RESUMEN
Clinical studies have found there still exists a lack of gene therapy dose-toxicity and dose-efficacy data that causes gene therapy dose selection to remain elusive. Model informed drug development (MIDD) has become a standard tool implemented throughout the discovery, development, and approval of pharmaceutical therapies, and has the potential to inform dose-toxicity and dose-efficacy relationships to support gene therapy dose selection. Despite this potential, MIDD approaches for gene therapy remain immature and require standardization to be useful for gene therapy clinical programs. With the goal to advance MIDD approaches for gene therapy, in this review we first provide an overview of gene therapy types and how they differ from a bioanalytical, formulation, route of administration, and regulatory standpoint. With this biological and regulatory background, we propose how MIDD can be advanced for AAV-based gene therapies by utilizing physiological based pharmacokinetic modeling and quantitative systems pharmacology to holistically inform AAV and target protein dynamics following dosing. We discuss how this proposed model, allowing for in-depth exploration of AAV pharmacology, could be the key the field needs to treat these unmet disease populations.
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Enfortumab vedotin is an antibody-drug conjugate (ADC) comprised of a Nectin-4-directed antibody and monomethyl auristatin E (MMAE), which is primarily eliminated through P-glycoprotein (P-gp)-mediated excretion and cytochrome P450 3A4 (CYP3A4)-mediated metabolism. A physiologically based pharmacokinetic (PBPK) model was developed to predict effects of combined P-gp with CYP3A4 inhibitor/inducer (ketoconazole/rifampin) on MMAE exposure when coadministered with enfortumab vedotin and study enfortumab vedotin with CYP3A4 (midazolam) and P-gp (digoxin) substrate exposure. A PBPK model was built for enfortumab vedotin and unconjugated MMAE using the PBPK simulator ADC module. A similar model was developed with brentuximab vedotin, an ADC with the same valine-citrulline-MMAE linker as enfortumab vedotin, for MMAE drug-drug interaction (DDI) verification using clinical data. The DDI simulation predicted a less-than-2-fold increase in MMAE exposure with enfortumab vedotin plus ketoconazole (MMAE geometric mean ratio [GMR] for maximum concentration [Cmax], 1.15; GMR for area under the time-concentration curve from time 0 to last quantifiable concentration [AUClast], 1.38). Decreased MMAE exposure above 50% but below 80% was observed with enfortumab vedotin plus rifampin (MMAE GMR Cmax, 0.72; GMR AUClast, 0.47). No effect of enfortumab vedotin on midazolam or digoxin systemic exposure was predicted. Results suggest that combination enfortumab vedotin, P-gp, and a CYP3A4 inhibitor may result in increased MMAE exposure and patients should be monitored for potential adverse effects. Combination P-gp and a CYP3A4 inducer may result in decreased MMAE exposure. No exposure change is expected for CYP3A4 or P-gp substrates when combined with enfortumab vedotin.ClinicalTrials.gov identifier Not applicable.
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ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 µM) and Mycobacterium abscessus (MIC, 0.4 µM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.
Asunto(s)
Proteasas ATP-Dependientes/antagonistas & inhibidores , Antituberculosos/farmacología , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Oligopéptidos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiologíaRESUMEN
Ensuring identity, purity, and reproducibility are equally essential during synthetic chemistry, drug discovery, and for pharmaceutical product safety. Many peptidic APIs are large molecules that require considerable effort for integrity assurance. This study builds on quantum mechanical 1H iterative Full Spin Analysis (HiFSA) to establish NMR peptide sequencing methodology that overcomes the intrinsic limitations of principal compendial methods in identifying small structural changes or minor impurities that affect effectiveness and safety. HiFSA sequencing yields definitive identity and purity information concurrently, allowing for API quality assurance and control (QA/QC). Achieving full peptide analysis via NMR building blocks, the process lends itself to both research and commercial applications as 1D 1H NMR (HNMR) is the most sensitive and basic NMR experiment. The generated HiFSA profiles are independent of instrument or software tools and work at any magnetic field strength. Pairing with absolute or 100% qHNMR enables quantification of mixtures and/or determination of peptide conformer populations. Demonstration of the methodology uses single amino acids (AAs) and peptides of increasing size, including the octapeptide, angiotensin II, and the nonapeptide, oxytocin. The feasibility of HiFSA coupled with automated NMR and qHNMR for use in QC/QA efforts is established through case-based examples and recommended procedures.
