Numerical study on the stimulation effect of boundary sealing and hot water injection in marine challenging gas hydrate extraction.

This study proposed a novel development mode combining boundary sealing and hot water injection to address the challenges of gas leakage, limited reservoir sensible heat, boundary water intrusion, and low productivity faced by challenging hydrate extraction, and the stimulation effect was numerically investigated with Shenhu hydrates as the geological background. The results showed that lower boundary permeability facilitated pressure propagation and achieved volumetric dissociation of hydrates, whereas insufficient formation energy resulted in substantial gas retention. Hot water injection was effective for stimulation, but open boundaries could not maintain the high injection pressure, leading to massive hot water losses and gas escapes. However, their combination achieved a synergistic stimulation like "1 + 1 > 2" because a piston water drive similar to secondary recovery in oil and gas development was formed. Relative to three-spot well patterns, the five-spot shortened the extraction cycle by 680 days and enhanced the gas-to-water ratio by 17%. Increasing injection pressure enhanced water yield more significantly while the improvement of gas yield was more significant by increasing hot water temperature. Overall, high-pressure and high-temperature injection was suggested for gas enhancement and water control. These findings provide important guidance for advancing the commercial development of challenging hydrates.

Disruption of the Na+/K+-ATPase-purinergic P2X7 receptor complex in microglia promotes stress-induced anxiety.

Na+/K+-ATPase (NKA) plays an important role in the central nervous system. However, little is known about its function in the microglia. Here, we found that NKAα1 forms a complex with the purinergic P2X7 receptor (P2X7R), an adenosine 5'-triphosphate (ATP)-gated ion channel, under physiological conditions. Chronic stress or treatment with lipopolysaccharide plus ATP decreased the membrane expression of NKAα1 in microglia, facilitated P2X7R function, and promoted microglia inflammatory activation via activation of the NLRP3 inflammasome. Accordingly, global deletion or conditional deletion of NKAα1 in microglia under chronic stress-induced aggravated anxiety-like behavior and neuronal hyperexcitability. DR5-12D, a monoclonal antibody that stabilizes membrane NKAα1, improved stress-induced anxiety-like behavior and ameliorated neuronal hyperexcitability and neurogenesis deficits in the ventral hippocampus of mice. Our results reveal that NKAα1 limits microglia inflammation and may provide a target for the treatment of stress-related neuroinflammation and diseases.

Injured sensory neurons-derived galectin-3 contributes to neuropathic pain via programming microglia in the spinal dorsal horn.

Emerging studies have demonstrated spinal microglia play a critical role in central sensitization and contribute to chronic pain. Although several mediators that contribute to microglia activation have been identified, the mechanism of microglia activation and its functionally diversified mechanisms in pathological pain are still unclear. Here we report that injured sensory neurons-derived Galectin-3 (Gal3) activates and reprograms microglia in the spinal dorsal horn (SDH) and contributes to neuropathic pain. Firstly, Gal3 is predominantly expressed in the isolectin B4 (IB4)-positive non-peptidergic sensory neurons and significantly up-regulated in dorsal root ganglion (DRG) neurons and primary afferent terminals in SDH in the partial sciatic nerve ligation (pSNL)-induced neuropathic pain model. Gal3 knockout (Gal3 KO) mice showed a significant decrease in mechanical allodynia and Gal3 inhibitor TD-139 produced a significant anti-allodynia effect in the pSNL model. Furthermore, pSNL-induced microgliosis was compromised in Gal3 KO mice. Additionally, intrathecal injection of Gal3 produces remarkable mechanical allodynia by direct activation of microglia, which have enhanced inflammatory responses with TNF-α and IL-1ß up-regulation. Thirdly, using single-nuclear RNA sequencing (snRNA-seq), we identified that Gal3 targets microglia and induces reprogramming of microglia, which may contribute to neuropathic pain establishment. Finally, Gal3 enhances excitatory synaptic transmission in excitatory neurons in the SDH via microglia activation. Our findings reveal that injured sensory neurons-derived Gal3 programs microglia in the SDH and contribute to neuropathic pain.

Microglial Cannabinoid CB2 Receptors in Pain Modulation.

