S. Stevens Negus

Preclinical Assessment of Candidate Analgesic Drugs: Recent Advances and Future Challenges

In analgesic drug development, preclinical procedures are widely used to assess drug effects on pain-related behaviors. These procedures share two principal components: 1) a manipulation intended to produce a pain-like state in the experimental subject and 2) measurement of behaviors presumably indicative of that pain state. Drugs can then be evaluated for their ability to attenuate pain-related behaviors. In the simplest procedures, the pain state is produced by delivery of an acute noxious stimulus (e.g., a warm thermal stimulus), and the primary dependent measures focus on withdrawal responses or other nocifensive behaviors that increase in rate, frequency, or intensity in response to the noxious stimulus. This approach has been refined in two ways. First, new methods have been developed to induce more clinically relevant pain states. In particular, pain requiring clinical intervention is often associated with inflammation or neuropathy, and novel procedures have emerged to model these conditions and their ability to produce hypersensitive pain states, such as allodynia and hyperalgesia. Second, studies are incorporating a broader array of pain-related behaviors as dependent measures. For example, pain not only stimulates nocifensive behaviors but also suppresses many adaptive behaviors, such as feeding or locomotion. Measures of pain-suppressed behaviors can provide new insights into the behavioral consequences of pain and the effects of candidate analgesics. In addition, functional magnetic resonance imaging has emerged as a noninvasive tool for investigating changes in neural activity associated with pain and analgesia. Integration of these complementary approaches may improve the predictive validity of analgesic drug development.

Effects of chronic d-amphetamine treatment on cocaine- and food-maintained responding under a second-order schedule in rhesus monkeys

Effective treatment of opioid dependence with methadone and of tobacco dependence with nicotine illustrates the potential usefulness of agonist medications for drug abuse treatment. The monoamine-releaser d-amphetamine is one drug under consideration as an agonist pharmacotherapy for cocaine dependence. To assess the concordance between preclinical evaluations and ongoing clinical trials, the present study examined the effects of chronic treatment with saline or d-amphetamine on cocaine- and food-maintained responding in rhesus monkeys. Cocaine injections and food pellets were available under a second-order schedule during alternating daily sessions of cocaine and food availability. d-Amphetamine (0.01-0.1 mg/kg per h i.v. for 7 consecutive days) dose-dependently decreased self-administration of a unit dose of cocaine (0.01 mg/kg per injection) at the peak of the cocaine self-administration dose-effect curve. d-Amphetamine (0.032-0.1 mg/kg per h for 7 days) also decreased self-administration of a broad range of cocaine doses (0.0032-0.1 mg/kg per injection) and produced rightward and downward shifts in the cocaine dose-effect curve. Food-maintained responding was usually decreased less than cocaine self-administration, and few signs of toxicity were noted. To evaluate the effects of a longer treatment regimen, d-amphetamine (0.1 mg/kg per h) was administered for 28 consecutive days. d-Amphetamine nearly eliminated self-administration of cocaine (0.01 mg/kg per injection) throughout this treatment, whereas food-maintained responding returned to baseline levels after approximately 9 days. These preclinical findings are concordant with recent clinical studies and suggest that chronic d-amphetamine may selectively decrease cocaine-taking behavior in rhesus monkeys, possibly by producing a selective decrease in the reinforcing effects of cocaine.

Interactions between kappa opioid agonists and cocaine. Preclinical studies.