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Péptidos/química , Teoría Cuántica , Análisis de Secuencia de Proteína , Péptidos/farmacología , Análisis de Componente Principal , Conformación Proteica , Espectroscopía de Protones por Resonancia Magnética , Control de CalidadRESUMEN
Residual complexity (RC) involves the impact of subtle but critical structural and biological features on drug lead validation, including unexplained effects related to unidentified impurities. RC commonly plagues drug discovery efforts due to the inherent imperfections of chromatographic separation methods. The new diketopiperazine, rufomyazine (6), and the previously known antibiotic, rufomycin (7), represent a prototypical case of RC that (almost) resulted in the misassignment of biological activity. The case exemplifies that impurities well below the natural abundance of 13C (1.1%) can be highly relevant and calls for advanced analytical characterization of drug leads with extended molar dynamic ranges of >1:1,000 using qNMR and LC-MS. Isolated from an actinomycete strain, 6 was originally found to be active against Mycobacterium tuberculosis with a minimum inhibitory concentration (MIC) of 2 µg/mL and high selectivity. As a part of lead validation, the dipeptide was synthesized and surprisingly found to be inactive. The initially observed activity was eventually attributed to a very minor contamination (0.24% [m/m]) with a highly active cyclic peptide (MIC â¼ 0.02 µM), subsequently identified as an analogue of 7. This study illustrates the serious implications RC can exert on organic chemistry and drug discovery, and what efforts are vital to improve lead validation and efficiency, especially in NP-related drug discovery programs.
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Antituberculosos/química , Antituberculosos/farmacología , Descubrimiento de Drogas , Mycobacterium tuberculosis/efectos de los fármacos , Oligopéptidos/química , Oligopéptidos/farmacología , Espectroscopía de Resonancia Magnética con Carbono-13 , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Pruebas de Sensibilidad Microbiana , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
This report describes an approach using 1H NMR iterative full-spin analysis (HiFSA) to extract definitive structural information on unknown peptides from 1D 1H NMR data. By comparing the experimental data and HiFSA fingerprint of a known analogue, it is possible to isolate the characteristic 1H subspectrum of the different amino acids and, thus, elucidate the structure of the peptide. To illustrate this methodology, a comprehensive analysis of five new anti-Mycobacterium tuberculosis peptides (2-6), all analogues of ecumicin (1), was carried out. The method was validated by demonstrating congruence of the HiFSA-based structures with all available data, including MS and 2D NMR. The highly reproducible HiFSA fingerprints of the new â¼1600 amu peptides were generated in this process. Besides oligo-peptides, the HiFSA sequencing approach could be extended to all oligomeric compounds consisting of chains of monomers lacking H-H spin-spin coupling across the moieties. HiFSA sequencing is capable of differentiating complex oligomers that exhibit minor structural differences such as shifted hydoxyl or methyl groups. Because it employs the basic and most sensitive 1D 1H NMR experiment, HiFSA sequencing enables the exploration of peptide analogues up to at least 2000 amu, even with basic contemporary spectrometers and when only sub-milligram amounts of isolates are available.
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Antituberculosos/aislamiento & purificación , Oligopéptidos/química , Protones , Antituberculosos/química , Antituberculosos/farmacología , Estructura Molecular , Mycobacterium tuberculosis/química , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificaciónRESUMEN
The eye, which is under constant exposure to environmental pathogens, has evolved various anatomic and immunological barriers critical to the protection of tissues lacking regenerative capacity, and the maintenance of a clear optic pathway essential to vision. By bypassing the ocular barriers, intravitreal (IVT) injection has become the mainstay for the delivery of drugs to treat conditions that affect the back of the eye. Both small molecules and biotherapeutics have been successfully administered intravitreally, and several drugs have been approved for the treatment of (wet) age-related macular degeneration and diabetic macular edema. However, IVT injection is an invasive procedure, which requires sufficient technical expertise from the healthcare professional administering the drug. Potential side effects include bleeding, retinal tear, cataracts, infection, uveitis, loss of vision, and increased ocular pressure. Pharmaceutical companies often differ in their drug development plan, including drug administration techniques, collection of ocular tissues and fluids, ophthalmology monitoring, and overall conduct of nonclinical and clinical studies. The present effort, under the aegis of the Innovation & Quality Ophthalmic Working Group, aims at understanding these differences, identifying pros and cons of the various approaches, determining the gaps in knowledge, and suggesting feasible good practices for nonclinical and early clinical IVT drug development.
