CA1157774A - Method for the reduction of mucin viscosity - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A method for reducing mucin viscosity which comprises administering an effective dose of a compound having protected sulfhydryl groups which metabolize in vivo to produce free sulfhydryl groups.

Description

BACKGROUND OF THE INVENTION
Field of the Invention:
The present invention is directed to a method for reducing mucin viscosity in mammals.
Description of the Prior Art:
me prior art techniques for treating mucus conditions have depended upon where the mucus was located in the body.
Mucus impaction of the gastrointestinal tract, biliary and pan-creatic ducts generally required surgical techniques for its removal since its viscosity was too high for the body to rid itself of the mucus by its normal path. Mucus in the respira-tory tract - often called sputum - has been treated by a variety of techniques including cough syrup and the like. One typical technique for respiratory obstructions has involved the aerosolization of N-acetylcysteine. While N-acetylcysteine is very effective in reducing sputum viscosity, it is extremely difficult, if not impossible, to obtain adequate delivery of this agent to the small airways of the lungs. Hence, its effectiveness in reducing the sputum viscosity is variable depending upon whether or not adequate delivery has been attained. In addition, N-acetylcysteine is extremely irritat-ing to gastrointestinal and respiratory mucosa.
The problem of elevated sputum viscosity is particu-larly aggravated in those individuals suffering from cystic fibrosis wherein the high viscosity of the sputum leads to clinical difficulties in clearance of the respiratory and gastrointestinal tracts and in delivery of gas and nutrients to their respective sites. Furthermore, in newborns, the cystic fibrosis may be present as meconium ileus, an intestinal

- 2 -obstructive syndrome due to the secretion of abnormally viscid gastrointestinal mucus. At the present time, the only method of treating these intestinal obstructions is through surgical techniques to physically remove the blockage.
Accordingly, there exists a need for a method of treating mucus impactions and for the reduction of sputum viscosity which does not involve either surgical intervention or the delivery of drugs in aerosol form.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a treatment technique which provides for the reduction in the viscosity of mucin solutions.
It is a further object of the present invention to provide a treatment for respiratory and gastrointestinal obstructions through the reduction of sputum and mucus viscosities.
It is yet another object of the present invention to achieve the reduction of sputum viscosity by a treatment involving either the oral or intravenous injection of a drug into the mammal.
These and other objects of the present invention which will become apparent have been attained through the oral or intravenous administration of a compound which is metabolized in vivo to produce compounds containing sulfhydryl groups.
BRIEF DESCRIPTION OF THE DRAWINGS
A more complete appreciation Gf the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
Figure 1 demonstrates the change in viscosity of G_ component I (~ ~ ~ ). Component IA (0-0-0), whole sputum (--0-0-0-), and fibrinogen (~ ) with protein concentration (A) Figure 2 demonstrates the change in viscosity of components I, IA and whole sputum with DTE additron (B), Figure 3 demonstrates the change in viscosity of whole sputum after addition of 5 mM MDP, and Figure 4 demonstrates the (A) appearance of rree sulfhydryls in the blood of mice after i.p. administration of 50 (~ ~ ), 100 (0-0-0), and 200 (~-0-0) mg/kg WR 2721. (B) Appearance of free sulfhydryls in the blood of mice after p.o. administration of 400 mg~kg. (C) Appearance of free sulfhydryl in the lungs of mice after i.p. administration of 200 mg~kg.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is based on the isolation of a mucin having a high molecular weight of 425,000 D from the sputum of a patient with cystic fibrosis. This protein may be responsible for the increased viscosity of the sputum since increasing its con-entration results in a rise in viscosity.
Also, as in the case of the crude sputum, the viscosity of the purified mucin can be decreased by treatment with sulfhydryl reducing agents. Similar results were also obtained with sputum obtained from patients suffering from pneumuccocal pneumonia, chronic bronchitis and the like. m e biochemical effect of the reducing agents on sputum mucin viscosity is to split the molecule into a component which retains most of the carbonydrate and ~t least two small peptides having mole-cular weights of 65,000 Daltons and 27,000 Daltons. This structural change leads to a dramatic alteration of the rheolo-gical properties of the molecùle. This alteration of the rheological properties of the molecule would allow the body to rid itself of the mucus secretions via the normal body processes. Although we did find the use of sulfhydryl compounds to be very effective in reducing the mucus viscosity, the administration of sulfhydryl compounds to mammals has not proven to be a satisfactory method for treating mucin with sulfhydryl compounds to be administered either intravenously or orally. In particular, fairly large doses of the sulfhydryl compound must be given to compensate for the reaction of the free thiol with plasma and gastrointestinal proteins while enroute to the lungs and other organs where mucus impaction may have occured. At the dosages which must be given, the sulfhydryl compounds can lead to toxic side effects. We have now discovered the problem which we encountered with the administration of sulfhydryl compounds to reduce mucin visco-sity may be overcome by administering the compounds which ; are converted to sulfhydryl groups in vivo. Suitable compounds include pharmaceutically acceptable thiosulfates, thiophosphates, disulfides and the like. ~he compounds which are suitable for use in the present invention are all characterized by containing a blocked sulfhydryl group wherein the blocking agent is removed in vivo to form a sulfhydryl group. Suitable compounds include aminoalkylthiosulfuric acids, aminoalkylphosphorothioates, thiosulfatoalkylamines, phenal-kylaminoalkylthiosulfuric acids, hydroxyalkylaminoalkylthio-sulfuric acids, hydroxyaminoalkylphosphorothioates, alkoxy-alkylaminoalkylthiosulfuric acids, cycloalkyloxyaminoalkyl-thiosulfuric acids, phenoxyalkylaminoalkylthiosulfuric acids, cycloalkylaminoalkylthiosulfuric acids, cycloalkylalkylamino-alkylphosphorothioates, cycloalkylalkylaminoalkyldisulfides, phenoxyalkylaminoethyldisulfides, hydroxyalkylaminoalkyldi-sulfides, alkylamidiniumthiosulfates, acetamidine derivativescontaining a blocked sulfhydryl group, arylalkylamidiniumthio-sulfates, aminoalkylaminoalkylphosphorothioates, quinolyloxy-alkylaminoalkylthiosulfuric acids, pyridyloxyalkylaminoalkyl-thiosulfuric acids, phenoxy- and phenylthioalkylamidinium-thiosulfates, cycloalkylamidiniumthiosulfates, and the like.
In general, any compound containing a blocked sulfhydryl group which is pharmaceutically acceptable may be employed in the present invention provided its blocked sulhydryl group is 0 converted to the free sulfhydryl group in vivo.
m e following compounds are preferred for use in the present invention:

