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Biomedical subjects

Robert B Raffa

Publications and source records attributed to Robert B Raffa.

At least 19 recordsLinked to original sources
PubMed2006

Nonlinear isobologram and superadditive withdrawal from cocaine: cannabinoid combinations in planarians.

Elucidation of interactions between drugs used in polydrug abuse is especially important. However, the necessary experimental conditions for precise quantitative analysis are difficult to establish. Because withdrawal effects of cocaine and the cannabinoid receptor agonist WIN 55212-2 are easily quantified in planarians, demonstration of synergistic effects (P<0.01) of certain ratios of this combination was possible. This synergy, here analyzed with the latest (nonlinear) isobolographic methodology, is now quantitatively established for the first time.

Animals↗
PubMed2006

Modified 'Joyce model' of opioid dependence/withdrawal.

By comprehensive and detailed measurement of the time course of withdrawal signs in rats, Joyce et al. (J. Theo. Biol. 240:531-537, 2006) recently provided a creative quantitative model of the onset of drug dependence based on the requirement of protein synthesis. Because the initial model fit the data imperfectly over the full time course, those authors postulated that additional features would be needed. We report excellent fit of the data (R(2)=0.96) by adding: (1) a transient early phase, and (2) a delay in the buildup of protein.

Analgesics, Opioid↗
PubMed2006

Subadditive withdrawal from cocaine/kappa-opioid agonist combinations in Planaria.

We have previously developed and extensively characterized a convenient and sensitive metric for the quantification of withdrawal responses using Planaria. Planaria are particularly valuable for these studies because of their permeable exteriors and their relevant neurotransmitter systems (e.g., dopaminergic, opioid, and serotonergic). In the present study, we used this metric and mathematically rigorous joint-action analysis to investigate poly-drug withdrawal from fixed-ratio cocaine/kappa-opioid agonist combinations. The D50 (concentration producing half-maximal effect) for cocaine and U-50,488H was 10.3 and 1.02 microg, respectively. The D50 for 19:1 or 1:19 combinations did not differ significantly (p>0.05) from expected additive values (11.6+/-3.0 vs. 9.9+/-1.4 and 1.1+/-0.2 vs. 1.5+/-0.1, respectively), but the 3:1, 1:1, and 1:3 ratios did (34.5+/-6.9 vs. 7.7+/-1.1; 55.1+/-10.0 vs. 5.7+/-0.7; and 40.8+/-8.9 vs. 3.3+/-0.4, respectively), indicating subadditive interaction at these ratios. The finding of subadditivity in this model suggests that abstinence-induced withdrawal from the combination is less intense than that predicted from the individual drug potencies. The concept that certain combinations of drugs leads to attenuated withdrawal might generalize to humans.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2006

A nitric oxide synthase inhibitor (L-NAME) attenuates abstinence-induced withdrawal from both cocaine and a cannabinoid agonist (WIN 55212-2) in Planaria.

We previously reported that planarians (Dugesia dorotocephala) that have been exposed to cocaine for 1 h undergo abstinence-induced withdrawal when placed into cocaine-free, but not cocaine-containing, water. We now report that planarians also display dose-related abstinence-induced withdrawal following exposure to the synthetic cannabinoid agonist WIN 55212-2, but not its inactive enantiomer (WIN 55212-3). The withdrawal from WIN 55212-2 was manifested as a significant (P < 0.05) decrease in the rate of planarian spontaneous locomotor activity over a 5-min observation period, using a recently designed metric (pLMV). We also report that withdrawal from cocaine (80 microM) or WIN 55212-2 (10 microM) was attenuated by the selective inhibitor of nitric oxide synthesis L-NAME (L-nitro-arginine methyl ester), which had no effect of its own on pLMV. These results suggest a common NO-dependent pathway of withdrawal from cocaine and WIN 55212-2 in Planaria.

Analysis of Variance↗
PubMed2006

Schild (apparent pA2) analysis of a kappa-opioid antagonist in Planaria.

Previous investigators have provided radioimmunological and immunocytochemical evidence for an enkephalinergic (opioid) system in Planaria and described naloxone-sensitive qualitative behavioral responses to kappa-opioid receptor agonists. We report the application of Schild-analysis to the antagonism of a selective kappa agonist (U-50,488H) by a selective kappa antagonist (nor-BNI) in a quantitative in vivo endpoint. The results provide further evidence of a kappa-opioid-like receptor in planarians.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2005

Measurement of glutamate and aspartate in Planaria.

