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PnPP-19 peptide has a primary sequence design based on molecular modeling studies of PnTx2-6 toxin. It comprises the amino acid residues that are potentially significant for the pharmacological action of PnTx2-6. Ex vivo and in vivo experiments in normotensive, hypertensive, or diabetic murine models have shown a significant improvement in penile erection after administration of PnPP-19. Given the potential use of PnPP-19 in pharmaceutical formulations to treat erectile dysfunction and the lack of information concerning its mode of action, the present work investigates its activities on the nitrergic system. PnPP-19 induced a significant increase in nitric oxide (NO) and cGMP levels in corpus cavernosum (cc). These effects were inhibited by l-NAME, a non-selective inhibitor of nitric oxide synthase (NOS); were partially inhibited by 7- Nitroindazole, a selective inhibitor of neuronal NOS (nNOS); and were abolished by L-NIL, a selective inhibitor of inducible NOS (iNOS). This potentiating effect was not affected by atropine. PnPP-19 also led to changes in mRNA levels, protein expression and phosphorylation at specific sites of NOS, in cc. Assays using cavernous tissue from knockout mice to endothelial NOS (eNOS), nNOS or iNOS showed that PnPP-19 potentiates relaxation only in eNOS-knockout mice, which suggests an essential role for nNOS. Surprisingly, iNOS enhanced the potentiation of erectile function evoked by PnPP-19. Our results demonstrate that this new synthetic peptide potentiates erectile function via nitric oxide activation and reinforce its role as a new pharmacological tool for the treatment of erectile dysfunction.

Introduction With the aim of overcoming the high toxicity of PnTx2-6 (or δ-CNTX-Pn2a), a toxin from the venom of the armed spider (Phoneutria nigriventer), the 19-aminoacid peptide, PnPP-19 (P nigriventer potentiator peptide), was synthesized based on molecular modeling studies of PnTx2-6. PnPP-19 improved the erectile function of normotensive rats and mice, without eliciting side effects, and no signs of toxicity were observed. In addition, PnPP-19 was able to potentiate the effect of sildenafil. Aim To evaluate the efficacy of PnPP-19 in hypertensive and diabetic mouse/rat models in restoring erectile function, after topical administration; verify the biodistribution of PnPP-19 administration (topical and intravenous), permeation, and cyclic guanosine monophosphate (cGMP)/nitric oxide via implication. Methods Corpus cavernosum relaxation was evaluated using cavernous strips from male spontaneous hypertensive rats (SHR) and from streptozotocin (STZ)-diabetic mice contracted with phenylephrine and submitted to electrical field stimulation before and after incubation with PnPP-19 (10−8 mol/L, 10 minutes) or vehicle. This procedure was also used to determine cGMP/nitric oxide levels, at 8 Hz and to check the effect of PnPP-19 with sildenafil citrate. Biodistribution assays were performed using iodine 123–radiolabeled PnPP-19. In vivo erectile function was evaluated using intracavernosal pressure/main arterial pressure ratio in STZ-diabetic rats after PnPP-19 topical administration. Main Outcome Measures PnPP-19 may become a new drug able to fill the gap in the pharmacologic treatment of erectile dysfunction, especially for hypertensive and diabetic individuals Results PnPP-19 potentiated corpus cavernosum relaxation, in both control and SHR rats. SHR-cavernosal tissue treated with PnPP-19 (1–32 Hz) reached the same relaxation levels as control Wistar rats (16 and 32 Hz). PnPP-19 treatment improved cavernosal tissue relaxation in STZ-diabetic mice and rats. PnPP-19 enhanced cGMP levels in STZ-diabetic mice corpus cavernosum strips. After topical or intravenous administration in rats, 123I-PnPP-19 was mainly recruited to the penis. When topically administered (400 μg/rat), PnPP-19 restores erectile function in STZ-diabetic rats, also improving it in healthy rats by increasing the intracavernosal pressure/main arterial pressure ratio. PnPP-19 exhibited an additive effect when co-administered with sildenafil, showing a novel mode of action regardless of phosphodiesterase type 5 inhibition. Clinical Implications PnPP-19 seems to be an indicated drug to be tested to treat ED in diabetic and hypertensive patients. Strength & Limitations PnPP-19, although active by topical application and showing safety to human beings (not shown), has low permeability, about 10% of the applied dose. Conclusion Our results showed that PnPP-19 may emerge as a potent new drug that can be topically administered, becoming a promising alternative for erectile dysfunction treatment.