1. (H2NCOCH2CH2NHCH2CH2S-)2 2 H ~COCH2CH2~HCH2CH2SS03H

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3. H2NcocH2cHzN~cH2cH2spo3 2

4. H2N(CH2)3NHCH2CH2SS03H

5, (H2N(CH2)3NHCH2CH2s-)2- '

6. CH3(CH2)9NHCH2CH2SS03H

7. CH3(CH2)9NHcH2cH2sPo3ff2 , ¦ 8- (CH3(CH2)9NHCH2CH2S )2 l 9. H2NCH2CH2SP03H2 l 10. H2N(C~2)sNH(CH2)2SP03~2 11. H2N(CH2)3NH(C~2)sPo3H2 ¦ Of these compounds, (11) is particularly preferred. This compound has ¦ been designated W~ 2721 in toxological tests conducted at the Walter Reed Army Medical Center. This compound has been proposed as a novel antiradiation ¦
¦ drug and is the subject of United States Patent 3,892,824.
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In the treatment of the present invention, the compound containing ~he ¦ blocked sulfhydryl group is administered to the patient at a dosage rate ¦ sufficient to reduce the mucin viscosity. Dosage rates ranging from l.~g/kg/
¦ day to an excess of 100 mg/kg/day have proven satisfactory. Dosage rates ir, the der of from 5 to 50 mg/kgJday are preferrbly employed. A