Pain, especially chronic pain, can strongly affect patients' quality of life. Cannabinoids ponhave been reported to produce potent analgesic effects in different preclinical pain models, where they primarily function as agonists of Gi/o protein-coupled cannabinoid CB1 and CB2 receptors. The CB1 receptors are abundantly expressed in both the peripheral and central nervous systems. The central activation of CB1 receptors is strongly associated with psychotropic adverse effects, thus largely limiting its therapeutic potential. However, the CB2 receptors are promising targets for pain treatment without psychotropic adverse effects, as they are primarily expressed in immune cells. Additionally, as the resident immune cells in the central nervous system, microglia are increasingly recognized as critical players in chronic pain. Accumulating evidence has demonstrated that the expression of CB2 receptors is significantly increased in activated microglia in the spinal cord, which exerts protective consequences within the surrounding neural circuitry by regulating the activity and function of microglia. In this review, we focused on recent advances in understanding the role of microglial CB2 receptors in spinal nociceptive circuitry, highlighting the mechanism of CB2 receptors in modulating microglia function and its implications for CB2 receptor- selective agonist-mediated analgesia.

OCP002, a Mixed Agonist of Opioid and Cannabinoid Receptors, Produces Potent Antinociception With Minimized Side Effects.

BACKGROUND: Increasing attention has been attracted to the development of bifunctional compounds to minimize the side effects of opioid analgesics. Pharmacological studies have verified the functional interaction between opioid and cannabinoid systems in pain management, suggesting that coactivation of the opioid and cannabinoid receptors may provide synergistic analgesia with fewer adverse reactions. Herein, we developed and characterized a novel bifunctional compound containing the pharmacophores of the mu-opioid receptor agonist DALDA and the cannabinoid peptide VD-Hpα-NH2, named OCP002. METHODS: The opioid and cannabinoid agonistic activities of OCP002 were investigated in calcium mobilization and western blotting assays, respectively. Moreover, the central and peripheral antinociceptive effects of OCP002 were evaluated in mouse preclinical models of tail-flick test, carrageenan-induced inflammatory pain, and acetic acid-induced visceral pain, respectively. Furthermore, the potential opioid and cannabinoid side effects of OCP002 were systematically investigated in mice after intracerebroventricular (ICV) and subcutaneous (SC) administrations. RESULTS: OCP002 functioned as a mixed agonist toward mu-opioid, kappa-opioid, and cannabinoid CB1 receptors in vitro. ICV and SC injections of OCP002 produced dose-dependent antinociception in mouse models of nociceptive (the median effective dose [ED50] values with 95% confidence interval [CI] are 0.14 [0.12-0.15] nmol and 0.32 [0.29-0.35] µmol/kg for ICV and SC injections, respectively), inflammatory (mechanical stimulation: ED50 values [95% CI] are 0.76 [0.64-0.90] nmol and 1.23 [1.10-1.38] µmol/kg for ICV and SC injections, respectively; thermal stimulation: ED50 values [95% CI] are 0.13 [0.10-0.17] nmol and 0.23 [0.08-0.40] µmol/kg for ICV and SC injections, respectively), and visceral pain (ED50 values [95% CI] are 0.0069 [0.0050-0.0092] nmol and 1.47 [1.13-1.86] µmol/kg for ICV and SC injections, respectively) via opioid and cannabinoid receptors. Encouragingly, OCP002 cannot cross the blood-brain barrier and exerted nontolerance-forming analgesia over 6-day treatment at both supraspinal and peripheral levels. Consistent with these behavioral results, repeated OCP002 administration did not elicit microglial hypertrophy and proliferation, the typical features of opioid-induced tolerance, in the spinal cord. Furthermore, at the effective analgesic doses, SC OCP002 exhibited minimized opioid and cannabinoid side effects on motor performance, body temperature, gastric motility, physical and psychological dependence, as well as sedation in mice. CONCLUSIONS: This study demonstrates that OCP002 produces potent and nontolerance-forming antinociception in mice with reduced opioid- and cannabinoid-related side effects, which strengthen the candidacy of bifunctional drugs targeting opioid/cannabinoid receptors for translational-medical development to replace or assist the traditional opioid analgesics.

OFP011 Cyclic Peptide as a Multifunctional Agonist for Opioid/Neuropeptide FF Receptors with Improved Blood-Brain Barrier Penetration.

