Esters of 21-thiol-steroids hydrocortisone and cortisone

BACKGROUND OF INVENTION
The present invention relates to new esters of the 21-thiol steroids having the general formula given at (I) below ##STR2## in which: R.sub.1 represents an alkyl radical comprising a number of carbon comprised between 4 and 9 atoms of carbon, or the parafluoro-phenyl radical;
R.sub.2 represents hydroxyl or the ketone function.
Several publications describe the modification of the group 21-hydroxy-methyl of the corticoids, and more particularly the replacement of the oxygen of this function by sulphur.
Thus, the 21-thio-acetate of hydrocortisone is synthetized and declared free of any interesting biological activity (J. Org. Chem. 26,1223, 1961).
Other derivatives have been proposed as anti-inflammatory products having solely a systemic action on the system or a local action and a systemic action:
The 21-thio-acetate and 21-thio propionate of prednisolone (U.S. Pat. No. 2,814,632) have been described as possessing an adreno-corticoid activity accompanied by considerable diuretic activity.
The 21-thio-acetate of dexamethasone (French Pat. No. 1187 M) has been proposed as an anti-inflammatory product with a local action and a systemic action.
The therapeutic use of corticoids having a systemic action generally gives rise to harmful "secondary effects" (Presse Medicale No. 31,1419-1423, 1970).
These secondary effects comprise mainly: endocrine troubles, sodium retention accompanied by a leakage of potassium, weakening of the defense reactions of the organism, which result in a pro-infection effect, digestive ulcers and disturbances of the glucidic, proteic and lipidic metabolisms.
The number and the variety of these secondary effects necessitate a certain prudence and careful supervision during the use of these products.
The present invention has for its object to find a remedy for these disadvantages.
STATEMENT OF INVENTION
It has been found that, in a surprising manner, the structures forming the object of the present invention comprising a thio-alkanoic group of higher molecular weight, possess a considerable anti-inflammatory activity but they have only small systemic effects. The therapeutic doses thus remain very remote from those capable of causing the appearance of the secondary effects previously described.
Thus, certain substances according to the invention possess a thymolytic activity 100 times less than that of the glyco-corticoid of reference, whereas in the other hand their local anti-inflammatory activity is greater than that of the same reference.
Generally speaking, the substances forming the object of the present invention have shown a strongly reduced or nul activity on the glucidic and proteic metabolisms, little or no regression of the adrenal glands, no sodium retention.
In consequence, these substances are therapeutic agents having a very high safety in use, and this applies even in heavy doses, which find their application in the local treatment of inflammatory affections, such as the following:
Cutaneous illnesses and mucous illnesses that can be treated by corticoids;
Auto-rhino-laryngological and opthalmological illnesses of an inflammatory and/or allergic nature;
Low digestive inflammations such as colities, recto-colities, and and recto-sigmoiditis;
Collagen troubles, articular and rhumatismal illnesses;
Asthma, emphysema and respiratory fibrosis.
In addition, and contrary to the corresponding non-sulphurous steroids, these products have a long period of action free from "rebound effect" which is of great interest in the treatment of chronic inflammatory illnesses.

DESCRIPTION OF PREFERRED EMBODIMENTS
According to the invention, the new esters of the 21-thiol steroids are prepared by condensation between:
on the one hand iodized derivatives having the general formula (II): ##STR3## in which R.sub.2 have the same signification as before;
on the other hand, S-thio-carboxylic acids utilised in their salified form, preferably in the form of the alkaline salts of these acids, especially the sodium salt. As S-thio-carboxylic acids, there are employed acids such as:
The thio-alkanoic acids comprising a number of carbon atoms comprised between 5 and 10, for example the S-thio-pivalic acid, the S-heptanethioic acid, the S-decanethioic acid and the S-pentanethioic acid, the S-methyl-2 butanethioic acid, the S-methyl-3 butanethioic acid, the S-hexanethioic acid, the S-methyl-4 pentanethioic acid, the S-dimethyl-3.3 butanethioic acid, the S-ethyl-2 butanethioic acid, the S-octanethioic acid, the S-ethyl-2 hexanethioic acid, the S-nonanethioic acid.
The parafluoro S-thiobenzoic acid is also used.
In order to salify the S-thio-carboxylic acid, the procedure is preferably as follows:
The solvent, preferably anhydrous acetone, and the S-thio-carboxylic acid are introduced into the reactor while stirring.
Then the sodium is introduced, preferably in the form of a methanol solution of sodium methylate, which is added drop by drop.
The operating conditions of the process of condensation of the iodized derivatives of Formula II and the alkaline S-thio-carboxylates are variable; however, the operation is generally carried out in the following manner:
There are introduced into a reactor comprising a reflux condenser, mechanical stirrer, the reaction solvent, especially anhydrous acetone, and then the iodized derivative of Formula II; to the suspension or solution thus formed there is added the acetone solution of the alkaline salt of S-thio-carboxylic acid previously prepared. The reaction medium is brought up to reflux and the solvent is then eliminated by distillation under vacuum.
It must be noted however that it is also possible to effect the condensation by introducing the iodized derivatives of Formula II in the powder form or in an acetone solution in the solution of sodium salt of S-thio-carboxylic acid and continuing the reaction as previously described.
The product obtained is purified depending on the case, either directly by crystallization from an alcohol having a low molecular weight or by column chromatography followed by a crystallization from the appropriate solvent or mixed solvents.
The molar ratio between the alkaline S-thio-carboxylate and the iodized derivative of Formula II employed is comprised between 1.4 mol of alkaline salt per mol of iodized derivative and 14 mols of alkaline salt per mol of iodized derivative.
The reaction temperatures are determined in dependence on the nature of the solvent and are in principle comprised between 56.degree. C. and 102.degree. C.
The time of the condensation reaction is favorably comprised between half an hour and 8 hours and preferably between 1 hour and 3 hours. For these reaction periods and according to the reactants utilized, the yields are substantially comprised between 12.5% and 90%. Generally speaking, the time of the reaction is determined in such a manner as to limit the formation of secondary derivatives.
In order to define the characteristics of the ester of the 21-thiol steroids thus prepared, analytic chemical methods are utilized, such as functional analysis and elementary centesimal analysis, and physico-chemical methods such as the ultra-violet and infra-red spectra.
The method which has just been described in general terms has been utilized for a whole variety of radicals R.sub.1 ; it will be seen in particular in connection with the non-limitative examples which follow below. These examples have been chosen in such manner as to define the utilization of the method according to the invention for at least one type of radical belonging to the families claimed hereinafter.
EXAMPLE 1
DIHYDROXY -11.beta., 17.alpha. THIOL -21 DIOXO -3.20 PREGNENE-4 21 PIVALATE (JO. 1016).
In a reactor of 50 liters equipped with a dropping funnel, a mechanical stirrer and a calcium chloride tube to protect the apparatus from moisture, sodium S-thiopivalate is prepared from 100 grams of S-thiopivalic acid (0.844 mol), 214 cu.cm. of solution of sodium methylate, 3.95 M (0.844 mol.) in 25 liters of anhydrous acetone.
There are then added 285 grams (0.063 mol.) of dihydroxy-11.beta., 17.alpha. iodo-21 dioxo-3.20 pregenene-4 and the mixture is brought up to the acetone reflux for two hours. The solvent is eliminated by distillation under vacuum until there is obtained a syrupy residue which is poured into 10 liters of iced water. The insoluble part is filtered and dried under vacuum.
The crude product is purified by recrystallization from ethanol; weight: 250 grams ; yield : 89.5%
Analysis :
C.sub.26 H.sub.38 O.sub.5 S : Calculated % : C,67.50; H,8.28; S,6.99. Found % : C,67.60; H,8.16; S,7.09.