I=

particularly preferred dosage rate involves the administration of 5 mg/kg four times a day. The compound may be administered either orally or intravenously in conjunction with a suitable pharmaceutical carrier.
Intravenous administration may involve the use of physiological saline solutions which may or may not contain a sodium carboxy methyl cellulose and if desired TWEEN 80 R. Obviously, simple physiological saline solutions may be employed; water alone can be used, or the like.
In the preferred method the compound is administered orally with a suitable solid carrier. If desired, adjuvants such as buffers and the like may be employed. It may be embodied in suitable tablet form, such as a WR 2721 containing tablet provided with an enteric coating, MR 2721 being present in major or minor amount. It may also be administered in a suitable capsùle, of geletin or the like. Suitable solid carriers include starch powder, lactose, glucose powder, sucrose, dextrose, manitol powder, avicel (microcrystalline cellulose), calcium phosphate anhydrous, calcium phosphate dihydrate, kaolin, talc, magnesium carbonate, magnesium hydroxide, calcium carbonate, sodium bicarbonate, sorbitol and the like.
U.S. Patent 3,892,824 reports on the toxicity of the compounds falling within formula I above. Animal studies conducted on these compounds revealed LD 50's ranging from 450 mg/kg up to 1,300 mg/kg. For the compound identified as WR 2721 no toxicity problems were encountered at regular doses of up to 100 mg/kg/day in these toxological tests. In tests on WR 2721 for its possible side effects, dosages were given to human volunteers at rates of up to 30 mg/kg/day without any adverse effects being noted. For this reason compound WR 2721 is the preferred compound in view of the extensive pharmaceutical tests in connection with its use as an antiradiation drug which have been performed to date.
Having generally described this invention, a further understanding can 57774~
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be obtained by reference to certain specific examples which are provided herein for purposes of il1ustration only and are not intended to be limiting unless otherwise specif,ied.
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EXAMPLE 1 , In the following examples, the following abbreviations have been used:
GSSG - reduced glutathione , GSH - oxidized glutathione SDS - sodium dodecyl sulphate DTE - dithioerythritol DMS - dimercaptosuccinic acid ~P - N-2-mercaptoethyl-1,3-d;am;nopropane MDP was prepared by boiling WR 2721 in 1 M HCl under nitrogen for . -fiYe minutes. The solution was neutralized with sodium ~icarbonate.

Sputa were obtained by postural drainage from a 7-year old male patient wlth a confirmed diagnosis of cystic fibrosis. .
. . ''.

! Samples were collected in jars containing ~.S ml of injectable . gentamycin sulfate. Upon receipt the samples were added to 0.1 ml of a 1 M
sodium a~ide solution and processed within 24 hours of collection.

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Isolation and Electrophoretic Characterization of Sputum Components:
A 10-ml aliquot of whole cystic fibros1s sputum was applied to a column (4 x 70 cm) of Bioge)~A5M (BioRad Corporation, Richmond, CA~ ¦
equilibrated with 0.01 M phosphate buffer (pH 7.0) containin~ 0.1 M NaCl and 1 mM sodium azide and eluted with the same buf~er. The material .
appearing in the void volume was pooled and designated component I.
After isolation on Biogel A5M, component I was passed successively through 0.8, 0.5 and 0.22 ~I filters (Millipore Corporation, Bedford, MA) to el;minate any possible bacterial contamination.
Component I was concentrated to 5 ml by volume dialysis in tubing o~
5/8" diameter (SGA Scientific, Bloomfield, NJ) and added to a final r~
concentration of 4% SDS. This sample was washed through an Arnicon~X~llOOA
filter (Amicon, Lexington, MA) under N2 pressure with 500 ml of 0.01 M
¦ phosphate buffer (pH 7.0) containing 0.1 M NaCl and 4% SDS. The material retained by the filter was designated component IA and was reconcentrated by vacuum dialysis for use in subsequent experiments.
. SDS-Polyacrylamide gradient gel electrophores;s (5-16% acrylamide) was performed by the method of Maizel, J.V. (1971) Methods Virol. 5:179.246.
Gels were stained with either Coomassie blue; Fairbanks, G;, T.L. Steck, D.F.H. Wallach, (1971) Biochem. 10:2606-2617; or periodic acid-Schiff.
reagent; Zacharias, R.J., T.E. Zell, J.H. Morrison, J.J. Woodlock, (1969) Anal, Biochem. 31:148-1~2.
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Purification and characterization of Component IA:
SDS-polyacrylamide gel electrophoresis was performed on component IA. The major constituent of component IA has an apparent molecular weight of 425KD. A number of other constituents, one of which (60KD) is the main contaminant, remain after the Amicon-SDS diafiltration, Efforts to remove these contaminants have not been successful.
Following treatment of component IA with reducing agents gel electrophoresis revealed several new Coomassie blue staining bands at 65KD (65,000 Daltons) and 27 KD. These peptides were apparently split from the large molecular weight mucin (425 KD) - since PAS staining of another gel revealed a PAS positive band in the same area as the original mucin.
VISCOMETRY:
The viscosity of crude sputum specimens before and after the addition of sulfhydryl agents was measured in a 0.2 ml pipette by determining the time required for 0.08 ml to run out. Purified mucin samples were concentrated by vacuum dialysis and dialyzed against 0.01 M phosphate buffer (pH 7.0) for several days. Their viscosity was then measured using a Beckman Low-Shear Rotary Viscometer (Model 250010). The viscosity of fibrinogen solutions of various concentrations was similarly measured. All viscometric studies were performed at room temperature. Changes in viscosity with the addition of 5mM DTE were measured by adding 50 ~ of a 500 mM aqueous solution of DTE to 5 ml of sample.

