Modulation of Morphine Analgesia, Antinociceptive Tolerance, and Mu-Opioid Receptor Binding by the Cannabinoid CB2 Receptor Agonist O-1966.

Acutely, non-selective cannabinoid (CB) agonists have been shown to increase morphine antinociceptive effects, and we and others have also demonstrated that non-selective CB agonists attenuate morphine antinociceptive tolerance. Activation of cannabinoid CB2 receptors reverses allodynia and hyperalgesia in models of chronic pain, and co-administration of morphine with CB2 receptor selective agonists has been shown to be synergistic. CB2 receptor activation has also been shown to reduce morphine-induced hyperalgesia in rodents, an effect attributed to CB2 receptor modulation of inflammation. In the present set of experiments, we tested both the acute and chronic interactions between morphine and the CB2 receptor selective agonist O-1966 treatments on antinociception and antinociceptive tolerance in C57Bl6 mice. Co-administration of morphine and O-1966 was tested under three dosing regimens: simultaneous administration, morphine pre-treated with O-1966, and O-1966 pre-treated with morphine. The effects of O-1966 on mu-opioid receptor binding were determined using [3H]DAMGO and [35S]GTPγS binding assays, and these interactions were further examined by FRET analysis linked to flow cytometry. Results yielded surprising evidence of interactions between the CB2 receptor selective agonist O-1966 and morphine that were dependent upon the order of administration. When O-1966 was administered prior to or simultaneous with morphine, morphine antinociception was attenuated and antinociceptive tolerance was exacerbated. When O-1966 was administered following morphine, morphine antinociception was not affected and antinociceptive tolerance was attenuated. The [35S]GTPγS results suggest that O-1966 interrupts functional activity of morphine at the mu-opioid receptor, leading to decreased potency of morphine to produce acute thermal antinociceptive effects and potentiation of morphine antinociceptive tolerance. However, O-1966 administered after morphine blocked morphine hyperalgesia and led to an attenuation of morphine tolerance, perhaps due to well-documented anti-inflammatory effects of CB2 receptor agonism.

Opioid-sparing effects of cannabinoids on morphine analgesia: participation of CB1 and CB2 receptors.

BACKGROUND AND PURPOSE: Much of the opioid epidemic arose from abuse of prescription opioid drugs. This study sought to determine if the combination of a cannabinoid with an opioid could produce additive or synergistic effects on pain, allowing reduction in the opioid dose needed for maximal analgesia. EXPERIMENTAL APPROACH: Pain was assayed using the formalin test in mice and the carrageenan assay in rats. Morphine and two synthetic cannabinoids were tested: WIN55,212-2 (WIN), which binds to both CB1 and CB2 receptors, and possibly TRPV1 channels; and GP1a, which has activity at CB2 receptors and is reported to inhibit fatty acid amide hydrolase, thus raising levels of endogenous cannabinoids. KEY RESULTS: Morphine in combination with WIN in the formalin test gave synergistic analgesia. Studies with selective antagonists showed that WIN was acting through CB1 receptors. Morphine in combination with GP1a in the formalin test was sub-additive. In the carrageenan test, WIN had no added effect when combined with morphine, but GP1a with morphine showed enhanced analgesia. Both WIN and Gp1a used alone had analgesic activity in the formalin pain test, but not in the carrageenan pain test. CONCLUSIONS AND IMPLICATIONS: The ability of a cannabinoid to produce an additive or synergistic effect on analgesia when combined with morphine varies with the pain assay and may be mediated by CB1 or CB2 receptors. These results hold the promise of using cannabinoids to reduce the dose of opioids for analgesia in certain pain conditions.

Single and combined effects of Δ9 -tetrahydrocannabinol and cannabidiol in a mouse model of chemotherapy-induced neuropathic pain.

BACKGROUND AND PURPOSE: The non-psychoactive phytocannabinoid cannabidiol (CBD) can affect the pharmacological effects of Δ9 -tetrahydrocannabinol (THC). We tested the possible synergy between CBD and THC in decreasing mechanical sensitivity in a mouse model of paclitaxel-induced neuropathic pain. We also tested the effects of CBD on oxaliplatin- and vincristine-induced mechanical sensitivity. EXPERIMENTAL APPROACH: Paclitaxel-treated mice (8.0 mg·kg-1 i.p., days 1, 3, 5 and 7) were pretreated with CBD (0.625-20.0 mg·kg-1 i.p.), THC (0.625-20.0 mg·kg-1 i.p.) or CBD + THC (0.04 + 0.04-20.0 + 20.0 mg·kg-1 i.p.), and mechanical sensitivity was assessed on days 9, 14 and 21. Oxaliplatin-treated (6.0 mg·kg-1 i.p., day 1) or vincristine-treated mice (0.1 mg·kg-1 i.p. days 1-7) were pretreated with CBD (1.25-10.0 mg·kg-1 i.p.), THC (10.0 mg·kg-1 i.p.) or THC + CBD (0.16 mg·kg-1 THC + 0.16 mg·kg-1 CBD i.p.). KEY RESULTS: Both CBD and THC alone attenuated mechanical allodynia in mice treated with paclitaxel. Very low ineffective doses of CBD and THC were synergistic when given in combination. CBD also attenuated oxaliplatin- but not vincristine-induced mechanical sensitivity, while THC significantly attenuated vincristine- but not oxaliplatin-induced mechanical sensitivity. The low dose combination significantly attenuated oxaliplatin- but not vincristine-induced mechanical sensitivity. CONCLUSIONS AND IMPLICATIONS: CBD may be potent and effective at preventing the development of chemotherapy-induced peripheral neuropathy, and its clinical use may be enhanced by co-administration of low doses of THC. These treatment strategies would increase the therapeutic window of cannabis-based pharmacotherapies.

Drug Combinations: Tests and Analysis with Isoboles.

Described in this unit are experimental and computational methods to detect and classify drug interactions. In most cases, this relates to two drugs or compounds with overtly similar effects, e.g., two analgesics or two anti-hypertensives. From the dose-response data of the individual drugs, it is possible to generate a curve, the isobole, which defines all dose combinations that are expected to yield a specified effect. The theory underlying the isobole involves the calculation of doses of drug A that are effectively equivalent to doses of drug B with that equivalence determining whether the isobole is linear or nonlinear. In either case, the isobole allows for a comparison with actual combination effects making it possible to determine whether the interaction is synergistic, additive, or sub-additive. Actual as well as illustrative data are employed to demonstrate experimental design and data analysis.

Levamisole enhances the rewarding and locomotor-activating effects of cocaine in rats.

BACKGROUND: The Drug Enforcement Agency estimates that 80% of cocaine seized in the United States contains the veterinary pharmaceutical levamisole (LVM). One problem with LVM is that it is producing life-threatening neutropenia in an alarming number of cocaine abusers. The neuropharmacological profile of LVM is also suggestive of an agent with modest reinforcing and stimulant effects that could enhance cocaine's addictive effects. METHODS: We tested the hypothesis that LVM (ip) enhances the rewarding and locomotor stimulant effects of cocaine (ip) using rat conditioned place preference (CPP) and locomotor assays. Effects of LVM by itself were also tested. RESULTS: LVM (0-10 mg/kg) produced CPP at 1mg/kg (P<0.05) and locomotor activation at 5mg/kg (P < 0.05). For CPP combination experiments, a statistically inactive dose of LVM (0.1 mg/kg) was administered with a low dose of cocaine (2.5 mg/kg). Neither agent produced CPP compared to saline (P > 0.05); however, the combination of LVM and cocaine produced enhanced CPP compared to saline or either drug by itself (P < 0.01). For locomotor experiments, the same inactive dose of LVM (0.1mg/kg, ip) was administered with low (10 mg/kg) and high doses (30 mg/kg) of cocaine. LVM (0.1 mg/kg) enhanced locomotor activation produced by 10mg/kg of cocaine (P < 0.05) but not by 30 mg/kg (P>0.05). CONCLUSIONS: LVM can enhance rewarding and locomotor-activating effects of low doses of cocaine in rats while possessing modest activity of its own.

Distinct interactions of cannabidiol and morphine in three nociceptive behavioral models in mice.

Cannabinoid and opioid agonists can display overlapping behavioral effects and the combination of these agonists is known to produce enhanced antinociception in several rodent models of acute and chronic pain. The present study investigated the antinociceptive effects of the nonpsychoactive cannabinoid, cannabidiol (CBD) and the µ-opioid agonist morphine, both alone and in combination, using three behavioral models in mice, to test the hypothesis that combinations of morphine and CBD would produce synergistic effects. The effects of morphine, CBD, and morphine/CBD combinations were assessed in the following assays: (a) acetic acid-stimulated stretching; (b) acetic acid-decreased operant responding for palatable food; and (c) hot plate thermal nociception. Morphine alone produced antinociceptive effects in all three models of acute nociception, whereas CBD alone produced antinociception only in the acetic acid-stimulated stretching assay. The nature of the interactions between morphine and CBD combinations were assessed quantitatively based on the principle of dose equivalence. Combinations of CBD and morphine produced synergistic effects in reversing acetic acid-stimulated stretching behavior, but subadditive effects in the hot plate thermal nociceptive assay and the acetic acid-decreased operant responding for palatable food assay. These results suggest that distinct mechanisms of action underlie the interactions between CBD and morphine in the three different behavioral assays and that the choice of appropriate combination therapies for the treatment of acute pain conditions may depend on the underlying pain type and stimulus modality.

Ethanol and cocaine: environmental place conditioning, stereotypy, and synergism in planarians.

More than 90% of individuals who use cocaine also report concurrent ethanol use, but only a few studies, all conducted with vertebrates, have investigated pharmacodynamic interactions between ethanol and cocaine. Planaria, a type of flatworm often considered to have the simplest 'brain,' is an invertebrate species especially amenable to the quantification of drug-induced behavioral responses and identification of conserved responses. Here, we investigated stereotypical and environmental place conditioning (EPC) effects of ethanol administered alone and in combination with cocaine. Planarians displayed concentration-related increases in C-shaped movements following exposure to ethanol (0.01-1%) (maximal effect: 9.9±1.1 C-shapes/5 min at 0.5%) or cocaine (0.1-5 mM) (maximal effect: 42.8±4.1 C-shapes/5 min at 5 mM). For combined administration, cocaine (0.1-5 mM) was tested with submaximal ethanol concentrations (0.01, 0.1%); the observed effect for the combination was enhanced compared to its predicted effect, indicating synergism for the interaction. The synergy with ethanol was specific for cocaine, as related experiments revealed that combinations of ethanol and nicotine did not result in synergy. For EPC experiments, ethanol (0.0001-1%) concentration-dependently increased EPC, with significant environmental shifts detected at 0.01 and 1%. Cocaine (0.001-1 µM) produced an inverted U-shaped concentration-effect curve, with a significant environmental shift observed at 0.01 µM. For combined exposure, variable cocaine concentrations (0.001-1 µM) were administered with a statistically ineffective concentration of ethanol (0.0001%). For each concentration of cocaine, the environmental shift was enhanced by ethanol, with significance detected at 1 µM. Cocaethylene, a metabolite of cocaine and ethanol, also produced C-shapes and EPC. Lidocaine (0.001-10 µM), an anesthetic and analog of cocaine, did not produce EPC or C-shaped movements. Evidence from planarians that ethanol produces place-conditioning effects and motor dysfunction, and interacts synergistically with cocaine, suggests that aspects of ethanol neuropharmacology are conserved across species.

Combining opioid and adrenergic mechanisms for chronic pain.

Chronic pain is a highly prevalent medical problem in the United States. Although opioids and serotonin-norepinephrine reuptake inhibitors (SNRIs) have demonstrated efficacy for relief of chronic pain, each has risks of adverse events in patients. Because of the risk of opioid abuse and addiction, combinations reducing opioid requirements are particularly valuable. Opioid and SNRI agents relieve pain by different pathways; concurrent use of each agent separately offers many potential benefits: complementary and possibly synergistic analgesic efficacy, separate titrations of opioid and SNRI effects, and the reduction of opioid requirements. However, few clinical studies have investigated the ideal ratios for combinations of opioids and SNRIs. A number of factors affect whether specific combinations have additive, synergistic, less than additive efficacy, or increase adverse events in patients, including general pharmacokinetic considerations, the potential for pharmacodynamic drug interactions, dose, and timing. Because there is little clinical evidence guiding combination therapy with separate opioid and SNRI agents, using single-molecule agents provides safe and effective therapy and should be the first option presented to patients. The use of empiric combinations of separate opioid and SNRI combinations needs to be considered in light of clinical cautions, including the lack of published evidence to guide dose conversion from any opioid to tramadol or to tapentadol, and vice versa; the need to avoid combinations with known drug interactions; and the need to titrate the dose when adding an SNRI to an opioid, and vice versa.

Levamisole and cocaine synergism: a prevalent adulterant enhances cocaine's action in vivo.

Levamisole is estimated by the Drug Enforcement Agency (DEA) to be present in about 80% of cocaine seized in the United States and linked to debilitating, and sometimes fatal, immunologic effects in cocaine abusers. One explanation for the addition of levamisole to cocaine is that it increases the amount of product and enhances profits. An alternative possibility, and one investigated here, is that levamisole alters cocaine's action in vivo. We specifically investigated effects of levamisole on cocaine's stereotypical and place-conditioning effects in an established invertebrate (planarian) assay. Acute exposure to levamisole or cocaine produced concentration-dependent increases in stereotyped movements. For combined administration of the two agents, isobolographic analysis revealed that the observed stereotypical response was enhanced relative to the predicted effect, indicating synergism for the interaction. In conditioned place preference (CPP) experiments, cocaine produced a significant preference shift; in contrast, levamisole was ineffective at all concentrations tested. For combination experiments, a submaximal concentration of cocaine produced CPP that was enhanced by inactive concentrations of levamisole, indicating synergism. The present results provide the first experimental evidence that levamisole enhances cocaine's action in vivo. Most important is the identification of synergism for the levamisole/cocaine interaction, which now requires further study in mammals.

Methylphenidate and impulsivity: a comparison of effects of methylphenidate enantiomers on delay discounting in rats.

RATIONALE: Current formulations of methylphenidate (MPH) used in treatment of attention-deficit/hyperactivity disorder (ADHD) result in significantly different bioavailability of MPH enantiomers. Daytrana®, a dl-MPH transdermal patch system, produces higher levels of l-MPH than when dl-MPH is administered orally (e.g., Ritalin®). One potential limitation of increased l-MPH was indicated in a preclinical study showing l-MPH may attenuate effects of d-MPH. OBJECTIVES: The objective of the study was to investigate the interactive effects of MPH enantiomers by (1) assessing drug effects via a preclinical model of "impulsivity" and (2) performing a quantitative dose equivalence analysis of MPH enantiomer interactions. METHODS: Sprague-Dawley rats were trained to emit either of two responses, one producing an immediate food pellet, the other producing four pellets delivered at increasing delays (0, 8, and 32 s). The percent selection of the larger food amount was graphed as a function of delay with the area under the curve (AUC) assessed. Increases in AUC are consistent with decreases in "impulsivity" (i.e., selection of the smaller, immediate over the larger, delayed reinforcer). RESULTS: Systemic administration of dl-MPH and d-MPH dose-dependently increased AUC, while l-MPH, morphine, and pentobarbital did not alter AUC. An analysis based upon dose equivalence indicated that dl-MPH produced additive effects that were not different from that predicted from effects of the enantiomers administered alone. CONCLUSIONS: The present results indicate pharmacologically selective effects in that only drugs prescribed for the treatment of ADHD symptoms decreased a measure of "impulsivity" and that l-MPH likely does not attenuate or enhance the effects of d-MPH in the current delay-discounting task.

Spinal-supraspinal and intrinsic µ-opioid receptor agonist-norepinephrine reuptake inhibitor (MOR-NRI) synergy of tapentadol in diabetic heat hyperalgesia in mice.

Tapentadol is a µ-opioid receptor (MOR) agonist and norepinephrine reuptake inhibitor (NRI) with established efficacy in neuropathic pain in patients and intrinsic synergistic interaction of both mechanisms as demonstrated in rodents. In diabetic mice, we analyzed the central antihyperalgesic activity, the occurrence of site-site interaction, as well as the spinal contribution of opioid and noradrenergic mechanisms in a hotplate test. Tapentadol (0.1-3.16 µg/animal) showed full efficacy after intrathecal as well as after intracerebroventricular administration (ED50 0.42 µg/animal i.t., 0.18 µg/animal i.c.v.). Combined administration of equianalgesic doses revealed spinal-supraspinal synergy (ED50 0.053 µg/animal i.t. + i.c.v.). Morphine (0.001-10 µg/animal) also showed central efficacy and synergy (ED50 0.547 µg/animal i.t., 0.004 µg/animal i.c.v., 0.014 µg/animal i.t. + i.c.v.). Supraspinal potencies of tapentadol and morphine correlated with the 50-fold difference in their MOR affinities. In contrast, spinal potencies of both drugs were similar and correlated with their relative systemic potencies (ED50 0.27 mg/kg i.p. tapentadol, 1.1 mg/kg i.p. morphine). Spinal administration of the opioid antagonist naloxone or the α2-adrenoceptor antagonist yohimbine before systemic administration of equianalgesic doses of tapentadol (1 mg/kg i.p.) or morphine (3.16 mg/kg i.p.) revealed pronounced influence on opioidergic and noradrenergic pathways for both compounds. Tapentadol was more sensitive toward both antagonists than was morphine, with median effective dose values of 0.75 and 1.72 ng/animal i.t. naloxone and 1.56 and 2.04 ng/animal i.t. yohimbine, respectively. It is suggested that the antihyperalgesic action of systemically administered tapentadol is based on opioid spinal-supraspinal synergy, as well as intrinsic spinally mediated MOR-NRI synergy.

Cocaine synergism with α agonists in rat aorta: computational analysis reveals an action beyond reuptake inhibition.

BACKGROUND: Cocaine has long been known to increase blood pressure, but the degree and mechanism of vasoconstricting action remain poorly understood. Here we examine the interaction between cocaine and alpha-adrenoceptor agonists, with the action of reuptake inhibition minimized. METHODS: Cocaine was administered to isolated rings of rat thoracic aorta, alone and in combination with three different adrenoceptor agonists: phenylephrine, methoxamine, and norepinephrine. Synergy analysis begins with the predicted additive effect of the combination of two agonists, based upon dose equivalence theory. This case where one agonist (cocaine) has no effect when administered alone requires only a t-test to demonstrate that a departure from additivity has occurred. RESULTS: At doses where cocaine alone produced no vasoconstriction, it potentiated the vasoconstriction produced by all three alpha agonists, a clear indication of synergism between cocaine and these agents. Higher doses of cocaine in combination with alpha adrenoceptor agents gave an inverted-U shaped (hormetic) dose-effect curve, i.e., dose-related relaxation at higher doses. The hormetic dose-effect relation was analyzed using computational methodology based on dose equivalence to derive the unknown second component of action that causes relaxation. CONCLUSIONS: Cocaine exhibits both vasoconstricting and vasorelaxant effects. This relaxing component, possibly related to activation of myosin light chain phosphatase, was quantified as a dose-effect curve. Most important is the synergism between cocaine and alpha-adrenoceptor stimulation which cannot be explained as an action due to reuptake inhibition, and has not been previously described.

Revisiting the isobole and related quantitative methods for assessing drug synergism.

The isobole is well established and commonly used in the quantitative study of agonist drug combinations. This article reviews the isobole, its derivation from the concept of dose equivalence, and its usefulness in providing the predicted effect of an agonist drug combination, a topic not discussed in pharmacology textbooks. This review addresses that topic and also shows that an alternate method, called "Bliss independence," is inconsistent with the isobolar approach and also has a less clear conceptual basis. In its simplest application the isobole is the familiar linear plot in cartesian coordinates with intercepts representing the individual drug potencies. It is also shown that the isobole can be nonlinear, a fact recognized by its founder (Loewe) but neglected or rejected by virtually all other users. Whether its shape is linear or nonlinear the isobole is equally useful in detecting synergism and antagonism for drug combinations, and its theoretical basis leads to calculations of the expected effect of a drug combination. Numerous applications of isoboles in preclinical testing have shown that synergism or antagonism is not only a property of the two agonist drugs; the dose ratio is also important, a fact of potential importance to the design and testing of drug combinations in clinical trials.

Fixed-dose combinations for emerging treatment of pain.

INTRODUCTION: Pain is a large and growing medical need that is not currently being fully met, primarily due to the shortcomings of existing analgesics (insufficient efficacy or limiting side-effects). Better outcomes might be achieved using a combination of analgesics. The ratio of the combinations matters and should therefore be evaluated using rigorous quantitative and well-documented analysis. AREAS COVERED: Advances have been made in understanding the normal physiology of pain processing, including the pathways and neurotransmitters involved. Insight has also been gained about physiological processes that can lead to different 'types' of pain and the transition from acute to chronic pain conditions. This 'multimechanistic' nature of most pains is better matched using a 'multimechanistic' rather than 'monomechanistic' analgesic approach. Such an approach - and the experimental design and data analysis to assess optimal combinations - is described and discussed. EXPERT OPINION: There are sound pharmacologic, as well as practical, reasons for using combinations of drugs to treat pain. Compared with single agents, they offer a potential better match to the underlying pain physiology and thus greater efficacy or reduced side effects. The optimal efficacy and side-effect ratio must be determined in a scientifically rigorous manner.

'Null method' determination of drug biophase concentration.

PK/PD modeling is enhanced by improvements in the accuracy of its metrics. For PK/PD modeling of drugs and biologics that interact with enzymes or receptors, the equilibrium constant of the interaction can provide critical insight. Methodologies such as radioliogand binding and isolated tissue preparations can provide estimates of the equilibrium constants (as the dissociation constant, K value) for drugs and endogenous ligands that interact with specific enzymes and receptors. However, an impediment to further precision for PK/PD modeling is that it remains a problem to convert the concentration of drug in bulk solution (A) into an estimate of receptor occupation, since A is not necessarily the concentration (C) of drug in the biophase that yields fractional binding from the law of mass action, viz., C/(C + K). In most experimental studies A is much larger than K, so the use of administered instead of biophase concentration gives fractional occupancies very close to unity. We here provide a simple way to obtain an estimate of the factor that converts the total drug concentration into the biophase concentration in isolated tissue preparation. Our approach is an extension of the now classic 'null method' introduced and applied by Furchgott to determination of drug-receptor dissociation constants.

On the quantitation of an agonist with dual but opposing components of action: application to vascular endothelial relaxation.

In this communication we show that the same principle that underlies the use of the isobolograph for assessing agonist interactions also leads to a method for analyzing the opposing effects of a single agonist. This is the principle of dose equivalence whose application is illustrated here and applied to the endothelium-dependent relaxing component of two putative vasoconstrictor peptides. These studies, employing angiotensin II and endothelin-1, were conducted with isolated preparations of rat aorta that were measured for agonist-induced isometric tension development in both endothelial-denuded and -intact vessels. The dose-effect relation of the relaxing component of each agonist, which should not be calculated from simple effect subtraction, was derived by the method described here.

Synergistic antihypersensitive effects of pregabalin and tapentadol in a rat model of neuropathic pain.

Neuropathic pain is a clinical condition which remains poorly treated and combinations of pregabalin, an antagonist of the α2δ-subunit of Ca(2+) channels, with tapentadol, a µ-opioid receptor agonist/noradrenaline reuptake inhibitor, or with classical opioids such as oxycodone and morphine might offer increased therapeutic potential. In the rat spinal nerve ligation model, a dose dependent increase in ipsilateral paw withdrawal thresholds was obtained using an electronic von Frey filament after IV administration of pregabalin (1-10mg/kg), tapentadol (0.316-10mg/kg), morphine (1-4.64 mg/kg) and oxycodone (0.316-3.16 mg/kg), with ED(50) values (maximal efficacy) of 4.21 (67%), 1.65 (94%), 1.70 (96%) and 0.63 mg/kg (100%), respectively. Equianalgesic dose combinations of pregabalin and tapentadol (dose ratio 2.5:1), morphine (2.5:1) or oxycodone (6.5:1) resulted in ED(50) values (maximal efficacy) of 0.83 (89%), 2.33 (97%) and 1.14 mg/kg (100%), respectively. The concept of dose-equivalence suggested an additive interaction of pregabalin and either oxycodone or morphine, while a synergistic interaction was obtained with pregabalin and tapentadol (demonstrated by isobolographic analysis). There was no increase in contralateral paw withdrawal thresholds and no locomotor impairment, as measured in the open field, for the combination of pregabalin and tapentadol; while a significant increase and impairment was demonstrated for the combinations of pregabalin and either morphine or oxycodone. Because combination of pregabalin and tapentadol resulted in a synergistic antihypersensitive activity, it is suggested that, beside the use of either drug alone, this drug combination may offer a beneficial treatment option for neuropathic pain.

Resveratrol in combination with other dietary polyphenols concomitantly enhances antiproliferation and UGT1A1 induction in Caco-2 cells.

AIMS: The only FDA approved medication for colorectal cancer (CRC) prevention is celecoxib. Its adverse effects underline the need for safer drugs. Polyphenols like resveratrol are in clinical trials for this purpose. This study aimed at examining effects of resveratrol alone and in combination with curcumin or chrysin on UGT induction in Caco-2 cells. Phytochemical combinations were selected using drug combination analyses of various anti-proliferation ratios of resveratrol+curcumin and resveratrol+chrysin. MAIN METHODS: Cell proliferation and UGT1A1 induction assays were carried out with individual polyphenols and combinations. Cell viability was determined with AlamarBlue assays. UGT1A1 mRNA was quantified via real time RT-PCR. UGT activity was determined with 4-methylumbelliferone (4MU) glucuronidation. KEY FINDINGS: Cell proliferation IC(50) estimates (± SE) for resveratrol, curcumin and chrysin were 20.8 ± 1.2, 20.1 ± 1.1 and 16.3 ± 1.3µM respectively. Combination of anti-proliferative effects showed additivity for resveratrol+chrysin and resveratrol+curcumin. Resveratrol at its IC(50) mediated a four-fold induction of UGT1A1 mRNA in a concentration independent manner. Chrysin at its IC(50) induced UGT1A1 expression seven-fold while Curcumin at its IC(90) mediated a two-fold induction. The 20 µM:40µ M resveratrol+curcumin and 20 µM :32 µM resveratrol+chrysin combinations mediated the greatest increases in mRNA expression (12 and 22 folds respectively). Significant increase in 4-MU glucuronidation was observed with combinations exhibiting maximal mRNA induction. SIGNIFICANCE: Phytochemical combinations can offer greater chemoprevention than single agents. These chemicals might offer safer options than present synthetic therapeutics for CRC prevention.

Quantitative methods for assessing drug synergism.

Two or more drugs that individually produce overtly similar effects will sometimes display greatly enhanced effects when given in combination. When the combined effect is greater than that predicted by their individual potencies, the combination is said to be synergistic. A synergistic interaction allows the use of lower doses of the combination constituents, a situation that may reduce adverse reactions. Drug combinations are quite common in the treatment of cancers, infections, pain, and many other diseases and situations. The determination of synergism is a quantitative pursuit that involves a rigorous demonstration that the combination effect is greater than that which is expected from the individual drug's potencies. The basis of that demonstration is the concept of dose equivalence, which is discussed here and applied to an experimental design and data analysis known as isobolographic analysis. That method, and a related method of analysis that also uses dose equivalence, are presented in this brief review, which provides the mathematical basis for assessing synergy and an optimization strategy for determining the dose combination.

Midazolam enhances the analgesic properties of dexmedetomidine in the rat.

OBJECTIVE: To investigate the analgesic properties of different dose combinations of midazolam and dexmedetomidine administered intraperitoneally (IP) in the rat. STUDY DESIGN: Prospective experimental trial. ANIMALS: Seventy adult male Sprague Dawley rats weighing 250-300 g. METHODS: Dexmedetomidine (D) 0.03, 0.06, 0.09, 0.12, 0.15, 0.18, 0.21 mg kg(-1) and midazolam (M) 5, 10, 25, 50 mg kg(-1) were administered IP, alone then in combinations ranging from 0.03 D:5 M to 0.18 D:30 M mg kg(-1). Analgesia was evaluated using the tail-flick test at time 0 (before injection), 15, 30, 45, 60 and 75 minutes. RESULTS: Midazolam at all doses administered (5-50 mg kg(-1)) did not significantly change tail-flick latencies from baseline values whereas D showed clear dose-dependent increases in tail-flick latency for doses administered in the range of 0.03-0.18 mg kg(-1). Tail-flick latencies in rats administered D+M combinations were significantly greater than D alone (p<0.05). CONCLUSIONS: A dose-related analgesic effect was demonstrated for D in the rat, which was enhanced by co-administration of M. CLINICAL RELEVANCE: The combination of D+M administered IP to rats at doses of 0.12:20 and 0.09:15 mg kg(-1) was shown to be a good combination to provide sedation/analgesia with a duration of action greater than 60 minutes. The onset of sedation was rapid (1-3 minutes), and onset of profound analgesia was reached within 5-10 minutes.