INTRODUCTION: The major excitatory neurotransmitters in the mammalian central nervous system are glutamate and aspartate. We developed a rapid and efficient method for the extraction and measurement of these amino acids in Planaria--a valuable model for mammalian processes because of their simple, centralized nervous system and similar neurotransmitter systems. METHOD: The method utilized buffer extraction (perchloric acid containing 0.025% of L-cystine and Na2EDTA), simple derivatization, high-pressure liquid chromatography (HPLC), and fluorescence detection. RESULTS: The mean+/-S.E.M. amounts of glutamate and aspartate were 322.6+/-43.6 and 188.6+/-27.6 pmol/mg-planarian, respectively. DISCUSSION: The method provides the ability to investigate changes in glutamate and aspartate in response to drug administration or withdrawal.

Animals↗
PubMed2005

Liquid chromatography-mass spectrometric analysis of buprenorphine and its N-dealkylated metabolite norbuprenorphine in rat brain tissue and plasma.

INTRODUCTION: A specific, accurate, and reproducible liquid chromatography-mass spectrometric (LC/MS) method was developed and validated that allows simultaneous measurement of the centrally acting analgesic buprenorphine and its major metabolite, norbuprenorphine, in rat brain and plasma samples. METHODS: A 96-well plate solid phase extraction (SPE) procedure was developed for buprenorphine and norbuprenorphine using mixed-mode cation-exchange reversed-phase sorbent. An LC method using a C8 column with isocratic mobile phase (80:20 water/acetonitrile with 20 mM ammonium acetate and 0.1% acetic acid) was developed for reproducible and selective separation. A quadrupole mass spectrometer with atmospheric electrospray ionization source under positive ion mode was used for detection. d4-Buprenorphine and d3-norbuprenorphine were used as internal standards. RESULTS: The calibration curves for buprenorphine and norbuprenorphine in plasma and brain tissue were linear within the range of 7 to 8333 ng/ml (plasma) and 5 to 5000 ng/g (brain). The lower limit of quantification for both buprenorphine and norbuprenorphine from brain tissue was 5 ng/g, and from plasma was 7 ng/ml. Assay accuracy and precision of back-calculated standards were within +/-15%. DISCUSSION: This method will be useful for investigation of buprenorphine's mechanism of action and clinical profile.

Analgesics, Opioid↗
PubMed2005

Description and quantification of cocaine withdrawal signs in Planaria.

Previous work provided indirect evidence that planarians undergo abstinence-induced withdrawal from cocaine. The present study's purpose was to determine if planarians display withdrawal signs and, if so, to quantify the behaviors. Planarians were soaked in cocaine then transferred to either the same cocaine concentration or cocaine-free water. Compared to the cocaine/cocaine group, the cocaine/water group displayed a significant number of atypical behaviors, providing direct evidence of a 'withdrawal phenomenon' in planarians.

Animals↗
PubMed2005

Amphetamine-induced increase in planarian locomotor activity and block by UV light.

The dopamine D2-receptor antagonist sulpiride decreases spontaneous locomotor velocity of planarians (pLMV) in an enantiomeric-selective and dose-dependent manner and is significantly attenuated by UV light (254 and 366 nm). We now report that amphetamine (10 microM) produced the opposite effect and was also reversed by UV light. These findings strengthen the hypothesis that the effects of dopaminergic ligands and UV light on pLMV relate to interaction with neurotransmitter transduction process(es).

Amphetamine↗
PubMed2005

Changes in aorta alpha1-adrenoceptor number and affinity during one year of streptozotocin-induced diabetes in rats.

alpha(1)-Adrenoceptor function and density in isolated thoracic aorta were measured during the course of streptozotocin-induced diabetes in rats. Diabetes was induced by a single tail vein injection of streptozotocin (60 mg/kg) and was verified by four measures (blood glucose level, increase in food intake, increase in water intake, and characteristic weight changes). Diabetes produced a significant increase in isolated aorta sensitivity to alpha(1)-adrenoceptor activation, manifested as a significant (p < 0.05) decrease in the A(50) value for phenylephrine and an increase in the A(50) (control)/A(50) (diabetic) ratio (1.2, 1.7, and 2.0, respectively) with increasing length of diabetes (4, 12 and 52 weeks). There was a large and biphasic change in receptor density (B(max)), without a significant change (p > 0.05) in either agonist (phenylephrine) or antagonist (prazosin) affinities (K(A) and pA(2) values, respectively). These results suggest compensatory mechanisms in receptor number and abnormalities in 2nd messenger transduction and can help direct efforts for improving antihypertensive or other pharmacological therapy for diabetic patients.