d~ 11 -Viscosity of Sputum, Component I and Component IA:
The relationship of protein concentration to viscosity is shown in Figure I for components I, IA and fibrinogen, a large molecular weight glycoprotein. The effect of ~mM DTE
on whole sputum, component I and component IA is shown in Figure 2. Both purified mucin fractions behave like sputum with respect to viscosity changes upon addition of DTE.
Changes in sputum vicosity upon the addition of various agents are shown in Table I. Of the sulfhydryl compounds tested, DTE was the most efficacious in vitro. me oxidized forms of glutathione and lipoic acid were very much less active than their corresponding thiols, oxidized lipoic acid being totally ineffective in reducing sputum viscosity in vitro. Since DTE might be acting as a metal chelator, and as a result of early reports the high concentration of EDTA could reduce sputum viscosity, nonsulfhydryl chelators were assayed in this system and found to be without effect.
Addition of 5 mM MDP to cystic fibrosis sputum reduced the viscosity by 70% in 15 minutes as shown in Figure 3. The parent thiophosphate (WR 2721) had no effect on sputum viscosity.

X

TABLE I
SHORT-TERM VISCOSITY CHANGES OF
WHOLE CYSTIC FIBROSIS SPUTUM

1 mM AgentMinimum Rel. Time Added Viscosity * (min.) DTE 21% 10 Antabuse 50% 10 Dimercaptosuccinate 50% 15 GSH 33% 40 GSSG 67% 15 Lipoate 100% 60 Dihydrolipoate 36% 10 D-penicilllamine 32% 20 WR 2721 30% 15 EDTA 100% 60 EGTA 100% 60 H202 100% 60 * Expressed as % of control viscosity.

Example 2 Tissue concentrations of MDP were determined at various times after intraperitoneal and oral administration of WR 2721 to mice. Lung and liver homogenates obtained by 5 minutes of hand bouncing in 1 ml saline, or 1 mm thick tissue slices, were incubated with 1 mg/ml of WR 2721 at 37C. Aliquots were taken at various times for assessment of conversion to MDP.
Blood was obtained by retro-orbital puncture, the mice sacri-ficed with CO2, and the lungs removed. The lung and liver homogenates (0.9% in saline w/v) and blood samples were treated with trichloroacetic acid to a final concentration of 10%, and allowed to stand at 4C for ten minutes. Samples were centrifuged at 3000 rpm for ten minutes and the supernatant neutralized with sodium bicarbonate. These neutralized super-natants were assayed for the presence of sulfhydryl groups by the method of Ellman et al, (1959) Arch. Biochem. Biophys. 82:
70-77.
,Metabolic Studies of WR 2721:
As is shown in Figure 4, WR 2721 appears in the blood of mice as the free thiol after both oral and i.p. administra-tion. The kinetics of appearance of MDP in the lungs of mice are similar to those for blood after oral (not shown) and i.p.
administration.
Oral administration of WR 2721 leads to a rapid appearance of MDP in the blood and lungs, whereas parenteral administration is followed by a rise in MDP concentration 24 hours later.
Homogenates of mouse lung, liver and small intestine incubated with 1 mg/ml of WR 2721 converted all of the compound ; 30 to MDP within 30 minutes:

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. '', thus, demonstrating in v;vo conversion of WR 2721 to its active~ free thiol derivative, for the reduction of mucin viscosity The administration of compounds containing protected sulfhydryl groups may be used in the treatment of any condition ~herein excessive mucin viscosity is present. Such conditions include cystic fibrosis, pneumonia, bronchitis, the common cold, mucin impaction of gastrointestinal tract, pancreas, liver, and the like.
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. " `~ - 15 -~ 157774 TABLE II
2-Aminoalkylthiosulfuric Aci~s R

