Investigating therapeutic usage of combined Ticagrelor and Aspirin through solid-state and analytical studies

The mainstay treatment for patients with acute coronary syndrome is an oral route dual antiplatelet therapy with a P2Y12-receptor antagonist and Aspirin (ASA). To improve patient adherence to such treatments, combination therapies (polypill) are envisioned. Physicochemical solid-state studies have been carried out to develop a preformulation strategy of ASA with the P2Y12-receptor antagonist Ticagrelor (TIC). The investigations were carried out using differential scanning calorimetry, liquid chromatography-high resolution-multistage mass spectrometry (LC-HR-MSn) and as complementary techniques Fourier transform infrared measurements and thermogravimetric analysis. A simple eutectic transition at 98 °C with a mole fraction for the eutectic liquid of 0.457 has been observed and the mixing of ASA and TIC molecules in each other's crystal structures appears to be limited. No cocrystals of TIC and ASA have been found. The appearance of the eutectic liquid was linked with a clear onset of chemical instability of the two pharmaceuticals. The decomposition mechanism in the liquid phase involves prior decomposition of ASA, whose residues react with well-identified TIC interaction sites. Seven interaction products were observed by LC-HR-MSn linked to corresponding degradation products. The most important degradation pathway is N-dealkylation. In conclusion, polypills of ASA and TIC are a viable approach, but the decomposition of ASA should be avoided by eliminating high temperatures and high humidity.Peer ReviewedPreprin

Identification of Roles for Peptide: N-Glycanase and Endo-β-N-Acetylglucosaminidase (Engase1p) during Protein N-Glycosylation in Human HepG2 Cells

BACKGROUND: During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo beta-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells. METHODS/PRINCIPAL FINDINGS: During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Delta deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man(8)GlcNAc(2), corresponding to approximately 70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) that corresponds to approximately 30% of total fOS. CONCLUSIONS/SIGNIFICANCE: As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming

Simple Approach to Enhance Green Tea Epigallocatechin Gallate Stability in Aqueous Solutions and Bioavailability: Experimental and Theoretical Characterizations

International audienceBecause of its antioxidant, antimutagenic, and anti-infectious properties, epigallocatechin gallate (EGCG) is the most interesting compound among the green tea catechins polyphenols. However, its health effects are inconclusive due to its very low bioavailability, largely due to a particular instability that does not allow EGCG to reach the potency required for clinical developments. Over the last decade, many efforts have been made to improve the stability and bioavailability of EGCG using complex delivery systems such as nanotechnology, but these efforts have not been successful and easy to translate to industrial use. To meet the needs of a large-scale clinical trial requiring EGCG in a concentrated solution to anticipate swallowing impairments, we developed an EGCG-based aqueous solution in the simplest way while trying to circumvent EGCG instability. The solution was thoroughly characterized to sort out the unexpected stability outcome by combining experimental (HPLC-UV-mass spectrometry and infrared spectroscopy) and computational (density functional theory) studies. Against all odds, the EGCG–sucrose complex under certain conditions may have prevented EGCG from degradation in aqueous media. Indeed, in agreement with the ICH guidelines, the formulated solution was shown to be stable up to at least 24 months under 2–8 °C and at ambient temperature. Furthermore, considerable improvement in bioavailability in rats, against EGCG powder formulated in hard-gel capsules, was shown after gavage. Thus, the proposed formulation may provide an easily implementable platform to administer EGCG in the context of clinical development

3D-Printed, Liquid-Filled Capsules of Concentrated and Stabilized Polyphenol Epigallocatechin Gallate, Developed in a Clinical Trial

