Development of 2nd generation aminomethyl spectinomycins that overcome native efflux in Mycobacterium abscessus.

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.

Development of a Minimalistic Physiologically Based Pharmacokinetic (mPBPK) Model for the Preclinical Development of Spectinamide Antibiotics.

Spectinamides 1599 and 1810 are lead spectinamide compounds currently under preclinical development to treat multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. These compounds have previously been tested at various combinations of dose level, dosing frequency, and route of administration in mouse models of Mycobacterium tuberculosis (Mtb) infection and in healthy animals. Physiologically based pharmacokinetic (PBPK) modeling allows the prediction of the pharmacokinetics of candidate drugs in organs/tissues of interest and extrapolation of their disposition across different species. Here, we have built, qualified, and refined a minimalistic PBPK model that can describe and predict the pharmacokinetics of spectinamides in various tissues, especially those relevant to Mtb infection. The model was expanded and qualified for multiple dose levels, dosing regimens, routes of administration, and various species. The model predictions in mice (healthy and infected) and rats were in reasonable agreement with experimental data, and all predicted AUCs in plasma and tissues met the two-fold acceptance criteria relative to observations. To further explore the distribution of spectinamide 1599 within granuloma substructures as encountered in tuberculosis, we utilized the Simcyp granuloma model combined with model predictions in our PBPK model. Simulation results suggest substantial exposure in all lesion substructures, with particularly high exposure in the rim area and macrophages. The developed model may be leveraged as an effective tool in identifying optimal dose levels and dosing regimens of spectinamides for further preclinical and clinical development.

Clinical and Translational Pharmacology Considerations for Anti-infectives Approved Under the FDA Animal Rule.

The US Food and Drug Administration's Animal Rule provides a pathway for approval of drugs and biologics aimed to treat serious or life-threatening conditions wherein traditional clinical trials are either not ethical or feasible. In such a scenario, determination of safety and efficacy are based on integration of data on drug disposition and drug action collected from in vitro models, infected animals, and healthy volunteer human studies. The demonstration of clinical efficacy and safety in humans based on robust, well-controlled animal studies is filled with challenges. This review elaborates on the challenges in the translation of data from in vitro and animal models to human dosing for antimicrobials. In this context, it discusses precedents of drugs approved under the Animal Rule, along with the approaches and guidance undertaken by sponsors.

Novel, Brain-Permeable, Cross-Species Benzothiazole Inhibitors of Microsomal Prostaglandin E Synthase-1 (mPGES-1) Dampen Neuroinflammation In Vitro and In Vivo.

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme of the cyclooxygenase (COX) cascade that generates prostaglandin E2 (PGE2) during inflammatory conditions. PGE2 is known to be a potent immune signaling molecule that mediates both peripheral and central inflammations. Inhibition of mPGES-1, rather than COX, may overcome the cardiovascular side effects associated with long-term COX inhibition by providing a more specific strategy to target inflammation. However, mPGES-1 inhibitor development is hampered by the large differences in cross-species activity due to the structural differences between the human and murine mPGES-1. Here, we report that our thiazole-based mPGES-1 inhibitors, compounds 11 (UT-11) and 19 derived from two novel scaffolds, were able to suppress PGE2 production in human (SK-N-AS) and murine (BV2) cells. The IC50 values of inhibiting PGE2 production in human and murine cells were 0.10 and 2.00 µM for UT-11 and 0.43 and 1.55 µM for compound 19, respectively. Based on in vitro and in vivo pharmacokinetic data, we selected UT-11 for evaluation in a lipopolysaccharide (LPS)-induced inflammation model. We found that our compound significantly suppressed proinflammatory cytokines and chemokines in the hippocampus but not in the kidney. Taken together, we demonstrated the potential of UT-11 in treating neuroinflammatory conditions, including epilepsy and stroke, and warrant further optimization.

Characterization of spectinamide 1599 efficacy against different mycobacterial phenotypes.

Spectinamides are a novel series of spectinomycin analogs being developed for the treatment of tuberculosis. The preclinical lead spectinamide 1599 is an antituberculosis drug that possesses robust in vivo efficacy, good pharmacokinetic properties, and excellent safety profiles in rodents. In individuals infected with Mycobacterium tuberculosis or Mycobacterium bovis, causative agents of tuberculosis, the host immune system is capable of restraining these mycobacteria within granulomatous lesions. The harsh microenvironmental conditions of these granuloma lead to phenotypic transformation of mycobacteria. Phenotypically transformed bacteria display suboptimal growth, or complete growth arrest and are frequently associated with drug tolerance. Here we quantified the effect of spectinamide 1599 on log-phase and phenotypically tolerant isoforms of Mycobacterium bovis BCG using various in vitro approaches as a first indicator of spectinamide 1599 activity against various mycobacterial isoforms. We also used the hollow fiber infection model to establish time-kill curves and deployed pharmacokinetic/pharmacodynamic modeling to characterize the activity differences of spectinamide 1599 towards the different phenotypic subpopulations. Our results indicate that spectinamide 1599 is more efficacious against log phase bacteria when compared to its activity against other phenotypically tolerant forms such as acid phase bacteria and hypoxic phase bacteria, a behavior similar to the established antituberculosis drug isoniazid.

