Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

Receptor mediated transcytosis harnessing the cellular uptake and transport of natural ligands across the blood-brain barrier (BBB) has been identified as a means for antibody delivery to the CNS. In this study, we characterized bispecific antibodies in which a BBB-crossing antibody fragment FC5 was used as a BBB carrier. Cargo antibodies were either a high-affinity, selective antibody antagonist of the metabotropic glutamate receptor-1 (BBB-mGluR1), a widely abundant CNS target, or an IgG that does not bind the CNS target (BBB-NiP). Both BBB-NiP and BBB-mGluR1 demonstrated a similar 20-fold enhanced rate of transcytosis across an in vitro BBB model compared with mGluR1 IgG fused to a control antibody fragment. All 3 bispecific antibodies exhibited identical pharmacokinetics in vivo Comparative assessment of BBB-NiP and BBB-mGluR1 revealed that, whereas their serum pharmacokinetics and BBB penetration were identical, their central disposition (brain levels) and elimination (cerebrospinal fluid levels) were widely different, due to central target-mediated removal of the mGluR1-engaging antibody. Central mGluR1 target engagement after systemic administration was demonstrated by a dose-dependent inhibition of mGluR-1-mediated thermal hyperalgesia and by colocalization of the antibody with thalamic neurons involved in mGluR1-mediated pain processing. We demonstrate the feasibility of targeting central G-protein-coupled receptors using a BBB-crossing bispecific antibody approach and emerging principles that govern brain distribution and disposition of these antibodies. These data will be important for designing safe and selective CNS antibody therapeutics.-Webster, C. I., Caram-Salas, N., Haqqani, A. S., Thom, G., Brown, L., Rennie, K., Yogi, A., Costain, W., Brunette, E., Stanimirovic, D. B. Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

Ultra-Low Doses of Naltrexone Enhance the Antiallodynic Effect of Pregabalin or Gabapentin in Neuropathic Rats.

Preclinical Research Treatment of neuropathic pain is an area of largely unmet medical need. Pregabalin and gabapentin are anticonvulsants widely used for the treatment of neuropathic pain. Unfortunately, these drugs are only effective in 50-60% of the treated patients. In addition, both drugs have substantial side effects. Several studies have reported that ultralow doses of opioid receptor antagonists can induce analgesia and enhance the analgesic effect of opioids in rodents and humans. The objective of the present study was to assess the antiallodynic synergistic interaction between gabapentinoids and naltrexone in rats. Oral administration of pregabalin (ED50 = 2.79 ± 0.16 mg/kg) or gabapentin (ED50 = 21.04 ± 2.87 mg/kg) as well as intrathecal naltrexone (ED50 = 0.11 ± 0.02 ng) reduced in a dose-dependent manner tactile allodynia in rats. Maximal antiallodynic effects (∼100%) were reached with 30 mg/kg of pregabalin, 300 mg/kg of gabapentin or 0.5 ng of naltrexone. Co-administration of pregabalin or gabapentin and naltrexone in a fixed-dose ratio (1:1) remarkably reduced spinal nerve ligation-induced tactile allodynia showing a synergistic interaction. The data indicate that combinations of pregabalin or gabapentin and ultra-low doses of naltrexone are able to reduce tactile allodynia in neuropathic rats with lower doses that those used when drugs are given individually and with an improved side effects profile. Drug Dev Res 78 : 371-380, 2017. © 2017 Wiley Periodicals, Inc.

A novel platform for engineering blood-brain barrier-crossing bispecific biologics.

The blood-brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic "arm" is combined with a BBB-transcytosing arm can significantly enhance their brain delivery. The BBB-permeable single-domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single-domain antibody phage display library. In this study, FC5 was engineered as a mono- and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30-fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5- compared with control domain antibody-Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB-impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5-Fc fusion proteins. Improved serum pharmacokinetics of Fc-fused FC5 contributed to a 60-fold increase in pharmacological potency compared with the single-domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB-carrier arm in bispecific antibodies or antibody-drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications.

