IE52497B1 - Antithrombotic treatment - Google Patents

Description

- 2 - 52 48 7 Sodium bisulphite (usually shown by the formula NaHSO^) has previously been used for many commercial purposes, for example, as a preservative, for preventing deterioration of liquids, such as 5 foodstuffs and pharmaceutical solids, and it has been used medically, externally for the treatment of parasitic skin diseases and internally as a gastrointestinal antiseptic.

The solid sodium bisulphite of commerce 10 reportedly consists chiefly of sodium metabisulphite, Na2S205, and sodium acid sulphite, NaHS03, and, for the purposes of this.invention, it is believed to possess the same properties as (and to be equivalent to) sodium bisulphite, when in the form of an aqueous 15 solution.

Anticoagulants and antithrombotic agents are a group of compounds with diversified pharmacological actions which are used in a variety of chemical thrombotic disorders. Thrombotic disorders axe generally 20 divided into venous thromboses and arterial occlusive disorders. Venous thrombosis of the lower extremities is important, because it can cause pulmonary embolisms which may be fatal. "Heparin" and "Warfarin" are commonly used in clinical medicine for the prevention \ - 3 - 52487 and treatment of deep venous thromboses and pulmonary embolisms. Their main pharmacological actions are to inhibit or interrupt blood coagulation activity.

Platelets play an important part in arterial thrombosis.

Drugs which inhibit platelet aggregation are generally regarded as potentially useful for the prophylactic therapy of arterial thrombotic disorders, including, for example, strokes, myocardial infarctions and peripheral vascular disease. Despite the availability 10 of many agents which possess platelet anti-aggregatory properties, only a few are currently under clinical trial (for example, "Aspirin", dipyridamole and sulphinpyrazone). None of these agents exhibits unequivocal efficacy. Compounds with more specific 15 pharmacological actions are urgently sought in order to provide better medical care for patients with these serious disorders.

An anticoagulant is a substance which inhibits coagulation of the blood. A platelet anti-aggregatory 20 agent is a substance which inhibits platelet aggregation.

An antithrombotic agent · is a substance which inhibits the formation or development of a thrombus (or thrombosis). For present purposes, the term "thrombus" or its equivalent includes the term 25 "embolus", unless otherwise specifically indicated. In general, an antithrombotic agent, in the presence of mammalian blood or appropriately-prepared plasma, may display anticoagulant activity and/or platelet antiaggregatory activity.

A class of active agents has now been dis covered, the members of which, when orally ingested and/or injected, produce amelioration of a thrombotic condition in mammals (including man), when used in antithrombotically-effective amounts as described below. 52487 - 4 - According to one aspect of this invention, a pharma-cuetical composition is provided for use in treating an actual or incipient thrombotic condition in a mammal, which contains at least one carrier substance and, as the active 5 treating agent, at least one compound selected from the following compounds: (A) compounds of formula: R? - NH® SO,H /. ό n R3 J m wherein: 10 R^ is an alkyl radical containing less than 11 carbon atoms, a cycloalkyl radical containing 6 to 10 carbon atoms or a monohydroxyalkyl radical containing - 5 - 52497 / less them 11 carbon atoms, R2 is a hydrogen atom, an alkyl radical containing less than 11 carbon atoms or a monohydroxyalkyl radical containing less than 11 carbon atoms, or 5 r and R together form an unsubstituted 1 * morpholine, piperidine or hexamethyleneimine ring or such a ring carrying on one of its carbon atoms an alkyl radical containing less than 11 carbon atoms, R^ is a hydrogen atom, an alkyl radical containing less than 11 carbon atoms, a monohydroxyalkyl radical containing less than 11 carbon atoms or a radical of the formula: “ *1 SO,H HNR.®-- on j 4 R2 l- 0 m wherein R^ and have the meanings defined above, 15 R4 is a divalent saturated aliphatic radical containing less than 11 carbon atoms, and mis 1 or 2, n is 0 or 1 and the sum of m and n is 2; (B) compounds of either of the formulae: \ 1 f\ * 20 X \ VX r6A N®1 S0_H s_0c2e> 7 / I ‘ / / I and / Λ > [V J2 wherein: Rg, Rg, Ry and Rg are the same or different 52497 * - ' ' :· - 6 - and each is an alkyl radical containing less than 11 carbon atoms, and m and n have the meanings defined above; (C) compounds of the formulas R—CHOH—S03M wherein: R is a hydrogen atom or a hydroxymethyl radical and M is an alkali metal or an ammonium group, but 10 excluding:- (a) 0.6M injectable aqueous solutions of ethanolamine bisulphite, and (b) compositions containing a formaldehyde bisulphite and a protamine.

