p-(4-Azipentyl)propofol: a potent photoreactive general anesthetic derivative of propofol

We synthesized 2,6-diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]phenol (p-(4-azipentyl)propofol), or p-4-AziC5-Pro, a novel photoactivable derivative of the general anesthetic propofol. p-4-AziC5-Pro has an anesthetic potency similar to that of propofol. Like propofol, the compound potentiates inhibitory GABA(A) receptor current responses and allosterically modulates binding to both agonist and benzodiazepine sites, assayed on heterologously expressed GABA(A) receptors. p-4-AziC5-Pro inhibits excitatory current responses of nACh receptors expressed in Xenopus oocytes and photoincorporates into native nACh receptor-enriched Torpedo membranes. Thus, p-4-AziC5-Pro is a functional general anesthetic that both modulates and photoincorporates into Cys-loop ligand-gated ion channels, making it an excellent candidate for use in identifying propofol binding sites.     

Activating and anesthetic effects of general depressants

The long-sleep (LS) and short-sleep (SS) lines of mice were derived by selective breeding with respect to ethanol sleep time. We found that in current generations LS mice also have longer sleep times than SS mice to trichloroethanol and paraldehyde. Two subsequent experiments tested our hypothesis that mice that are relatively insensitive to the hypnotic effects of depressant drugs might be relatively activated by low doses of these drugs. Both experiments failed to support the hypothesis. First, although SS mice were more activated than LS mice by subhypnotic doses of paraldehyde, the lines did not differ in the degree of activation produced by low doses of trichloroethanol. Second, among mice from a genetically heterogeneous population (HS), there was no relation between the degree of activation induced by a low dose of ethanol and sensitivity to the hypnotic effects of a higher dose.     

Pharmacological profile of RWJ 20085: A new,potent, long-acting local anesthetic

RWJ 20085 is a potent, long-acting local anesthetic that has been studied in vivo and in vitro relative to several clinically active agents. In mice, RWJ 20085 [ED₅₀ = 0.0078% (0.0053–0.011%)] was more potent by perineural infiltration than bupivacaine [ED₅₀ = 0.035% (0.026–0.046%)], etidocaine [ED₅₀ = 0.025% (0.016–0.035%)], or lidocaine [ED₅₀ = 0.18% (0.13–0.26%)]. The onset of action of each compound was 5 minutes, and the duration was 90 minutes for RWJ 20085 and 30 minutes for bupivacaine or etidocaine while lidocaine was active for only 15 minutes. In the rabbit corneal assay, RWJ 20085 [ED₅₀ = 0.012% (0.0049–0.023%)] was more potent than bupivacaine [ED₅₀ = 1.4% (0.47–3.05%)]. The lowest topical local anesthetic ED₅₀ of each agent was observed within 5 or 15 minutes after administration, and RWJ 20085 was active for 300 minutes, whereas bupivacaine and etidocaine were active for 90 or 45 minutes, respectively. In the guinea pig intradermal assay RWJ 20085 [ED50 = 0.017% (0.0094–0.028%)], bupivacaine [ED₅₀ = 0.016% (0.0078–0.028%)], and etidocaine [ED₅₀ = 0.02% (0.0093–0.042%)] were equipotent while lidocaine [ED₅₀ = 0.072% (0.040–0.12%)] was less potent. The onset of action of each compound was 5 minutes. The duration of action of RWJ 20085 and etidocaine was 60 minutes, whereas bupivacaine and lidocaine were active for 45 and 15 minutes, respectively. In the frog isolated-sciatic nerve preparation, similar concentrations of RWJ 20085 (2.5 × 10^?3M) and lidocaine (5.0 × 10^?3M) produced a 50% reduction of the amplitude of the pretreatment action potential; however, bupivacaine and etidocaine were each effective at a lower concentration (5.0 × 10^?4M). When their intramuscular therapeutic ratios (TR = lethal dose LD₅₀/local anesthetic ED₅₀) were calculated in mice, the therapeutic ratio of RWJ 20085 (82) was less than that of etidocaine (103) but was larger than that of either lidocaine (36) or bupivacaine (29). Calculation of the therapeutic ratios by using the intravenous LD₅₀ values suggests that lidocaine would have the smallest margin of safety if inadvertently administered by the intravenous route. The therapeutic ratios of the other agents were larger than that of lidocaine; however, there was little difference among them. RWJ 20085 has a low potential for dermal irritation as shown in rabbits and has no liability for contact sensitization as shown in guinea pigs. RWJ 20085 may have clinical utility as an injectable and/or topical local anesthetic.     

