A Phase I clinical trial of lodenafil carbonate, a new phosphodiesterase Type 5 (PDE5) inhibitor, in healthy male volunteers

Lodenafil carbonate is a new phosphodiesterase Type 5 (PDE5) inhibitor used in treatment of erectile dysfunction. Objective: The present study was conducted to evaluate the safety, tolerability, and pharmacokinetics of lodenafil carbonate after administering ascending (1 - 100 mg) single oral doses to healthy male volunteers (n = 33). Methods: The study was an open-label, dose-escalation, Phase I clinical trial involving the administration of single oral doses of lodenafil carbonate. Lodenafil carbonate was administered sequentially, escalating in single doses of 1 mg - 100 mg with a washout period of at least 1 week between each dose. The progression to the next dose was allowed after clinical and laboratory exams, Ambulatory Monitoring of Arterial Pressure (AMAP) without relevant clinical modifications and adverse events without clinical relevancy. Blood samples were collected at pre-dose, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 14, 16, 20 and 24 h post-dosing. Plasma samples for measurement of lodenafil carbonate and lodenafil were analyzed by liquid chromatography coupled to tandem mass spectrometry. Results: No serious adverse events were observed, and none of the subjects discontinued the study due to intolerance. The AMAP measurements, clinical and laboratory exams and ECG revealed no significant changes even at higher doses. Lodenafil carbonate was not detected in any samples, indicating that it acts as a prodrug. The mean lodenafil pharmacokinetic parameters for t(max) and t(1/2) were 1.6 (+/- 0.4) h and 3.3 (+/- 1.1) h, respectively. This study demonstrated that lodenafil carbonate was well tolerated and showed a good safety profile in healthy male volunteers.

Rôle du monoxyde d'azote dans la calcification vasculaire et la rigidité artérielle dans un modèle d'hypertension systolique isolée

L’hypertension systolique isolée (HSI) est le résultat de changements au niveau de la paroi vasculaire qui ont pour conséquence d’augmenter la rigidité artérielle. Ces modifications surviennent surtout au niveau des grosses artères comme l’aorte et sont associées au vieillissement. La fragmentation des fibres élastiques, leur calcification (élastocalcinose) et la fibrose font partie des changements majeurs observés avec l’âge. En plus de ces changements, le vieillissement vasculaire provoque des modifications au niveau des cellules qui composent la paroi. Les cellules endothéliales sécrètent moins de monoxyde d’azote (NO) provoquant une dysfonction endothéliale et les cellules musculaires lisses vasculaires (CMLVs) synthétisent maintenant des protéines matricielles et osseuses. Situé entre le sang et les CMLVs, l’endothélium contrôle le tonus vasculaire par la sécrétion de plusieurs substances vasoactives qui interagissent entre elles afin de maintenir l’homéostasie du système vasculaire. Parmi celles-ci, on note l’endothéline (ET), un puissant vasoconstricteur et le NO, un gaz vasorelaxants. Ce dernier est aussi reconnu pour bloquer la production d’ET par un mécanisme dépendant du guanosine monophosphate cyclique (GMPc). Comme il y a une interaction entre le NO et l’ET, et que cette dernière est impliquée dans la calcification artérielle, le NO pourrait être impliqué dans la modulation de l’élastocalcinose et de la rigidité artérielle par l’inhibition de l’ET et la modification de la composition de la paroi. Cet effet, qui se produirait au delà des effets vasorelaxants du NO, offre un potentiel thérapeutique intéressant pour l’HSI. Afin d’évaluer l’implication du NO dans la calcification vasculaire et la rigidité artérielle, un modèle animal d’HSI a été utilisé (modèle warfarine vitamine K, WVK). Ce modèle d’élastocalcinose est basé sur l’inhibition de la maturation d’une protéine anti-calcifiante, la matrix Gla protein (MGP), par la warfarine. Afin de déterminer l’implication physiologique du NO dans l’initiation et la progression de l’élastocalcinose, sa production a été inhibée par un analogue de la L-arginine, le L-NG-nitroarginine methyl ester (L-NAME). Lors des processus d’initiation de la calcification, le L-NAME a prévenu l’élastocalcinose sans toutefois modifier la vitesse de l’onde de pouls (PWV). Suite au traitement L-NAME, l’expression de la NO synthase inductible (iNOS) a été diminuée alors qu’elle a été augmentée lors du traitement WVK. Elle pourrait donc être impliquée dans les processus de calcification vasculaire. De plus, la NO synthase endothéliale (eNOS) semble également impliquée puisqu’elle a été augmentée dans le modèle WVK. Cette hausse pourrait être bénéfique pour limiter l’élastocalcinose alors que l’expression de la iNOS serait délétère. Lors de la progression de la calcification, le L-NAME a augmenté l’élastocalcinose et le PWV. Dans ce contexte, l’ET serait impliquée dans l’amplification de la calcification vasculaire entrainant une hausse de la rigidité artérielle. Comme le NO endogène limite la progression de la calcification et conséquemment la rigidité artérielle, il semble être protecteur. L’efficacité d’une modulation de la voie du NO dans le modèle WVK a été étudiée par l’administration d’un donneur de NO, le sinitrodil, ou d’un inhibiteur de la phosphosdiestérase 5 (PDE5), le tadalafil. La modulation de la voie du NO semble être bénéfique sur la rigidité artérielle, mais seulement de façon aiguë. En effet, le sinitrodil a modifié de transitoirement la rigidité au niveau de l’aorte possiblement par la modulation du tonus vasculaire sans toutefois avoir des effets sur la composition de la paroi. Comme le modèle WVK n’affecte pas la fonction endothéliale, les concentrations endogènes de NO semblent être optimales puisque le sinitrodil provoque une augmentation de l’élastocalcinose possiblement par le développement d’une tolérance. Tout comme le sinitrodil, le tadalafil a modulé de manière aiguë la rigidité artérielle sans modifier la composition de la paroi. Globalement, ces travaux ont permis de mettre en évidence les effets bénéfiques du NO endogène pour limiter le développement de l’HSI, suggérant qu’une dysfonction endothéliale, tel qu’observé lors du vieillissement, a un impact négatif sur la maladie.

