Synthesis, Kinetic Evaluation and Utilization of a Biotinylated Dipeptide Proline Diphenyl Phosphonate for the Disclosure of Dipeptidyl Peptidase IV-like Serine Proteases

In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.

Ischemic preconditioning before lower limb ischemia-reperfusion protects against acute lung injury

Objective: Prolonged limb ischemia followed by reperfusion (I/R) is associated with a systemic inflammatory response syndrome and remote acute lung injury. Ischemic preconditioning (IPC), achieved with repeated brief periods of I/R before the prolonged ischemic period, has been shown to protect skeletal muscle against ischemic injury. The aim of this study was to ascertain whether IPC of the limb before I/R injury also attenuates systemic inflammation and acute lung injury in a fully resuscitated porcine model of hind limb I/R. Methods: This prospective, randomized, controlled, experimental animal study was performed in a university-based animal research facility with 18 male Landrace pigs that weighed from 30 to 35 kg. Anesthetized ventilated swine were randomized (n = 6 per group) to three groups: sham-operated control group, I/R group (2 hours of bilateral hind limb ischemia and 2.5 hours of reperfusion), and IPC group (three cycles of 5 minutes of ischemia/5 minutes of reperfusion immediately preceding I/R). Plasma was separated and stored at -70° C for later determination of plasma tumor necrosis factor-a and interleukin-6 with bioassay as markers of systemic inflammation. Circulating phagocytic cell priming was assessed with a whole blood chemiluminescence assay. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were markers of edema and neutrophil sequestration, respectively. The alveolar-arterial oxygen gradient and pulmonary artery pressure were indices of lung function. Results: In a porcine model, bilateral hind limb (I/R) injury significantly increased plasma interleukin-6 concentrations, circulating phagocytic cell priming, and pulmonary leukosequestration, edema, and impaired gas exchange. Conversely, pigs treated with IPC before the onset of the ischemic period had significantly reduced interleukin-6 levels, circulating phagocytic cell priming, and experienced significantly less pulmonary edema, leukosequestration, and respiratory failure. Conclusion: Lower limb IPC protects against systemic inflammation and acute lung injury in lower limb I/R injury.

Identification of protease and ADP-ribose 1''-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3.

The replicase polyproteins, pp1a and pp1ab, of porcine Transmissible gastroenteritis virus (TGEV) have been predicted to be cleaved by viral proteases into 16 non-structural proteins (nsp). Here, enzymic activities residing in the amino-proximal region of nsp3, the largest TGEV replicase processing product, were characterized. It was shown, by in vitro translation experiments and protein sequencing, that the papain-like protease 1, PL1pro, but not a mutant derivative containing a substitution of the presumed active-site nucleophile, Cys1093, cleaves the nsp2|nsp3 site at 879Gly|Gly880. By using an antiserum raised against the pp1a/pp1ab residues 526–713, the upstream processing product, nsp2, was identified as an 85 kDa protein in TGEV-infected cells. Furthermore, PL1pro was confirmed to be flanked at its C terminus by a domain (called X) that mediates ADP-ribose 1''-phosphatase activity. Expression and characterization of a range of bacterially expressed forms of this enzyme suggest that the active X domain comprises pp1a/pp1ab residues Asp1320–Ser1486.

Structure-activity studies on position 14 of human alpha-calcitonin gene-related peptide

A structure-activity study was performed to examine the role of position 14 of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) in activating the CGRP receptor. Interestingly, position 14 of h-alpha-CGRP contains a glycyl residue and is part of an alpha-helix spanning residues 8-18. Analogues [Ala(14)]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, [Asn(14)]-h-alpha-CGRP, and [Pro(14)]-h-alpha-CGRP were synthesized by solid phase peptide methodology and purified by RP-HPLC. Secondary structure was measured by circular dichroism spectroscopy. Agonist activities were determined as the analogues' ability to stimulate amylase secretion from guinea pig pancreatic acini and to relax precontracted porcine coronary arteries. Analogues [Ala(1)4]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, and [Asn(14)]-h-alpha-CGRP, all containing residues with a high helical propensity in position 14, were potent full agonists compared to h-alpha-CGRP in both tissues. Interestingly, replacement of Gly(14) of h-alpha-CGRP with these residues did not substantially increase the helical content of these analogues. [Pro(14)]-h-alpha-CGRP, predictably, has significantly lower helical content and is a 20-fold less potent agonist on coronary artery, known to contain CGRP-1 receptor subtypes, and an antagonist on pancreatic acini, known to contain CGRP-2 receptor subtypes. In conclusion, the residue in position 14 plays a structural role in stabilizing the alpha-helix spanning residues 8-18. The alpha-helix is crucial for maintaining highly potent agonist effects of h-alpha-CGRP at CGRP receptors. The wide variety of functional groups that can be tolerated in position 14 with no substantial modification of agonist effects suggests the residue in this position is not in contact with the CGRP receptor. [Pro(14)]-h-alpha-CGRP may be a useful pharmacological tool to distinguish between CGRP-1 and CGRP-2 receptor subtypes.