Genotoxic effects of anesthetic agents: an update

INTRODUCTION: Exposure to anesthetics in the health environment may entail a health risk for patients and operating room personnel. Knowing the effects of anesthetic agents on genetic material could be a valuable basic support for anesthesia care providers to improve treatment performance, increase patient safety and reduce the risks for patients and staff in the operating room. AREAS COVERED: Relevant literature was identified using MEDLINE, CINAHL? and Cochrane Library databases. Over 200 abstracts for articles published from 1980 to 2010 were examined. Original articles were reviewed and relevant citations from these articles were also considered. EXPERT OPINION: Despite some conflicting results, the current available data indicate that exposure to anesthetics, especially nitrous oxide and halogenated agents, is associated with general and genotoxic risks, whereas intravenous agents, such as propofol and its metabolites are not associated with genotoxic effects. Moreover, given that different anesthetic drugs are used in combination it is, thus, very difficult to understand whether the observed effects or absence of effects are due to an individual agent action or linked to a synergy action of different anesthetics involved. Further clinical and experimental evidence is warranted.     

Genotoxic effects of anesthetic agents: an update

Introduction: Exposure to anesthetics in the health environment may entail a health risk for patients and operating room personnel. Knowing the effects of anesthetic agents on genetic material could be a valuable basic support for anesthesia care providers to improve treatment performance, increase patient safety and reduce the risks for patients and staff in the operating room.

Areas covered: Relevant literature was identified using MEDLINE, CINAHL® and Cochrane Library databases. Over 200 abstracts for articles published from 1980 to 2010 were examined. Original articles were reviewed and relevant citations from these articles were also considered.

Expert opinion: Despite some conflicting results, the current available data indicate that exposure to anesthetics, especially nitrous oxide and halogenated agents, is associated with general and genotoxic risks, whereas intravenous agents, such as propofol and its metabolites are not associated with genotoxic effects. Moreover, given that different anesthetic drugs are used in combination it is, thus, very difficult to understand whether the observed effects or absence of effects are due to an individual agent action or linked to a synergy action of different anesthetics involved. Further clinical and experimental evidence is warranted.     

Chiral discrimination and enantioselective analysis of drugs: an overview

Molecular recognition of different enantiomers of a drug has become of increasing importance in the last decade due to the racemic switch strategy adapted by the pharmaceutical industry. Different analytical techniques to carry out enantioselective analysis of chiral compounds have been suggested in the literature. In the following, a brief overview of different techniques used for enantioselective analysis is given. Challenging aspects of these techniques, such as the quality of analytical information received from each technique, advantages, and disadvantages are discussed. Alternatives (enantioselective membranes, amperometric biosensors, molecularly imprinted polymers (MIPs)), capable of meeting the requirements of industrial processes, in terms of productivity, cost-effectiveness, and environmental issues are critically reviewed.     

Dose-response and biased set study of an amphetamine and a barbiturate

Three dosage levels of barbiturate and three of amphetamine were compared with a placebo, all under three set conditions (neutral, stimulant and sedative). Drug reactions were assessed by performance measures and by self-rating scales administered at a series of standard times following the drug administration in order to provide drug-time-response patterns.The self-rating scales yielded clear dosage-time-response curves for both drugs at all dosage levels. There was no systematic evidence of set or drug-by-set-effects. Research strategies in human drug studies are discussed.     

Methoxyalkyl thiazoles: a novel series of potent, orally active and enantioselective inhibitors of 5-lipoxygenase.

