Cumulative Dose-response Studies Research Articles

Truncated μ-Opioid Receptors With 6 Transmembrane Domains Are Essential for Opioid Analgesia.

Most clinical opioids act through μ-opioid receptors. They effectively relieve pain but are limited by side effects, such as constipation, respiratory depression, dependence, and addiction. Many efforts have been made toward developing potent analgesics that lack side effects. Three-iodobenzoyl-6β-naltrexamide (IBNtxA) is a novel class of opioid active against thermal, inflammatory, and neuropathic pain, without respiratory depression, physical dependence, and reward behavior. The μ-opioid receptor (OPRM1) gene undergoes extensive alternative precursor messenger ribonucleic acid splicing, generating multiple splice variants that are conserved from rodents to humans. One type of variant is the exon 11 (E11)-associated truncated variant containing 6 transmembrane domains (6TM variant). There are 5 6TM variants in the mouse OPRM1 gene, including mMOR-1G, mMOR-1M, mMOR-1N, mMOR-1K, and mMOR-1L. Gene-targeting mouse models selectively removing 6TM variants in E11 knockout (KO) mice eliminated IBNtxA analgesia without affecting morphine analgesia. Conversely, morphine analgesia is lost in an exon 1 (E1) KO mouse that lacks all 7 transmembrane (7TM) variants but retains 6TM variant expression, while IBNtxA analgesia remains intact. Elimination of both E1 and E11 in an E1/E11 double KO mice abolishes both morphine and IBNtxA analgesia. Reconstituting expression of the 6TM variant mMOR-1G in E1/E11 KO mice through lentiviral expression rescued IBNtxA but not morphine analgesia. The aim of this study was to investigate the effect of lentiviral expression of the other 6TM variants in E1/E11 KO mice on IBNtxA analgesia. Lentiviruses expressing 6TM variants were packaged in HEK293T cells, concentrated by ultracentrifugation, and intrathecally administered 3 times. Opioid analgesia was determined using a radiant-heat tail-flick assay. Expression of lentiviral 6TM variant messenger ribonucleic acids was examined by polymerase chain reaction (PCR) or quantitative PCR. All the 6TM variants restored IBNtxA analgesia in the E1/E11 KO mouse, while morphine remained inactive. Expression of lentiviral 6TM variants was confirmed by PCR or quantitative PCR. IBNtxA median effective dose values determined from cumulative dose-response studies in the rescued mice were indistinguishable from wild-type animals. IBNtxA analgesia was maintained for up to 33 weeks in the rescue mice and was readily antagonized by the opioid antagonist levallorphan. Our study demonstrated the pharmacological relevance of mouse 6TM variants in IBNtxA analgesia and established that a common functional core of the receptors corresponding to the transmembrane domains encoded by exons 2 and 3 is sufficient for activity. Thus, 6TM variants offer potential therapeutic targets for a distinct class of analgesics that are effective against broad-spectrum pain models without many side effects associated with traditional opioids.

Continuous morphine produces more tolerance than intermittent or acute treatment

Dosing protocol and analgesic efficacy have been proposed to be important determinants of the magnitude of opioid tolerance. The present study examined the effect of acute, intermittent and continuous treatment with the low analgesic efficacy agonist morphine on analgesic tolerance. Mice were implanted s.c. with a 25 mg morphine pellet for 1–7 days. Other mice were implanted s.c. with two 25 mg, or one 75 mg morphine pellet for 7 days. The release of morphine from subcutaneous implanted pellets was quantitated using a spectrophotometric assay. In other studies, mice were injected with morphine once (18.5–185 mg/kg/day; ≈ 10–100 times ED 50 for morphine analgesia) or once/day for 7 days. Controls were implanted with a placebo pellet or injected with saline. Analysis of drug release from a 25 mg pellet indicated that release was greatest during the first 24 h, declined and then remained relatively constant. The amount of morphine released over 7 days by a 75 mg pellet (23.9 mg) was more than that of a single 25 mg pellet (15.4 mg) but less than two 25 mg pellets (30.8 mg). Following treatment, morphine cumulative dose–response studies were conducted (tailflick). Continuous treatment with morphine using pellet implantation produced a dose-dependent shift in the morphine ED 50 by 3.3, 5.8 and 8.5 fold for one 25 mg pellet, one 75 mg pellet and two 25 mg pellets, respectively. Acute and intermittent morphine administration produced substantially less analgesic tolerance than continuous release of morphine by implant pellets. The maximum shift in the ED 50 was 1.6 for acute treatment and 2.7 for 7 day intermittent treatment; despite a larger total daily dose. The present results indicate that continuous treatment with morphine results in greater analgesic tolerance than acute or intermittent morphine treatment even at comparable daily doses. These results are consistent with the suggestion that intermittent dosing has reduced risk of producing opioid tolerance.

