Estudo visando a produção de uma cápsula para transplantação fecal: da seleção de doadores de fezes aos ensaios laboratoriais
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Data
2022-08-29
Autores
Missio, Aline Parisoto [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Inúmeros estudos apontam a importância da microbiota intestinal na manutenção da homeostase do organismo, de modo que alterações em sua composição de espécies podem estar associadas a causa de várias doenças. Em muitos casos, estas alterações - conhecidas como disbiose - podem ser revertidas pela administração de probióticos, que são microrganismos com algum papel modulador, com efeitos benéficos para além do intestino. Uma estratégia de restauração da normobiose intestinal é a aplicação direta de fezes de indivíduos saudáveis naqueles apresentando algum tipo de patologia associada com disbiose. Esta abordagem, que tem surtido efeito em muitos ensaios clínicos, sendo até mesmo recomendada para doenças como a diarreia por Clostridioides difficile refratária à antibioticoterapia, apresenta uma série de desafios, tendo em vista a necessidade de se evitar a transmissão de patógenos intestinais (segurança) e de garantia (eficácia) de restauração da normobiose. Este trabalho teve como propósito explorar pontos que assegurem o alcance destes objetivos, aplicando-os ao formato de transplantação fecal menos invasivo - a cápsula. A primeira etapa do trabalho consistiu na seleção de doadores de fezes, iniciando-se com uma chamada pública que teve alcance nacional, seguida de um processo de seleção, compreendendo uma triagem por entrevista, exames clínicos e laboratoriais para atestar a saúde e ausência de patógenos nas fezes de potenciais candidatos. Após ou em paralelo a esta fase de avaliação do nível de segurança das fezes de interessados em participar do processo de seleção, foi feita a análise do microbioma fecal, para eventualmente atestar sua eficácia. Além de atestar a segurança do candidato para ser um doador, excluindo, por exemplo, aqueles apresentando doenças crônicas, a entrevista permitiu coletar informações que poderiam dificultar ou comprometer a colheita e manutenção da viabilidade microbiana nas fezes. As questões foram divididas em duas categorias: aquelas visando a obtenção de informações de natureza transitória ou aquelas que poderiam comprometer definitivamente a participação do interessado. De um total de 221 candidatos, 196 foram eliminados por apresentarem limitações tais como, diagnóstico de doenças crônicas ou residirem em local impeditivo de realização de colheita (fora da cidade de Botucatu). Aos 25 candidatos que passaram na triagem da entrevista, foi solicitado o encaminhamento para os exames laboratoriais preliminares. Destes, 11 desistiram do processo de seleção e 6 foram desqualificados, por terem se mudado de Botucatu. Os oito remanescentes incluíram 2 que vieram a se mudar de Botucatu, nesta etapa, 1 que teve restrição nos exames laboratoriais, 1 que passou a apresentar constipação intestinal crônica e 1 que iniciou um regime terapêutico com possível implicação no equilíbrio na microbiota. De modo que restaram apenas 3 indivíduos elegíveis e que forneceram amostras fecais para análise de microbioma, que também foram utilizadas para a avaliação de protocolos de produção de cápsulas de administração oral. Foram coletadas 3 amostras, de cada um destes indivíduos, em dias diferentes, que serviram para a comparar a diferença na riqueza e abundância de OTUs (Operational Taxonomic Units) do microbioma deles, bem como do microbioma das fezes e das cápsulas correspondentes, durante a estocagem a -80°C, visando a definição de um protocolo para produção de cápsulas em série. A análise do microbioma das triplicatas de cada um dos três potenciais doadores não mostrou variação estatisticamente significativa na diversidade alfa entre eles, medida pelo índice de Shannon. Foram avaliados 4 protocolos de produção de cápsulas, 3 dos quais tinham como ingrediente básico, além das fezes, mistura de manteiga de cacau e glicerol, acrescidas ou não de salina 0,9% e que eram dispensados diretamente em invólucros de liberação tardia (DRcaps), de tamanho 00 ou em invólucros de gelatina, tamanho 0, que seriam transferidos posteriormente para os invólucros 00 DRcaps. Além de diferenças na composição de ingredientes, estes protocolos variavam quanto ao procedimento de remoção de resíduos das fezes e homogeneização da mistura. Todos os protocolos implicaram em perda na riqueza e abundância de OTUs, que chegaram a -98%, na comparação entre fezes e cápsulas correspondentes, um deles, adicionalmente, produziu um concentrado fecal sem fluidez insuficiente para a dispensação no invólucro, um deles apresentou baixo rendimento e um terceiro comprometia a estrutura dos invólucros. O protocolo que não apresentou estas limitações técnicas foi testado e aprovado quanto à capacidade de manter a abundância e riqueza de OTUs, durante estocagem das cápsulas por 124 dias, tendo sido o escolhido para a produção em série de cápsulas. Dois dos 3 potenciais doadores de fezes desistiram do processo de seleção, após a análise de seu microbioma fecal. De modo que um deles – na verdade uma potencial doadora – se submeteu aos exames finais para atestar a segurança de seu material fecal, tendo apresentado apenas restrições temporárias, posteriormente revertidas, passou a colaborar com o projeto. Seu microbioma fecal foi confrontado com o microbioma de mucosa de dois pacientes com retocolite ulcerativa, vindo a apresentar profundas diferenças no sentido de apresentar OTUs característicos de uma microbiota saudável, tais como Christensenellaceae, Faecalibacterium e Lachnospiraceae, sendo que estas duas últimas fizeram parte do core microbiome de suas fezes e das cápsulas correspondentes estocadas. A experiência de seleção de doadores, em que, ao final do processo, se conseguiu apenas 1 entre 221 (0,45%) candidatos, mostra uma taxa inferior aos estudos já realizados segundo os quais ~3% ao final são elegíveis.