Sildenafil na hipertensão pulmonar persistente do recém-nascido associada a hérnia diafragmática congénita

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Comparison of clinical trials with sildenafil, vardenafil and tadalafil in erectile dysfunction

Erectile dysfunction (ED) affects up to 50% of men, between 40 and 70 years of age. In the first major trial of sildenafil in ED, at 24 weeks, improved erections were reported by 77 and 84% of men taking sildenafil 50 and 100 mg, respectively. Subsequently, sildenafil has been reported to be effective in men with ED associated with diabetes and prostate cancer, and in psychogenic ED. Sildenafil is safe in men with coronary artery disease, provided it is not used with the nitrates (a contraindication). The most commonly reported adverse effects with sildenafil are headache, flushing and dyspepsia. Vardenafil is more potent and more selective than sildenafil at inhibiting phosphodiesterase-5. Vardenafil is similarly effective to sildenafil in the treatment of ED. The only advantage that vardenafil has over sildenafil is that it does not inhibit phosphodiesterase-6 to alter colour perception, a rare side effect which sometimes occurs with sildenafil. Tadalafil has a longer duration of action than sildenafil and vardenafil. Tadalafil is similarly effective as sildenafil in the treatment of ED. In comparison studies, tadalafil is preferred to sildenafil (50/100 mg) by men with ED, possibly because of its longer duration of action. of the phosphodiesterase inhibitors, tadalafil may displace sildenafil as the drug of choice among men with ED.

Safety and tolerability considerations in the use of sildenafil for children with pulmonary arterial hypertension.

Sildenafil is a phosphodiesterase type-5 inhibitor approved for treatment of pulmonary arterial hypertension (PAH) in adults. Data from pediatric trials demonstrate a similar acute safety profile to the adult population but have raised concerns regarding the safety of long-term use in children. Interpretation of these trials remains controversial with major regulatory agencies differing in their recommendations - the US Food and Drug Administration recommends against the use of sildenafil for treatment of PAH in children, while the European Medicines Agency supports its use at "low doses". Here, we review the available pediatric data regarding dosing, acute, and long-term safety and efficacy of sildenafil for the treatment of PAH in children.

Isolation of bovine herpesvirus type 5 from the semen of a healthy bull in Australia.

Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed.

Safety and immunogenicity of a replication-defective adenovirus type 5 HIV vaccine in Ad5-seronegative persons: a randomized clinical trial (HVTN 054).

BACKGROUND: Individuals without prior immunity to a vaccine vector may be more sensitive to reactions following injection, but may also show optimal immune responses to vaccine antigens. To assess safety and maximal tolerated dose of an adenoviral vaccine vector in volunteers without prior immunity, we evaluated a recombinant replication-defective adenovirus type 5 (rAd5) vaccine expressing HIV-1 Gag, Pol, and multiclade Env proteins, VRC-HIVADV014-00-VP, in a randomized, double-blind, dose-escalation, multicenter trial (HVTN study 054) in HIV-1-seronegative participants without detectable neutralizing antibodies (nAb) to the vector. As secondary outcomes, we also assessed T-cell and antibody responses. METHODOLOGY/PRINCIPAL FINDINGS: Volunteers received one dose of vaccine at either 10(10) or 10(11) adenovector particle units, or placebo. T-cell responses were measured against pools of global potential T-cell epitope peptides. HIV-1 binding and neutralizing antibodies were assessed. Systemic reactogenicity was greater at the higher dose, but the vaccine was well tolerated at both doses. Although no HIV infections occurred, commercial diagnostic assays were positive in 87% of vaccinees one year after vaccination. More than 85% of vaccinees developed HIV-1-specific T-cell responses detected by IFN-γ ELISpot and ICS assays at day 28. T-cell responses were: CD8-biased; evenly distributed across the three HIV-1 antigens; not substantially increased at the higher dose; and detected at similar frequencies one year following injection. The vaccine induced binding antibodies against at least one HIV-1 Env antigen in all recipients. CONCLUSIONS/SIGNIFICANCE: This vaccine appeared safe and was highly immunogenic following a single dose in human volunteers without prior nAb against the vector. TRIAL REGISTRATION: ClinicalTrials.gov NCT00119873.