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Retinopatía Diabética , Edema Macular , Humanos , Edema Macular/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Preparaciones Farmacéuticas , Inyecciones IntravítreasRESUMEN
Direct oral anticoagulants, such as apixaban and rivaroxaban, are important for the treatment and prophylaxis of venous thromboembolism and to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. Because apixaban and rivaroxaban are predominantly eliminated by cytochrome P450 (CYP) 3A and P-glycoprotein (P-gp), concomitant use of combined P-gp and strong CYP3A4 inhibitors and inducers should be avoided. Physiologically-based pharmacokinetic models for apixaban and rivaroxaban were developed to estimate the net effect of CYP3A induction, P-gp inhibition, and P-gp induction by rifampicin. The disposition of rivaroxaban is more complex compared with apixaban because both hepatic and renal P-gp is considered to contribute to rivaroxaban elimination. Furthermore, organic anion transporter-3, a renal uptake transporter, may also contribute the elimination of rivaroxaban from systemic circulation. The models were verified with observed clinical drug-drug interactions with CYP3A and P-gp inhibitors. With the developed models, the predicted area under the concentration time curve and maximum concentration ratios were 0.43 and 0.48, respectively, for apixaban, and 0.50-0.52 and 0.72-0.73, respectively, for rivaroxaban when coadministered with 600 mg multiple doses of rifampicin and that were very close to observed data. The impact of each of the elimination pathways was assessed for rivaroxaban, and inhibition of CYP3A led to a larger impact over intestinal and hepatic P-gp. Inhibition of renal organic anion transporter-3 or P-gp led to an overall modest interaction. The developed apixaban and rivaroxaban models can be further applied to the investigation of interactions with other P-gp and/or CYP3A4 inhibitors and inducers.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Fibrilación Atrial/tratamiento farmacológico , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Factor Xa/farmacocinética , Transporte Biológico , Inductores del Citocromo P-450 CYP3A/farmacocinética , Inhibidores del Citocromo P-450 CYP3A/farmacocinética , Interacciones Farmacológicas , Embolia/prevención & control , Inhibidores del Factor Xa/administración & dosificación , Inhibidores del Factor Xa/uso terapéutico , Humanos , Pirazoles/administración & dosificación , Pirazoles/farmacocinética , Pirazoles/uso terapéutico , Piridonas/administración & dosificación , Piridonas/farmacocinética , Piridonas/uso terapéutico , Rifampin/administración & dosificación , Rifampin/farmacocinética , Rivaroxabán/administración & dosificación , Rivaroxabán/farmacocinética , Rivaroxabán/uso terapéutico , Accidente Cerebrovascular/prevención & control , Tromboembolia Venosa/prevención & controlRESUMEN
Whereas generic, LC-based pharmaceutical control quality procedures depend largely on the detection mode and can be particularly 'blind' to certain impurities, NMR is a more versatile and, thus, often more judicious detector. While adulteration presents ever-evolving challenges for the analysis of active pharmaceutical ingredients (APIs) and finished products sold in the worldwide (online) marketplace, research chemicals are usually trusted rather than being considered flawed or even adulterated. This report shows how NMR analysis uncovered the unanticipated presence of substantial amounts of mannitol (20 and 43% w/w) as undeclared constituent in two custom synthetic peptides, DR and DRVYI, that were sourced commercially. Quantitative 1H NMR (qHNMR) readily detected the contaminant, even on a 60â¯MHz benchtop instrument, and quantified the highly polar and UV-transparent adulterant. Quantum-mechanical 1H iterative Full Spin Analysis (HiFSA) not only achieved unambiguous identification of both the mannitol and the peptides, but also confirmed the quantitative results. The cases show that experimental verification supersedes trust in both pharmaceutical and research QC. They also highlight the promising utility of both established high-field and recently re-evolving low-field benchtop qHNMR. The unanticipated findings remind manufacturers and researchers alike about the advantages of including/performing NMR and qNMR with routine CofA documentation and/or verification of research grade chemicals. Especially when done jointly, this can greatly improve confidence in research and help streamline the pharmaceutical QC toolbox.
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Péptidos/química , Preparaciones Farmacéuticas/química , Contaminación de Medicamentos , Imagen por Resonancia Magnética/métodos , Control de CalidadRESUMEN
The rise of multi- and extensively drug-resistant Mycobacterium tuberculosis (M. tb) strains and co-infection with human immunodeficiency virus has escalated the need for new anti-M. tb drugs. Numerous challenges associated with the M. tb, in particular slow growth and pathogenicity level 3, discouraged use of this organism in past primary screening efforts. From current knowledge of the physiology and drug susceptibility of mycobacteria in general and M. tb specifically, it can be assumed that many potentially useful drug leads were missed by failing to screen directly against this pathogen. This review discusses recent high-throughput phenotypic screening strategies for anti-M. tb drug discovery. Emphasis is placed on prioritization of hits, including their extensive biological and chemical profiling, as well as the development status of promising drug candidates discovered with phenotypic screening.