Methoxyalkyl thiazoles are novel 5-lipoxygenase inhibitors which are neither redox agents nor iron chelators and are exemplified by ICI211965 [1-(3-(naphth-2-ylmethoxy)phenyl)-1-(thiazol-2-yl)prop yl methyl ether]. ICI211965 potently inhibits LTC4 synthesis in murine macrophages (IC50 = 0.0085 microM) and its selectivity with respect to cyclo-oxygenase (greater than 5800) is greater than any previously reported lipoxygenase inhibitor. ICI211965 also selectively inhibits LTB4 synthesis by human blood in vitro (IC50 = 0.45 microM) and rat blood ex vivo (ED50 = 10 mg/Kg, p.o.). Methoxyalkyl thiazoles exhibit a tight structure activity relationship and resolution of a chiral member of the series demonstrates that 5-lipoxygenase inhibition resides largely in one enantiomer. Methoxyalkyl thiazoles represent the first class of agents for which 5-lipoxygenase inhibition is mediated by specific, enantioselective interaction with the enzyme.     

Synthesis and pharmacological evaluation of potent and enantioselective sigma 1, and sigma 2 ligands.

In a previous study we found that substitutions of the (+)-cis-N-normetazocine nucleus of (+)-MPCB with 1-adamantanamine provide the compound (+/-)-10 with high affinity and selectivity for sigma receptors. Starting with this result we have synthesized a new series of eight 1-phenyl-2-cyclopropylmethylamines structurally related to (+/-)-10, and binding affinities, with respect to sigma 1, sigma 2, opioid and dopaminergic D2 receptors, have been reported. All compounds showed a negligible opioid and dopaminergic affinity and high selectivity for sigma receptors. Modifications on the amino moiety and methylcarboxyester group of 10 provide compounds with different sigma 1 and sigma 2 binding affinity and selectivity. Moreover, we have also synthesized the respective enantiomers of componds (+/-)-10 and (+/-)-18 in order to evaluate the enantioselectivity for sigma 1 and sigma 2 receptors. The binding data showed that carboxymethylester on the cyclopropane ring was more critical for enantioselectivity than the hydroxymethylenic group. In fact, the (-)-10 enantiomer showed a preference for sigma 1 whereas (+)-10 showed a preference for sigma 2.     

11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors

Rationale

While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABA_A) receptors, the binding sites that mediate these actions have not been definitively identified.

Objectives

This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABA_A receptors.

Results

Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C–H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABA_A receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α₁β₂γ_2L subunits, modulated [³⁵S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [³H]CW12 and [³H]CW14 showed that these reagents covalently label GABA_A receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α₁ and β₂ subunits or β₃ subunits of the GABA_A receptor. Photolabeling of rat brain GABA_A receptors was shown to be both concentration-dependent and stereospecific.

Conclusions

CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABA_A receptors.     

Highly potent 5-aminotetrahydropyrazolopyridines: enantioselective dopamine D3 receptor binding, functional selectivity, and analysis of receptor-ligand interactions

Heterocyclic dopamine surrogates of types 5 and 7 were synthesized and investigated for their dopaminergic properties. The enantiomerically pure biphenylcarboxamide (S)-5a displayed an outstanding K(i) of 27 pM at the agonist-labeled D(3) receptor and significant selectivity over the D(2) subtype. Measurement of [(35)S]GTPγS incorporation in the presence of a coexpressed PTX-insensitive G(α0-1) subunit indicated highly efficient G-protein coupling. Comparison of ligand efficacy data from cAMP accumulation and [(3)H]thymidine incorporation experiments revealed that ligand biased signaling is exerted by the test compound (S)-5a. Starting from the D(3) crystal structure, a combination of homology modeling and site directed mutagenesis gave valuable insights into the binding mode and the intermolecular origins of stereospecific receptor recognition. According to these data, the superior affinity of the eutomer 5a is caused by the favorable binding energy that results from interaction between the ligand's central ammonium unit and the aspartate residue in position 3.32 of the receptor.     

