Journal of Pharmacology and Experimental Therapeutics最新文献

Painful bladder syndrome and irritable bowel syndrome affect 10%-15% of the global population. Current treatment options for these syndromes are ineffective in severe disease progression and are fraught with adverse effects. Prolonged use of conventional opioids causes constipation, respiratory depression, tolerance, and addiction. Bifunctional opioid ligands with mixed agonist/antagonist profiles at 2 types of opioid receptors (ORs) possess therapeutic advantages. Eluxadoline (ELX, Viberzi), a drug for diarrhea-predominant irritable bowel syndrome, has an agonistic effect on μ-opioid receptor (MOR) and an antagonistic effect on δ-opioid receptor (DOR). ELX alleviates pain and normalizes peristalsis by activating MOR without causing constipation, a DOR antagonistic effect. However, its mechanism of analgesic action is not known. We investigated the analgesic mechanism of ELX in colon and bladder pain by recording the visceromotor responses (VMRs) to painful distension of the organ and identified the site of action of the drug in pain signaling pathways. ELX inhibited VMRs via the activation of spinal MOR. The peripherally restricted MOR antagonist naloxone-methiodide (meth-NLX) did not reverse the VMR inhibition by ELX, whereas centrally acting NLX reversed it. ELX did not inhibit the excitation of mechanosensitive afferent fibers in lumbar 6 (L6) and sacral 1 (S1) dorsal root innervating the bladder or colon. In contrast, ELX inhibited excitation of bladder distension-responsive L6 and S1 spinal neurons. The inhibition was reversed by NLX, but not by meth-NLX. Electrophysiology results reinforce behavioral experiments suggesting that ELX produces analgesia by attenuating responses of spinal neurons, but not visceral sensory afferents. SIGNIFICANCE STATEMENT: The bifunctional opioid ligand eluxadoline is an FDA-approved drug for the treatment of diarrhea-predominant irritable bowel syndrome to normalize bowel movement and to relieve abdominal pain. This study documents for the first time to our knowledge that unlike its peripheral action to normalize diarrhea, the drug alleviates colon and bladder pain centrally by modulating responses of lumbo-sacral (L6-S1) spinal neurons.

Ferroptosis is a form of regulated cell death closely associated with glutathione depletion and accumulation of reactive lipid peroxides. In this study, we seek to determine whether 2-hydroxyestrone (2-OH-E₁) and 2-hydroxyestradiol (2-OH-E₂), 2 major metabolites of endogenous estrone (E₁) and 17β-estradiol (E₂) formed by cytochrome P450 in the liver, can protect against erastin- and RSL3-induced ferroptosis in hepatoma cells (H-4-II-E and HuH-7) in vitro and acetaminophen-induced mouse liver injury in vivo. We find that 2-OH-E₁ and 2-OH-E₂ can protect, in a dose-dependent manner, H-4-II-E hepatoma cells against erastin/RSL3-induced ferroptosis. A similar protective effect of 2-OH-E₁ and 2-OH-E₂ against erastin- and RSL3-induced ferroptosis is also observed in HuH-7 hepatoma cells. These 2 estrogen metabolites can strongly abrogate erastin- and RSL3-induced accumulation of cellular NO, reactive oxygen species (ROS), and lipid-ROS. Mechanistically, 2-OH-E₁ and 2-OH-E₂ protect cells against chemically induced ferroptosis by binding to cellular protein disulfide isomerase and then inhibiting its catalytic activity and reducing protein disulfide isomerase-mediated activation (dimerization) of inducible nitric oxide synthase, abrogating cellular NO, ROS, and lipid-ROS accumulation. Animal studies show that 2-OH-E₁ and 2-OH-E₂ also exhibit strong protection against acetaminophen-induced liver injury in mice. Interestingly, although E₁ and E₂ have a very weak protective effect in cultured hepatoma cells, they exhibit a similarly strong protective effect as 2-OH-E₁ and 2-OH-E₂ in vivo, suggesting that the metabolic conversion of E₁ and E₂ to 2-OH-E₁ and 2-OH-E₂ contributes importantly to their hepatoprotective effect. This study reveals that 2-OH-E₁ and 2-OH-E₂ are important endogenous factors for protection against chemically induced liver injury in vivo. SIGNIFICANCE STATEMENT: Ferroptosis is an iron-dependent and lipid reactive oxygen species-dependent form of regulated cell death. Recent evidence has shown that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for ferroptosis protection. This study shows that 2-hydroxyestrone and 2-hydroxyestradiol are 2 inhibitors of PDI that can strongly protect against chemically induced ferroptotic hepatocyte death in vitro and in vivo. This work supports a PDI-mediated, estrogen receptor-independent mechanism of hepatocyte protection by 2-hydroxyestrone and 2-hydroxyestradiol.