Abstract: Kappa opioid agonists inhibit dopamine release from mesolimbic dopaminergic neurons and attenuate some behavioral effects of cocaine in rodents. Evidence that kappa opioid agonists may act as functional antagonists of cocaine led us to examine their interactions with cocaines abuse‐related effects in rhesus monkeys. In cocaine self‐administration studies, four arylacetamides (U50,488, enadoline, (−) spiradoline and PD117302) and four benzomorphans (ethylketocyclazocine [EKC], bremazocine, Mr2033 and cyclazozine) each were administered as continuous infusions over 10 days. EKC, Mr2033, bremazocine, U50,488 and enadoline produced significant dose‐dependent and sustained decreases in cocaine self‐administration and also decreased food‐maintained responding at some doses. Emesis and sedation were occasionally observed during the first two days of kappa agonist treatment, but tolerance developed rapidly to these effects. Cyclazocine, PD117302 and spiradoline did not significantly alter cocaine self‐administration. The behavioral effects of EKC and U50,488 were antagonized by both the kappa opioid antagonist nor‐binaltorphimine and the non‐selective opioid antagonist naloxone. In general, compounds with mixed activity at both kappa and mu opioid receptors (e.g. EKC, Mr2033) decreased cocaine self‐administration more consistently and with fewer or less severe undesirable side effects than more selective kappa agonists (e.g. U50,488, spiradoline). Although several kappa agonists decreased cocaine self‐administration, EKC and U50,488 did not consistently block the discriminative stimulus effects of cocaine in monkeys trained to discriminate cocaine from saline. The extent to which kappa agonist‐induced decreases in cocaine self‐administration reflect an antagonism of cocaines abuse‐related effect remains to be determined.

Effects of dopamine D1-like and D2-like agonists on cocaine self-administration in rhesus monkeys: rapid assessment of cocaine dose-effect functions

Abstract  Rationale: The reinforcing effects of cocaine have been most compellingly related to its action as an indirect dopamine agonist. Although it is generally believed that both D1-like and D2-like receptor mechanisms may be involved, recent studies suggest that D1-like and D2-like agonists have differing profiles of cocaine-related actions. Objective: To develop a procedure for rapid assessment of complete dose–effect functions for cocaine self-administration in rhesus monkeys and to compare the effects of D1-like and D2-like agonists on cocaine self-administration using this procedure. Methods: Responding was maintained by various doses of cocaine or by food under a multiple-component schedule [fixed ratio (FR) 30; time out period (TO) 10 s] in 2-h sessions. After responding stabilized, the effects of pretreatment with D1-like and D2-like agonists (administered i.m., 10 min or 30 min prior to the session) were assessed. Results: Complete inverted U-shaped dose–effect functions for cocaine self-administration were obtained in all five rhesus monkeys trained with the rapid assessment procedure. Both the position and shape of the cocaine dose– effect function remained stable in repeated assessments, and levels of responding were controlled by the unit dose of cocaine rather than by other variables (e.g., infusion duration and volume) that were used to vary the cocaine dose. Pretreatment with the D1-like agonists SKF 82958 (0.32–1.8 mg/kg) and R-6-Br-APB (0.1–1.0 mg/kg) produced downward shifts in the cocaine dose–effect function at doses that also markedly decreased food-maintained responding. In contrast, pretreatment with the D2-like agonists quinelorane (0.001–0.01 mg/kg) and 7-OH-DPAT (0.01–0.10 mg/kg) shifted the cocaine dose–effect function to the left. D2-like agonists also increased responding maintained by the cocaine-associated cue lights alone, and moderately decreased food-maintained responding. Conclusions: The results suggest that D1-like and D2-like agonists produce qualitatively different effects on cocaine self-administration that may influence their usefulness for the treatment of cocaine abuse and dependence.

Effects of Pain- and Analgesia-Related Manipulations on Intracranial Self-Stimulation in Rats: Further Studies on Pain-Depressed Behavior

ABSTRACT Pain stimulates some behaviors (e.g., withdrawal responses) but depresses many other behaviors (e.g., feeding). Pain‐stimulated behaviors are widely used in preclinical research on pain and analgesia, but human and veterinary medicine often rely on measures of functional impairment and pain‐depressed behavior to diagnose pain or assess analgesic efficacy. In view of the clinical utility of measures of pain‐depressed behaviors, our laboratory has focused on the development of methods for preclinical assays of pain‐depressed behavior in rodents. The present study compared the effects of a chemical noxious stimulus (IP lactic acid injections) and an opioid analgesic (morphine) administered alone or in combination on the stretching response (a pain‐stimulated behavior) and intracranial self‐stimulation (ICSS; a behavior that may be depressed by pain) in rats. In the ICSS procedure, rats implanted with electrodes in the lateral hypothalamus responded to electrical stimulation across a range of current frequencies to permit rapid determination of frequency‐rate curves and evaluation of curve shifts following treatment. Lactic acid alone produced a concentration‐dependent stimulation of stretching and depression of ICSS, expressed as rightward shifts in ICSS frequency‐rate curves. Morphine had little effect alone, but it produced a dose‐dependent blockade of both acid‐stimulated stretching and acid‐depressed ICSS. Both lactic acid and morphine were equipotent in the stretching and ICSS procedures. These results suggest that ICSS may be useful as a behavioral baseline for studies of pain‐depressed behavior.