Background PnPP-19 is a 19-amino-acid synthetic peptide previously described as a novel drug for the treatment of erectile dysfunction. Objective The aim of this work was to evaluate the physicochemical properties of cationic transfersomes containing PnPP-19 and the skin permeation of free PnPP-19 and PnPP-19-loaded transfersomes. Methods Three different liposomal preparation methods were evaluated. Cationic transfersomes contained egg phosphatidyl choline: stearylamine (9:1 w/w) and Tween 20 (84.6:15.4 lipid:Tween, w/w). Lipid concentration varied from 20 to 40 mM. We evaluated the entrapment percentage, mean diameter, zeta potential and stability at 4 °C of the formulations. The skin permeation assays were performed with abdominal human skin using Franz diffusion cell with 3 cm2 diffusion area at 32 °C and a fluorescent derivative of the peptide, containing 5-TAMRA, bound to PnPP-19 C-terminal region, where an extra lysine was inserted. Results Our results showed variable entrapment efficiencies, from 6% to 30%, depending on the preparation method and the lipid concentration used. The reverse phase evaporation method using a total lipid concentration equal to 40 mM led to the best entrapment percentage (30.2 + 4.5%). Free PnPP-19 was able to permeate skin at a rate of 10.8 ng/cm2/h. However, PnPP-19 was specifically hydrolyzed by skin proteases, generating a fragment of 15 amino acid residues. Encapsulated PnPP-19 permeated the skin at a rate of 19.8 ng/cm2/h. Conclusion The encapsulation of PnPP-19 in cationic transfersomes protected the peptide from degradation, favoring its topical administration.

Antimicrobial peptides (AMPs) have been found in all organism taxa and may play an essential role as a host defense system. AMPs are organized in various conformations, such as linear peptides, disulfide bond-linked peptides, backbone-linked peptides and circular peptides. AMPs apparently act primarily on the plasma membrane, although an increasing number of works have shown that they may also target various intracellular sites. Spider venoms are rich sources of biomolecules that show several activities, including modulation or blockage of ion channels, anti-insect, anti-cancer, antihypertensive and antimicrobial activities, among others. In spider venoms from the Lycosidae family there are many linear AMPs with a wide range of activities against several microorganisms. Due to these singular activities, some Lycosidae AMPs have been modified to improve or decrease desirable or undesirable effects, respectively. Such modifications, especially with the aim of increasing their antibiotic activity, have led to the filing of many patent applications. This review explores the abundance of Lycosidae venom AMPs and some of their derivatives, and their use as new drug models.