In vitro studies have shown that epigallocatechin gallate (EGCG), the most potent antioxidant of the green tea polyphenol catechins, is able to effectively prevent the formation of amyloid plaques and induce their clearance. However, its high chemical reactivity promotes high chemical instability, which represents a major obstacle for the development of pharmaceutical forms containing solubilized EGCG, an essential condition for a better systemic passage via the oral route. After discovering that EGCG forms a deep eutectic with choline chloride, we exploited this property to formulate and patent liquid-filled capsules containing 200–800 mg of soluble EGCG in easy-to-administer sizes. The gelatin envelopes used are of the conventional type and their filling has been achieved using 3D printing technology. Not only did the EGCG-choline complex allow the formulation of hydrophilic solutions with a high concentration of active substance but it also contributed significantly to its chemical stability, since after at least 18 months of storage at 25 °C/60% RH and one year at 40 °C/75% RH, the capsules show unchanged hardness, chromatographic profiles and antioxidant activity compared to T0. Preclinical studies in monkeys showed that bioavailability was increased by a factor of 10 compared to marketed capsules comprising EGCG powder. This pharmaceutical development was conducted in the context of upcoming clinical trials to evaluate EGCG alone or in combination when treating transthyretin and light-chain cardiac amyloidosis

Density functional theory and liquid chromatography-multistage mass spectrometry to characterize raltitrexed photo-degradation mechanisms

International audienc

3D-Printed, Liquid-Filled Capsules of Concentrated and Stabilized Polyphenol Epigallocatechin Gallate, Developed in a Clinical Trial

In vitro studies have shown that epigallocatechin gallate (EGCG), the most potent antioxidant of the green tea polyphenol catechins, is able to effectively prevent the formation of amyloid plaques and induce their clearance. However, its high chemical reactivity promotes high chemical instability, which represents a major obstacle for the development of pharmaceutical forms containing solubilized EGCG, an essential condition for a better systemic passage via the oral route. After discovering that EGCG forms a deep eutectic with choline chloride, we exploited this property to formulate and patent liquid-filled capsules containing 200–800 mg of soluble EGCG in easy-to-administer sizes. The gelatin envelopes used are of the conventional type and their filling has been achieved using 3D printing technology. Not only did the EGCG-choline complex allow the formulation of hydrophilic solutions with a high concentration of active substance but it also contributed significantly to its chemical stability, since after at least 18 months of storage at 25 °C/60% RH and one year at 40 °C/75% RH, the capsules show unchanged hardness, chromatographic profiles and antioxidant activity compared to T0. Preclinical studies in monkeys showed that bioavailability was increased by a factor of 10 compared to marketed capsules comprising EGCG powder. This pharmaceutical development was conducted in the context of upcoming clinical trials to evaluate EGCG alone or in combination when treating transthyretin and light-chain cardiac amyloidosis

Arccan Excellencies: a dialectical analysis of the relation between secrecy and transparency in the Legislative Power of São Paulo

Este trabalho apresenta uma discussão acerca do acesso à informação nos marcos do Poder Legislativo, com o objetivo de analisar a interação da população da cidade de São Paulo com o direito de acesso à informação, tendo como estudo de caso a Ouvidoria da Câmara Municipal de São Paulo. Utiliza como método o Materialismo Histórico-Dialético e metodologias variadas, sendo elas: a pesquisa bibliográfica, a análise documental e o estudo de caso. No primeiro capítulo apresenta uma discussão teórica sobre a relação Estado e sociedade civil, com base nas ideias de autores que contribuíram para a formulação do Estado Moderno e os ideais liberais. O segundo capítulo busca posicionar a discussão do acesso à informação pública como um direito humano, apresentando a evolução deste conceito ao longo dos anos. Já no capítulo três busca-se analisar a legislação brasileira e os marcos legais produzidos no âmbito do parlamento paulistano, para verificar seu alinhamento com os princípios de um regime de acesso à informação. O capítulo quarto é dedicado a verificar a atenção aos princípios da transparência passiva no âmbito do poder legislativo do município de São Paulo, analisando o banco protocolos de acesso à informação da Câmara Municipal de São Paulo, no período de 2015 a 2017, para verificar como se dá a interação entre o poder público e a sociedade civil nos marcos da LAI. Por fim, as considerações finais pontuam questões identificadas ao longo da pesquisa e apresentam sugestões que possam dialogar com estes desafiosThis paper presents a discussion about the access to information within the framework of the Legislature, aiming to analyze the interaction between the population of the city of São Paulo and the right of access to information, having as its case study the Ombudsman\'s Office of São Paulo. This paper uses the Historical-Dialectical Materialism method and other methodologies, as: bibliographical research, documentary analysis and the case study. The first chapter introduces a theoretical discussion of the relation between the State and the Civil Society, based on the ideas of authors who contributed to the formulation of the Modern State and the liberal ideals. The second chapter seeks to place the discussion of access to public information as a human right, presenting the evolution of this concept over the years. In chapter three, the aim is to analyze the Brazilian legislation and the legal frameworks produced within the scope of São Paulo City Council to verify its alignment with the principles of an access to information regime. The fourth chapter is dedicated to verify the attention to the principles of passive transparency in the scope of the legislative power of the city of São Paulo, analyzing the bank protocols of access to information of the São Paulo City Council, from 2015 to 2017, to verify how there is the interaction between the public power and civil society in the framework of Information Access Act. Lastly, the final considerations point out issues identified throughout the research and present suggestions that can dialogue with these challenge