Innumerous studies support the role of gut microbiota in maintaining the body homeostasis, so that oscillations in its species composition can be associated with the cause of many diseases. In some cases, these alterations, known as dysbiosis, can be reversed by the administration of probiotics, which comprise microorganism exerting some modulatory role, with beneficial effects extending to extra-intestinal parts of the body. A strategy for restoring the intestinal normobiosis comprise of the direct administration of stools from healthy subjects in the gut of those bearing some kind of dysbiosis associated disease. This approach, which has shown positive results in many clinical trials and has even been recommended for pathologies, such as Clostridioides difficile diarrhea refractory to antibiotic therapy, presents with many challenges, in view of the need of avoiding the cross-transfer of intestinal pathogens (safety) and the assurance of potential (efficacy). The aim of the present work was to investigate some issues relevant to assess the two points above, focusing in the formulation of an oral capsules for fecal transplantation (FT), considering a less invasive approach, by using a less invasive administration route. The first phase of the study comprised of the selection of candidates for stool donation, starting with a public call of national reach, followed by the selection process, which consisted of an interview, clinical and laboratory examinations to assure the healthy condition and absence of pathogens in the stools of potential candidates. Following or in parallel to this phase of evaluation for the safety level of the subjects’ stools to qualify he or she for donation, the fecal microbiome was analyzed, to attest its efficacy. In addition to attest the safety condition of the candidate to be considered for donation, eliminating, for example, those diagnosed with chronic diseases, the interview enabled the gathering of information that could difficult or compromise routine stool collection and maintenance of the bacterial viability in the stools. The questions were divided in two categories: those with the aim of getting information of transient nature and those that could definitely compromise the eligibility of the subject. Of a total of 221 candidates, 196 were not qualified for reasons such as diagnosis of chronic diseases, living in places distant of the laboratory (out of Botucatu). The 25 candidates who passed the interview were requested to submit to preliminary laboratory examinations. Of these, 11 desisted the selection process and 6 declared to live out of Botucatu. The remaining 8 subjects comprised of one subject who was found to have restrictions in the laboratory examinations, one that displayed intestinal constipation and one which started a therapeutic regimen which could interfere with the species composition equilibrium of the gut microbiota. Therefore, only 3 subjects were eligible at the end of this phase and from whom 3 stool samples were collected for microbiome analysis and also used for the evaluation of protocols for the production of capsules. Comparative analyses were performed in between the fecal microbiome and the corresponding capsules of the 3 subjects, by assessing the richness and abundance of their operational taxonomic units (OTU), after storage at -80ºC. Microbiome analyses of the 3 samples from each of the candidates did not show statistically significant variations in the alpha diversity among them as determined by the Shannon index. A number of 4 protocols for capsule production were evaluated, 3 of which included cocoa butter and glycerol, in addition to stools, added or not with 0,9% saline in its formulation. The mixture was dispensed in DRcaps (00) or gelatin caps (0) involucres. In addition to the differences in the formulation, these protocols differed as to the procedure involved in the elimination of the stool residues and homogenization of the mixture. In all protocols an OTU loss was detected up to -90%, in the comparison between stools samples and the corresponding capsules. The formulation of one protocol was not fluid enough for dispensation in the involucres; that of second protocol produced a low yield, and of the third one induced crack in the involucres. The protocol which did not presented with any of the above technical limitations was tested and approved as to the ability of maintaining the richness and abundance of the OTUs in the capsules under storage in deep freezer, for 124 days, Two of the 3 candidates who still were considered for stool donation desisted of the selection process, after the analyses of their fecal microbiome. Therefore, a single donor remained, which then submitted to the final laboratory examinations for evaluation the safety of her stools. The examination results revealed only temporary restrictions, so that it came to be qualified for the for the donation. Her fecal microbiome was compared with those of the gut mucosa of two ulcerative colitis patients and profound difference was found, consisting in the presence of OTUs typical of a healthy organism in the donor (e.g. Faecalibacterium, Christensenellaceae and Lachnospiraceae). The proportion of selected donor among the candidates (1/221, 0.45%) matches those of similar studies conducted in the past, according to which less than ~3% of the candidates are eligible for stool donation.