., WR No. Rl R2 ALD
mq/kg 1826l9 C2H5 H

1903 CH3(CH2)2 (DL) H 325 1904 CH3(CH2)2 (L) H 250 2085 CH3(CH2)2 (D) H 250 2176 CH3(CH2)2 CH3 300 4619 (CH3)2CH H 500 2293 (CH3)2CH CH3 >400 1663 CH3~CH2)3 H 175 2177 CH3~CH2)3 CH3 300 1661 (CH3)2cHcH2 H 200 2178 (CH3)2CHCH2 ~ CH3 240 1662 CH3CH2CH(CH3) H 250 2649 -(CH2~4- 800 2579 -(CH2)5- 300 2381 CH3(CH2)5 CH3 180 I

TABLE III
2-Aminoalkylphosphorothioates IR~

WR No. Rl R2 ALD50 mg/kg ~, 5137 (CH3)2CH H 6vû
2378 (CH3)2CHCH2 H 250 3311 (CH2)5 300 2819 CH3 2 2 CH3 >400 . . I

1 1S777~

TABLE IV
2-Thiosulfatoalkylamines . R

WR No. R ALD50 mg/kg . -- ~

2516 CH3 >800 1090 CH3CH2CH2 . 188 1081 CH3(CH2~3 150 1144 CH3(CH2)4 150 1250 CH3~CH2)7 300 . I
.

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. ~ i 157774 TABLE V
2-Aminoethylphosphorothioates R~

R2 '' WR No. Rl R2 ALD50 mg/kg 176241 CH20H CH2~H >1250 194423 H2NCH2CH2 (L) H >1250 179209 H2N(CH2)3 ~D~) H >1000 187094 H2N~CH2)3 (L) H > 800 176542 H2N(CH2)4 (DL) H 900a 187093 H2N(CH2)4 (L+) H 275 183977 H2N(CH2)5 H 500 . I
a Oral ad~inistration ~ _19_ I _ ~, I

~ 15777~

TABLE YI
Al kylaminoethanethiosulfates WR No. R ALD50 mg/kg 1033 CH3(CH2)2 350 2244 CH3(CH2)3 300 2341 CH3(CH2)4 200 2137 CH3(CH2)5 100 2078 . CH3(CH2)6 125 1618 CH3(CH2)7 125 t818 - CH3(CH2)8 40 1607 CH3(CH2)9 13 2111 CH3( CH2)10 8 2080 CH3(CH2)11 10 2236 CH3(CH2)12 10 Z281 CH3(CH2)13 . 10 2345 CH3(CH2)14 40 241 2 ~ C~3(CH2?15 . ` ; . 150 23 56 CH3(CH2)16 ~ 200 2362 CH3(CH2?17 . 400 . ...
. - 20 -TABLE YII
Branched - Alkylaminoethanethiosul~uric Acids RNHCH2CH2SS03H , . ', WR No. R ALD50 mg/kg 259 (CH3)2CH 270 874 (CH3)3C 250 8165 (CH3)3CCH2 475 2856 (CH3)2CHCH2CHCH3 175 2231 CH3(CH2)4CHCH3 175 1606 CH3(CH2)5CHCH3 138 2079 CH3(cH2)3cH(c2Hs)cH2 180 2246 CH (CH ) CHCH CH 125 2402 CH3(CH2~3CHCH2CH2CH3 140 2690 (C2H5)3CCH2 121J
(CH3)3CcH2c(cH3)2 2077 (CH3)2CH(cH2)5cH2 140 2386 - CH3(CH2)4CHCH2CH2CH3 200 2926 CH3(CH2)5CHCH2CH3 125 9528 (CH3)3CCH2CH(CH3)CH2CH2 100 2245 CH3(CH2)7CHCH3 25 2709 . CH3(CH2)6cHcH3 50 2390 CH3(CH2)6CHCH2CH3 100 3358 (CH3)2CH(CH2)3CH(CH3)CH2CH3 125 2411 CH3(CH2)8 3 20 Il ` .

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TABLE VIII
Phenalkylami~noethanethiosulfuric Acids A ~ (cH2)n-NHcH2cH2sso3H

W~ No. n A
3552 -- H ~800 4701 CH2 4-oCH3 180 2456 (CH2)2 H 150 2229 (CH2)3 H 125 3300 (CH2)3 2-OCH3 200 2144 (CH2)3 3-OCH3 300 454?. (CH2)3 4-OCH3 200 2754 (CH2)4 H 300 71462 (CH2)4 4-Br 43 3086 (CH2)4 4-CH3 15 . 3337 (CH2)4 2,5-(CH3)2 20 3340 (CH2)4 2,4-(CH3)2 38 3342 (CH2)4 4-C2~5 15 4102 (CH2)4 2-OH 300 3050 (CH2)4 4-oCH3 175 4109,(CH2)4 3-OCH3 150 3338 (Ch2)4 3,4-(oCH3)2 350 4103 (CH2)4 4-oC2H5 22 3611 (CH2)4 4-C6Hll 15 6138CH(c2Hs)cH2 3-OC~3 100 .

