Pharmacokinetics of Quinacrine Efflux from Mouse Brain via the P-glycoprotein Efflux Transporter

The lipophilic cationic compound quinacrine has been used as an antimalarial drug for over 75 years but its pharmacokinetic profile is limited. Here, we report on the pharmacokinetic properties of quinacrine in mice. Following an oral dose of 40 mg/kg/day for 30 days, quinacrine concentration in the brain of wild-type mice was maintained at a concentration of ∼1 µM. As a substrate of the P-glycoprotein (P-gp) efflux transporter, quinacrine is actively exported from the brain, preventing its accumulation to levels that may show efficacy in some disease models. In the brains of P-gp–deficient Mdr10/0 mice, we found quinacrine reached concentrations of ∼80 µM without any signs of acute toxicity. Additionally, we examined the distribution and metabolism of quinacrine in the wild-type and Mdr10/0 brains. In wild-type mice, the co-administration of cyclosporin A, a known P-gp inhibitor, resulted in a 6-fold increase in the accumulation of quinacrine in the brain. Our findings argue that the inhibition of the P-gp efflux transporter should improve the poor pharmacokinetic properties of quinacrine in the CNS

Comparative costs and activity from a sample of UK clinical trials units

Background: The costs of medical research are a concern. Clinical Trials Units (CTUs) need to better understand variations in the costs of their activities. Methods: Representatives of ten CTUs and two grant-awarding bodies pooled their experiences in discussions over 1.5 years. Five of the CTUs provided estimates of, and written justification for, costs associated with CTU activities required to implement an identical protocol. The protocol described a 5.5-year, nonpharmacological randomized controlled trial (RCT) conducted at 20 centres. Direct and indirect costs, the number of full time equivalents (FTEs) and the FTEs attracting overheads were compared and qualitative methods (unstructured interviews and thematic analysis) were used to interpret the results. Four members of the group (funding-body representatives or award panel members) reviewed the justification statements for transparency and information content. Separately, 163 activities common to trials were assigned to roles used by nine CTUs; the consistency of role delineation was assessed by Cohen's κ. Results: Median full economic cost of CTU activities was £769,637 (range: £661,112 to £1,383,323). Indirect costs varied considerably, accounting for between 15% and 59% (median 35%) of the full economic cost of the grant. Excluding one CTU, which used external statisticians, the total number of FTEs ranged from 2.0 to 3.0; total FTEs attracting overheads ranged from 0.3 to 2.0. Variation in directly incurred staff costs depended on whether CTUs: supported particular roles from core funding rather than grants; opted not to cost certain activities into the grant; assigned clerical or data management tasks to research or administrative staff; employed extensive on-site monitoring strategies (also the main source of variation in non-staff costs). Funders preferred written justifications of costs that described both FTEs and indicative tasks for funded roles, with itemised non-staff costs. Consistency in role delineation was fair (κ = 0.21-0.40) for statisticians/data managers and poor for other roles (κ < 0.20). Conclusions: Some variation in costs is due to factors outside the control of CTUs such as access to core funding and levels of indirect costs levied by host institutions. Research is needed on strategies to control costs appropriately, especially the implementation of risk-based monitoring strategies

