Effects of Seocalcitol (EB1089) on nitrosomethyl urea-induced rat mammary tumors.

Although 1,25-dihydroxyvitamin D3 is a potent cell-differentiating agent, its use in cancer prevention or therapy is precluded because it induces hypercalcemia. Synthetic analogs have been developed which inhibit tumor progression in animal models of breast cancer. One analog, Seocalcitol (EB1089) has been shown to be effective in causing regression of N-methyl-nitrosourea-induced rat mammary tumors. However, at the most effective oral dose, a significant increase in serum and urinary calcium levels were observed. In order to compare the efficacy of different dosing schedules of Seocalcitol, rats were treated either 6 times weekly (1 microg/kg) or by intermittent dosing to achieve the same total weekly dose. All dosing schedules of Seocalcitol were effective in inhibiting tumor progression. Once daily dosing was significantly more effective than intermittent dosing but was associated with a greater rise in serum calcium concentration. In order to evaluate alternative treatment strategies to limit calcemic effects, we assessed the efficacy of limiting vitamin D-induced hypercalcemia using bisphosphonates. Seocalcitol (2.5 microg/kg daily p.o. for 4 weeks) alone and in combination with pamidronate (APD 0.4 mg/kg per day s.c.) or the same dose of the bisphosphonate EB 1053 caused substantial tumor regression. No statistically significant difference was seen between combination treatment and Seocalcitol treatment alone. Co-treatment with APD or EB 1053 did not limit the rise in serum calcium induced by Seocalcitol alone. Cessation of treatment or administration of a lower dose (1microg/kg twice weekly) reversed hypercalcemia, hypercalciuria and weight loss induced by high dose Seocalcitol. However, reduction in tumor volume was maintained in the majority of animals.

CHS 828, a novel pyridyl cyanoguanidine with potent antitumor activity in vitro and in vivo.

A new class of recently discovered antineoplastic agents, the pyridyl cyanoguanidines, exert a potent antitumor activity in rodents after oral administration. Optimization in vitro and in vivo has resulted in the selection of the lead candidate CHS 828 (N-(6-chlorophenoxyhexyl)-N'cyano-N"-4-pyridylguanidine). CHS 828 was found to exert potent cytotoxic effects in human breast and lung cancer cell lines, with lesser effects on normal fibroblasts and endothelial cells. In a study using a panel of cell lines with different resistance patterns, the effects of CHS 828 showed a low correlation with the activity patterns of known anticancer agents, and no sensitivity to known mechanisms of multidrug resistance was observed. In nude mice bearing human tumor xenografts, CHS 828, at doses from 20 to 50 mg/kg/day p.o., inhibited the growth of MCF-7 breast cancer tumors and caused regression of NYH small cell lung cancer tumors. Oral administration of CHS 828 once weekly improved efficacy without increasing toxicity. CHS 828 was found to compare favorably with established chemotherapeutic agents such as cyclophosphamide, etoposide, methotrexate, and paclitaxel. In mice with NYH tumors, long-term survival (>6 months) was observed after treatment with CHS 828 was stopped. In conclusion, CHS 828 is an effective new antitumor agent, with a potentially new mechanism of action. CHS 828 is presently being tested in Phase I clinical trials in collaboration with the European Organization for Research and Treatment of Cancer.

Discovery of OT4003, a novel, potent, and orally active cys-LT1 receptor antagonist.

The present paper describes the structural modifications leading to the discovery of a new series of quinoline-containing cys-LT1 receptor (LTD4 receptor) antagonists. A structural optimization with respect to the in vitro receptor binding, the in vivo brochoconstriction, and the toxicological effect in the form of peroxisomal proliferation was performed in order to achieve the target compound OT4003. OT4003 ((S)-(+)-E-2-(3-(2-(7- chloroquinolin-2-yl)ethenyl)phenylaminomethyl)-phenoxyl++ +-hexanoic acid) was found to be a potent and selective inhibitor of [3H]LTD4 specific binding to guinea pig lung membranes (IC50 2.4 +/- 1.0 nM), and also a potent, orally active, antagonist of LTD4 induced bronchoconstriction in guinea pigs [ED50 0.14 (ED16 0.1-ED84 0.4) mg/kg; 4 h pretreatment]. The enantiomerically pure OT4003 was prepared using a short convergent synthesis, including an enzymatic resolution step.

Effect of 1alpha-vitamin D3 and estrogen therapy on cortical bone mechanical properties in the ovariectomized rat model.

It is well documented that both bone mass and size of ovariectomized rats can be increased by 1alpha-vitamin D3 therapy. The repercussion of this therapy on bone mechanical competence is far less clear. Therefore, the objective of this study was to examine the mechanical properties of the shaft femur in ovariectomized rats (3 months old) receiving estrogen (0.25 mg/kg-week) and /or 1alpha-vitamin D3 (0.5 microgram/kg-day). The medication was given during 6 months starting immediately after ovariectomy or starting 3 months later. Torsional testing was performed from which the parameters strength, stiffness, maximum angular displacement, and energy-absorbing capacity (toughness) were derived. Multiple regression models were generated to estimate the relative importance of the therapies on bone mechanical properties. Bone stiffness increased with age. Ovariectomy improved bone mechanical parameters until 6 months postovariectomy, whereas estrogen treatment resulted in similar mechanical properties as those in intact age-matched controls. A significant improvement of all mechanical parameters was observed after 1alpha-vitamin D3 therapy. The combined therapy of 1alpha-vitamin D3 and estrogen was less effective than 1alpha-vitamin D3 alone, but better than estrogen therapy alone, suggesting interactive effects between both therapies. We conclude that 1alpha-vitamin D3 treatment of ovariectomized rats improves bone mechanical competence, which might be partially related to alterations in both bone mass and size.

Effect of 1 alpha-vitamin D3 on bone strength and composition in growing rats with and without corticosteroid treatment.

The effects of 1 alpha-vitamin D3 were studied for 6 months in 2-month-old male and female rats on a moderately low calcium diet with or without low-dose prednisolone treatment. Both cortical bone mechanical and biochemical properties were examined. Femoral bone specimens were subjected to torsional loading tests. With age, bone strength and stiffness increased in both sexes, accompanied by an increased degree of mineralization (bone ash and calcium concentrations). During growth, strength and stiffness increased more in male than in female rats. When 1 alpha-vitamin D3 (0.5 micrograms/kg/day) was given alone, bone mechanical competence improved significantly whereas insulin-like growth factor-I (IGF-I) and calcium concentrations in the bone matrix were significantly reduced. Treatment with low-dose prednisolone (0.5 mg/kg/day) alone did not influence bone mechanical properties compared with intact control rats (without prednisolone) although a significant reduction in calcium concentration and an increased phosphorus concentration were measured. A combined therapy with prednisolone and 1 alpha-vitamin D3 significantly increased bone strength, toughness, and stiffness compared with control bones. Both mineralization degree (ash and calcium concentration) and IGF-I concentration were decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Disodium 1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphonate (EB-1053) is a potent inhibitor of bone resorption in vitro and in vivo.

The ability of the new nitrogen-containing bisphosphonate disodium-1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphona te (EB-1053) to inhibit osteoclastic resorption was examined in vitro and in vivo. Results were compared to those obtained with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (pamidronate or APD). In vitro, when tested in osteoclast precursor-dependent systems (fetal mouse metacarpals and a coculture system), EB-1053 suppressed 45Ca release effectively and was found to be about 10 times more potent than pamidronate (ED50 = 2.5 x 10(-7) versus 2.5 x 10(-6) M, respectively). The EB-1053-inhibited osteoclastic resorption could be reversed by treatment with parathyroid hormone (PTH). In vivo, daily subcutaneous injections of EB-1053 to young growing rats for 7 days increased metaphyseal bone mass in tibiae dose dependently. In these experiments EB-1053 was about 50 times more potent than pamidronate. These studies show that EB-1053 is a very potent bisphosphonate that has potential use in the treatment of skeletal disorders.

Prevention and treatment of osteopenia in the ovariectomized rat: effect of combined therapy with estrogens, 1-alpha vitamin D, and prednisolone.

The effects of estrogens and 1-alpha were studied in young animals after ovariectomy (OVX) and/or prednisolone (PDN). These medications were given separately or in combination as preventive therapy from the start of the experiment, and as curative therapy starting 3 months later. Changes in bone mass were evaluated by single photon absorptiometry of the femur at the diaphysis (containing mostly cortical bone) and at the distal end of the femur (containing mostly trabecular bone). Radiogrammetry was performed at 50% of the length of the femur. Estrogens prevented further bone loss after OVX and OVX + PDN, given either at the beginning of the experiment or started 3 months later, except for trabecular bone loss immediately after OVX + PDN. After 1-alpha vitamin D, a highly significant increase in BMC and BMD was found in controls, in animals treated with PDN, and after OVX and OVX + PDN. The combination of 1-alpha with estrogens was less effective than 1-alpha but more effective than estrogens alone. After correction for body weight changes globally the same results were found. We conclude that (1) estrogens prevent bone changes after ovariectomy and ovariectomy + prednisolone; and (2) 1-alpha vitamin D highly significantly increased bone mass in male and female rats, and after prednisolone treatment, ovariectomy, and ovariectomy + prednisolone treatment.

Effect of ovariectomy and prednisolone on bone mineral content in rats: evaluation by single photon absorptiometry and radiogrammetry.

The effects of ovariectomy and prednisolone were studied for 9 months in 3-month-old rats on a moderately low calcium diet. Measurements were made by single photon absorptiometry of the femur at cortical and trabecular bone sites. Radiogrammetry was performed at the midshaft of the femur. During growth, body weight, bone size, and bone mineral content (BMC) increased in male rats more than in females. After ovariectomy, body weight increase was more pronounced, but bone mineral increase was lower than in controls. At the distal end of the femur, bone mineral density decreased after 3 months, and at the midshaft of the femur, medullary width increased significantly from 6 months on. Prednisolone in a dose of 0.5 mg/kg/day did not influence BMC. However, prednisolone treatment after ovariectomy induced a more pronounced effect on bone than ovariectomy alone. BMC increase was lower than in ovariectomy, and bone mineral density decreased significantly, especially at the distal end of the femur. After correction for differences in body weight, globally the same results were found. We conclude that (1) the combination of single photon absorptiometry and radiogrammetry allows the evaluation of growth and the effect of ovariectomy and prednisolone treatment in rats; (2) ovariectomy results in bone loss first at the distal end and later in the midshaft of the femur; (3) prednisolone in a dose of 0.5 mg/kg/day alone did not affect bone mass; and (4) prednisolone profoundly enhanced the effects of ovariectomy.

Effects of a novel vitamin D analogue MC903 on cell proliferation and differentiation in vitro and on calcium metabolism in vivo.

MC 903 is a novel vitamin D analogue which has been tested for its effects on cell differentiation and cell proliferation in vitro using the human histiocytic lymphoma cell line U937, and on calcium metabolism in rats in vivo. In the present investigation MC 903 was compared to the natural metabolite of vitamin D3, 1 alpha,25-dihydroxycholecalciferol [1,25(OH)2D3] and to its synthetic analogue 1 alpha-hydroxycholecalciferol [1 alpha (OH)D3]. MC 903 was found to be a potent inducer of cell differentiation and to inhibit cell proliferation and DNA-synthesis in concentrations comparable to those observed with 1,25(OH)2D3. 1 alpha (OH)D3, which is only active after metabolic conversion to 1,25(OH)2D3, was more than 100 times less potent. Oral or intraperitoneal administration of MC 903 to rats showed that the compound was at least 100 times less active than 1,25(OH)2D3 and 1 alpha (OH)D3 in causing hypercalciuria, hypercalcemia and bone calcium mobilisation. The low vitamin D activity of MC 903 was further confirmed by administration of the compound to rachitic rats. The strong direct effects of MC 903 on cell proliferation and cell differentiation, coupled with its decreased activity as a classical vitamin D makes this compound an interesting candidate for studies in human proliferative disorders such as psoriasis.

Captopril(SQ 14,225): in vitro and in vivo influence on the proliferative response of rat lymphocytes.

Captopril in vitro (50-500 micrograms/ml) increased 3H-TdR incorporation in unstimulated and mitogen-stimulated cultures of rat lymphocytes. Unseparated spleen and lymph node cells of rats orally treated with captopril (50 mg/kg/day x 4)) showed decreased basal and mitogen stimulated 3H-TdR incorporation. The removal of macrophages abrogated this inhibitory effect. Leucine aminopeptidase activity of macrophages was reduced - in vivo and in vitro - by captopril.

Antirheumatic drugs and macrophage function: effects on tumour cell growth in vitro.