Adrenergic alpha-1 Receptor Agonists↗
PubMed2004

Bacterial communication ("quorum sensing") via ligands and receptors: a novel pharmacologic target for the design of antibiotic drugs.

The purpose of the present Perspectives is to present a synopsis of the literature on bacterial "quorum sensing" as a background for the proposal that interference with this communication system offers potential targets for the design of novel antibiotic drugs. Quorum sensing is the recently discovered chemical communication system among bacteria (both Gram-positive and -negative). It is vital for intra- and interbacterial gene regulation and for keeping bacterial colonies ("biofilms") intact, allowing resident bacteria to assume specialized roles that contribute to enhanced survival of the group. There are several processes involved in quorum sensing that are familiar to pharmacologists; i.e., specific signaling molecules bind to and activate receptors that transduce the quorum-sensing signal into intracellular second messenger responses. We highlight herein the similarity between quorum-sensing communication to ligand-receptor interactions, suggesting that inhibitor drugs could be designed using current standard pharmacologic principles. Such drugs would have novel mechanisms of action and might therefore be more effective against antibiotic-resistant strains of bacteria.

Anti-Bacterial Agents↗
PubMed2004

Opioid receptors and acetaminophen (paracetamol).

We report that the acetaminophen (paracetamol)-induced spinal (intrathecal, i.t.)/supraspinal (intracerebroventricular, i.c.v.) site/site antinociceptive 'self-synergy' in mice is attenuated by the opioid receptor subtype selective antagonists beta-funaltrexamine hydrochloride (beta-FNA; mu), naltrindole (delta), and nor-binaltorphine hydrochloride (nor-BNI; kappa). These findings further implicate endogenous opioids (viz., endorphins, enkephalins, and dynorphins) and their pathways as contributors to the central antinociceptive action of acetaminophen.

Acetaminophen↗
PubMed2004

Cocaine and kappa-opioid withdrawal in Planaria blocked by D-, but not L-, glucose.

Planarians (Dugesia dorotocephala) that were exposed for 1 h to cocaine (80 microM) or to the kappa-selective opioid receptor agonist U-50,488H (1 microM) displayed an abstinence-induced withdrawal syndrome, indicative of the development of physical dependence, when they were tested in cocaine- (or U-50,488H-) free water, but not when they were tested in cocaine- (or U-50,488H-) containing water. The withdrawal was manifested as a significant (P<0.05) decrease in the rate of planarian spontaneous locomotor activity over a 5-min observation period, using a recently designed metric. Co-exposure of the planarians to D-glucose (1 microM) or to 2-deoxy-D-glucose (2-DG, 1 microM), but not to L-glucose (1 microM), significantly attenuated (P<0.05) the development of physical dependence, shown by an attenuated withdrawal syndrome, from cocaine and U-50,488H. These results suggest that either D-glucose and 2-deoxy-D-glucose compete with a common cocaine and kappa-opioid transport mechanism or that the development of physical dependence (or the inhibition of abstinence-induced withdrawal) in planarians requires energy supplied from glucose metabolism.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2004

Protonation effect on drug affinity.

Pharmacologic ligand-macromolecule interactions are commonly characterized by affinity (dissociation) constants such as K(d) or K(i) without regard to the protonation effect of the buffer used in the measurement. The protonation effect is demonstrated here using isothermal titration microcalorimetry measurements of the competitive inhibitor binding of cytidine 2'-monophosphate (2'-CMP) to RNase-A as a model system in buffers of different ionization Delta H(buffer). The results demonstrate the importance of protonation in measures of affinity.

Buffers↗
PubMed2004

Isothermal titration calorimetric study of RNase-A kinetics (cCMP --> 3'-CMP) involving end-product inhibition.