Microneedle-mediated intradermal delivery of 5-aminolevulinic acid: potential for enhanced topical photodynamic therapy

Photodynamic therapy of deep or nodular skin tumours is currently limited by the poor tissue penetration of the porphyrin precursor 5-aminolevulinic acid (ALA). In this study, silicon microneedle arrays were used, for the first time, to enhance skin penetration of ALA in vitro and in vivo. Puncturing excised murine skin with 6x7 arrays of microneedles 270 mum in height, with a diameter of 240 mum at the base and an interspacing of 750 mum led to a significant increase in transdermal delivery of ALA released from a bioadhesive patch containing 19 mg ALA cm(-2). Microneedle puncture enhanced ALA delivery to the upper regions of excised porcine skin but, at mean depths of 1.875 mm, ALA concentrations were similar to control values, possibly reflecting binding of ALA by tissue components. However, and importantly, in vivo experiments using nude mice showed that microneedle puncture could reduce application time and ALA dose required to induce high levels of the photosensitiser protoporphyrin IX in skin. This clearly has implications for clinical practice, as shorter application times would mean improved patient and clinician convenience and also that more patients could be treated in the same session. As ALA is expensive and degrades rapidly via a second order reaction, reducing the required dose is also a notable advantage.

Characterisation of antibodies to chloramphenicol, produced in different species by enzyme-linked immunosorbent assay and biosensor technologies

Six polyclonal antisera to chloramphenicol (CAP) were successfully raised in camels, donkeys and goats. As a comparison of sensitivity, IC50 values ranged from 0.3 ng mL(-1) to 5.5 ng mL(-1) by enzyme-linked immunosorbent assay (ELISA) and from 0.7 ng mL(-1) to 1.7 ng mL(-1) by biosensor assay. The introduction of bovine milk extract improved the sensitivity of four of the antisera by ELISA and two by biosensor assay; a reduction in sensitivity of the remaining antisera ranged by a factor of 1.1-2.6. Porcine kidney extract reduced the sensitivity of all the antisera by a factor ranging from 1.1 to 7 by ELISA and a factor of 1.5 to 4 by biosensor. A low cross-reactivity with thiamphenicol (TAP) and florfenicol (FF) was displayed by antiserurn G2 (1.2% and 18%, respectively) when a homologous ELISA assay format was employed. No cross-reactivity was displayed by any of the antisera when a homologous biosensor assay format was employed. Switching to a heterologous ELISA format prompted three of the antisera to display more significant cross-reactivity with TAP and FF (53% and 82%, respectively, using Dl). The heterologous biosensor assay also increased the cross-reactivity of D1 for TAP and FF (56% and 129%, respectively) and of one other antiserum (Gl) to a lesser degree. However, unlike the ELISA, the heterologous biosensor assay produced a substantial reduction in sensitivity (by a factor of 6 for D1). (C) 2007 Elsevier B.V. All rights reserved.

Biochemical characterization of arterivirus nonstructural protein 11 reveals the nidovirus-wide conservation of a replicative endoribonuclease

Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.

Bystander-induced differentiation: a major response to targeted irradiation of a urothelial explant model.

