RESUMO
The 505(b)(2) NDA pathway can reduce drug development costs and accelerate the time to market by leveraging existing public data using clinical bridging and regulatory strategies. Whether or not a drug qualifies for the 505(b)(2) pathway depends on the active ingredient, drug formulation, clinical indication and other factors. Clinical programs can be streamlined and accelerated, and confer unique marketing benefits, such as exclusivity, depending on the regulatory strategy and product. Considerations for chemistry, manufacturing and controls (CMC) and the unique manufacturing issues that can arise owing to the accelerated development of 505(b)(2) drug products are also discussed.
Assuntos
Desenvolvimento de Medicamentos , Estados Unidos , United States Food and Drug AdministrationRESUMO
Indoximod has shaped our understanding of the biology of IDO1 in the control of immune responses, though its mechanism of action has been poorly understood. Previous studies demonstrated that indoximod creates a tryptophan (Trp) sufficiency signal that reactivates mTOR in the context of low Trp concentrations, thus opposing the effects caused by IDO1. Here we extend the understanding of indoximod's mechanism of action by showing that it has pleiotropic effects on immune regulation. Indoximod can have a direct effect on T cells, increasing their proliferation as a result of mTOR reactivation. Further, indoximod modulates the differentiation of CD4+ T cells via the aryl hydrocarbon receptor (AhR), which controls transcription of several genes in response to different ligands including kynurenine (Kyn). Indoximod increases the transcription of RORC while inhibiting transcription of FOXP3, thus favoring differentiation to IL-17-producing helper T cells and inhibiting the differentiation of regulatory T cells. These indoximod-driven effects on CD8+ and CD4+ T cells were independent from the activity of IDO/TDO and from the presence of exogenous Kyn, though they do oppose the effects of Kyn produced by these Trp catabolizing enzymes. Indoximod can also downregulate expression of IDO protein in vivo in murine lymph node dendritic cells and in vitro in human monocyte-derived dendritic cells via a mechanism that involves signaling through the AhR. Together, these data improve the understanding of how indoximod influences the effects of IDO, beyond and distinct from direct enzymatic inhibition of the enzyme.
RESUMO
A series of different prodrugs of indoximod, including estesrs and peptide amides were synthesized with the aim of improving its oral bioavailability in humans. The pharmacokinetics of prodrugs that were stable in buffers, plasma and simulated gastric and intestinal fluids was first assessed in rats after oral dosing in solution or in capsule formulation. Two prodrugs that produced the highest exposure to indoximod in rats were further tested in Cynomolgus monkeys, a species in which indoximod has oral bioavailability of 6-10% and an equivalent dose-dependent exposure profile as humans. NLG802 was selected as the clinical development candidate after increasing oral bioavailability (>5-fold), Cmax (6.1-3.6 fold) and AUC (2.9-5.2 fold) in monkeys, compared to equivalent molar oral doses of indoximod. NLG802 is extensively absorbed and rapidly metabolized to indoximod in all species tested and shows a safe toxicological profile at the anticipated therapeutic doses. NLG802 markedly enhanced the anti-tumor responses of tumor-specific pmel-1 T cells in a melanoma tumor model. In conclusion, NLG802 is a prodrug of indoximod expected to increase clinical drug exposure to indoximod above the current achievable levels, thus increasing the possibility of therapeutic effects in a larger fraction of the target patient population.
Assuntos
Antineoplásicos/síntese química , Neoplasias/tratamento farmacológico , Pró-Fármacos/síntese química , Triptofano/análogos & derivados , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Composição de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Haplorrinos , Humanos , Absorção Intestinal/fisiologia , Camundongos , Conformação Molecular , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacocinética , Ratos , Triptofano/administração & dosagem , Triptofano/síntese química , Triptofano/farmacocinéticaRESUMO
A novel class of 5-substituted 5H-imidazo[5,1-a]isoindoles are described as potent inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1). A structure-based drug design approach was used to elaborate the 5H-imidazo[5,1-a]isoindole core and to improve potency and pharmacological properties. Suitably placed hydrophobic and polar functional groups in the lead molecule allowed improvement of IDO1 inhibitory activity while minimizing off-target liabilities. Structure-activity relationship studies focused on optimizing IDO1 inhibition potency and a pharmacokinetic profile amenable to oral dosing while controlling CYP450 and hERG inhibitory properties.