Physical characteristics :
M.P = 195.degree. - 200.degree. C; (.alpha.).sub.D.sup. 20 = + 145.degree., (dioxanne : c = 1%; .lambda.max. (methanol) at 229 nm, log..sub.10 .epsilon. = 4.259; Main absorptions of infra-red spectrum (KBr pellet) : 1722, 1688, 1660, 1623, 1368, 1237, 1116, 1038, 858 and 720 cm.sup.-1.
EXAMPLE 2
DIHYDROXY - 11.beta. , 17.alpha. THIOL--21 DIOXO - 3.20 PREGNENE-4 HEPTANOATE (J01027)
In a one liter three necked round bottomed flask equipped with a dropping funnel, a mechanical stirrer and a calcium chloride tube to protect the apparatus from moisture, there are introduced successively 400 cu.cm. of anhydrous acetone and 13.22 grams of S-heptanethioic acid (90 mmols).
22.8 cu.cm. of a methanol solution of sodium methylate 3.90 M (89 mmols) are introduced drop by drop in 12 minutes.
There is no modification of temperature, but the initial pale yellow colour becomes darker. After the introduction, stirring is continued for 5 minutes.
On the other hand, in a reactor of 10 liters fitted with a mechanical stirrer, a dropping funnel and a thermometer and reflux condenser protected from moisture by a calcium chloride tube, there are introduced 4 liters of anhydrous acetone followed by 30 grams (63.5 mmols) of dihydroxy-11.beta. -17.alpha. iodo-21 dioxo-3.20 pregnene-4.
To this suspension, the acetone solution of sodium S-heptanethioate prepared above is introduced while stirring in 30 minutes. There is no change in temperature, the medium turns yellow and the product dissolves gradually.
The solution is brought up to the acetone reflux for 2 hours and then the solvent is eliminated by distillation under vacuum. The yellow oily residue is poured into 1.2 liters of water filtered and dried under vacuum at 40.degree. C.
The crude product is isolated by the usual method and purified by column chromatography on 300 grams of mixture of magnesium oxide and silicon oxide (15-85) W/W (80-100 mesh). After elution with benzene which enables the by-products to be eliminated, elution by chloroform gives 22.4 grams of product which are crystallized from ethanol-hexane mixture, weight : 13.6 grams; yield : 43.6%.
Analysis : C.sub.28 H.sub.42 O.sub.5 S : Calculated : C,68.53; H,8.63; S,6.53. Found % : C,68.66; H,8.47; S,6.44.
Physical characteristics : M.P 118.degree. C; (.alpha.).sub.D.sup.20 = + 135.degree., (dioxanne : c = 1,2%); .gamma. max. (methanol) at 237.5 nm, log.sub.10 .epsilon. = 4.287; Main absorptions of intra-red spectrum (KBr pellet) : 1728, 1692, 1640, 1605, 1360, 1225, 1122, 1035, 868 and 712 cm.sup.-1
EXAMPLE 3
DIHYDROXY - 11.beta. , 17.alpha. THIOL-21 DIOXO-3.20 PREGNENE-4 21 DECANOATE (J01048)
Carrying out the operation under the same conditions as in Example 2, from 5.13 grams of S - decanethioic acid (27.24 mmols) 7.2 cu.cm. of sodium methylate solution 3.8 N (27.24 mmols) and 10.72 grams of dihydroxy-11.beta. , 17.alpha. iodo-21 dioxo-3.20 pregnene-4 (22.7 mmols) there are obtained after treatment and crystallisation from ethanol 9.9 grams of white crystals; yield : 81.8%.
Analysis : C.sub.31 H.sub.48 0.sub.5 S : Calculated % : C,69,89; H,9,08; S,6,02. Found % : C,69,81; H,9,04; S,6,01.
Physical characteristics : M.P. = 136.degree. - 139.degree. C; (.alpha.).sub.D.sup.20 = + 133.degree. (dioxanne, c = 1%); .lambda. max. (methanol) at 238 nm, log..sub.10 .epsilon. = 5,29; Main absorptions of infr-red spectrum (KBr pellet) : 2920, 2850, 1780, 1625, 1270, 1235, 1130, 1110, 880 and 720 cm.sup.-1
By proceeding in the manner of EXAMPLE 2, but with S-thioalkanoics acids indicated in Column 1 below the thio esters of Column 2 are obtained.
______________________________________COLUMN 1 COLUMN 2______________________________________S - pentanethioic acid dihydroxy-11.beta., 17.alpha. thiol-21 dioxo-3.20 pregnene-4 21 pentanoateS - methyl-2 dihydroxy-11.beta., 17.alpha. thiol-21 butanethioic acid dioxo-3.20 pregnene-4 21 methyl-2 butanoateS - methyl-3 dihydroxy-11.beta., 17.alpha. thiol-21 butanethioic acid dioxo-3.20 pregnene-4 21 methyl-3 butanoateS - hexanethioic acid dihydroxy-11.beta., 17.alpha. thiol-21 dioxo-3.20 pregnene-4 21 hexanoateS - methyl-4 dihydroxy-11.beta., 17.alpha.thiol-21 pentanethioic acid dioxo-3.20 pregnene-4 21 methyl-4 pentanoateS - dimethyl-3.3 dihydroxy-11.beta., 17.alpha. thiol-21 butanethioic acid dioxo-3.20 pregnene-4 21 dimethyl-3.3 butanoateS - ethyl-2 dihydroxy-11.beta., 17.alpha. thiol-21 butanethioic acid dioxo-3.20 pregnene-4 21 ethyl-2 butanoateS - octanethioic acid dihydroxy-11.beta., 17.alpha. thiol-21 dioxo-3.20 pregnene-4 21 octanoateS - ethyl-2 dihydroxy-11.beta., 17.alpha. thiol-21 hexanethioic acid dioxo-3.20 pregnene-4 21 ethyl-2 hexanoateS - nonanethioic acid dihydroxy-11.beta.,17.alpha. thiol-21 dioxo-3.20 pregnene-4 21 nonanoate______________________________________
EXAMPLE 4
DIHYDROXY - 11.beta. , 17.alpha. THIOL - 21 DIOXO - 3.20 PREGNENE - 4 - 21 p. FLUORO-BENZOATE (J 01026)
Carrying out the operation under the same conditions as in Example 2, from 42 grams of S-.sub.p. fluoro-thio-benzoic acid (0.269 mol.), 73.5 cu.cm. of sodium methylate solution 3.66 M (0.269 mol) and 72 grams of dihydroxy-11.beta. , 17.alpha. iodo-21 dioxo-3.20 pregnene-4 (0.153 mol), there are obtained after treatment and crystallization from methanol 22 grams of pale pink crystals. Yield 32 28.8%.
Analysis : C.sub.18 H.sub.33 FO.sub.5 S : Calculated % : C,67.17; H,6.64; F,3.80; S,6.41. Found % : C,67.31; H,6.34; F,3.73; S,6.43.
Physical characteristics : M.P = 225.degree. - 230.degree. C; (.alpha.).sub.D.sup.20 = + 165.degree., (dioxanne : c = 1,2%); .lambda. max. (methanol) at 236,5 nm, log.sub.10 .epsilon. = 4.516; Main absorptions of infra-red spectrum (KBr pellet) : 1717, 1670, 1635, 1600, 1508, 1235, 1210, 1115, 920, 848 and 720 cm.sup.-1.
