The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for ...enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of peptides without compromising their potency. Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo. We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.
This work describes the enhancement of a novel antitumor therapeutic platform that combines advantages from small-molecule drug conjugates (SMDCs) and antibody drug conjugates (ADCs). ...Valine–citrulline (VCit) dipeptide linkers are commonly used cathepsin B cleavable linkers for ADCs. However, the instability of these linkers in mouse serum makes translating efficacy data from mouse to human more challenging. Replacing the VCit linker with glutamic acid–valine–citrulline (EVCit) has been reported to enhance the stability of ADCs in mouse serum. However, the effect of EVCit linker on the stability of SMDCs has not been reported. Here, we report that incorporating the EVCit linker in prostate-specific membrane antigen-targeting SMDCs, equipped with the transthyretin ligand AG10, resulted in conjugates with lower toxicity, an extended half-life, and superior therapeutic efficacy to docetaxel in a xenograft mouse model of prostate cancer. This should make SMDCs’ preclinical toxicity and efficacy data from mice more reliable for predicting human results.
Metabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate ...in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors.
Herein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors.
Efficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors.
GA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10- fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment.
These studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.
The rising rate of antimicrobial resistance continues to threaten global public health. Further hastening antimicrobial resistance is the lack of new antibiotics against new targets. The bacterial ...enzyme, 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), is thought to play important roles in central metabolism, including processes required for pathogen adaptation to fluctuating host environments. Thus, impairing DXPS function represents a possible new antibacterial strategy. We previously investigated a DXPS-dependent metabolic adaptation as a potential target in uropathogenic
(UPEC) associated with urinary tract infection (UTI), using the DXPS-selective inhibitor butyl acetylphosphonate (BAP). However, investigations of DXPS inhibitors
have not been conducted. The goal of the present study is to advance DXPS inhibitors as
probes and assess the potential of inhibiting DXPS as a strategy to prevent UTI
. We show that BAP was well-tolerated at high doses in mice and displayed a favorable pharmacokinetic profile for studies in a mouse model of UTI. Further, an alkyl acetylphosphonate prodrug (homopropargyl acetylphosphonate, pro-hpAP) was significantly more potent against UPEC in urine culture and exhibited good exposure in the urinary tract after systemic dosing. Prophylactic treatment with either BAP or pro-hpAP led to a partial protective effect against UTI, with the prodrug displaying improved efficacy compared to BAP. Overall, our results highlight the potential for DXPS inhibitors as
probes and establish preliminary evidence that inhibiting DXPS impairs UPEC colonization in a mouse model of UTI.IMPORTANCENew antibiotics against new targets are needed to prevent an antimicrobial resistance crisis. Unfortunately, antibiotic discovery has slowed, and many newly FDA-approved antibiotics do not inhibit new targets. Alkyl acetylphosphonates (alkyl APs), which inhibit the enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), represent a new possible class of compounds as there are no FDA-approved DXPS inhibitors. To our knowledge, this is the first study demonstrating the
safety, pharmacokinetics, and efficacy of alkyl APs in a urinary tract infection mouse model.
Cognitive decline in Alzheimer's disease (AD) is associated with hyperphosphorylated tau (pTau) propagation between neurons along synaptically connected networks, in part via extracellular vesicles ...(EVs). EV biogenesis is triggered by ceramide enrichment at the plasma membrane from neutral sphingomyelinase2 (nSMase2)-mediated cleavage of sphingomyelin. We report, for the first time, that human tau expression elevates brain ceramides and nSMase2 activity.
To determine the therapeutic benefit of inhibiting this elevation, we evaluated PDDC, the first potent, selective, orally bioavailable, and brain-penetrable nSMase2 inhibitor in the transgenic PS19 AD mouse model. Additionally, we directly evaluated the effect of PDDC on tau propagation in a mouse model where an adeno-associated virus (AAV) encoding P301L/S320F double mutant human tau was stereotaxically-injected unilaterally into the hippocampus. The contralateral transfer of the double mutant human tau to the dentate gyrus was monitored. We examined ceramide levels, histopathological changes, and pTau content within EVs isolated from the mouse plasma.
Similar to human AD, the PS19 mice exhibited increased brain ceramide levels and nSMase2 activity; both were completely normalized by PDDC treatment. The PS19 mice also exhibited elevated tau immunostaining, thinning of hippocampal neuronal cell layers, increased mossy fiber synaptophysin immunostaining, and glial activation, all of which were pathologic features of human AD. PDDC treatment reduced these changes. The plasma of PDDC-treated PS19 mice had reduced levels of neuronal- and microglial-derived EVs, the former carrying lower pTau levels, compared to untreated mice. In the tau propagation model, PDDC normalized the tau-induced increase in brain ceramides and significantly reduced the amount of tau propagation to the contralateral side.
PDDC is a first-in-class therapeutic candidate that normalizes elevated brain ceramides and nSMase2 activity, leading to the slowing of tau spread in AD mice.
Alzheimer's disease (AD) is characterized by the progressive accumulation of amyloid-β and hyperphosphorylated tau (pTau), which can spread throughout the brain via extracellular vesicles (EVs). ...Membrane ceramide enrichment regulated by the enzyme neutral sphingomyelinase 2 (nSMase2) is a critical component of at least one EV biogenesis pathway. Our group recently identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), the most potent (30 nM) and selective inhibitor of nSMase2 reported to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), modest brain penetration, and rapid clearance, limiting its clinical translation. To enhance its PK properties, we conjugated DPTIP to a hydroxyl-PAMAM dendrimer delivery system, creating dendrimer-DPTIP (D-DPTIP). In an acute brain injury model, orally administered D-DPTIP significantly reduced the intra-striatal IL-1β-induced increase in plasma EVs up to 72 h post-dose, while oral DPTIP had a limited effect. In a mouse tau propagation model, where a mutant hTau (P301L/S320F) containing adeno-associated virus was unilaterally seeded into the hippocampus, oral D-DPTIP (dosed 3× weekly) significantly inhibited brain nSMase2 activity and blocked the spread of pTau to the contralateral hippocampus. These data demonstrate that dendrimer conjugation of DPTIP improves its PK properties, resulting in significant inhibition of EV propagation of pTau in mice. Dendrimer-based delivery of DPTIP has the potential to be an exciting new therapeutic for AD.
Glutamate carboxypeptidase-II (GCPII) is a zinc-dependent metalloenzyme implicated in numerous neurological disorders. The pharmacophoric requirements of active-site GCPII inhibitors makes them ...highly charged, manifesting poor pharmacokinetic (PK) properties. Herein, we describe the discovery and characterization of catechol-based inhibitors including L-DOPA, D-DOPA, and caffeic acid, with sub-micromolar potencies. Of these, D-DOPA emerged as the most promising compound, with good metabolic stability, and excellent PK properties. Orally administered D-DOPA yielded high plasma exposures (AUCplasma = 72.7 nmol·h/mL) and an absolute oral bioavailability of 47.7%. Unfortunately, D-DOPA brain exposures were low with AUCbrain = 2.42 nmol/g and AUCbrain/plasma ratio of 0.03. Given reports of isomeric inversion of D-DOPA to L-DOPA via D-amino acid oxidase (DAAO), we evaluated D-DOPA PK in combination with the DAAO inhibitor sodium benzoate and observed a >200% enhancement in both plasma and brain exposures (AUCplasma = 185 nmol·h/mL; AUCbrain = 5.48 nmol·h/g). Further, we demonstrated GCPII target engagement; orally administered D-DOPA with or without sodium benzoate caused significant inhibition of GCPII activity. Lastly, mode of inhibition studies revealed D-DOPA to be a noncompetitive, allosteric inhibitor of GCPII. To our knowledge, this is the first report of D-DOPA as a distinct scaffold for GCPII inhibition, laying the groundwork for future optimization to obtain clinically viable candidates.
The 2023 Generic Drug Science and Research Initiative Public Workshop organized by the U.S. Food and Drug Administration (FDA) discussed the research needs to improve and enhance bioequivalence (BE) ...approaches for generic drug development. FDA takes such research needs and panel discussions into account to develop its Generic Drug User Fee Amendments III (GDUFA III) Science and Research Initiatives specific to generics. During the five workshop sessions, presentations and panel discussions focused on identifying and addressing scientific gaps and research needs related to nitrosamine impurity issues, BE assessment for oral products, innovative BE approaches for long-acting injectable products, alternative BE approaches for orally inhaled products, and advanced BE methods for topical products. Specifically, this report highlights the discussions on how to improve BE assessment for developing generic drug products based on research priorities for leveraging quantitative methods and modeling, as well as artificial intelligence/machine learning (AI/ML).
Graphical Abstract
•Extracellular vesicles (EVs) are potent vehicles of intercellular communication.•EVs can transport pathological cargo that contributes to disease.•One pathway of EV biogenesis is dependent upon ...ceramides generated by nSMase2.•Inhibition of nSMase2 shows promise in treating diseases that propagate via EVs.•Potent and selective nSMase2 inhibitors have recently been discovered.
Extracellular vesicles (EVs) are indispensable mediators of intercellular communication, but they can also assume a nefarious role by ferrying pathological cargo that contributes to neurological, oncological, inflammatory, and infectious diseases. The canonical pathway for generating EVs involves the endosomal sorting complexes required for transport (ESCRT) machinery, but an alternative pathway is induced by the enrichment of lipid membrane ceramides generated by neutral sphingomyelinase 2 (nSMase2). Inhibition of nSMase2 has become an attractive therapeutic strategy for inhibiting EV biogenesis, and a growing number of small-molecule nSMase2 inhibitors have shown promising therapeutic activity in preclinical disease models. This review outlines the function of EVs, their potential role in disease, the discovery and efficacy of nSMase2 inhibitors, and the path to translate these findings into therapeutics.
Extracellular vesicles (EVs) can carry pathological cargo and play an active role in disease progression. Neutral sphingomyelinase-2 (nSMase2) is a critical regulator of EV biogenesis, and its ...inhibition has shown protective effects in multiple disease states. 2,6-Dimethoxy-4-(5-phenyl-4-thiophen-2-yl-1H-imidazol-2-yl)phenol (DPTIP) is one of the most potent (IC50 = 30 nM) inhibitors of nSMase2 discovered to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), limiting its clinical development. To overcome DPTIP’s PK limitations, we synthesized a series of prodrugs by masking its phenolic hydroxyl group. When administered orally, the best prodrug (P18) with a 2′,6′-diethyl-1,4′-bipiperidinyl promoiety exhibited >fourfold higher plasma (AUC0–t = 1047 pmol·h/mL) and brain exposures (AUC0–t = 247 pmol·h/g) versus DPTIP and a significant enhancement of DPTIP half-life (2 h vs ∼0.5 h). In a mouse model of acute brain injury, DPTIP released from P18 significantly inhibited IL-1β-induced EV release into plasma and attenuated nSMase2 activity. These studies report the discovery of a DPTIP prodrug with potential for clinical translation.
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