RESUMO
Infusion site reactions are common following subcutaneous infusion of drugs. Such reactions can lead to discontinuation of the treatment. Therefore, assessment of such reactions is essential during preclinical safety studies, and magnetic resonance imaging (MRI) can assist in evaluation. Here, in vivo and ex vivo MRI evaluations were used in addition to classical histopathology to assess the infusion site reaction to ND0701, a novel formulation of apomorphine base developed for the treatment of Parkinson's disease, in comparison to the commercial apomorphine hydrochloride (HCl) formulation. Both formulations, each at two concentrations, were continuously administered subcutaneously for 20 hr to each of 3 male and 3 female domestic pigs. Based on MRI evaluations, there was a gradual decrease in the volume of the subcutaneous lesions over 4 weeks, with smaller lesions and quicker resolution with ND0701 at concentrations 2.5- to 5-fold higher when compared to the commercial apomorphine HCl formulation. Histopathological evaluation of ND0701 revealed only minimal inflammation at the sites of infusion, whereas the commercial apomorphine HCl caused persistent inflammatory reactions and necrosis. This study provides support to the use of MRI in preclinical testing of subcutaneous drugs when evaluating local site reactions.
Assuntos
Antiparkinsonianos/efeitos adversos , Apomorfina/efeitos adversos , Reação no Local da Injeção/diagnóstico por imagem , Injeções Subcutâneas/efeitos adversos , Imageamento por Ressonância Magnética , Animais , Antiparkinsonianos/administração & dosagem , Apomorfina/administração & dosagem , Biomarcadores/sangue , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Masculino , Doença de Parkinson/tratamento farmacológico , SuínosRESUMO
A 90-day study in Göttingen minipigs was conducted to test the local tolerability and systemic toxicity of ND0612, a novel aqueous solution of carbidopa (CD)/levodopa (LD) intended for the treatment of Parkinson's disease by continuous subcutaneous administration using a discrete infusion pump. To evaluate tissue site reactions, we used a unique study design involving multiple infusion sites to evaluate the effect of dose per site (270/63, 360/45, and 360/84 mg LD/CD), volume of infusion per site (4.5 and 6 ml per site), formulation concentration (60/14 and 60/7.5 mg/ml LD/CD), daily rate of infusion per site (240 µl/hr for16 hr and 80 µl/hr for 8 hr, 320 µl/hr for 16 hr and 100 µl/hr for 8 hr, or 750 µl/hr for 8 hr), frequency (once every 5, 10, 15, or 20 days), and number of infusions (4, 6, or 9) to the same infusion site. No systemic adverse effects were observed. Histopathological changes at infusion sites started with localized minimal necrosis and acute inflammation that progressed to subacute and chronic inflammatory and reparative changes with evidence of progressive recovery following the final infusion. None of the infusion site effects were judged to be adverse, and clinical exposures to ND0612 are not expected to result in adverse responses.
Assuntos
Carbidopa/toxicidade , Agonistas de Dopamina/toxicidade , Tolerância a Medicamentos , Reação no Local da Injeção/etiologia , Levodopa/toxicidade , Animais , Carbidopa/administração & dosagem , Carbidopa/sangue , Agonistas de Dopamina/administração & dosagem , Agonistas de Dopamina/sangue , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Feminino , Infusões Subcutâneas , Reação no Local da Injeção/patologia , Levodopa/administração & dosagem , Levodopa/sangue , Masculino , Necrose , Suínos , Porco Miniatura , Testes de Toxicidade CrônicaRESUMO
Amyloid deposits are pathological hallmark of a large group of human degenerative disorders of unrelated etiologies. While accumulating evidence suggests that early oligomers may account for tissue degeneration, most detection tools do not allow the monitoring of early association events. Here we exploit bimolecular fluorescence complementation analysis to detect and quantify the dimerization of three major amyloidogenic polypeptides; islet amyloid polypeptide, ß-amyloid and α-synuclein. The constructed systems provided direct visualization of protein-protein interactions in which only assembled dimers display strong fluorescent signal. Potential inhibitors that interfere with the initial intermolecular interactions of islet amyloid polypeptide were further identified using this system. Moreover, the identified compounds were able to inhibit the aggregation and cytotoxicity of islet amyloid polypeptide, demonstrating the importance of targeting amyloid dimer formation for future drug development.