Conformationally restricted peptidomimetics

Peptidomimetic compounds are becoming increasing important as medicinal agents. This thesis describes the design and synthesis of a novel class of non-hydrolysable peptidomimetic isosteres, the tetrazole-based amide bond isosteres. These new structural mimics have been developed for incorporation into enzyme inhibitors and biological probes where a non-hydrolysable cis- amide bond mimic is required. Chapter one provides a general outline of conformational restriction and how this concept has been advantageously applied to the design of bioactive peptidomimetics. Conformational restriction, when used to pre-organise peptidomimetic ligands into a desired bioactive conformation, gives ligands that bind to target receptors with a greater affinity. The tetrazole heterocycle has obvious structural similarities to the cis-conformation of the amide bond and has been used to lock amide bonds in an equivalent conformation. Hence, the 1,5-disubstituted tetrazole ring is a popular constrained, planar cis- amide bond mimic and its use as such is reviewed in chapter one. Chapter two outlines the design of the tetrazole-based isosteres, the α-methylene tetrazole (2.5.1), α-hydroxymethylene tetrazole (2.5.2), and α-keto tetrazole isostere (2.5.3). The tetrazole-based isosteres incorporate the design features of a non-hydrolysable amide bond isostere and the conformational restriction of the 1,5-disubstituted tetrazole ring, to generate the first examples of non-hydrolysable tetrazole-based cis- amide bond mimics. Chapter three reviews the synthesis of N-Z-(2RS,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid [N-Z-AHPBA, 3.1.1], a key amide bond isostere used in potent protease inhibitors and a synthetic building block of the α-hydroxymethylene tetrazole isostere (2.5.2). We have investigated the synthesis of 3.1.1 by functionalisation of N-Z-L- phenylalaninal, 3.2.3. Due to shortcomings in this published procedure we have developed a new, generally applicable synthesis of this important amide bond isostere by hydroxylation of an enolate derived from methyl (3S)-N-Z-3-amino-4-phenylbutanoate (3.4.2) with the oxodiperoxymolybdenum (pyridine) (hexamethyl phosphoric triamide) complex (MoOPH). Chapter four describes the synthesis and spectral analysis of diastereomeric tetrazole-based compounds. We have synthesised two series of α-methylene tetrazolebased compounds, 4.1.4a-4.1.7a and 4.1.4b-4.1.7b, derived from L-alanine and D-alanine, respectively. We have used this analysis to establish trends in the 1H and 13C NMR of the diastereomeric series as an aid to the assignment of configuration of tetrazole-based ligands. We have also been able to monitor epimerisation of the C6 stereocentre adjacent to the tetrazole by 1H NMR under the conditions of peptide coupling. Chapter five outlines the synthesis of (2S)-1-(benzyl ethanoate)-5-[2-(N-benzyloxycarbonylamino)-3-phenylpropane]-tetrazole, 5.2.1, an α-methylene tetrazolebased dipeptide mimic. We have incorporated 5.2.1 into extended substrate sequences of HIVp and tested these compounds for in vitro activity against HIVp. The modestly potent HlVp inhibitors 5.4.1, 5.4.2, and 5.4.3 represent a step-wise elongation of the C-terminal, and gave IC50 values against HIVp of 94 µM, 47 µM and 18 µM, respectively. Chapter six describes the synthesis of the α-keto tetrazole isostere, by direct alkylation of (1RS,2S)-5-[2-(N-tert-butyloxycarbonylamino)-1-hydroxyl-3-phenylpropane]-1H-tetrazole, 6.3.13, with benzyl bromoacetate, followed by a TEMPO oxidation. The desired 1,5-disubstituted dipeptide mimic, (2S)-1-(benzyl ethanoate)-5-[2-(N-tert-butyloxycarbonylamino)-1-oxo-3-phenylpropane]-tetrazole, 6.3.16, was isolated from the 2,5-disubstitiuted dipeptide mimic, (2S)-2-(benzyl ethanoate)-5-[2-(N-tertbutyloxycarbonylamino)-1-oxo-3-phenylpropane J-tetrazole, 6.3.17. This outlines a new and generally applicable synthesis of the tetrazole-based isosteres. Chapter seven describes the solid state structures of three tetrazole-based dipeptide mimics. The α-methylene tetrazole based isostere has been observed in the X-ray structure of the dipeptide mimic, (2S)-1-(benzyl ethanoate)-5-[2-(N-benzyloxycarbonylamino)-3-phenylpropane]-tetrazole and the cyclic tetrazolodiazepine analogue, cyclo-{[(2S)-5-(2-amino-3-phenylpropane)-1-ethanamide] tetrazole}, These are the first examples of the solid state structure of the α-methylene tetrazole isostere. We have also observed the solid state structure of the 2,5-disubstituted tetrazole, (2S)-2-(Benzyl ethanoate )-5-[2-(N-tertbutyloxycarbonylamino)-1-oxo-3-phenylpropane]-tetrazole, 6.3.17

Developing therapeutics for the diseases of protein misfolding.

Our current structural and biologic understanding of the misfolding diseases has restricted the development of therapies that target these diseases at a molecular level. The prion diseases are illustrative of this group of misfolding disorders and provide a model system for therapeutic intervention. Strategies to inhibit the replication and accumulation of the prion protein are being developed and have entered animal and clinical studies. Due to the underlying molecular basis of this disease class, many of the therapeutic approaches used to target prion misfolding have parallels in other misfolding diseases.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tReviewinfo:eu-repo/semantics/publishe

Site-directed mutagenesis demonstrates the plasticity of the beta helix: implications for the structure of the misfolded prion protein.

The left-handed parallel beta helix (LbetaH) fold has recently received attention as a possible structure for the prion protein (PrP) in its misfolded state. In light of this interest, we have developed an experimental system to examine the structural requirements of the LbetaH fold, using a known LbetaH protein, UDP-N-acetylglucosamine acyltransferase (LpxA), from E. coli. We showed that the beta helix can tolerate nonhydrophobic residues at interior positions and prolines were important, but not critical, in folding of the beta helix. Using our structural studies of the LbetaH, we threaded the sequence of the amyloidogenic fragment of the prion protein (residues 104-143) onto the structure of LpxA. Based on the threading result, we constructed the recombinant PrP-LpxA and tested its functional activity in an E. coli antibiotic sensitivity assay. The results of these experiments suggest that the amyloidogenic PrP fragment may fold into a beta helix in the context of a larger beta-helical structure.Journal ArticleResearch Support, N.I.H. ExtramuralSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Prions: so many fibers, so little infectivity.

Journal ArticleReviewinfo:eu-repo/semantics/publishe

Discovery of 2-Aminothiazoles as Potent Antiprion Compounds▿ ‡

Prion diseases are fatal, untreatable neurodegenerative diseases caused by the accumulation of the misfolded, infectious isoform of the prion protein (PrP), termed PrPSc. In an effort to identify novel inhibitors of prion formation, we utilized a high-throughput enzyme-linked immunosorbent assay (ELISA) to evaluate PrPSc reduction in prion-infected neuroblastoma cell lines (ScN2a). We screened a library of ∼10,000 diverse small molecules in 96-well format and identified 121 compounds that reduced PrPSc levels at a concentration of 5 μM. Four chemical scaffolds were identified as potential candidates for chemical optimization based on the presence of preliminary structure-activity relationships (SAR) derived from the primary screening data. A follow-up analysis of a group of commercially available 2-aminothiazoles showed this class as generally active in ScN2a cells. Our results establish 2-aminothiazoles as promising candidates for efficacy studies of animals and validate our drug discovery platform as a viable strategy for the identification of novel lead compounds with antiprion properties

Ionic silver functionalized ovine forestomach matrix – a non-cytotoxic antimicrobial biomaterial for tissue regeneration applications

Abstract Background Antimicrobial technologies, including silver-containing medical devices, are increasingly utilized in clinical regimens to mitigate risks of microbial colonization. Silver-functionalized resorbable biomaterials for use in wound management and tissue regeneration applications have a narrow therapeutic index where antimicrobial effectiveness may be outweighed by adverse cytotoxicity. We examined the effects of ionic silver functionalization of an extracellular matrix (ECM) biomaterial derived from ovine forestomach (OFM-Ag) in terms of material properties, antimicrobial effectiveness and cytotoxicity profile. Methods Material properties of OFM-Ag were assessed by via biochemical analysis, microscopy, atomic absorption spectroscopy (AAS) and differential scanning calorimetry. The silver release profile of OFM-Ag was profiled by AAS and antimicrobial effectiveness testing utilized to determine the minimum effective concentration of silver in OFM-Ag in addition to the antimicrobial spectrum and wear time. Biofilm prevention properties of OFM-Ag in comparison to silver containing collagen dressing materials was quantified via in vitro crystal violet assay using a polymicrobial model. Toxicity of ionic silver, OFM-Ag and silver containing collagen dressing materials was assessed toward mammalian fibroblasts using elution cytoxicity testing. Results OFM-Ag retained the native ECM compositional and structural characteristic of non-silver functionalized ECM material while imparting broad spectrum antimicrobial effectiveness toward 11 clinically relevant microbial species including fungi and drug resistant strains, maintaining effectiveness over a wear time duration of 7-days. OFM-Ag demonstrated significant prevention of polymicrobial biofilm formation compared to non-antimicrobial and silver-containing collagen dressing materials. Where silver-containing collagen dressing materials exhibited cytotoxic effects toward mammalian fibroblasts, OFM-Ag was determined to be non-cytotoxic, silver elution studies indicated sustained retention of silver in OFM-Ag as a possible mechanism for the attenuated cytotoxicity. Conclusions This work demonstrates ECM biomaterials may be functionalized with silver to favourably shift the balance between detrimental cytotoxic potential and beneficial antimicrobial effects, while preserving the ECM structure and function of utility in tissue regeneration applications

Correction: A novel chemotactic factor derived from the extracellular matrix protein decorin recruits mesenchymal stromal cells in vitro and in vivo.

[This corrects the article DOI: 10.1371/journal.pone.0235784.]