A Parte I deste trabalho descreve o desenvolvimento de uma formulação lipossomal contendo PnPP-19, derivado da toxina PnTx2-6 da aranha Phoneutria nigriventer, para o tratamento tópico da disfunção erétil (DE). PnPP-19 é um peptídeo sintético de 19 aminoácidos, previamente descrito como uma nova substância bioativa para o tratamento da disfunção erétil. O objetivo desse trabalho foi avaliar as propriedades físico-químicas de lipossomas deformáveis catiônicos contendo PnPP-19 e a permeação cutânea de PnPP19, tanto livre quanto encapsulado nesses lipossomas. Três preparações lipossomais diferentes foram avaliadas. Os lipossomas deformáveis catiônicos continham fosfatidilcolina de ovo, estearilamina e Tween 20. A concentração lipídica variou de 20 mM a 40 mM. O teor de encapsulação, o diâmetro médio, o potencial zeta e a estabilidade a 4oC das formulações foram avaliados. Os experimentos de permeação cutânea foram realizados com pele abdominal humana em células de Franz, com área de difusão de 3 cm2 a 32ºC. Um derivado fluorescente do peptídeo, contendo 5-TAMRA ligado à região Cterminal de PnPP-19, onde uma lisina adicional foi inserida, foi utilizado para estes experimentos. Os resultados mostram que os diferentes métodos de preparação apresentaram diferentes teores de encapsulação, de 6% a 30%, e o método de evaporação em fase reversa na concentração lipídica de 40 mM apresentou o melhor teor de encapsulação (30,2 + 4,5 %). PnPP-19 livre foi capaz de permear a pele, na velocidade de 10,8 ng/cm2/h. Porém, PnPP-19 sofreu possivelmente uma hidrólise específica pelas enzimas proteolíticas presentes na pele, gerando um fragmento de 15 resíduos de aminoácidos. PnPP-19 encapsulado permeou a pele na velocidade de 12,6 ng/cm2/h. A encapsulação de PnPP-19 em lipossomas deformáveis catiônicos protegeu o peptídeo contra degradação, favorecendo sua administração tópica. O desenvolvimento de fármacos a partir da exploração da biodiversidade brasileira vem passando por uma fase de regulamentação no Brasil. A aplicação da medida provisória MP 2.186-16, de agosto de 2001, revogada pela lei Nº 13.123, de 20 de maio de 2015, gerou discussões importantes, com reflexos na produção científica e tecnológica brasileira. Nesse contexto, a Parte II do presente trabalho compreende o levantamento das patentes produzidas no Brasil e no exterior, entre 2000 e 2011, que descrevem fármacos ou medicamentos desenvolvidos a partir de venenos e toxinas da fauna brasileira. Nossos resultados mostraram que apenas 8 gêneros da fauna brasileira estão envolvidos em documentos de patente. As invenções geradas envolvem, em sua maioria, composições antitumorais, antimicrobianas, vacinas e agentes hipotensivos. Os inventores brasileiros detêm 49% dos documentos de patente, sendo os outros documentos depositados por inventores da América do Norte e Europa. Desta forma, ainda há um grande arsenal de moléculas provenientes de venenos da rica fauna brasileira a serem exploradas para o desenvolvimento de novas substânias bioativas. As leis brasileiras que regem a investigação e a exploração comercial de produtos provenientes do patrimônio genético brasileiro são também discutidas. In Part I, this work describes the development of a liposomal formulation containing PnPP-19, derived from the toxin PnTx2-6 from Phoneutria nigriventer spider, for the topical treatment of erectile dysfunction (ED). PnPP-19 is a 19-amino-acid synthetic peptide previously described as a novel bioactive compound for the treatment of erectile dysfunction. The aim of this work was to evaluate the physicochemical properties of cationic transfersomes containing PnPP-19 and the skin permeation of PnPP-19, whether free or encapsulated. Three different liposomal preparation methods were evaluated. Cationic transfersomes contained egg phosphatidyl choline, stearylamine and Tween 20. Lipid concentration varied from 20mM to 40mM. We evaluated the entrapment efficiency, mean diameter, zeta potential and stability at 4oC of the formulations. The skin permeation assays were performed with abdominal human skin using Franz diffusion cell with 3 cm2 diffusion area at 32oC and a fluorescent derivative of the peptide, containing 5-TAMRA, bound to PnPP-19 C-terminal region, where an extra lysine was inserted. Our results showed variable entrapment efficiencies, from 6% to 30%, depending on the preparation method and the lipid concentration used, and reversed phase evaporation at 40 mM lipid concentration led to the best entrapment efficiency (30,2 + 4,5 %). Free PnPP-19 was able to permeate skin at a rate of 10.8 ng/cm2/h. However, PnPP-19 was specifically hydrolyzed by skin proteases, generating a fragment of 15 amino acid residues. Encapsulated PnPP-19 permeated skin at a rate of 12.6 ng/cm2/h. The encapsulation in cationic transfersomes protected the peptide from degradation, favoring its topical administration. The use of drugs developed from the exploitation of the Brazilian biodiversity, as in the present work, is in a stage of regulamentation in Brazil. The provisional measure MP 2.186-16, into force since August 2001, revogated by the law 13.123, on May 20, 2015, was extensively questioned by Brazilian researchers, and has affected scientific and technological production. In this context, Part II involves a search at patent banks for inventions developed in Brazil and abroad, filed between 2000 and 2011, that describe drugs or medicines developed after venoms, toxins or their derivatives from the Brazilian fauna. Our results show that only 8 Brazilian genera are involved in patent applications. The inventions mostly involve anticancer compositions, antimicrobials, vaccines and hypotensives. Brazilian inventors hold 49% of the patent applications, and the other applications were filed by American and European inventors. Therefore, there is still a great arsenal of venom molecules from the rich Brazilian fauna to be explored for the development of new bioactive compounds. Brazilian laws that rule the investigation and commercial exploitation of products comprising the use of Brazilian genetic resources are also discussed herein.

