FORMULATION OF POPULATION PHARMACOKINETIC MODELS OF ANTI-CANCER AGENTS

The primary objective of the study is to assemble population pharmacokinetic models from the cancer pharmacokinetics literature for different types of anti-cancer drugs and to formulate them in ways suitable for input into cancer simulation programs. To fulfill the objectives, a step-based approach is adopted: 1)To catalogue the types of pharmacokinetic models through general review articles and books 2)To develop a search strategy for defining a body of research literature related to cancer pharmacokinetics in clinical trials for a limited set of drugs (Taxol, Platinum compounds. Fluoropyrimidine and Topoisomerase inhibitors) 3)To collect pharmacokinetic articles according to defined search criteria4)To gather information from the collected PK articles 5)To synthesize the information separately for each drug, using a questionnaire instrument and present them in template form for each class of antineoplastic agent.6)To formulate population pharmacokinetic models for each anti-cancer drug, from the constituent submodels for components of the overall model. This work will promote public health, specifically in support of the development of anti-cancer drug regimens for cancer patients, byproviding standardized information about pharmacokinetics for input into simulations

Randomized Clinical Trial of High-Dose Rifampicin With or Without Levofloxacin Versus Standard of Care for Pediatric Tuberculous Meningitis: The TBM-KIDS Trial

Background. Pediatric tuberculous meningitis (TBM) commonly causes death or disability. In adults, high-dose rifampicin may reduce mortality. The role of fluoroquinolones remains unclear. There have been no antimicrobial treatment trials for pediatric TBM. Methods. TBM-KIDS was a phase 2 open-label randomized trial among children with TBM in India and Malawi. Participants received isoniazid and pyrazinamide plus: (i) high-dose rifampicin (30 mg/kg) and ethambutol (R30HZE, arm 1); (ii) high-dose rifampicin and levofloxacin (R30HZL, arm 2); or (iii) standard-dose rifampicin and ethambutol (R15HZE, arm 3) for 8 weeks, followed by 10 months of standard treatment. Functional and neurocognitive outcomes were measured longitudinally using Modified Rankin Scale (MRS) and Mullen Scales of Early Learning (MSEL). Results. Of 2487 children prescreened, 79 were screened and 37 enrolled. Median age was 72 months; 49%, 43%, and 8% had stage I, II, and III disease, respectively. Grade 3 or higher adverse events occurred in 58%, 55%, and 36% of children in arms 1, 2, and 3, with 1 death (arm 1) and 6 early treatment discontinuations (4 in arm 1, 1 each in arms 2 and 3). By week 8, all children recovered to MRS score of 0 or 1. Average MSEL scores were significantly better in arm 1 than arm 3 in fine motor, receptive language, and expressive language domains (P < .01). Conclusions. In a pediatric TBM trial, functional outcomes were excellent overall. The trend toward higher frequency of adverse events but better neurocognitive outcomes in children receiving high-dose rifampicin requires confirmation in a larger trial. Clinical Trials Registration. NCT02958709

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Influence of protein-micelle ratios and cysteine residues on the kinetic stability and unfolding rates of human mitochondrial VDAC-2.

Delineating the kinetic and thermodynamic factors which contribute to the stability of transmembrane β-barrels is critical to gain an in-depth understanding of membrane protein behavior. Human mitochondrial voltage-dependent anion channel isoform 2 (hVDAC-2), one of the key anti-apoptotic eukaryotic β-barrel proteins, is of paramount importance, owing to its indispensable role in cell survival. We demonstrate here that the stability of hVDAC-2 bears a strong kinetic contribution that is dependent on the absolute micellar concentration used for barrel folding. The refolding efficiency and ensuing stability is sensitive to the lipid-to-protein (LPR) ratio, and displays a non-linear relationship, with both low and high micellar amounts being detrimental to hVDAC-2 structure. Unfolding and aggregation process are sequential events and show strong temperature dependence. We demonstrate that an optimal lipid-to-protein ratio of 2600∶1 - 13,000∶1 offers the highest protection against thermal denaturation. Activation energies derived only for lower LPRs are ∼17 kcal mol(-1) for full-length hVDAC-2 and ∼23 kcal mol(-1) for the Cys-less mutant, suggesting that the nine cysteine residues of hVDAC-2 impart additional malleability to the barrel scaffold. Our studies reveal that cysteine residues play a key role in the kinetic stability of the protein, determine barrel rigidity and thereby give rise to strong micellar association of hVDAC-2. Non-linearity of the Arrhenius plot at high LPRs coupled with observation of protein aggregation upon thermal denaturation indicates that contributions from both kinetic and thermodynamic components stabilize the 19-stranded β-barrel. Lipid-protein interaction and the linked kinetic contribution to free energy of the folded protein are together expected to play a key role in hVDAC-2 recycling and the functional switch at the onset of apoptosis