Unequal Behaviour between Hydrolysable Functions of Nirmatrelvir under Stress Conditions: Structural and Theoretical Approaches in Support of Preformulation Studies

International audienceNirmatrelvir is an antiviral drug approved for the treatment of COVID-19. The available dosage form consists of tablets marketed under the brand name PAXLOVID®. Although knowledge of nirmatrelvir’s intrinsic stability may be useful for any potential development of other pharmaceutical forms, no data regarding this matter is available to date. Preliminary forced degradation studies have shown that the molecule is stable under oxidative and photolytic conditions, while hydrolytic conditions, both acidic and basic, have proven deleterious. Indeed, the molecule presents a priori several functions that can undergo hydrolysis, i.e., three amide moieties and a nitrile function. However, considering the degradation products formed under forced conditions and which were detected and identified by LC-UV-HRMSn, the hydrolysis process leading to their formation is selective since it involved only 2 of the 4 hydrolysable functions of the molecule. Ab initio studies based on density functional theory (DFT) have helped better understand these reactivity differences in aqueous media. Some hydrolyzable functions of nirmatrelvir differ from others in terms of electrostatic potential and Fukui functions, and this seems to correlate with the forced degradation outcomes

Investigating therapeutic usage of combined Ticagrelor and Aspirin through solid-state and analytical studies

The mainstay treatment for patients with acute coronary syndrome is an oral route dual antiplatelet therapy with a P2Y12-receptor antagonist and Aspirin (ASA). To improve patient adherence to such treatments, combination therapies (polypill) are envisioned. Physicochemical solid-state studies have been carried out to develop a preformulation strategy of ASA with the P2Y12-receptor antagonist Ticagrelor (TIC). The investigations were carried out using differential scanning calorimetry, liquid chromatography-high resolution-multistage mass spectrometry (LC-HR-MSn) and as complementary techniques Fourier transform infrared measurements and thermogravimetric analysis. A simple eutectic transition at 98 °C with a mole fraction for the eutectic liquid of 0.457 has been observed and the mixing of ASA and TIC molecules in each other's crystal structures appears to be limited. No cocrystals of TIC and ASA have been found. The appearance of the eutectic liquid was linked with a clear onset of chemical instability of the two pharmaceuticals. The decomposition mechanism in the liquid phase involves prior decomposition of ASA, whose residues react with well-identified TIC interaction sites. Seven interaction products were observed by LC-HR-MSn linked to corresponding degradation products. The most important degradation pathway is N-dealkylation. In conclusion, polypills of ASA and TIC are a viable approach, but the decomposition of ASA should be avoided by eliminating high temperatures and high humidity.Peer Reviewe