Efficacy of a brain-penetrant antiviral in lethal Venezuelan and eastern equine encephalitis mouse models.

Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 µM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg-1 per day fully protected against a 10× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 EEEV FL93-939-infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg-1 per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD50 VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg-1 per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.

Use of Clinical Trial Simulations to Compare the Performance of Different Approaches for Population Analyses of Pediatric Pharmacokinetic Data.

The adequate characterization of the pharmacokinetics of a drug used in pediatrics is a mainstay of pediatric development programs and is critical for accurate dose selection in pediatrics. Analysis approaches can impact the estimation and characterization of pediatric pharmacokinetic parameters. Simulations were conducted to compare the performance of different approaches for analyzing pediatric pharmacokinetic data in the presence of extensive data from adult studies. Simulated clinical trial datasets were generated encompassing different scenarios that might be encountered in pediatric drug development. For each scenario, 250 clinical trials were simulated and analyzed using each of the following approaches: (1) estimating pediatric parameters using only pediatric data; (2) fixing specific parameters to adult estimates and estimating the remaining pediatric parameters using only pediatric data; (3) estimating pediatric parameters using adult parameters as informative Bayesian priors; (4) estimating pediatric parameters using combined adult and pediatric datasets with exponents for body weight effects estimated using adult and pediatric data; and (5) estimating pediatric parameters using combined adult and pediatric datasets with exponents for body weight effects estimated using pediatric data only. Each analysis approach was evaluated for its success in the estimation of true pediatric pharmacokinetic parameter values. Results demonstrated that analyzing pediatric data using a Bayesian approach generally performed best and had the lowest probability of significant bias in the estimated pediatric pharmacokinetic parameters among different scenarios evaluated. This clinical trial simulation framework can be used to inform the optimal approach for analyses of pediatric data for other pediatric drug development program scenarios beyond the cases evaluated in these analyses.

Alcohol inhibits the metabolism of dimethyl fumarate to the active metabolite responsible for decreasing relapse frequency in the treatment of multiple sclerosis.

Dimethyl fumarate (DMF) is a first-line prodrug for the treatment of relapsing-remitting multiple sclerosis (RRMS) that is completely metabolized to monomethyl fumarate (MMF), the active metabolite, before reaching the systemic circulation. Its metabolism has been proposed to be due to ubiquitous esterases in the intestines and other tissues, but the specific enzymes involved are unknown. We hypothesized based on its structure and extensive presystemic metabolism that DMF would be a carboxylesterase substrate subject to interaction with alcohol. We sought to determine the enzymes(s) responsible for the extensive presystemic metabolism of DMF to MMF and the effect of alcohol on its disposition by conducting metabolic incubation studies in human recombinant carboxylesterase-1 (CES1), carboxylesterase-2 (CES2) and human intestinal microsomes (HIM), and by performing a follow-up study in an in vivo mouse model. The in vitro incubation studies demonstrated that DMF was only metabolized to MMF by CES1. Consistent with the incubation studies, the mouse pharmacokinetic study demonstrated that alcohol decreased the maximum concentration and area-under-the-curve of MMF in the plasma and the brain after dosing with DMF. We conclude that alcohol may markedly decrease exposure to the active MMF metabolite in the plasma and brain potentially decreasing the effectiveness of DMF in the treatment of RRMS.

Preclinical Evaluation of Inhalational Spectinamide-1599 Therapy against Tuberculosis.

The lengthy treatment time for tuberculosis (TB) is a primary cause for the emergence of multidrug resistant tuberculosis (MDR-TB). One approach to improve TB therapy is to develop an inhalational TB therapy that when administered in combination with oral TB drugs eases and shortens treatment. Spectinamides are new semisynthetic analogues of spectinomycin with excellent activity against Mycobacterium tuberculosis (Mtb), including MDR and XDR Mtb strains. Spectinamide-1599 was chosen as a promising candidate for development of inhalational therapy. Using the murine TB model and intrapulmonary aerosol delivery of spectinamide-1599, we characterized the pharmacokinetics and efficacy of this therapy in BALB/c and C3HeB/FeJ mice infected with the Mtb Erdman strain. As expected, spectinamide-1599 exhibited dose-dependent exposure in plasma, lungs, and ELF, but exposure ratios between lung and plasma were 12-40 times higher for intrapulmonary compared to intravenous or subcutaneous administration. In chronically infected BALB/c mice, low doses (10 mg/kg) of spectinamide-1599 when administered thrice weekly for two months provide efficacy similar to that of higher doses (50-100 mg/kg) after one month of therapy. In the C3HeB/FeJ TB model, intrapulmonary aerosol delivery of spectinamide-1599 (50 mg/kg) or oral pyrazinamide (150 mg/kg) had limited or no efficacy in monotherapy, but when both drugs were given in combination, a synergistic effect with superior bacterial reduction of >1.8 log10 CFU was observed. Throughout the up to eight-week treatment period, intrapulmonary therapy was well-tolerated without any overt toxicity. Overall, these results strongly support the further development of intrapulmonary spectinamide-1599 as a combination partner for anti-TB therapy.

Model-Based Exposure-Response Assessment for Spectinamide 1810 in a Mouse Model of Tuberculosis.

Despite decades of research, tuberculosis remains a leading cause of death from a single infectious agent. Spectinamides are a promising novel class of antituberculosis agents, and the lead spectinamide 1810 has demonstrated excellent efficacy, safety, and drug-like properties in numerous in vitro and in vivo assessments in mouse models of tuberculosis. In the current dose ranging and dose fractionation study, we used 29 different combinations of dose level and dosing frequency to characterize the exposure-response relationship for spectinamide 1810 in a mouse model of Mycobacterium tuberculosis infection and in healthy animals. The obtained data on 1810 plasma concentrations and counts of CFU in lungs were analyzed using a population pharmacokinetic/pharmacodynamic (PK/PD) approach as well as classical anti-infective PK/PD indices. The analysis results indicate that there was no difference in the PK of 1810 in infected compared to healthy, uninfected animals. The PK/PD index analysis showed that bacterial killing of 1810 in mice was best predicted by the ratio of maximum free drug concentration to MIC (fCmax/MIC) and the ratio of the area under the free concentration-time curve to the MIC (fAUC/MIC) rather than the cumulative percentage of time that the free drug concentration is above the MIC (f%TMIC). A novel PK/PD model with consideration of postantibiotic effect could adequately describe the exposure-response relationship for 1810 and supports the notion that the in vitro observed postantibiotic effect of this spectinamide also translates to the in vivo situation in mice. The obtained results and pharmacometric model for the exposure-response relationship of 1810 provide a rational basis for dose selection in future efficacy studies of this compound against M. tuberculosis.

Pediatric Dose Selection for Therapeutic Proteins.

In selecting optimal dosing regimens in support of the clinical use of monoclonal antibodies and other therapeutic proteins in pediatric indications, the unique pharmacokinetic properties of this class of biologics, as well as the underlying physiologic and pathophysiologic processes and their modulation by childhood growth and development, needs to be appreciated. During drug development, first-in-pediatric dose selection is a capstone event in the pediatric investigation plan that relies heavily on extrapolation of pharmacokinetic and pharmacodynamic data from adult to pediatric populations. It is facilitated by combinations of pharmacometric approaches, including allometry, physiologically based pharmacokinetic modeling, and population pharmacokinetic analyses, although data on reliability and qualification of some of these tools in the context of therapeutic proteins are still limited but emerging. Presented data suggest nonlinear relationships between body weight and both clearance and volume of distribution for therapeutic proteins in pediatric populations, with allometric exponents of 0.75 and 0.8, respectively. For newborns and infants (<1 year), even higher nonlinearity seems to occur. Translation of the quantitative characterization of the pediatric pharmacokinetics of therapeutic proteins into dosing regimens for the drug label requires compromising between precision dosing and clinical practicability, with tiered dosing algorithms based on size or age strata being the currently most frequently applied methodology.

Pharmacokinetics and Clinical Pharmacology of Monoclonal Antibodies in Pediatric Patients.

Monoclonal antibodies (mAbs) and their derivatives are increasingly used in pediatric pharmacotherapy, and the number of antibody-based drug products with approved pediatric indications is continuously growing. In most instances, pediatric use is being pursued after the efficacy and safety of novel antibody medications have been established in adult indications. The pediatric extrapolation exercise that is frequently used in this context to bridge efficacy and safety from adults to children is oftentimes challenged through uncertainties and knowledge gaps in how to reliably extrapolate pharmacokinetics and clinical pharmacology of mAbs to different pediatric age groups, and how to derive age-appropriate dosing regimens that strike a balance between precision dosing and practicability. The article highlights some of the pharmacokinetic and clinical pharmacology challenges with regard to therapeutic use of mAbs and antibody derivatives in children, including immunogenicity events. Although considering body size-based differences in drug disposition can account for many of the perceived and actual differences in the distribution and elimination of antibody-based therapeutics between children and adults, increasing evidence suggests potential or actual age-associated differences beyond size differences, especially for young pediatric patients such as newborns and infants. To overcome age-associated differences in antibody disposition, various different dosing approaches have been applied to ensure safe and efficacious antibody exposure for pediatric populations of different ages. The development of such dosing regimens and the associated pathway to pediatric indication approval is illustrated in more detail for two antibody-based biologics, the fusion protein abatacept and the mAb tocilizumab.