IL36587A - Compositions containing pyridine derivatives and their use in a method of modifying cellular surface reactions - Google Patents

Description

COMPOSITIONS CONTAINING PYRIDINE DERIVATIVES AND THEIR USE IN A OF CELLULAR SURFACE REACTIONS It has been discovered that cellular surface reaction can be by bringing the coll into reactive a reagent which is capable of blocking the sulfhydryl groups of the cell It has been more that the of tumor metastasis and invasiveness in living mammal can be inhibited by administering to said mammals a eagent which is capable of preferentially reacting with peripheral sulfhydryl groups present about mammalian cells rath with those sulfhydryl groups are present within the the reagent employed is one in additi to blocking the peripheral sulfhydryl groups of the also has the effect of modifying the electric charge cell It has also been discovered various types of dithio all having the structure each represents an organic have the ability to react preferentially the peripheral sulfhydryl groups present about the those of said dithio compounds wherei the R radicals incorporate groups for as which have a negative charge under pH conditions above about have the ability to modify the electric charge cell It has previously been disclosed that in experiments homogenates of Ehrlich ascites cells were found to react with acid to an ximately that of intact It has also been disclosed that in experiments basic thione forming disulfides were found to have an effect on the carbohydrate metabolism of said cells and specifically to inhibit glycolysis and respiration while acidic disulfides and most of the have little or no It has now been discovered that cotinic aoid and their sodium salts have unusual characteristics which make them particularly well adapted to be employed in mammalian systems for the reversible blocking of peripheral cellular sulfhydryl More said compounds appear to be essentially while also having the ability to react tially with peripheral rather than with intracellular hydryl said compounds have the desired ability to modify the electric charge of the treated sell The afiresaid according to the are capable of reacting with a sulfhydryl group to a thione said compounds thus being referred to as typical reaction which takes place between acid and a peripheral group on a cell can be represented as where the reaction roduct not linked to the cell is It has also been disoovered that when treating mammaliar cells with acid or their it is possible to reversibly block or essentially of the peripheral sulfhydryl groups without altering the rate of cellular respiration and glycolysis to any Significant the blocking reaction is a reversible when said reaction occurs the body of a living mammal the blocked groups return to their original form within a relatively short period of time several as a result of conventional metabolic assuming no further amounts of the cyclic reagent are available for reaction in the The tion of a thiol of the type found in mammalian systems for as will ration of free peripheral groups at a rapid deblocking wherein glutathione is indioated by oan be illustrated as follows using cotinic acid as the reagents Repeated continuous administration of the sonicotinio acid and acid or other organic disulfide reagent to the mammal of maintain the cellular system in any desired condition of peripheral aulfhydryl group blocking without damage to the order to determine which reagents are capable of preferentially reacting with peripheral eellular an excess of the reagent can be reacted first with a mass of cells and then with a lime mass of or comminuted cells of the same When the relative amount of the product compound the thione which is formed by reaction with whole cells very substantially lower than the amount formed during reaetion with the the desired preferential attribute is This method will be described below in Example Ehrlich ascites tumor cells were harvested from to transplants in female Swiss mice and then washed free of blood in the manner described below in Example One portion of cells was then left as while the other was verted to a homogenate in which essentially no whole cells were 15 samples of the intact and of the homogenized cells were incubated for 1 hour at with 5 of buffer containing 5 of one or another of the disulfide reagents enumerated below in Table with shaking in This amount was sufficient to provide a system containing the reagents a concentration of 1 X 10 After filtration through glass paper the amount of thione produc was determined from the absorbance at the wavelength indicated the In the case of 2 2 the amount of thione product formed by reaction with whole cells wa at least as high as that formed from the homogena the higher value obtained in the case of cells being attributable to enzymatic action occurrring in inta or active Each of these values was then as in Table 1 The other two reagents gave approximately this same amount of thione product when reacted w the the amount of thione provided by these two reagents when reacted with whole cells was but 20 percent o value obtained the 2 Ti indicates that these selectively thenselves to peripheral rather than to intracellular ulfhydryl These data are expressed bel in Table 1 Reagent Cha V or r 6 r 2 In op rations conducted in the but using sodium thiod nicotinic acid and of 2 2 rather than the free the thione product values obtained are essentially the same as those reported in Table 1 2 Ehrlich ascites fluid was harvested from to transplants in female Swiss the cells were separated by pentrifugation at low then suspended in KRP buffer Ringer phosphate buffer at centrifuged The cells were then KRP buffer for several times in the presence of heparin to remove red bloo The cells were in the KRP buffer to yield X cells in 10 of buffer and then incubated for 5 minutes at in the absence and presence mol of 6 6 this being sufficient to provide the system with a 10 concentration of said A portion of the resulting mixture was then used as such in the manometric The remainder of the cells were then separated by washed twice with the buffer and then resuspended in the same buffer to form a suspensio containing approximately 150 X which was then used for manometric In said the experiments were run fo one at using the method described in detail in the Effect of Some Pyridine Derivativ on the Carbohydrate Metabolism of Ehrlich Ascites et Journal of Medicinal Chemistry except that the present tests no further heparin was employed after the initial as described The results EFFECT TREATMENT ITH 6 ACID ON RESPIRATION AND O ASCITES TUMOR CELLS Ehrlich ascites cells 4 Ehrlich ascites presence of CPDS 8 ehrlich ascites treated 7 5 with then washed The data of the above Table 2 show that of Ehrlich Ascites cells with acid at concentration of 10 followed by does not alter rates of respiration and Respiration and glycolysis of Ehrlich ascites cells in the presence of acid are also essentially When the foregoing operation is repeated using thiodipyridine as the evolution of and stantially ceases afte several thus indicating that his reagent is strongly inhibitory of cell glycolysis and respiration and thus is cell Example 3 This operation was conducted to the degree persistence of the action in the living Swiss mice which had been inoculated with ascites cells 6 days before the were each injected intraperitoneal with aqueous solution containing 6 of acid sodium this dosage being equivalent to 300 of the reagent per lcilo of body This salt had been prepared by adding the acid o an aqueous solution of sodium bicarbonate present in the amount stoichiometrically required to neutralize the acid and thus the same in the aqueous The latter was then brought to a of by addition of as before being injected into each As indicated in Table 3 a certain number of mice were sacrificed at intervals of 0 and 20 hours after the reagent was After the indicated the ascites fluid was withdrawn from animal and ccntrifuged to separate the cells then 5 with lot of recovered number of which in mou e averaged 1 2 X treated with a solution of 5μ mole of cysteine resulting product was centrifuged minutes at 800 X following which the supernatant was denatured for 5 at bears a negative charge at said physiological pH The mice forming the subject of the above experiment showed no signs of discomfort when injected with the acid in a test conducted in same but using a of the acid a gum arabic solution in buffer at the mice employed in the test exhibited drowsiness for several at the end of 4 hours after it was found that 65 percent of the peripheral about the ascites cells still remained This is attributable to the fact that injected being was taken up at a slow rate and thus was able to effect a continuing reaction with the peripheral cell groups as it was slowly converted in vivo to the soluble In still another test run as a the mice were injected with a saline of and here again the symptoms of drowsiness were thus showing that the gum arabic bove was nota causative Each of 6 healthy free of any tumor or other were injected intraperitoneally with 10 mg per day of thiodinicotinic acid sodium salt for 21 consecutive equivalent to a daily dosage of 450 mg per kilo of body The injected chemical was in the as described above in Example 3 The mice exhibited no visible of drowsiness or other ill effect during the period of the test or thereafte appetites remained good and no weight loss Ehrlich ascites tumor cells were withdrawn from one or more day ascites the cells counted and diluted with physiological saline to an appropriate centrations between about and cells per ml were The preferred concentration was about Cell suspension was injected into the brain of using a Hamilton syringe and 25 gauge needle Part of the needle was vered by polyethylene tubing so as to limit the penetration of the needle into the brain to 6 The needle was inserted into the midbrain through squamopet osal fissure The mice were divided into two groups of 24 mice Those of group A received an injection IP of 2 thiodinicotinic acid in sodium bicarbonate solution every 4 hours starting within not less than one hour and not more than 3 hours after the intracerebral and for 7 consecutive days and or 4 injections Each mouse of the second group received an injection of physiological saline solution every 4 given at the same time as the injections of group for 7 consecutive days and or 42 At the end of 7 the mice were sacrificed and their lungs excised and Two portions of about 80 milligrams of minced lung tissue from each mouse were implanted neously in the retroscapular area of a healthy on both We thus had 24 mice containing implants of lungs of group A and 24 mice containing 48 implants of lungs of group B These mice were kept without any special treatment for 30 then sacrificed and any tumors that had formed at the site of the lung implants were excised and A tumor weighing 100 or more was considered a the lung tissue from which it originated contained a The results obtained in this experiments are given of Takes Average Weight 1 of Implants Takes of Tumors Group B 67 179 Group A 17 59 Average total weight of all including those weighing less than or more than 100 These results show that 6 when administered as had the effect of decreasing the number of takes to that of untreated and of creasing the total tumor mass to that of untreated It is known that still higher doses of the 6 6 tinic acid which may lead to further are well Example 6 The experiment of Example 5 was and the following results were of Takes Average Weight of Implants Takes of Tumors Group B 64 547 Group A 15 177 Example 7 o of Takes Average Weight of Implants Takes of Tumors Group B 29 133 Group A 7 79 dithionicotinic 2 dithiobisisonico The dosage rate of employed in a practice of this invention can be varied within wide limits and still be effective for the intended In good results can be obtained by nistering the reagent in amounts calculated to maintain a 6 3 concentration in blood of to So the peripheral bonding reagents have utility in modifying and moderating various undesirable reactions involving the cell periphery such as those encountered in tumor metastasis and For an advantageous use of the 6 acid herein described consists in the administration of quate doses at the time of surgery for It is known that manipulation of a tumor during surgery causes numbers of cer cells to enter the blood stream with concomitant high danger of formation of Treatment with the reagent should be continued for an adequate period of time both fore and following surgery until the natural defenses of the organism have destroyed the remaining circulating cancer or 2 acid The reagent employed in a practice of this invention to react preferentially with peripheral groups of cells can be administered to living mammals in any desired fashion for as by injection into the blood stream or into muscle or body Oral nistration may also be The chemical can be employed per se or along with an oleaginous or other vehicle which will show down its rate of absorption into the It is also possible to use a reagent of the foregoing character which is prepared in radioactive form by incorporating one more sulfur or tritium By use of this practice the efficiency and efficacy of the ve elements are greatly enhanced inasmuch as they become insufficientOCRQuality

Claims (1)

1. t. A composition consisting essentially of a solution of a compound selected from the group consisting of 6,6'^ithiodinicetinic acid, 2,2'-dithiobisisoniootinio acid and their sodium salts in a pharmaceutically acceptable diluent, being formulated for use as an agent for blocking tumor metastasis and invasiveness. 2· A composition, as claimed in Claim 1 » vherein the diluent is a physiologically acceptable buffer solution having a pH within the range 7.2 ± 0.2» Tel-Aviv, April 7, 1971