The Journal of Pharmacology and Experimental Therapeutics

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan, with important roles in chronic inflammation, cancer and autoimmunity. 4-methylumbelliferone (4-MU), a small molecule inhibitor of HA synthases, is widely used to study HAs interactions with the surrounding tissues and the immune cells. There is substantial experimental and therapeutic interest in using oral 4-MU to inhibit HA synthesis, but pharmacokinetic and pharmacodynamic data on treatment routes have been lacking. Moreover, it recently became clear that the main metabolite of 4-MU, 4-methlyumbelliferyl glucuronide (4-MUG), is bioactive. We therefore sought to define the pharmacokinetics and pharmacodynamics of 4-MU and its active metabolite 4-MUG in mice. Single dose mouse studies showed that 4-MU administered intravenously (i.v.) resulted in 100-fold higher 4-MU exposure compared to oral (p.o.) administration. The 4-MU ratio AUC i.v./AUC p.o. was 96/1. 4-MUG exposures were much higher than 4-MU exposures after both 4-MU i.v. and p.o. administration, but only small differences in 4-MUG exposure were seen after 4-MU i.v. versus p.o. administration. The 4-MUG metabolite was also administered as a single dose both i.v. and p.o. and showed a 25.9% bioavailability. Compared to 4-MUG p.o. dosing, 1.14 higher 4-MUG exposures were seen after 4-MU p.o. dosing. 4-MU exposure after 4-MUG p.o. administration was minimal but similar to 4-MU exposure after 4-MU p.o. administration. In mice treated for several weeks with 4-MU in chow, the 4-MU concentration immediately drops after treatment was stopped, whereas the 4-MUG concentration showed a peak 1 hour after treatment stop. In a build-up study, 4-MU and 4-MUG treatment in mice lead to a plateau of 4-MU concentration starting at 4 days post treatment start. These 4-MU and 4-MUG concentration findings in vivo will inform future clinical studies and experimental work with 4-MU.

Previous studies show ATP-sensitive potassium (KATP) channel openers can reduce hypersensitivity associated with chronic pain models in rodents, and reduce morphine tolerance. Many agonists of KATP channels are not soluble in physiologically relevant vehicles, requiring adaptation for clinical use. This study compared the antinociception activity of novel KATP channel targeting prodrugs, CKLP1, CKLP2, and CF3-CKLP. These prodrugs are activated by endogenous alkaline phosphatase enzymes present in the peripheral and central nervous systems. Analgesic capabilities of intrathecally injected prodrugs were tested in rodent models of spinal nerve ligation (SNL) and Complete Freunds Adjuvant (CFA) as models for neuropathic and inflammatory pain, respectively. CKLP1 and CKLP2 significantly increased mechanical paw withdrawal thresholds 1-2 hours after intrathecal administration in the SNL model, but all three prodrugs were able to attenuate hypersensitivity up to 7 days after CFA treatment. The reduction of opioid tolerance and opioid-induced hypersensitivity in mice treated chronically with morphine was significantly reduced in CKLP1 and CKLP2 treated animals. Prodrug cleavage was confirmed in mouse spinal cords using liquid chromatography. These studies may aid in the further development of KATP channel prodrugs for use in treatments of chronic pain, opioid tolerance, and withdrawal.

4-chlorokynurenine (4-Cl-KYN) is in clinical development for potential CNS indications. We have sought to further understand the distribution and metabolism of 4-Cl-KYN as this information might provide a strategy to enhance the clinical development of this drug. We used excretion studies in rats, in vitro transporter assays and pharmacogenetic analysis of clinical trial data to determine how 4-Cl-KYN and metabolites are distributed. Our data indicated that a novel acetylated metabolite (N-acetyl-4-Cl-KYN) did not affect the uptake of 4-Cl-KYN across the blood-brain barrier via LAT1. 4-Cl-KYN and metabolites were found to be renally excreted in rodents. In addition, we found that N-acetyl-4-Cl-KYN inhibited renal and hepatic transporters involved in excretion. Thus, this metabolite had the potential to limit the excretion of a range of compounds. Our pharmacogenetic analysis found that a SNP in N-acetyltransferase 8 (NAT8, rs13538) was linked to levels of N-acetyl-4-Cl-KYN relative to 4-Cl-KYN found in the plasma and that a SNP in SLC7A5 (rs28582913) was associated with the plasma levels of the active metabolite, 7-Cl-KYNA. Thus, we have a pharmacogenetics-based association for plasma drug level that could aid in the drug development of 4-Cl-KYN and have investigated the interaction of a novel metabolite with drug transporters.