Anti-Apoptotic Effects of Dapsone After Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a condition producing irreversible damage to the neurological function. Among the leading mechanisms associated to cell death after SCI, excitotoxicity, oxidative stress, inflammatory response and apoptosis are considered potential targets to prevent tissue damage. We recently reported that dapsone an anti-inflammatory drug, decreases the activity of myeloperoxidase, lipid peroxidation, improve neurological function and increase the amount of spared tissue after SCI in rats. In this study, we characterized the anti-apoptotic effect of dapsone administered at 12.5 mg/kg/24 h dose, starting at 3 and 5 h after SCI. We monitored the activity of caspases-8, 9, and 3 and quantitated Annexin V and TUNEL positive cells in the core of the lesion. Results showed increased activities of caspase-8, 9 and 3 at 72 h by SCI to reach increments of 69, 143 and 293 %, respectively, as compared to sham group. Meanwhile, dapsone, administered at 3 and 5 after SCI, reduced caspase-8 activity by 36 and 44 % respectively, whereas the activity of caspase-9 was diminished by 37 %. Likewise, the activity of caspase-3 showed a decrease of 38 %. Finally, both Annexin V and TUNEL-positive cells were significantly reduced by DDS as compared to untreated SCI animals. Results showed that dapsone exerted anti-apoptotic effect after SCI.

Multiplexed evaluation of serum and CSF pharmacokinetics of brain-targeting single-domain antibodies using a NanoLC-SRM-ILIS method.

FC5 and FC44 are single-domain antibodies (VHHs), selected by functional panning of phage-display llama VHH library for their ability to internalize human brain endothelial cells (BEC) and to transmigrate the in vitro BBB model. Quantification of brain delivery of FC5 and FC44 in vivo was challenging using classical methods because of their short plasma half-life and their loss of functionality with radioactive labeling. A highly sensitive (detection limit <2 ng/mL) and specific SRM-ILIS method to detect and quantify unlabeled VHHs in multiplexed assays was developed and applied to comparatively evaluate brain delivery of FC5 and FC44, and two control VHHs, EG2 and A20.1. FC5 and FC44 compared to control VHHs demonstrated significantly (p < 0.01) enhanced transport (50-100-fold) across rat in vitro BBB model as well as in vivo brain targeting assessed by optical imaging. The multiplexed SRM-ILIS analyses of plasma and CSF levels of codosed VHHs demonstrated that while all 4 VHHs have similar blood pharmacokinetics, only FC5 and FC44 show elevated CSF levels, suggesting that they are potential novel carriers for delivery of drugs and macromolecules across the BBB.

Sex-dependent antiallodynic effect of α2 adrenergic receptor agonist tizanidine in rats with experimental neuropathic pain.

The purpose of this study was to investigate the mechanism of antiallodynic effect of tizanidine in neuropathic rats. Spinal nerve ligation reduced withdrawal threshold which was interpreted as tactile allodynia. Increasing doses of tizanidine induced a dose-dependent antiallodynic effect in nerve injured rats. Tizanidine was more effective in female than male neuropathic rats. This drug induced a lower antiallodynic effect in ovariectomized, compared with non-ovariectomized, neuropathic rats, while systemic reconstitution of estradiol (E2) levels in ovariectomized neuropathic females fully restored the antiallodynic effect of tizanidine. Naloxone reduced the antiallodynic effect of tizanidine in male but not in female neuropathic rats. Ovariectomy restored the antagonizing effect of naloxone in the antiallodynic effect of tizanidine, whereas treatment with E2 abolished the effect of naloxone on tizanidine activity. Rauwolscine (α2 antagonist) and imiloxan (α2B antagonist) completely abated tizanidine-induced antiallodynic effect in female neuropathic rats. In contrast, BRL-44408 (α2A antagonist) partially decreased the effect of tizanidine while JP-1302 (α2C antagonist) was ineffective. Rauwolscine, imiloxan and BRL-44408 decreased withdrawal threshold in naïve female rats. Rauwolscine did not modify withdrawal threshold in naïve male rats. AGN192403 (I1 antagonist), BU224 (I2 antagonist), prazosin (α1 antagonist) and methiothepin (5-HT antagonist) did not modify tizanidine-induced antiallodynia in neuropathic females and males. These data indicate that tizanidine exhibits a sex-dependent antiallodynic effect in neuropathy. Data also suggest that activation of adrenergic α2B and α2A and opioid receptors participate in the antiallodynic effect of tizanidine in female and male, respectively, neuropathic rats.

Interactions of Echinacea spp. Root Extracts and Alkylamides With the Endocannabinoid System and Peripheral Inflammatory Pain.