Mounting evidence indicates that the neuropeptide FF (NPFF) system is involved in the side effects of opioid usage, including antinociceptive tolerance, hyperalgesia, abuse, constipation, and respiratory depression. Our group recently discovered that the multitarget opioid/NPFF receptor agonist DN-9 exhibits peripheral antinociceptive activity. To improve its metabolic stability, antinociceptive potency, and duration, in this study, we designed and synthesized a novel cyclic disulfide analogue of DN-9, OFP011, and examined its bioactivity through in vitro cyclic adenosine monophosphate (cAMP) functional assays and in vivo behavioral experiments. OFP011 exhibited multifunctional agonistic effects at the µ-opioid and the NPFF1 and NPFF2 receptors and partial agonistic effects at the δ- and κ-opioid in vitro, as determined via the cAMP functional assays. Pharmacokinetic and pharmacological experiments revealed improvement in its blood-brain barrier permeability after systemic administration. In addition, subcutaneous OFP011 exhibited potent and long-lasting antinociceptive activity via the central µ- and κ-opioid receptors, as observed in different physiological and pathological pain models. At the highest antinociceptive doses, subcutaneous OFP011 exhibited limited tolerance, gastrointestinal transit, motor coordination, addiction, reward, and respiration depression. Notably, OFP011 exhibited potent oral antinociceptive activities in mouse models of acute, inflammatory, and neuropathic pain. These results suggest that the multifunctional opioid/NPFF receptor agonists with improved blood-brain barrier penetration are a promising strategy for long-term treatment of moderate to severe nociceptive and pathological pain with fewer side effects.

NOP01, a NOP receptor agonist, produced potent and peripherally restricted antinociception in a formalin-induced mouse orofacial pain model.

Orofacial pain is one of the most common medical challenges. A preliminary report indicates that the NOP receptor may act as a therapeutic target in orofacial pain. Previous studies have shown that [(pF)Phe4, Aib7, Aib11, Arg14, Lys15]N/OFQ-NH2 (NOP01) functions as a potent NOP receptor peptide agonist. This work aims to investigate the antinociception of NOP01 and its possible action mechanisms in a formalin-induced mouse orofacial pain model at different levels. Our results demonstrated that local, intraperitoneal (i.p.) or intrathecal (i.t.) injection of NOP01 produced dose-related antinociception in both phases of the formalin pain, which could be inhibited by the NOP receptor antagonist but not the classical opioid receptor antagonist. Furthermore, the antinociception induced by systemic NOP01 was blocked by local but not spinal pretreatment with the NOP receptor antagonist, suggesting the involvement of the peripheral NOP receptor in NOP01-induced systemic antinociception. Moreover, local injection of NOP01 markedly suppressed the expression of c-Fos protein induced by formalin in ipsilateral trigeminal ganglion (TG) neurons. In conclusion, this work suggests that NOP01 exerts significant antinociception on orofacial pain at both peripheral and spinal levels via the NOP receptor. Notably, NOP01 cannot readily penetrate the blood-brain barrier. Thus, NOP01 may behave as a potential compound for developing peripherally restricted analgesics.

Development of Multifunctional and Orally Active Cyclic Peptide Agonists of Opioid/Neuropeptide FF Receptors that Produce Potent, Long-Lasting, and Peripherally Restricted Antinociception with Diminished Side Effects.

We previously reported that a multifunctional opioid/neuropeptide FF receptor agonist, DN-9, achieved peripherally restricted analgesia with reduced side effects. To develop stable and orally bioavailable analogues of DN-9, eight lactam-bridged cyclic analogues of DN-9 between positions 2 and 5 were designed, synthesized, and biologically evaluated. In vitro cAMP assays revealed that these analogues, except 7, were multifunctional ligands that activated opioid and neuropeptide FF receptors. Analogue 1 exhibited improved potency for κ-opioid and NPFF2 receptors. All analogues exhibited potent, long-lasting, and peripherally restricted antinociception in the tail-flick test without tolerance development after subcutaneous administration and produced oral analgesia. Oral administration of the optimized compound analogue 1 exhibited powerful, peripherally restricted antinociceptive effects in mouse models of acute, inflammatory, and neuropathic pain. Remarkably, orally administered analogue 1 had no significant side effects, such as tolerance, dependence, constipation, or respiratory depression, at effective analgesic doses.