Effects of the putative dopamine D3 receptor agonist 7-OH-DPAT in rhesus monkeys trained to discriminate cocaine from saline

These studies were designed to evaluate the effects of the putative dopamine D3 receptor agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT), alone and in combination with cocaine, in four rhesus monkeys trained to discriminate cocaine (0.4 mg/kg, IM) from saline under a fixed-ratio 30 schedule of food presentation. Under these conditions, cumulative doses of cocaine (0.013–1.3 mg/kg) produced a dose-dependent and complete generalization to the training dose of cocaine in all monkeys, while producing only minimal effects on response rates. The discriminative stimulus effects of cocaine were antagonized by the non-selective dopamine receptor antagonist flupenthixol (0.018 mg/kg, IM) in all four monkeys. The effects of 7-OH-DPAT (0.01–1.8 mg/kg) were inconsistent across monkeys. In two of the four monkeys (monkeys L990 and L958), 7-OH-DPAT consistently and completely generalized to cocaine and decreased response rates in a dose-dependent manner. Both the cocaine-like discriminative stimulus effects and rate-decreasing effects of 7-OH-DPAT were antagonized by flupenthixol in these two monkeys. Pretreatment with low doses of 7-OH-DPAT (0.01–0.032 mg/kg) had no effect on the cocaine dose-effect curve in monkeys L990 and L958; however, higher doses of 7-OH-DPAT (0.032–0.32 mg/kg) shifted the cocaine dose-effect curve to the left. In the other two monkeys (monkeys 150F and 89B036), 7-OH-DPAT produced a dose-dependent decrease in response rates but did not consistently generalize to cocaine. Flupenthixol did not antagonize the rate-decreasing effects of 7-OH-DPAT in these two monkeys, and pretreatment with 7-OH-DPAT (0.1–0.32 mg/kg) produced a decrease in response rates but had no effect on the cocaine dose-effect curve. Time-course experiments revealed that 7-OH-DPAT (0.32 mg/kg) displayed a slower onset and a longer duration of effect than the training dose of cocaine. Finally, the D3/D2 dopamine agonist quinpirole completely generalized to cocaine in three monkeys, and partially in the fourth monkey. Quinpirole showed the highest potency in those monkeys in which 7-OH-DPAT consistently generalized to cocaine. The results of the present study suggest that, in rhesus monkeys, 7-OH-DPAT produces cocaine-like effects and may modulate the discriminative stimulus effects of cocaine in some monkeys.

COCAINE VERSUS FOOD CHOICE PROCEDURE IN RATS: ENVIRONMENTAL MANIPULATIONS AND EFFECTS OF AMPHETAMINE

We have adapted a nonhuman primate model of cocaine versus food choice to the rat species. To evaluate the procedure, we tested cocaine versus food choice under a variety of environmental manipulations as well as pharmacological pretreatments. Complete cocaine-choice dose-effect curves (0-1.0 mg/kg/infusion) were obtained for each condition under concurrent fixed ratio schedules of reinforcement. Percentage of responding emitted on the cocaine-reinforced lever was not affected significantly by removal of cocaine-associated visual or auditory cues, but it was decreased after removal of response-contingent or response-independent cocaine infusions. Cocaine choice was sensitive to the magnitude and fixed ratio requirement of both the cocaine and food reinforcers. We also tested the effects of acute (0.32, 0.56, 1.0, 1.8 mg/kg) and chronic (0.1, 0.32 mg/kg/hr) d-amphetamine treatment on cocaine choice. Acute and chronic d-amphetamine had opposite effects, with acute increasing and chronic decreasing cocaine choice, similar to observations in humans and in nonhuman primates. The results suggest feasibility and utility of the choice procedure in rats and support its comparability to similar procedures used in humans and monkeys.