Historical ethnobotanies of indigenous peoples of the North American prairies reveal treatment of many painful conditions by Echinacea spp. Recent evidence suggests a pharmacological basis for such use as the bioactivity of E. angustifolia and E. purpurea is mediated, in part, through activation of the endocannabinoid system (ECS). Whereas the cannabimimetic effects of individual echinacea products and alkylamides have been described, the activity of crude extracts has not been compared between cannabinoid (CB) receptors or across species or genotypes. Moreover, few studies have explored echinacea's engagement of the ECS for historic treatments or new therapeutic applications in peripheral inflammatory pain. We hypothesized that 1) the in vitro effects of root extracts on CB receptor internalization would vary with species and phytochemistry, and that echinacea root extracts would reduce inflammatory pain in vivo through activation of the ECS. Root extracts of different E. angustifolia and E. purpurea accessions were prepared, analyzed by HPLC-DAD to quantify caffeic acid derivatives and alkylamides (AKA), and tested for agonist and antagonist activities using receptor redistribution assays. Linear regression of activity relative to phytochemistry identified predictive compounds that were assessed individually in redistribution assays. Extracts were evaluated in the Hargreaves model of chronic inflammatory pain in rats with co-administration of selective CB1/2 antagonists to gauge involvement of the ECS. CB receptor agonist activity varied among accessions of both species with linear regression revealing a significant relationship between CB1 activity and AKA2 for E angustifolia, and AKA 9 + 10 for E purpurea. CB2 activity was positively related with AKA 9 + 10 and total AKAs in E. angustifolia. Four isolated AKA demonstrated agonist activity in the CB2, but not CB1, assay. In the inflammatory pain model, oral administration of either E angustifolia or E. purpurea root extract produced dose-dependent analgesic effects that were partially reversed by co-administration of CB receptor antagonists. This study demonstrates that in vitro effects of crude echinacea root extracts on CB receptors is predicted by phytochemistry. In vivo, echinacea has potential applications for peripheral inflammatory pain such as arthritis and burns, reflecting the traditional uses of Indigenous North Americans.

l-Proline as a Valuable Scaffold for the Synthesis of Novel Enantiopure Neonicotinoids Analogs.

In this research, six neonicotinoid analogs derived from l-proline were synthesized, characterized, and evaluated as insecticides against Xyleborus affinis. Most of the target compounds showed good to excellent insecticidal activity. To the best of our knowledge, this is the first report dealing with the use of enantiopure l-proline to get neonicotinoids. These results highlighted the compound 9 as an excellent candidate used as the lead chiral insecticide for future development. Additionally, molecular docking with the receptor and compound 9 was carried out to gain insight into its high activity when compared to dinotefuran. Finally, the neurotoxic evaluation of compound 9 showed lower toxicity than the classic neonicotinoid dinotefuran.

Venom components of the scorpion Centruroides limpidus modulate cytokine expression by T helper lymphocytes: Identification of ion channel-related toxins by mass spectrometry.

The study of the effector mechanisms of T helper cells has revealed different phenotypic characteristics that can be manipulated for designing new therapeutic schemes in different pathological scenarios. Ion channels are significant targets in T lymphocyte modulation since they are closely related to their effector activity. Remarkably, some toxins produced by scorpions specifically affect the function of these membrane proteins. For that reason, these toxins are important candidates in the search for new immunomodulators. Here, the effect of two venom fractions of the scorpion Centruroides limpidus was assessed on T lymphocyte proliferation and cytokine production. The venom fractions ClF8 and ClF9 were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and cultured at 25 and 35 µg/ml with murine T lymphocytes. The results indicate that the fraction ClF8 increased both production and secretion levels of IFN-γ, IL-4, IL-17A and IL-10 by CD4+ T cells at 24 h. In contrast, fraction ClF9 only promoted the secretion of IL-17A and IL-10 at its highest concentration (35 µg/ml). Both fractions did not show any effect on T cell proliferation. Subsequent analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed seventeen toxins in the fraction ClF8 and five toxins in the fraction ClF9, most of them with voltage-gated sodium (NaScTx) and potassium (KScTx) channels as molecular targets. These toxins might probably interact with ion channels involved in T lymphocyte activity. Our findings suggest that the difference in composition between the two fractions could be related to the observed effects, and the components identified could be isolated to search for possible immunomodulatory molecules.