EXAMPLE 5
HYDROXY - 17.alpha. THIOL - 21 TRIOXO - 3,11,20 PREGNENE - 4 21 - PIVALATE (J 01034)
The sodium S-thiopivalate is prepared in the usual manner from 2.21 grams (18,7 mmols) of S-thiopivalic acid, 4.8 cu. cm. of methanol solution of sodium methylate 3.9 M (18.7 mmols) of anhydrous acetone.
The solution is introduced into an acetone solution of hydroxy-17.alpha. iodo-21 trioxo-3,11,20 pregnene-4 (13.4 mmols): 6,3 grams.
After reaction and treatments, the product is purified by crystallization from 300 cu. cm. of methanol; weight = 32.5 grams; yield = 53%.
Analysis: C.sub.26 H.sub.36 O.sub.5 S: Calculated %: C,67.79; H, 7.88; S, 6.96. Found %: C, 67.93; H, 7.69; S, 6.78.
Physical characteristics: M.P = 213.degree. - 215.degree. C; (.alpha.).sub.D.sup.20 = + 187.5.degree., (dioxanne: c = 1%); .lambda. max. (methanol) at 235 nm, log..sub.10 .epsilon. = 4.307; Main absorptions of infra-red spectrum (KBr pellet) 1700, 1675, 1650, 1365, 1230, 955 and 870 cm.sup.-1
EXAMPLE 6
HYDROXY - 17.alpha. THIOL - 21 TRIOXO - 3,11,20 PREGNENE - 4 21 HEPTANOATE (JO 1035)
Under the same conditions as in the preceding example, from 8.25 grams (56,5 mmols) of S-heptanethioic acid and 14.5 cu. cm. of methanol solution of sodium methylate 3.9 M (56.5 mmols) on the one hand and 19 grams (4.4 mmols) of hydroxy-17.alpha. iodo-21 trioxo-3,11,20 pregnene-4 on the other hand, there is obtained a crude product which is purified by crystallisation from 100 ml of methanol: weight: 12.6 grams; yield: 63.9%.
Analysis: C.sub.28 H.sub.40 O.sub.5 S: Calculated %: C, 68.82; H, 8.25; S, 6.56. Found %: C, 68.98; H, 8.31; S, 6.47.
Physical characteristics: M.P = 125.degree. - 126.degree. C; (.alpha.).sub.D.sup.20 = + 175.degree., (dioxanne: c = 1%); .lambda. max. (methanol) at 234 nm, log..sub.10 .epsilon. = 4.286; Main absorptions of infra-red spectrum (KBr pellet) 1700, 1655, 1275, 1050, 935 and 865 cm.sup.-1
EXAMPLE 7
HYDROXY - 17.alpha. THIOL - 21 TRIOXO - 3,11,20 PREGNENE - 4 21 DECANOATE (JO 1049)
Under the same conditions as in Example 5, from 5.16 g of S - decanethioic acid (27.4 mmols), 7.2 cu. cm. of sodium methylate solution 3.8 N (27.4 mmols) and 10.75 g of hydroxy-17.alpha. iodo-21 trioxo-3,11,20 pregnene-4 (22.8 mmols), there are obtained after treatment and crystallisation from methanol 9.4 g of white product; yield = 77.7%.
Analysis: C.sub.31 H.sub.46 O.sub.5 S: Calculated %: C, 70,16; H, 8,74; S, 6,04. Found %: C, 69,89; H, 8.61; S, 6,04.
Physical characteristics M.P = 115.degree. - 117.degree. C; (.alpha.).sub.D.sup.20 = + 175.degree., (dioxanne, c = 1%); .lambda. max. (methanol) at 236 nm, log.sub.10 .epsilon. = 5,291; Main absorptions of infra-red spectrum (KBr pellet): 2920, 1680, 1650, 1360, 1270, 1220, 1050 and 935 cm.sup.-1
By proceeding in the manner of Example 5, but with S-thioalkanoics acids indicated in Column 1 below the thio esters of Column 2 are obtained.
______________________________________COLUMN 1 COLUMN 2______________________________________S - pentanethioic acid Hydroxy-17.alpha. thiol-21 trioxo-3,11,20 pregnene-4 21 pentanoateS - methyl - 2 Hydroxy-17.alpha. thiol-21 butanethioic acid trioxo-3,11,20 pregnene-4 21 methyl-2 butanoateS - methyl - 3 Hydroxy-17.alpha. thiol-21 butanethioic acid trioxo-3,11,20 pregnene-4 21 methyl-3 butanoateS - hexanethioic acid Hydroxy-17.alpha. thiol-21 trioxo-3,11,20 pregene-4 21 hexanoateS - methyl -4- Hydroxy-17.alpha. thiol-21 pentanethioic acid trioxo-3,11,20 pregnene-4 21 methyl-4 pentanoateS - dimethyl - 3,3 Hydroxy-17.alpha. thiol-21 butanethioic acid trioxo-3,11,20 pregnene-4 21 dimethyl-3,3 butanoateS - ethyl - 2 Hydroxy-17.alpha. thiol-21 butanethioic acid trioxo-3,11,20 pregnene-4 21 ethyl-2 butanoateS - octanethioic acid Hydroxy-17.alpha. thiol-21 trioxo-3,11,20 pregene-4 21 octanoateS - ethyl - 2 Hydroxy-17.alpha. thiol-21 hexanethioic acid trioxo-3,11,20 pregnene-4 21 ethyl-2 hexanoateS - nonaethioic acid Hydroxy-17.alpha. thiol-21 trioxo-3,11,20 pregnene-4 21 nonanoate______________________________________
EXAMPLE 8
HYDROXY - 17.alpha. THIOL - 21 TRIOXO - 3,11,20 PREGNENE - 4 21 p. FLUOROBENZOATE (JO 1046)
Carrying out the operation under the same conditions as in Example 5, from 4.28 grams of S-p. fluoro-thiobenzoic acid (27.4 mmols), 7.2 cu.cm. of sodium methylate solution 3.8 N (27.4 mmols) and 10.72 grams of hydroxy-17.alpha. iodo-21 trioxo-3,11,20 pregnene-4 (22.8 mmols), there are obtained after treatment and crystallisation from ethanol 5.5 grams of product. Yield = 48.4%.
Analysis: C.sub.28 H.sub.31 FO.sub.5 S: Calculated %: C, 67.45; H, 6.26; F, 3,81; S, 6,43. Found %: C, 67.35; H, 6.24; F, 3,75; S, 6,33.
Physical characteristics: M.P = 166.degree. - 169.degree. C; (.alpha.).sub.D.sup.20 = + 196.degree., (dioxanne, c = 1%); .lambda. max. (methanol) at 239 nm, log..sub.10 .epsilon. = 5,426; Main absorptions of infra-red spectrum (KBr pellet): 2930, 1700, 1595, 1500, 1230, 1205, 920 and 850 cm.sup.-1
PHARMACOLOGICAL STUDY
There will now be described the tests which have enabled the determination of the pharmaco-dynamic properties of the esters of the 21-thiols steroids according to the invention.
ANTI-INFLAMMATORY ACTIVITY
The experimental local anti-inflammatory activity of the compounds presented was estimated in rats by their anti-proliferative (anti-granulomatous) action, and for one of these, by its anti-arthritic activity and its anti-exudative activity.
a. Anti-proliferative Activity
The anti-proliferative (anti-granulomatous) activity has been brought into evidence by means of a test, the principle of which is as follows.
The introduction of a foreign body into an organism produces a set of inflammatory reactions which results, in the chronic stage, in the formation of a defence granuloma around the foreign body. The proliferation of this granuloma is eliminated or attenuated by anti-inflammatory agents.