Hydromorphone efficacy and treatment protocol impact on tolerance and μ-opioid receptor regulation

This study examined the antinociceptive (analgesic) efficacy of hydromorphone and hydromorphone-induced tolerance and regulation of μ-opioid receptor density. Initially s.c. hydromorphone's time of peak analgesic (tail-flick) effect (45 min) and ED 50 using standard and cumulative dosing protocols (0.22 mg/kg, 0.37 mg/kg, respectively) were determined. The apparent analgesic efficacy ( τ) of hydromorphone was then estimated using the operational model of agonism and the irreversible μ-opioid receptor antagonist clocinnamox. Mice were injected with clocinnamox (0.32–25.6 mg/kg, i.p.) and 24 h later, the analgesic potency of hydromorphone was determined. The τ value for hydromorphone was 35, which suggested that hydromorphone is a lower analgesic efficacy opioid agonist. To examine hydromorphone-induced tolerance, mice were continuously infused s.c. with hydromorphone (2.1–31.5 mg/kg/day) for 7 days and then morphine cumulative dose response studies were performed. Other groups of mice were injected with hydromorphone (2.2–22 mg/kg/day) once, or intermittently every 24 h for 7 days. Twenty-four hours after the last injection, mice were tested using morphine cumulative dosing studies. There was more tolerance with infusion treatments compared to intermittent treatment. When compared to higher analgesic efficacy opioids, hydromorphone infusions induced substantially more tolerance. Finally, the effect of chronic infusion (31.5 mg/kg/day) and 7 day intermittent (22 mg/kg/day) hydromorphone treatment on spinal cord μ-opioid receptor density was determined. Hydromorphone did not produce any change in μ-opioid receptor density following either treatment. These results support suggestions that analgesic efficacy is correlated with tolerance magnitude and regulation of μ-opioid receptors when opioid agonists are continuously administered. Taken together, these studies indicate that analgesic efficacy and treatment protocol are important in determining tolerance and regulation of μ-opioid receptors.

The analgesic efficacy of fentanyl: Relationship to tolerance and μ-opioid receptor regulation

This study determined if fentanyl analgesic efficacy predicts the magnitude of tolerance and μ-opioid receptor regulation. To estimate efficacy, mice were injected i.p. with saline or clocinnamox (CCAM), an irreversible μ-opioid receptor antagonist, (0.32–25.6 mg/kg) and 24 h later fentanyl cumulative dose–response studies were conducted. CCAM dose dependently shifted the fentanyl dose–response function to the right. The apparent efficacy ( τ) of fentanyl, based on the operational model of agonism, was estimated as 58, indicating that fentanyl is a high analgesic efficacy agonist. Next, mice were infused with fentanyl (1, 2 or 4 mg/kg/day) for 7 days. Controls were implanted with placebo pellets. At the end of 7 days, morphine cumulative dose–response studies or μ-opioid receptor saturation binding studies were conducted. Fentanyl infusions dose dependently decreased morphine potency with the highest fentanyl dose reducing morphine potency by ≈ 6 fold. Chronic infusion with fentanyl (4 mg/kg/day) significantly reduced μ-opioid receptor density by 28% without altering affinity, whereas lower infusion doses had no effect. Taken together, the present results strengthen the proposal that opioid analgesic efficacy predicts μ-opioid receptor regulation and the magnitude of tolerance.

In Vivo Efficacy of 3 Opioid Analgesics in the Mouse.

It has been suggested that opioid agonist efficacy is a determinant of μ-opioid receptor (μOR) density regulation. In this study, the irreversible μOR antagonist Clocinnamox (CCAM) was used to alkylate receptors in the intact mouse to examine the in vivo efficacy of 3 opioid analgesics. CCAM has been shown to dose-dependently reduce μOR density in the mouse CNS and can be employed to estimate agonist efficacy using receptor depletion. Initially, the time of peak effect (15min) and the ED50 (0.6ug/kg, 16.6mg/kg, 1.0mg/kg; etorphine, propoxyphene, oxycodone) was estimated using the tailflick assay. To reduce animal use, efficacy studies were conducted using a cumulative dose-response protocol which yields ED50s similar to those determined using a standard dosing protocol. Mice were injected ip with saline or CCAM (0.32–12.8mg/kg) and 24hr later, cumulative dose-response studies were conducted with each agonist. CCAM dose-dependently increased the ED50 for each agonist. The mean rightward shift in the ED50 following 12.8mg/kg of CCAM was 5-fold for etorphine and propoxyphene, and 12-fold for oxycodone. Initial analysis using the operational model of agonism indicated that CCAM dose-dependently reduced μOR by 25–80% and that the relative efficacy ranking was etorphine propoxyphene > oxycodone. These results suggest that oxycodone is a relatively low intrinsic efficacy agonist. Previous studies have shown that chronic etorphine, but not oxycodone, reduces μOR density. Taken together, these data are consistent with suggestions that chronic treatment with a low intrinsic efficacy μ-opioid agonist does not downregulate μOR density.

Enhancement by Parainfluenza 3 Infection of Contractile Responses to Substance P and Capsaicin in Airway Smooth Muscle from the Guinea Pig

Guinea pigs were inoculated by nasal insufflation with parainfluenza 3 (P-3) or virus growth medium 4 days before performing in vitro pharmacologic studies on left bronchial ring segments. Cumulative dose-response studies with capsaicin revealed an enhanced contractile response after P-3 infection. The sensitivity and magnitude of contractile effects of substance P in the left bronchi were also enhanced by P-3 infection. After pretreatment of the isolated tissues with phenoxybenzamine to block histamine H1 (with metiamide to block histamine H2), muscarinic, serotonergic, and alpha adrenergic receptors, or indomethacin to block the cyclooxygenase pathway of arachidonic acid metabolism, P-3 remained effective in enhancing contractile responses, even though these pretreatments altered the sensitivity and/or magnitude of contractions produced by substance P. When ETYA or NDGA were combined with indomethacin to also block the lipoxygenase pathway of arachidonic acid metabolism, the sensitizing effect of P-3 infection was diminished or abolished, especially at larger concentrations of substance P. With combination of FPL55712 and indomethacin, the sensitizing effect of P-3 was not abolished. Contractile responses to LTC4 and LTD4 were not enhanced by P-3 infection. The data suggest a selective influence of P-3 infection on the substance P system and provide evidence for a role of the lipoxygenase pathway of arachidonic acid metabolism in the sensitizing action. Peptide leukotrienes do not appear to be the lipoxygenase products involved in this effect of virus.

Effect of particle size of bronchodilator aerosols on lung distribution and pulmonary function in patients with chronic asthma.

The particle size of bronchodilator aerosols may be important in determining the site of deposition in the lung and their therapeutic effect. The distribution of aerosols (labelled with technetium-99m diethylene triamine pentacetic acid) of two different particle sizes has been studied by gamma camera imaging. The particles had mass median aerodynamic diameters (geometric standard deviations) of 1.4 (1.4) and 5.5 (2.3) micron, and they were administered from a jet nebuliser to eight patients with chronic severe stable asthma. There was no significant difference in peripheral lung deposition with the two aerosols in any patient. The bronchodilator effect of the two aerosols was determined from cumulative dose-response studies. To avoid large doses that might mask possible differences in effect due to aerosol size, small, precisely determined incremental amounts of salbutamol (25-250 micrograms total lung dose) were used. The two doses were given via a nebuliser on separate occasions and the bronchodilator response was measured from FEV1, forced vital capacity, and peak expiratory flow 30 minutes after each dose. Bronchodilatation was similar with the two aerosols at each dose of salbutamol. There was therefore no difference in distribution within the lung or any difference in bronchodilator effect between an aerosol of small (1.4 micron) particle size and an aerosol of 5.5 microns in patients with severe but stable asthma.