Opioid analgesics like morphine and fentanyl induce mu-opioid receptor (MOR)-mediated hyperactivity in mice. Here we show that morphine, fentanyl, SR-17018, and oliceridine have submaximal intrinsic efficacy in the mouse striatum using 35S-GTP{gamma}S binding assays. While all of the agonists act as partial agonists for stimulating G protein coupling in striatum, morphine, fentanyl and oliceridine are fully efficacious in stimulating locomotor activity; meanwhile, the noncompetitive biased agonists, SR-17018 and SR-15099 produce submaximal hyperactivity. Moreover, the combination of SR-17018 and morphine attenuates hyperactivity while antinociceptive efficacy is increased. The combination of oliceridine with morphine increases hyperactivity which is maintained over time. These findings provide evidence that noncompetitive agonists at MOR can be used to suppress morphine-induced hyperactivity while enhancing antinociceptive efficacy; moreover, they demonstrate that intrinsic efficacy measured at the receptor level is not directly proportional to drug efficacy in the locomotor activity assay.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (Ki 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3H and 14C labelled RNB-61 showed apparent Kd values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

Pentilludin is a novel, potent (690 nM) irreversible inhibitor of actions of the receptor type protein tyrosine phosphatase D (PTPRD). Pentilludin displays no in vitro activities in Ames or micronucleus tests, at hERG channels or at targets for currently-licensed drugs. Rats treated with pentilludin doses up to 100 mg/kg/day for two weeks have not been found to display behavioral, hematologic or serum chemistry abnormalities. Treatment with 20 mg/kg sc pentilludin prior to every other M-W-F self-administration session substantially reduces self-administration of amphetamine and more modestly reduces self-administration of remifentanil. Pentilludin provides a novel means for reducing self-administration of psychostimulant and, modestly, opiate drugs in ways that could enhance abstinence in humans.