Spinal endomorphins attenuate burn-injury pain in male mice by inhibiting p38 MAPK signaling pathway through the mu-opioid receptor.

Burn injury is one of the main causes of mortality worldwide and frequently associated with severe and long-lasting pain that compromises the quality of patient life. Several studies have shown that the mu-opioid system plays an important role in burn pain relief. In this study, we investigated the spinal antinociception induced by the endogenous mu-opioid receptor (MOR) agonists endomorphins and explored their mechanisms of actions in burn injury-induced pain model. Our results showed that intrathecal injection of endomorphin-1 and -2 dose-dependently attenuated mechanical allodynia and thermal hyperalgesia via the mu-opioid receptor in mice on day 3 after burn injury, which was consistent with the data obtained from the mu-opioid receptor knockout mice. Western blot showed that the phosphorylation levels of extracellular signal-regulated kinase1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) in ipsilateral spinal cord tissues were significantly up-regulated after burn injury. Intrathecal injection of endomorphins selectively inhibited the activation of p38 MAPK on day 3 after burn injury via the mu-opioid receptor. Further studies found that repeated application of the specific p38 MAPK inhibitor SB203580 dose-dependently inhibited burn-injury pain, as well as the activation of spinal p38 MAPK. Taken together, our present study demonstrates that intrathecal injection of endomorphins attenuates burn-injury pain in male mice by affecting the spinal activation of p38 MAPK via the mu-opioid receptor.

Synthesis and Biological Characterization of Cyclic Disulfide-Containing Peptide Analogs of the Multifunctional Opioid/Neuropeptide FF Receptor Agonists That Produce Long-Lasting and Nontolerant Antinociception.

In a previously described chimeric peptide, we reported that the multifunctional opioid/neuropeptide FF (NPFF) receptor agonist 0 (BN-9) produced antinociception for 1.5 h after supraspinal administration. Herein, four cyclic disulfide analogs containing l- and/or d-type cysteine at positions 2 and 5 were synthesized. The cyclized analogs and their linear counterparts behaved as multifunctional agonists at both opioid and NPFF receptors in vitro and produced potent analgesia without tolerance development. In comparison to 0, cyclized peptide 6 exhibited sevenfold more potent µ-opioid receptor agonistic activity in vitro. Interestingly, the cyclized analog 6 possessed an improved stability in the brain and an increased blood-brain barrier permeability compared to the parent peptide 0 and produced more potent analgesia after supraspinal or subcutaneous administration with improved duration of action of 4 h. In addition, antinociceptive tolerance of analog 6 was greatly reduced after subcutaneous injection compared to fentanyl, as was the rewarding effect, withdrawal reaction, and gastrointestinal inhibition.

VF-13, a chimeric peptide of VD-hemopressin(α) and neuropeptide VF, produces potent antinociception with reduced cannabinoid-related side effects.

Pharmacological evidence indicated a functional interaction between neuropeptide FF (NPFF) and cannabinoid systems, and the cannabinoids combined with the NPFF receptor agonist neuropeptide VF (NPVF) produced antinociception without tolerance. In the present study, VF-13, a chimeric peptide containing the pharmacophores of the endogenous cannabinoid peptide VD-hemopressin(α) (VD-Hpα) and NPVF, was synthesized and pharmacologically evaluated. In vitro, VF-13 significantly upregulated the phosphorylated level of extracellular signal-regulated kinase 1/2 (ERK1/2) in CHO cells stably expressing CB1 receptors and inhibited forskolin-induced cAMP accumulation in HEK293 cells stably expressing NPFF1 or NPFF2 receptors. Moreover, VF-13 induced neurite outgrowth in Neuro 2A cells via CB1 and NPFF receptors. These results suggest that VF-13 exhibits multifunctional agonism at CB1, NPFF1 and NPFF2 receptors in vitro. Interestingly, intracerebroventricular VF-13 produced dose-dependent antinociception in mouse models of tail-flick and carrageenan-induced inflammatory pain via the TRPV1 receptor. In contrast, the reference compound (m)VD-Hpα-NH2 induced CB1 receptor-mediated supraspinal antinociception. Additionally, subcutaneous injection of (m)VD-Hpα-NH2 and VF-13 produced significant antinociception in carrageenan-induced inflammatory pain model. In the tetrad assay, our data demonstrated that VF-13 elicited hypothermia, but not catalepsy and hypoactivity after intracerebroventricular injection. Notably, VF-13 produced non-tolerance forming antinociception over 6 days treatment in both acute and inflammatory pain models. Furthermore, VF-13 had no apparent effects on gastrointestinal transit, pentobarbitone-induced sedation, food intake, and motor coordination at the supraspinal level. In summary, VF-13, a novel chimeric peptide of VD-Hpα and NPVF, produced non-tolerance forming antinociception in preclinical pain models with reduced cannabinoid-related side effects.

