Alan Cowan

pA2 and receptor differentiation: A statistical analysis of competitive antagonism

Abstract The pA 2 of a competitive antagonist was defined by Schild as the negative logarithm of the molar concentration of an antagonist which reduces the effect of a dose of agonist to that of half the dose. The relationship of the pA 2 to the dissociation constant, K, of the antagonist-receptor complex is discussed within the framework of competitive theory. Various methods for the determination of pA 2 , and the accuracy and precision of these methods, are presented. Special problems associated with the determination of pA 2 in vivo are also discussed, with particular attention being given to narcotic analgesics and their antagonists.

STANDARDIZATION OF THE RAT PAW FORMALIN TEST FOR THE EVALUATION OF ANALGESICS

Administration of 5% formalin into the rat or guinea pig hind paw evokes two spontaneous responses: flinching/shaking and licking/biting of the injected paw. The temporal and behavioral characteristics of these objective endpoints are described. Additionally, several practical suggestions aimed at standardizing this test for the evaluation of analgesics are presented. The early/acute and late/tonic (0–10 and 20–35 min post-formalin, respectively) phases of flinching were used to quantitate antinociception in the rat. PD 117302, the kappa selective agonist, was three times more potent than morphine against tonic flinching after SC administration. Formalin may therefore be a noxious stimulus of choice in the evaluation of kappa agonists. Morphine was only twice as potent against tonic flinching as against acute flinching or the tail-dip reflex to water (50° C). In contrast, PD 117302 was 27 times less potent on early phase and was inactive in the tail-dip test. Thus, while morphine is essentially equipotent across tests, PD 117302 shows a spectrum of activity with impressive potency and efficacy being obtained against tonic pain. Kappa receptors may therefore be prominently involved in tonic pain states. Aspirin given orally was not consistently antinociceptive in either phase of the formalin test. Spinal transection completely abolished late phase responding but only partly attenuated flinching in the early phase. This suggests that the relative involvement of spinal (as opposed to supraspinal) processing of noxious inputs may, at least in part, be a function of stimulus intensity and underlie the differences in antinociceptive potency observed in this work.

The rat paw formalin test: comparison of noxious agents

&NA; A comparison was made of the spontaneous nociceptive behaviors elicited by s.c. injection into the rat hind paw of the following 8 irritants: acetic acid, carrageenan, formalin, kaolin, platelet‐activating factor, mustard oil (given topically), serotonin, and yeast. Two distinct quantifiable behaviors indicative of pain were identified: flinching/shaking of the paw and hindquarters and licking/biting of the injected paw. These behaviors were prolonged and intense after formalin and acetic acid. Formalin‐induced flinching was biphasic across time, a finding potentially useful for the study of both acute and tonic pain. Of the remaining test agents, only yeast caused significant spontaneous behavioral activity, which was of low intensity but long duration. Different time‐courses for nociceptive behavior and development of edema were demonstrated for formalin, acetic acid and yeast. It is therefore unlikely that these endpoints are causally related. Overall, the present data strongly support the use of formalin as a noxious stimulus in tonic pain research.

Methodological refinements to the mouse paw formalin test. An animal model of tonic pain.

The formalin tonic pain test has recently been adapted for use in mice. We have observed two problems with this procedure. First, compared to a vehicle control group, the majority of mice injected in a hind paw with dilute formalin exhibited a significant nociceptive response (paw licking), including peaks of activity, well beyond the end of the recommended observation period; and, second, an unacceptably wide variation in response occurred. We report two modifications that eliminate these difficulties: a) extending the observation period to 20-45 min postformalin injection, and b) lightly anesthetizing the mice with ether before injection of formalin into the paw. The modified protocol was tested by evaluating morphine, pentazocine and zomepirac (analgesic A50 values were 2.1, 23.8, and greater than 100 mg/kg, s.c., respectively). An intrathecal or intracerebroventricular injection of saline, given to the lightly anesthetized mouse, did not interfere with the nociceptive behavior; this finding widens the applicability and usefulness of the test. Collectively, these refinements significantly improve the mouse formalin model of tonic pain.