Dose-response characteristics of nebulized fenoterol in asthmatic children

Fenoterol hydrobromide, a beta 2-selective bronchodilator, was administered by aqueous nebulization to 31 children with stable asthma. An initial comparison of 0, 100, 300, and 1000 micrograms drug in 20 of these patients showed a significant change in forced expiratory volume in 1 second for all three doses compared with change after placebo (P less than 0.0001). However, the differences in peak pulmonary response from 100 to 1000 micrograms were not large (P greater than 0.2). Assessment of spirometric responses of 11 children to 3, 10, 30, or 100 micrograms nebulized fenoterol clearly revealed the dose-response effect (P less than 0.01). When all the FEV1 data were plotted over the entire range of 3 to 1000 micrograms, the resultant log dose vs response curve could be characterized by the ED50, the amount of drug producing half-maximal response. At 15, 30, or 60 minutes, the ED50 was in the range 8 to 10 micrograms. With increasing time there was a parallel shift of the entire dose response curve to the right, manifested by ED50 of 47 and 150 micrograms at two and three hours, respectively, after administration. This decreasing potency of a sympathomimetic drug with time shows that duration of effect and dosage are interdependent variables and must be evaluated simultaneously. Such considerations cannot be derived from cumulative dose-response studies. In our patients, 100 to 300 micrograms fenoterol delivered by aqueous nebulization achieved optimal bronchodilation with no detectable cardiovascular side effects.

A Difference in Sensitivity to Alpha-Adrenergic Agonists Exhibited by Detrusor and Bladder Neck of Rabbit

In cumulative dose-response studies, strips from bladder neck of rabbit were significantly more sensitive to stimulation with noradrenaline, phenylephrine, and methoxamine than were strips from detrusor. There was no difference between the two regions in sensitivity to isoprenaline or carbachol. From the known characteristics of these agents, it seemed unlikely that metabolic destruction or uptake could account for the different sensitivities seen. Also, neither normetanephrine nor desmethylimipramine could alter significantly the potency of noradrenaline in either area of the bladder. It seems likely that the difference in sensitivity to alpha-adrenoceptor stimulation in the bladder neck and detrusor is due to factors at the receptor level.

STUDIES ON PHARMACOLOGICAL AND BIOCHEMICAL PROPERTIES OF DEAMINO-DICARBA-[GLY7]-OXYTOCIN (Y-5350)

AbstractPharmacological and biochemical properties were investigated on de-amino-dicarba-[Gly7]-oxytocin (Y-5350). This is a newly developed compound, in which the disulfide bond and [Pro7] of deamino-oxytocin are substituted by an ethylene linkage and glycine respectively. Bioassayed Y-5350 exhibited 202.6 u/mg of oxytocic activity, 4.6 u/mg of avian depressor activity, 441.2 u/mg of rat milk-ejecting activity and 0.02 u/mg of rat antidiuretic activity, however, a depressor activity was also evident in rats. In particular, the duration of antidiuretic activity was short. Moreover, the oxytocic activity in pregnant rats and rabbits was weak in comparison with oxytocin. Cumulative dose-response studies were carried out on the isolated rat uterus using van Dyke-Hasting solution. The intrinsic activity was the same level as that of oxytocin, and the pD2 value was 8.62. Oxytocic activity was much enhanced by the existence of 0.5 or 2 mM magnesium ion in vitro.However, the agreement between in vivoand in vitrooxytocic activity was not complete when assay was carried out in the presence of 2 mM magnesium ion. Oxytocin was inactivated by the serum of pregnant women. In the experiment using rats, oxytocin was also destroyed by the extracts of uterus, kidney and liver. In contrast, Y-5350 was not destroyed by any of the enzyme solutions mentioned above.

Dose-response of cholinergic agonists on cat irides.

In vitro cumulative dose-response studies were performed on cat iris strips with a variety of cholinergic agonists. Nearly a one thousandfold difference in potency was found between carbachol, the most potent, and acetylcholine, the least potent of these agents. On the other hand, differences in maximum efficacy did not exceed 50%. The results emphasize the need to distinguish between efficacy and potency of pharmacologic agents. Pilocarpine differed substantially from all of the other agonists studied in having a latent onset of action, decreased maximum efficacy, and in proving difficult to wash from the tissues. These in vitro studies demonstrate ceiling effects similar to those seen in clinical studies of antiglaucoma therapeutic agents.