Chiral dioxolane inhibitors of leukotriene biosynthesis: structure-activity relationships and syntheses using asymmetric dihydroxylation.

1,3-Dioxolanes have been described as chiral inhibitors of 5-lipoxygenase (5LO). In the present work, this series has been developed further to provide agents which showed comparable or superior potency in vivo to ZD2138, a methoxytetrahydropyran inhibitor of 5LO, which is currently undergoing clinical evaluation. An asymmetric synthesis was developed to these dioxolanes based on asymmetric dihydroxylation. (S)-N-Methyl-4'-[[4-(2,2,4- trimethyl-1,3-dioxolan-4-yl)thien-2-yl]thio]acetanilide ((S)-10d) inhibited leukotriene B4 (LTB4) synthesis in A23187-stimulated human whole blood in vitro with IC50 0.039 microM, 25-fold more potent than (R)-10d. In vivo, (S)-10d inhibited LTB4 synthesis by 70% in zymosan-inflamed air pouch exudate in rat 10 h after an oral dose of 1.5 mg/kg. Structure-activity relationship considerations suggested that the dioxolane and methoxytetrahydropyran series are related, a conclusion which can be supported by molecular modeling.

(Methoxyalkyl)thiazoles: a new series of potent, selective, and orally active 5-lipoxygenase inhibitors displaying high enantioselectivity.

(Methoxyalkyl)thiazoles are novel 5-lipoxygenase (5-LPO) inhibitors that are neither redox agents nor iron chelators. Consideration of a hypothetical model of the enzyme active site led to this series which is exemplified by 1-[3-(naphth-2-ylmethoxy)phenyl]-1-(thiazol-2-yl)propy l methyl ether (2d, ICI211965). 2d inhibits cell-free guinea pig 5-LPO activity, LTC4 synthesis in plasma free mouse macrophages, and LTB4 synthesis in rat and human blood (IC50s 0.1 microM, 8 nM, 0.5 microM, and 0.4 microM, respectively) but does not inhibit the synthesis of cyclooxygenase products at concentrations up to 50 microM in macrophages and 100 microM in blood. 2d is orally active in rat (ex vivo ED50 10 mg/kg in blood taken in 1 h after dosing). SAR studies show that high in vitro potency requires methoxy, thiazolyl, and naphthyl groups and depends critically on the substitution pattern. (Methoxyalkyl)thiazoles are chiral. Resolution of 1-methoxy-6-(naphth-2-ylmethoxy)-1-(thiazol-2-yl)indan (2j, ICI216800) shows that (+)-2j is 50-150-fold more potent than (-)-2j in in vitro assays. Thus, (methoxyalkyl)thiazoles are a new series of orally active, selective 5-LPO inhibitors and represent the first class of inhibitors in which inhibition is mediated by specific, enantioselective interactions with the enzyme.

Methoxytetrahydropyrans. A new series of selective and orally potent 5-lipoxygenase inhibitors.

Investigation of the SAR of the lead (methoxyalkyl)thiazole 1-[3-(naphth-2-ylmethoxy)phenyl]-1-thiazol-2-ylprop yl methyl ether (1, ICI 211965) led to the methoxytetrahydropyrans, a new series of 5-lipoxygenase (5-LPO) inhibitors exemplified by the parent compound 4-[3-(naphth-2-ylmethoxy)phenyl]-4- methoxy-3,4,5,6-tetrahydro-2H-pyran (4f). In vitro 4f inhibited leukotriene C4 (LTC4) synthesis in zymosan-stimulated plasma-free mouse macrophages and LTB4 synthesis in A-23187-stimulated human whole blood (IC50s 0.5 nM and 0.07 microM, respectively). In the rat 4f inhibited LTB4 synthesis in blood ex vivo and in zymosan-inflamed air pouch exudate with an ED50 3 h after oral dosing of 10 mg/kg in each system. In seeking more potent orally active compounds, strategies were explored in congeners of 4f for reducing lipophilicity without sacrificing potency. For example, replacement of 2-naphthyl of 4f by various aza- and oxoheterocycles afforded compounds in which log P is reduced by 1.7-2.3 units while potency in human whole blood in vitro was maintained or enhanced relative to 4f. In addition, the oxoheterocyclic replacements provided compounds with improved oral potency and the preferred compound from this group is 6-[[3-fluoro-5-(4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4- yl)phenoxy]methyl]-1-methylquinol-2-one (4y). In the in vitro systems, 4y inhibited LT formation with IC50s in mouse macrophages and human whole blood of 3 nM and 0.02 microM, respectively. 4y did not inhibit the synthesis of cyclooxygenase (CO) products at concentrations up to 500 microM in human blood, a selectivity for 5-LPO over CO of greater than 20,000-fold. In the rat 4y inhibited the formation of LTB4 in blood ex vivo and in inflammatory exudate with ED50s 3 h after oral dosing of 0.9 and 0.3 mg/kg, respectively. 4y was more potent in vitro in human whole blood and in rat blood ex vivo at 3 h than either the 5-LPO inhibitor A-64077 or the FLAP antagonist MK-886. Based on these data 4y (ICI D2138) has been entered into development as an orally active, selective 5-LPO inhibitor for clinical evaluation in inflammatory conditions in which LTs are believed to play a role.