The technique employed is very similar to that described by Winter and Porter (J. Am. Pharm. Ass. 46/9. 515 1957) with rats.
Homogeneous groups of 10 male adult rats of the Wistar Strain were used, distributed at random and having weights comprised between 180 and 200 grams.
The implants or pellets were prepared from rolls of dental cotton; the weight of the pellets was between 35 and 40 mg.
Immediately before their introduction, the pellets were soaked with an antibiotic solution (0.1 ml. of a solution of penicillin G and streptomycin containing 200 000 UI of penicillin G and 0.1 gram of streptomycin sulphate per cc.)
Each animal received two pellets in the sub-cutaneous dorsal tissue on each side of the spinal column, at the costo-lumbar angle, under light anaesthesia with ether. The day of the operation and for three days after, the animals received by sub-cutaneous injection, 0.1 ml. of the antibiotic solution in the caudal region.
Six days after the introduction, the animals were killed by inhalation of chloroform and the granulomae were extracted and weighed, (wet and dry), and then the initial weights of the cotton pellets were substracted from the total weight.
Certain non-sulphured steroids causing a large increase in protein catabolism which can influence the formation of the granuloma independently of their anti-inflammatory action, the weights of the granulomae were expressed as a percentage of the body weight (technique proposed by G. Dipasquale and A. Meli: J. Pharm. Pharmacol. (1965), 17, 367-382) and the anti-proliferative effect of the various compounds as a percentage inhibition with respect to the reference granulomae. The ED.sub.50 were calculated by transferring the results on semi-logarithmic paper.
a - 1. Local anti-inflammatory Activity
For this study, the products to be tested were dissolved in chloroform or in dimethylsuphoxide (DMSO) and the solutions obtained, deposited on the pellets at a volume of 0.2 ml per pellet. The solvant was then evaporated under high vacuum at ambient temperature, the complete elimination of the solvent being checked by weighing the pellets. "Reference" pellets, soaked with the solvent alone, were treated in the same manner.
a - 2. Systemic anti-inflammatory Activity
For this study, each animal received two untreated pellets. The product are given by oral route, daily during six days; the first administration began about 2 hours after the implantation.
The products were administered in suspension in a solution of arabic gum at 5% (w/v), at a volume of 1 ml/rat.
b. Anti-exudative Action
The anti-exudative activity was brought into evidence by means of a test, the principle of which is as follows.
This test consists of creating, under the dorsal skin of the rat an air pouch in which an irritant product is injected. An inflammatory reaction appears rapidly and is shown by the accumulation of liquid in the pouch of air.
The introduction of an anti-inflammatory product into the air pouch, reduced more or less completely, the accumulation of exudate.
This study was carried out with male rats of the Wistar strain, the initial weight of which was between 160 and 180 grams, following the technique described by M. Fukuhara and S. Tsurufuji (Biochem. Pharmac. 18, 475-484, 1969). Each lot comprised ten animals distributed at random.
Twenty-four hours before the beginning of the test, each animal received a sub-cutaneous injection of 6 ml. of air in the dorsal region; previously shaved.
The following day, 4 ml. of a 2% carrageenan solution in the physiological solution (NaCL at 0.9%), were injected in the pouch, the solution being kept lukewarm to prevent setting in a lump.
An injection of 0.1 ml. of a solution of penicillin and streptomycin containing 200 000 UI of penicillin and 0.1 gram of streptomycin per ml. was carried out immediately afterwards in the caudal region by sub-cutaneous methods.
Ninety-six hours after the administration of the carrageenan, the products under study were injected into the pouch in a volume of 0.2 ml, in suspension in carboxy-methyl-cellulose at 0.5%.
Ninety-six hours after this last injection, the animals were killed and the exudate contained in the pouch was collected through a small incision made with a scalpel, and its volume was measured in a test tube.
The difference in volume of the exudate between the animals treated and the control lot was expressed as a percentage inhibition. The ED.sub.50 were determined by transferring these results on to semi-logarithmic paper.
c. Anti-arthritic Activity
The anti-arthritic activity was brought out by means of a test, the principle of which is as follows:
This test was carried out following a method derived from that described by Foldi-Borcsok and Coll.(Arzneimittel Forschung 21, 2025-2030, 1971).
The injection of kaolin in the tibio-metatarsal joint of the rat causes an inflammation which develops in two successive phases:
an acute phase characterized by an oedema of the joint;
a chronic phase which follows, characterized by the proliferation of an inflammatory granuloma.
The intensity of the inflammatory reaction is estimated following the width of the articulation.
Male rats of Wistar stock were utilized, the initial weight of which was between 180 and 200 grams. Each group comprised ten animals taken at random, in which the width of the right-paw tibio-metatarsal joint was measured to the nearest 1/20th of a millimeter.
All the animals received 0.05 ml. of suspension of kaolin at 10% in a 0.9% physiological solution by intra-articular injection in the right-paw tibio-metatarsal joint.
Eighteen hours after this injection, the width of the joint was measured (initial inflammation) and there was then carried out an intra-articular injection of the products under study, in suspension in 0.5% carboxy-methyl-cellulose at a volume of 0.05 ml. The animals belonging to the control lot received 0.05 ml of the vehicle by the same method.
Twenty-four hours after this last injection, the width of the joint treated was again measured and then daily for 9 or 10 days, according to the evolution of the animals of the control group. The variation of width of the joints treated, representing the anti-arthritic activity of the products under study, were expressed as a percentage of the initial inflammation according to the formula: ##EQU1## in which: .DELTA..sub.1 = increase in width of the joint with respect to its initial width, during the initial inflammation;
.DELTA..sub.n = increase in width of the joint with respect to its initial width, on the day considered.
The calculations were carried out by using the averages of the individual results of each lot.
SYSTEMIC EFFECTS
The systemic effects of the compounds according to the invention were evaluated through the intermediary of their thymolytic activity and for some of these, their possible influence on the glucidic metabolism, the hydro-mineral equilibrium, the weight increase, the endocrine glands and the genital tractus was examined and also a possible ulcerogenic effect.
d. Thymolytic effect
The thymolytic effects have been examined by means of a test, the principle of which is as follows:
The repeated administration of a gluco-corticoid having a systemic activity causes an involution of the defence system of the organism, of which two organs belong to the reticuloendothelial system, the spleen and the thymus, this latter being the most sensitive to this action, especially with young animals. The thymic involution is estimated by weighing. The thymolytic effects was studied by two routes:
1. local route (local effects)
2. oral route (systemic effects)
d - 1. Local Effects
For this study, the products are administered locally on pellets, following the procedure a.sub.1.
Six days after inplantation, the thymus are taken off and quickly weighed.
d - 2. Systemic Effects
For this study, the various products were injected daily by the oral or the sub-cutaneous route for four days, to young male rats of Wistar-strain, the initial weight of which was between 45 and 55 drams, distributed at random by groups of ten.
The products under study were administered at a volume of 0.2 ml. per animal for both routes, in suspension in:
Carboxy-methyl-cellulose at 0.5% for sub-cutaneous injection;
Gum arabic at 5% for the oral route.
The animals of the control groups received the same volume of the corresponding vehicle.
Ninety-six hours after the first administration, the animals were killled, the thymus glands being taken and weighed immediately.