Assuntos
Amiloide/química , Multimerização Proteica , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/ultraestrutura , Descoberta de Drogas , Fluorescência , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Modelos Moleculares , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/patologia , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/química , alfa-Sinucleína/ultraestruturaRESUMO
The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.
Assuntos
Antiparkinsonianos/química , Barreira Hematoencefálica/efeitos dos fármacos , Manitol/química , Doença de Parkinson/tratamento farmacológico , alfa-Sinucleína/metabolismo , Amiloide/antagonistas & inibidores , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Animais , Antiparkinsonianos/farmacologia , Benzotiazóis , Drosophila , Feminino , Corantes Fluorescentes/química , Proteínas de Choque Térmico HSP70/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Locomoção , Masculino , Manitol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Neocórtex/metabolismo , Neocórtex/patologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Tiazóis/química , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/química , alfa-Sinucleína/ultraestruturaRESUMO
BACKGROUND: The oligomeriztion of α-synuclein (α-syn) into ordered assemblies is associated with the symptoms of Parkinson's Disease (PD). Yet, it is still debatable whether oligomers are formed as part of a multistep process towards amyloid fibril formation or alternatively as "off-pathway" aggregates. METHODS: 100µM α-syn was incubated with decreasing amounts of cinnamon extract precipitation (CEppt). The fibril formation was measured using spectroscopy and microscopy analyses and oligomers were detected using western blot analysis. The secondary structure of the protein was analyzed using CD. Drosophila brains were studied using immunostaining and confocal microscopy. RESULTS: Here we probed the inhibition pattern of oligomeric and fibrillar forms of α-syn, using a natural substance, CEppt which was previously shown to effectively inhibit aggregation of ß-amyloid polypeptide. We demonstrated that CEppt has a differential inhibitory effect on the formation of soluble and insoluble aggregates of α-synuclein in vitro. This inhibition pattern revokes the possibility of redirection to "off-pathway" oligomers. When administering to Drosophila fly model expressing mutant A53T α-syn in the nervous system, a significant curative effect on the behavioral symptoms of the flies and on α-syn aggregation in their brain was observed. CONCLUSIONS: We conclude that CEppt affects the process of aggregation of α-syn without changing its secondary structure and suggest that increasing amounts of CEppt slow this process by stabilizing the soluble oligomeric phase. When administered to Drosophila fly model, CEppt appears to have a curative effect on the defective flies. GENERAL SIGNIFICANCE: Our results indicate that CEppt can be a potential therapeutic agent for PD.
