Jean Cottney

Comparative effects of drugs on four paw oedema models in the rat

The development of novel anti-inflammatory drugs (AID) has been claimed to be dependent on the discovery of models of inflammation that differ from those currently used for drug screening, e.g. carrageenen paw oedema and u.v. erythema. We have thus evaluated the effect of a variety of drugs in a number of novel models of inflammation in the rat produced in the hind paw. We have utilized kaolin, zymosan, anti-rat IgG (anti-IgG) and the Reversed Passive Arthus (RPA) reaction to produce these oedema models.We found that the non-steroidal AIDs, e.g. aspirin, flufenamic acid, indomethacin, naproxen and phenylbutazone, were active in all four tests. Of the nine novel AID examined, levamisole and tetramisole demonstrated considerable activity in all four tests and dapsone was especially active in the anti-IgG and RPA tests. In contrast, the anti-rheumatic d-penicillamine was inactive in all four models. Each of the ten compounds tested which has been claimed to influence complement function, was active in the RPA but not in the kaolin model. These results are discussed in the context of the aetiology of each oedema and the suspected mode of action of the various drugs.

Action of gold salts in some inflammatory and immunological models

Several gold salts were compared in kaolin-induced rat paw oedema, u.v. erythema in guinea pigs, delayed type hypersensitivity and humoral immunity in mice, and adjuvant-induced arthritis in the rat. In the latter the additional parameters of serum gold and copper levels and lysosomal enzyme activity were determined. In addition, the in vitro inhibition of several lysosomal enzymes derived from mouse macrophages was studied. The gold compounds examined were aurothiomalate, aurothioglucose, triethylphosphine gold chloride (SK & F 36914) and its glucopyranoside derivative (SK & F D-39162), triphenylphosphine gold chloride and sodium gold chloride dihydrate.SK & F 36914 and SK & F D-39162 had significant activity after oral dosage upon paw kaolin and u.v. erythema in rats and guinea pigs, respectively. Gastric swelling also occurred.In Wistar rats, adjuvant arthritis was little affected by the gold salts but in the Lewis rats there was suppression. In both strains there was less elevation in serum copper levels with treatment by SK & F 36914 and SK & F D-39162, but not by aurothiomalate.None of the compounds had any measurable effect on delayed hypersensitivity or humoral antibody levels in mice.The in vitro activities of cathepsin B1 and cathepsin D were inhibited by all the gold compounds. Reactivity of gold dependent on compound solubility and the nature of the gold ligand.Considerable differences exist between the profiles of activity for the different gold salts evaluated.These observations indicate that some gold salts do possess anti-inflammatory activity with a potency similar to that of indomethacin.

Mechanisms of phytohaemagglutinin-P-, concanavalin-A- and kaolin-induced oedemas in the rat

Subplantar administration of either Phytohaemagglutinin-P (PHA), Concanavalin-A (Con A) or kaolin into the rat hind paw produced a dose related oedema which was still present at 48 h. Both of the lectins were more inflammagenic than kaolin on a weight per weight basis. As a result of studies using mediator inhibitors and depletors it appears that 5HT, but not histamine, may play a role in the early phases (0.5-1.5 h) of both PHA and Con A responses. Neither mediator appears to be involved in the kaolin oedema. Kinins are also likely mediators of the inflammatory response to all three irritants and could be detected in irritant injected air blebs in the rat. Prostaglandins are unlikely to play a significant role in PHA or Con A oedema since indomethacin-induced inhibition of their synthesis has only a slight inhibitory effect on the lectin induced paw oedemas and only small amounts of prostaglandin-like material could be detected in PHA or Con A blebs. However, kaolin oedema appears to have a significant prostaglandin component since large amounts of prostaglandin-like materials were detected in kaolin blebs and also indomethacin reduced the kaolin induced paw oedema. Other mediators of the inflammatory process such as complement are likely to be involved in all three irritant induced oedemas.

Parallel synthesis and biological activity of a new class of high affinity and selective δ-opioid ligand

A considerable number of research papers describing the synthesis and testing of the delta opioid receptor (DOR) ligands, SNC-80 and TAN-67, and analogues of these two compounds, have been published in recent years. However, there have been few reports of the discovery of completely new structural classes of selective DOR ligand. By optimising a hit compound identified by high throughput screening, a new series of tetrahydroisoquinoline sulphonamide-based delta opioid ligands was discovered. The main challenge in this series was to simultaneously improve both affinity and physicochemical properties, notably aqueous solubility. The most active ligand had an affinity (IC(50)) of 6 nM for the cloned human DOR, representing a 15-fold improvement relative to the original hit 1 (IC(50) 98 nM). Compounds from this new series show good selectivity for the DOR over mu and kappa opioid receptors. However the most active and selective compounds had poor aqueous solubility. Improved aqueous solubility was obtained by replacing the phthalimide group in 1 by basic groups, allowing the synthesis of salt forms. A series of compounds with improved affinity and solubility relative to 1 was identified and these compounds showed activity in an in vivo model of antinociception, the formalin paw test. In the case of compound 19, this analgesic activity was shown to be mediated primarily via a DOR mechanism. The most active compound in vivo, 46, showed superior potency in this test compared to the reference DOR ligand, TAN-67 and similar potency to morphine (68% and 58% inhibition in Phases 1 and 2, respectively, at a dose of 10 mmol/kg i.v.).

Synthesis of novel analogues of the delta opioid ligand SNC-80 using AlCl3-promoted aminolysis

Two focused libraries of delta opioid ligands were synthesised using AlCl3 facilitated aminolysis. Several compounds were identified with DOR binding affinities higher or similar to SNC-80. A novel acyclic derivative of SNC-80 produced antinociception in the acetic acid abdominal constriction test, which is at least partially mediated via the delta-opioid receptor.

Synthesis of novel analogues of the delta opioid ligand SNC-80 using REM resin.

Focused libraries of delta opioid ligands were synthesised using REM resin methodology. Several high affinity compounds were identified with good selectivity over the mu opioid receptor. Automated REM resin recycling was used to synthesise larger amounts of ligand for in vivo studies.

Modulation of the immune system in the mouse. 2. Drug administration following antigen sensitization

A model has been developed in the mouse in which metylated bovine serum albumin (MBSA) and sheep red blood cells have been used to produced delayed type hypersensitivity (DTH) and humoral immunity (HI), respectively.The time coruse between antigen sensitization and challenge was chosen such that cyclophosphamide (CY), administered prior to sensitization, produced DTH enhancement and also produced suppression of HI when administered at high doses. A number of other drugs have been examined in this model but only CY-like drugs, viz. alkylating agents, produced similar DTH enhancement. DTH was suppressed in a few cases.The HI response was enhanced or suppressed by a number of unrelated drugs. CY was the only alkylatng agent to suppress the antibody titre.The mechanisms for these drug effects are uncertain. However, a number of drugs elicit effects on either DTH or HI which suggests there is selective removal of certain cell populations rather than non-specific cytotoxicity. Possible theories for the effects observed are discussed.These studies also suggest that CY possesses unique properties in this particular model.

A comparison of tissue gold levels in guinea-pigs after treatment with myocrisin injected intramuscularly and triethylphosphine gold chloride and myocrisin administered orally.

A comparative study of tissue gold levels produced in guinea-pigs after the oral administration of either triethyl-phosphine gold chloride or Myocrisin (sodium aurothiomalate) or after the injection of Myocrisin intramuscularly is reported. Gold concentrations were determined 5, 24 and 168 hours after administration in stomach, small intestine, large intestine, kidney, liver and spleen and 5 and 24 hours after administration in skin, adrenals, heart, lung and brain. In gastrointestinal tissues, tissue gold concentrations were highest with triethylphosphine gold chloride. The stomach gold level 5 hours after oral administration of triethyl-phosphine gold chloride is particularly high and, taken in conjunction with the other gastrointestinal gold levels measured, suggests that a stomach rather than an intestinal absorption mechanism may predominate. A more extensive time-course study on kidney and liver is reported and the possible relationship between tissue concentration and toxicity is discussed.

Copper distribution and reactivity in serum following administration of cuprous oxide to rats and guinea pigs.

Total serum copper levels produced by the administration of a single dose of cuprous oxide (500 mg/kg) both s.c. and orally to rats and guinea pigs are reported 1, 5, 7 and 24 h after administration, together with caeruloplasmin oxidase activities and changes in the sulphydryl group concentrations. The highest serum copper levels were obtained one hour after oral administration to the guinea pig. Both animal species exhibited increased serum copper levels after s.c. administration and this increase persisted for a longer time than after oral administration. Caeruloplasmin oxidase activity varied in a complex manner and sulphydryl group activity was significantly depressed in all cases except after s.c. administration to the guinea pig. The latter results must be treated cautiously since they may indicate interactions between copper and the test procedures. Analysis of serum fractions separated by electrophoresis indicated that the absorbed copper is mainly transported on albumin. The dissolution of cuprous oxide by amino acids and hydrochloric acid and the absorption of cuprous oxide and some related copper complexes by albumin were investigated in vitro. The implications of the results are discussed in terms of their relevance to pharmacological and clinical studies.

Pharmacological and biochemical effects of the cardiotonic agent Org10325 in isolated cardiac and vascular tissue preparations

1 The pharmacological and biochemical effects of a novel cardiotonic agent, Org10325 have been studied in isolated cardiac and vascular tissue preparations. 2 Org10325 produced concentration‐dependent (0.15–4.8 mm) positive inotropic, positive chronotropic and vascular relaxant responses in rabbit isolated papillary, atrial and aortic preparations, respectively. The maximal chronotropic effect (45%) was significantly less than the isoprenaline maximum. The inotropic effects of Org10325 were not modified by α‐ or β‐adrenoceptor blockade or by pretreatment with reserpine. Org10325 was at least 23 times more potent at relaxing aortic strips pre‐contracted with phenylephrine than with KCl. 3 Org10325 (74 μm) potentiated (10–14 fold) the positive inotropic effects of isoprenaline in rabbit isolated papillary muscles. Carbachol inhibited the positive inotropic effect of Org10325. Both the positive inotropic and vasorelaxant effects of Org10325 were accompanied by increases in cyclic AMP but not cyclic GMP. 4 In rat perfused heart preparation Org10325 increased phosphorylase a, cyclic AMP‐dependent protein kinase activities and stimulated phosphorylation of contractile proteins (troponin‐I and C‐protein). 5 Org10325 selectively inhibited the cyclic AMP hydrolytic activity of cyclic AMP high affinity cyclic nucleotide phosphodiesterase (PDE) isoenzymes, PDE III (IC50 65 μm) and PDE IV (IC50 71 μm), from rabbit cardiac ventricle. Weak inhibition (IC50 > 250μm) of PDE I and PDE II was observed. 6 The results show that the cardiac and vascular effects of Org10325 are mediated by an increase in cellular cyclic AMP due to inhibition of PDE III and PDE IV activities. However, in contrast to other PDE‐inhibitors Org10325 produced a marked increase in relaxation time of isolated papillary muscle suggesting the involvement of additional cyclic AMP‐independent mechanisms of action.