Spinal administration of the multi-functional opioid/neuropeptide FF agonist BN-9 produced potent antinociception without development of tolerance and opioid-induced hyperalgesia.

Chronic opioids treatment is impeded by the development of analgesic tolerance and opioid-induced hyperalgesia. Recent studies have shown that multi-functional opioid compounds produce analgesic activities with limited side effects. We developed a novel multi-functional peptide targeting opioid and neuropeptide FF receptors named BN-9, which produced potent and non-tolerance forming antinociceptive effect after supraspinal and systemic administrations. In the present study, the analgesic properties and potential side effects of intrathecal BN-9 were investigated in a range of preclinical rodent models. In complete Freund's adjuvant-induced inflammatory pain model, intrathecal BN-9 dose-dependently produced analgesic effect via opioid receptors, and the spinal antinociceptive effect was augmented by the neuropeptide FF receptor antagonist RF9. In contrast, in plantar incision-induced postoperative pain model, BN-9 exhibited potent anti-allodynic effect via opioid receptors and, at least partially, neuropeptide FF receptors. In mouse models of acetic acid-induced visceral pain and formalin pain, BN-9-induced spinal antinociception was mainly mediated by opioid receptors, independent of neuropeptide FF receptors. Furthermore, at the spinal level, chronic treatments with BN-9 did not lead to analgesic tolerance and cross-tolerance to morphine. Moreover, opioid-induced hyperalgesia was observed after repeated administration of morphine, but not BN-9. Taken together, our present study suggests that intrathecal BN-9 produces potent and non-tolerance forming antinociception, and does not cause opioid-induced hyperalgesia. Thus, BN-9 might serve as a promising lead compound in the development of multi-functional opioid analgesics with minimized side effects.

Central and peripheral modulation of gastrointestinal transit in mice by DN-9, a multifunctional opioid/NPFF receptor agonist.

BACKGROUND: The nonapeptide DN-9 functions as a multifunctional agonist to opioid and neuropeptide FF (NPFF) receptors and exhibits antinociceptive effects at the central and peripheral levels. METHODS: The effects of DN-9 on small and colonic intestinal transit were evaluated using the upper gastrointestinal (GI) transit test and colonic bead expulsion assay, respectively. Opioid and NPFF receptor antagonists were used to investigate the mechanisms of DN-9-induced GI inhibition. Furthermore, the agonism of the DN-9 analog [Phg9 ]-DN-9 to opioid and NPFF receptors was tested by the cAMP assay. KEY RESULTS: Intracerebroventricular administration of DN-9 dose-dependently slowed upper GI transit and colonic expulsion via mu- and kappa-opioid receptors in the brain, independent of the delta-opioid receptor. Similarly, intraperitoneal injection of DN-9 dose-dependently inhibited GI propulsion via the peripheral opioid receptors. DN-9-induced GI transit inhibitions were significantly aggravated by the NPFF receptor antagonist RF9. Moreover, the DN-9 analog [Phg9 ]-DN-9, an agonist at mu-, delta-, and kappa-opioid receptors but not NPFF receptors, inhibited GI more potently than DN-9. In addition, intracerebroventricular NPFF significantly attenuated the central inhibitory effects induced by [Phg9 ]-DN-9 and morphine. However, central and peripheral injections of NPFF or RF9 almost had no significant effects on GI transit by itself. CONCLUSION AND INFERENCES: Intracerebroventricular and intraperitoneal administrations of DN-9 inhibit GI transit via opioid receptors in mice by central and peripheral mechanisms, respectively. In addition, the NPFF agonism of DN-9 possesses antiopioid effects on GI transit, which might explain the reduced constipation at the antinociceptive doses.

