extracted from the ultrafiltrate (200 microl) using C18 solid-phase extraction. Detection was by selected reactant monitoring of mycophenolic acid (m/z 318.9-->190.9) and the internal standard (m/z 356.0-->297.1) with an atmospheric pressure chemical ionisation interface. The total chromatographic analysis time was 12 min. The method was found to be linear over the range investigated, 2.5-200 microg/l (r>0.990, n=6). The relative recovery of the method for the control samples studied (7.5, 40.0 and 150 microg/l) ranged from 95 to 104%. The imprecision of the method, expressed in terms of intra- and inter-day coefficients of variation, was <8 and <9%, respectively. Further, analysis of pooled patient plasma produced an intra-day imprecision of 6.6%. The signal-to-noise ratio at the limit of quantification (2.5 microg/l) was approximately 5:1. The mean absolute recovery (n=6) of mycophenolic acid and the internal standard were 76.0+/-13.5% and 86.0+/-9.1%, respectively. The method reported provides an accurate and precise quantification of free mycophenolic acid over a wide analytical range and thus can be used for routine monitoring and pharmacokinetic studies.

Characterization of a Serine Protease Homologous to House Dust Mite Group 3 Allergens from the Scabies Mite Sarcoptes scabiei

The scabies mite, Sarcoptes scabiei var. hominis, infests human skin, causing allergic reactions and facilitating bacterial infection by Streptococcus sp., with serious consequences such as rheumatic fever and rheumatic heart disease. To identify a possible drug target or vaccine candidate protein, we searched for homologues of the group 3 allergen of house dust mites, which we subsequently identified in a cDNA library. The native protein, designated Sar s 3, was shown to be present in the mite gut and excreted in fecal pellets into mite burrows within the upper epidermis. The substrate specificity of proteolytically active recombinant rSar s 3 was elucidated by screening a bacteriophage library. A preference for substrates containing a RS(G/A) sequence at the P1-P2′ positions was revealed. A series of peptides synthesized as internally quenched fluorescent substrates validated the phage display data and high performance liquid chromatography/mass spectrometry analysis of the preferred cleaved substrate and confirmed the predicted cleavage site. Searches of the human proteome using sequence data from the phage display allowed the in silico prediction of putative physiological substrates. Among these were numerous epidermal proteins, with filaggrin being a particularly likely candidate substrate. We showed that recombinant rSar s 3 cleaves human filaggrin in vitro and obtained immunohistological evidence that the filaggrin protein is ingested by the mite. This is the first report elucidating the substrate specificity of Sar s 3 and its potential role in scabies mite biology.

Bayesian forecasting and prediction of tacrolimus concentrations in pediatric liver and adult renal transplant recipients.

Aim To test the predictive capacity of two recently derived population pharmacokinetic models and the usefulness of Bayesian forecasting to predict tacrolimus blood concentrations in pediatric liver and adult kidney transplant recipients. Materials and Methods New databases were added to the Abbottbase PKS (Bayesian dosage prediction) program to incorporate the population pharmacokinetic models developed for tacrolimus. Two independent populations of transplant recipients were used to predict tacrolimus trough concentrations. Pharmacokinetic, demographic, and covariate data were collected from patient records. Different time weighting factors were tested (1, 1.005, 1.01) and the influence of excluding data collected in the first 5 days post-transplant examined. Concentrations were predicted until the 10th tacrolimus measurement. Actual tacrolimus concentrations were compared with those predicted by the PKS program and bias and precision determined. Results Tacrolimus concentrations predicted by the PKS program were, on average, unbiased for the pediatric liver population, but were over-predicted (9%) for the adult renal population. In both populations predictions were not precise (imprecision ranged from 39 to 50%). Conclusions Due to the imprecision seen in this study, these models could not be used in clinical practice in the immediate post-transplant period. Poor precision may be due to reliance on routine drug monitoring data alone, difficulties with expression of covariates in continuous modeling relationships in the PKS program, lack of accurate quantitative measures of liver function, or large, random intraindividual variability in the bioavailability of tacrolimus.

Quantitative PCR-based genome size estimation of the astigmatid mites Sarcoptes scabiei, Psoroptes ovis and Dermatophagoides pteronyssinus

BackgroundThe lack of genomic data available for mites limits our understanding of their biology. Evolving high-throughput sequencing technologies promise to deliver rapid advances in this area, however, estimates of genome size are initially required to ensure sufficient coverage.MethodsQuantitative real-time PCR was used to estimate the genome sizes of the burrowing ectoparasitic mite Sarcoptes scabiei, the non-burrowing ectoparasitic mite Psoroptes ovis, and the free-living house dust mite Dermatophagoides pteronyssinus. Additionally, the chromosome number of S. scabiei was determined by chromosomal spreads of embryonic cells derived from single eggs.ResultsS. scabiei cells were shown to contain 17 or 18 small (< 2 μM) chromosomes, suggesting an XO sex-determination mechanism. The average estimated genome sizes of S. scabiei and P. ovis were 96 (± 7) Mb and 86 (± 2) Mb respectively, among the smallest arthropod genomes reported to date. The D. pteronyssinus genome was estimated to be larger than its parasitic counterparts, at 151 Mb in female mites and 218 Mb in male mites.ConclusionsThis data provides a starting point for understanding the genetic organisation and evolution of these astigmatid mites, informing future sequencing projects. A comparitive genomic approach including these three closely related mites is likely to reveal key insights on mite biology, parasitic adaptations and immune evasion.

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2

Hookworm activation-associated secreted proteins can be structurally classified into at least three different groups. The hallmark feature of Group 1 activation-associated secreted proteins is a prominent equatorial groove, which is inferred to form a ligand binding site. Furthermore, a conserved tandem histidine motif is located in the centre of the groove and believed to provide or support a yet to be determined catalytic activity. Here, we report three-dimensional crystal structures of Na-ASP-2, an L3-secreted activation-associated secreted protein from the human hookworm Necator americanus, which demonstrate transition metal binding ability of the conserved tandem histidine motif. We further identified moderate phosphohydrolase activity of recombinant Na-ASP-2, which relates to the tandem histidine motif. By panning a random 12-mer peptide phage library, we identified a peptide with high similarity to the human calcium-activated potassium channel SK3, and confirm binding of the synthetic peptide to recombinant Na-ASP-2 by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex.