Geoffrey D. Reynolds

In vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus.

Chrysophanic acid (1,8-dihydroxy-3-methylanthraquinone), isolated from the Australian Aboriginal medicinal plant Dianella longifolia, has been found to inhibit the replication of poliovirus types 2 and 3 (Picornaviridae) in vitro. The compound inhibited poliovirus-induced cytopathic effects in BGM (Buffalo green monkey) kidney cells at a 50% effective concentration of 0.21 and 0.02 microgram/ml for poliovirus types 2 and 3, respectively. The compound inhibited an early stage in the viral replication cycle, but did not have an irreversible virucidal effect on poliovirus particles. Chrysophanic acid did not have significant antiviral activity against five other viruses tested: Coxsackievirus types A21 and B4, human rhinovirus type 2 (Picornaviridae), and the enveloped viruses Ross River virus (Togaviridae) and herpes simplex virus type 1 (Herpesviridae). Four structurally-related anthraquinones--rhein, 1,8-dihydroxyanthraquinone, emodin and aloe-emodin were also tested for activity against poliovirus type 3. None of the four compounds was as active as chrysophanic acid against the virus. The results suggested that two hydrophobic positions on the chrysophanic acid molecule (C-6 and the methyl group attached to C-3) were important for the compounds activity against poliovirus.

Screening of Australian medicinal plants for antiviral activity.

Extracts of 40 different plant species used in the traditional medicine of the Australian Aboriginal people have been investigated for antiviral activity. The extracts have been tested for activity against one DNA virus, human cytomegalovirus (HCMV) and two RNA viruses, Ross River virus (RRV) and poliovirus type 1, at non-cytotoxic concentrations. The most active extracts were the aerial parts of Pterocaulon sphacelatum (Asteraceae) and roots of Dianella longifolia var. grandis (Liliaceae), which inhibited poliovirus at concentrations of 52 and 250 microg/ml, respectively. The extracts of Euphorbia australis (Euphorbiaceae) and Scaevola spinescens (Goodeniaceae) were the most active against HCMV. Extracts of Eremophila latrobei subsp. glabra (Myoporaceae) and Pittosporum phylliraeoides var. microcarpa (Pittosporaceae) exhibited antiviral activity against RRV.

High-performance liquid chromatographic determination of morphine and its 3- and 6-glucuronide metabolites: improvements to the method and application to stability studies

Improvements to previously reported methods for the determination of morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in human plasma are described. The improved methods involve the use of a solid-phase extraction cartridge and a chromatographic system which uses paired-ion reversed-phase high-performance liquid chromatography with a radially compressed column. Only one cartridge is used to prepare each sample for chromatography and each cartridge may be used for at least fourteen 1-ml plasma samples. The recovery is greater than 85%. The improvements to the method of sample pretreatment and in the chromatographic conditions have allowed determination of morphine, M3G and M6G in human plasma down to 13.3 nmol/l (coefficient of variation = 9.3%), 108 nmol/l (6.6%) and 41 nmol/l (6.7%), respectively, using ultraviolet detection alone. It was shown that all three compounds were stable in plasma for up to 101 weeks when stored at -20 degrees C.

Influence of polarity on dose-response relationships of intrathecal opioids in rats

&NA; Dose‐response curves were constructed for intrathecal morphine (M), oxymorphone (OM), hydromorphone (HM), diamorphine (DM), 14‐hydroxydihydromorphine (OHM), oxycodone (OC), hydrocodone (HC) and fentanyl (F). Intrathecal catheters were placed in 69 rats under halothane/N2O anaesthesia. After recovery, baseline hot plate and tail flick latencies were measured, and a dose of opioid was given. Hot plate and tail flick latencies were assessed at 5, 15, 30, 60, 90, 120 min and then hourly until they returned to within 25% of baseline. Response latencies were converted to per cent of maximum possible effect (% MPE) and the area under the % MPE × time curve was taken as the response. This measure includes information about both potency and duration of action. Each rat received 3 opioids and saline at intervals of 2–3 days. On a fifth occasion, the animals first treatment was repeated. Each opioid was studied over an 8‐fold dose range. Results of both hot plate and tail flick were best described by a model including log(dose), a component due to development of tolerance over the 5 experimental days, and an among‐rat variation term. In the hot plate test, doses equieffective in producing a response (AUC) over the dose range studied were in the order OHM < OM < HM < M < F < DM < HC < OC. Slopes of the log(dose)‐response curves were similar for all drugs except OHM, which had a steeper slope. A model is proposed in which hot plate and tail flick latencies are prolonged while CSF concentrations of a drug are above its minimum effective concentration, and drug is cleared from the CSF by a first‐order process, possibly uptake into the spinal cord and removal via the blood. This model predicts that log(dose)‐response curves will be linear, as was observed, with slopes inversely proportional to the rate constant for clearance from CSF. According to this model the steeper slope of the OHM log(dose)‐response may be interpreted as indicating slower clearance from CSF. OHM has the lowest octanol/pH 7.4 buffer distribution coefficient (0.34) of all opioids studied, possibly leading to a lower rate of uptake into the spinal cord.

The effect of old age on the disposition and antinociceptive response of morphine and morphine-6β-glucuronide in the rat

Abstract The aims of this study were to examine the effect of old age on the pharmacokinetics of morphine and morphine‐6&bgr;‐glucuronide (M6G) and their relationships to antinociceptive activity. Morphine (21.0 &mgr;mol/kg) or M6G (21.7 &mgr;mol/kg) were administered s.c. to young adult and aged male Hooded‐Wistar rats. Antinociceptive effect was measured by the tail‐flick method at various times up to 2.5 h or 6.5 h after morphine or M6G administration, respectively, and concentrations of morphine, morphine‐3&bgr;‐glucuronide (M3G) and M6G in plasma and brain were determined by HPLC. Creatinine clearance was significantly lower by 33% or 21% in aged compared to young adult rats receiving morphine or M6G, respectively. After morphine administration, the areas under the (i) antinociceptive effect‐time curve, (ii) plasma morphine concentration‐time curve, and (iii) brain morphine concentration‐time curve were not different between young adult and aged rats. However, the AUC for plasma M3G was five‐fold higher in the aged relative to young adult rats, which could not be accounted for by only a 33% lower creatinine clearance. M6G was not detected in any plasma or brain sample from rats administered morphine and no M3G was detected in brain. For M6G administration, the areas under the (i) antinociceptive effect‐time curve, and (ii) plasma M6G concentration‐time curve were 1.8‐ and 1.6‐fold higher in aged compared to young adult rats, respectively. Concentrations of M6G in brain were below the limit of quantification. No morphine or M3G was detected in any of the plasma or brain samples of rats administered M6G. The results demonstrate no change in morphine antinociception and pharmacokinetics with age, and suggest that blood‐brain barrier permeability and receptor sensitivity to morphine are not altered in aged rats. Accumulation of M3G in plasma of aged rats is probably due to diminished renal clearance of M3G in addition to a reduction in the biliary excretion of M3G. The heightened sensitivity of the aged rats to M6G is probably due to the observed altered kinetics of M6G rather than a pharmacodynamic change.

CONCENTRATION‐EFFECT RELATIONSHIPS OF MORPHINE AND MORPHINE‐6β‐GLUCURONIDE IN THE RAT

1. The aims of the present study were to determine the relationship between the antinociceptive effect and concentrations of morphine and morphine‐6β‐glucuronide (M6G) in plasma and in the brain.

The reactions of 3-glycidoxypropyltrimethoxysilane in acidic solutions on polymerization and in the presence of silica

The epoxy group in 3-glycidoxypropyltrimethoxysilane (GPS) readily undergoes hydrolysis in acidic conditions to form a solution containing principally the vicinal diol or a reaction product between the vicinal diol and the silanol groups formed during the hydrolysis (an alkoxysilane). Additional reaction products include alkene, enol, low concentrations of carbonyl compounds, intermolecular condensation products, e.g. ethers, and reaction products specific to the acid used to promote the hydrolysis of the alkoxysilane and epoxy groups. On evaporation of acidified GPS solutions at room temperature, a polymerized form of GPS is produced. This polymer is found to be readily oxidized on heating in air and from simulated outdoor weathering trials (SODW), but not when formed on silica powders from a 1-2 wt% solution to form a coating. In the absence of silica, unreacted hydroxy groups are readily available for oxidation, a result that is consistent with the reported molecular structure of polymerized GPS.

Direct determination of codeine-6-glucuronide in plasma and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A sensitive and selective method was developed for the direct determination of codeine-6-glucuronide in plasma and urine using high-performance liquid chromatography (HPLC) with fluorescence detection. Codeine-6-glucuronide was synthesised and its purity estimated using acid and enzyme hydrolysis. The hydrolysis of codeine-6-glucuronide by beta-glucuronidase was incomplete and urine reduced the extent of hydrolysis. Codeine-6-glucuronide was recovered from plasma using a solid-phase extraction column and separated on a reversed-phase C18 HPLC column. The assay showed good reproducibility and accuracy (within 10%), and standard curves were linear between 32 and 1600 ng/ml in plasma and between 0.32 and 160 micrograms/ml in urine. The assay has been applied to the study of the pharmacokinetics and metabolism of codeine in patients.

The effects of phytoestrogenic isoflavones on the formation and disposition of paracetamol sulfate in the isolated perfused rat liver.

This study examines the potential for the phytoestrogenic isoflavones, a type of complementary medicine, to be involved in pharmacokinetic interactions in the liver. Rat livers were isolated and perfused to steady state, in single‐pass mode, with either 5 μm paracetamol (n=6), or 5 μm paracetamol with a 50:50 molar mixture of genistein and biochanin A or daidzein and formononetin, at a total isoflavone concentration of 1 and 10 μm (n = 6 for each mixture at each concentration). At 1 μm, neither isoflavone mixture had any effect, while at 10 μm both mixtures decreased the clearance of paracetamol and the formation clearance to paracetamol sulfate. Genistein and biochanin A (10 μm) also increased the biliary extraction of hepatically‐generated paracetamol sulfate. Additional livers were perfused with an infusion of 5 μm 14C‐paracetamol in the absence (n = 4), or presence, of a 10 μm genistein and biochanin A mixture (n = 4). Analysis of washout perfusate and bile samples (up to 30min after stopping the infusion) revealed that the isoflavones reduced the first‐order rate constant for paracetamol sulfate transport into perfusate, but not for transport into bile. The results indicate that isoflavones can reduce the formation of paracetamol sulfate and that its enhanced excretion into bile arises from the inhibition of sinusoidal efflux transport.