We designed a peptide, PnPP-19, comprising the potential active core of the Phoneutria nigriventer native toxin PnTx2-6. We investigated its role on erectile function, and its toxicity and immunogenicity.Erectile function was evaluated by the intracavernous pressure-to-mean arterial pressure ratio during electrical field stimulation on rat pelvic ganglia. Cavernous strips were contracted with phenylephrine and relaxation was induced by electrical field stimulation with or without PnPP-19 (10(-8) M). Activity on sodium channels was evaluated by electrophysiological screening of transfected channels on Xenopus oocytes and dorsal root ganglion cells. Antibodies were detected by indirect enzyme-linked immunosorbent assay in mice previously treated with the peptide. Histopathological studies were performed with mouse organs treated with different doses of PnPP-19.PnPP-19 was able to potentiate erection at 4 and 8 Hz in vivo and ex vivo. It showed no toxicity and low immunogenicity in mice, and did not affect sodium channels or rat hearts. PnPP-19 increased cyclic guanosine monophosphate levels at 8 Hz. This effect was inhibited by L-NAME (10(-4) M). Erectile function was partially inhibited by 7-nitroindazole (10(-5) M), a selective inhibitor of neuronal nitric oxide synthase.PnPP-19 potentiates erection in vivo and ex vivo via the nitric oxide/cyclic guanosine monophosphate pathway. It does not affect sodium channels or rat hearts and shows no toxicity and low immunogenicity. These findings make it a promising candidate as a novel drug in the therapy of erectile dysfunction.

The venom of Phoneutria nigriventer spider has been extensively studied. It is highly neurotoxic, with clinical manifestations occurring a few minutes after the bite, mainly in children. Among the intoxication signs that may lead to neurogenic shock are agitation, somnolence, sweating, nausea, profuse vomiting, lacrimation, excessive salivation, hypertension, tachycardia, tachypnea, tremors, muscle spasms, and priapism. Many toxins from this venom have been purified and characterized for their structure and/or function. The most studied Phoneutria nigriventer toxins are PnTx1, PnTx2-5, and PnTx2-6, which show complex effects on sodium channel kinetics. PnTx2-5 and PnTx2-6 are both described as responsible for priapism; PnTx4(6-1) and PnTx4(5-5) show insecticidal activities and act on insect sodium channels; PnTkPs are muscle-active peptides; and nigriventrine is a piperidine derivative that has neuroactive properties and causes convulsive spells. PnTx3-3 and PnTx3-4 toxins were demonstrated to be effective on preventing cell death after ischemia injury. On the other hand, PnTx3-6 was shown to be efficient in the treatment of persistent pathological pain. The present chapter compiles biochemical, physiological, and pharmacological studies of fractions and purified toxins from Phoneutria nigriventer venom, showing their great potential as new tools for pharmacological studies and drug development.

Enzymes with gelatinolytic activity were detected in Tityus bahiensis and Tityus serrulatus venom. Their activity was optimal at pH 8.0 in SDS-PAGE-gelatin. They were inhibited by PMSF but not by iodoacetamide, pepstatin or phenantrolin in the assay conditions used. This suggests that these enzymes are serine proteases. The presence of metal ions did not affect the proteolytic activity of these enzymes. Several possible functions may be envisaged for these enzymes: in tissue permeabilization, pancreatitis and toxin processing.

