The role of morphine-6-glucuronide (M6G) in pain control

Morphine-6beta-D-glucuronide (M6G) is an analgesically active metabolite of morphine, accounting for approximate to10% of the morphine dose when administered by systemic routes to humans. Although M6G is more hydrophilic than morphine, it crosses the blood-brain barrier, albeit relatively slowly. For this reason, it is generally thought that, after chronic dosing, M6G contributes significantly to the analgesic effects of systemically administered morphine. Owing to its polar nature, M6G is cleared from the systemic circulation primarily via renal elimination. As M6G accumulates in patients with renal impairment, there is an increased risk of M6G-induced respiratory depression in renal failure patients who are being dosed chronically with systemic morphine. Consistent with its analgesic and respiratory depressant properties, M6G binds to the p-opioid receptor in a naloxone-reversible manner. Although the affinity of M6G for the mu-opioid receptor is similar to or slightly less than that of morphine, preclinical studies in rodents show that M6G is one to two orders of magnitude more potent than morphine when administered by central routes. This major discrepancy between the markedly higher intrinsic antinociceptive potency of M6G relative to morphine, despite their similar p-opioid receptor binding affinities, is difficult to reconcile. It has been proposed that M6G mediates its pain-relieving effects through a novel 'M6G opioid receptor', while others have argued that M6G may have higher efficacy than morphine for transduction of intracellular events. When administered by parenteral routes to rodents, M6G's antinociceptive potency is no more than twofold higher than morphine. In humans, the analgesic efficacy and respiratory depressant potency of M6G relative to morphine have been assessed in a number of short-term studies involving the intrathecal or intravenous routes of administration. For example, in hip replacement patients, intrathecal M6G provided excellent postoperative analgesia but the occurrence of late respiratory depression in 10% of these patients raised serious concern about safety. In postoperative patients, intravenous M6G administered by means of patient-controlled analgesia (PCA), or bolus plus PCA, produced no analgesia in one study and limited analgesia in another. Similarly, there was a lack of significant analgesia in healthy volunteers who received intravenous M6G for the alleviation of experimental pain (carbon dioxide applied to the nasal mucosa). In contrast, satisfactory analgesia was produced by bolus doses of intravenous M6G administered to patients with cancer pain, and to healthy volunteers with experimentally-induced ischaemic, electrical or thermal (ice water) pain. Studies to date in healthy volunteers suggest that intravenous M6G may be a less potent respiratory depressant and have a lower propensity for producing nausea and vomiting than morphine. However, it is unclear whether equi-analgesic doses of M6G and morphine were compared. Clearly, more extensive short-term trials, together with studies involving chronic M6G administration, are necessary before the potential clinical utility of M6G as an analgesic drug in its own right can be determined.

Ethical issues in using a cocaine vaccine to treat and prevent cocaine abuse and dependence

A cocaine vaccine'' is a promising immunotherapeutic approach to treating cocaine dependence which induces the immune system to form antibodies that prevent cocaine from crossing the blood brain barrier to act on receptor sites in the brain. Studies in rats show that cocaine antibodies block cocaine from reaching the brain and prevent the reinstatement of cocaine self administration. A successful phase 1 trial of a human cocaine vaccine has been reported. The most promising application of a cocaine vaccine is to prevent relapse to dependence in abstinent users who voluntarily enter treatment. Any use of a vaccine to treat cocaine addicts under legal coercion raises major ethical issues. If this is done at all, it should be carefully trialled first, and only after considerable clinical experience has been obtained in using the vaccine to treat voluntary patients. There will need to be an informed community debate about what role, if any, a cocaine vaccine may have as a way of preventing cocaine addiction in children and adolescents.

Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys

Vascular endothelial growth factor (VEGF) is one of the major mediators of retinal ischemia-associated neovascularization. We have shown here that adeno-associated virus (AAV)-mediated expression of sFIt-1, a soluble form of the Flt-1 VEGF receptor, was maintained for up to 8 and 17 months postinjection in mice and in monkeys, respectively. The expression of sFIt-1 was associated with the long-term (8 months) regression of neovascular vessels in 85% of trVEGF029 eyes. In addition, it resulted in the maintenance of retinal morphology, as the majority of the treated trVEGF029 eyes (75%) retained high numbers of photoreceptors, and in retinal function as measured by electroretinography. AAV-mediated expression of sFIt-1 prevented the development of laser photocoagulation-incluced choroidal neovascularization in all treated monkey eyes. There were no clinically or histologically detectable signs of toxicity present in either animal model following AAV.sFlt injection. These results suggest that AAV-mediated secretion gene therapy could be considered for treatment of retinal and choroidal neovascularizations.