TABLE V I I I
(Conti nued ) WR No. n A
5983 CH(C2H5)CH2 4-OCH3 110 120767 CH2CH ( C2Hs ) 4 -OCH3 130 4114 CH(c2H5)cH2 H 125 7468 CH(c2H5)cH2 4-C1 140 3427 (CH2)5 H . 140 4548 (CH2)S 4-OCH3 45 3313 (CH2)6 H 38 4783 (CH2)6 4-OCH3~ 40 976~ (C~2)~ 5 ':
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TABLE IX
Hydroxyalkylam~noethanethiosulfuric Acids RNHcH2c~l2s~io3H

WR N0. R

2g86 HocH2cHoHcH2 1750 3569 (HCH2)2cH 1750 3273 HocH2cHoHcHoHcH21800 3093 (CH3)2C(OH)CHOHCH21400 2970 H0CH2 (CH2)6 550 2971 ! H0CH2(CH2)7 625 2865 CH3~CH2)5CHCH2~ 140 2908 HocH2~cH2)8 3 . 2907 H0CH2 (CH2) 9 > 200 2909 C~3 (CH2) 7C~C~20H25 3024 HOCH2 (CH2)8CHOHCH2 27748 HOCH2CHOH(CH2)9 400 ,, .. , . '.

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TABLE X
Hydro~yalkylaminoethylphosphorothioates . RNHCH2CH2sPo3~2 WR NO.

8192 CH3CE~OHCH2 1250 27736 CH3CH2cH2(cHOH)2cH2 450 27077 (CH3)2CH~CHOH)2CH2 275 42597 (cH3cH2)2cHcHoHcH2 240 25077 CH3(CH2)4(CHOH)2CH2 36978 (cH3)3ccH2cH(cH3)cHoHcH2 125 52114 CH3(cN2)4cHoNcH2cNoNcN2 1800 ., ~ , Ij 1 157774 ~ - ' TABLE XI
Hydroxyalkylaminoethyldisulfldes RNHcH2cH2sscH2cH2NHR

WR N0. R

3568 (H0CH2)2CH 1250 39748 HOCH2(CH2)4 600 36951 HOCH2(CH2)5 220 4853 CH3(cH2)4cHoHcHoHcH2(rac. 1) 75 5027 CH3(CH2)4cHOHc~oHcH2(rac. 2) 75 30462 H0CH2CHOH(CH2)g 175 3331 CH3(CH2)7(CHOH)2(CH2)8 133 ~ ~¦ 3333 CU3(CH2)sCU03(C~2~11 133 ''.
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TABLE XII
Alkoxyalkylaminoethanethiosulfuric Acids R-0-(CH2)nNHCH2CH2~S03H

WR No. R n ALD50 mg/kg 3604 HOCH2CH 2 900 .
3607 HOCH2CHOHCH2 2 . 2000 983 CH2 = CH 2 63 159244 CH3(CH2)~ 5 . - i .1 II -~
. I , TABLE XIII
Cycloalkyloxyaminoethanethiosulfuric Acids R-0 -(cH2)nNHcHzcH2sso3H

K N0. R n .
3069 (CH2)5CH 3 lS0 4111 (CH2)4CH 4 150 2972 (CH2)5CH 4 150 1500 4444 (CH2)6CH 4 lS0 4446 (CH2)7C~ 4 250 4541 (CH2)4C~ 5 150 1300 2973 (CH2)5CH 5 lS0 >800 4538 2-CH3C6Hlo . 75 1100 ~781. 1-CH3C6Hlo lS0 1000 4540 4-CH3C6Hlo 5 18 1300 _~8415 2-(CH3)2CCH 5 CP3 60 >900 4542 ~CH2)6CH 5 35 4545 (CH2)4CH 6 200 1800 2990 (CH2)5CH 6 150 4544 (CH2)5CH 8 15 3610 (cH2)scHcH2cH2 4 38 5331 .(CH2)5CHCH(Cz~5~ 96 >600 3567 (C~2)5CHCH2 5 18 3716 (CH2)7CH 5 180 .. __.

