Sadhana Patel

The role of central and peripheral Cannabinoid1 receptors in the antihyperalgesic activity of cannabinoids in a model of neuropathic pain.

&NA; We have examined the effects of cannabinoid agonists on hyperalgesia in a model of neuropathic pain in the rat and investigated the possible sites of action. The antihyperalgesic activity of the cannabinoids was compared with their ability to elicit behavioural effects characteristic of central cannabinoid activity. WIN55,212‐2 (0.3–10 mg kg−1), CP‐55,940 (0.03–1 mg kg−1) and HU‐210 (0.001–0.03 mg kg−1) were all active in a ‘tetrad’ of tests consisting of tail‐flick, catalepsy, rotarod and hypothermia following subcutaneous administration, with a rank order of potency in each of HU‐210>CP‐55,940>WIN55,212‐2. The effects of WIN55,212‐2 in each assay were blocked by the Cannabinoid1 (CB1) antagonist SR141716A. In the partial sciatic ligation model of neuropathic pain WIN55,212‐2, CP‐55,940 and HU‐210 produced complete reversal of mechanical hyperalgesia within 3 h of subcutaneous administration with D50 values of 0.52, 0.08 and 0.005 mg kg−1, respectively. In this model WIN55,212‐2 was also effective against thermal hyperalgesia and mechanical allodynia. WIN55,212‐2 produced pronounced reversal of mechanical hyperalgesia following intrathecal administration that was blocked by the CB1 antagonist SR141716A. Following intraplantar administration into the ipsilateral hindpaw, WIN55,212‐2 produced up to 70% reversal of mechanical hyperalgesia, although activity was also observed at high doses following injection into the contralateral paw. The antihyperalgesic effect of WIN55,212‐2 injected into the ipsilateral paw was blocked by subcutaneously administered SR141716A, but was not affected by intrathecally administered SR141716A. These data show that cannabinoids are highly potent and efficacious antihyperalgesic agents in a model of neuropathic pain. This activity is likely to be mediated via an action in both the CNS and in the periphery.

The effects of GABAB agonists and gabapentin on mechanical hyperalgesia in models of neuropathic and inflammatory pain in the rat

&NA; We have examined the effects of a novel GABAB agonist, CGP35024, in models of chronic neuropathic (partial sciatic ligation) and inflammatory (Freunds complete adjuvant) pain in the rat, and its inhibitory action on spinal transmission in vitro. The effects of CGP35024 were compared with L‐baclofen and gabapentin. CGP35024 and L‐baclofen reversed neuropathic mechanical hyperalgesia following single subcutaneous or intrathecal administration, but did not affect inflammatory mechanical hyperalgesia. Gabapentin only moderately affected neuropathic hyperalgesia following a single administration by either route, but produced significant reversal following daily administration for 5 days. It was only weakly active against inflammatory hyperalgesia following single or repeated administration. The antihyperalgesic effects of L‐baclofen and CGP35024, but not gabapentin, were blocked by the selective GABAB receptor antagonist CGP56433A. CGP35024 was seven times more potent against neuropathic hyperalgesia than in the rotarod test for motor co‐ordination, whilst L‐baclofen was approximately equipotent in the two tests. In the isolated hemisected spinal cord from the rat, CGP35024, L‐baclofen and gabapentin all inhibited capsaicin‐evoked ventral root potentials (VRPs). CGP35024 and L‐baclofen, but not gabapentin, also inhibited the polysynaptic and monosynaptic phases of electrically‐evoked VRPs, as well as the ‘wind‐up’ response to repetitive stimulation. These data indicate that CGP35024 and L‐baclofen modulate nociceptive transmission in the spinal cord to inhibit neuropathic hyperalgesia, and that CGP35024 has a therapeutic window for antihyperalgesia over spasmolysis.

Disease modifying and anti-nociceptive effects of the bisphosphonate, zoledronic acid in a model of bone cancer pain.

&NA; Inoculation of syngeneic MRMT‐1 mammary tumour cells into one tibia of female rats produced tumour growth within the bone associated with a reduction in bone mineral density (BMD) and bone mineral content (BMC), severe radiological signs of bone destruction, together with the development of behavioural mechanical allodynia and hyperalgesia. Histological and radiological examination showed that chronic treatment with the bisphosphonate, zoledronic acid (30 &mgr;g/kg, s.c.), for 19 days significantly inhibited tumour proliferation and preserved the cortical and trabecular bone structure. In addition, BMD and BMC were preserved and a dramatic reduction of tartrate resistant acid phosphatase‐positive polykaryocytes (osteoclasts) was observed. In behavioural tests, chronic treatment with zoledronic acid but not the significantly less effective bisphosphonate, pamidronate, or the selective COX‐2 inhibitor, celebrex, attenuated mechanical allodynia and hyperalgesia in the affected hind paw. Zoledronic acid also attenuated mechanical hyperalgesia associated with chronic peripheral neuropathy and inflammation in the rat. In contrast, pamidronate or clodronate did not have any anti‐hyperalgesic effect on mechanical hyperalgesia in the neuropathic and inflammatory pain models. We conclude that zoledronic acid, in addition to, or independent from, its anti‐metastatic and bone preserving therapeutic effects, is an anti‐nociceptive agent in a rat model of metastatic cancer pain. This unique property of zoledronic acid amongst the bisphosphonate class of compounds could make this drug a preferred choice for the treatment of painful bone metastases in the clinic.