A ureter primary explant technique, using porcine tissue sections was developed to study bystander effects under in vivo like conditions where dividing and differentiated cells are present. Targeted irradiations of ureter tissue fragments were performed with the Gray Cancer Institute charged particle microbeam at a single location (2 microm precision) with 10 3He2+ particles (5 MeV; LET 70 keV/microm). After irradiation the ureter tissue section was incubated for 7 days allowing explant outgrowth to be formed. Differentiation was estimated using antibodies to Uroplakin III, a specific marker of terminal urothelial differentiation. Even although only a single region of the tissue section was targeted, thousands of additional cells were found to undergo bystander-induced differentiation in the explant outgrowth. This resulted in an overall increase in the fraction of differentiated cells from 63.5+/-5.4% to 76.6+/-5.6%. These changes are much greater than that observed for the induction of damage in this model. One interpretation of these results is that in the tissue environment, differentiation is a much more significant response to targeted irradiation and potentially a protective mechanism.

Autoradiographic and scintillation analysis of 5-aminolevulinic acid permeation through epithelialised tissue: Implications for topical photodynamic therapy of superficial gynaecological neoplasias

Purpose. Aminolevulinic acid (5-ALA) diffusion through both keratinised and non-keratinised tissue, used as a model tissue substrates, was evaluated, together with the depth of permeation and the concentration achieved following delivery from bioadhesive patch and proprietary cream formulations. Materials and Methods. Moisture-activated, bioadhesive patches loaded with 5-ALA at concentrations of 19.0, 38.0 and 50.0 mg cm(-2) and an o/w cream (20% w/w 5-ALA) were radiolabelled with C14 5-ALA and applied to excised human vaginal tissue and porcine skin. After 1, 2 and 4 h, tissue was sectioned in two orientations and the 5-ALA concentration at specific depths determined using autoradiography and liquid scintillation counting (LSC). Results. The stratum corneum was a significant barrier to 5-ALA permeation, with concentrations in tissue dependent on application time and drug loading. 5-ALA was detected at 6 mm using autoradiography after 2 h, with LSC showing phototoxic concentrations at 2.375 mm after 4 h of application. Inclusion of oleic acid and dimethyl sulphoxide in bioadhesive patches increased 5-ALA significantly in neonate porcine tissue, but only for patches cast from blends containing 5% w/w oleic acid. Conclusions. The bioadhesive patch described delivered 5-ALA to depths of at least 2.5 mm in tissue types indicative of vulval skin, suggesting that photodynamic therapy of deep vulval intraepithelial neoplasia is feasible using this means of bioadhesive 5-ALA delivery.

Microneedle Arrays Allow Lower Microbial Penetration Than Hypodermic Needles In Vitro

Methods: In this study we determined, for the first time, the ability of microorganisms to traverse microneedle-induced holes using two different in vitro models.

Results: When employing Silescol® membranes, the numbers of Candida albicans, Pseudomonas aeruginosa and Staphylococcus epidermidis crossing the membranes were an order of magnitude lower when the membranes were punctured by microneedles rather than a 21G hypodermic needle. Apart from the movement of C. albicans across hypodermic needle-punctured membranes, where 40.2% of the microbial load on control membranes permeated the barrier over 24 h, the numbers of permeating microorganisms was less than 5% of the original microbial load on control membranes. Experiments employing excised porcine skin and radiolabelled microorganisms showed that the numbers of microorganisms penetrating skin beyond the stratum corneum were approximately an order of magnitude greater than the numbers crossing Silescol® membranes in the corresponding experiments. Approximately 103?cfu of each microorganism adhered to hypodermic needles during insertion. The numbers of microorganisms adhering to MN arrays were an order of magnitude higher in each case.

Conclusion: We have shown here that microneedle puncture resulted in significantly less microbial penetration than did hypodermic needle puncture and that no microorganisms crossed the viable epidermis in microneedle—punctured skin, in contrast to needle-punctured skin. Given the antimicrobial properties of skin, it is, therefore, likely that application of microneedle arrays to skin in an appropriate manner would not cause either local or systemic infection in normal circumstances in immune-competent patients. In supporting widespread clinical use of microneedle-based delivery systems, appropriate animal studies are now needed to conclusively demonstrate this in vivo. Safety in patients will be enhanced by aseptic or sterile manufacture and by fabricating microneedles from self-disabling materials (e.g. dissolving or biodegradable polymers) to prevent inappropriate or accidental reuse.