For each animal, the weight of the thymus has been brought to 100 grams of body weight. The thymolytic activity of the products under study was then expressed as a percentage of regression of the thymus with respect to the animals of the control group and the ED.sub.50 of each product tested was estimated by transferring the percentage inhibition obtained for each does on semi-logarithmic paper.
e. Action on the Glucidic Metabolism
The action on the glucidic metabolism was investigated by means of a test, the principle of which is as follows:
Gluco-corticoid reduce the peripheral needs of the organism in glucose, they also increase the synthesis by the liver of glycogen (neo-glycogenesis). Their action on the glucidic metabolism is thus indicated:
1. By an increase of glycemia;
2. By a glycogenic overload on the liver and especially by the persistence of the hepatic stock of glycogen in the food-deprived animal, and treated with a gluco-corticoid by comparison with the non-treated animal.
e - 1. Action on Glycemia
This study was done after the subchronic administration of JO 1016.
There were utilised groups of ten male rats or ten female rats of SPRAGUE DAWLEY I. O. P. S. Strain which have received the products under study by subcutaneous methods, daily for four weeks.
The products were administered in suspension in 0.5% carboxy-methyl-cellulose under a volume of 0.5 ml per 100 grams of body weight in three different doses; the control animals receiving the same volume of the vehicle alone.
e - 2. Action on the Glycogenic Overload on the Liver
For this study, Wistar male rats were used; the initial weight of which was between 150 and 160 grams, distributed at random in groups of ten.
The products studied were administered by the oral route in suspension in 5% gum arabic, under a volume of 0.5ml. per 100 grams, the animals of the control group receiving the same volume of vehicles under the same condition.
The treatments were carried out daily for four days, a fifth administration being given 7 hours before killing the animals, which took place 96 hours after the first administration.
Eighteen hours before being killed, all the animals were put on a water diet, this period of fasting having proved sufficient to eliminate the hepatic stock of glycogen in the animals of the control group.
Immediately after killing the animals, a sample of the hepatic tissue was taken from the central lobe and weighed. Immediately afterwards, it was subjected to digestion in 30% potassium hydroxyde in a boiling water-bath for 20 minutes. The glycogen contained in the digestate was then dosed, following the technique described by R. O. Stafford, L.E. Barnes and Coll. (Proc. Soc. Exp. Med. 39/3, 371-374, 1955).
The content of glycogen in the liver samples has been expressed in grams of glycogen per 100 grams of hepatic tissue.
f. Action on the Hydro-mineral Metabolism
The action on the hydro-mineral metabolism was examined by means of a test, the principle of which is as follows:
The gluco-corticoids are not all free from mineral-corticoid effects (for example Hydro-cortisone). These latter are indicated by:
A reduced renal elimination of sodium (sodium retention)
A loss of potassium (potassium leakage),
these two effects reproducing the action of aldosteron.
f - 1. After a Short-term Study (96 hours)
This study has been carried out on male Wistar rats having weights between 180 and 190 grams.
The animals were distributed at random in groups of ten.
The products under study were administered by the oral route in suspension in 5% gum arabic. The animals of the control group received the same volume of vehicle under the same conditions.
The treatments were carried out daily for four days. The renal functional exploratory test was effected 90 hours after the first administration of the product tested.
Eighteen hours before the test, the animals were completely deprived of food and water, this being subsequently maintained up to the end of the test.
Immediately before the renal functional test, the animals received an overdose by the oral route of physiological solution equal to 5% by volume of their body weight.
The urine passed during five hours was collected for each animal in a polyethylene tube.
The concentration of sodium was determined in each sample by flame photometry; the total quantity excreted was then calculated as a function of the corresponding volume of urine. The values obtained for each group were then compared following the Fisher Student method with the quantity of sodium excreted by the animals of the control group.
f - 2. After a Long Term Study
(Daily administration for 4 weeks following the same procedure described in e.sub.1.).
At the end of the treatment, the urine sodium excretion in 5 hours, and the serum ionogram were determined.
g. Action on the Protein Metabolism
The action on the protein metabolism was examined by means of a test, the principle of which is as follows:
The administration of gluco-corticoids results in a disturbance of the protein metabolism, which is shown by an exaggerated protein catabolism resulting in shrinking of the tissues objectified by an inhibition of growth in weight in the young animal and a loss of body weight in the adult animal.
e - 1. After a Short Term Study
For this study, immature rats of Wistar strain were utilized, the initial weight of which was between 45 and 55 grams, which were distributed at random groups of ten.
g - 1.1. Local Activity
For this study, the products are administered locally on pellets, like the described procedure in a.sub.1. The animals are weighed before the implantation and six days after.
g -1.2. Systemic Activity
The products were administered daily for four days.
The animals were weighed daily during the four days of treatment and 24 hours after the last dose.
The products tested were administered either orally or by sub-cutaneous injection, in suspension in 5% gum arabic for the oral route or in carboxy-methyl-cellulose at 5% for the sub-cutaneous method, the animals of the control group receiving the same volume of vehicle by the same method as the corresponding animals treated.
The average variation of the body weight during the 96 hours treatment was calculated for each group of animals.
g - 2. After a Long Term Study
The average variation of the body weight between the initial weight and the final weight after 4 weeks of treatment by JO 1016 by subcutaneous route are calculated.
h. Action on the Adrenals
The administration of corticoids may result in an inhibition of the secretion of the ante-hypophysiary hormone which is shown by a lowering of the effectivness of the target glands:adrenals. This effect was evaluated after a short or a long term study.
h - 1. Short Term Study
h - 1.1. Local Effect
The products are administered locally on pellets following the described procedure a.sub.1. Six days after the implantation, the adrenals are taken off and quickly weighed.
h - 1.2. Systemic Effects
The products are administered daily during six days. The products given are in suspension in a solution of arabic gum at 5% (w/v) by oral route, under a volume of 1 ml/rat. The day after the last administration, the adrenals are taken off and quickly weighed.
h - 2. Long Term Study
(Daily administration for 4 weeks following the same procedure described in e.sub.1). At the end of the study, the adrenals are taken off and quickly weighed, then are fixed in Bouin Holland for an histological examination The results are expressed in mg/100 gram s of bodyweight; the mean weights are calculated for each group.
i. Action on the Genital Tractus
The repeated administration of corticoids may result in an indirect action on organs which are under the dependance of an hormone secretion.
For this reason, after a sub-chronic administration with one product of the invention (cf e.sub.1), the gonads (testicles or ovaries), the seminal tubes or the uterus are taken off and weighed. The results are expressed in mg/100 grams of bodyweight.
j. Action on the Digestive Tractus, and Pro-infectious Effect
The repeated administration of certain corticoids produced ulceration generally localised at the stomach.
After a sub-chronic administration with one product, the stomach, the duodenum, and some portions of the small intestine, ileon and colon are preleved, examined and fixed in the Bouin Holland for histological examination. Liver, kidneys, thymus and spleen are also preleved and examined.
RESULTS OF THE PHARMACOLOGICAL STUDY
There will now be described the results of the pharmacological study.
a. Anti-proliferative Activity
a - 1. Local Anti-inflammatory Activity
The ED.sub.50 obtained for each of the products presented and the corresponding steroids are indicated in Tables I and II below.
TABLE I______________________________________DERIVATIVES OF HYDRO-CORTISONETREAT WET WEIGHT OF DRY WEIGHT OFMENT PELLET PELLET______________________________________Hydro-cortisone ED.sub.50 = 10 mg/pellet ED.sub.50 = 4,1 mg/pellet(base)Hydro-cortisone ED.sub.50 = 1,05 mg/pellet ED.sub.50 = 0,80 mg/pellet(acetate)JO 1016 ED.sub.50 = 0,35 mg/pellet ED.sub.50 = 0,18 mg/pelletJO 1026 ED.sub.50 = 0,40 mg/pellet ED.sub.50 = 0,23 mg/pellet______________________________________
in considering the dry weight of the granulomae, it is clear that the local anti-inflammatory activity of compounds JO 1016 and JO 1026 respectively is approximatively 4,5 times and 3,5 times greater than the activity of the reference compound, hydrocortisone acetate.
TABLE II______________________________________DERIVATIVES OF CORTISONE WET WEIGHT OF DRY WEIGHT OFTREATMENT PELLET PELLET______________________________________Cortisone ED.sub.50 = 17 mg/pellet ED.sub.50 = 0,70 mg/pelletacetateJO 1034 ED.sub.50 = 7 mg/pellet ED.sub.50 = 0,40 mg/pellet______________________________________
When administered by oral route, compound JO 1034 exerts an antiinflammatory activity nearly twice that of the corresponding activity of the reference
a - 2. Systemic Anti-inflammatory Activity
The ED.sub.50 obtained are indicated in Table III
TABLE III______________________________________DERIVATIVES OF CORTISONE (ORAL ROUTE) WET WEIGHT OF DRY WEIGHT OFTREATMENT PELLET PELLET______________________________________Cortisone acetate ED.sub.50 = 150 mg ED.sub.50 = 19,8 mgmg/RatJO 1034 ED.sub.50 > 750 mg ED.sub.50 > 750 mgmg/Rat______________________________________
The ED.sub.50 are expressed as the total doses administered during six days, per rat.
It can be considered that, when administered by oral route, compound JO 1034 exerts an antiinflammatory activity about 40 times less than that of the corresponding activity of the reference compound, cortisone acetate.
b. Anti-exudative Activity
The results indicated on TABLE IV were obtained with the JO 1016.
TABLE IV______________________________________ Anti-exudative activity______________________________________Hydrocortisone acetate ED.sub.50 = 1.5 mgJO 1016 ED.sub.50 = 1.5 mg______________________________________
When administered by local route, compound JO 1016 exerts the same anti-exsudative activity as hydrocortisone acetate.
c. Anti-arthritic Activity
For example, the results obtained are referred to:
A derivative of hydrocortisone: JO 1016
and also to the corresponding reference steroid.
The figures indicated in TABLE V below, represent the anti-arthritic activity of the products, estimated from the diminution in width of the joint (as a percentage of the initial inflammation), 24 hours after their injection and 120 hours after the injection. JO 1016 and its corresponding reference steroid were administered in a ratio of doses equal to the ratio of their respective molecular weights.
TABLE V______________________________________ DOSE 24 HOURS 120 HOURS______________________________________Hydrocortisone acetate 2,91 mg -65,0% -76,2%JO 1016 3,34 mg - 58,9% - 81,2%______________________________________
When administered by local route, compound JO 1016 exerts an antiarthritic activity similar to that of hydrocortisone acetate.
d. Thymolytic Activity
d - 1. Local Activity
The thymolytic activity by local route is expressed in TABLE VI
TABLE VI______________________________________ WEIGHT OF No. THYMUS % AND OF MEAN PROBA-TREATMENT RATS (MG) .+-. e.s. BILITIES______________________________________CONTROL 10 308,7 .+-. 13,10 --CORTISONE ACETATE0,1 mg/pellet 10 303,1 .+-. 10,90 -1,8 N.S.CORTISONE ACETATE0,5 mg/pellet 10 313,1 .+-. 13,62 + 1,4 N.S.CORTISONE ACETATE - 39,22,5 mg/pellet 10 187,7 .+-. 12,21 p < 0,001CORTISONE ACETATE - 91,312,5 mg/pellet 10 26,9 .+-. 2,72 p < 0,001JO 1034 .+-. 3,80,01 mg/pellet 10 320,3 .+-. 13,16 N.S.JO 1034 - 3,70,05 mg/pellet 10 297,3 .+-. 6,87 N.S.JO 1034 - 4,10,25 mg/pellet 10 296,1 .+-. 9,07 N.S.JO 1034 + 1,21,25 mg/pellet 10 312,3 .+-. 7,88 N.S.______________________________________
When administered in efficient doses for local antiinflammatory activity, compound JO 1034 is completely free of thymolytic effect, whereas this is not the case for the reference compound, cortisone acetate.
d - 2. Systemic Activity
The ED.sub.50 obtained for each products presented and for the corresponding steroids are indicated in Tables VII and VIII which follow. (The calculated ED.sub.50 are the total doses administered during four days per rat of about 50 grams at the beginning of the study.
TABLE VII______________________________________DERIVATIVES OF HYDROCORTISONE ORAL SUB-CUTANEOUSTREATMENT ROUTE ROUTE______________________________________Hydrocortisone (base) : 4 mg 1.8 mgHydrocortisone (acetate) : 4 mg 0.68 mgJO 1016 400 mg 120 mgJO 1026 >40 mg >40 mg______________________________________
When administered by oral route, compounds JO 1026 and JO 1016 exert a thymolytic activity 10 to 100 times less than the activity of the reference compound, hydro cortisone acetate. By subcutaneous route, the same two compounds have a thymolytic activity 60 to 180 times less than the activity of the same reference compound.
TABLE VIII______________________________________DERIVATIVES OF CURTISONETREATMENT ORAL ROUTE______________________________________CORTISONEACETATE ED.sub.50 = 12 mgJO 1034 ED.sub.50 > 750 mg______________________________________
When administered by oral route, compound JO 1034 exerts a thymolytic activity at least 60 times less than the corresponding activity of the reference compound, cortisone acetate.
Ratio of the Local Anti-inflammatory Activity to the Thymolytic Activity
The ratio of the local anti-inflammatory activity to the thymolytic activity is all the greater as these products possess a low anti-inflammatory activity (ED.sub.50 high in the numerator) and a large thymolytic activity (ED.sub.50 low in the denominator).
The ratio is indicated in TABLES IX AND X which follow:
TABLE IX______________________________________DERIVATIVES OF HYDROCORTISONE ED.sub.50 Anti-proliferative activity ED.sub.50 Thymolytic activity Thymolytic Thymolytic acti- activity vity determined determined by by the sub- the oral route cutaneous route______________________________________HYDROCORTISONE 2.5 5.5(BASE)HYDROCORTISONE 0.25 1.2(ACETATE)JO 1016 <0.003 0.0008JO 1026 <0.01 <0.01______________________________________
This chart shows, on one hand, how significant the local antiproliferative activity of the compounds according to the invention is and, on the other hand, that these compounds are free of systemic thymolytic effect.
TABLE X______________________________________DERIVATIVES OF CORTISONE ED.sub.50 Anti-proliferative activity ED.sub.50 Thymolytic activity/oral Route______________________________________CORTISONE ACETATE 1,4JO 1034 <0,009______________________________________
This chart shows the local antiproliferative activity of JO 1034 and the absence of systemic thymolytic effect.
e. Action on the Glucidic Metabolism
e - 1. Action on Glycemia
The following TABLES XI AND XII indicate the value of glycemia in the control animals and in the animals treated for 4 weeks following and described procedure.
TABLE XI______________________________________ Number Percentage of Mean (g/litre) of variation andMale animals .+-. standard error Animals Probabilities______________________________________CONTROL 1.1.+-.0.05 10JO 1016-100 mg/kg 1.2.+-.0.04 10 +16% p<0.01JO 1016-250 mg/kg 1.2.+-.0.05 10 +15% 0.02<p<0.05JO 1016-500 mg/kg 1.1.+-.0.03 10 N.S.______________________________________ N.S. : insignificant
TABLE XII______________________________________ Number Percentage of Mean (g/litre) of variation andFemale animals .+-.standard error animals Probabilities______________________________________Control 1.00 .+-. 0.04 10JO 1016-100 mg/kg 1.10 .+-. 0.04 10 +10% N.S.JO 1016-250 mg/kg 1.21 .+-. 0.03 10 +21% p 0.01JO 1016-500 mg/kg 1.10 .+-. 0.04 10 +10% N.S.______________________________________
The charts XI et XII show that, after a repeated administration, compound JO 1016 does not modify glycemia; the meaningful variation which is noted remain within the normal physiological range of the animal having undergone the test.
e - 2. Action on the Glycogenic Overload on the Liver
Table XIII which follows, indicates the content of glycogen in the liver of the control animals and the treated animals for 96 hours, after a water diet of 18 hours.
