RESUMO
During 2020, The European Chemicals Agency (ECHA) began evaluating the OECD Test Guideline 443: Extended One Generation Reproductive Toxicity Study (EOGRTS) to analyze specific aspects related to study design, conduct and toxicological findings. A significant outcome of this ECHA evaluation focused on adequate dose level selection. Subsequently, ECHA published recommendations for DART studies, however, these recommendations seemingly do not align with the principles of the 3Rs, animal welfare or human safety goals, specifically, regarding three aspects. First, the requirement to segregate testing for sexual function and fertility from the ability to produce normally developing offspring increases the risk of inadequate identification of postnatal hazards for development and sexual function and fertility, therefore failing human health protection goals. Second, the current ECHA high-dose level setting recommendations for EOGRTS exceed the MTD (Maximum Tolerated Dose), and therefore compromise the interpretation of the biological response relative to the intrinsic effect of the chemical under evaluation. Third, the combination of these aspects will result in an increase in the number of animals tested, increasing animal welfare concerns. This paper reflects the consensus of subject matter experts, professional, and scientific societies who have authored and signed on to this statement. The signatories encourage ECHA to adopt a revised science-driven approach to the dose selection criteria that strikes a balance between regulatory vigilance and scientific pragmatism.
Assuntos
Relação Dose-Resposta a Droga , Reprodução , Testes de Toxicidade , Animais , Reprodução/efeitos dos fármacos , Testes de Toxicidade/métodos , Testes de Toxicidade/normas , Humanos , Organização para a Cooperação e Desenvolvimento Econômico , Bem-Estar do Animal , Feminino , Medição de Risco , Guias como Assunto , Substâncias Perigosas/toxicidadeRESUMO
Drug hypersensitivity reactions (DHRs) are a type of adverse drug reaction that can occur with different classes of drugs and affect multiple organ systems and patient populations. DHRs can be classified as allergic or non-allergic based on the cellular mechanisms involved. Whereas nonallergic reactions rely mainly on the innate immune system, allergic reactions involve the generation of an adaptive immune response. Consequently, drug allergies are DHRs for which an immunological mechanism, with antibody and/or T cell, is demonstrated. Despite decades of research, methods to predict the potential for a new chemical entity to cause DHRs or to correctly attribute DHRs to a specific mechanism and a specific molecule are not well-established. This review will focus on allergic reactions induced by systemically administered low-molecular weight drugs with an emphasis on drug- and patient-specific factors that could influence the development of DHRs. Strategies for predicting and diagnosing DHRs, including potential tools based on the current state of the science, will also be discussed.
Assuntos
Hipersensibilidade a Drogas , Humanos , Hipersensibilidade a Drogas/diagnóstico , Hipersensibilidade a Drogas/imunologiaRESUMO
Cytotoxic T-lymphocytes (CTL) are a subset of T-cells that play a critical role in protecting against intracellular infections and cancer, and have the ability to identify and kill infected or transformed cells expressing non-self peptides associated with major histocompatibility (MHC) Class I molecules. Conversely, aberrant CTL activity can contribute to immune-related pathology under conditions of overwhelming infection or autoimmunity. Disease-modifying therapeutics can have unintended effects on CTL, and a growing number of therapeutics are intended to either suppress or enhance CTL or their functions. The susceptibility of CTL to unintended effects from common therapeutic modalities underscores the need for a better understanding of the impact that such therapies have on CTL function and the associated safety implications. While there are reliable ways of quantifying CTL, notably via flow cytometric analysis of specific CTL markers, it has been a greater challenge to implement fit-for-purpose methods measuring CTL function in the context of safety studies of therapeutics. This review focuses on methods for measuring CTL responses in the context of drug safety and pharmacology testing, with the goals of informing the reader about current approaches, evaluating their pros and cons, and providing perspectives on the utility of these approaches for safety evaluation.
Assuntos
Neoplasias , Linfócitos T Citotóxicos , Animais , Primatas , Neoplasias/terapia , Citotoxicidade ImunológicaRESUMO
Motivation: The availability of databases identifying allergenic proteins via a transparent and consensus-based scientific approach is of prime importance to support the safety review of genetically-modified foods and feeds, and public safety in general. Over recent years, screening for potential new allergens sequences has become more complex due to the exponential increase of genomic sequence information. To address these challenges, an international collaborative scientific group coordinated by the Health and Environmental Sciences Institute (HESI), was tasked to develop a contemporary, adaptable, high-throughput process to build the COMprehensive Protein Allergen REsource (COMPARE) database, a publicly accessible allergen sequence data resource along with bioinformatics analytical tools following guidelines of FAO/WHO and CODEX Alimentarius Commission. Results: The COMPARE process is novel in that it involves the identification of candidate sequences via automated keyword-based sorting algorithm and manual curation of the annotated sequence entries retrieved from public protein sequence databases on a yearly basis; its process is meant for continuous improvement, with updates being transparently documented with each version; as a complementary approach, a yearly key-word based search of literature databases is added to identify new allergen sequences that were not (yet) submitted to protein databases; in addition, comments from the independent peer-review panel are posted on the website to increase transparency of decision making; finally, sequence comparison capabilities associated with the COMPARE database was developed to evaluate the potential allergenicity of proteins, based on internationally recognized guidelines, FAO/WHO and CODEX Alimentarius Commission.