Pre-clinical pharmacology of ICI D2138, a potent orally-active non-redox inhibitor of 5-lipoxygenase.

1. This paper describes the pre-clinical pharmacology of ICI D2138, a potent orally-active non-redox inhibitor of 5-lipoxygenase which is undergoing clinical evaluation. 2. ICI D2138 potently inhibited leukotriene synthesis in murine peritoneal macrophages (IC50 = 3 nM) and human blood (IC50 = 20 nM). In human and dog blood, ICI D2138 did not inhibit thromboxane B2 synthesis at a concentration of 500 microM, thus the selectivity ratio (cyclo-oxygenase: 5-lipoxygenase) was greater than 20,000. In contrast, zileuton (a 5-lipoxygenase inhibitor also undergoing clinical evaluation) exhibited a selectivity ratio of 15-100. 3. ICI D2138 potently and dose-dependently inhibited ex vivo leukotriene B4 (LTB4) synthesis by rat blood with ED50 values of 0.9, 4.0 and 80.0 mg kg-1 p.o. at 3, 10 and 20 h respectively after dosing. Similar activity was observed for inhibition of LTB4 production in a zymosan-inflamed rat air pouch model. Zileuton produced ED50 values of 5 and 20 mg kg-1 at 3 and 10 h respectively. 4. Oral administration of 1, 3 or 10 mg kg-1 ICI D2138 to dogs produced maximal inhibition of ex vivo LTB4 synthesis by blood for 5, 9 and 31 h respectively. A dose of 5 mg kg-1 p.o. of zileuton caused maximal inhibition of LTB4 for 24 h. 5. Oral administration of 10 mg kg-1 ICI D2138 caused total inhibition of LTB4 production in zymosan-inflamed rabbit knee joint. 6. Topical administration of ICI D2138 to rabbit skin caused a dose-related inhibition of arachidonic acid-induced plasma extravasation with an ID30 of 1.08 nmol per site. Zileuton was approximately 40 times less potent.7. Oral anti-inflammatory activity was assessed in an arachidonic acid-induced mouse ear oedema model in animals treated with indomethacin to block pro-inflammatory prostanoids. ICI D2138, given orally, caused dose-dependent inhibition of oedema with an approximate ID50 of 1.8 mg kg'. Zileuton was approximately 10 times less potent.8. ICI D2138 caused a dose-dependent inhibition of antigen-induced broncho-constriction in guineapigs with an approximate ID50 of 0.1 mg kg-', i.v. Zileuton was approximately 10 times less potent.9. In view of the pharmacological profile described here, ICI D2138 has the potential to provide improved clinical efficacy compared to existing lipoxygenase inhibitors such as zileuton.

Long-term follow-up of partial limb amputation in 13 horses.

Thirteen cases of partial limb amputation in horses were reviewed. Nine horses (69%) survived 18 to 111 months (mean, 41 months). Four unsuccessfully treated horses were euthanatized within 21 days of surgery. The horses' ages at amputation were 4 to 13 years (mean, 7.3 years). There was no predilection for the left or right limb nor for fore or hind limb involvement. Osteomyelitis in six horses (46%) was the most common cause for amputation. Severe, open, comminuted fractures of the third metacarpal bone that were impossible to stabilize by any currently available technique in four horses (31%) were the second most common cause for amputation. One gelding was salvaged because of his sentimental value, two horses were used as breeding stallions, and six were broodmares. One stallion lived 30 months, completed two breeding seasons, and started a third. The other stallion began breeding, but became sterile. Three of the mares have produced five foals. One mare died while in foal. One mare aborted near-term twin fetuses and died of uterine hemorrhage. One mare aborted twice before the femoral head ligament on the contralateral limb ruptured, and she was euthanatized.

Clinical chemistry of staphylococcal enterotoxin poisoning in monkeys.

Clinical chemistry values were examined in 90 monkeys administered a purified preparation of staphylococcal enterotoxin, type B, intravenously. These studies showed an early release of epinephrine accompanied by a mild increase in blood glucose. This was followed by progressively developing prolonged hypoglycemia. An early increase in bloodurea nitrogen occurred, presumably as a result of both prerenal azotemia and functional renal failure seen in association with the observed hypotension. Serum protein, Ca, and Cl concentrations decreased with time. Pi levels increased, whereas Na and K concentrations in serum remained unchanged. Serum enzyme concentrations were unchanged, with the exception of serum glutamic oxaloacetic transaminase, which rose rapidly when compared with prechallenge control observations or with values from sham-challenged monkeys. These changes were statistically significant. These results suggested that enterotoxin administered intravenously produced early change in glucose metabolism, possibly related initially to catecholamine release and later to increased utilization of glucose and metabolic acidosis. Other findings were compatible with tissue breakdown at as yet undetermined locations and with loss of endothelial membrane integrity, as evidenced by loss of protein from the vascular space.