TABLE XIII______________________________________ Glycogen in g/100 g of hepatic tissue______________________________________CONTROL 0.15Hydrocortisone acetate 2 mg/kg 0.19Hydrocortisone acetate 5 mg/kg 0.45Hydrocortisone acetate 10 mg/kg 0.46Hydrocortisone acetate 20 mg/kg 1.26Hydrocortisone acetate 50 mg/kg 2.49JO 1016 25 mg/kg 0.25JO 1016 50 mg/kg 0.39JO 1016 100 mg/kg 0.42______________________________________
Compound JO 1016 does not cause the formation of glycogen when administered in a dose of up to 100 mg/kg/. On the contrary, when administered in a dose 10 times less, hydrocortisone acetate causes a significant neoglycogenesis.
F. action on the Hydro-mineral Metabolism
f - 1. After a Short Term Study
The results obtained with hydrocortisone acetate and JO 1016 following the procedure described in f.sub.1 are indicated in TABLE XIV.
TABLE XIV______________________________________ mEq of sodium excreted Number Percentage of Mean .+-. of variation and standard error Animals Probabilities______________________________________CONTROL 0.816 .+-. 0.0493 4Hydrocortisone 0.371 .+-. 0.0827 5 -55%acetate - 16 mg/kg 0.001<p<0.01JO 1016 16 mg/kg 0.820 .+-. 0.1040 5 0.4% N.S.JO 1016 32 mg/kg 0.754 .+-. 0.0465 4 -8% N.S.JO 1016 80 mg/kg 0.684 .+-. 0.1591 4 -16% N.S.JO 1016 160 mg/kg 0.664 .+-. 0.0484 5 -19% N.S.______________________________________
When administered in a dose 10 times greater than ED.sub.50 of hydrocortisone acetate, compound JO 1016 does not induce sodium retention.
f - 2. After a Long Term Study
The result obtained in the case of a treatment lasting 4 weeks following the procedure described, are indicated in the following TABLE XV.
TABLE XV______________________________________ mEq of sodium excreted Number Percentage of Mean .+-. of variation and standard error Animals Probabilities______________________________________CONTROLMALE RAT 1.77 .+-. 0.089 10 --CONTROLFEMALE RAT 1.77 .+-. 0.065 10 --JO 1016 male rat -20%500 mg/kg 1.41 .+-. 0.116 10 0.02<p<0.05JO 1016 female rat -22%500 mg/kg 0.91 .+-. 0.98 10 0.02<p<0.05______________________________________
After having administered a dose of 500 mg/kg/day, for four weeks, a slight sodium retention is noted.
g. Action on the Protein Metabolism
g - 1. After a Short Term Study
g - 1.1.. Local Action
The local action on the protein metabolism are expressed in TABLE XVI.
______________________________________ INCREASE No. OF BODY- % AND OF WEIGHT PROBA-TREATMENT RATS Mean(g).+-.e.s. BILITIES______________________________________CONTROL 10 40,4 .+-. 2,09 --CORTISONE ACETATE 10 39,5 .+-. 1,77 - 2,20,1 mg/pellet N.S.CORTISONE ACETATE 10 37,0 .+-. 2,35 - 8,40,5 mg/pellet N.S.CORTISONE ACETATE 10 33,4 .+-. 2,05 - 17,32,5 mg/pellet p < 0,01CORTISONE ACETATE 10 5,1 .+-. 2,02 - 87,412,5 mg/pellet p < 0,001JO 1034 10 36,6 .+-. 2,12 - 9,40,01 mg/pellet N.S.JO 1034 10 40,6 .+-. 1,78 + 0,50,05 mg/pellet N.S.JO 1034 10 38,9 .+-. 1,70 - 3,70,25 mg/pellet N.S.JO 1034 10 35,8 .+-. 1,60 - 11,41,25 mg/pellet N.S.______________________________________
The doses of compounds JO 1034 which exert a local antiinflammatory activity do not cause any proteolytic effect, this distinguishing this compound from the reference compound, cortisone acetate.
g - 1.2.. Systemic Action
The increases in weight of immature rats (45 to 55 grams) treated for 96 hours following the procedure g) by JO 1016, by the oral or the sub-cutaneous routes, or by JO 1034 and cortisone acetate by the oral rote, (average weight at the end of the study, less the average weight at the beginning) are indicated in the following TABLES XVII and XVIII.
TABLE XVII______________________________________ ORAL SUB-CUTANEOUS ROUTE ROUTE______________________________________CONTROL 13 g 14 gJO 1016 1 mg/rat/day -- 17 gJO 1016 3 mg/rat/day 14 g --JO 1016 10 mg/rat/day 11 g 16 gJO 1016 30 mg/rat/day 13 g 16 gJO 1016 100 mg/rat/day 8 g --______________________________________
When administered by oral route, compound JO 1016 starts to show a proteolytic activity only from 2g/kg/day.
TABLE XVIII______________________________________ INCREASETREATMENT No. OF BODY % AND(ORAL) OF WEIGHT PROBA-ROUTE RATS Mean(g).+-.e.s BILITIES______________________________________CONTROL 10 21,4 .+-. 1,00 --CORTISONE ACETATE 10 20,6 .+-. 0,91 - 3,70,1 mg/Rat/jour N.S.CORTISONE ACETATE 10 21,4 .+-. 1,06 0,00,5 mg/Rat/jour N.S.CORTISONE ACETATE 10 17,4 .+-. 0,75 -18,72,5 mg/Rat/jour p<0,01CORTISONE ACETATE 10 14,0 .+-. 1,11 -34,612,5 mg/Rat/jour p<0,001JO 1034 10 24,0 .+-. 1,12 +12,21 mg/Rat/jour N.S.JO 1034 10 23,8 .+-. 1,20 +11,25 mg/Rat/jour N.S.JO 1034 10 24,5 .+-. 0,95 +14,525 mg/Rat/jour p<0.01JO 1034 10 24,7 .+-. 1,73 +15,4125 mg/Rat/jour N.S.______________________________________
This chart confirms that compound JO 1034 is free of activity, when administered by systemic route, contrary to the reference compond, cortisone acetate.
g - 2. After a Long Term Study
The weights of the animals at the end of the 4 weeks of treatment by JO 1016 by the sub-cutaneous route are indicated in the following TABLES XIX and XX.
TABLE XIX______________________________________ PERCENT- AGES OF NUMBER VARIATION MEAN (G) .+-. OF AND PROBA-MALE RATS Standard error ANIMALS BILITIES______________________________________CONTROL 296 .+-. 7.6 10JO 1016 100 mg/kg 299 .+-. 5.2 10 N.S.JO 1016 250 mg/kg 289 .+-. 7.7 10 N.S.JO 1016 500 mg/kg 274 .+-. 2.5 10 - 7% 0.02<p<0.05______________________________________
TABLE XX______________________________________ PERCENT- AGES OF NUMBER VARIATION MEAN (G) .+-. OF AND PROBA-FEMALE RATS standard error ANIMALS BILITIES______________________________________CONTROL 218 .+-. 3.22 10JO 1016 100 mg/kg 219 .+-. 3.06 10 N.S.JO 1016 250 mg/kg 216 .+-. 3.18 10 N.S.JO 1016 500 mg/kg 198 .+-. 4.85 10 -9% 0.001<p<0.01______________________________________
When administered for 4 weeks, compound JO 1016 does not modify the weight increase or the animals; the modification which is noted remains within the normal physiological range of the animals having undergone the test.