Synergistic antiallodynic interaction between gabapentin or carbamazepine and either benfotiamine or cyanocobalamin in neuropathic rats.

Anticonvulsants, including gabapentin and carbamazepine, have shown activity against several types of neuropathic pain; however, they have limiting side effects that may minimize their use. In this study the possible synergistic interaction between anticonvulsants and benfotiamine or cyanocobalamin on spinal nerve ligation-induced tactile allodynia was assessed. Oral administration of gabapentin (15-300 mg/kg), carbamazepine (10-300 mg/kg), benfotiamine (30-600 mg/kg) or cyanocobalamin (0.3-6.0 mg/kg) significantly reduced tactile allodynia in rats. Maximal antiallodynic effects were reached with gabapentin 300 mg/kg (approximately 70%), carbamazepine 300 mg/kg (approximately 66%), benfotiamine 600 mg/kg (approximately 51%) and cyanocobalamin 6 mg/kg (approximately 59%). At the highest tested doses, gabapentin, but not carbamazepine, benfotiamine or cyanocobalamin, significantly reduced motor coordination. Coadministration of gabapentin or carbamazepine with benfotiamine or cyanocobalamin in a fixed ratio markedly reduced spinal nerve ligation-induced tactile allodynia, showing a synergistic interaction between anticonvulsants and B vitamins. Data indicate that combinations of anticonvulsants with benfotiamine or cyanocobalamin are able to reduce tactile allodynia without affecting motor coordination in rats, and suggest the possible clinical use of these combinations in the treatment of neuropathic pain in humans.

Melatonin reduces formalin-induced nociception and tactile allodynia in diabetic rats.

The purpose of this study was to assess the antinociceptive and antiallodynic effect of melatonin as well as its possible mechanism of action in diabetic rats. Streptozotocin (50 mg/kg) injection caused hyperglycemia within 1 week. Formalin-evoked flinching was increased in diabetic rats as compared to non-diabetic rats. Oral administration of melatonin (10-300 mg/kg) dose-dependently reduced flinching behavior in diabetic rats. In addition, K-185 (a melatonin MT(2) receptor antagonist, 0.2-2 mg/kg, s.c.) completely blocked the melatonin-induced antinociception in diabetic rats, whereas that naltrexone (a non-selective opioid receptor antagonist, 1 mg/kg, s.c.) and naltrindole (a selective delta opioid receptor antagonist, 0.5 mg/kg, s.c.), but not 5'-guanidinonaltrindole (a selective kappa opioid receptor antagonist, 1 mg/kg, s.c.), partially reduced the antinociceptive effect of melatonin. Given alone K-185, naltrexone, naltrindole or 5'-guanidinonaltrindole did not modify formalin-induced nociception in diabetic rats. Four to 8 weeks after diabetes induction, tactile allodynia was observed in the streptozotocin-injected rats. On this condition, oral administration of melatonin (75-300 mg/kg) dose-dependently reduced tactile allodynia in diabetic rats. Both antinociceptive and antiallodynic effects were not related to motor changes as melatonin did not modify number of falls in the rotarod test. Results indicate that melatonin is able to reduce formalin-induced nociception and tactile allodynia in streptozotocin-injected rats. In addition, data suggest that melatonin MT(2) and delta opioid receptors may play an important role in these effects.

Subcutaneous, intrathecal and periaqueductal grey administration of asimadoline and ICI-204448 reduces tactile allodynia in the rat.