Venom variability in specimens of Tityus serrulatus scorpion was assessed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analyses. An expanded time lag venom extraction protocol was carried out using ten scorpions to study individual variations that might occur due to different rates in protein expression and/or processing. The first extraction of venom was made from the animals after 20 days of starvation, which allowed the venom gland to be filled up. The second extraction event was carried out 24 hours after the first one. The third was 8 days after the first extraction. By means of MALDI-TOF analyses, important variations were observed in venoms of a single specimen extracted at different times, especially in latter extraction events. These variations are most probably related to dynamics in cell gland production. Since T. serrulatus is a parthenogenetic species, sexual variations are naturally excluded and we did not expect intra-specific variations, which was confirmed. Knowledge of individual venom variability is extremely important to avoid misunderstandings in the use of venom proteomic analysis as a taxonomic tool.

A alopécia é uma doença caracterizada por perda de cabelo, que pode ter fisiopatologia autoimune ou relacionada ao metabolismo hormonal de andrógenos, sendo denominada alopécia areata e androgenética, respectivamente. Os tratamentos medicamentosos descritos têm se mostrado eficazes na indução do crescimento capilar. Porém, é necessário o uso contínuo, sendo relatado reaparecimento dos sintomas caso o tratamento seja interrompido, não sendo raros os casos em que o tratamento é descontinuado em função de efeitos adversos graves. Nesse trabalho foi formulado e caracterizado um gel-creme transdérmico, contendo uma substância química vasodilatadora, cuja readequação farmacológica é uma proposta de nova alternativa para proporcionar aos pacientes um tratamento mais eficaz. A caracterização fisíco-química, contemplou aferição de cremação, pH, densidade, viscosidade, espalhabilidade, assim como doseamento e teste de liberação in vitro por método validado. Foi encontrado pH levemente ácido, compatível com a aplicação na pele, assim como perfil reológico adequado às condições de armazenamento e uso propostas. O perfil de liberação encontrado demonstra a capacidade do sistema de veiculação de cerca de 90% do ativo durante um período de aproximadamente 10 horas, com velocidade de liberação adequada para efeito local. Alopecia is a disease characterized by hair loss, whosepathophysiology can be related to hormonal metabolism of androgens or autoimmunity, being called androgenetic or areata alopecia, respectively. The drug treatments previously described have been effective in capillary growth induction. However, continuous use is required, and the symptoms tend to recur if the treatment is discontinued, and treatment is commonly discontinued because of serious adverse effects. In this work, a transdermal gel-cream containing a vasodilating chemical substance was formulated and characterized. The pharmacological re-adaptation proposed herein may be a new alternative to provide a more effective treatment option for patients. The physico-chemical characterization included determination of pH, cremation, density, viscosity, spreadability, as well as dosing and in vitro release profile tested by a validated method. A slightly acidic pH was found, which is compatible with the application on the skin, and the rheological profile was adequate for the proposed storage and use conditions. The release profile demonstrates the capacity of the delivery system to liberate about 90% of the active compound over a period of approximately 10 hours with a suitable release rate for a local effect.

A presente dissertação trata da possibilidade de aplicação da arbitragem como meio alternativo de resolução de litígios e controvérsias em contratos de transferência de tecnologia celebrados com instituições científicas e tecnológicas públicas. Foram analisados o Novo Marco Legal de Inovação e os Contratos de Transferência e Licenciamento de Tecnologia, levando em consideração o incentivo a parcerias envolvendo Instituições Científicas e Tecnológicas Públicas. Foram apresentados os aspectos gerais da nova Lei de Inovação, dos Contratos de Licenciamento e Transferência de Tecnologia, bem como a aplicação da arbitragem, demonstrando a importância de cada ponto e destacando aspectos fundamentais que possibilitam as ICTs públicas a aplicarem a arbitragem como meio alternativo de resolução de conflitos nos Contratos de Transferência e Licenciamento de Tecnologia. This dissertation deals with the possibility of application of arbitration as an alternative means of resolving disputes and controversies in technology transfer contracts with scientific and technological public institutions. The New Regulatory Framework of Innovation and Technology Transfer and Licensing Agreements were analyzed, taking into account the encouragement of partnerships involving Public Institutions of Science and Technology. The general aspects of the new Innovation Act, Technology Transfer and Licensing Agreements and the application of arbitration were presented, demonstrating the importance of each point and highlighting key aspects that enable public ICTs to apply arbitration as an alternative means for dispute resolution in Technology Transfer and Licensing Agreements.