Assuntos
Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indóis/farmacologia , Animais , Cães , Desenho de Fármacos , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Imidazóis/química , Imidazóis/farmacocinética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indóis/química , Indóis/farmacocinética , Camundongos , Simulação de Acoplamento Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
Global conformational transitions of the hexameric RepA helicase of plasmid RSF1010, induced by the nucleoside tri and di-phosphate binding, have been examined using analytical ultracentrifugation and dynamic light scattering techniques. The global structure of the RepA hexamer in solution, modeled as an oblate ellipsoid of revolution, is very different from its crystal structure, with the axial ratio of the ellipsoid being approximately 4.5 as compared to only approximately 2.4 in the crystal structure. The large axial ratio and the experimentally determined partial specific volume strongly suggest that, in solution, the diameter of the cross-channel of the hexamer is larger than approximately 17 A seen in the crystal. The global conformation of the helicase is modulated by a specific number of bound nucleotides. The enzyme exists in at least four conformational states, occurring sequentially as a function of the number of bound cofactors. These conformational states are different for ADP, as compared to beta,gamma-imidoadenosine 5'-triphosphate (AMP-PNP). Modulation of the global structure is separated into two phases, different for complexes with up to three bound nucleotides, from the effect observed at the saturating level of cofactors. This heterogeneity indicates different functional roles of the two modulation processes. Nucleotide control of helicase - single-stranded (ss)DNA interactions occurs through affecting the enzyme structure and the ssDNA affinity prior to DNA binding. Only one conformational state of the helicase, with two AMP-PNP molecules bound, has dramatically higher ssDNA-affinities than the complexes with ADP. Moreover the same state also has an increased site-size of the enzyme - ssDNA complexes. The implications of these findings for functional activities of a hexameric helicase are discussed.
Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Luz , Plasmídeos/metabolismo , Conformação Proteica , Espalhamento de Radiação , Ultracentrifugação , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Matemática , Modelos Moleculares , Modelos Estatísticos , Plasmídeos/genética , Ligação Proteica , TermodinâmicaRESUMO
The structure of the complex of the hexameric replicative helicase RepA protein of plasmid RSF1010 with ssDNA has been examined using the fluorescence energy transfer and analytical ultracentrifugation methods. We utilized the fact that the RepA monomer contains a single, natural cysteine residue. The cysteine residue has been modified with a fluorescent marker, which serves as the donor to the acceptor placed in different locations on the DNA. Using the two independent fluorescence donor-acceptor pairs and different DNA oligomers, we provide direct evidence that, in the complex with the enzyme, the ssDNA passes through the inner channel of the RepA hexamer. In the stationary complex, the RepA hexamer assumes a strictly single orientation with respect to the polarity of the sugar-phosphate backbone of the nucleic acid, with the large domain of protomers facing the 3' end of the bound DNA. Interactions with the helicase induce profound changes in the structure of the bound DNA, and these changes are predominantly localized in the proper DNA-binding site. The heterogeneity of the structure of the bound DNA reflects the heterogeneous structure of the total RepA helicase DNA-binding site. This is in excellent agreement with the thermodynamic data. The structure of the RepA hexamer, in solution, differs considerably from the crystal structure of the enzyme. Both fluorescence energy transfer and analytical ultracentrifugation data indicate a significant conformational flexibility of the RepA hexamer. Implications of these results for the mechanism of interactions of the hexameric helicase with the DNA are discussed.
Assuntos
Coenzimas/metabolismo , DNA Helicases/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Plasmídeos/metabolismo , Sequência de Bases , Sítios de Ligação , Coenzimas/química , Cisteína/química , Cisteína/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Plasmídeos/genética , Conformação Proteica , SoluçõesRESUMO
Interactions of polymerase X from African swine fever virus with single-stranded DNA (ssDNA) have been studied, using quantitative fluorescence titration and analytical ultracentrifugation techniques. Experiments were performed with a fluorescent etheno-derivative of ssDNA oligomers. Studies of unmodified ssDNA oligomers were carried out using the competition titration method. The total site-size of the pol X-ssDNA complex is 16(+/-1) nucleotide residues. The large total ssDNA-binding site has a complex heterogeneous structure. It contains the proper ssDNA-binding site that encompasses only 7(+/-1) residues. As the length of the ssDNA increases, the enzyme engages an additional binding area in interactions with the DNA, at a distance of approximately 7-8 nucleotides from the proper site, which is located asymmetrically within the polymerase molecule. As a result, the net ion release accompanying the interactions with the DNA, increases from approximately 1 for the proper DNA-binding site to approximately 6 for the total DNA-binding site. Unlike in the case of the mammalian polymerase beta that belongs to the same polymerase X family, the DNA-binding areas within the total DNA-binding site of pol X are not autonomous. Consequently, the enzyme does not form different binding modes with different numbers of occluded nucleotide residues, although the interacting areas are structurally separated. The statistical thermodynamic model that accounts for the engagement of the proper and the total DNA-binding site in interactions with the DNA provides an excellent description of the binding process. Pol X binds the ssDNA without detectable cooperativity and with very modest base specificity.