Frank Porreca

Statistical analysis of drug-drug and site-site interactions with isobolograms.

The use of more than one drug to achieve a desired effect has been a common practice in pharmacologic testing and in clinical practice. For example, combinations of analgesics are frequently prescribed with a view to enhancing pain relief and reducing adverse effects. It is also well established that administration of more than one drug may give effects that are greater than, or less than, the additive effect of each drug given individually. A non-mechanistic method of characterizing the effect resulting from the administration of two compounds is the isobologram. It is relatively simple to draw and interpret isobolograms. However, this graphical technique, which employs equieffective concentrations of individual drugs and combinations of these, obtains the concentrations as random variables from concentration-effect data, usually transformed to a parallel line assay. Thus, statistical confidence limits from such assays, as well as from non-parallel designs, must be expressed on the isobologram if this diagram is to establish superadditive, subadditive, or merely additive effects. We now present a detailed statistical analysis of the isobolographic method illustrated with examples of the statistical procedures, a rational basis for selecting proportions of each drug in the combination, and a relatively novel application of the isobolographic concept, i.e., interactions involving different anatomical sites.

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.

Ketazocines and morphine: Effects on gastrointestinal transit after central and peripheral administration

The mu agonist, morphine, and the prototype kappa agonists, ketocyclazocine and ethylketocyclazocine (EK), were studied for their effects on gastrointestinal transit. Following s.c. administration, both morphine (0.3-3 mg/kg) and ketocyclazocine (0.3-10 mg/kg) antagonized transit of an opaque marker through the small intestines of mice. Morphine (0.1-1 microgram) was also effective after intracerebroventricular (icv) administration in mice whereas ketocyclazocine (0.3-30 micrograms) was not. Similarly, while both morphine (0.3-5 mg/kg) and EK (0.6-10 mg/kg) slowed transit after s.c. injection to rats, only morphine (1-10 micrograms), but not EK (0.3-300 micrograms), was active following icv administration. Icv infusion of the mu benzomorphan, phenazocine (10-100 micrograms), slowed transit in a dose-related manner. These results indicate that there may be an anatomically distinct distribution of receptors for benzomorphan kappa agonists in both the mouse and rat, with these opiate receptors not being located near the lateral cerebral ventricles. The difference in efficacy between morphine and ketazocines in slowing gastrointestinal transit after icv administration to rodents suggests that (a) inactivity in this endpoint is a characteristic of benzomorphan kappa compounds and (b) the model may serve as a useful screen when establishing in vivo profiles of kappa agonists in mice and rats.

Tolerance and cross-tolerance studies with morphine and ethylketocyclazocine

It is generally believed that the in vivo effects of opioids are mediated by subclasses of opiate receptors that have been designated as p, K and u (Gilbert & Martin 1976; Martin et al 1976). In this model, agonists are specific for particular receptor subtypes, and the ultimate effects are initiated by an interaction of agonist with receptor subtype. For example, the benzomorphan, ethylketocyclazocine (EK), is thought to cause analgesia in pressure and writhing tests by interacting with K receptors whereas morphine is active in the same tests through an interaction with p receptors (Tyers 1980). The view that an effect, in a particular test for analgesia, can be initiated through two distinct receptor subtypes is inconsistent with conventional models of drug action. Traditionally, a pharmacological effect has been associated with a receptor rather than the agonist, with a single effect being mediated through only one receptor subtype. Our previous (Cowan et al 1978) and present studies (involving pressure and heat stimuli, respectively) with EK and morphine suggest that this more traditional view is also applicable to the opioids. Specifically, we believe that morphine and E K , prototype ligands at p and K receptors, respectively, cause analgesia in rats through agonist actions on a common receptor.

Morphine-receptor dissociation constant and the stimulus-effect relation for inhibition of gastrointestinal transit in the rat

The dissociation constant (KA) of morphine for its receptors was determined by the method of partial irreversible blockade of the receptor population using inhibition of gastrointestinal transit of a forced charcoal meal as the pharmacological endpoint. The anti-motility effect of morphine was antagonized when rats were pretreated with buprenorphine (0.3 mg/kg s.c.), a narcotic antagonist analgesic, 30 min before morphine and the extent of gastrointestinal transit was estimated a further 45 min later. With this schedule of drug administration, the agonist action of buprenorphine is minimal and its antagonist action predominates. The value of KA was (1.1 +/- 0.2) x 10(-5) mol/kg, a value close to that previously reported (2.9 x 10(-5) mol/kg) by us with these compounds in the rat tail flick test. The value of [A50], found here was 2.15 x 10(-6) mol/kg, approximately 1/5 of that of KA. Also, the stimulus-effect relation of the tissue, defined in Stephensons theory, was plotted and found to be nonlinear. This result, when coupled with the inequality of KA and [A50], argues against the application of classical drug-receptor theory to this system. The apparent agreement between KA values for antinociception and inhibition of gastrointestinal transit is interesting, but does not necessarily prove equivalent receptors mediating the two different effects.

Estimation in vivo of the receptor constants of morphine in naive and morphine-tolerant rats

The efficacy and dissociation constant of morphine in naive and morphine-tolerant rats were estimated by the method of partial irreversible blockade of a fraction of the receptor population with buprenorphine. The dissociation constant (KA) of morphine increased from 3.3 x 10(-5) M in naive to 1.4 x 10(-4) M in morphine-tolerant animals, indicating a decrease in the affinity of morphine for its receptor in the tolerant state. The efficacy of morphine (KA/A50 + 1) was constant in naive and tolerant animals (4.23 and 4.46, respectively). When the data were recalculated following conversion of administered dose to brain morphine concentration, the value of KA was 1.7 x 10(-7) M in naive and 7.7 x 10(-7) M in morphine-tolerant rats, while the efficacy was 2.5 and 3.4, respectively. In addition, the stimulus-effect relationship varied in the two states, with the curve in the tolerant animal being of different shape and broader range than in the naive rat. The present results suggest that (a) tolerance to opiate agonists may involve affinity changes and (b) post-receptor events leading to the measured effect may also be affected.