The purpose of this study was to assess the possible antiallodynic effect of asimadoline ([N-methyl-N-[1S)-1-phenyl)-2-(13S))-3-hydroxypyrrolidine-1-yl)-ethyl]-2,2-diphenylacetamide HCl]) and ICI-20448 ([2-[3-(1-(3,4-Dichlorophenyl-N-methylacetamido)-2-pyrrolidinoethyl)-phenoxy]acetic acid HCl]), two peripheral selective kappa opioid receptor agonists, after subcutaneous, spinal and periaqueductal grey administration to neuropathic rats. Twelve days after spinal nerve ligation tactile allodynia was observed, along with an increase in kappa opioid receptor mRNA expression in dorsal root ganglion and dorsal horn spinal cord. A non-significant increase in periaqueductal grey was also seen. Subcutaneous (s.c.) administration of asimadoline and ICI-204448 (1-30 mg/kg) dose-dependently reduced tactile allodynia. This effect was partially blocked by s.c., but not intrathecal, naloxone. Moreover, intrathecal administration of asimadoline or ICI-204448 (1-30 mug) reduced tactile allodynia in a dose-dependent manner and this effect was completely blocked by intrathecal naloxone. Microinjection of both kappa opioid receptor agonists (3-30 mug) into periaqueductal grey also produced a naloxone-sensitive antiallodynic effect in rats. Our results indicate that systemic, intrathecal and periaqueductal grey administration of asimadoline and ICI-204448 reduces tactile allodynia. This effect may be a consequence of an increase in kappa opioid receptor mRNA expression in dorsal root ganglion, dorsal horn spinal cord and, to some extent, in periaqueductal grey. Finally, our data suggest that these drugs could be useful to treat neuropathic pain in human beings.

Benfotiamine relieves inflammatory and neuropathic pain in rats.

Benfotiamine has shown therapeutic efficacy in the treatment of painful diabetic neuropathy in human beings. However, so far there is no evidence about the efficacy of this drug in preclinical models of pain. The purpose of this study was to assess the possible antinociceptive and antiallodynic effect of benfotiamine in inflammatory and neuropathic pain models in the rat. Inflammatory pain was induced by injection of formalin in non-diabetic and diabetic (2 weeks) rats. Reduction of flinching behavior was considered as antinociception. Neuropathic pain was induced by either ligation of left L5/L6 spinal nerves or administration of streptozotocin (50 mg/kg, i.p.) in Wistar rats. Benfotiamine significantly reduced inflammatory (10-300 mg/kg) and neuropathic (75-300 mg/kg) nociception in non-diabetic and diabetic rats. Results indicate that oral administration of benfotiamine is able to reduce tactile allodynia from different origin in the rat and they suggest the use of this drug to reduce inflammatory and neuropathic pain in humans.

In vitro and in vivo methods for assessing FcRn-mediated reverse transcytosis across the blood-brain barrier.

The neonatal Fc receptor, FcRn, mediates endocytic recycling pathway that prevents degradation of IgG and is expressed in most endothelial cells. The blood-brain barrier (BBB), formed by brain endothelial cells sealed with tight junctions, restricts transport of IgG from the blood to the brain. In contrast, it has been suggested that IgG undergoes efflux from the brain parenchyma via reverse transcytosis across the BBB mediated by FcRn. The fast elimination of therapeutic antibodies from the brain via this route may limit their therapeutic potency. In vitro and in vivo methods described in this chapter were developed to facilitate research into mechanisms and dynamics of brain efflux of compounds, including FcRn-mediated reverse transcytosis across the BBB. The in vitro model uses immortalized adult rat brain endothelial cells which express high levels of FcRn. In vivo models use Prospective optical imaging to measure the clearance rate of intracerebrally injected FcRn-transported molecules tagged with near-infrared fluorescent probes.

Sildenafil and glyceryl trinitrate reduce tactile allodynia in streptozotocin-injected rats.

The possible antiallodynic effect of phosphodiesterase 5 inhibitor sildenafil and nitric oxide donor glyceryl trinitrate as well as the changes in phosphodiesterase 5A2 mRNA expression in dorsal root ganglion and spinal cord of allodynic diabetic rats was assessed. Diabetes was induced by streptozotocin (50mg/kg, i.p.) in male Wistar rats. Streptozotocin injection produced hyperlglycemia, polydipsia, polyphagia and polyuria as well as long-term tactile allodynia (12 weeks) and a reduction of phosphodiesterase 5A2 mRNA expression in spinal cord of diabetic rats. Systemic administration of sildenafil (1-5.6 mg/kg, i.p.) reduced tactile allodynia in a dose-dependent manner in diabetic rats. Likewise, glyceryl trinitrate patches (0.2mg/h) also reduced tactile allodynia in diabetic rats. Moreover, both drugs reversed streptozotocin-induced phosphodiesterase 5A2 mRNA expression reduction. Our results indicate that glyceryl trinitrate and sildenafil reduce tactile allodynia in diabetic rats suggesting that nitric oxide and cyclic GMP supply is an important step in their mechanism of action of these drugs in diabetic animals. Data suggest that nitric oxide donors (as glyceryl trinitrate) and drugs which increase cyclic GMP levels (as sildenafil) could have a role in the pharmacotherapy of tactile allodynia in diabetic patients.