Neurokinin and NMDA antagonists (but not a kainic acid antagonist) are antinociceptive in the mouse formalin model

&NA; While much evidence implicates substance P (SP), an endogenous neurokinin (NK), as a primary sensory transmitter of acute pain in mammalian spinal cord, its role in continuous (tonic) pain is less clear. Although glutamate is co‐localized with SP in dorsal root ganglion neurons, its role in nociceptive processing is uncertain. While antagonists of NKs and excitatory amino acids (EAAs) have been found to be antinociceptive in some acute assays, they have not been tested against tonic pain. We hypothesize that:NKs and EAAs contribute to signaling of tonic chemogenic nociceptioninteraction between NK and EAA systems is important in determining the perceived intensity of a continuous noxious stimulus. We therefore evaluated two NK antagonists ([d‐Pro2,d‐Trp7,9] SP (DPDT‐SP, 0.26–6.6 nmoles, non‐specific) and [d‐Pro4,d‐Trp7,9,10,Phe11]‐SP(4–11) (DPDTP‐octa, 1.6–12.3 nmoles, somewhat NK‐1 selective)), as well as dl‐2‐amino‐5‐phosphonovalerate (dl‐AP5, NMDA antagonist, 0.05–1 nmole) and urethane (a kainic acid (KA) antagonist at 2.5 &mgr;moles) for antinociceptive activity in the mouse formalin model. Administered intrathecally (i.t.), dl‐AP5 and both NK antagonists were significantly antinociceptive while urethane (2.5 &mgr;moles) and naloxone (2.7 nmoles) were inactive. A50 values for mean % analgesia, nmoles/mouse i.t. (95% CLs) were: DPDT‐SP, 1.1 (0.79–1.6); DPDTP‐octa, 3.9 (2.4–6.1); dl‐AP5, 0.29 (0.16–0.71). The antinociception associated with 1.3 nmoles of DPDT‐SP was not reversed by co‐administering 2.7 nmoles of naloxone. Co‐administration of 0.1 nmoles of dl‐AP5 with either 1.3 nmoles of DPDT‐SP or 3.3 nmoles of DPDTP‐octa did not lead to additive antinociception. One nmole of dl‐AP5 also produced significant antinociception in the mouse tail‐flick test. These data support the contention that NK (probably NK‐1) and NMDA (but not KA) receptors are involved in spinal processing of tonic chemical/inflammatory nociception in mice.

Rat cold water tail-flick: A novel analgesic test that distinguishes opioid agonists from mixed agonist-antagonists

The models currently used to assess antinociceptive efficacy in animals are far from ideal. Those procedures that detect both opioid agonists and mixed agonist-antagonists fail to differentiate between them unless the noxious stimulus is adjusted. Furthermore, changes in the sensitivity of the test often result in positive responses being elicited from agents that are either not analgesics or only weak ones, at best. The technique described in this report uses cold water as the noxious stimulus in rats. It is simple, requires no complicated instrumentation or training, correlates well with clinical efficacy in man, and allows separation of opioid agonists from mixed agonist-antagonists without detecting non-opioid agents.

Targeting of the Orphan Receptor GPR35 by Pamoic Acid: A Potent Activator of Extracellular Signal-Regulated Kinase and β-Arrestin2 with Antinociceptive Activity