Innumerous studies support the role of gut microbiota in maintaining the body homeostasis, so that oscillations in its species composition can be associated with the cause of many diseases. In some cases, these alterations, known as dysbiosis, can be reversed by the administration of probiotics, which comprise microorganism exerting some modulatory role, with beneficial effects extending to extra-intestinal parts of the body. A strategy for restoring the intestinal normobiosis comprise of the direct administration of stools from healthy subjects in the gut of those bearing some kind of dysbiosis associated disease. This approach, which has shown positive results in many clinical trials and has even been recommended for pathologies, such as Clostridioides difficile diarrhea refractory to antibiotic therapy, presents with many challenges, in view of the need of avoiding the cross-transfer of intestinal pathogens (safety) and the assurance of potential (efficacy). The aim of the present work was to investigate some issues relevant to assess the two points above, focusing in the formulation of an oral capsules for fecal transplantation (FT), considering a less invasive approach, by using a less invasive administration route. The first phase of the study comprised of the selection of candidates for stool donation, starting with a public call of national reach, followed by the selection process, which consisted of an interview, clinical and laboratory examinations to assure the healthy condition and absence of pathogens in the stools of potential candidates. Following or in parallel to this phase of evaluation for the safety level of the subjects’ stools to qualify he or she for donation, the fecal microbiome was analyzed, to attest its efficacy. In addition to attest the safety condition of the candidate to be considered for donation, eliminating, for example, those diagnosed with chronic diseases, the interview enabled the gathering of information that could difficult or compromise routine stool collection and maintenance of the bacterial viability in the stools. The questions were divided in two categories: those with the aim of getting information of transient nature and those that could definitely compromise the eligibility of the subject. Of a total of 221 candidates, 196 were not qualified for reasons such as diagnosis of chronic diseases, living in places distant of the laboratory (out of Botucatu). The 25 candidates who passed the interview were requested to submit to preliminary laboratory examinations. Of these, 11 desisted the selection process and 6 declared to live out of Botucatu. The remaining 8 subjects comprised of one subject who was found to have restrictions in the laboratory examinations, one that displayed intestinal constipation and one which started a therapeutic regimen which could interfere with the species composition equilibrium of the gut microbiota. Therefore, only 3 subjects were eligible at the end of this phase and from whom 3 stool samples were collected for microbiome analysis and also used for the evaluation of protocols for the production of capsules. Comparative analyses were performed in between the fecal microbiome and the corresponding capsules of the 3 subjects, by assessing the richness and abundance of their operational taxonomic units (OTU), after storage at -80ºC. Microbiome analyses of the 3 samples from each of the candidates did not show statistically significant variations in the alpha diversity among them as determined by the Shannon index. A number of 4 protocols for capsule production were evaluated, 3 of which included cocoa butter and glycerol, in addition to stools, added or not with 0,9% saline in its formulation. The mixture was dispensed in DRcaps (00) or gelatin caps (0) involucres. In addition to the differences in the formulation, these protocols differed as to the procedure involved in the elimination of the stool residues and homogenization of the mixture. In all protocols an OTU loss was detected up to -90%, in the comparison between stools samples and the corresponding capsules. The formulation of one protocol was not fluid enough for dispensation in the involucres; that of second protocol produced a low yield, and of the third one induced crack in the involucres. The protocol which did not presented with any of the above technical limitations was tested and approved as to the ability of maintaining the richness and abundance of the OTUs in the capsules under storage in deep freezer, for 124 days, Two of the 3 candidates who still were considered for stool donation desisted of the selection process, after the analyses of their fecal microbiome. Therefore, a single donor remained, which then submitted to the final laboratory examinations for evaluation the safety of her stools. The examination results revealed only temporary restrictions, so that it came to be qualified for the for the donation. Her fecal microbiome was compared with those of the gut mucosa of two ulcerative colitis patients and profound difference was found, consisting in the presence of OTUs typical of a healthy organism in the donor (e.g. Faecalibacterium, Christensenellaceae and Lachnospiraceae). The proportion of selected donor among the candidates (1/221, 0.45%) matches those of similar studies conducted in the past, according to which less than ~3% of the candidates are eligible for stool donation.
Descrição
Palavras-chave
Cápsulas, Microbiota fecal, Transplantação fecal