The corticosteroid dexamethasone, which is used to treat numerous health conditions, remains the first-line treatment for patients hospitalized with COVID-19 requiring oxygen. Current British National Formulary prescribing advice warns of a "severe theoretical" or "severe anecdotal" risk of drug-drug interactions between dexamethasone and 138 different medications. In humans, dexamethasone is eliminated via the cytochrome P450 monooxygenase system, particularly CYP3A4. It is also described as a human cytochrome P450-inducing agent. To establish factors that affect concomitant therapy and dexamethasone efficacy in the treatment of COVID-19, we used a unique mouse model humanized for cytochrome P450s and the transcription factors that regulate their expression, the pregnane X receptor, and the constitutive androstane receptor. We found that induction of CYP3A4 with the anticancer drug dabrafenib or the herbal medicine St John's wort profoundly reduced dexamethasone exposure. These data suggest that comedications that induce cytochrome P450 expression can have a marked effect on dexamethasone exposure and, potentially, clinical efficacy. We also observed that rather than increasing CYP3A4 expression, dexamethasone at doses equivalent to or higher than those used in the treatment of COVID-19 reduced CYP3A4 expression and increased exposure to dabrafenib. These data indicate the need for a clinical trial to establish the risk of overexposure to comedications during dexamethasone treatment, including the treatment of COVID-19. SIGNIFICANCE STATEMENT: Current prescribing advice identifies a potential theoretical risk of severe side effects when dexamethasone, one of the most widely used drugs in clinical practice, is coadministered with many other drugs; it is, however, difficult to define the magnitude of this risk for specific drug combinations. We describe the use of cytochrome P450-humanized 8HUM mice to predict drug-drug interactions in patients on polypharmacy, a means of generating data that could better inform clinicians regarding foreseeable drug-drug interactions involving dexamethasone.

Streptolysin O (SLO), a bacterial toxin produced by common hemolytic streptococci, including Streptococcus pyogenes and resident microbiota, may be associated with inflammation in the cardiovascular system. We previously reported that short-term treatment with SLO at relatively high concentrations (10-1000 ng/mL) diminished acetylcholine-induced, endothelial-dependent relaxation in a concentration-dependent manner. However, the vascular function effects of long-term exposure to SLO at lower concentrations are poorly understood. In this study, treatment of rat aorta with endothelium with SLO (0.1-10 ng/mL) for 72 hours inhibited contractions in response to norepinephrine and phenylephrine in a concentration-dependent manner, and this effect was abolished by endothelium denudation. We also observed decreased endothelium-dependent relaxation in aorta treated with a lower concentration of SLO (10 ng/mL) for 72 hours. Long-term treatment with SLO (10 ng/mL) increased the expression of inducible nitric oxide synthase (iNOS) in aorta with endothelium but not aorta without endothelium, and the SLO-induced decrease in contraction was restored by treatment with iNOS inhibitors. Pharmacologic and gene-mutant analyses further indicated that SLO-induced vascular dysfunction and iNOS upregulation are mediated through the toll-like receptor 4/NADPH oxidase 2/reactive oxygen species/p38 mitogen-activated protein kinase pathways. In vivo SLO treatment (46.8 pg/kg per minute) for 7 days also diminished vascular contraction and relaxation activity in aorta with endothelium. We concluded that long-term treatment with SLO inhibits vascular contractile responses, primarily due to increased iNOS expression in the endothelium through toll-like receptor 4-mediated pathways. Our present results, together with those of our previous study, suggest that endothelial cells play a key role in the pathophysiologic changes in cardiovascular function associated with long-term exposure to SLO. SIGNIFICANCE STATEMENT: In the present study, we showed that long-term exposure to streptococcal exotoxin streptolysin O (SLO) inhibits agonist-induced contraction in rat aorta with endothelium, driven primarily by elevated nitric oxide synthase production via NADPH oxidase 2-mediated reactive oxygen species production through toll-like receptor 4 activation on endothelial cells. In vivo treatment with SLO for 7 days also diminished vascular contraction and relaxation, providing evidence of possible pathophysiologic roles of SLO in endothelium-dependent vascular homeostasis.

Parkinson's disease (PD) treatment focuses mainly on the augmentation of dopamine transmission, but to alleviate adverse motor effects accompanying L-DOPA use, additional treatments with serotonergic (5-HT) medications may be considered. We propose a novel concept based on the simultaneous activation of 5-HT_1BR and 5-HT₆R blockade as a putative therapeutic option for PD treatment. We have developed PZKKN-94, a dual human 5-HT_1BR agonist (EC₅₀ = 39 nM) and human 5-HT₆R antagonist (K_B = 7.7 nM), with selectivity over 43 targets, favorable drug-like properties, and brain penetration. Importantly, PZKKN-94 potency was increased or retained at rat 5-HT_1B and 5-HT₆ orthologs but not at mouse 5-HT₆. Therefore, PZKKN-94 was tested in 2 rat disease models: haloperidol-induced catalepsy and 6-hydroxydopamine-induced sensorimotor deficits in rats, showing antiparkinsonian-like effects in both. Of note, PZKKN-94 did not affect the therapeutic effects of L-DOPA and attenuated L-DOPA-induced motor fluctuation ("on-off" phenomena) in the stepping and vibrissae tests. PZKKN-94 had no effect on L-DOPA-induced contralateral rotation, suggesting no impact on dopamine-mimetic medication effects. In addition, PZKKN-94 reversed scopolamine-, phencyclidine-, and aged-induced learning deficits in the rat novel object recognition test, increased cognitive flexibility in the attention set-shifting task, and displayed antidepressant-like actions in the forced swim test in rats. Our data suggest that dual-acting 5-HT_1BR agonists/5-HT₆R antagonists provide a novel therapeutic approach to alleviate both motor symptoms and accompanying cognitive and depression comorbidities in PD. Our present findings highlight the dual 5-HT_1BR agonist/5-HT₆R antagonist strategy to simultaneously spare L-DOPA's action and alleviate motor fluctuations related to L-DOPA treatment. SIGNIFICANCE STATEMENT: The commonly used L-DOPA-based medications for Parkinson's disease, though effective in alleviating initial disease states, are limited in long-term use due to the motor (dyskinesia and on-off phenomena) and nonmotor (psychotic-like) side effects. A novel nondopaminergic strategy for treatment of Parkinson's disease based on simultaneous activation of the 5-HT_1B receptor and blockade of the 5-HT₆ receptor is proposed. The compound PZKKN-94 produces an antiparkinsonian-like effect and attenuates motor fluctuations, preserving the efficacy of L-DOPA. In addition, PZKKN-94 demonstrates procognitive and antidepressant-like properties.

Secondary spinal cord injury (SCI) is characterized by increased cytokines and chemokines at the site of injury that have been associated with the development of neuropathic pain. Nearly 80% of SCI patients report suffering from chronic pain, which is poorly managed with available analgesics. While treatment with the US Food and Drug Administration-approved β₂-adrenergic receptor agonist formoterol improves various aspects of recovery post-SCI in vivo, its effects on cytokines, chemokines, and neuropathic pain remain unknown. Female mice were subjected to moderate (60 kilodynes [kdyn]) or severe (80 kdyn) SCI followed by daily treatment with vehicle or formoterol (0.3 mg/kg, i.p.) beginning 8 hours after injury. The expression of proinflammatory cytokines/chemokines, such as interferon gamma-induced protein 10, macrophage inflammatory protein 1a, monocyte chemoattractant protein 1, B-cell attracting chemokine 1, and nuclear factor kappa-light-chain-enhancer of activated B-cells, was increased in the injury site of vehicle-treated mice 24 hours post-SCI, which was ameliorated with formoterol treatment, regardless of injury severity. Thermal hyperalgesia and mechanical allodynia, as measured by Hargreaves infrared apparatus and von Frey filaments, respectively, were assessed prior to SCI and then weekly beginning 21 days post-injury (DPI). While all injured mice exhibited decreased withdrawal latency following thermal stimulation compared with baseline, formoterol treatment reduced this response ∼15% by 35 DPI. Vehicle-treated mice displayed significant mechanical allodynia, as evidenced by a 55% decrease in withdrawal threshold from baseline. In contrast, mice treated with formoterol maintained a consistent withdrawal time at all times tested. These data indicate that formoterol reduces inflammation post-SCI, likely contributing to mitigation of neuropathic pain and further supporting the therapeutic potential of this treatment strategy. SIGNIFICANCE STATEMENT: Chronic pain is a detrimental consequence of spinal cord injury (SCI). We show that treatment with the US Food and Drug Administration-approved drug formoterol after SCI decreases injury site proinflammatory chemo-/cytokines and alters markers of glial cell activation and infiltration. Additionally, formoterol treatment improves locomotor function and body composition, and decreases lesion volume. Finally, formoterol treatment decreased mechanical allodynia and thermal hyperalgesia post-SCI. These data are suggestive of the mechanism of formoterol-induced recovery, and further indicate its potential as a therapeutic strategy for SCI.

The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) established S7B and E14 guidelines in 2005 to prevent drug-induced torsade de pointes (TdP), effectively preventing the development of high-risk drugs. However, those guidelines hampered the development of some potentially valuable drug candidates despite not being proven to be proarrhythmic. In response, comprehensive in vitro proarrhythmia assay and exposure-response modeling were proposed in 2013 to reinforce proarrhythmic risk assessment. In 2022, the ICH released E14/S7B questions and answers (stage 1), emphasizing a "double negative" nonclinical scenario for low-risk compounds. For "non-double negative" compounds, new questions and answers are expected to be enacted as stage 2 shortly, in which more detailed recommendations for proarrhythmia models and proarrhythmic surrogate markers will be provided. This review details the onset mechanisms of drug-induced TdP, including I_Kr inhibition, pharmacokinetic factors, autonomic regulation, and reduced repolarization reserve. It also explores the utility of proarrhythmic surrogate markers (J-T_peak, T_peak-T_end, and terminal repolarization period) besides QT interval. Finally, it presents various in silico, in vitro, ex vivo, and in vivo models for proarrhythmic risk prediction, such as comprehensive in vitro proarrhythmia assay in silico model, induced pluripotent stem cell-derived cardiomyocyte sheet, Langendorff-perfused heart preparation, chronic atrioventricular block animals (dogs, monkeys, pigs, and rabbits), acute atrioventricular block rabbits, methoxamine-sensitized rabbits, and genetically engineered rabbits for specific long QT syndromes. Those models along with the surrogate markers can play important roles in quantifying TdP risk of new compounds, impacting late-phase clinical design and regulatory decision-making, and preventing adverse events on postmarketing clinical use. SIGNIFICANCE STATEMENT: Since ICH S7B/E14 guidelines hampered the development of some potentially valuable compounds with unproven proarrhythmic risk, comprehensive in vitro proarrhythmia assay and exposure-response modeling were proposed in 2013 to reinforce proarrhythmic risk assessment of new compounds. In 2022, the ICH released questions and answers (stage 1), emphasizing a "double negative" nonclinical scenario for low-risk compounds, and new questions and answers (stage 2) for "non-double negative" compounds are expected. This review delves into proarrhythmic mechanisms with surrogate markers and explores various models for proarrhythmic risk prediction.

Genetic loss-of-function mutations of the Na_V1.7 channel, abundantly expressed in peripheral nociceptive neurons, cause congenital insensitivity to pain in humans, indicating that selective inhibition of the channel may lead to potential therapy for pain disorders. In this study, we investigated a novel compound, 5-chloro-N-(cyclopropylsulfonyl)-2-fluoro-4-(2-(8-(furan-2-ylmethyl)-8-azaspiro [4.5] decan-2-yl) ethoxy) benzamide (QLS-278) that inhibits Na_V1.7 channels and exhibits antinociceptive activity. Compound QLS-278 exhibits inactivation- and concentration-dependent inhibition of macroscopic currents of Na_V1.7 channels stably expressed in HEK293 cells with an IC₅₀ of 1.2 ± 0.2 μM. QLS-278 causes a hyperpolarization shift of the channel inactivation and delays recovery from inactivation, without any noticeable effect on voltage-dependent activation. In mouse dorsal root ganglion neurons, QLS-278 suppresses native tetrodotoxin-sensitive Na_V currents and also reduces neuronal firings. Moreover, QLS-278 dose-dependently relieves neuropathic pain induced by spared nerve injury and inflammatory pain induced by formalin without significantly altering spontaneous locomotor activity in mice. Therefore, our identification of the novel compound QLS-278 may hold developmental potential in chronic pain treatment. SIGNIFICANCE STATEMENT: QLS-278, a novel voltage-gated sodium Na_V1.7 channel blocker, inhibits native tetrodotoxin-sensitive Na⁺ current and reduces action potential firings in dorsal root ganglion sensory neurons. QLS-278 also exhibits antinociceptive activity in mouse models of pain, demonstrating the potential for the development of a chronic pain treatment.

The active ingredient of Gastrodia elata, 4-hydroxybenzaldehyde (4-HBd), can rapidly enter the brain and undergo massive oxidation to produce the metabolite 4-hydroxybenzoic acid, which has no significant activity after equal dose gavage. It is crucial to clarify the metabolic pathway of 4-HBd and its correlation with the anti-ischemic stroke mechanism. The objective of this study was to explore the possible mechanism of 4-HBd in clearing reactive oxygen species (ROS) and protecting blood-brain barrier from oxidative stress damage during brain metabolism from the perspective of ROS response. A rat model of cerebral ischemia-reperfusion injury and a cellular oxidative stress response model were replicated to simulate the accumulation process of ROS in the brain. The changes in ROS and peroxidation products before and after 4-HBd intervention were detected, and the changes in oxidative metabolism were also measured to confirm the correlation between antioxidant stress damage and ROS capture/clearance in oxidative metabolism. 4-HBd has significant antioxidant stress resistance both in vitro and in vivo, and can reduce the levels of malondialdehyde and 4-hydroxy-2-nonenal in ischemic brain tissue. It can capture O₂^⋅- and ⋅OH in vitro and use the captured ROS to oxidize and metabolize 4-hydroxybenzoic acid. The oxidative metabolism process of 4-HBd in the brain is one of its mechanisms for exerting antioxidant stress damage and protecting blood-brain barrier. SIGNIFICANCE STATEMENT: The active ingredient 4-hydroxybenzaldehyde of Gastrodia elata can be converted into metabolite 4-hydroxybenzoic acid in the brain mainly through oxidative metabolic pathway. The mechanism of its action against oxidative stress damage of blood-brain barrier is related to the oxidative metabolic process in the brain that traps/clears reactive oxygen species and forms stable intermediates to terminate the free radical chain reaction. This is one of the main mechanisms of 4-hydroxybenzaldehyde's anti-ischemic stroke effect in the brain.