Fatal opioid overdoses in the United States have nearly tripled during the past decade, with greater than 92% involving a synthetic opioid like fentanyl. Fentanyl potently activates the -opioid receptor to induce both analgesia and respiratory depression. The danger of illicit fentanyl has recently been exacerbated by adulteration with xylazine, an 2-adrenergic receptor agonist typically used as a veterinary anesthetic. In 2023, over a 1,000% increase in xylazine-positive overdoses was reported in some regions of the U.S. Xylazine has been shown to potentiate the lethality of fentanyl in mice, yet a mechanistic underpinning for this effect has not been defined. Herein, we evaluate fentanyl, xylazine, and their combination in whole-body plethysmography (to measure respiration) and pulse oximetry (to measure blood oxygen saturation and heart rate) in male and female CD-1 mice. We show that xylazine decreases breathing rate more than fentanyl by increasing the expiration time. In contrast, fentanyl primarily reduces breathing by inhibiting inspiration, and xylazine exacerbates these effects. Fentanyl but not xylazine decreased blood oxygen saturation, and when combined, xylazine did not change the maximum level of fentanyl-induced hypoxia. Xylazine also reduced heart rate more than fentanyl. Finally, loss in blood oxygen saturation correlated with the frequency of fentanyl-induced apneas, but not breathing rate. Together, these findings provide insight into how the addition of xylazine to illicit fentanyl may increase the risk of overdose. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/608310v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1a3aabeorg.highwire.dtl.DTLVardef@1eac11org.highwire.dtl.DTLVardef@1b2ceb9org.highwire.dtl.DTLVardef@220f06_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundMicroglial reactivity, a hallmark of many neurodegenerative diseases, is thought to contribute significantly to disease pathology. In experimental models, colony stimulating factor 1 receptor (CSF1R) inhibitors transiently deplete microglia to resolve inflammation, leading to improved neuropathology. In oncology, CSF1R inhibitors modulate tumor-associated macrophages (TAMs) toward a tumor-suppressive phenotype by silencing CSF1-CSF1R signaling. As for any therapeutic, target engagement depends on effective drug delivery. In the brain a major hurdle is the limited drug delivery caused by the presence of the blood brain barrier (BBB) containing drug efflux transporters. However, the affinity to these transporters of most CSF1R inhibitors is unknown. MethodsWe assessed the brain penetrance of two CSF1R inhibitors, pexidartinib (PLX3397) and sotuletinib (BLZ945), in the absence and presence of drug transporters ABCB1 and ABCG2. We further assessed their impact on peripheral immune populations, tissue-resident macrophages, microglia and oligodendrocyte progenitor cells (OPCs). ResultsBoth compounds have a limited brain permeability (brain-to-plasma ratio: 0.1). Sotuletinib was a substrate for both ABCB1 and ABCG2, whereas pexidartinib was transported primarily by ABCB1. Despite low brain exposure, both are able to ablate microglia when given to mice at high doses, accompanied by marked depletion of OPCs and macrophage populations in the liver, intestine, and kidney, as well as non-classical monocytes in blood. Pexidartinib additionally altered splenic immune composition, increasing T cells and neutrophils, and reducing dendritic cells and non-classical monocytes. ConclusionThese findings highlight that high-dose CSF1R inhibition rapidly depletes microglia, but induces substantial off-target effects. Such systemic impacts, as well as the impact on OPCs, should be considered when interpreting experimental outcomes or translating CSF1R inhibition into clinical contexts where brain targeting is required. Key messagesO_LICSF1R inhibitors pexidartinib and sotuletinib show poor brain penetrance (brain-to-plasma ratio 0.1) C_LIO_LISotuletinib is a substrate to ABCB1 and ABCG2, pexidartinib is a substrate to ABCB1. C_LIO_LIBoth drugs rapidly deplete microglia, despite poor brain penetration C_LIO_LIMicroglia depletion is accompanied by loss of OPCs and tissue macrophages C_LI

An acetaminophen (APAP) overdose is the leading cause of drug-induced hepatotoxicity and acute liver failure (ALF) in the United States. While N-acetylcysteine (NAC), is highly effective when administered early after an overdose, its efficacy decreases with delayed administration. Since most patients present late to the clinic, there is an urgent need for novel late-acting therapeutic options to prevent progression to ALF. We previously demonstrated the benefit of delayed activation of the Adenosine A2B Receptor (A2BAR) in attenuating APAP-induced hepatotoxicity and this study focuses on its effects on liver recovery after injury. Fasted male C57BL/6J mice were treated with 300 mg/kg APAP, followed by activation of A2BAR 6 or 9 h later and sacrifice 24, 48 or 72 h post-APAP with evaluation of liver injury, the innate immune response and liver regeneration. Delayed activation of A2BAR significantly enhanced liver recovery, with accelerated repopulation of the liver by Kupffer cells, increased macrophage migration to the necrotic areas and their faster resolution. A2BAR activation also upregulated lipid metabolism related genes in non-parenchymal cells and cell proliferation and metabolism genes in hepatocytes. Remarkably, genes such as Cidec and Plin2, crucial for lipid droplet formation, were upregulated, indicating that A2ABR activation enhances lipid metabolism which plays a key role in providing energy for liver regeneration. Overall, these findings highlight the potential of A2BAR activation not only in protecting against liver injury, but also in promoting and accelerating liver regeneration by modulating the innate immune responses and metabolic pathways.