Comparative activity of the anti-convulsants oxcarbazepine, carbamazepine, lamotrigine and gabapentin in a model of neuropathic pain in the rat and guinea-pig

Anti‐epileptic drugs (AEDs) are increasingly used for the treatment of neuropathic pain. Oxcarbazepine is a recently introduced AED that is effective in treating epilepsy and has an improved side‐effect profile compared to existing therapies. Here we have examined the effect of oxcarbazepine and other AEDs in a model of neuropathic pain in the rat and guinea‐pig. Oxcarbazepine and carbamazepine (3–100 mg kg−1) did not affect mechanical hyperalgesia or tactile allodynia induced by partial sciatic nerve ligation in the rat following oral administration. However, in the same model in the guinea‐pig, both drugs produced up to 90% reversal of mechanical hyperalgesia with respective D50 values of 10.7 and 0.8 mg kg−1. The active human metabolite of oxcarbazepine, monohydroxy derivative, was similarly active against mechanical hyperalgesia in the guinea‐pig but not the rat. Lamotrigine (3–100 mg kg−1, p.o.) was effective against mechanical hyperlagesia in both species although it showed greater efficacy and potency in the guinea‐pig (D50 4.7 mg kg−1) compared to the rat (D50 27 mg kg−1). Lamotrigine produced slight inhibition of tactile allodynia in the rat only at the highest dose tested of 100 mg kg−1. Gabapentin was poorly active against mechanical hyperalgesia in both the rat and guinea‐pig following a single oral administration (100 mg kg−1), although upon repeated administration it produced up to 70 and 90% reversal in rat and guinea‐pig, respectively. Gabapentin did however produce significant dose‐related reversal of tactile allodynia in the rat following a single administration. These data show that oxcarbazepine and other AEDs are effective anti‐hyperalgesic or anti‐allodynic agents in an animal model of neuropathic pain, and provide further support for their use in the treatment of neuropathic pain in the clinic.

Role of the cysteine protease cathepsin S in neuropathic hyperalgesia

Abstract Using a gene expression analysis approach we found that the mRNA encoding the lysosomal cysteine protease cathepsin S (CatS) was up‐regulated in rat dorsal root ganglia (DRG) following peripheral nerve injury. CatS protein was expressed in infiltrating macrophages in DRG and near the site of injury. At both sites CatS expression progressively increased from day 3 to day 14 after injury. In naïve rats, intraplantar injection of activated rat recombinant (rr) CatS (0.3, 1 μg/rat) induced a mechanical hyperalgesia that developed within half‐an‐hour, diminished by 3 h and was absent after 24 h. Activated rrCathepsin B (CatB) and non‐activated rrCatS injected intraplantarly at the same or higher doses than activated rrCatS had no effect on rat nociceptive thresholds. In nerve‐injured rats, mechanical hyperalgesia, but not allodynia, was significantly reversed for up to 3 h by systemic administration of a non‐brain penetrant, irreversible CatS inhibitor (LHVS, 3–30 mg/kg s.c.). Depletion of peripheral macrophages by intravenous injection of liposome encapsulate clodronate (1 ml, 5 mg/ml) partially reduced established mechanical hyperalgesia but not allodynia, and abolished the anti‐hyperalgesic effect of LHVS. Our results demonstrate a pro‐nociceptive effect of CatS and indicate that endogenous CatS released by peripheral macrophages contributes to the maintenance of neuropathic hyperalgesia following nerve injury.