Assuntos
Amiloide/antagonistas & inibidores , Cinnamomum zeylanicum , Drosophila , Doença de Parkinson/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Amiloide/genética , Amiloide/metabolismo , Animais , Animais Geneticamente Modificados , Células CHO , Cinnamomum zeylanicum/química , Cricetinae , Cricetulus , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Drosophila/genética , Feminino , Humanos , Masculino , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fitoterapia/métodos , Multimerização Proteica/genética , alfa-Sinucleína/genéticaRESUMO
Amyloid formation is associated with several human diseases including Alzheimer's disease (AD), Parkinson's disease, Type 2 Diabetes, and so forth, no disease modifying therapeutics are available for them. Because of the structural similarities between the amyloid species characterizing these diseases, (despite the lack of amino acid homology) it is believed that there might be a common mechanism of toxicity for these conditions. Thus, inhibition of amyloid formation could be a promising disease-modifying therapeutic strategy for them. Aromatic residues have been identified as crucial in formation and stabilization of amyloid structures. This finding was corroborated by high-resolution structural studies, theoretical analysis, and molecular dynamics simulations. Amongst the aromatic entities, tryptophan was found to possess the most amyloidogenic potential. We therefore postulate that targeting aromatic recognition interfaces by tryptophan could be a useful approach for inhibiting the formation of amyloids. Quinones are known as inhibitors of cellular metabolic pathways, to have anti- cancer, anti-viral and anti-bacterial properties and were shown to inhibit aggregation of several amyloidogenic proteins in vitro. We have previously described two quinone-tryptophan hybrids which are capable of inhibiting amyloid-beta, the protein associated with AD pathology, both in vitro and in vivo. Here we tested their generic properties and their ability to inhibit other amyloidogenic proteins including α-synuclein, islet amyloid polypeptide, lysozyme, calcitonin, and insulin. Both compounds showed efficient inhibition of all five proteins examined both by ThT fluorescence analysis and by electron microscope imaging. If verified in vivo, these small molecules could serve as leads for developing generic anti-amyloid drugs.
Assuntos
Proteínas Amiloidogênicas/antagonistas & inibidores , Proteínas Amiloidogênicas/química , Naftoquinonas/química , Triptofano/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Calcitonina/química , Humanos , Insulina/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Muramidase/química , alfa-Sinucleína/químicaRESUMO
OBJECTIVES: Although commercially available levodopa (LD) formulations include carbidopa (CD) or benserazide for gastrointestinal L-aromatic amino acid decarboxylase inhibition, little is known how manipulating CD delivery affects the pharmacokinetics of LD. Our research systematically evaluated the peripheral and central pharmacokinetics of LD during continuous subcutaneous CD delivery. METHODS: We conducted pharmacokinetic experiments in pigs, mice, and humans to characterize effects of continuous subcutaneous CD delivery co-administered with LD as compared with oral LD/CD administration on LD pharmacokinetics. The porcine and human studies compared peripheral LD pharmacokinetic parameters (area under the curves [AUCs], peak plasma concentrations [Cmax], and plasma elimination half-life [t1/2]) and the mouse studies compared brain LD and dopamine concentrations. RESULTS: In the pig, supplementary subcutaneous CD delivery significantly increased the LD t1/2 and AUC versus LD/CD alone and versus additional oral CD administration. In mice, administration of supplementary subcutaneous CD substantially increased mean plasma concentrations of both LD and CD versus oral LD/CD alone at all time points. These increases were mirrored by increased brain dopamine levels for at least the 7 hours of study. In healthy human subjects, continuous subcutaneous CD administration, 3.33 mg/h x24h, increased the plasma LD t1/2, Cmax, and AUC by 17.4%, 40.5%, and 22.3%, respectively (P < 0.003). CONCLUSIONS: This series of studies demonstrates that small continuous dosing of subcutaneous CD has an unexpected effect on LD pharmacokinetics greater than the extent of decarboxylase inhibition achieved by additional oral CD administration.
Assuntos
Antiparkinsonianos/administração & dosagem , Inibidores das Descarboxilases de Aminoácidos Aromáticos/administração & dosagem , Carbidopa/administração & dosagem , Levodopa/farmacocinética , Adulto , Animais , Quimioterapia Combinada , Feminino , Voluntários Saudáveis , Humanos , Levodopa/administração & dosagem , Levodopa/sangue , Masculino , Camundongos , Pessoa de Meia-Idade , SuínosRESUMO
Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy.