The dopamine transporter (DAT) is an essential protein in the maintenance of dopamine homeostasis in the brain. Thus, single amino acid changes in the gene that encodes for DAT can be sufficient to induce disease, such as Dopamine Transporter Deficiency Syndrome (DTDS). DTDS-associated variants are posited to cause DAT protein misfolding, retention in the endoplasmic reticulum, and a consequent depletion or loss of DAT at the cell surface. In turn, proper dopaminergic regulation is lost. Current treatments for DTDS are largely ineffective, and improved therapeutic options are greatly needed. To this end, we have created a novel mouse model of DTDS harboring the A313V knock-in DAT variant, a proxy for the DTDS-causing A314V variant in humans. We show that the A313V knock-in DAT mice are hyperactive, have increased striatal tissue content of dopamine and its metabolites homovanillic acid (HVA) and DOPAC, and impaired dopamine uptake. We demonstrate that FDA approved compounds alpha-methyl-para-tyrosine ([a]MPT) and amphetamine (AMPH) ameliorate hyperactivity in this mouse model. Moreover, [a]MPT may be a disease-modifying treatment by addressing the hyperdopaminergic tone underlying this hyperactivity. In contrast, noribogaine, a pharmacological chaperone for DAT, is unable to rescue DAT expression. Taken together, these findings show that the A313V knock-in DAT variant mice recapitulate several defining phenotypes seen in patients with DTDS, and provide evidence for two novel treatments for the disease.

Background and PurposeThe use of "Bath Salts" drug preparations has been associated with high rates of toxicity and death. Preparations often contain mixtures of drugs including multiple synthetic cathinones or synthetic cathinones and caffeine; however, little is known about whether interactions among "Bath Salts" constituents contribute to the adverse effects often reported in users. Experimental ApproachThis study used adult male Sprague-Dawley rats to characterize the cardiovascular effects, locomotor effects, and pharmacokinetics of methylone, MDPV, and caffeine, administered alone and as binary mixtures. Dose-addition analyses were used to determine the effect levels predicted for a strictly additive interaction for each dose pair. Key ResultsMethylone, MDPV, and caffeine increased heart rate and locomotion, with methylone producing the largest increase in heart rate, MDPV producing the largest increase in locomotor activity, and caffeine being the least effective in stimulating heart rate and locomotor activity. MDPV and caffeine increased mean arterial pressure, with caffeine being more effective than MDPV. The nature of the interactions between methylone and MDPV tended toward sub-additivity for all endpoints, whereas interactions between MDPV or methylone and caffeine tended to be additive or sub-additive for cardiovascular endpoints, and additive or supra-additive for increases in locomotion. No pharmacokinetic interactions were observed between individual constituents, but methylone displayed non-linear pharmacokinetics at the largest dose evaluated. Conclusion and ImplicationsThese findings demonstrate that the composition of "Bath Salts" preparations can impact both cardiovascular and locomotor effects and suggest that such interactions among constituent drugs could contribute to the "Bath Salts" toxidrome reported by human users. What is already known"Bath Salts" preparations are associated with a sympathomimetic toxidrome in human users. What this study addsCharacterization of both pharmacokinetic and pharmacodynamic interactions between common "Bath Salts" constituents with regard to cardiovascular and locomotor effects. Clinical SignificanceThe vast majority of drug overdose deaths involve more than one substance. Though these studies focused on combinations of stimulant drugs, they provide direct evidence that the toxidrome resulting from multi-drug overdoses can be significantly different than would be expected for a single drug.