Regulation and function of spinal and peripheral neuronal B1 bradykinin receptors in inflammatory mechanical hyperalgesia

Activation of either B1 or B2 bradykinin receptors by kinins released from damaged tissues contributes to the development and maintenance of inflammatory hyperalgesia. Whereas B2 agonists activate sensory neurones directly, B1 agonists were thought only to have indirect actions on sensory neurones. The recent discovery of constitutive B1 receptor expression in the rat nervous system lead us to re‐investigate the role of neuronal B1 receptors in inflammatory hyperalgesia. Therefore we have examined B1 bradykinin receptor regulation in rat dorsal root ganglia in a model of inflammatory hyperalgesia, and correlated it with hyperalgesic behaviour. Twenty‐four hours after injection of Freunds complete adjuvant into one hindpaw, there was a significant increase in B1 protein expression (measured by immunohistochemistry) in both ipsilateral and contralateral dorsal root ganglion neurones, whereas axotomy resulted in reduction of B1 protein in ipsilateral dorsal root ganglia. In behavioural experiments, the B1 antagonist desArg10HOE140, administered by either intrathecal or systemic routes, attenuated Freunds complete adjuvant‐induced mechanical hyperalgesia in the inflamed paw, but did not affect mechanical allodynia. The B1 agonist, desArg9BK, did not affect paw withdrawal thresholds in naïve rats following intraplantar administration into the paw, whilst intrathecal administration elicited mechanical hyperalgesia. However, after Freunds complete adjuvant‐induced inflammation, desArg9BK caused a marked mechanical hyperalgesia, by either route, of the contralateral, uninflamed hindpaw, correlating with the observed contralateral and ipsilateral increases in receptor levels. Our results suggest a functional role for B1 receptors expressed both in the periphery and in the spinal cord, in mechanical hyperalgesia during inflammation.

In vivo pharmacology of SDZ 249-665, a novel, non-pungent capsaicin analogue

&NA; Capsaicin and analogues are valuable analgesic agents when administered to mammals, including humans. However, their pungency and the effects on the cardiovascular and respiratory systems through their general activation of small calibre (nociceptive) primary afferents severely limit their use. Recently, structure activity analysis revealed that the initial pungent and general excitatory effects can be prevented by structural modifications in such a way that the analgesic activity is retained. In this paper we present SDZ 249‐665, a capsaicin analogue which produced analgesia in the mouse and anti‐hyperalgesic effects in the rat and guinea pig. SDZ 249‐665 was administered p.o., s.c. and i.v. in models of nociceptive pain, such as tail flick latency in response to a noxious thermal stimulus and acetic acid‐induced writhing in mice, and in models of inflammatory mechanical hyperalgesia induced by turpentine or carrageenan in the rat and guinea pig, respectively. SDZ 249‐665 was effective in the tail flick and the writhing assays and produced significant anti‐hyperalgesic effects in the inflammatory models. The efficacy of SDZ 245‐665 was similar to that of capsaicin, however, it was significantly more potent. SDZ 249‐665 did not produce any irritancy in a nose wipe assay in guinea pigs or an eye irritancy assay in rats, while capsaicin was clearly irritant in both cases. Furthermore, unlike capsaicin, SDZ 249‐665 did not produce unwanted side effects such as bronchoconstriction and blood pressure changes in the analgesic/anti‐hyperalgesic dose range. Thus, a clear analgesic therapeutic window exists for SDZ 249‐665. In summary, SDZ 249‐665 is a potent orally active, analgesic/anti‐hyperalgesic agent in mouse, rat and guinea pig. It lacks the excitatory effects associated with capsaicin and other close analogues, and therefore provides a clear therapeutic window for use in painful conditions. In addition to this favourable profile, no sign of tolerance was detected after a 5 day repeated dose treatment.

Selective neurokinin-1 receptor antagonists are anti-hyperalgesic in a model of neuropathic pain in the guinea-pig

Neuropathic pain is poorly managed by conventional analgesic therapy, such as non-steroidal anti-inflammatory drugs and opiates. The development of animal models of peripheral neural damage has aided in our understanding of the pathology and pharmacology of neuropathic pain. This report is the first clear demonstration using selective neurokinin-1 receptor antagonists of a potentially novel therapeutic approach to the treatment of neuropathic pain resulting from peripheral nerve damage in a guinea-pig model. The neurokinin-1 receptor antagonists, SDZ NKT 343 and LY 303,870 significantly reduced mechanical hyperalgesia following oral and intrathecal administration. (R,R)-SDZ NK T343, the enantiomer of SDZ NKT 343 did not show anti-hyperalgesic activity. RPR 100,893 showed significant anti-hyperalgesic activity only following intrathecal administration suggesting poor absorption or low level penetration of the blood-brain barrier. These results imply that neurokinin-1 receptor antagonists offer a new class of anti-hyperalgesic drugs with a largely central site of action in neuropathic pain.

p38 stress-activated protein kinase inhibitor reverses bradykinin B1 receptor-mediated component of inflammatory hyperalgesia