Examples of suitable alkylamine starting materials for making compounds of the formula in (A) include methyl-amine, dimethylamine, trimethylamine, ethylamine, tripentyl-amine, monocyclohexylamine, dirnethylcyclohexylamine, dimethyl-ethylamine, dirnethylcyclohexylamine and tri-n-butylamine.

Trimethylamine salts tend to be undesirable because of a strong associated odour, however with this sole exception, tri(lower alkyl)amines are one currently preferred class of starting materials for the preparation of compounds of the formula in (A). Another currently preferred class of 25 starting materials for such a preparation comprises mono-alkylamines having 5 to 10 carbon atoms per molecule, such as mono-n-oetylamine. Examples of suitable diamine starting materials for making compounds of the formula in (A) include ethylene-diamine, hexamethylene-diamine, 1,2-propylene-diamine, 30 tetramethyl-ethylene-diamine and N^, ^-dimethyl-ethylene-diamine.

Examples of quaternary ammonium bisulphite compounds include tetramethy1-ammonium-bisulphite, tetraethyl-ammonium-bisulphite, tetrabuty1-ammonium-bisulphite, trimethylethyl-35 ammonium-bisulphite and tetramethy1-ammonium-metabisulphite. Tetra-(lower alkyl)-ammonium-sulphites are also active agents, however.

Antithrombotic agents of this invention are believed to be usable in both arterial thrombosis and 5249? - 7 - venous thrombosis.

Examples of clinical thrombotic conditions include stroke (as a cerebral vascular thrombosis), myocardial infarction (coronary artery disease), 5 peripheral vascular disease, cardiac valve replacement, deep vein thrombosis and pulmonary embolism.

The mechanism(s) by which the active agents function is currently unknown; however, an inhibition of platelet aggregation and a prolongation of normal 10 blood coagulation time appear to be associated with their use in the manner disclosed herein.

In one aspect, the present invention is concerned with certain organic bisulphite and sulphite pharmaceutical compositions for use as antithrombotic, 15 anticoagulant and platelet anti-aggregatory agents in mammalian medicine (including human medicine).

In another aspect, compositions ·ο£ the present invention can be used for the control of and/or the prevention of an embolus or a thrombus in mein by oral 20 ingestion and/or by injection or the use of suppositories of a pharmaceutically-effective amount of one or more compounds comprising the active agents of this invention.

In another aspect, the compositions of the 25 present invention can provide syjptanatie and objective inprovotent in thrombotic (including cardiovascular) disease conditions, for example, abnormal coagulation or intravascular thrombosis, in man. The term "symptomatic improvement", as used herein, means an improvement in a patient's 30 subjective symptoms (e.g., as reported by the patient).

The term "objective improvement", as used herein, means a measurable change in a patient's condition.

Mare particularly, the compositions of this.indention can be used in, a process for, treating a human or other mannal wherein 35 there is introduced orally and/or by injection into - 8 - . 52497 *» l ' such mammal a pharmaceutic.ally-effective amount of an active agent of this invention as an antithrombotic agent.

Sulphite and/or bisulphite anions do not 5 normally occur in human tissues or blood, so far as is now known.

In medicine, arterial thrombosis is diagnosable, for example, by clinical manifestations by arteriography and, recently, by an indium 111 platelet labelling 10 technique (see, .for example, the article entitled "Differential Effects of Two Doses of Aspirin on Platelet-Vessel Wall Interaction In Vivo" by K.K. Wu et al being published in the Journal of Clinical Investigation, August, 1981).

Also, a thrombosis is detectable, for example, from a patient's conditions symptomatically perceivable by a skilled medical practitioner and well known to the art of medicine. Objectively, various methods are available, including venography, impedance plethys- 125 20 mography, doppler ultrasound and the I-fibrinogen test (see, for example, the articles by Kakkar, "Archives of Surgery", 104, page 152 (1972) and by Kelton, J.G. et al, Journal of Clinical Investigation, Vol. 62, pgs. 892-895, (1978).

The present invention does not contemplate feeding a normal patient (that is, one not suffering from a thrombotic condition) an active agent of this invention at a pharmaceutically-effective dosage as indicated herein.

The term "thrombotic condition" as used herein, means both: (a) an existing thrombus (including an embolus); (b) an incipient thrombus (including an incipient embolus), 35 An "incipient thrombus" or "incipient thrombotic 52497 - 9 - condition", as used herein, is a condition which can exist in a patient who is predisposed to the development of a thrombotic condition. For example, diabetes mellitus and hyperlipidemia are conditions which 5 predispose a patient to arterial thrombosis. On the other hand, surgery, trauma and bed rest, for example, predispose a patient to venous thrombosis.

Those skilled in the practice of medicine routinely determine the presence of a thrombotic 10 condition (including an actual thrombus in a patient).

Preferably, the practice of this invention in vivo involves introducing into the blood of a patient, such as a human, the equivalent of 1 to 100 milligrams per kilogram of mammal body weight (including 15 human) per day, though larger or smaller dosage rates may be employed, if desired, within the scope of this invention. The exact amount or dose in any given case is selected to be sufficient and appropriate for achieving a desired antithrombotic effect.

In general such an introduction· may be commenced at a dosage rate.within the.range above indicated as soon as a thrombotic condition (or a thrombus) is found in a patient.

For example*, it is preferred that 25 as a first step, a determination is made that a patient suffers from a thrombotic condition. Then, one starts orally feeding and/or injecting such patient with at least one active agent of the present invention at an effective dosage rate in the rarige indicated 30 above. Currently, an especially preferred dosage rate is 20 to 50 mg/kg per day. Preferably, at least two or three spaced doses per day are given, each such dose being conveniently administered around a meal-time.

Any convenient dosage arrangement can be employed. 52497 - 10 - Not uncommonly, it is desirable or necessary to start treatment immediately upon the discovery o£ a patient's thrombotic condition, in order to avoid damage or injury or perhaps even death of the patient, 5 as from an embolus. If oral administration is not convenient or rapid enough, the active agent can be directly introduced by injection into the patient, if desired, such as intravenously, intramuscularly or subcutaneously. When an active agent is directly 10 introduced, it is preferably dissolved in an aqueous medium, the total amount of active agent introduced into such medium preferably being within the range from 1 to 11 weight percent (based on the total solution weight). Distilled water is preferred'as the aqueous medium.

If desired, conventional (standardized) aqueous media can be used as vehicles for such introduction; for example, standard saline solutions can be used as vehicles.

In order to evaluate an antithrombotic effect 20 it is preferable to withdraw samples of blood from a patient undergoing treatment and measure platelet aggregation. One method is described by Born in Nature 194, pp. 927-929 (1962) and may be used for this purpose, if desired.

After administration has started, the dosage rate is preferably adjusted to a value which is sufficient to disrupt platelet function and/or coagulation factors and thereby achieve a desired antithrombotic effect.