. - 28 -TABLE XIV

Phenoxyalkylaminoethanethiosulfuric Acids . A ~ -(CH2)nNHCH2CH~SSO3H

.INTRAPERITONEAL DAT~ ~ ORAL DATA

WR_NO _ A n _............. _ 4381a H 2200 3071 3-C1 2lS0 4543b H 2700 5146a H 3175 ~301 3-CH3 3125 3305 H 4>250 3431a H 4 75 . 3087 2-CH3 4150 3121 4-CH3 . 4 35 1400 .

33712 2-(CH3)2CN .4 320 4537 4-C6Hll j 4 50 3830 4-CH30 4200 ~2000 3828 4-C2H5O 4 95 ~ 100 TABLE XIV
( Con ti nued ) . INT~A~ERITONEAL DATA ORAL D~TA

,;R No. A n - ALD50 ~ ALD50 5981 4-Br 4 >150 4106 4-C1 4 175 _ 3819 3-C1 4 150 ~1250 4792 2-C1 4 150 >3200 111489 3-(C2Hs)2N 4 175 > 900 4447 4-N02 4 150 ~2400 3335 3-CF3 4 45 ~1200 4769 2,3-(CH3)2 4 22 3336 2,4-(CH3)2 4 20 '1000 4805 2,5-(CH3)2 4 40 >2000 478.8 2,6-(CH3)2 4 250 >1500 3343 3,4-(CH3)2 4 45 >1200 124940 3-CH3-4-(CH3)2N - 4 150 75234 3-(CH3)2N-4-~H3 4 . 180 > 900 150640 3-NH2-4-CH3 4 150 . > 300 3122 2,4-C12 4 125 >2000 43899 2,4-~(CH3)2CH)2 4 75 3832 2-CH30-4-C2H5 4 125 ~2400 36958. 2~3,6-(CH3~3 4 ~ >100 .
39750 2-Cl-4-Br 475 40640 2-(CH3)3C 438 _ 3822 H 5 ~ 50 >2000 _ . , TABLE XIV - .
( Conti nued ) . -INTRAPERITONE~L VATA ORAL DATA
~LD50 - ALD ~~~
NO. A n mg/kg 3826a H 5 300 _ 4S46 2-CH3 5 200 >3200 ¦ 136181 3-(CH3)2N-4-CH3 5 135 _ 4777 2-CH3 6 150 >1500 ~' . ,1 . The phenylthio analog '~. 2-Naphthyloxyethylaminoethanethiosulfuric acid . . i.

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TABLE XV
Phenoxyalkylaminoethyldisulfides [ ~o--(C112)nNHC~2c1l2s~2 WR N0. A n _ 6368 H ` 3 120 5141a H 3 lS0 36159 2,5-(CH3)3 4 45 '.
33714 2~6-(cH3)3 4 S60 . 40644 2,3,6-(CH3)3 4 56 43893 2-(C~3)2CH 4 100 . a. The phenylthio analog .

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Cycloalkylalkyla~inoethyldisulfides . ~ ' . I
~R-(CH2)n-NHCH2cH2s-12 WR N0. R n 692 (CH2)5CH 0 92 48680 2-bornyl . 0 180 I, 148667 l-adamantyl 1 75 3023 (CH2)5CH 4 22 33713 (CH2)5CH CH(C2Hs)C~245 36952 (Cuz)scu CH(CU3)(CI12~3 45 .
, `~

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TABLE XVIII
Cycloalkylalkylaminoethylphosphorothioates R-(CH2)n-~cH2cH2sPo3H2 ALDso WR No. R n D~/kg 40335 2-bornyl 0 430 35979 l-adamantyl 0 240 12;696 2-adamantyl 0 150 133567 (CH2)11CH 0 70 74171 2-nrobornyl 0 240 142076 l-adamantyl 2 70 4481 4-CH3-2(=0)c6H8 C(CH3)2 375 151321 l-adamantyl 3 115 159742 (CH2)5CH 4. 45 117909 (CH2)5CH CH(C2H5)CH2 125 4480 2(=O)C6Hg CH(n-C3H7)88 : 75235 4-C6Hll-C6Hll >1800 .
. , i, TABLE XIX
2-Pyri~3yloxyalkylaminoethanethiosulfuric Acids A
O (cH2)nNHcH2cH2sso3~
.INTRAPERITONEAL DATAORAL DATA