Known agonists of the orphan receptor GPR35 are kynurenic acid, zaprinast, 5-nitro-2-(3-phenylproplyamino) benzoic acid, and lysophosphatidic acids. Their relatively low affinities for GPR35 and prominent off-target effects at other pathways, however, diminish their utility for understanding GPR35 signaling and for identifying potential therapeutic uses of GPR35. In a screen of the Prestwick Library of drugs and drug-like compounds, we have found that pamoic acid is a potent GPR35 agonist. Pamoic acid is considered by the Food and Drug Administration as an inactive compound that enables long-acting formulations of numerous drugs, such as the antihelminthics oxantel pamoate and pyrantel pamoate; the psychoactive compounds hydroxyzine pamoate (Vistaril) and imipramine pamoate (Tofranil-PM); and the peptide hormones triptorelin pamoate (Trelstar) and octreotide pamoate (OncoLar). We have found that pamoic acid induces a Gi/o-linked, GPR35-mediated increase in the phosphorylation of extracellular signal-regulated kinase 1/2, recruitment of β-arrestin2 to GPR35, and internalization of GPR35. In mice, it attenuates visceral pain perception, indicating an antinociceptive effect, possibly through GPR35 receptors. We have also identified in collaboration with the Sanford-Burnham Institute Molecular Libraries Probe Production Center new classes of GPR35 antagonist compounds, including the nanomolar potency antagonist methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID2745687). Pamoic acid and potent antagonists such as CID2745687 present novel opportunities for expanding the chemical space of GPR35, elucidating GPR35 pharmacology, and stimulating GPR35-associated drug development. Our results indicate that the unexpected biological functions of pamoic acid may yield potential new uses for a common drug constituent.

Studies on bombesin-induced grooming in rats

The gross behavior induced by centrally administered bombesin in rats was compared to that elicited by ACTH-(1-24) and the somatostatin analog, des AA1,2,4,5,12,13[D-Trp8]-somatostatin (ODT8-SS). Bombesin (0.001-1 microgram, ICV) caused dose-related excessive scratching which was qualitatively different from that associated with the other two groom-inducing agents. Bombesin-induced grooming was not markedly affected by behaviorally nondepressant doses of haloperidol, morphine, naloxone or neurotensin. Bombesin was active in genetically hypotrichotic (essentially furless) rats; and, again in such animals, even after numbing the area caudal to the shoulders with lidocaine. Tolerance and cross-tolerance studies with bombesin and ODT8-SS indicated that they produce scratching through different mechanisms. Bombesin caused scratching when injected directly into the periaqueductal gray, but not when administered intravenously. Neither hypophysectomy nor adrenalectomy markedly affected bombesin-induced grooming. This behavior appears to be initiated in the central nervous system and is produced independently of the pituitary-adrenal axis.

Comparison of the anticonvulsant effects of opioid peptides and etorphine in rats after icv administration.

The effect of acute icv administration of β-endorphin (5–160 μg), D-ala2-D-leu5-enkephalin (DADL; 5–160 μg), D-ala2-met-enkephalinamide (DAME; 10–160 μg), and etorphine (0.05–1.6 μg) on brain excitability was studied by measuring flurothyl seizure thresholds in rats. Each test compound produced a behavioral stupor characterized by muscle rigidity, exophthalmos, and the absence of spontaneous movement. Wet-dog shakes occured only after injection of the opioid peptides. All four compounds produced a dose-related increase in seizure threshold. Naloxone antagonized the behavioral and anticonvulsant effects; the increase in seizure threshold induced by β-endorphin was the most resistant to naloxone. These results indicate that the opioid peptides, in addition to their known EEG epileptogenic potential, are also anticonvulsant in the rat, thus raising the possibility of a dual action for the opioid peptides on central nervous system excitability.

Naloxone antagonizes behavioural effects of d-amphetamine in mice and rats

Abstract The influence of naloxone on two characteristic behavioural effects of d -amphetamine has been studied in rodents. Naloxone (0.3, 1 and 3 mg/kg, s.c.) antagonized the increase in spontaneous locomotor activity that is associated with administration of d -amphetamine (2 mg/kg, s.c.) to mice. Naloxone, at 0.3, 1 and 3 mg/kg, subcutaneously (but not at 10 and 30 mg/kg), antagonized d -amphetamine-induced ipsilateral turning in rats lesioned unilaterally with 6-hydroxydopamine in the substantia nigra. It may be postulated that the effect of d -amphetamine in these behavioural models is brought about partially by its activity in releasing an endogenous opiate which acts via an opiate receptor influencing dopaminergic activity (naloxone sensitive) and partially by its activity on dopaminergic neurones (naloxone insensitive).