EphA4 regulates central nervous system vascular formation

Molecules involved in axon guidance have recently also been shown to play a role in blood vessel guidance. To examine whether axon guidance molecules, such as the EphA4 receptor tyrosine kinase, might also play a role in development of the central nervous system (CNS) vasculature and repair following CNS injury, we examined wild-type and EphA4 null mutant (-/-) mice. EphA4-/- mice exhibited an abnormal CNS vascular structure in both the cerebral cortex and the spinal cord, with disorganized branching and a 30% smaller diameter. During development, EphA4 was expressed on endothelial cells. This pattern of expression was not maintained in the adult. After spinal cord injury in wild-type mice, expression of EphA4 was markedly up-regulated on activated astrocytes, many of which were tightly associated with blood vessels. In EphA4-/- spinal cord following injury, astrocytes were not as tightly associated with blood vessels as the wild-type astrocytes. In uninjured EphA4-/- mice, the blood-brain barrier (BBB) appeared normal, but it showed prolonged leakage following spinal cord injury. These results support a role for EphA4 in CNS vascular formation and guidance during development and an additional role in BBB repair.

Comparison of the binding of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines with their isosteric sulfonamides to the active site of phenylethanolamine N-methyltransferase

3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines (14, 16, and 18-22) are highly potent and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT). Molecular modeling studies with 3-fluoromethyl-7-(N-alkyl aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines, such as 16, suggested that the sulfonamide -NH-could form a hydrogen bond with the side chain of Lys57. However, SAR studies and analysis of the crystal structure of human PNMT (hPNMT) in complex with 7 indicated that the sulfonamide oxygens, and not the sulfonamide -NH-, formed favorable interactions with the enzyme. Thus, we hypothesized that replacement of the sulfonamide -NH-with a methylene group could result in compounds that would retain potency at PNMT and that would have increased lipophilicity, thus increasing the likelihood they will cross the blood brain barrier. A series of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines (23-30) were synthesized and evaluated for their PNMT inhibitory potency and affinity for the R2-adrenoceptor. A comparison of these compounds with their isosteric sulfonamides (14, 16, and 18-22) showed that the sulfones were more lipophilic but less potent than their corresponding sulfonamides. Sulfone 24 (hPNMT K-i = 1.3 mu M) is the most potent compound in this series and is quite selective for PNMT versus the R2-adrenoceptor, but 24 is less potent than the corresponding sulfonamide, 16 (hPNMT K-i = 0.13 mu M). We also report the crystal structure of hPNMT in complex with sulfonamide 15, from which a potential hydrogen bond acceptor within the hPNMT active site has been identified, the main chain carbonyl oxygen of Asn39. The interaction of this residue with the sulfonamide -NH-is likely responsible for much of the enhanced inhibitory potency of the sulfonamides versus the sulfones.

Enhancement of drug affinity for cell membranes by conjugation with lipoamino acids II. Experimental and computational evidence using biomembrane models

Lipoamino acids (LAAs) are promoieties able to enhance the amphiphilicity of drugs, facilitating their interaction with cell membranes. Experimental and computational studies were carried out on two series of lipophilic amide conjugates between a model drug (tranylcypromine, TCP) and LAA or alkanoic acids containing a short, medium or long alkyl side chain (C-4 to C-16). The effects of these compounds were evaluated by monolayer surface tension analysis and differential scanning calorimetry using dimyristoylphosphatidylcholine nnonolayers and liposomes as biomembrane models. The experimental results were related to independent calculations to determine partition coefficient and blood-brain partitioning. The comparison of TCP-LAA conjugates with the related series of TCP alkanoyl amides confirmed that the ability to interact with the biomembrane models is not due to the mere increase of lipophilicity, but mainly to the amphipatic nature and the kind of LAA residue. (C) 2005 Elsevier B.V. All rights reserved.

