Pre-treatment with Depo-Provera modifies the pharmacokinetics of CMPD167 in rhesus macaques following vaginal ring administration

BACKGROUND: Vaginal ring devices are being developed to provide sustained release of HIV microbicides. To date, only limited pharmacokinetic data is available from animal or human studies. Here we report the effect of Depo-Provera (DP) pre- treatment, commonly used to thin the vaginal epithelium in challenge experiments, on the pharmacokinetic profile of CMPD167 (a small molecule CCR5 co-receptor antagonist) in rhesus macaques following vaginal ring administration.

METHODS: A single 400mg CMPD167 silicone elastomer vaginal ring was inserted into each of twelve female rhesus macaques. Six macaques were treated with (DP) 30 days before ring placement; the other six macaques were untreated. Blood, vaginal fluid and vaginal biopsies were collected prior to and at various times during 28 days of ring placement and assayed for CMPD167 levels by HPLC. Rings were assayed for residual CMPD167 at the end of the study and the calculated amount of CMPD167 released in vivo compared with in vitro release data.

RESULTS: Vaginal fluid, plasma and tissue levels of CMPD167 were detectable throughout ring placement. Significant differences were observed in mean daily vaginal fluid levels between the DP-treated (16–56 mcg/mL) and untreated groups (48–181 mcg/mL). Plasma CMPD167 levels were significantly higher peaking at 4 ng/mL and maintaining levels of 1–2 nM throughout the 14 days of testing in animals pre-treated with DP compared to non DP-treated macaques (<1 ng/mL maintained). Tissue levels were varied between 2–10 g/mL CMPD167 with no significant difference between the DP-treated and untreated macaques.

CONCLUSIONS: The study demonstrates that clinically relevant, and possibly protective doses of CMPD167 are released in the vaginal vault of rhesus macaques from vaginal rings through 28 days duration. DP is known to induce vaginal epithelial thinning and lower vaginal fluid levels, which accounts for the increased plasma levels of CMPD167. In contrast, macaques not treated with DP had minimal absorption into plasma compartments and significantly higher levels of CMPD167 in the vagina, similar to those previously shown to be protective against vaginal challenge.

Adenosine induces histamine release from human bronchoalveolar lavage mast cells

Previous studies have shown that in vitro adenosine enhances histamine release from activated human lung mast cells obtained by enzymic dispersion of lung parenchyma. However, adenosine alone has no effect on histamine release from these cells. Given the evidence for direct activation of mast cells after endobronchial challenge with adenosine and previous studies indicating that mast cells obtained at bronchoalveolar lavage are a better model for asthma studies than those obtained by enzymic dispersion of lung tissue, the histamine-releasing effect of adenosine was examined on lavage mast cells. Bronchoalveolar lavage fluid was obtained from patients attending hospital for routine bronchoscopy (n = 54). Lavage cells were challenged with adenosine or adenosine receptor agonists (20 min, 37 degrees C) and histamine release determined using an automated fluorometric assay. Endogenous adenosine levels were also measured in lavage fluid (n = 9) via an HPLC method. Adenosine alone caused histamine release from ravage mast cells in 37 of 54 patients with a maximal histamine release of 20.56 +/- 2.52% (range 5.2-61 %). The adenosine receptor agonists (R)-N-6-(2-phenylisopropyl)adenosine, 5'-N-ethylcarboxamido-adenosine and CGS21680 also induced histamine release from lavage mast cells. Preincubation of lavage mast cells with the adenosine receptor antagonist xanthine amine congener caused significant inhibition of the response to adenosine (P = 0.007). There was an inverse correlation between endogenous adenosine levels in the lavage fluid and the maximal response to in vitro adenosine challenge of the lavage cells. The findings of the present study indicate a means by which adenosine challenge of the airways can induce bronchoconstriction and support a role for adenosine in the pathophysiology of asthma. The results also suggest that cells obtained from bronchoalveolar ravage fluid may provide the ideal model for the testing of novel, adenosine receptor, targeted therapies for asthma.

Neurokinin A is the predominant tachykinin in human bronchoalveolar lavage fluid in normal and asthmatic subjects

BACKGROUND—Multiple sensory neuropeptides are present in human airways and may contribute to diseases such as asthma. This study quantified and characterised substance P (SP), neurokinin A (NKA), and calcitonin gene related peptide (CGRP) immunoreactivity in bronchoalveolar lavage fluid in asthmatic and normal subjects.
METHODS—Using specific radioimmunoassay (RIA), SP, NKA and CGRP were measured in bronchoalveolar lavage fluid from asthmatic subjects (n = 5), normal subjects (n = 5), atopic non-asthmatic subjects (n = 6), and asthmatic subjects four hours after allergen challenge (n = 12). Peptide immunoreactivity was characterised using high performance liquid chromatography (HPLC) and RIA.
RESULTS—No SP or CGRP immunoreactivity was detected in any of the fractions from samples after extraction, HPLC, and RIA. Non-specific binding resulted in spurious SP immunoreactivity being detected in bronchoalveolar lavage fluid when no extraction process was employed. NKA was detected in significant amounts in asthmatic (median 550, range 425-625 pg/ml) and normal subjects (median 725, range 350-1425 pg/ml). The level of NKA was significantly higher in the asthmatic subjects after allergen challenge (median 750, range 350-1250 pg/ml) than in unchallenged asthmatic subjects (median 600, range 425-600 pg/ml, p<0.01).
CONCLUSIONS—Extraction and characterisation of peptides from bronchoalveolar lavage fluid must be performed to ensure that the measured immunoreactivity represents target peptide. NKA is present in bronchoalveolar lavage fluid in high concentrations and is the predominant tachykinin. The concentrations of NKA are similar in normal subjects and subjects with mild asthma.

Apolipoprotein E genotype is associated with macular pigment optical density.

PURPOSE: Age-related macular degeneration (AMD) is the most common cause of blindness in older people in developed countries, and risk factors for this condition may be classified as genetic and environmental. Apolipoprotein E is putatively involved in the transport of the macular pigment (MP) carotenoids lutein (L) and zeaxanthin (Z) in serum and may also influence retinal capture of these compounds. This study was designed to investigate the relationship between macular pigment optical density (MPOD) and ApoE genotype. METHODS: This was a cross-sectional study of 302 healthy adult subjects. Dietary intake of L and Z was assessed by food frequency questionnaire, and MPOD was measured by customized heterochromatic flicker photometry. Serum L and Z were measured by HPLC. ApoE genotyping was performed by direct polymerase chain reaction amplification and DNA nucleotide sequencing from peripheral blood. RESULTS: Genotype data were available on 300 of the 302 (99.3%) subjects. The mean (+/- SD) age of the subjects in this study was 47.89 +/- 11.05 (range, 21-66) years. Subjects were classed into one of three ApoE genotype groups, as follows: group 1, epsilon2epsilon2 or epsilon2epsilon3; group 2, epsilon3epsilon3; group 3, epsilon2epsilon4 or epsilon3epsilon4 or epsilon4epsilon4. All three groups were statistically comparable in terms of age, sex, body mass index, cigarette smoking, and dietary and serum levels of L and Z. There was a statistically significant association between ApoE genotype and MPOD. Subjects who had at least one epsilon4 allele had a higher MPOD across the macula than subjects without this allele (group 1 MPOD area, 0.70 +/- 0.40; group 2 MPOD area, 0.67 +/- 0.42; group 3 MPOD area, 0.85 +/- 0.46; one-way ANOVA, P = 0.014. CONCLUSIONS: These results suggest that ApoE genotype status is associated with MPOD. This association may explain, at least in part, the putative protective effect of the epsilon4 allele for AMD and is consistent with the view that apolipoprotein profile influences the transport and/or retinal capture of circulating L and/or Z.

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2

Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5 Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys18 and Cys24 and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133–160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr6)-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.

Antimicrobial peptides in the venom of the European hornet, Vespa crabro, identified as mastoparan C and crabrolin

Amphibian skin secretions are rich sources of cationic amphipathic peptides which often possess potent and broad-spectrum antimicrobial activity. However, the venoms of other animals such as hymenopteran insects, also contain peptides with these characteristics and the literature is unclear as to their antimicrobial potential. Here we subjected the venom of the European hornet, Vespa crabro, to reverse phase HPLC fractionation followed by screening of aliquots of individual fractions in bacterial zonal inhibition assays. Two major peptides possessing activity in these assays were further purified by HPLC and subjected to MALDI-TOF MS analysis and MS/MS fragmentation using an ESI mass spectrometer. The peptides were identified as mastoparan C (LNLKALLAVAKKILamide) and crabrolin (FLPLILRKIVTALamide). Replicates of both peptides were synthesised by solid-phase methodology and mean inhibitory concentrations (MICs) established against Staphylococcus aureus and Escherichia coli. Mastoparan C was found to be a potent antimicrobial with MIC values of 2 µM and 4 µM against S. aureus and E. coli, respectively. Crabrolin was found to be less potent with MIC values of > 160 µM and 40 µM for S. aureus and E. coli. Hornet venom thus contains a potent antimicrobial peptide that has been unambiguously identified as mastoparan C, a peptide that is known to affect profound histamine release from mast cells and to generally activate membrane G protein-linked receptors. It is thus highly probable that its antimicrobial effects, like those previously documented, are a result of a generalized membrane interactive and disruptive function — perhaps reflective of the authentic role of amphibian skin antimicrobials.

Peptide DV-28 amide: Prototype of a novel class of bradykinin receptor antagonist isolated from the defensive skin secretion of bombinid toads

Kinestatin, isolated from the skin of the Chinese toad, Bombina maxima, was the first bradykinin B2 receptor antagonist identified in amphibians. Molecular cloning established that it is co-encoded with the bradykinin-related peptide, maximakinin, within one of several skin kininogens. To examine other species within the genus Bombina for the presence of structural homologues of kinestatin, we subjected skin secretion of the toad, Bombina orientalis, to HPLC fractionation with subsequent bioassay of fractions for antagonism of bradykinin activity using an isolated rat tail artery smooth muscle preparation. A single fraction was located that inhibited bradykinin-induced relaxation of rat arterial smooth muscle and MALDI-TOF analysis of this fraction revealed that it contained a single peptide of molecular mass 3198.5 Da. Further primary structural analysis of this peptide showed that it was a 28-mer with an N-terminal Asp (D) residue and a C-terminal Val (V) residue that was amidated. The peptide was named DV-28 amide in accordance with these primary structural attributes. Synthetic DV-28 amide replicated the observed bradykinin antagonistic effect within the smooth muscle bioassay in a dose-dependent manner. In addition, it was observed to inhibit the proliferation of human microvessel endothelial cells (HMECs) as assessed by MTT assay. Bioinformatic analysis revealed that DV-28 amide was, like kinestatin, co-encoded with a bradykinin receptor agonist on one of two skin kininogens identified in B. orientalis. DV-28 amide thus represents a novel class of bradykinin antagonist from skin secretions of bombinid toads that appear to be a rich source of such novel peptides.

Molecular cloning of the helokinestatin/CNP precursor from a venom-derived cDNA library of the Mexican beaded lizard, Heloderma horridum, and identification of a novel encoded bradykinin-antagonist peptide, helokinestatin-6

Helokinestatins 1–5 represent a novel family of bradykinin antagonist peptides originally isolated from the venom of the Gila Monster, Heloderma suspectum. We found that they were encoded in tandem along with a single copy of C-type natriuretic peptide (CNP), by two different but almost identical biosynthetic precursors that were cloned from a venom-derived cDNA library. Here we have applied the same strategy to the venom of a related species, the Mexican beaded lizard, Heloderma horridum. Lyophilised venom was used as a surrogate tissue to generate a cDNA library that was interrogated with primers from the previous study and for reverse phase HPLC fractionation. The structure of a single helokinestatin precursor was obtained following sequencing of 20 different clones. The open-reading frame contained 196 amino acid residues, somewhat greater than the 177–178 residues of the corresponding helokinestatin precursors in H. suspectum. The reason for this difference in size was the insertion of an additional domain of 18 amino acid residues encoding an additional copy of helokinestatin-3. Helokinestatin-6 (GPPFNPPPFVDYEPR) was a novel peptide from this precursor identified in venom HPLC fractions. A synthetic replicate of this peptide antagonised the relaxation effect of bradykinin on rat arterial smooth muscle. The novel peptide family, the helokinestatins, have been shown to be present in the venom of H. horridum and to be encoded by a single precursor of different structure to those from H. suspectum. Studies such as this reveal the naturally-selected structures of bioactive peptides that have been optimised for purpose and provide the scientist with a natural analogue library for pharmacological investigation.

A novel and potent trypsin inhibitor peptide, AC-17, from the skin secretion of the edible frog, Rana esculenta

Protease inhibitors are found in many venoms and evidence suggests that they occur widely in amphibian skin secretions. Kunitz inhibitors have been found in the skin secretions of bombinid toads and ranid frogs, Kazal inhibitors in phyllomedusine frogs and Bowman–Birk inhibitors in ranid frogs. Selective protease inhibitors could have important applications as therapeutics in the treatment of diseases in which discrete proteases play an aetiologcal role. Here we have examined the skin secretion of the edible frog, Rana esculenta, for protease inhibitors using trypsin as a model. HPLC fractions of secretions were screened for inhibitory activity using a chromogenic substrate as reporter. Three major peptides were resolved with trypsin inhibitory activity in HPLC fractions — one was a Kunitz-type inhibitor, a second was a Bowman–Birk inhibitor but the third represented a novel class of trypsin inhibitor in European frog skin. Analysis of the peptide established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17. Peptide AC-17 resembled a typical “Rana box” antimicrobial peptide but while it was active against Escherichia coli (MIC 30 µM) it was devoid of activity against Staphylococcus aureus and of haemolytic activity. In contrast, the peptide was a potent inhibitor of trypsin with a Ki of 5.56 µM. AC-17 represents the prototype of a novel trypsin inhibitor from the skin secretion of a European ranid frog that may target a trypsin-like protease present on the surface of Gram-negative bacteria.

Antimicrobial peptides from the skin secretion of the Wuyi Mountain torrent frog, Amolops wuyiensis: An inhabitant of a pristine aquatic environment

The antimicrobial peptides of amphibian skin secretions are proposed to aid survival in microbe-rich environments. While many amphibians inhabit such environments, other such as the Wuyi Mountain torrent frog, Amolops wuyiensis, live in pristine waters flowing from underground mountain springs. This species thus represents an interesting model in which to study antimicrobial peptides. “Shotgun” cloning of a skin-derived cDNA library from this species identified transcripts encoding a brevinin-1 and a ranatuerin-2. Peptides with coincident molecular masses to both predicted mature peptides were identified in HPLC fractions of skin secretion. Synthetic replicates of both peptides were generated by solid-phase peptide synthesis and tested for activity using Staphylococcus aureus, Escherichia coli and Candida albicans. The brevinin was found to be broad-spectrum and potent with minimum inhibitory concentrations (MICs) of 24 µM (Sa), 5 µM (Ec) and 20 µM (Ca). In contrast, the ranatuerin was less effective and of narrower spectrum with an MIC > 200 µM for Sa, 40 µM (Ec) and 120 µM (Ca). Thus this species of amphibian that lives in a pristine environment does indeed possess at least one potent and broad-spectrum antimicrobial peptide in its skin secretion arsenal. This phenomenon could be explained in several ways. Firstly, it may represent an ancestral peptide required when the stem species inhabited microbe-rich environments. However, there is mounting evidence for the second reason, that suggests the function of such peptides is not primarily in antimicrobial defence.

Up-regulation of GABA transporters and GABAA Receptor α1 subunit in tremor rat hippocampus

The loss of GABAergic neurotransmission has been closely linked with epileptogenesis. The modulation of the synaptic activity occurs both via the removal of GABA from the synaptic cleft and by GABA transporters (GATs) and by modulation of GABA receptors. The tremor rat (TRM; tm/tm) is the parent strain of the spontaneously epileptic rat (SER; zi/zi, tm/tm), which exhibits absence-like seizure after 8 weeks of age. However, there are no reports that can elucidate the effects of GATs and GABAA receptors (GABARs) on TRMs. The present study was conducted to detect GATs and GABAR a1 subunit in TRMs hippocampus at mRNA and protein levels. In this study, total synaptosomal GABA content was significantly decreased in TRMs hippocampus compared with control Wistar rats by high performance liquid chromatography (HPLC); mRNA and protein expressions of GAT-1, GAT-3 and GABAR a1 subunit were all significantly increased in TRMs hippocampus by real time PCR and western blot, respectively; GAT-1 and GABAR a1 subunit proteins were localized widely in TRMs and control rats hippocampus including CA1, CA3 and dentate gyrus (DG) regions whereas only a wide distribution of GAT-3 was observed in CA1 region by immunohistochemistry. These data demonstrate that excessive expressions of GAT-1 as well as GAT-3 and GABAR a1 subunit in TRMs hippocampus may provide the potential therapeutic targets for genetic epilepsy.

mVps34 is activated following high-resistance contractions

Following resistance exercise in the fasted state, both protein synthesis and degradation in skeletal muscle are increased. The addition of essential amino acids potentiates the synthetic response suggesting that an amino acid sensor, which is involved in both synthesis and degradation, may be activated by resistance exercise. One such candidate protein is the class 3 phosphatidylinositol 3OH-kinase (PI3K) Vps34. To determine whether mammalian Vps34 (mVps34) is modulated by high-resistance contractions, mVps34 and S6K1 (an index of mTORC1) activity were measured in the distal hindlimb muscles of rats 0.5, 3, 6 and 18 h after acute unilateral high-resistance contractions with the contralateral muscles serving as a control. In the lengthening tibialis anterior (TA) muscle, S6K1 (0.5 h = 366.3 +/- 112.08%, 3 h = 124.7 +/- 15.96% and 6 h = 129.2 +/- 0%) and mVps34 (3 h = 68.8 +/- 15.1% and 6 h = 36.0 +/- 8.79%) activity both increased, whereas in the shortening soleus and plantaris (PLN) muscles the increase was significantly lower (PLN S6K1 0.5 h = 33.1 +/- 2.29% and 3 h = 47.0 +/- 6.65%; mVps34 3 h = 24.5 +/- 7.92%). HPLC analysis of the TA demonstrated a 25% increase in intramuscular leucine concentration in rats 1.5 h after exercise. A similar level of leucine added to C2C12 cells in vitro increased mVps34 activity 3.2-fold. These data suggest that, following high-resistance contractions, mVps34 activity is stimulated by an influx of essential amino acids such as leucine and this may prolong mTORC1 signalling and contribute to muscle hypertrophy.

Development of ELISAs for detecting domoic acid, okadaic acid, and saxitoxin and their applicability for the detection of marine toxins in samples collected in Belgium

Okadaic acid, a diarrhetic shellfish poison, domoic acid, an amnesic shellfish poison, and saxitoxin, a paralytic shellfish poison, are three of the best-known marine biotoxins. The mouse bioassay is the method most widely used to detect many of these toxins in shellfish samples, but animal welfare concerns have prompted researchers to seek alternative methods of detection. In this study, three direct competitive enzyme-linked immunosorbent assays (ELISAs), each based on antibodies raised in rabbits against a conjugate of the analyte of interest, were developed for marine biotoxin detection in mussel, oyster, and scallop. One assay was for okadaic acid, one for saxitoxin, and one for domoic acid usually detected and quantified by high-performance liquid chromatography-ultraviolet light (HPLC-UV). All three compounds and a number of related toxins were extracted quickly and simply from the shellfish matrices with a 9 : 1 mixture of ethanol and water before analysis. The detection capabilities (CC values) of the developed ELISAs were 150 mu g kg-1 for okadaic acid, 50 mu g kg-1 for domoic acid, and 5 mu g kg-1 or less for saxitoxin. The assays proved satisfactory when used over a 4-month period for the analysis of 110 real samples collected in Belgium.

Single Laboratory Validation of a Surface Plasmon Resonance Biosensor Screening method for Paralytic Shellfish Poisoning Toxins

A research element of the European Union (EU) sixth Framework project BioCop focused on the development of a surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins in shellfish as an alternative to the increasingly ethically unacceptable mouse bioassay. A biosensor assay was developed using both a saxitoxin binding protein and chip surface in tandem with a highly efficient simple extraction procedure. The present report describes the single laboratory validation of this immunological screening method, for this complex group of toxins with differing toxicities, according to the European Decision 2002/657/EC in conjunction with IUPAC and AOAC single laboratory validation guidelines. The different performance characteristics (detection capability CC beta, specificity/selectivity, repeatability, reproducibility, stability, and applicability) were determined in relation to the EU regulatory limit of 800 mu g of saxitoxin equivalents (STX eq) per kg of shellfish meat. The detection capability CC beta was calculated to be 120 mu g/kg. Intra-assay repeatability was found to be between 2.5 and 12.3% and interassay reproducibility was between 6.1 and 15.2% for different shellfish matrices. Natural samples were also evaluated and the resultant data displayed overall agreements of 96 and 92% with that of the existing AOAC approved methods of mouse bioassay (MBA) and high performance liquid chromatography (HPLC), respectively.

The application of NMR to study changes in polar metabolite concentrations in beef longissimus dorsi stored for different periods post mortem

In this study data generated by H-1 NMR spectroscopy were combined with chemometrics to analyse beef samples aged over a 21 day period. In particular, the amino acids, of which 12 were identified were found to increase over the ageing period with samples matured for 3 days having notably lower concentrations than carcasses aged for 21 days. This is believed to be a result of increased proteolysis within the muscle. This novel approach of using high resolution NMR spectrometry to analyse beef samples has not previously been reported and these findings demonstrate the potential of this technique linked with HPLC to be used as a suitable method for profiling meat samples.