An active agent of this invention is characteristically capable of exhibiting platelet aggregation both in vitro and in vivo. Also, such an active agent ia characteristically capable of lengthening both PT (prothrombin time) and PTT (blood partial 35 thromboplastin time) in. vitro. The dosage rate of the - 11 - 82497 active agent is currently believed to be related to the resulting effects upon blood factors, such as inhibition of platelet aggregation. Consequently, under this preferred procedure, use of an active agent at a 5 suitable dose for an individual patient ameliorates that patient's thrombotic condition.

Selected blood parameters of a patient are preferably determined before dosing with an active agent is started, when time permits. Preferably, dosage 10 rate adjustment is made while administration of an active agent is continuing. The amount of adjustment (or incremental change in dosage) is determinable by comparing a patient's measured values during administration of active agent to desired values (such as the patient's .15 own corresponding starting values or normal species, e.g. human, values). Inhibition of platelet aggregation can be used for such measurements. Then, the deviation, if any, from the patient's measured values is compared to the desired values (the patient's starting values 20 or normal species values, for example)-. Then, a change in dosage rate may be made to correct any deviation so determined.

For instance, in humans, normal values for platelet aggregation are dependent upon the particular 25 agent used for stimulation. For example, when adenosine diphosphate (ADP) at 3 micromolar concentration is employed, platelet aggregation.values fall typically in the range from 50% to' 100%. of light transmission.

Other stimulation agents include collagen, epinephrine 30 and arachidonic acid.

Also, in humans, normal PT values fall in the range from U to 13 seconds, while normal PTT values fall in the range from. 25 to 41 seconds. If PT values and/or PTT values could be measured in a given patient, 35 for the purpose of achieving a desired antithrombotic : 5^497 - 12 - & effectiveness, it is currently estimated, that a lengthening of PTT value to 1.5 to 2 times a PIT value in. such normal range in a given starting patient is appropriate or suitable for antithrombotic effectiYe-5 ness, which amounts to a PTT value for a given patient of 45 to 60 seconds; such an estimate is consistent, for example, with the lengthened. PTT values achieved in the human use of heparin, sometimes employed previously as an antithrombotic agent. Similarly, it is currently estimated that a lengthening of PT value to 2,0 times a PT value in such normal range in a given starting patient is appropriate or suitable for antithrombotic effectiveness, which amounts to a PT value for a given patient of 22 to 26 seconds; such 15 an estimate is consistent, for example, with the lengthened PT values achieved in the human use of coumadin (warfarin), sometimes employed previously as-an antithrombotic agent. The active agents of the present invention, contrary to such prior art agents, 20. surprisingly appear to affect both PT and PTT values.

The mechanism by which the present active agents work is apparently substantially different from, and not comparable to, the prior art agents. Study and evaluation of the active agents of this invention 25 continues.

Contrary to prior agents (such as heparin and coumadin), the active agents of the present invention appear to affect both blood coagulation factors and platelet aggregation. Conveniently and preferably, 30 measurements of blood factors are carried out periodically, such as every 3 to 7 days, on. a patient undergoing .treatment under the practice of this invention.

An active agent can be given orally, in the form of a capsule or tablet, or in the form, of a solution 35 (e.g. aqueous). Also, an active agent can be injected 52497 - 13 - in the £orm of an aqueous solution.

A particularly preferred antithrombotic field of use is in post-operative treatment, as when arteries or deep veins may be involved in, or threatened by, 5 a thrombotic condition.

By way of explanation, as those familiar with mammalian anatomy appreciate, the venous system in the lower extremities consists of superficial and deep veins. Because of the manner in which the deep 10 veins interconnect and supply blood to the heart and lungs, a thrombus occurring in the deep veins, but not in the superficial veins, can become the source of a blood clot which is moved through the veins and becomes lodged in the lungs, resulting in a pulmonary embolus, 15 which can have obvious catastrophic effects (including causing death). Examples of deep veins include the iliac, the femoral, the popliteal and the calf veins.

The prevention of pulmonary emboli following surgery affecting the deep veins in the lower extremities is a 20 significant medical problem. One solution to this prohlem is to prevent thrombi from occurring and/or developing in deep veins. To achieve this, active agents of this invention appear to be well suited.

Thus, in carrying out this invention, one may achieve 25 a symptomatic and objective improvement of a deep vein thrombotic complication in a patient during postoperative care, inhibiting intravascular thrombus formation (including embolism).

In one preferred practice of this invention, 30 an aqueous solution of 1 to 10 percent by weight of an active agent of this invention is prepared. Then, this solution is orally consumed by a human or is injected at a total (or accumulated) dosage rate ranging from 1.0 to 50 mg per kg of body weight per 35 day, more preferably in the form, of at least two 52497 - 14 spaced doses per day, and still more preferably in the form. of. at least three spaced doses per day, each dose preferably being taken around a meal-time.

Instead, solid or encapsulated active agents may be 5 orally consumed.

Because of a tendency for bisulphites to undergo oxidation when in aqueous solution, it is currently preferred to minimize contact of active agents with oxygen before use. However, the compounds of 10 formula (C) appear to be stable towards oxygen.

The water used in such a solution is preferably purified (e.g., filtered, deionized or distilled). After preparation, the solution is preferably stored in a closed container to reduce 15 oxidation.

Such an aqueous solution can be directly used in carrying out this invention, in which case such a solution can be dispensed dropwise or it can be encapsulated, for instance, and used as measured dosage 20 units, as desired. For.example, an aqueous solution can be injected into a patient, or it can be directly consumed by a patient as drops (e.g., from 5 to 9 drops per meal for each of the two or three meals eaten by such patient per day, depending upon an individual 25 patient's body weight).

Symptomatic improvement in varicose veins and in haemorrhoids may be observable when using an active agent of this invention.

Compounds which are active agents of the fomilae 30 (A) and (B) are conveniently prepared by preparing an aqueous solution (preferably using purified or distilled water) of a corresponding lower (or corresponding quaternary ammonium, hydroxide) alkylamine compound, present at a specified or calculated concentration, 35 such as 10 weight percent. Then, S02 gas is bubbled 5249 7 • V.* - 15 - through this solution, until the resulting solution increases in weight to an extent sufficient to correspond to the desired sulphite or bisulphite salt.

For example, to prepare a solution which is sub-5 stantially a bisulphite salt, twice as much weight increase is needed as for the corresponding sulphite salt. If the amine or quaternary ammonium compound is not fully soluble (or fully in a dissolved form) at the start of the sulphur dioxide gas addition (but is 10 partially dispersed or suspended in the aqueous phase), it becomes completely dissolved as the S02 addition continues. The solutions made from the various corresponding amines appear to exist most conveniently in solution form, although some if desired may be 15 obtained as solids, e.g., monoethyl ammonium sulphite. Solutions may be diluted to 1 to 10 percent and are preferably stored in closed containers to reduce oxidation. An alternative synthesis procedure is to use an ion exchange resin in the sulphite form, if 20 desired. Some of the solutions made from various corresponding quaternary compounds, alter preparation, can subsequently be evaporated to dry solid form, such as tetramethyl ammonium bisulphite! others appear to exist only in solution form.

Salts of carbonyl sulphur dioxide adducts can be prepared hy any convenient techniques. One currently preferred starting material for making a compound of the formula in (C) is formaldehyde.- Sodium formaldehyde bisulphite may be purchased 30 commercially.

One convenient technique of preparation is to agitate a saturated aqueous solution of the desired bisulphite salt with less than a stoichiometric amount of a carbonyl compound which corresponds to the carbonyl 35 sulphur dioxide adduct desired. Solutions containing - 16 - . 52497 « . v , more than about 60 weight percent of water are preferred* Other known synthesis methods may be used, if desired.