Kappa antagonist properties of buprenorphine in non-tolerant and morphine-tolerant rats

Buprenorphine was evaluated for its ability to act as a kappa opioid antagonist in rats responding under a fixed-ratio 30 schedule of food presentation both before and after the induction of morphine tolerance. Before the induction of morphine tolerance, both buprenorphine and the selective kappa agonist bremazocine decreased rates of responding in a dose-dependent manner, and buprenorphine (0.03 and 0.3 mg/kg) failed to antagonize bremazocines rate-decreasing effects. Following the induction of morphine tolerance, the bremazocine dose-effect curve was unaffected, but a profound cross-tolerance developed to buprenorphine. Furthermore, buprenorphine (0.03, 0.3 and 1.0 mg/kg) produced a dose-dependent antagonism of the rate-decreasing effects of bremazocine in the morphine-tolerant rats. These results support the hypothesis that buprenorphine has antagonist activity at kappa opioid receptors.

Interactions between mu and kappa opioid agonists in the rat drug discrimination procedure.

The present study was designed to explore the nature of the interaction between mu and kappa opioid agonists in the rat drug discrimination procedure. In rats trained to discriminate the kappa agonist U50,488 (5.6 mg/kg) from water, the other kappa agonist bremazocine substituted completely for the U50,488 training stimulus, and the additional kappa agonist tifluadom substituted in three of five of rats tested. In contrast, the mu agonists morphine, fentanyl, and buprenorphine produced primarily vehicle-appropriate responding. When morphine, fentanyl, and buprenorphine were combined with the training dose of U50,488, all three mu agonists reduced U50,488-appropriate responding. In rats trained to discriminate the mu agonist morphine (10.0 mg/kg) from saline, the other mu agonists morphine and buprenorphine all substituted in a dose-dependent manner for the morphine training stimulus, whereas U50,488, bremazocine, and tifluadom produced primarily vehicle-appropriate responding. When combined with the training dose of morphine, bremazocine antagonized morphines discriminative stimulus effects, whereas U50,488 and tifluadom had no effect. The barbiturate pentobarbital neither substituted for, nor antagonized, the discriminative stimulus effects of either U50,488 or morphine. These results suggest that mu agonists and kappa agonists produce interacting effects in the drug discrimination procedure in rats.

Targeting pain-depressed behaviors in preclinical assays of pain and analgesia: Drug effects on acetic acid-depressed locomotor activity in ICR mice

AIMS Pain depresses expression of many behaviors, and one goal of analgesic treatment is to restore pain-depressed behaviors. Assays that focus on pain-depressed behaviors may contribute to preclinical assessment of candidate analgesics. MAIN METHODS This study compared effects of the mu opioid receptor agonist morphine (an acknowledged analgesic), the dopamine receptor antagonist haloperidol (a non-analgesic sedative), the adenosine receptor antagonist caffeine (a non-analgesic stimulant) and the neurokinin-1 receptor antagonist CJ 11,974-01 (a candidate analgesic) on acetic acid-induced writhing (a traditional pain-stimulated behavior) and acetic acid-induced suppression of locomotor activity (a pain-depressed behavior) in male ICR mice. Drug effects on non-depressed (baseline) locomotor activity were also examined. KEY FINDINGS I.P. administration of acetic acid (0.18-1%) was equipotent in stimulating writhing and depressing locomotor activity. Morphine blocked both acid-induced stimulation of writhing and depression of locomotion, although it was 56-fold less potent in the assay of acid-depressed locomotion. Haloperidol and CJ 11,974-01 decreased acid-stimulated writhing, but failed to block acid-induced depression of locomotion. Caffeine had no effect on acid-stimulated writhing or acid-depressed locomotor activity, although it did increase non-depressed locomotion. Thus, morphine was the only drug to block both acid-stimulated writhing and acid-depressed locomotion. SIGNIFICANCE Complementary assays of pain-stimulated and pain-depressed behaviors may improve the predictive validity of preclinical studies that assess candidate analgesic drugs. The low potency of morphine to block acid-induced depression of locomotion suggests that locomotor activity may be a relatively insensitive measure for studies of pain-depressed behavior.