After 14 days of treatment by hydrocortisone acetate at corresponding ratio, by the sub-cutaneous route, weights of the animals are indicated in the following TABLE XXI.
TABLE XXI______________________________________ PERCENT- AGES OF NUMBER VARIATION MEAN (G) .+-. OF AND PROBA-MALE RATS standard error ANIMALS BILITIES______________________________________CONTROL 264 .+-. 4.33 10Hydrocortisone 110 .+-. 2.10 8 - 58.4 %acetate 218 mg/kg 0.001<p<0.01Hydrocortisone 105 .+-. 4.12 2 - 60.2 %acetate 436 mg/kg 0.001<p<0.01______________________________________
When administered for 14 days, the reference steroid (hydrocortisone acetate) causes the body weight to drop very significantly.
h. Action on the Adrenals
h - 1. After a Short Term Study
h - 1.1.. Local Effect
The results are reported in the TABLE XXII.
TABLE XXII______________________________________ ADRENAL PERCENT- WEIGHT AGES OF (mg) Mean VARIATION NUMBER .+-. standard AND PROBA-TREATMENT OR RATS error BILITIES______________________________________CONTROL 10 25,8 .+-. 1,31 --CORTISONE ACETATE 10 24,6 .+-. 0,87 - 4,70,1 mg/pellet N.S.CORTISONE ACETATE 10 26,2 .+-. 0,99 + 1,60,5 mg/pellet N.S.CORTISONE ACETATE 10 24,3 .+-. 0,52 - 5,82,5 mg/pellet N.S.CORTISONE ACETATE 10 19,3 .+-. 1,03 - 25,212,5 mg/pellet p< 0,001JO 1034 10 26,5 .+-. 0,88 + 2,70,01 mg/pellet N.S.JO 1034 10 25,2 .+-. 1,09 - 2,30,05 mg/pellet N.S.JO 1034 10 26,7 .+-. 0,97 + 3,50,25 mg/pellet N.S.JO 1034 10 25,4 .+-. 0,39 - 1,61,25 mg/pellet N.S.______________________________________
The doses of compounds JO 1034 which exert a local antiinflammatory activity do not cause any adrenolytic effect, this distinguishing this compound from the reference compound, cortisone acetate.
h - 1.2. Systemic Effect
The results are reported in the TABLE XXIII
TABLE XXIII______________________________________ ADRENAL PERCENT- WEIGHT AGES OF (mg) Mean VARIATION NUMBER .+-. standard AND PROBA-TREATMENT OR RATS error BILITIES______________________________________CONTROL 10 28,1 .+-. 1,02 --CORTISONE ACETATE 10 28,5 .+-. 1,44 + 1,4 N.S.0,1 mg/Rat/jourCORTISONE ACETATE 10 27,4 .+-. 1,07 - 2,5 N.S.0,5 mg/Rat/jourCORTISONE ACETATE 10 23,0 .+-. 1,34 - 18,22,5 mg/Rat/jour p<0,01CORTISONE ACETATE 10 22,2 .+-. 1,43 - 21,012,5 mg/Rat/jour p<0,01JO 1034 10 29,0 .+-. 1,98 + 3,2 N.S.1 mg/Rat/jourJO 1034 10 27,8 .+-. 0,66 - 1,1 N.S.5 mg/Rat/jourJO 1034 10 28,8 .+-. 1,52 + 2,5 N.S.25 mg/Rat/jourJO 1034 10 27,1 .+-. 0,96 - 3,6 N.S.125 mg/Rat/jour______________________________________
This chart confirms that compound JO 1034 is free of systemic activity.
h - 2. After a Long Term study
The results are reported in TABLE XXIV. Weights are expressed per 100 grams of bodyweight. At the histological examination, neither atrophy nor hypertrophy was seen.
TABLE XXIV______________________________________ JO 1016 100 250 500 CONTROL mg/kg mg/kg mg/kg______________________________________ADRENAL 15.2 mg 14.9 mg 12.3 mg 13.5 mgGLANDES N.S. N.S. N.S.(male rats) mgADRENAL 25.1 mg 26.7 mg 24.5 mg 27.0 mgGLANDES N.S. N.S. N.S.(female rats)______________________________________
After having administered compound JO 1016, for 4 weeks, in a dose up to 500 mg/kg/day, no adrenolytic is noted.
i. Action on the Genital Tractus
The results are expressed on the TABLE XXV. Neither modification of the functional aspect of the male and female genital organs are appeared.
TABLE XXV______________________________________ JO 1016 100 250 500 CONTROL mg/kg mg/kg mg/kg______________________________________Testicles 1.131 g 1.143 g 1.144 g 1.207 g N.S. N.S. N.S.Ovaries 38.5 g 38.4 mg 38.3 mg 39.3 mg N.S. N.S. N.S.Seminal 284.3 g 328.0 mg 296.0 mg 274.6 mgvesicles N.S. N.S. N.S.Uterins tubes 150.4 g 211.8 mg 184.7 mg 165.4 mg N.S. N.S. N.S. N.S.______________________________________
At the end of the same long term study, no activity on the genital tractus is noted.
j. Action on the Digestive Tractus and Pro-infectious Effects
The results of the macroscopic observations in indicated in TABLE XXVI
TABLE XXVI__________________________________________________________________________ 14 days of treatment with hydrocortisone 4 weeks of treatment acetate with JO 1016 DOSES DOSESOrgans Macroscopic 218 mg/kg 436 mg/kg 250 mg/kg 500 mg/kgexamined observation Case n.degree. Case n.degree. Case n.degree. Case n.degree.__________________________________________________________________________LIVER Presence of 7/8 1/2 0/10 0/10 abscessKIDNEYS " 2/8 1/2 0/10 0/10SPLEEN Atrophied 8/8 2/2 0/10 0/10THYMUS Atrophied 8/8 2/2 0/10 0/10STOMACH Ulcerated 6/8 2/2 0/10 0/10 Ulcerated 4/8 2/2 0/10 0/10 and infected__________________________________________________________________________
This chart shows the difference between the indesirable general effects caused by hydrocortisone acetate when administered for 14 days, and the absence of corresponding effects after the administration of compound JO 1016 for 4 weeks.
POSOLOGY
The products described in the present invention are preferably utilized by local methods on the skin, the mucous membranes of the O.R.L. organ, the mucous membranes of the respiratory organs and the high and low digestive mucous.
The products are also used locally in the form of intra-articular injectable suspensions.
Generally speaking, the products are presented in the form of injectable, oral, nasal and auricular suspensions, of mouth-washes, gels and pomades, of suppositories, tablets and aerosols.
For the forms in which the product is in suspension, or may be considered as such, the active principle is utilized in the "micronized" form, the mean dimension of the particles being 2 microns.
The useful posology of the structures described by the present invention, as a function of their method of administration, extends between 0.25 and 50 mg. per unit taken and 1 to 200 mg. per day in adult animals.
The pharmaceutical forms may contain the products according to the invention, alone or associated with other therapeutic agents.

Number	Name	Date
2814632	Nussbaum	Nov 1957
3803133	Vogt	Apr 1974
3959260	Phillipps et al.	May 1976

Esters of 21-thiol-steroids hydrocortisone and cortisone

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