Cysteine Residues Impact the Stability and Micelle Interaction Dynamics of the Human Mitochondrial β-Barrel Anion Channel hVDAC-2

<div><p>The anti-apoptotic 19-stranded transmembrane human voltage dependent anion channel isoform 2 (hVDAC-2) β-barrel stability is crucial for anion transport in mitochondria. The role of the unusually high number of cysteine residues in this isoform is poorly understood. Using a Cys-less construct of hVDAC-2, we haveinvestigated the contribution of cysteines to channel function, barrel stability and its influence on the strength of protein-micelle interactions. We observe that despite the overall preservation in barrel structure upon cysteine mutation, subtle local variations in the mode of interaction of the barrel with its refolded micellar environment arise, which may manifest itself in the channel activity of both the proteins.Fluorescence measurements of the Trp residues in hVDAC-2 point to possible differences in the association of the barrel with lauryldimethylamine oxide (LDAO) micelles. Upon replacement of cysteines in hVDAC-2, our data suggests greater barrel rigidity by way of intra-protein interactions. This, in turn, lowers the equilibrium barrel thermodynamic parameters in LDAOby perturbingthe stability of the protein-micelle complex. In addition to this, we also find a difference in the cooperativity of unfolding upon increasing the LDAO concentration, implying the importance of micelle concentration and micelle-protein ratios on the stability of this barrel. Our results indicate that the nine cysteine residues of hVDAC-2 are the key in establishing strong(er) barrel interactions with its environment and also impart additional malleability to the barrel scaffold.</p></div

Thermal denaturation (T-scans) of refolded hVDAC-2 in various LDAO concentrations.

<p>Representative profiles of refolded hVDAC-2 WT (left) and C0 (right) in 5 mM (<b>○</b>, black), 13 mM (<b>□</b>, red), 30 mM (<b>⋄</b>, green), 65 mM (<b>▵</b>, blue), 80 mM (<b>▿</b>, dark pink) and 100 mM (<b>▹</b>, dark yellow) are shown here as unfolded fractions obtained at the various temperatures. The solid lines represent fits to a two-state equation. Note that hVDAC-2 C0 (right) shows substantial increase (∼30%) in the unfolded fraction by ∼80°C in 80 mM and 100 mM LDAO, even before commencement of the unfolding transition. Insets show the fits for samples directly refolded in 5 mM LDAO, to achieve an LPR of 2600∶1 (purple) or 1000∶1 (brown), and are compared with the 5 mM LDAO sample prepared by dilution (black) of the refolding stock to also achieve an LPR of 2600∶1 (see text for details). Actual data points have been omitted for clarity. The directly refolded samples, despite exhibiting a similar <i>T_m</i>, show a distinct loss in unfolding cooperativity, when compared with samples prepared by dilution.</p

Comparison of the rate of unfolding of hVDAC-2 WT and C0.

<p>Rate of unfolding of refolded hVDAC-2 WT and C0 as a function of LDAO concentrations (A) and temperature (B) derived from isothermal kinetics experiments. Panel (A) highlights the decrease in unfolding rate with increasing LDAO concentration. Panel (B) highlights the increase in the unfolding rate with increase in temperature. Values for the solid symbols have been derived from the <i>k</i>_u1 of the double exponential fits whereas the hollow symbols are for the single exponential fits. Figure legends in panel (B) are distributed in the left and right graphs. Error bars denote the goodness of the fit obtained by fitting the mean data of three independent experiments.</p

Representative far-UV circular dichroism profiles of refolded hVDAC-2 WT and C0 with increasing LDAOconcentrations.

<p>Both WT (left) and C0 (right) display CD spectra corresponding to an extended conformation, with a negative maximum at ∼215 nm. Both proteins exhibit comparable secondary structure content, despite a 20-fold change in the LDAO concentration from 5 mM to 100 mM. The inset shows the CD spectra of control samples prepared by ‘direct’ refolding in LPRs of 2600∶1 (5 mM D2) and 1000∶1 (5 mM D5), and are compared with ‘refolded’ protein in 5 mM LDAO, also having an LPR of 2600:1. Molar ellipticity (ME) values are lower for the control samples, suggesting the importance of absolute LDAO concentrations to mediate optimal refolding.</p

Cartoon representations of hVDAC-2 WT (left) and C0 (right).

<p>The structures were modeled using I-TASSER <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087701#pone.0087701-Roy1" target="_blank">[55]</a> using the crystal (2JK4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087701#pone.0087701-Hiller1" target="_blank">[29]</a>) and NMR (2K4T <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087701#pone.0087701-Bayrhuber1" target="_blank">[56]</a>) structure of hVDAC-1 as the template. The cysteine residues have been highlighted as yellow spheres for the WT protein and they are largely oriented towards the intermembrane space in the 19-stranded model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087701#pone.0087701-Maurya1" target="_blank">[22]</a>. The corresponding mutated residues in C0 have been represented as spheres and colored according to the chemical characteristics of the amino acid.</p