Assessment of the potential role of muscle spindle mechanoreceptor afferents in chronic muscle pain in the rat masseter muscle.

BACKGROUND: The phenotype of large diameter sensory afferent neurons changes in several models of neuropathic pain. We asked if similar changes also occur in "functional" pain syndromes. METHODOLOGY/PRINCIPAL FINDINGS: Acidic saline (AS, pH 4.0) injections into the masseter muscle were used to induce persistent myalgia. Controls received saline at pH 7.2. Nocifensive responses of Experimental rats to applications of Von Frey Filaments to the masseters were above control levels 1-38 days post-injection. This effect was bilateral. Expression of c-Fos in the Trigeminal Mesencephalic Nucleus (NVmes), which contains the somata of masseter muscle spindle afferents (MSA), was above baseline levels 1 and 4 days after AS. The resting membrane potentials of neurons exposed to AS (n = 167) were hyperpolarized when compared to their control counterparts (n = 141), as were their thresholds for firing, high frequency membrane oscillations (HFMO), bursting, inward and outward rectification. The amplitude of HFMO was increased and spontaneous ectopic firing occurred in 10% of acid-exposed neurons, but never in Controls. These changes appeared within the same time frame as the observed nocifensive behaviour. Ectopic action potentials can travel centrally, but also antidromically to the peripheral terminals of MSA where they could cause neurotransmitter release and activation of adjacent fibre terminals. Using immunohistochemistry, we confirmed that annulospiral endings of masseter MSA express the glutamate vesicular transporter VGLUT1, indicating that they can release glutamate. Many capsules also contained fine fibers that were labelled by markers associated with nociceptors (calcitonin gene-related peptide, Substance P, P2X3 receptors and TRPV1 receptors) and that expressed the metabotropic glutamate receptor, mGluR5. Antagonists of glutamatergic receptors given together with the 2(nd) injection of AS prevented the hypersensitivity observed bilaterally but were ineffective if given contralaterally. CONCLUSIONS/SIGNIFICANCE: Low pH leads to changes in several electrical properties of MSA, including initiation of ectopic action potentials which could propagate centrally but could also invade the peripheral endings causing glutamate release and activation of nearby nociceptors within the spindle capsule. This peripheral drive could contribute both to the transition to, and maintenance of, persistent muscle pain as seen in some "functional" pain syndromes.

Role of opioid receptors in the reduction of formalin-induced secondary allodynia and hyperalgesia in rats.

This study assesses the effects of peripheral or intrathecal pre-treatment or post-treatment with micro, delta, kappa and nociceptin/orphanin FQ (NOP) opioid receptor agonists (morphine, U-50488 [trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide hydrochloride], DADLE [D-Ala2-Leu5-enkephalin] and nociceptin, respectively) on formalin-induced secondary mechanical allodynia and hyperalgesia in rats. 1% Formalin injection produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term tactile secondary allodynia and hyperalgesia. Neither peripheral (into the formalin-injected paw) nor intrathecal morphine post-treatment reversed formalin-induced secondary allodynia and hyperalgesia. In contrast, morphine pre-treatment prevented the development of these pain behaviors. Intrathecal and peripheral post- but not pre-treatment with U-50488 or DADLE significantly reduced secondary allodynia and hyperalgesia. Interestingly, nociceptin reduced both pain behaviors regardless of the administration site or treatment time. Local antinociceptive effects of morphine, DADLE, U-50488 or nociceptin were blocked by naltrexone, naltrindole, 5-guanidinonaltrindole and [Nphe(1)]nociceptin(1-13)NH(2), respectively. These results suggest that the long-term nociceptive behaviors induced by formalin are differentially modulated by selective opioid receptor agonists. In addition, data suggest that peripheral and spinal delta and kappa opioid receptors are important when nociceptive behaviors are established. In contrast, micro opioid receptors are more important at the beginning of the injury when the sensory system has not changed. NOP receptors participate diminishing both the development and maintenance of nociceptive behaviors. Results suggest that a barrage of afferent input induced by formalin injection initiates a long-term differential change in peripheral and spinal processing that affect the efficacy of opioid receptor agonists.

Isobolographic analyses of the gabapentin-metamizol combination after local peripheral, intrathecal and oral administration in the rat.