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Antituberculosos/aislamiento & purificación , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Pruebas de Sensibilidad MicrobianaRESUMEN
Addressing the urgent need to develop novel drugs against drug-resistant Mycobacterium tuberculosis ( M. tb) strains, ecumicin (ECU) and rufomycin I (RUFI) are being explored as promising new leads targeting cellular proteostasis via the caseinolytic protein ClpC1. Details of the binding topology and chemical mode of (inter)action of these cyclopeptides help drive further development of novel potency-optimized entities as tuberculosis drugs. ClpC1 M. tb protein constructs with mutations driving resistance to ECU and RUFI show reduced binding affinity by surface plasmon resonance (SPR). Despite certain structural similarities, ECU and RUFI resistant mutation sites did not overlap in their SPR binding patterns. SPR competition experiments show ECU prevents RUFI binding, whereas RUFI partially inhibits ECU binding. The X-ray structure of the ClpC1-NTD-RUFI complex reveals distinct differences compared to the previously reported ClpC1-NTD-cyclomarin A structure. Surprisingly, the complex structure revealed that the epoxide moiety of RUFI opened and covalently bound to ClpC1-NTD via the sulfur atom of Met1. Furthermore, RUFI analogues indicate that the epoxy group of RUFI is critical for binding and bactericidal activity. The outcomes demonstrate the significance of ClpC1 as a novel target and the importance of SAR analysis of identified macrocyclic peptides for drug discovery.
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Antituberculosos/química , Proteínas Bacterianas/química , Proteínas de Choque Térmico/química , Mycobacterium tuberculosis/efectos de los fármacos , Oligopéptidos/química , Antituberculosos/farmacología , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Ligandos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Oligopéptidos/farmacología , Dominios ProteicosRESUMEN
Natural Deep Eutectic Solvent (NADES) species can exhibit unexpected solubilizing power for lipophilic molecules despite their simple composition: hydrophilic organic molecules and water. In the present study, the unique properties of NADES species were applied in combination with a model polymer system: a hydrophilic chitosan/alginate hydrogel. Briefly, NADES species (e.g., mannose-dimethylurea-water, 2:5:5, mole/mole) formed matrices to 1) dissolve lipophilic molecules (e.g., curcumin), 2) load lipophilic molecule(s) into the hydrogel, and 3) spontaneously vacate from the system. NADES species ubiquitously occur in natural sources, and a crude extract is a mixture of the NADES species and bioactive metabolites. Based on these ideas, we hypothesized that the crude extract may also allow the loading of natural bioactive molecules from a natural NADES species into (bio)hydrogel systems. To evaluate this hypothesis in vitro, Schisandra chinensis fruit extract was chosen as a representative mixture of lipophilic botanical molecules and hydrophilic NADES species. The results showed that the NADES matrix of S. chinensis was capable of loading at least three bioactive lignans (i.e., gomisin A, gomisin J, and angeloylgomisin H) into the polymer system. The lipophilic metabolites can subsequently be released from the hydrogel. The outcomes suggest that a unique drug delivery mechanism may exist in nature, thereby potentially improving the bioavailability of lipophilic metabolites through physicochemical interactions with the NADES.
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Lignanos/química , Fitoquímicos/química , Schisandra/química , Solventes/química , Disponibilidad Biológica , Frutas/química , Hidrogeles/química , Extractos Vegetales/químicaRESUMEN
While natural products constitute an established source of lead compounds, the classical iterative bioassay-guided isolation process is both time- and labor-intensive and prone to failing to identify active minor constituents. (HP)TLC-bioautography-MS/NMR, which combines cutting-edge microbiological, chromatographic, and spectrometric technologies, was developed to accelerate anti-tuberculosis (TB) drug discovery from natural sources by acquiring structural information at a very early stage of the isolation process. Using the avirulent, bioluminescent Mtb strain mc27000 luxABCDE, three variations of bioautography were evaluated and optimized for sensitivity in detecting anti-TB agents, including established clinical agents and new leads with novel mechanisms of action. Several exemplary applications of this approach to microbial extracts demonstrate its potential as a routine method in anti-TB drug discovery from natural sources.