We examined the effects of ML3000 and several non-steroidal antiinflammatory drugs (NSAIDs) on the synthesis of products of 5-LOX (LTB4, LTC4) and COX-1/2 (TXB2, PGE2) in vitro and ex vivo in order ...to further elucidate the mechanism of action of ML3000.
Using a human whole blood assay the effect of ML3000 on the shunt of arachidonic acid to the lipoxygenase pathway when COX is blocked was studied. ML3000 (0.3, 1, 3, 10, 30 microg/ml) and indomethacin (0.3, 1, 3, 10, 30 microg/ml) concentration-dependently inhibited the synthesis of PGE2 (IC50 = 3.9 and 4.5 microM). In contrast to ML3000, indomethacin produced an increase of LTC4 of up to 155.5% of control. 5-lipoxygenase inhibition was further tested in a basophilic leukemia cell assay using RBL-1 cells. ML3000 (1-10 microM) inhibited the synthesis of LTB4 in a concentration related manner (IC50: 3.6 microM). In carrageenan induced rat paw edema, ML3000 and indomethacin completely blocked the formation of PGE2 in the inflamed tissue. The LTB4 production in the inflamed paw was reduced to basal levels by ML3000 (10 +/- 1.4 pg/paw saline control and 7.5 +/- 1.3-5.9 +/- 3.2 pg/paw ML3000), whereas LTB4 levels remained markedly elevated as compared to saline control by indomethacin (30.7 pg/paw). 5-LOX inhibition in the inflamed rat colon was investigated by measuring LTB4 synthesis. MK-886 and ML3000 at 10 mg/kg p.o. reduced LTB4 production to 29.8 +/- 4.9 and 30.1 +/- 2.8 pg/mg tissue as compared to control (54.2 +/- 7.4 mg/kg tissue). LTB4 levels in the rat stomach were comparable to control (2.5 +/- 0.4 pg/mg protein) after oral administration of ML3000 (10, 30, 100 mg/kg), whereas oral treatment with indomethacin (0.3, 1, 3 mg/kg) or diclofenac (1, 3 mg/kg) increased LTB4 up to 9.2 +/- 2.3 or 8.9 +/- 1.6 pg/mg protein. This effect was significant at 1 mg/kg diclofenac and 0.3 mg/kg indomethacin.
These results provide further evidence, that ML3000 inhibits 5-LOX as well as COX-1 and COX-2 in vitro and in animal experiments. The favourable gastrointestinal (GI) tolerability of the compound is believed to be linked to the mechanism of combined 5-LOX and COX-1/2 inhibition of ML3000.
To search for potential new therapies to inhibit the progression of joint destruction in patients with rheumatoid arthritis.
We evaluated the dual acting antiinflammatory drug ML3000 ...(2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro- H-pyrrolizine-5-yl) acetic acid, a dual inhibitor of 5-lipoxygenase (5-LOX) as well as both cyclooxygenases (COX-1 and COX-2) in the rat model of adjuvant arthritis. On Day 0, female Lewis rats (5 per group) were injected intradermally with complete Freund's adjuvant at base of the tail. Treatment began on Day 2; the rats received ML3000 (20 or 80 mg/kg/day) twice daily 7 h apart for 28 days and were then sacrificed. To reduce pain, the positive control group and 2 treatment groups received paracetamol (3 mg/ml water). Joint histology was scored for synovial cell proliferation, fibroproliferative pannus, and cartilage and bone erosions, as well as diffuse leukocyte infiltrates.
Daily doses of 20 or 80 mg/kg ML3000 significantly reduced the arthritis associated deficiency of body growth, the edema/erythema score, and splenomegaly. In the ankle joint, ML3000 significantly reduced the overall histological score, synovial cell proliferation, and bone/cartilage erosions, and inhibited the appearance of fibroproliferative pannus. The addition of paracetamol in the drinking water had no influence. No side effects were noted.
ML3000 is an antiarthritic drug with a high gastrointestinal tolerability, which can reduce synovial cell proliferation and joint erosion and is capable of markedly suppressing prostaglandin synthesis.
The studies reported were designed to evaluate the effects of ML3000 on platelet aggregation and platelet-induced thrombin generation in human platelet rich plasma and its antithrombotic effect in a ...rat thrombosis model. ML3000 is a potent inhibitor of both COX-1/2 and 5-LOX with demonstrated antiinflammatory activity and a low incidence of GI mucosal injury in animal and human studies.
The antithrombotic activity of ML3000 (10, 30 and 100 mg/kg) and aspirin (30 and 100 mg/kg) was measured in the mesenteric venules of rats using the laser-induced thrombus model. Both ML3000 and aspirin, at all doses tested, showed significant antithrombotic activity. The mean number of laser injuries necessary to induce a thrombus that blocked the vessel was 1.93 +/- 0.28 in the control group, 3.3 +/- 0.53, 3.6 +/- 0.14 or 4.07 +/- 0.37 in the groups treated with ML3000 at 10, 30 or 100 mg/kg p.o. and 3.4 +/- 0.55 or 3.9 +/- 0.3 in the groups treated with Aspirin at 30 or 100 mg/kg p.o. The antithrombotic activity in this model was significant up to 12 h post-administration of 100 mg/kg ML3000 or Aspirin. The aggregation inhibiting activity of ML3000 (1-100 microg/ml) and indomethacin (1 microg/ml) was studied using the following inducing agents: ADP (1 and 2 microM), epinephrine (25 and 50 microM), collagen (0.5 and 1 microg/ml), and the thromboxane mimetic U46619 (0.8 and 1.6 microM). Aggregation inhibitory activity was observed with ML3000 in all assays except with the higher concentration of U46619 at 1.6 microM. Indomethacin (1 microg/ml) inhibited aggregation in all assays.
ML3000 has significant antithrombotic activity and a marked platelet aggregation inhibiting effect. Given its demonstrated antiinflammatory activity, platelet function inhibition, and antithrombotic effects along with a lack of effect on the GI mucosa, ML3000 may offer an alternative to the combination of a COX-2 inhibitor and aspirin in arthritis patients at risk for cardiovascular disease.
In this study we characterized the effects of a novel anti-inflammatory drug, ML 3000 (2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine- 5-yl-acetic acid), on the gastric mucosa ...and attempted to determine the mechanism responsible for its apparent stomach-sparing properties. Acute gastric damaging properties of ML 3000 versus indomethacin were examined in the rat, while chronic-type gastric ulcer was examined in the rabbit. At doses of up to 100 mg/kg p.o., ML 3000 did not produce significant acute gastric injury, while indomethacin at 5-20 mg/kg p.o. caused mucosal necrosis and bleeding. ML 3000 significantly inhibited gastric and blood prostaglandin E2 synthesis, with the higher doses tested (30 and 100 mg/kg) producing comparable effects to that seen with indomethacin at 10 or 20 mg/kg. Gastric and blood leukotriene B4 synthesis were not significantly affected by either drug. While indomethacin caused a significant increase in leukocyte adherence to mesenteric venules, ML 3000 did not. When administered repeatedly to rabbits, diclofenac caused penetrating ulcer formation in the gastric antrum of the majority of the animals. ML 3000 did not produce any detectable damage at doses of 10 or 30 mg/kg, but an ulcer was observed in one of five rabbits given the 100 mg/kg dose. Prior administration of ML 3000 (10-100 mg/kg) did not significantly affect the extent of gastric damage induced by subsequent oral administration of ethanol. These studies demonstrate that ML 3000 spares the gastric mucosa despite significantly suppressing gastric prostaglandin synthesis.
To elucidate if the planar chiral paracyclophane moiety conveys pharmacological activity to arylacetic acid analogs in two animal models.
Female NMRI mice (6 mice/group); female Wistar rats (8 ...rats/group); thrombocytes from human blood.
The enantiomers of 2.2paracyclophaneacetic acid were applied locally (10(-7) and 10(-6) mol/ear) and orally (10-100 mg/kg).
(a) Phorbol myristyl acetate model of acute inflammation of the inner auricle. (b) Oxazolone model of allergic contact dermatitis. (c) Carrageenan model of acute inflammation. (d) Inhibition of cyclooxygenase-1 and 12-lipoxygenase (in vitro).
(a) PMA model: pR-(-)-2.2paracyclophaneacetic acid (10(-6) mmol/ear): 58% inhibition after 24 h (p < 0.05). (b) Oxazolone model: pR-(-)-2.2paracyclophaneacetic acid (10(-6) mmol/ear): 42% inhibition after 24 h (p < 0.05). (c) Carrageenan model: pR-(-)-2.2paracyclophaneacetic acid (10 mg/kg): 31.4% inhibition (paw volume 0.48 +/- 0.13 ml). (d) Cyclooxygenase-1 and 12-lipoxygenase: no inhibition at concentrations up to 10 microM.
The easily accessible 2.2paracyclophane moiety should find its use in medicinal chemistry as it is a pharmacophoric substituent with the interesting feature of planar chirality.
ML 3000 is a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (5-LO), two enzymes that contribute to the airway inflammation in asthma. When administered as an aerosol at a dose of 100 mg, ...0.5 h before antigen challenge in allergic sheep, ML 3000 provided significant inhibition against the early bronchial response (EAR, mean 33% protection,P<0.05), completely blocked the late antigen-induced bronchoconstriction (LAR, mean 81% protection,P<0.05) and the airway hyperresponsiveness (AHR,P<0.05) to aerosolized carbachol that occurs 24 h after antigen challenge in this model. Consistent with this functional protection was a small but significant reduction in the percentage of neutrophils recovered in bronchoalveolar lavage (BAL) at 8 h and 24 h after challenge. These findings are similar to previous data obtained in this animal model with other 5-LO inhibitors (blockade of the LAR and AHR) and COX inhibitors (blockade of AHR). These results suggest that aerosol administration of a dual inhibitor of COX and 5-LO may have beneficial effects in the treatment of allergic airway disease.
Recently published results provide evidence of the importance of oxidatively modified LDL in the development of atherosclerosis. Several typical characteristics of this disease can be ascribed to the ...effects of oxidized LDL on the different cells involved in lesion formation. In various cell culture systems oxidized LDL was found to be cytotoxic. Therefore we were interested in its influence on parameters of energy metabolism such as glycogen and ATP content as determined for aortic segments in vitro. The results show that oxidized LDL leads to sharp decreases in both parameters, indicating an activation of cellular energy metabolism. Findings obtained from contraction experiments in which oxidized LDL shows a contractionenhancing effect on arterial segments suggest that the oxidized lipoprotein facilitates cellular Ca
2+
liberation. This seems to be a common signal leading to its effects on energy metabolism and contraction and could also explain its cytotoxicity if cells are exposed to it for longer periods.
To evaluate in vivo therapeutic efficacy of licofelone, a novel competitive dual inhibitor of 5-lipoxygenase (5-LOX) and cyclooxygenase (COX) in chondrocyte death in the canine ligament transection ...model of osteoarthritis (OA), and to explore its effect on factors involved in the apoptotic phenomenon, i.e., caspase-3, COX-2, and inducible nitric oxide synthase (iNOS).
Cartilage specimens were obtained from 3 experimental groups of dogs: Group 1, dogs subjected to sectioning of the anterior cruciate ligament of the right knee and given placebo treatment; Groups 2 and 3, operated dogs that received oral treatment with licofelone (2.5 or 5.0 mg/kg/day, respectively) for 8 weeks starting immediately after surgery. All dogs were killed 8 weeks postsurgery. The cartilage level of chondrocyte death was detected by TUNEL reaction. Cartilage distribution of caspase-3, COX-2, and iNOS was documented by immunohistochemistry using specific antibodies, and other levels were quantified by morphometric analysis.
In cartilage specimens from placebo treated dogs a large number of chondrocytes in the superficial layers stained positive for TUNEL reaction. Treatment with therapeutic concentrations of licofelone (2.5 and 5.0 mg/kg/day) markedly reduced the level of chondrocyte apoptosis to the same extent in both therapeutic groups (p < 0.0001, p < 0.002, respectively). In these groups, the levels of caspase-3, COX-2, and iNOS in cartilage from both condyles and plateaus were also significantly decreased (p < 0.0001, p < 0.0001, p < 0.0002, respectively) compared to the control (placebo) group.
Licofelone is an effective treatment in vivo, capable of reducing the level of OA chondrocyte death. This effect is likely mediated by a decrease in the level of caspase-3 activity, which may be related to the reduced production of 2 major factors involved in chondrocyte apoptosis, NO and prostaglandin E2. These findings may explain some of the mechanisms by which licofelone reduces the progression of experimental OA.
2,2-Dimethyl-6-(4-chlorophenyl)-7-phenyl, 2,3-dihydro-1H-pyrrolizine-5-yl)-acetic acid (ML 3000) is a dual inhibitor of the enzymes cyclo-oxygenase and 5-lipoxygenase in bovine and human thrombocytes ...and granulocytes. In animal experiments the compound has antiphlogistic, analgesic, antipyretic, antiasthmatic and antiaggregative activity at a dosage that causes no gastrointestinal damage.