The ability of the antirheumatic drugs D-penicillamine, chloroquine and levamisole to modify macrophage-mediated inhibition of tumour cell growth in vitro was investigated. Increasing numbers of rat peritoneal macrophages were cocultured with a fixed number of ascites hepatoma AH-13 rat tumour cells. Tumour cell growth was assessed as the uptake of 3H-thymidine (3H-TdR) by AH-13 cells at the end of a 24 h period of coculture with macrophages treated in vitro or in vivo with the various drugs. In vitro, preincubation of macrophages with D-penicillamine or chloroquine (50 - 250 micrograms/ml) increased tumour cell 3H-TdR incorporation, compared to cultures with untreated macrophages. Macrophages from rats treated with D-penicillamine or chloroquine (50 mg/kg/day orally) for 4 days similarly increased tumour cell 3H-TdR incorporation, compared to cultures with macrophages from untreated rats. These effects persisted for at least 3 to 4 weeks of treatment. Preincubation with levamisole (10 - 100 micrograms/ml) in vitro had no effect on macrophage-mediated inhibition of tumour cells, whereas increased tumour cell 3H-TdR incorporation was observed in cultures with macrophages from rats treated with levamisole (5 mg/kg/day orally) in vivo. Macrophages from rats with experimentally induced chronic inflammation, i.e. adjuvant arthritis, were found to increase tumour cell 3H-TdR incorporation, compared to macrophages from nonarthritic rats. This trend was further enhanced by treatment with D-penicillamine, chloroquine or levamisole.

The effect of some antirheumatic drugs in vivo on the response of spleen cells to concanavalin A in rats with chronic inflammation.

During the course of adjuvant arthritis in rats adherent spleen cells inhibited the response of spleen lymphocytes to the T-cell mitogen concanavalin A (Con A). The effects of 14 days treatment with various antirheumatic drugs on spleen cell responsiveness to Con A were investigated. Two nonsteroidal anti-inflammatory drugs, indomethacin (1 mg/kg/day p.o.) and acetylsalicylic acid (200 mg/kg/day p.o.) did not modify the spleen cell response, whereas treatment with chloroquine (50 mg/kg/day p.o.) or levamisole (5 mg/kg/day p.o.) further increased the inhibitory effects of the adherent suppressive spleen cells. On the contrary, treatment with sodium aurothiomalate (10 mg/kg/day i.m.), D-penicillamine (50 mg/kg/day p.o.) or pyritinol (50 mg/kg/day p.o.) significantly enhanced the response of the lymphocytes to Con A. In addition to the effects on spleen cell responsiveness, the ability of the various drug treatments to modify the polyarthritic lesions of the disease was investigated. It is suggested that this model may provide a valuable approach for evaluating the effects of antirheumatic drugs in vivo on immunological responsiveness during chronic inflammatory disease.

Chronic inflammation in adjuvant arthritic rats correlates with enhancement of 12-L-HETE-synthesis.

The development of chronic inflammation in adjuvant arthritic rats was found to be strongly correlated with the appearance in serum of a factor (HSF) which enhanced the formation of 12-L-HETE by platelet-lipoxygenase, and with the serum-concentration of 12-L-HETE. The latter was determined by scanning at 235 nm after extraction and high performance thin-layer chromatography. Arthritic rat platelet-rich plasma (PRP) converted exogenous arachidonic acid to 12-L-HETE at a rate 2.6-fold higher than control rat PRP. By resuspending arthritic rat platelets in normal rat plasma, and normal rat platelets in arthritic rat plasma, this increase in conversion rate was found to be caused by HSF present in the arthritic rat plasma. Treatment of arthritis with non-steroidal anti-inflammatory drugs inhibited HSF activity as well as the increase in serum-12-L-HETE concentration, which indicates a prostaglandin-mediated mechanism of HSF synthesis or release.

An unusual profile of activity of a new basic anti-inflammatory drug, timegadine.

Timegadine (SR 1368, N-cyclohexyl-N"-4-(2-methylguinolyl)-N'-2-thiazolylguanidine) dose-dependently inhibited carrageenan-, nystatin-, and concanavalin A-induced edema. Detailed studies in adjuvant arthritic rats showed: (a) long dosing regimen with timegadine inhibited primary and secondary lesions, leukocytosis and hyperfibrinogemia, (b) timegadine was significantly active in reducing the severity of the already established disease, (c) a short course of dosing with timegadine at the time of adjuvant injection permanently prevented the development of secondary lesions. The tuberculin hypersensitivity reaction was enhanced by timegadine in both adjuvant arthritic and normal rats. Experimental allergic encephalomyelitis in rats and guinea pigs was not affected. It is concluded that timegadine has a profile or activity which differs from that of known anti-inflammatory drugs.

Inhibition of adjuvant arthritis by intraperitoneal administration of low doses of silica.

Daily intraperitoneal administration from day 3 to day 10 post-adjuvant of crystalline silica in doses 1.5-3.1 mg/kg per day or of amorphous silica in doses of 12.5 mg/kg per day inhibited the development of adjuvant arthritis mostly suppressing the swelling of the non-injected paw. These doses of silica did not impair the carbon clearance. Higher doses of silica (25 mg/kg per day of the crystalline form and 50 mg/kg of the amorphous form) administered from day 17 to day 24 post-adjuvant had no effect on the already established disease.

Macrophages from adjuvent arthritic rats preferentially synthetize prostacyclin.

Peritoneal macrophages obtained from rats 21 days after induction of adjuvant arthritis and maintained in culture for 20 h in presence of [14C]-arachidonic acid and 10% foetal calf serum were found to have increased capacity for synthetizing prostacyclin and diminished capacity for synthetizing PGE2 compared with macrophages from normal rats. Similar results were obtained when foetal calf serum was replaced by either normal or arthritic rat serum. Orally administered indomethacin inhibited the increased synthesis of prostacyclin.

Immunological effects of D-penicillamine during experimental induced inflammation in rats.

Administration of D-penicillamine (50 mg/kg/day orally) to rats with adjuvant arthritis for up to 42 days significantly modified the incorporation of 3H-thymidine (3H-TdR) in concanavalin A (Con A)-stimulated lymph node cells. Treatment with D-penicillamine abolished the ability of macrophages from arthritic rats to inhibit lymphocyte responsiveness to Con A and lipopolysaccharide (LPS) 14 days after the induction of the disease. Increased T-cell responsiveness to Con A was found from day 14 to 35 in cultures of unseparated and adherent-cell-depleted lymph node cells from D-penicillamine-treated arthritic rats. B-cell responsiveness to LPS was not affected. Experiments with bovine serum albumin gradient-separated lymph node cells confirmed these findings and indicated that treatment with D-penicillamine may specifically enhance T-helper cell responsiveness to Con A. It is suggested that administration of D-penicillamine may interfere with macrophage function during the course of an immunologically induced chronic inflammation, leading to an increased response of T-helper cells. The theoretical implications of these findings are discussed.

Splenic suppressor cells in adjuvant arthritic rats: effect of D-penicillamine.

Adherent spleen cells from rats with adjuvant arthritis inhibit the incorporation of 3H-thymidine into DNA and 3H-leucine into protein in nonadherent spleen lymphocytes, stimulated by the mitogens Concanavalin A and E. coli Lipopolysaccharide. This suppressive activity was abolished by pretreatment of the adherent cells with the selective macrophage toxin silica. It is suggested that suppressor macrophages directly, or through interaction with suppressor T cells, inhibit the response of lymphocytes to mitogens by inhibition of cellular protein synthesis, followed by inhibition of DNA-synthesis and death of about 30% of the lymphocytes. Treatment of adjuvant arthritis rats with D-penicillamine resulted in significantly increased incorporation of 3H-thymidine in spleen lymphocytes, compared to cultures from untreated, arthritic rats. This approach may prove useful in the investigation of cellular interactions in a model of immunologically induced inflammation and provide a tool for the evaluation of the effects of immunoregulatory drugs.

Basic antiinflammatory compounds. N,N',N''-Trisubstituted guanidines.

A variety of basic N,N',N'',-trisubstituted guanidines was prepared and tested for antiinflammatory activity. Compounds with a thiazolylguanidine moiety linked to the 4 position of the 2-methylquinoline ring exhibited fairly high antiinflammatory activity. Optimal activity was associated with the presence of N-cycloalkyl substituents on N''-4-(2-methylquinolyl)-N'-2-thiazolylguanidine. Pharmacological data on N-cyclohexyl-N''-4-(2-methylquinolyl)-N'-2-thiazolylguanidine (SR 1368, 44) are presented and discussed.