Spinal DN-9, a Peptidic Multifunctional Opioid/Neuropeptide FF Agonist Produced Potent Nontolerance Forming Analgesia With Limited Side Effects.

The development of multitarget opioid drugs has emerged as an attractive therapeutic strategy to eliminate opioid-related side effects. Our previous study developed a series of opioid and neuropeptide FF pharmacophore-containing chimeric peptides, including DN-9 (Tyr-D.Ala-Gly-NMe.Phe-Gly-Pro-Gln-Arg-Phe-NH2), which produced potent nontolerance forming analgesia at the supraspinal level. In the present study, the antinociceptive effects of DN-9 in a series of preclinical pain models and the potential side-effects were investigated at the spinal level in mice. In the tail-flick test, intrathecal injection of DN-9 produced potent analgesia with an ED50 value at 1.33 pmol, and the spinal antinociception of DN-9 was mainly mediated by µ- and κ-opioid receptors. In addition, DN-9-induced spinal antinociception was augmented by the neuropeptide FF receptors antagonist. Furthermore, DN-9 could decrease both the frequency and amplitude of sEPSCs in lamina IIo neurons of the spinal cord, which were mediated by opioid receptors. In contrast to morphine, chronic intrathecal treatments with DN-9 did not induce analgesic tolerance, c-Fos expression or microglial activation. Intrathecal injection of DN-9 showed potent analgesia with antinociceptive ED50 values between .66 and 55.04 pmol in different pain models, including the formalin test, acetic acid-induced writhing test, carrageenan-induced inflammatory pain and neuropathic pain. Moreover, DN-9 did not show side effects in locomotor function and coordination, gastrointestinal transit inhibition, the cardiovascular system, and body temperature regulation at antinociceptive doses. Taken together, the present study showed DN-9 produced effective, nontolerance forming analgesia with reduced side effects at the spinal level. DN-9 might be a promising compound for developing multifunctional opioid analgesics with limited adverse effects. PERSPECTIVE: This article presents the potent and nontolerance forming analgesia effects of DN-9 in a series of preclinical pain models with less opioid related adverse effects at the spinal level in mice. This study also demonstrates that DN-9 has translational potential into an intrathecal analgesic.

The multifunctional peptide DN-9 produced peripherally acting antinociception in inflammatory and neuropathic pain via µ- and κ-opioid receptors.

BACKGROUND AND PURPOSE: Considerable effort has recently been directed at developing multifunctional opioid drugs to minimize the unwanted side effects of opioid analgesics. We have developed a novel multifunctional opioid agonist, DN-9. Here, we studied the analgesic profiles and related side effects of peripheral DN-9 in various pain models. EXPERIMENTAL APPROACH: Antinociceptive effects of DN-9 were assessed in nociceptive, inflammatory, and neuropathic pain. Whole-cell patch-clamp and calcium imaging assays were used to evaluate the inhibitory effects of DN-9 to calcium current and high-K+ -induced intracellular calcium ([Ca2+ ]i ) on dorsal root ganglion (DRG) neurons respectively. Side effects of DN-9 were evaluated in antinociceptive tolerance, abuse, gastrointestinal transit, and rotarod tests. KEY RESULTS: DN-9, given subcutaneously, dose-dependently produced antinociception via peripheral opioid receptors in different pain models without sex difference. In addition, DN-9 exhibited more potent ability than morphine to inhibit calcium current and high-K+ -induced [Ca2+ ]i in DRG neurons. Repeated treatment with DN-9 produced equivalent antinociception for 8 days in multiple pain models, and DN-9 also maintained potent analgesia in morphine-tolerant mice. Furthermore, chronic DN-9 administration had no apparent effect on the microglial activation of spinal cord. After subcutaneous injection, DN-9 exhibited less abuse potential than morphine, as was gastroparesis and effects on motor coordination. CONCLUSIONS AND IMPLICATIONS: DN-9 produces potent analgesia with minimal side effects, which strengthen the candidacy of peripherally acting opioids with multifunctional agonistic properties to enter human studies to alleviate the current highly problematic misuse of classic opioids on a large scale.