RESUMO
A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6-11 with varying chain lengths (n = 1-6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds 6-11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8-10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure-activity relationship for transport and dual target inhibition.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fosforribosilaminoimidazolcarboxamida Formiltransferase/antagonistas & inibidores , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Células CHO , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cricetulus , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Células KB , Estrutura Molecular , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Fosforribosilglicinamido Formiltransferase/metabolismo , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Relação Estrutura-AtividadeRESUMO
2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or ß (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and ß over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.
Assuntos
Antineoplásicos/química , Receptor 1 de Folato/antagonistas & inibidores , Antagonistas do Ácido Fólico/química , Transportador de Folato Acoplado a Próton/antagonistas & inibidores , Pirimidinas/química , Pirróis/química , Tiofenos/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Transporte Biológico , Células CHO , Linhagem Celular Tumoral , Cricetulus , Cristalografia por Raios X , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Receptor 1 de Folato/química , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Xenoenxertos , Humanos , Camundongos SCID , Modelos Moleculares , Transplante de Neoplasias , Pemetrexede/farmacologia , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Fosforribosilglicinamido Formiltransferase/química , Transportador de Folato Acoplado a Próton/química , Pirimidinas/síntese química , Pirimidinas/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/farmacologiaRESUMO
We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5-10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.
Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Hidroximetil e Formil Transferases/antagonistas & inibidores , Nucleotídeos de Purina/biossíntese , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Células CHO , Proliferação de Células/efeitos dos fármacos , Cricetulus , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Humanos , Hidroximetil e Formil Transferases/metabolismo , Células KB , Modelos Moleculares , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Pirróis/síntese química , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
A new series of 6-substituted straight side chain pyrrolo[2,3-d]pyrimidines 3a-d with varying chain lengths (n = 5-8) was designed and synthesized as part of our program to provide targeted antitumor agents with folate receptor (FR) cellular uptake specificity and glycinamide ribonucleotide formyltransferase (GARFTase) inhibition. Carboxylic acids 4a-d were converted to the acid chlorides and reacted with diazomethane, followed by 48% HBr to generate the α-bromomethylketones 5a-d. Condensation of 2,4-diamino-6-hydroxypyrimidine 6 with 5a-d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines 7a-d. Hydrolysis and subsequent coupling with diethyl l-glutamate and saponification afforded target compounds 3a-d. Compounds 3b-d showed selective cellular uptake via FRα and -ß, associated with high affinity binding and inhibition of de novo purine nucleotide biosynthesis via GARFTase, resulting in potent inhibition against FR-expressing Chinese hamster cells and human KB tumor cells in culture. Our studies establish, for the first time, that a side chain benzoyl group is not essential for tumor-selective drug uptake by FRα.
Assuntos
Receptor 1 de Folato/antagonistas & inibidores , Antagonistas do Ácido Fólico/farmacologia , Ácido Fólico/metabolismo , Nucleotídeos de Purina/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Células CHO , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cricetulus , Relação Dose-Resposta a Droga , Receptor 1 de Folato/metabolismo , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Humanos , Células KB , Modelos Moleculares , Estrutura Molecular , Nucleotídeos de Purina/biossíntese , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Relação Estrutura-AtividadeRESUMO
We previously reported the selective transport of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl-for-benzoyl-substituted side chain and a three- (3a) and four-carbon (3b) bridge. Compound 3a was more potent than 3b against tumor cells. While 3b was completely selective for transport by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over the reduced folate carrier (RFC), 3a was not. To determine if decreasing the distance between the bicyclic scaffold and l-glutamate in 3b would preserve transport selectivity and potency against human tumor cells, 3b regioisomers with [1,3] (7 and 8) and [1,2] (4, 5, and 6) substitutions on the thienoyl ring and with acetylenic insertions in the four-atom bridge were synthesized and evaluated. Compounds 7 and 8 were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with complete selectivity for FRα and PCFT over RFC.
Assuntos
Antineoplásicos/síntese química , Receptor 1 de Folato/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Purinas/antagonistas & inibidores , Pirimidinas/síntese química , Pirróis/síntese química , Proteína Carregadora de Folato Reduzido/metabolismo , Tiofenos/síntese química , Alcinos/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Purinas/biossíntese , Pirimidinas/química , Pirimidinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologiaRESUMO
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1-3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with l-glutamate diethyl ester, followed by saponification, afforded 1-3. Compound 3 selectively inhibited the proliferation of cells expressing folate receptors (FRs) α or ß, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, much more potently than 4. Compound 3 was more inhibitory than 4 toward ß-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1-, 2-, and 4-atom bridge lengths for the activity of this series.