A comparison of the receptor constants of morphine and ethylketocyclazocine for analgesia and inhibition of gastrointestinal transit in the rat

The efficacies and dissociation constants of proposed mu and kappa receptor agonists (morphine and ethylketocyclazocine, respectively) were compared using the method of partial irreversible blockade (with buprenorphine) and Stephensons theory of drug action. While there was good agreement between the dissociation constant (KA) of morphine in analgesia (3.3 x 10(-5) M) and in inhibition of gastrointestinal transit (1.1 x 10(-5) M), the KA of ethylketocyclazocine differed by an order of magnitude in these endpoints (3.2 x 10(-6) M and 6.7 x 10(-5) M, respectively). The efficacies of morphine were found to be similar for the two effects studied (4.23 and 5.26), while those for ethylketocyclazocine differed markedly (2.06 and 10.39). The fraction of receptors remaining unblocked after buprenorphine was consistent for the test but not for the agonist, indicating a different distribution of receptors for the two endpoints. Our results strongly suggest that morphine induces analgesia, and slows transit in the small intestine, through the same type of receptor. The same conclusion cannot be drawn for ethylketocyclazocine.

Differentiation of apomorphine from bromocriptine, piribidel and TRH by chronic administration in rats

The dopaminergics, apomorphine (1 mg/kg), bromocriptine (1, 10 and 20 mg/kg), piribidel (5, 20 and 40 mg/kg) and thyrotropin releasing hormone (TRH) (1, 10 and 20 mg/kg), were each injected SC in rats at 09.00 h and 18.00 h daily for at least 20 days. Only apomorphine induced a behavioral syndrome (AIBS) in both male and female rats, characterized by tail-vibration (within 5 days) and a hyperexcitement which included defecation, mounting (in males only), jumping, cliff-jumping, climbing and, after 9–11 days, sham-boxing. Chronically-treated rats were placed, individually, in a small glass cylinder; only those rats on apomorphine displayed an increase in excitement and constant circling, almost exclusively in a counterclockwise direction. Acute pretreatment with d-butaclamol (0.64 mg/kg, SC), but not with the l-enatiomer, blocked the AIBS. No sham-boxing occurred when d-amphetamine (1 and 5 mg/kg), bromocriptine (10 and 20 mg/kg), piribidel (1, 5, 10, 20 and 40 mg/kg) and TRH (1 and 20 mg/kg) were substituted for apomorphine; in the presence of noise, substitution of piribidel (5, 10, 20 and 40 mg/kg) elicited mild displays of sham-boxing. When apomorphine (1 mg/kg) was substituted for chronically-administered bromocriptine (20 mg/kg) bidirectional circling, but no AIBS, was seen. Apomorphine, when substituted for piribidel or TRH, did not induce circling or the AIBS. Tolerance did not develop to the AIBS over 90 days; 31 days after the final injection, the AIBS could again be elicited by a single dose of apomorphine (1 mg/kg).The behavioral syndrome linked with chronic administration of apomorphine, and the associated reverse tolerance, differentiate apomorphine from the other test compounds. Nevertheless, the partial cross-sensitization between apomorphine and piribidel, and between bromocriptine and apomorphine, provides evidence of common mechanisms at the functional level. The AIBS represents an additional model that may be used in the study of dopamine receptors in vivo.

Estimation of the dissociation constant of naloxone in the naive and morphine-tolerant guinea-pig isolated ileum: Analysis by the constrained schild plot

The pA2 values for naloxone-morphine were determined using ilea taken from naive and morphine-tolerant guinea-pigs. This constant decreased from 8.56 (+/- 0.22) in the naive to 7.95 (+/- 0.12) in tolerant tissues. The values of the pA2s were based on Schild plots using conventional regression techniques; this regression resulted in values of -log KB of 8.79 (+/- 1.5) and 8.18 (+/- 1.2) in naive and morphine-tolerant ilea, respectively. While pA2 differed, the difference in -log KB was not statistically significant. Since these two values are theoretically equivalent, the above finding appears paradoxical. Constraining the slope of the Schild plot to the theoretically required -1 resolved this discrepancy by reducing the error inherent in the calculation of -log KB determined by conventional regression.

Time course of antagonism of morphine antinociception by intracerebroventricularly administered naloxone in the rat

The kinetic profile and half-life of naloxone were studied for possible use in determination of pA2 and KB in vivo. Rats were given morphine subcutaneously and after 15 min naloxone or saline, intracerebroventricularly. A further 15 min later, and at 15 min intervals up to 135 min after morphine, the animals were tested for analgesia in the tail flick test. The dose-response curves of the naloxone group were shifted to the right of those for the saline group. The amount of displacement decreased with time, indicative of the disappearance of naloxone. The graph of log (dose ratio-1) vs. time was linear with negative slope, in agreement with the time-dependent form of the equation for competitive antagonism. From this slope, the half-life of naloxone was calculated to be 13.3 min. These results demonstrate that the time-dependent method is useful in obtaining the kinetics of centrally acting opiate antagonists.