Allyl isothiocyanate: comparative disposition in rats and mice

Allyl isothiocyanate (AITC), the major component of volatile oil of mustard, was recently reported to induce transitional-cell papillomas in the urinary bladder of male Fischer 344 rats, but not in the bladders of female rats or B6C3F1 mice. The present investigation of comparative disposition in both sexes of each species was designed to detect sex or species differences in disposition which might explain susceptibility to AITC toxicity. AITC was readily cleared from all rat and mouse tissues so that within 24 hr after administration less than 5% of the total dose was retained in tissues. The highest concentration of AITC-derived radioactivity was observed in male rat bladder. Clearance of AITC-derived radioactivity by each species was primarily in urine (70 to 80%) and in exhaled air (13 to 15%) with lesser amounts in feces (3 to 5%). Rats excreted one major and four minor metabolites in urine. The major metabolite from rat urine was identified by NMR spectroscopy to be the mercapturic acid N-acetyl-S-(N-allylthiocarbamoyl)-L-cysteine. Mice excreted in urine the same major metabolite identified in rat urine as well as three other major and two minor metabolites. Sex-related variations were observed in the relative amounts of these metabolites. Both species excreted a single metabolite in feces. Metabolism of AITC by male and female rats was similar, but female rats excreted over twice the urine volume of male rats. Results of the present study indicate that excretion of a more concentrated solution of AITC metabolite(s) in urine may account for the toxic effects of AITC on the bladder of male rats.     

Pyridoxamine: an extremely potent scavenger of 1,4-dicarbonyls

1,4-Dicarbonyl compounds, which include 2,5-hexanedione and recently discovered endogenous 4-ketoaldehydes (levuglandins, isoketals, and neuroketals), exhibit severe toxicity. The key step in the toxicity of these compounds is their reaction with the lysyl residues of proteins to form pyrrole adducts. To screen for effective scavengers of these toxic compounds, we determined the reaction rates of pyrrole formation for a series of primary amines with a model 4-ketoaldehyde, 4-oxopentanal (OPA). We found pyridoxamine (PM) to react extremely rapidly, with a second-order rate constant at physiological pH being approximately 2300 times faster than that of Nalpha-acetyllysine. The extreme reactivity of PM was unique to 1,4-dicarbonyls, as its reactions with methylglyoxal and 4-hydroxy-2(E)-nonenal were much slower and only slightly faster than with Nalpha-acetyllysine. The phenolic group of PM was found to be essential to its high reactivity, and the rate constant for pyrrole formation with OPA exhibited a maximum at pH 7.5, close to the second pKa of PM. We therefore propose a mechanism involving transfer of the phenolic proton to the carbonyl of the initially formed hemiacetal, which facilitates subsequent nucleophilic attack and ring closure. Only 1,4-dicarbonyls are likely to participate in the proposed mechanism, thereby conferring unique sensitivity of this class of compounds to scavenging by PM.     

Design of benzophenone-containing photoactivatable linear vasopressin antagonists: pharmacological and photoreactive properties

We designed and synthesized new photoactivatable linear vasopressin analogues containing benzophenone photophores. All compounds were monitored and purified using RP-HPLC and characterized by mass spectrometry. Affinity and selectivity were determined in CHO cells expressing either human V(1a), V(1b) or V(2) receptor subtypes. Within the series, compounds 6 (PhCH(2)CO-lBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I)-NH(2)) and 9 (PhCH(2)CO-dBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I)-NH(2)), containing a benzoylphenylalanine residue (Bpa), were selected and their antagonistic properties determined (K(inact) = 1.87 and 0.35 nM, respectively). The dissociation constant of the most potent candidate (compound 9) was further calculated from saturation experiments using the (125)I derivative (K(d) = 0.07 +/- 0.01 nM). Photolabeling experiments using radioactive compound 9 as a probe were specific and UV-dependent and allowed the identification of two bands at molecular masses around 85-90 kDa and 46 kDa, respectively, as previously described by Phalipou et al., using two photoreactive linear azidopeptide antagonists. The results suggest therefore that compound 9 is a potent new tool for the accurate mapping of the human V(1a) receptor antagonist binding site.     