PURPOSE: Isothermal titration calorimetry (ITC) and progress curve analysis was used to measure the enzyme kinetic parameters (KM and kcat) of the hydrolysis of cCMP by RNase-A, a reaction that includes end-product competitive inhibition by 3'-CMP. METHODS: The heat generated from injection of 9-15 microl cCMP (20 mM) into bovine pancreatic RNase-A (600 nM) in 50 mM Na+ acetate buffer (pH 5.5; 37 degrees C) was monitored for 1500-2000 s. Thermal power (dQ/dt), equal to (1)/deltaH(app) x d(cCMP)/dt was recorded every 1 s. The end-product inhibition constant (Kp) and enthalpy of the inhibitor binding interaction was obtained from the saturation data of 60 sequential injections of 3'-CMP (1.2 mM) into 0.05 mM RNase-A. The data of the plot of -d[cCMP]/dt against [cCMP] were fitted to kinetic equations incorporating Kp to yield KM and kcat. RESULTS: DeltaH(app) for each run was obtained by integration of the progress curve. The plot of -d[cCMP]/dt against [cCMP] yielded the kinetic parameters KM = 105.3 microM, 121.6 microM, and 131.3 microM; kcat = 1.63 s(-1), 1.56 s(-1), and 1.71 s(-1). The end-product bound with 1:1 stoichiometry and Kp = 53.2 microM. CONCLUSIONS: The combination of progress curve analysis and ITC allowed rapid and facile measurement of the kinetic parameters for catalytic conversion of cCMP to 3'-CMP by RNase-A, a reaction complicated by end-product inhibition.

Animals↗
PubMed2003

kappa-Opioid withdrawal in Planaria.

Many drug-abusers engage in poly-drug abuse, but there has been relatively little quantification of withdrawal from poly-drug use. Planarians are an advantageous model for these studies due to mammalian-relevant neurotransmitter systems (e.g. dopamine, opioid, and 5-HT). We recently developed a metric that quantified an acute cocaine withdrawal phenomenon in planarians. However, despite much indirect evidence, we lacked direct evidence of a receptor- or carrier-mediated effect. We now report dose-related, naloxone- and nor-binaltorphine-sensitive acute abstinence-induced withdrawal and naloxone-precipitated withdrawal from the kappa-opioid agonist U-50,488H (trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)-benzeneacetamide). The less active enantiomer [1R,2R]U-50,488 produced significantly less withdrawal and U-50,488H withdrawal was not due to pH or osmolarity. These data provide pharmacologic evidence of a kappa-opioid receptor-mediated withdrawal phenomenon and neuroadaptation to a pharmacologic stimulus (adaptations in transduction mechanisms) in this model.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2003

Antinociceptive synergy, additivity, and subadditivity with combinations of oral glucosamine plus nonopioid analgesics in mice.

Glucosamine (2-amino-2-deoxy-d-glucose) and glucosamine-containing products have been reported to have efficacy in the treatment of various musculoskeletal disorders. Glucosamine's efficacy, including reduction of pain, is attributed to disease-modifying properties, specifically to cartilage-rebuilding associated with modulation of interleukin-1-induced activation of chondrocytes and to inhibition of proinflammatory effects of the nuclear factor-kappaB pathway. However, glucosamine has not been shown to have direct analgesic activity. We report here that commercial glucosamine (90.4% glucosamine sulfate + 9.6% excipients) administered as the sole agent (up to 500 mg/kg p.o.) was inactive in the mouse abdominal irritant test but that certain combinations of glucosamine with nonopioid analgesics at the oral doses and ratios tested resulted in a synergistic (ibuprofen and ketoprofen), additive (diclofenac, indomethacin, naproxen, and piroxicam), or subadditive (aspirin and acetaminophen) antinociceptive interaction. In the specific case of ibuprofen, the racemate (standard ibuprofen) produced dose-related antinociception with ED50 = 26.1 +/- 3.4 mg/kg. Combinations containing racemic ibuprofen and glucosamine in greater than 1:1 ratio (glucosamine/ibuprofen) were synergistic in the test (e.g., ED50 = 11.0 +/- 2.1 for the 9:1 ratio; p < 0.01, analysis of variance). Combinations containing glucosamine and ibuprofen (2:1 and 9:1) yielded plasma levels of ibuprofen that were no different from administration of ibuprofen alone. The possibility that combinations containing certain fixed ratios of glucosamine and certain nonsteroidal anti-inflammatory drugs (NSAIDs) might enhance pain relief in patients with pain or might achieve acceptable levels of pain relief with lower doses of NSAIDs (reduced adverse effects) is presently being pursued in clinical trials.

Acetaminophen↗