Pathogenesis of lethal shock after intravenous staphylococcal enterotoxin B in monkeys.

The pathogenesis of shock in the rhesus monkey given intravenous staphylococcal enterotoxin B (SEB) is not understood. Several cardiovascular changes produced by a highly purified preparation of SEB were studied after administration of doses ranging from 50 to 1,000 mug/kg. Irreversible arterial hypotension was found consistently at the higher doses. Arterial blood pressure and cardiac output declined substantially as shock developed. Total peripheral vascular resistance did not rise at any time, but showed a significant fall during the late stages of shock. Portal and central venous pressures remained essentially unchanged. Venous O(2) content and pO(2) declined gradually throughout the period of toxemia, but arterial O(2) content remained constant until just prior to death, when a slight fall was noted in some monkeys. These changes were consistent with a pooling of blood in the peripheral vascular beds and seemed to resemble cardiovascular responses reported to occur in monkeys during shock due to bacterial endotoxin. Epinephrine, administered in the late stages of shock, caused arterial pressure to increase almost immediately and cardiac output to return to normal about 1 min later. Although life could occasionally be prolonged for several hours by continuous or intermittent epinephrine infusions, this therapy never succeeded in reversing the lethal effects of high doses of SEB.

A modified Cloward's technique for arthrodesis of the normal metacarpophalangeal joint in the horse.

A modified Cloward's technique was performed for arthrodesis of one metacarpophalangeal (MCP) joint in eight horses. Dorsal arthrotomies were performed medial and lateral to the common digital extensor tendon and two 16 mm holes were drilled through the joint. A perforated cylindrical stainless steel basket filled with cancellous bone was impacted into each hole. The limbs were supported in casts for 8 weeks. The joints were examined and radiographed at 4 weeks, 8 weeks, 6 months, and 10 months. One horse was euthanatized at week 14 to assess the progress of the arthrodesis. In the other seven horses, there was clinical fusion at month 6. Dynamographic evaluations were performed 11 months after surgery at the walk and trot. The maximum vertical forces exerted during weight bearing by treated and control limbs were compared. No difference was detected at the walk; however, a significant difference was present at the trot (p less than 0.05). It was calculated that at the trot the horses placed 90% as much force on the treated limb as on the control limb. Eleven months after surgery, the baskets contained compact and cancellous bone. Ingrowth of bone occurred through all openings, completely filling the baskets and fusing the joints.

Suspensory apparatus prosthesis in the horse. Part 1: In vitro mechanical properties.

Mechanical properties of equine suspensory apparatus preparations and three braided synthetic prostheses were evaluated in vitro. Force versus displacement plots and failure modes were recorded from single load-to-failure testing in 18 cadaver limbs before and after replacement of each suspensory apparatus with a prosthesis. Mean load at failure, energy to failure, and stiffness values of polytetrafluoroethylene (PTFE) prostheses were lower than those of the suspensory apparatus and aramid prosthesis. The PTFE prosthesis failed by elongation or rupture of the prosthesis. Mechanical properties of the aramid prosthesis with collar augmentation were not significantly different from the suspensory apparatus. The main site of failure in both aramid prostheses was at the screw fixation to bone.

Cryotherapy precision. Clinician's estimate of cryosurgical iceball lateral spread of freeze.

OBJECTIVE: To examine physicians' ability to estimate the lateral spread of freeze (LSF) of a cryosurgical iceball using three techniques. DESIGN: A nonrandomized control trial of in vitro nitrous oxide cryosurgical procedures. SETTING: Primary care residency training programs. PARTICIPANTS: A convenience sample of 80 resident and faculty physicians from four family practice residency programs and one obstetrics and gynecology residency program. INTERVENTIONS: After performing cryosurgery with standard naked-eye and colposcopic-assisted techniques, subjects used a new experimental cryosurgical iceball gauge (CIG) to estimate the LSF during cryotherapy. MAIN OUTCOME MEASURES: LSF estimations reported physicians were compared simultaneously with those measured by an observer. RESULTS: The mean (+/- SD) LSF estimation errors at the termination of freeze were as follows: 2.62 +/- 2.42 mm for the colposcopy technique, 2.00 +/- 2.16 mm for the naked-eye method, and 1.28 +/- 0.87 mm for the CIG technique. The range of maximum error was 6.5 to 11 mm for the colposcopic technique, 5.5 to 12.5 mm for the naked-eye method, and 3.0 to 4.0 mm for the CIG technique. CONCLUSIONS: Overestimation of the LSF, which increases the risk of undertreatment and residual disease, was more common than underestimation. The CIG minimized perceptual error and provided the best cryosurgical precision.

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.

Structure and activity relationships leading to the discovery of ICI D2138, a selective, potent and orally active inhibitor of 5-lipoxygenase.

Structure and activity relationships of (methoxyalkyl)thiazole and 4-methoxytetrahydropyran series of 5-lipoxygenase inhibitors are reviewed. One member of the 4-methoxytetrahydropyran series, 6-([fluoro-5-(4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl)phenoxy]methyl) -1- methylquinol-2-one (ICI D2138), is undergoing clinical evaluation.