Valproic acid: an anticonvulsant drug with potent antinociceptive and anti-inflammatory properties

Valproic acid (VA) is a major antiepileptic drug, used for several therapeutic indications. It has a wide activity spectrum, reflecting on mechanisms of action that are not fully understood. The objectives of this work were to study the effects of VA on acute models of nociception and inflammation in rodents. VA (0.5, 1, 10, 25, and 50 mg/kg, p.o.) effects were evaluated on the carrageenan-induced paw edema, carrageenan-induced peritonitis, and plantar tests in rats, as well as by the formalin test in mice. The HE staining and immunohistochemistry assay for TNF-α in carrageenan-induced edema, from paws of untreated and VA-treated rats, were also carried out. VA decreased paw edema after carrageenan, and maximum effects were seen with doses equal to or higher than 10 mg/kg. VA also preserved the tissue architecture as assessed by the HE staining. Immunohistochemical studies revealed that VA significantly reduced TNF-α immunostaining in carrageenan-inflamed rat paws. In addition, the anti-inflammatory action of VA was potentiated by pentoxifylline (a phosphodiesterase inhibitor, known to inhibit TNF-α production), but not by sodium butyrate or by suberoylanilide hydroxamic acid (SAHA), nonspecific and specific inhibitors, respectively, of histone deacetylase. However, the decrease in the number of positive TNF-α cells in the rat paw was drastically potentiated in the VA?+?SAHA associated group. VA also reduced leukocytes and myeloperoxidase (MPO) releases to the peritoneal exudate, in the carrageenan-induced peritonitis. Although in the formalin test, VA inhibited both phases, the inhibition was mainly on the second phase. Furthermore, VA significantly increased the reaction time to thermal stimuli, as assessed by the plantar test. VA is a multi-target drug, presenting potent antinociceptive and anti-inflammatory properties at a lower dose range. These effects are partly dependent upon its inhibitory action on TNF-α-related pathways. However, the participation of the HDAC inhibition with the VA anti-inflammatory action cannot be ruled out. Inflammatory processes are associated with free radical damage and oxidative stress, and their blockade by VA could also explain the present results.     

Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor.

Pravadoline (1) is an (aminoalkyl)indole analgesic agent which is an inhibitor of cyclooxygenase and, in contrast to other NSAIDs, inhibits neuronally stimulated contractions in mouse vas deferens (MVD) preparations (IC50 = 0.45 microM). A number of conformationally restrained heterocyclic analogues of pravadoline were synthesized in which the morpholinoethyl side chain was tethered to the indole nucleus. Restraining the morpholine diminished the ability of these pravadoline analogues to inhibit prostaglandin synthesis in vitro. In contrast, mouse vas deferens inhibitory activity was enhanced in [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(4-methoxyphenyl)methano ne (20). Only the R enantiomer of 20 was active (IC50 = 0.044 microM). An optimal orientation of the morpholine nitrogen for MVD inhibitory activity within the analogues studied was in the lower right quadrant, below the plane defined by the indole ring. A subseries of analogues of 20 and a radioligand of the most potent analogue, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (21) were prepared. Inhibition of radioligand binding in rat cerebellar membranes was observed to correlate with functional activity in mouse vas deferens preparations. Binding studies with this ligand (Win 55212-2) have helped demonstrate that the (aminoalkyl)indole binding site is functionally equivalent with the CP-55,940 cannabinoid binding site. These compounds represent a new class of cannabinoid receptor agonists.     

Transcutaneous immunization: an emerging route of immunization and potent immunostimulation strategy.

Transcutaneous immunization (TCI) has emerged recently as a new method of vaccination that uses the skin. The simplicity of a patch-based immunization may obscure the potency of this strategy for immunostimulation because TCI allows the safe use of a wide variety of potent adjuvants. It is thought that these adjuvants activate Langerhans cells in the skin, which migrate to the draining lymph to orchestrate robust systemic immune responses. TCI represents a novel combination using established knowledge relating to skin penetration, the potency of adjuvant-based immunostimulation, and data showing that Langerhans cells are highly desirable targets because of their antigen-presenting cell function. The near-term challenge will be to take this promising insight into successful product development.