Two new blood flukes (Digenea: Sanguinicolidae) from epinephelinae (Perciformes: Serranidae) of the Pacific Ocean

Pearsonellum pygmaeus n. sp. is described from Cromileptes altivelis (Serranidae), the Barramundi Cod, from Heron Island (southern Great Barrier Reef) and Lizard Island (northern Great Bat-Her Reef). This new species differs from Pearsonellum eorventum (type and only species) in the combination of smaller overall body size, the relative distance of the brain from the anterior end, the relative lengths of both the oesophagus and the testis, the degree to which the testis extends outside the intercaecal field, the shape of the testis, the shape and size of the ovary and the extent to which the uterzus loops around the ovary. There are in addition, 20 base pair differences between the ITS2 rDNA sequence of P. pygmaeus n. sp. and that of P corventum. Three new host records for P. corventum are reported. Adelomyllos teenae n. g., n. sp. is described from Epinephelus coioides (Serranidae), the Estuary Cod, from Moreton Bay, southeast Queensland. The new genus differs from the 22 other sanguinicolid genera in the combined possession of two testes, a cirrus-sac, separate genital pores, a post-ovarian uterus and an H-shaped intestine. A. teenae n. sp. is the third sanguinicolid described from the Epinephelinae. Sanguinicolids have now been reported from 11 species of Serranidae. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Parasitic isopods (Gnathia sp.) reduce haematocrit in captive blackeye thicklip (Labridae) on the Great Barrier Reef

Captive Hemigymnus melapterus exposed to large numbers of cultured juvenile parasitic isopods (Gnathia sp.) had significantly lower haematocrit (median 27-62% +/- 5-83% inter-quartile range) than uninfected, control fish (median 32-73% +/- 4-90%). This study is the first to show that juvenile Gnathia sp. reduce total blood volume in H. melapterus. The low haematocrit in infected fish was most likely due to plasma replacing erythrocytes lost as a result of isopods feeding on fish blood. (c) 2005 The Fisheries Society of the British Isles.

An exceptionally rich complex of Sanguinicolidae von Graff, 1907 (Platyhelminthes : Trematoda) from Siganidae, Labridae and Mullidae (Teleostei : Perciformes) from the Indo-West Pacific Region

We describe an unprecedented radiation of sanguinicolid blood flukes ( Digenea: Sanguinicolidae) from two species of Labridae (Choerodon venustus and C. cauteroma), seven species of Mullidae (Mulloidichthys vanicolensis, Parupeneus barberinoides, P. barberinus, P. bifasciatus, P. cyclostomus, P. indicus and P. multifasciatus) and ten species of Siganidae (Siganus argenteus, S. corallinus, S. doliatus, S. fuscescens, S. lineatus, S. margaritiferus, S. puellus, S. punctatus, S. virgatus and S. vulpinus) from sites off Australia and Palau. The flukes were morphologically similar in having the combination of a long thread-like body, tegumental spines in lateral transverse rows, a vestigial oral sucker bearing concentric rows of fine spines, an H-shaped intestine, a cirrussac, a notch level with the male genital pore, a lateral or post-ovarian uterus, a uterine chamber and separate genital pores. These species are divided into two genera on the basis of testis number. Sanguinicolids from Siganus fuscescens have a single large testis between the intestinal bifurcation and the ovary and are placed in Ankistromeces Nolan & Cribb, 2004. Species from the remaining nine species of Siganidae, Labridae and Mullidae are placed in Phthinomita n. g.; these species have two testes, the anterior testis being large and between the intestinal bifurcation and the ovary whereas the small posterior testis is at the posterior end of the body and appears rudimentary or degenerate and probably non-functional. The second internal transcribed spacer (ITS2) of ribosomal DNA ( rDNA) from 29 host/parasite/location combinations (h/p/l) was sequenced together with that of Ankistromeces mariae Nolan & Cribb, 2004 for comparison. From 135 samples we found 19 distinct genotypes which were interpreted as representing at least that many species. Replicate sequences were obtained for 25 of 30 h/p/l combinations ( including A. mariae); there was no intraspecific variation between replicates sequences for any of these. Interspecific variation ranged from 1 - 41 base differences (0.3 - 12.7% sequence divergence). The 19 putative species were difficult to recognise by morphological examination. We describe 13 new species; we do not describe (= name) six species characterised solely by molecular sequences and three putative species for which morphological data is available but for which molecular data is not. We have neither morphological nor molecular data for sanguinicolids harboured in five hosts species ( Siganus margaritiferus, S. puellus, Choerodon cauteroma, Parupeneus indicus and P. multifasciatus) in which we have seen infections. Where host species were infected in different localities they almost always harboured distinct species. Some host species ( for example, S. argenteus and S. lineatus from Lizard Island) harboured two or three species in a single geographical location. This suggests that, for parts of this system, parasite speciation has outstripped host speciation. Distance analysis of ITS2 showed species from each host family ( Siganidae, Mullidae and Labridae) did not form monophyletic clades to the exclusion of species from other host families. However, a host defined clade was formed by the sequences from sanguinicolids from S. fuscescens.

Cardicola Short, 1953 and Braya n. gen. (Digenea : Sanguinicolidae) from five families of tropical Indo-Pacific fishes

A survey of Pacific coral reef fishes for sanguinicolids revealed that two species of Lutjanidae (Lutjanus argentimaculatus, L. bohar), six species of Siganidae (Siganus corallinus, S. fuscescens, S. lineatus, S. margaritiferus, S. punctatus, S. vulpinus), seven species of Chaetodontidae (Chaetodon aureofasciatus, C. citrinellus, C. flavirostris, C. lineolatus, C. reticulatus, C. ulietensis, C. unimaculatus), three species of Scombridae (Euthynnus affinis, Scomberomorus commerson, S. munroi) and three species of Scaridae (Chlorurus microrhinos, Scarus frenatus, S. ghobban) were infected with morphologically similar sanguinicolids. These flukes have a flat elliptical body, a vestigial oral sucker, a single testis, separate genital pores and a post-ovarian uterus. However, these species clearly belong in two genera based on the position of the testis and genital pores. Sanguinicolids from Lutjanidae, Siganidae, Chaetodontidae and Scombridae belong in Cardicola Short, 1953; the testis originates anteriorly to, or at the anterior end of, the intercaecal field and does not extend posteriorly to it, the male genital pore opens laterally to the sinistral lateral nerve chord and the female pore opens near the level of the ootype ( may be anterior, lateral or posterior to it) antero-dextral to the male pore. Those from Scaridae are placed in a new genus, Braya; the testis originates near the posterior end of the intercaecal field and extends posteriorly to it, the male pore opens medially at the posterior end of the body and the female pore opens posterior to the ootype, antero-sinistral to the male pore. The second internal transcribed spacer (ITS2) of ribosomal DNA from these sanguinicolids and a known species, Cardicola forsteri Cribb, Daintith & Munday, 2000, were sequenced, aligned and analysed to test the distinctness of the putative new species. Results from morphological comparisons and molecular analyses suggest the presence of 18 putative species; 11 are described on the basis of combined morphological and molecular data and seven are not because they are characterised solely by molecular sequences or to few morphological specimens (n= one). There was usually a correlation between levels of morphological and genetic distinction in that pairs of species with the greatest genetic separation were also the least morphologically similar. The exception in this regard was the combination of Cardicola tantabiddii n. sp. from S. fuscescens from Ningaloo Reef ( Western Australia) and Cardicola sp. 2 from the same host from Heron Island ( Great Barrier Reef). These two parasite/ host/location combinations had identical ITS2 sequences but appeared to differ morphologically ( however, this could simply be due to a lack of morphological material for Cardicola sp. 2). Only one putative species ( Cardicola sp. 1) was found in more than one location; most host species harboured distinct species in each geographical location surveyed ( for example, S. corallinus from Heron and Lizard Islands) and some ( for example, S. punctatus, S. fuscescens and Chlorurus microrhinos) harboured two species at a single location. Distance analysis of ITS2 showed that nine species from siganids, three from scombrids and five from scarids formed monophyletic clades to the exclusion of sanguinicolids from the other host families. Cardicola milleri n. sp. and C. chaetodontis Yamaguti, 1970 from lutjanids and chaetodontids, respectively, were the only representatives from those families that were sequenced. Within the clade formed by sanguinicolids from Siganidae there wasa further division of species; species from the morphologically similar S. fuscescens and S. margaritiferus formed a monophyletic group to the exclusion of sanguinicolids from all other siganid species.