4,439,521 3/1984 Archer et al. ...................... 435/240.23 4,743,552 5/1988 Friedman et al. ... . 435/240.23 4,829,000 5/1989 Kleinman et al. ... 435.240.23 4,902,508 2/1990 Badylak et al. ...... ... 424.795 4.958.178 9/1990 Badylak et al. ........................ 424,551 5,266.430 11/1993 Naughton et al. .... 435/240.243 5,275.828 1/1994 Badylak et al. ........................ 424/55 5,281,422 1/1994 Badylak et al. ...... ... 424/55 5,352.483. 10/1994 Badylak et al. ...... ... 424,551 5,372,821 12/1994 Badylak et al. ...... ...... 424/55 5,478,739 12/1995 Slivka et al........... 435.240.23 5,518.915 5/1996 Naughton et al. ................. 435/240.23

Diarylsulfonylureas, such as N-(4-chlorophenyl)aminocarbonyl-2,3-dihydro-1-indene-5-sulfonamide (LY186641, Sulofenur) and N-(4-chlorophenyl)aminocarbonyl-4-methylbenzene sulfonamide (LY181984), have been shown to be effective antitumor agents in a variety of in vivo and in vitro animal models. Their mechanism of action is unknown but does not appear to be the result of nonselective destruction of actively dividing cell populations. Mitochondria have been shown to accumulate Sulofenur and therefore may be targets of drug action. The purpose of these investigations was to examine the effects of a variety of diarylsulfonylureas in mitochondria and attempt to determine the relevance of these changes to antitumor activity. Many of the diarylsulfonylureas which were effective antitumor agents in animal models were also uncouplers of mitochondrial oxidative phosphorylation. They increased state 4 respiration and dissipated the mitochondrial membrane potential in a concentration-related fashion. The mechanism of uncoupling appeared to be related to a dissociable hydrogen ion as these molecules had pK(a) values that ranged from 6.0 to 6.2 and were highly lipophilic. Thus, the uncoupling action appears to be the result of hydrogen ion translocation. The mechanism of antitumor activity does not appear to be the result of uncoupling as no correlation was evident between inhibition of cell growth and uncoupling action of a variety of active and inactive diarylsulfonylureas. In vitro, Sulofenur is cytotoxic at high concentrations and inhibits cell growth at lower concentrations in the absence of any overt cell kill. The inhibition of cell growth also did not appear to be related to the uncoupling action of these drugs. In contrast, uncoupling may have played a partial role in the early, high exposure cell kill that can occur with these compounds.

Cell populations derived from adult rat bone were grown in cell culture and characterized with respect to their morphology and response to hormones. The cells were isolated from adult rat calvaria by mechanical rather than enzymatic methods. Cultures were initiated in modified BGJb medium supplemented with fetal bovine serum. These cultures and several cloned populations derived from them retained the ability to mineralize in vitro even after extended serial passage.

Vlndesine(VDS; deacetyl vinblastineamide sulfate) giveni.p. dailycompletelyinhibitedthe growthof boththe Ridgewayosteogenic sarcomaat 0.4mg/kgandthe Gard ncrIymphosarcóma at 0.25mg/kg.Incontrast,the parent alkaloid, vinbbastine (VLB), at 0.5 mg/kg was inactive againstthesetwotumors.VDScausedinhibition ofgrowth of the Meccalymphosarcoma thatwascomparableto the inhibitionby vincristine(VCR),(60versus68%)whileVLB caused 41% inhibition.The Ca115 Shlonogimammary carcinomadidnotrespondto VDS,butbothVLBandVCR causedinhibitions of 62and67%,respectively. TheCa755 mammaryadenocarcinoma showeda moderateresponse to VDS (46%)and VCR (39%),and no responseto VLB. The Sarcoma180 ascitestumor(solid,s.c.), the X5563 myeloma,and the C3H mammarycarcinomawere not responsive to VDS,VLB,or VCR. VDScauseda 113%prolongation of life in micewiththe B16melanoma(i.p.), and there were three of ten 45-day survivorsin one study,andten of ten 45-daysurvivorsin anotherstudy.VLBhadtwoof ten andeightof ten 45-day survivorsin the same studies.Animalsinoculatedwith the P388leukemiaandthe Walker256andEhrlichascites tumorshad greater than 100% prolongation of life with manyindefinitesurvivorswhentreatedwithVDS,VLB,or VCR. The L1210, AKR, L5178Y,and C1498 leukemias showsdnoresponseto VDSgivendailyat I.0 mg/kg. In tissueculturesof Chinesehamsterovarycells, the minimum effectiveconcentration of VDSthatcaused10to 15%accumulation of cellsin mitosiswas2.3 x 1O@N, for VLBit was 2.2 x 1O@M,andfor VCRft was 7.3 x 1O@M. The concentration of VDSneededfor 40 to 50%accumu lationof cells in mitosiswas 2.8 x 1O@M;for VLBit was 8.3 x 1O-@ M; and for VCR ft was 2.4 x 1O@N.

Beating myocardial cells from 1 to 5-day old mice maintained in tissue culture can convert [U-14C]glucose, [2-14C]pyruvate and [1-14C]octanoate to carbon dioxide. Single component porcine insulin stimulated only the oxidation of pyruvate without enhancing glycogen or lactic acid formation. The effect of insulin on pyruvate does not require glucose. It is suggested that insulin increases the oxidation of pyruvate by the activation of pyruvic dehydrogenase in these beating heart cells.