Functional peptidomics of amphibian skin secretion: A novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina senegalensis

Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete "families", some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function. Among the components of these secretory peptidomes is an array of protease inhibitors. Inhibitors of trypsin are of widespread occurrence in different taxa and are representative of many established structural classes, including Kunitz, Kazal and Bowman-Birk. However, few protease inhibitors with activity against other specific proteases have been described from this source. Here we report for the first time, the isolation and structural characterisation of an inhibitor of chymotrypsin of Kunitz-type from the skin secretion of the African hyperoliid frog, Kassina senegalensis. To this end, we employed a functional peptidomic approach. This scheme involves fractionation of the peptidome, functional end-point screening, structural characterisation of resultant actives followed by molecular cloning of biosynthetic precursor-encoding cDNA(s). The novel mature and active polypeptide identified consisted of 62 amino acid residues (average molecular mass 6776.24 Da), of which 6 were positionally-conserved cysteines. The P(1) position within the active site was occupied by a phenylalanyl residue. Bioinformatic analysis of the sequence using BLAST, revealed a structural similarity to Kunitz-type chymotrypsin inhibitors from other organisms, ranging from silkworms to snakes.

Identification and molecular cloning of a novel amphibian Bowman Birk-type trypsin inhibitor from the skin of the Hejiang Odorous Frog; Odorrana hejiangensis.

In this study, an amphibian (Odorrana hejiangensis) skin extract was fractionated by reverse phase HPLC and fractions were screened for trypsin inhibitory activity. Using this initial approach, a novel trypsin inhibitory peptide was detected with an apparent protonated molecular mass of 1804.83Da, as determined by MALDI-TOF mass spectrometry. It was named Hejiang trypsin inhibitor (HJTI) in accordance. The primary structure of the biosynthetic precursor of HJTI was deduced from a cDNA sequence cloned from a skin-derived cDNA library. The primary structure of the encoded predicted mature active peptide was established as: GAPKGCWTKSYPPQPCS (non-protonated monoisotopic molecular mass - 1802.81Da). On the basis of this unequivocal amino acid sequence, a synthetic replicate was synthesized by solid phase Fmoc chemistry. This replicate displayed a moderately potent trypsin inhibition with a K(i) of 388nM. Bioinformatic analysis of the primary structure of this peptide indicated that it was a member of the Bowman-Birk family of protease inhibitors. The substitutions of Gln-14 and Ser-17 by Lys, resulted in an increase in cationicity and a small increase in potency to a K(i) value of 218nM. Neither HJTI nor its synthetic analog, possessed any significant antimicrobial activity.

A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas.

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.

pLR-HL: a novel amphibian Bowman-Birk-type trypsin inhibitor from the skin secretion of the broad-folded frog, Hylarana latouchii

In this study, we report a novel heptadecapeptide (LIGGCWTKSIPPKPCLV) of the pLR/ranacyclin family, named pLR-HL, whose structure was deduced from its biosynthetic precursor-encoding cDNA cloned from the skin secretion-derived cDNA library of the broad-folded frog, Hylarana latouchii, by employing a "shotgun" cloning technique. It contains a disulphide loop between Cys5 and Cys15 which is consistent with Bowman-Birk-type protease inhibitors. The primary structure of pLR-HL deduced from the cDNA sequence was confirmed by fractionating the skin secretion using reverse phase HPLC and subsequent analysis using MALDI-TOF mass spectrometry and LC/MS/MS fragmentation sequencing. On the basis of the establishment of unequivocal amino acid sequence, a synthetic replicate was synthesised by solid-phase Fmoc chemistry, and it displayed a moderately potent trypsin inhibition with a Ki of 143 nM. The substitution of Lys-8 by Phe (Phe8 -pLR-HL) resulted in abolition of trypsin inhibition but generation of modest inhibition on chymotrypsin with a Ki of 2.141 μM. Additionally, both the disulphide loops of pLR-HL and Phe8 -pLR-HL were synthesised and tested. Both of the catalytic loops retained similar inhibitory potencies towards trypsin or chymotrypsin in comparison with the original intact molecules. Thus, the replacement of reactive site residues could alter the specificity of these protease inhibitors, while the canonical reactive loop alone can independently constitute biologically-active moiety.

Type 5 Design H Port Side; KI Luckenbach

Print No:77.

Type 5 Design H Starboard Side; KI Luckenbach

Print No:69.

Type 5 Design G Port Side; KI Luckenbach

Print No:200. ; Initials Lower Right: ELW (Everett Longley Warner)

Type 5 Design G Starboard Side; KI Luckenbach

Print No:74.; Initials Lower Right: ELW (Everett Longley Warner)