Assuntos
Anticorpos Neutralizantes/farmacologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Multimerização Proteica , Anticorpos/imunologia , Anticorpos Neutralizantes/imunologia , Autoanticorpos/imunologia , Autoanticorpos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/imunologia , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
Parkinson's disease is a common neurodegenerative disorder with the pathology of α-synuclein aggregation in Lewy bodies. Currently, there is no available therapy that arrests the progression of the disease. Therefore, the need of animal models to follow α-synuclein aggregation is crucial. Drosophila melanogaster has been researched extensively as a good genetic model for the disease, with a cognitive phenotype of defective climbing ability. The assay for climbing ability has been demonstrated as an effective tool for screening new therapeutic agents for Parkinson's disease. However, due to the assay's many limitations, there is a clear need to develop a better behavioral test. Courtship, a stereotyped, ritualized behavior of Drosophila, involves complex motor and sensory functions in both sexes, which are controlled by large number of neurons; hence, behavior observed during courtship should be sensitive to disease processes in the nervous system. We used a series of traits commonly observed in courtship and an additional behavioral trait-nonsexual encounters-and analyzed them using a data mining tool. We found defective behavior of the Parkinson's model male flies that were tested with virgin females, visible at a much younger age than the climbing defects. We conclude that this is an improved behavioral assay for Parkinson's model flies.
RESUMO
The development of generic inhibitors in order to control the formation of amyloid fibrils and early oligomers is still an unmet medical need. As it is hypothesized that amyloid assemblies represent a generic protein supramolecular structure of low free energy, targeting the key molecular recognition and self-assembly events may provide the route for the development of such potential therapeutic agents. We have previously demonstrated the ability of hybrid molecules composed of an aromatic moiety and the α-aminoisobutyric acid ß-sheet breaker elements to act as excellent inhibitors of amyloid fibril formation. Specifically, the D-Trp-Aib was shown to be a superb inhibitor of the formation of Alzheimer's disease ß-amyloid fibrils and oligomers both in vitro and in vivo. Here, we demonstrate that the rationally designed molecule has the generic ability to inhibit amyloid fibril formation by calcitonin, α-synuclein, and the islet amyloid polypeptide. Moreover, we demonstrate the inability of two modified peptides, D-Ala-Aib and D-Trp-Ala, to inhibit and disassemble amyloid fibril formation, a fact that provides an additional evidence for the suggested structural basis of the inhibitor activity. Taken together, we believe that the use of ß-breaker elements combined with aromatic moiety may present a promising approach for the development of fibrillization inhibition drug candidate.
Assuntos
Aminoácidos Aromáticos/química , Peptídeos beta-Amiloides/antagonistas & inibidores , Dipeptídeos/química , Desenho de Fármacos , Fragmentos de Peptídeos/química , Multimerização Proteica , Peptídeos beta-Amiloides/genética , Bactérias/genética , Dipeptídeos/síntese química , Fragmentos de Peptídeos/síntese química , Conformação ProteicaRESUMO
The distribution of endomorphins (EM) 1 and 2 in the human brain inversely correlates with cerebral neurodegeneration in Alzheimer's disease (AD), implying a protective role. These endogenous opioid peptides incorporate aromatic residues and a ß-breaker motif, as seen in several optimized inhibitors of Aß aggregation. The activity of native endomorphins was studied, as well as the rationally designed analogue Aib-1, which includes a remarkably efficient ß-breaker, α-aminoisobutyric acid (Aib). In vitro and GFP fusion protein assays showed that Aib-1 interacted with Aß and markedly inhibited the formation of toxic oligomer and fibril growth. Moreover, Aib-1 prevented the toxicity of Aß toward neuronal PC12 cells and markedly rectified reduced longevity of an AD fly model. Atomistic simulations and NMR-derived solution structures revealed that Aib-1 significantly reduced the propensity of Aß to aggregate due to multimode interactions including aromatic, hydrophobic, and polar contacts. We suggest that hindering the self-assembly process by interfering with the aromatic core of amyloidogenic peptides may pave the way toward developing therapeutic agents to treat amyloid-associated diseases.
Assuntos
Peptídeos beta-Amiloides/química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Multimerização Proteica/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Animais , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Drosophila melanogaster/efeitos dos fármacos , Feminino , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Células PC12 , Fragmentos de Peptídeos/metabolismo , Estabilidade Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we identified molecular interaction domains within the ß-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease.