Application of Pharmacokinetic Prediction Platforms in the Design of Optimized Anti-Cancer Drugs.

Cancer is the second most common cause of death in the United States, accounting for 602,350 deaths in 2020. Cancer-related death rates have declined by 27% over the past two decades, partially due to the identification of novel anti-cancer drugs. Despite improvements in cancer treatment, newly approved oncology drugs are associated with increased toxicity risk. These toxicities may be mitigated by pharmacokinetic optimization and reductions in off-target interactions. As such, there is a need for early-stage implementation of pharmacokinetic (PK) prediction tools. Several PK prediction platforms exist, including pkCSM, SuperCypsPred, Pred-hERG, Similarity Ensemble Approach (SEA), and SwissADME. These tools can be used in screening hits, allowing for the selection of compounds were reduced toxicity and/or risk of attrition. In this short commentary, we used PK prediction tools in the optimization of mitogen activated extracellular signal-related kinase kinase 1 (MEK1) inhibitors. In doing so, we identified MEK1 inhibitors with retained activity and optimized predictive PK properties, devoid of hERG inhibition. These data support the use of publicly available PK prediction platforms in early-stage drug discovery to design safer drugs.

The C-C Chemokine Receptor Type 4 Is an Immunomodulatory Target of Hydroxychloroquine.

The emergence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; COVID-19) in China, reported to the World Health Organization on December 31, 2019, has led to a large global pandemic and is a major public health issue. As a result, there are more than 200 clinical trials of COVID-19 treatments or vaccines that are either ongoing or recruiting patients. One potential therapy that has garnered international attention is hydroxychloroquine; a potent immunomodulatory agent FDA-approved for the treatment of numerous inflammatory and autoimmune conditions, including malaria, lupus, and rheumatoid arthritis. Hydroxychloroquine has demonstrated promise in vitro and is currently under investigation in clinical trials for the treatment of COVID-19. Despite an abundance of empirical data, the mechanism(s) involved in the immunomodulatory activity of hydroxychloroquine have not been characterized. Using the unbiased chemical similarity ensemble approach (SEA), we identified C-C chemokine receptor type 4 (CCR4) as an immunomodulatory target of hydroxychloroquine. The crystal structure of CCR4 was selected for molecular docking studies using the SwissDock modeling software. In silico, hydroxychloroquine interacts with Thr-189 within the CCR4 active site, presumably blocking endogenous ligand binding. However, the CCR4 antagonists compound 18a and K777 outperformed hydroxychloroquine in silico, demonstrating energetically favorable binding characteristics. Hydroxychloroquine may subject COVID-19 patients to QT-prolongation, increasing the risk of sudden cardiac death. The FDA-approved CCR4 antagonist mogalizumab is not known to increase the risk of QT prolongation and may serve as a viable alternative to hydroxychloroquine. Results from this report introduce additional FDA-approved drugs that warrant investigation for therapeutic use in the treatment of COVID-19.

Neuropsychological Deficits Chronically Developed after Focal Ischemic Stroke and Beneficial Effects of Pharmacological Hypothermia in the Mouse.

Stroke is a leading cause of human death and disability, with around 30% of stroke patients develop neuropsychological/neuropsychiatric symptoms, such as post-stroke depression (PSD). Basic and translational research on post-stroke psychological disorders is limited. In a focal ischemic stroke mouse model with selective damage to the sensorimotor cortex, sensorimotor deficits develop soon after stroke and spontaneous recovery is observed in 2-4 weeks. We identified that mice subjected to a focal ischemic insult gradually developed depression/anxiety like behaviors 4 to 8 weeks after stroke. Psychological/psychiatric disorders were revealed in multiple behavioral examinations, including the forced swim, tail suspension, sucrose preference, and open field tests. Altered neuronal plasticity such as suppressed long-term potentiation (LTP), reduced BDNF and oxytocin signaling, and disturbed dopamine synthesis/uptake were detected in the prefrontal cortex (PFC) during the chronic phase after stroke. Pharmacological hypothermia induced by the neurotensin receptor 1 (NTR1) agonist HPI-363 was applied as an acute treatment after stroke. A six-hr hypothermia treatment applied 45 min after stroke prevented depression and anxiety like behaviors examined at 6 weeks after stroke, as well as restored BDNF expression and oxytocin signaling. Additionally, hypothermia induced by physical cooling also showed an anti-depression and anti-anxiety effect. The data suggested a delayed beneficial effect of acute hypothermia treatment on chronically developed post-stroke neuropsychological disorders, associated with regulation of synaptic plasticity, neurotrophic factors, dopaminergic activity, and oxytocin signaling in the PFC.

Pharmacological hypothermia induced neurovascular protection after severe stroke of transient middle cerebral artery occlusion in mice.

Therapeutic hypothermia is a potential protective strategy after stroke. The present study evaluated the neurovascular protective potential of pharmacological hypothermia induced by the neurotensin receptor 1 agonist HPI-201 after severe ischemic stroke. Adult C57BL/6 mice were subjected to filament insertion-induced occlusion of the middle cerebral artery (60 min MCAO). HPI-201 was i.p. injected 120 min after the onset of MCAO to initiate and maintain the body temperature at 32-33°C for 6 hrs. The infarct volume, cell death, integrity of the blood brain barrier (BBB) and neurovascular unit (NVU), inflammation, and functional outcomes were evaluated. The hypothermic treatment significantly suppressed the infarct volume and neuronal cell death, accompanied with reduced caspase-3 activation and BAX expression while Bcl-2 increased in the peri-infarct region. The cellular integrity of the BBB and NVU was significantly improved and brain edema was attenuated in HPI-201-treated mice compared to stroke controls. The hypothermic treatment decreased the expression of inflammatory factors including tumor necrosis factor-α (TNF-α), MMP-9, interleukin-1ß (IL-1ß), the M1 microglia markers IL-12 and inducible nitric oxide synthase (iNOS), while increased the M2 marker arginase-1 (Arg-1). Stroke mice received the hypothermic treatment showed lower neurological severity score (NSS), performed significantly better in functional tests, the mortality rate in the hypothermic group was noticeably lower compared with stroke controls. Taken together, HPI-201 induced pharmacological hypothermia is protective for different neurovascular cells after a severely injured brain, mediated by multiple mechanisms.

Targeting peripheral Ï°-opioid receptors for the non-addictive treatment of pain.

Drug addiction to prescription mu-opioid agonists used in the setting of pain is a major public health threat, affecting millions of Americans. Kappa opioid agonists (KOAs) may serve as a possible solution. KOAs have demonstrated indistinguishable analgesic activity relative to mu-opioid agonists in models of acute and chronic pain; however, conventional KOAs suffer from central nervous system-mediated psychoactive side-effects. In this review, we discuss our efforts, as well as other's efforts, in developing peripherally-restricted kappa opioid agonists with retained or improved efficacy in rodent models of pain. Results indicate that our lead compound JT09 acts as efficacious as morphine in alleviating peripheral pain, while failing to produce undesired central nervous system-mediated side-effects. In this review, we discuss our former results and future directions.

Diuretic Activity of a Novel Peripherally-Restricted Orally-Active Kappa Opioid Receptor Agonist.

Kappa-opioid agonists (KOAs) enhance cardiac performance, as well as reduce infarct size and prevent deleterious cardiac remodeling following myocardial infarction. Additionally, KOAs promote diuresis; however, there has been limited development of KOAs as a class due to the promotion of untoward central nervous system (CNS)-mediated side effects. Our laboratory has developed a peripherally-restricted, orally-active, KOA (JT09) for the treatment of pain and cardiovascular disease. Peripherally-restricted KOAs possess a limited side-effect profile and demonstrate potential in preventing heart failure. The aim of this study was to assess the diuretic activity of lead compound JT09 relative to vehicle control and Tolvaptan through single oral administration to adult male Sprague-Dawley rats. JT09-administered rats demonstrated significantly increased urine output relative to vehicle control. However, the effect persisted for 8 h, whereas Tolvaptan-administered rats demonstrated diuretic activity for 24 h. Relative to Tolvaptan, urine output was significantly reduced in JT09 administered animals at all-time points, suggesting that the overall diuretic effect of JT09 is less profound than Tolvaptan. Additionally, JT09-administered rats demonstrated alterations in clinical chemistry; reduced urine specific gravity; and increased urine pH relative to vehicle control. The following study establishes a preliminary diuretic profile for JT09.

Therapeutic Potential of Kappa Opioid Agonists.

Many original research articles have been published that describe findings and outline areas for the development of kappa-opioid agonists (KOAs) as novel drugs; however, a single review article that summarizes the broad potential for KOAs in drug development does not exist. It is well-established that KOAs demonstrate efficacy in pain attenuation; however, KOAs also have proven to be beneficial in treating a variety of novel but often overlapping conditions including cardiovascular disease, pruritus, nausea, inflammatory diseases, spinal anesthesia, stroke, hypoxic pulmonary hypertension, multiple sclerosis, addiction, and post-traumatic cartilage degeneration. This article summarizes key findings of KOAs and discusses the untapped therapeutic potential of KOAs in the treatment of many human diseases.

Non-addictive orally-active kappa opioid agonists for the treatment of peripheral pain in rats.

Addiction to conventional opioid pain analgesics is a major societal problem that is increasing at an alarming rate. New drugs to combat the effects of opioid abuse are desperately needed. Kappa-opioid agonists are efficacious in peripheral pain models but suffer from centrally-mediated effects. In this article, we discuss our efforts in developing peripheral kappa-based opioid receptor agonists that have the potential analgesic activity of opioids but do not manifest the negative side-effects of opioid use and abuse. Further, derivatives of the tetra-peptide D-Phe-D-Phe-D-Nle-D-Arg-NH2, such as CR665, exhibit high peripheral to central selectivity in analgesic models when administered intravenously (i.v.); however, they are inactive when administered orally. Application of our laboratory's proprietary non-natural amino acid technology to CR665 produced derivatives that exhibit peripheral analgesic activity when dosed orally but do not promote CNS-based effects. Lead compound JT09 activates the kappa-opioid receptor with EC50s in the low nM range, while agonist selectivity for kappa over other peripheral opioid receptors was >33,400 fold. Results indicate that JT09 is approximately as efficacious as morphine in alleviating peripheral pain, while failing to produce undesired CNS-mediated activity. Additionally, JT09 did not promote other CNS-mediated effects associated with morphine (addiction, sedation, dysphoria, tolerance, addiction). Thus, we propose that JT09 has potential for development as a novel analgesic. PERSPECTIVE: This article presents data supporting the analgesic properties of an orally available, peripherally-restricted, kappa-opioid agonist for peripheral pain. A potential out-patient pharmaceutical that acts as efficacious as morphine in alleviating peripheral pain, while failing to produce undesired CNS-mediated effects, could help reduce the current health care burden associated with prescription opioids.

The Effects of Pharmacological Hypothermia Induced by Neurotensin Receptor Agonist ABS 201 on Outcomes of CPR.

Neurotensin is an endogenous tridecapeptide that binds to neurotensin receptors in the brain, which induce hypothermia. The aim of this study was to investigate whether the receptor agonist ABS 201 could induce therapeutic hypothermia and improve postresuscitation outcomes in a ventricular fibrillation cardiac arrest (VFCA) rat model. VF was electrically induced in 12 rats. Defibrillation was achieved after 6 min of cardiopulmonary resuscitation. After successful resuscitation, animals were randomized to receive ABS 201 (8 mg/kg/h) or placebo. Postresuscitation myocardial function and neurological deficit scores (NDS) were assessed, and postresuscitation survival duration was observed for up to 72 h. After administration of ABS 201, blood temperature decreased significantly from 37°C to 34°C, and was maintained for 2.5 h. There was a significant improvement of postresuscitation myocardial dysfunction, NDS, and survival duration in animals treated with ABS 201. These results demonstrated that ABS 201 induces therapeutic hypothermia in a VFCA rat model, ameliorates postresuscitation myocardial-neurological dysfunction, and prolongs survival duration. ABS 201 may therefore be an alternative method to induce therapeutic hypothermia with current cooling methods and improve postresuscitation outcomes.

Targeting Peripheral Kappa Opioid Receptors for the Treatment of Chronic Pain: Review Article.

Addiction to conventional opioid pain analgesics is a major societal problem that is increasing at an alarming rate. New drugs to combat the effects of opioid abuse are desperately needed. Kappa-opioid agonists are efficacious in peripheral pain models but suffer from centrally-mediated effects. In this review, we discuss our efforts, as well as other's efforts in developing peripheral kappa-based opioid receptor agonists that have the potential analgesic activity of opioids but do not manifest the negative side-effects of opioid use and abuse. Further, derivatives of the tetra peptide D-Phe-D-Phe-D-Nle-D-Arg-NH2, such as CR665, exhibit high peripheral to central selectivity in analgesic models when administered intravenously (IV); however, they are inactive when administered orally. Application of the JT Pharmaceuticals non-natural amino acid technology to CR665 produced derivatives that exhibit peripheral analgesic activity when dosed orally but do not promote CNS-based effects. Lead compound JT09 activates the kappa-opioid receptor with EC50s in the low nM range, while agonist selectivity for kappa over other peripheral opioid receptors was >33,400 fold. Results indicate that JT09 acts as efficacious as morphine in alleviating peripheral pain, while failing to produce undesired CNS-mediated activity. Additionally, JT09 did not promote other CNS-mediated effects associated with morphine (addiction, sedation, dysphoria, tolerance). Thus, we propose that JT09 has potential for development as a novel analgesic.

Longitudinal MRI evaluation of neuroprotective effects of pharmacologically induced hypothermia in experimental ischemic stroke.

Pharmacologically induced hypothermia (PIH) shows promising neuroprotective effects after stroke insult. However, the dynamic evolution of stroke infarct during the hypothermic therapy has not been understood very well. In the present study, MRI was utilized to longitudinally characterize the infarct evolution in a mouse model of ischemic stroke treated by PIH using the neurotensin agonist HPI201. Adult male C57BL/6 mice underwent permanent occlusion of the right middle cerebra artery (MCA). Each animal received a vehicle or HPI201 intraperitoneal injection. The temporal changes of stroke lesion were examined using T2-weighted imaging and diffusion-weighted imaging (DWI) in the acute phase (1-3h) and 24h post stroke. Significantly reduced infarct and edema volumes were observed in PIH treated stroke mice, in agreement with TTC staining findings. Also, the TUNEL staining results indicated apoptotic cells were widely distributed among the ischemic cortex in control group but limited in PIH treated mice. Dramatically reduced growth rate of infarction was seen in PIH treated stroke mice. These results demonstrate HPI201 has strong neuroprotection effects during acute stroke. In particular, MRI with the numerical modelling of temporal infarct evolution could provide a unique means to examine and predict the dynamic response of the PIH treatment on infarct evolution.

Regulation of therapeutic hypothermia on inflammatory cytokines, microglia polarization, migration and functional recovery after ischemic stroke in mice.

Stroke is a leading threat to human life and health in the US and around the globe, while very few effective treatments are available for stroke patients. Preclinical and clinical studies have shown that therapeutic hypothermia (TH) is a potential treatment for stroke. Using novel neurotensin receptor 1 (NTR1) agonists, we have demonstrated pharmacologically induced hypothermia and protective effects against brain damages after ischemic stroke, hemorrhage stroke, and traumatic brain injury (TBI) in rodent models. To further characterize the mechanism of TH-induced brain protection, we examined the effect of TH (at ±33°C for 6h) induced by the NTR1 agonist HPI-201 or physical (ice/cold air) cooling on inflammatory responses after ischemic stroke in mice and oxygen glucose deprivation (OGD) in cortical neuronal cultures. Seven days after focal cortical ischemia, microglia activation in the penumbra reached a peak level, which was significantly attenuated by TH treatments commenced 30min after stroke. The TH treatment decreased the expression of M1 type reactive factors including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-12, IL-23, and inducible nitric oxide synthase (iNOS) measured by RT-PCR and Western blot analyses. Meanwhile, TH treatments increased the expression of M2 type reactive factors including IL-10, Fizz1, Ym1, and arginase-1. In the ischemic brain and in cortical neuronal/BV2 microglia cultures subjected to OGD, TH attenuated the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α), two key chemokines in the regulation of microglia activation and infiltration. Consistently, physical cooling during OGD significantly decreased microglia migration 16h after OGD. Finally, TH improved functional recovery at 1, 3, and 7days after stroke. This study reveals the first evidence for hypothermia mediated regulation on inflammatory factor expression, microglia polarization, migration and indicates that the anti-inflammatory effect is an important mechanism underlying the brain protective effects of a TH therapy.

Improved Therapeutic Benefits by Combining Physical Cooling With Pharmacological Hypothermia After Severe Stroke in Rats.

BACKGROUND AND PURPOSE: Therapeutic hypothermia is a promising strategy for treatment of acute stroke. Clinical translation of therapeutic hypothermia, however, has been hindered because of the lack of efficiency and adverse effects. We sought to enhance the clinical potential of therapeutic hypothermia by combining physical cooling (PC) with pharmacologically induced hypothermia after ischemic stroke. METHODS: Wistar rats were subjected to 90-minute middle cerebral artery occlusion by insertion of an intraluminal filament. Mild-to-moderate hypothermia was induced 120 minutes after the onset of stroke by PC alone, a neurotensin receptor 1 (NTR1) agonist HPI-201 (formally ABS-201) alone or the combination of both. The outcomes of stroke were evaluated at 3 and 21 days after stroke. RESULTS: PC or HPI-201 each showed hypothermic effect and neuroprotection in stroke rats. The combination of PC and HPI-201 exhibited synergistic effects in cooling process, reduced infarct formation, cell death, and blood-brain barrier damages and improved functional recovery after stroke. Importantly, coapplied HPI-201 completely inhibited PC-associated shivering and tachycardia. CONCLUSIONS: The centrally acting hypothermic drug HPI-201 greatly enhanced the efficiency and efficacy of conventional PC; this combined cooling therapy may facilitate clinical translation of hypothermic treatment for stroke.

Pharmacologically induced hypothermia attenuates traumatic brain injury in neonatal rats.

Neonatal brain trauma is linked to higher risks of mortality and neurological disability. The use of mild to moderate hypothermia has shown promising potential against brain injuries induced by stroke and traumatic brain injury (TBI) in various experimental models and in clinical trials. Conventional methods of physical cooling, however, are difficult to use in acute treatments and in induction of regulated hypothermia. In addition, general anesthesia is usually required to mitigate the negative effects of shivering during physical cooling. Our recent investigations demonstrate the potential therapeutic benefits of pharmacologically induced hypothermia (PIH) using the neurotensin receptor (NTR) agonist HPI201 (formerly known as ABS201) in stroke and TBI models of adult rodents. The present investigation explored the brain protective effects of HPI201 in a P14 rat pediatric model of TBI induced by controlled cortical impact. When administered via intraperitoneal (i.p.) injection, HPI201 induced dose-dependent reduction of body and brain temperature. A 6-h hypothermic treatment, providing an overall 2-3°C reduction of brain and body temperature, showed significant effect of attenuating the contusion volume versus TBI controls. Attenuation occurs whether hypothermia is initiated 15min or 2h after TBI. No shivering response was seen in HPI201-treated animals. HPI201 treatment also reduced TUNEL-positive and TUNEL/NeuN-colabeled cells in the contusion area and peri-injury regions. TBI-induced blood-brain barrier damage was attenuated by HPI201 treatment, evaluated using the Evans Blue assay. HPI201 significantly decreased MMP-9 levels and caspase-3 activation, both of which are pro-apototic, while it increased anti-apoptotic Bcl-2 gene expression in the peri-contusion region. In addition, HPI201 prevented the up-regulation of pro-inflammatory tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6. In sensorimotor activity assessments, rats in the HPI201 treated group exhibited improved functional recovery after TBI versus controls. These data support that PIH therapy using our NTR agonist is effective in reducing neuronal and BBB damage, attenuating inflammatory response and detrimental cellular signaling, and promoting functional recovery after TBI in the developing brain, supporting its potential for further evaluation towards clinical development.

Therapeutic effects of pharmacologically induced hypothermia against traumatic brain injury in mice.

Preclinical and clinical studies have shown therapeutic potential of mild-to-moderate hypothermia for treatments of stroke and traumatic brain injury (TBI). Physical cooling in humans, however, is usually slow, cumbersome, and necessitates sedation that prevents early application in clinical settings and causes several side effects. Our recent study showed that pharmacologically induced hypothermia (PIH) using a novel neurotensin receptor 1 (NTR1) agonist, HPI-201 (also known as ABS-201), is efficient and effective in inducing therapeutic hypothermia and protecting the brain from ischemic and hemorrhagic stroke in mice. The present investigation tested another second-generation NTR1 agonist, HPI-363, for its hypothermic and protective effect against TBI. Adult male mice were subjected to controlled cortical impact (CCI) (velocity=3 m/sec, depth=1.0 mm, contact time=150 msec) to the exposed cortex. Intraperitoneal administration of HPI-363 (0.3 mg/kg) reduced body temperature by 3-5°C within 30-60 min without triggering a shivering defensive reaction. An additional two injections sustained the hypothermic effect in conscious mice for up to 6 h. This PIH treatment was initiated 15, 60, or 120 min after the onset of TBI, and significantly reduced the contusion volume measured 3 days after TBI. HPI-363 attenuated caspase-3 activation, Bax expression, and TUNEL-positive cells in the pericontusion region. In blood-brain barrier assessments, HPI-363 ameliorated extravasation of Evans blue dye and immunoglobulin G, attenuated the MMP-9 expression, and decreased the number of microglia cells in the post-TBI brain. HPI-363 decreased the mRNA expression of tumor necrosis factor-α and interleukin-1ß (IL-1ß), but increased IL-6 and IL-10 levels. Compared with TBI control mice, HPI-363 treatments improved sensorimotor functional recovery after TBI. These findings suggest that the second generation NTR-1 agonists, such as HPI-363, are efficient hypothermic-inducing compounds that have a strong potential in the management of TBI.

Development of a Peptide-derived orally-active kappa-opioid receptor agonist targeting peripheral pain.

Kappa-opioid agonists are particularly efficacious in the treatment of peripheral pain but suffer from central nervous system (CNS)-mediated effects that limit their development. One promising kappa-agonist is the peptidic compound CR665. Although not orally available, CR665 given i.v. exhibits high peripheral to CNS selectivity and benefits patients with visceral and neuropathic pain. In this study we have generated a series of derivatives of CR665 and screened them for oral activity in the acetic acid-induced rat writhing assay for peripheral pain. Five compounds were further screened for specificity of activation of kappa receptors as well as agonism and antagonism at mu and delta receptors, which can lead to off-target effects. All active derivatives engaged the kappa receptor with EC50s in the low nM range while agonist selectivity for kappa over mu or delta was >11,000-200,000-fold. No antagonist activity was detected. One compound was chosen for further analysis (Compound 9). An oral dose response of 9 in rats yielded an EC50 of 4.7 mg/kg, approaching a druggable level for an oral analgesic. To assess the peripheral selectivity of this compound an i.v. dose response in rats was assessed in the writhing assay and hotplate assay (an assay of CNS-mediated pain). The EC50 in the writhing assay was 0.032 mg/kg while no activity was detectable in the hotplate assay at doses as high as 30 mg/kg, indicating a peripheral selectivity of >900-fold. We propose that compound 9 is a candidate for development as an orally-available peripherally-restricted kappa agonist.

A novel stroke therapy of pharmacologically induced hypothermia after focal cerebral ischemia in mice.

Compelling evidence from preclinical and clinical studies has shown that mild to moderate hypothermia is neuroprotective against ischemic stroke. Clinical applications of hypothermia therapy, however, have been hindered by current methods of physical cooling, which is generally inefficient and impractical in clinical situations. In this report, we demonstrate the potential of pharmacologically induced hypothermia (PIH) by the novel neurotensin receptor 1 (NTR1) agonist ABS-201 in a focal ischemic model of adult mice. ABS-201 (1.5-2.5 mg/kg, i.p.) reduces body and brain temperature by 2-5°C in 15-30 min in a dose-dependent manner without causing shivering or altering physiological parameters. Infarct volumes at 24 h after stroke are reduced by ∼30-40% when PIH therapy is initiated either immediately after stroke induction or after 30-60 min delay. ABS-201 treatment increases bcl-2 expression, decreases caspase-3 activation, and TUNEL-positive cells in the peri-infarct region, and suppresses autophagic cell death compared to stroke controls. The PIH therapy using ABS-201 improves recovery of sensorimotor function as tested 21 d after stroke. These results suggest that PIH induced by neurotensin analogs represented by ABS-201 are promising candidates for treatment of ischemic stroke and possibly for other ischemic or traumatic injuries.

Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic.

The neurotensin hexapapetide fragment NT(8-13) is a potent analgesic when administered directly to the central nervous system but does not cross the blood-brain barrier. A total of 43 novel derivatives of NT(8-13) were evaluated, with one, ABS212 (1), being most active in four rat models of pain when administered peripherally. Compound 1 binds to human neurotensin receptors 1 and 2 with IC(50) of 10.6 and 54.2 nM, respectively, and tolerance to the compound in a rat pain model did not develop after 12 days of daily administration. When it was administered peripherally, serum levels and neurotensin receptor binding potency of 1 peaked within 5 min and returned to baseline within 90-120 min; however, analgesic activity remained near maximum for >240 min. This could be due to its metabolism into an active fragment; however, all 4- and 5-mer hydrolysis products were inactive. This pharmacokinetic/pharmacodynamic dichotomy is discussed. Compound 1 is a candidate for development as a first-in-class analgesic.

Comparison of N-terminal modifications on neurotensin(8-13) analogues correlates peptide stability but not binding affinity with in vivo efficacy.

Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.