17R NO. _ A n 37679 ~l ,2 99590 S-Br . 2175 98080 H 3l9S
80859 3-~1 3160 ~4203 5-Br 3125 ~ 900 : 91493 3-N02 3175 ;91489 5-N02 3180 108504 5-C1 4190 >1200 .104695 5-Br 4180 > 900 82422 5-Cl- 5225 ~1000 138412 3,5-C12- 5175 900 99582 3-Br 5170 43900 5-Br 590 . 50480 6-Br 5140 99581~ 3,5-Br2 S160 ~1200 . - 40 -,_ , TABLE XIX ~continued) i 2-Pyridyloxyalkylaminoethanethiosulfuric Acids ~ ~ 0(CH2)nNHCH2SSO3H

INTRAPERITONEAL DATA ORAI DATA

WR NO. A n mg/kq 104696 5-NO2 5 150 ~ 900 94533 5-CH3 5 125 ~900 106162 5-C1 6 120 >800 91494 5-Br 6 225 1 124950 3,5-Br2 6 250 i 145723 6-Br 7 50 94531 5-Br 10 160 TABLE XX
2-QuinolyloxyalkylaminoetHanethiosulfuric Acids A ~ (cH2)n~cH2c~2sso3H
INTRAPERITONEAL DATA ORAL DATA
.._ WR No. A n ~ - ..

122960 4-CH3,6-OCH3 2100 106156 H . 3220 ¦25930a H 34SO
9463b H 3125 122968 4-OCH3 3175 . .
54204 4-CH3 3180 >900 120769 4-CH3,8-C1 3300 .108502 4-CH3,6-C1 3 75 >600 . 111490 4-CH3,6-OCH3 3100 122970 4,6-(CH3)2 3125 >900 126456 4-C3H7 3125 >600 104706 4-CH3 4225 >750 111486 4-Cl. 5275 122959 4-OCH3 5225 >900 42761 .4-CH3 5740 123953 4,6-(CH3)2 5115 .1492 4-CH3,6-C1 5 ¦ 175 ' ~1 ` ` 1 157774 TABLE XX
( Conti nued ) . INTRAPERITONEAL DATA
. _ .

W~ No. A n mg/kg 120768 4-CH3,8-C1 5325 i~4951 4-C3H7 5120 369S5c 7-Cl S250 1~;1491d H 5175 ._ _ !
2 5-Quinolyloxypropylaminoethanethiosulfu-ic acid.
b 8-Quinolyloxypropylaminoethanethiosulfuric acid.
7-Chloro-4-quinolyloxypen~ylaminoethanethiosulfuric acid. I
d 1 ~oquino1yloxypenty1a=iooethanethio~u1furic acid.
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TABLE XXIII

Parent Acetamidine Derivatives H2NC ~ =1~ ) CH25Y

INTRAPERITONEAL DATA ORAL DATA

WR NO. Y mq/kq mq/kq ¦166817 SCH2C(----NH)NH2 60 125 ¦108250 P3H2 100 135 ''' ?

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Claims (7)

The embodiments of the invention is which an exclusive property or privilege is claimed are defined as follows:-

1. A pharmaceutical composition comprising a compound having protected sulfhydryl groups which metabolize in vivo to produce free sulfhydryl groups and a pharmaceutically acceptable solid carrier.

2. A pharmaceutical composition according to claim 1, which also comprises an adjuvant.

3. A pharmaceutical composition according to claim 2, wherein said adjuvant comprises a buffer.

4. A pharmaceutical composition according to claims 1, 2 or 3, wherein the compound containing protected sulfhydryl groups is selected from the group consisting of:

(H2NCOCH2CH2NHCH2CH2S-)2 H2NCOCH2cH2NHCH2cH2SPO3H2 H2N(CH2)3NHCH2CH2SSO3H
(H2N(CH2)3NHCH2CH2S-)2 CH3(CH2)9NHCH2CH2SSO3H
CH3(CH2)9NHCH2CH2SpO3H2 (CH3(CH2)9NHCH2CH2S-)2 H2N(CH2)5NH(CH2)2SPO3H2 H2N(CH2)3NH(cH2)SPO3H2 and their pharmaceutically acceptable salts.

5. A pharmaceutical composition according to claims 1, 2 or 3, wherein said compound containing protected sulfhydryl group has the formula:
H2N(CH2)3NH(CH2)SPO3H2.

6. A pharmaceutical composition according to claims 1, 2 or 3 in tablet form.

7. A pharmaceutical composition according to claims 1, 2 or 3, in tablet form, said tablet provided with an enteric coating.