Influence of solution viscosity and injection protocol on distribution patterns of jet injectors: Application to photodynamic tumour targeting

Photodynamic therapy of deep or nodular skin tumours is currently limited by the poor tissue penetration of the porphyrin precursor 5-aminolevulinic acid (ALA) and preformed photosensitisers. In this study, we investigated the potential of jet injection to deliver both ALA and a preformed photosensitiser (meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate, TMP) into a defined volume of skin. Initial studies using a model hydrogel showed that as standoff distance is increased, injection depth decreases. As the ejected volume is increased, injection depth increases. It was also shown, for the first time, that, as injection solution viscosity was increased, for a given injection setting and standoff distance, both total depth of jet penetration, L-t, and depth at which the maximum width of the penetration pattern occurred, L-m, decreased progressively. For a standoff distance of zero, the maximum width of the penetration pattern, L-w, increased progressively with increasing viscosity at each of the injection settings. Conversely, when the standoff distance was 2.5 mm, L-w decreased progressively with increasing viscosity. Studies with neonate porcine skin revealed that an injection protocol comprising an 8.98 mPas solution, an arbitrary injection setting of 8 and a standoff distance of zero was capable of delivering photosensitisers to a volume of tissue (L-t of 2.91 mm, L-m of 2.14 mm, L-w of 5. 10 mm) comparable to that occupied by a typical nodular basal cell carcinoma. Both ALA and TMP were successfully delivered using jet injection, with peak tissue concentrations (67.3 mg cm(-3) and 5.6 mg cm(-3), respectively) achieved at a depth of around 1.0 mm and substantial reductions in drug concentration seen at depths below 3.0 mm. Consequently, jet injection may be suitable for selective targeting of ALA or preformed photosensitisers to skin tumours. (c) 2007 Elsevier B.V. All rights reserved.

cAMP/PKA-dependent increases in Ca Sparks, oscillations and SR Ca stores in retinal arteriolar myocytes after exposure to vasopressin.

PURPOSE: To investigate the effects of arginine vasopressin (AVP) on Ca(2+) sparks and oscillations and on sarcoplasmic reticulum (SR) Ca(2+) content in retinal arteriolar myocytes. METHODS: Fluo-4-loaded smooth muscle in intact segments of freshly isolated porcine retinal arteriole was imaged by confocal laser microscopy. SR Ca(2+) store content was assessed by recording caffeine-induced Ca(2+) transients with microfluorimetry and fura-2. RESULTS: The frequencies of Ca(2+) sparks and oscillations were increased both during exposure to, and 10 minutes after washout of AVP (10 nM). Caffeine transients were increased in amplitude 10 and 90 minutes after a 3-minute application of AVP. Both AVP-induced Ca(2+) transients and the enhancement of caffeine responses after AVP washout were inhibited by SR 49059, a V(1a) receptor blocker. Forskolin, an activator of adenylyl cyclase, also persistently enhanced caffeine transients. Rp-8-HA-cAMPS, a membrane-permeant PKA inhibitor, prevented enhancement of caffeine transients by both AVP and forskolin. Forskolin, but not AVP, produced a reversible, Rp-8-HA-cAMPS insensitive reduction in basal [Ca(2+)](i). CONCLUSIONS: AVP activates a cAMP/PKA-dependent pathway via V(1a) receptors in retinal arteriolar smooth muscle. This effect persistently increases SR Ca(2+) loading, upregulating Ca(2+) sparks and oscillations, and may favor prolonged agonist activity despite receptor desensitization.

Hexyl Aminolaevulinate Is a More Effective Topical Photosensitiser Precursor than Methyl Aminolaevulinate and 5-Aminolaevulinic Acids When Applied in Equimolar Doses

Aminolaevulinic acid (ALA) is known to poorly penetrate into thick lesions, such as nodular basal cell carcinomas Short chain ALA esters, possessing increased lipophilicity relative to their hydrophilic parent, have previously been shown to be highly efficient at inducing protoporphyrin IX (PpIX) production in cell culture, at equimolar concentrations. In contrast, in vitro skin permeation and in vivo animal studies, which up to now have compared prodrugs on a % w/vv basis, have failed to demonstrate such benefits For the first time, equimolar concentrations of ALA, methyl-ALA (m-ALA) and hexyl-ALA (h-ALA) have been incorporated into an o/w cream preparation. In vitro penetration studies into excised porcine skin revealed that increased levels of h-ALA, compared to ALA and m-ALA were found in the upper skin layers, at all drug loadings studied. Topical application of the formulations to nude murine skin in vivo, revealed that creams containing h-ALA induced significantly higher levels of peak PpIX fluorescence (F-max = 289.0) at low concentrations compared to m-ALA (F-max = 159.2) and ALA (F-max = 191 9). Importantly, this study indicates that when compared on an equimolar basis, h-ALA has improved skin penetration, leading to enhanced PpIX production compared to the parent drug and m-ALA (C) 2010 Wiley-Liss, Inc and the American Pharmacists Association J Pharm Sci 99 3486-3498, 2010