This study was designed to evaluate the possible antinociceptive interaction between gabapentin and metamizol on formalin-induced nociception. Gabapentin, metamizol or a fixed dose-ratio combination of both drugs were assessed after local peripheral, intrathecal and oral administration in rats. Isobolographic analyses were employed to define the nature of the interaction between drugs. Gabapentin, metamizol and gabapentin-metamizol combinations yielded a dose-dependent antinociceptive effect when administered by the three different routes. ED30 values were estimated for the individual drugs and isobolograms were constructed. Theoretical ED30 values for the combination estimated from the isobolograms were 21.11 +/- 1.17 microg/paw, 104.6 +/- 5.5 microg/rat and 78.8 +/- 5.5 mg/kg for the local peripheral, intrathecal and oral administration routes, respectively. These values were significantly higher than the experimentally obtained ED30 values which were 11.3 +/- 1.5 microg/paw, 36.8 +/- 3.1 microg/rat and 15 +/- 1.2 mg/kg indicating a synergistic interaction. Systemic administration resulted in the highest synergism. Data confirm that low doses of the gabapentin and metamizol can interact synergistically to reduce formalin-induced nociceptive behavior suggesting that this combination could be useful to treat inflammatory pain in humans.

Thiamine and cyanocobalamin relieve neuropathic pain in rats: synergy with dexamethasone.

Treatment of neuropathic pain is an area of largely unmet medical need. Therefore, this pain may require the development of novel drug entities. In the search for alternatives, B vitamins have been found to be a clinically useful pharmacological tool for patients with neuropathic pain. However, preclinical studies supporting this use are lacking. In this study, we assessed the possible antiallodynic effects of thiamine, pyridoxine, and cyanocobalamin as well as dexamethasone and their combination on spinal nerve ligation induced allodynia. Sub cutaneous administration of thiamine (75-600 mg/kg), pyridoxine (75-600 mg/kg), cyanocobalamin(0.75-6 mg/kg), and dexamethasone (4-32 mg/kg) significantly reduced tactile allodynia in rats. Maximal antiallodynic effects were reached with 600 mg/kg of thiamine (approximately 58%), 600 mg/kg of pyridoxine (approximately 22%), 6 mg/kg of cyanocobalamin (approximately 73%), and 32 mg/kg of dexamethasone (approximately 68%). Since a small antiallodynic effect was observed with pyridoxine, this drug was not further analyzed in the combinations. Coadministration of thiamine or cyanocobalamin and dexamethasone remarkably reduced spinal nerve ligation induced allodynia (approximately 90%), showing a synergistic interaction between either thiamine or cyanocobalamin and dexamethasone. Our data indicate that thiamine and pyridoxine as well as the combination of B vitamins and dexamethasone are able to reduce tactile allodynia in rats and suggest the possible clinical use of these drugs in the treatment of neuropathic pain in human beings.

Oral administration of B vitamins increases the antiallodynic effect of gabapentin in the rat.

The purpose of this study was to assess the possible synergistic interaction between gabapentin and B-vitamins (100:100:1 of vitamin B1, B6 and B12, respectively) in a neuropathic pain model in the rat. Neuropathic pain was induced by ligation of the left L5 and L6 spinal nerves of female Wistar rats. Tactile allodynia was determined by measuring paw withdrawal in response to probing with a series of calibrated von Frey filaments. Gabapentin (30-300 mg/kg), B-vitamins (75-600 mg/kg), or a combination of gabapentin and B-vitamins were administered orally and the antiallodynic effect was determined. Isobolographic analyses were used to define the nature of the functional interactions between gabapentin and B-vitamins in a fixed-dose ratio (0.5:0.5). Gabapentin (ED30 23.0+/-5.3 mg/kg), B-vitamins (ED30 524.0+/-97.0 mg/kg), or a fixed-dose ratio combination of gabapentin-B vitamins combinations dose-dependently reduced tactile allodynia. The theoretical ED30 value for the combination estimated from the isobologram was 273.5+/-48.6 mg/kg. This value was significantly higher than the experimental ED30 value which was 18.7+/-1.7 mg/kg. Results indicate that systemic administration of gabapentin and B vitamins can interact synergistically to reduce neuropathic pain in the rat and suggest the use of this combination to relieve neuropathy in humans.