The effects of a p38 stress-activated protein kinase inhibitor, 4-(4-fluorophenyl)-2-(-4-methylsulfonylphenyl)-5-(4-pyridynyl) imidazole (SB203580), were evaluated in a rat model of inflammatory hyperalgesia. Oral, but not intrathecal, administration of SB203580 significantly reversed inflammatory mechanical hyperalgesia induced by injection of complete Freunds adjuvant into the hindpaw. SB203580 did not, however, affect the increased levels of interleukin-1beta and cyclo-oxygenase 2 protein observed in the hindpaw following complete Freunds adjuvant injection. Intraplantar injection of interleukin-1beta into the hindpaw elicited mechanical hyperalgesia in the ipsilateral paw, as well as in the contralateral paw, following intraplantar injection of the bradykinin B(1) receptor agonist des-Arg(9)-bradykinin. Oral administration of SB203580 1 h prior to interleukin-1beta administration prevented the development of hyperalgesia in the ipslateral paw and the contralateral bradykinin B(1) receptor-mediated hyperalgesia. In addition, following interleukin-1beta injection into the ipsilateral paw, co-administration of SB203580 with des-Arg(9)-bradykinin into the contralateral paw inhibited the bradykinin B(1) receptor-mediated hyperalgesia. In human embryonic kidney 293 cells expressing the human bradykinin B(1) receptor, its agonist des-Arg(10)-kallidin produced a rapid phosphorylation of endogenous p38 stress-activated protein kinase. Our data suggest that p38 stress-activated protein kinase is involved in the development of inflammatory hyperalgesia in the rat, and that its pro-inflammatory effects involve the induction of the bradykinin B(1) receptor as well as functioning as its downstream effector.

Oral anti-hyperalgesic and anti-inflammatory activity of NK1 receptor antagonists in models of inflammatory hyperalgesia of the guinea-pig

&NA; The oral analgesic and anti‐inflammatory activity of NK1 antagonists with species preference for the human receptor were assessed in (1) the carrageenan‐induced inflammatory hyperalgesia and (2) Freunds complete adjuvant (FCA)‐induced extravasation in the knee joint models of the guinea‐pig, respectively. Mechanical hyperalgesia was determined by measuring the withdrawal threshold to a noxious mechanical stimulus applied to the paw and thermal hyperalgesia as the withdrawal latency to a noxious thermal stimulus applied to the plantar surface. A concentration of 1.0% carrageenan (intraplantar) reduced mechanical thresholds from 124±5 to 63±3 g and thermal latencies from 19±0.4 to 4.7±0.9 s as determined 4 h after injection. The hyperalgesia persisted for over 24 h. The NK1 receptor antagonists, SDZ NKT 343, RPR100893 and SR140333, reduced mechanical hyperalgesia by 68, 36 and 27% at a dose of 30 mg kg−1 p.o., respectively. No further reduction was noted at higher doses (maximum 100 mg kg−1 p.o.). The anti‐hyperalgesic effect of SDZ NKT 343 and RPR100893 peaked at 3 h while SR140333 produced maximal reversal at 1 h after oral administration. D30 values indicated significant differences between the potency of these compounds. SDZ NKT 343 was by far the most potent anti‐hyperalgesic agent (D30: 1.1 mg kg−1). The D30 values for RPR100893 and SR140333 were estimated to be 17 and >100 mg kg−1, respectively. In thermal hyperalgesia, SDZ NKT 343 produced a significantly weaker anti‐hyperalgesic effect with a peak of 25% reversal. The D30 value for SDZ NKT 343 was 3.89 mg kg−1. For comparison, morphine inhibited the carrageenan‐induced mechanical and thermal hyperalgesia with an ED50 of 1.85 and 2.51 mg kg−1 s.c., respectively. When tested up to 300 mg kg−1 p.o., aspirin reduced carrageenan‐induced mechanical and thermal hyperalgesia by 55.0 and 45.2%, respectively. In addition to the anti‐hyperalgesic effects of NK1 receptor antagonists, the effects of SDZ NKT 343 and RPR100893 on plasma protein extravasation were measured in the FCA‐treated knee joint of the guinea‐pig. SDZ NKT 343 reversed plasma protein extravasation 2 h after administration by 60% at the oral dose of 30 mg kg−1. RPR100893 was significantly less effective with a maximum reversal of 30% at 100 mg kg−1. In comparison, indomethacin produced a 50% reversal at a 10 mg kg−1 dose. These experiments indicate that the carrageenan‐induced hyperalgesia in the guinea‐pig may be predictive of analgesic activity of NK1 receptor antagonists in man. NK1 receptor antagonists are active anti‐hyperalgesic drugs in both mechanical and thermal hyperalgesia in the guinea‐pig. In addition they inhibit plasma protein extravasation in the same species. The variability of in vivo potency and efficacy of the NK1 receptor antagonists in the mechanical hyperalgesia model is difficult to interpret as all compounds are highly effective at blocking the NK1 receptor in guinea‐pig tissues. Amongst several possibilities, differences in pharmacokinetics may explain discrepancies.