Optical coherence tomography is a valuable tool in the study of the effects of microneedle geometry on skin penetration characteristics and in-skin dissolution.

In this study, we used optical coherence tomography (OCT) to extensively investigate, for the first time, the effect that microneedle (MN) geometry (MN height, and MN interspacing) and force of application have upon penetration characteristics of soluble poly(methylvinylether-co-maleic anhydride, PMVE/MA) MN arrays into neonatal porcine skin in vitro. The results from OCT investigations were then used to design optimal and suboptimal MN-based drug delivery systems and evaluate their drug delivery profiles cross full thickness and dermatomed neonatal porcine skin in vitro. It was found that increasing the force used for MN application resulted in a significant increase in the depth of penetration achieved within neonatal porcine skin. For example, MN of 600 µm height penetrated to a depth of 330 µm when inserted at a force of 4.4 N/array, while the penetration increased significantly to a depth of 520 µm, when the force of application was increased to 16.4 N/array. At an application force of 11.0 N/array it was found that, in each case, increasing MN height from 350 to 600 µm to 900 µm led to a significant increase in the depth of MN penetration achieved. Moreover, alteration of MN interspacing had no effect upon depth of penetration achieved, at a constant MN height and force of application. With respect to MN dissolution, an approximate 34% reduction in MN height occurred in the first 15 min, with only 17% of the MN height remaining after a 3-hour period. Across both skin models, there was a significantly greater cumulative amount of theophylline delivered after 24 h from an MN array of 900 µm height (292.23 ± 16.77 µg), in comparison to an MN array of 350 µm height (242.62 ± 14.81 µg) (p < 0.001). Employing full thickness skin significantly reduced drug permeation in both cases. Importantly, this study has highlighted the effect that MN geometry and application force have upon the depth of penetration into skin. While it has been shown that MN height has an important role in the extent of drug delivered across neonatal porcine skin from a soluble MN array, further studies to evaluate the full significance of MN geometry on MN mediated drug delivery are now underway. The successful use of OCT in this study could prove to be a key development for polymeric MN research, accelerating their commercial exploitation.

Design, optimisation and characterisation of polymeric microneedle arrays prepared by a novel laser-based micromoulding technique.

PURPOSE:
Design and evaluation of a novel laser-based method for micromoulding of microneedle arrays from polymeric materials under ambient conditions. The aim of this study was to optimise polymeric composition and assess the performance of microneedle devices that possess different geometries.
METHODS:
A range of microneedle geometries was engineered into silicone micromoulds, and their physicochemical features were subsequently characterised.
RESULTS:
Microneedles micromoulded from 20% w/w aqueous blends of the mucoadhesive copolymer Gantrez® AN-139 were surprisingly found to possess superior physical strength than those produced from commonly used pharma polymers. Gantrez® AN-139 microneedles, 600 µm and 900 µm in height, penetrated neonatal porcine skin with low application forces (>0.03 N per microneedle). When theophylline was loaded into 600 µm microneedles, 83% of the incorporated drug was delivered across neonatal porcine skin over 24 h. Optical coherence tomography (OCT) showed that drug-free 600 µm Gantrez® AN-139 microneedles punctured the stratum corneum barrier of human skin in vivo and extended approximately 460 µm into the skin. However, the entirety of the microneedle lengths was not inserted.
CONCLUSION:
In this study, we have shown that a novel laser engineering method can be used in micromoulding of polymeric microneedle arrays. We are currently carrying out an extensive OCT-informed study investigating the influence of microneedle array geometry on skin penetration depth, with a view to enhanced transdermal drug delivery from optimised laser-engineered Gantrez® AN-139 microneedles.