RA-0002034 (1) is a potent covalent inhibitor targeting the alphavirus nsP2 cysteine protease. The species-dependent pharmacokinetics and metabolism of 1 were investigated to evaluate its therapeutic potential. Pharmacokinetic profiling revealed rapid clearance in mice, predominantly mediated by glutathione S-transferase (GST)-catalyzed conjugation. This metabolic liability contrasted with slower clearance observed in human hepatocytes and preclinical species such as rats, dogs, and monkeys. Cross-species studies confirmed the dominance of GST-driven metabolism in mice, whereas oxidative pathways were more pronounced in dogs. Despite rapid systemic clearance, 1 achieved antiviral efficacy in mice, reducing CHIKV viral loads in multiple tissues. Initial estimations of human hepatic clearance and half-life extrapolated from animal data indicate that b.i.d. dosing of 1 will be possible to maintain concentrations sufficient for antiviral activity in humans. These cross-species pharmacokinetic and metabolism studies support the continued evaluation of 1 as a promising anti-alphaviral therapeutic. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/632788v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@8c1558org.highwire.dtl.DTLVardef@cd7e21org.highwire.dtl.DTLVardef@113e88aorg.highwire.dtl.DTLVardef@7c7dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF), with acute kidney injury (AKI) contributing substantially to morbidity and mortality in those patients. To determine whether APAP-induced AKI depends on hepatic CYP2E1-mediated bioactivation, we used CYP2E1^flox/flox^ mice treated with AAV8-TBG-Cre to selectively delete hepatic CYP2E1 while preserving renal metabolism. Male and female mice received APAP (600 mg/kg) and were evaluated up to 48 hours for liver and kidney injury. Liver-specific CYP2E1 deletion reduced APAP hepatotoxicity, confirming the absence of hepatic NAPQI formation. Despite this protection, both male and female mice treated with AAV8-TBG-Cre and APAP developed progressive renal injury, with marked increases in blood urea nitrogen (BUN) and creatinine, tubular vacuolation, and strong induction of KIM-1 and osteopontin, along with apoptotic cell death at 48 hours. Notably, female mice, lacking renal CYP2E1 and displaying no detectable renal protein adducts, still progressed to AKI, demonstrating that kidney injury can occur through CYP-independent mechanisms. Given that APAP-induced AKI is a delayed injury, we further considered p-aminophenol (PAP), a deacetylation product of APAP, as a potential CYP-independent contributor. These findings support the concept that non-CYP pathways, including PAP formation, may contribute to kidney injury during the later phase of toxicity, although this pathway likely represents only one component of a multifactorial injury process. Together, these results demonstrate that APAP-induced AKI is a kidney-intrinsic process that can develop independently of both hepatic and renal CYP2E1 activity, emphasizing the need for kidney-specific therapeutic strategies for preventing APAP-induced renal injury.

FC-12738, a retro-inverso pentapeptide developed by Neurodegenerative Disease Research, Inc., is currently under investigation for treating neuroinflammation associated with amyotrophic lateral sclerosis (ALS). This study aimed to evaluate the pharmacokinetic properties of FC-12738, including absorption, distribution, metabolism, excretion, and drug-drug interactions. Pharmacokinetics were assessed in Sprague-Dawley rats and beagle dogs following intravenous and subcutaneous administration. Our findings suggest that FC-12738 demonstrates many favorable pharmacokinetic properties, although further optimization may be required to improve CNS penetrance.

PurposeIfosfamide (IFO) is an effective chimiotherapeutic agent for sarcomas and germ-cell tumors but its clinical use is limited by severe toxicities, particularly hemorrhagic cystitis and encephalopathy. We recently demonstrated that IFO displays a circadian rhythm of lethal toxicity at LD50, with survival, body weight loss and core temperature strongly dependent on dosing time (Chennoufi MM & Boughattas NA, 2025). That study established the presence of an intrinsic chronotolerance rhythm, but lethality prevented organ-specific analyses. Here, we extend this work by assessing whether sublethal IFO exposure (LD30). Methods160 male Swiss albinos mice were synchronized to a 12:12 h light-dark cycle. IFO LD30 was administered at four circadian times: 1, 7, 13, and 19 Hours After Light Onset (HALO). Endpoints included hematology, hepatic enzymes, histopathology (brain, liver, kidney, and bladder) and neurobehavioral function. Organ-specific chronotoxicity patterns were compared to the LD50 chronotolerance rhythm previously reported. ResultsMarked circadian rhythms were observed across all endpoints. Dosing at 7 HALO induced the most severe overall toxicity, while administration at 13 HALO significantly reduced hepatic, renal, and bladder injury, encephalopathy-related lesions appeared only at 19 HALO. Hematological suppression and hepatic enzyme elevations also varied by dosing time, with lowest toxicity consistently at 13 HALO. Neurobehavioral impairment followed the same pattern. The optimal tolerance window overlapped with the LD50 peak tolerance found in our earlier study. ConclusionSublethal IFO toxicity is strongly time-of-day dependent. Administration at 13 HALO (early active phase) minimizes brain and multi-organ injury and aligns with the chronotolerance peak previously defined at LD50. These complementary findings support the development of circadian-based IFO chronotherapy to improve clinical safety.

Parkinsons disease (PD) is the second most common neurodegenerative disease. Despite a concerted effort on the part of the scientific community, there is no proven strategy for slowing PD progression. Nevertheless, there are several potential drug targets that if functionally modified could alter disease course. Preclinical, epidemiological and clinical trial data suggest that Cav1 Ca2+ channels are one such target. Dihydropyridines (DHPs) are voltage-dependent, negative allosteric modulators of Cav1 Ca2+ channels that are approved for human use. However, the brain concentration of DHPs that can be safely achieved in humans with oral dosing is limited because of the widespread distribution of these channels, particularly in the vasculature. Intranasal administration of DHPs is a potential alternative delivery strategy that has been used with compounds that have similar limitations. To test the viability of this drug administration strategy, mice were intranasally or orally administered the DHP isradipine mixed in one of three vehicles. Plasma and brain concentrations of isradipine were then determined using liquid chromatography/mass spectroscopy at subsequent times. These studies demonstrated that intranasal administration of isradipine was able to achieve higher brain concentrations than those in the plasma, and these differences persisted for hours. Thus, intranasal administration of DHPs could be used to achieve high levels of Cav1 Ca2+ channel inhibition in the brain without producing unwanted peripheral side-effects.

Coronary heart disease is caused by the accumulation of atherosclerotic plaques which narrow the arteries over time. The plaques are formed by cholesterol deposits in the arterial intima and lead to the symptom of angina pectoris. Borneolum syntheticum (Bingpian) has been extensively used as a component in Chinese herbal medicines for cardiovascular diseases. This investigation aimed to examine Bingpian metabolism and its effects on anti-atherosclerotic activities. Major circulating Bingpian compounds were detected in human subjects who received a Bingpian-containing medicine. In vitro and rat studies were also conducted to facilitate the understanding of disposition factors that govern the systemic exposure to Bingpian compounds. Although Bingpian constituents, borneol (1) and isoborneol (2), are efficiently absorbed in the intestine, extensive hepatic first-pass glucuronidation, which is mediated predominantly by UGT2B7, coupled with MRP3 and MRP4-mediated efflux of the glucuronides into the blood, and oxidation, which is mediated by CYP2A6, CYP2B6, and CYP3A, result in the formation of metabolites borneol-2-O-glucuronide (M1G), isoborneol-2-O-glucuronide (M2G), and camphor (3) as the major circulating Bingpian compounds instead of the unchanged 1 and 2. Glucuronides are predominantly eliminated through renal excretion, which involves both glomerular filtration and OAT3- and OAT4-mediated tubular secretion. Furthermore, M1G, M2G, and 3, as well as 1 and 2, displayed inhibitory effects on oxidized low-density lipoprotein-induced foam-cell formation in macrophages. The findings emphasized that the metabolites must be given priority in pharmacodynamic studies of Bingpian. Comprehensive integration of pharmacokinetic and pharmacodynamic studies facilitates understanding how Bingpian functions in the body to provide therapeutic benefits.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by rapid progression and high mortality. With genetic mutations, particularly in the SOD1 gene, playing a significant role in ALS pathogenesis, targeted therapies have become a primary focus. This study introduces RD-12500 (RAG-17), a novel siRNA-ACO (Accessory Oligonucleotide) conjugate designed to address the challenges of delivering duplex RNAs to the central nervous system (CNS). RD-12500 exhibits remarkable in vitro stability and target specificity with minimal immunostimulation. In vivo studies demonstrate its extensive CNS biodistribution, sustained accumulation post-intrathecal administration, and a robust dose-exposure-activity correlation. Notably, RD-12500 significantly reduces cerebrospinal fluid (CSF) SOD1 protein levels, indicating potent SOD1 mRNA and protein knockdown in cynomolgus monkeys. Most notably, our study breaks new ground by demonstrating the effectiveness of RD-12500 in late-stage treatment scenarios. In SOD1G93A ALS mice, post-onset administration of RD-12500 significantly delayed disease progression, improved motor function, and extended survival, marking a significant advancement over other treatments which are typically initiated pre-symptomatically in the same model mice. These findings suggest RD-12500s potential to provide therapeutic benefits not only to pre-symptomatic but also to post-symptomatic and late-stage SOD1-ALS patients.

11{beta}-hydroxysteroid dehydrogenase type 1 (HSD-1) inhibitors represent a potential therapeutic approach for patients with Cushings syndrome, autonomous cortisol secretion (ACS), or iatrogenic glucocorticoid (GC) excess. We assessed whether the HSD-1 inhibitor SPI-62 prevents GC-associated morbidity in a mouse Cushings syndrome model. Corticosterone (CORT) was administered to mice for 5 weeks. Animals who received CORT were randomized between vehicle and 3 SPI-62 regimens. A control group received neither CORT nor SPI-62. Body weight was measured daily to enable weight-based SPI-62 administration. Food consumption by pair-housed animals was reported weekly. Insulin sensitivity was evaluated by fasting homeostatic model assessment of insulin resistance. Adiposity and skeletal myopathy were measured using magnetic resonance imaging and post-mortem weights of fat depots and skeletal muscles. Grip strength was measured with a digital meter. Ambulation behavior in an open field maze was assessed. Post-mortem dermal thickness was quantified. Skin structure was evaluated by histology. CORT was associated with decreased insulin sensitivity, increased adiposity, skeletal myoatrophy, reduced grip strength, decreased dermal thickness, and skin structural changes. A trend of increased food consumption and an unexpected biphasic weight change were observed with CORT. SPI-62 attenuated all observed adverse effects in a dose-dependent manner. In general, results for the high SPI-62 regimen were similar as those in animals who received no CORT, suggesting that full HSD-1 inhibition should be maintained throughout a dose interval to mitigate the effects of glucocorticoid excess.

The opioid epidemic has led to a devastating loss of life nationwide. Of those dependent on opioids, many individuals desire to quit or reduce use, but their efforts are often unsuccessful given the powerful reinforcing properties associated with opioid drugs, especially fentanyl given its high potency and speed of onset. Here, we developed a novel theraputic based on a newly developed artificial intelligence (AI)-based platform, which was rationally designed to identify markers of dysregulation from human drug user postmortem brain tissue. The GATC-021 compound was synthesized and validated with in vitro screening for target specificity. Thereafter, GATC-021 was examined for its effectiveness in modulating opioid dependence with an animal model of addiction. We found that GATC-021 substantially reduced fentanyl intake in both male and female rats, as assessed with intravenous self-administration. However, given drug soluability challenges, additional studies are needed to better develop drug formulations to permit translation into clinical populations more effectively. Taken together, these findings validate our AI-based platform for novel therapeutic development with a polypharmacy approach and further support the effectiveness of such target modulation as a promising therapeutic approach for those suffering from opioid use disorder.