Since aqueous solutions of sulphur dioxide display a capacity to lengthen PT and PTT values at 5 least to the extent indicated above, aqueous solutions of sulphur dioxide could be used in combination with, e.g. in admixture with, aqueous solutions of compounds of the formulae (Ά), (B) and (C) above identified.

A preferred process for treating a thrombotic 10 condition in a mammal (including man), with a composition of the present invention, includes the following steps: (A) determining from a sample of blood removed from the mammal (1) one or both of the blood coagulation-15 factors consisting of prothrombin time and partial thromboplastin time and (2) the platelet aggregation factor, (B) when such factors fall within normal ranges for such mammal, administering to such mammal 20 a pharmaceutical composition according to the invention, at a dosage rate and for a time sufficient to inhibit .the blood coagulation and platelet aggregation factors to an extent which is at least sufficient to inhibit thrombus development in the mammal but 25 insufficient to cause spontaneous internal bleeding, (C) at intervals during administration, removing further samples of blood from the mammal and determining tne lengthened blood coagulation factor(s) and the inhibited platelet aggregation factor, 30 (D) adjusting administration of the compo sition so as to achieve and maintain predetermined desired values for the lengthened blood coagulation factor(s) and the inhibited platelet aggregation factor in the mammal, and 35 (E) continuing steps (B), (C) and (D) until 5 2 4 9 7 - 17 - the mammal has recovered from the thrombotic condition.

The compositions of the present invention can also be used in vivo or in vitro for inhibiting blood coagulation factors and for inhibiting platelet aggregation involving 5 adding to blood and/or plasma derived from such blood an active agent of this invention.

Unless otherwise indicated herein, plasma is prepared by centrifuging whole blood at 2,000 r.p.m. Also, unless otherwise indicated, the term "control" in relation to plasma 10 indicates a prothrombin time (ΡΓ) or a partial thromboplastin time (PTT) as determined with normal saline solution (equal volume).

The present invention is further illustrated by the following Examples. Reference may.also be made to the dis-15 closures, including specific Examples, contained in Patent Specification No. f/ and European Application No. 822 000 91.5 (EP-A-55702) relating respectively to inorganic compounds and monosaccharide complex compounds.. It is considered that a fuller understanding of the respective 20 inventions can be obtained from the various disclosures when taken in conjunction.

EXAMPLE A A 10 weight percent aqueous solution of triethyl-amine-bisulphite was prepared by bubbling S02 through an 25 appropriate solution or dispersion of trlethylamine in water.

EXAMPLE B ' Using the procedure of Example A, a 10 weight percent aqueous solution of diethylamine-bisulphite was prepared from diethylamine.

EXAMPLE C Using the procedure of Example A, a 10 weight percent aqueous solution of isobutylamino-bisulphite was prepared from isobutylamine.

.EXAMPLE D Using the procedure of Example A, a 5 weight percent aqueous solution of n-octylamine-bisulphite was prepared from n-octylamine. • 52497 - 18 - EXAMPLE E Using the procedure of Example A, a 5 weight percent solution of tributylamine-bisulphite was prepared from tributylamine.

EXAMPLE F Using the procedure of Example A, a 5 weight percent solution of ethylene-diamine-bisulphite was prepared from, ethylene-diamine.

EXAMPLE G Using the procedure of Example A, a 5 weight percent solution of morpholine-bisulphite was prepared from morpholine, EXAMPLE H Using the procedure of Example A, a 5 weight 15 percent solution of cyclohexylamine-bisulphite was prepared from cyclohexylamine.

EXAMPLE X Using the procedure of Example A,' a 10 weight percent solution of triethylamine-sulphite was 20 prepared from triethylamine. Half as much S02 was consumed as in the preparation of Example A.

EXAMPLE J A 10 weight percent aqueous solution of tetramethyl-ammonium-bisulphite was prepared by bubbling 25 SC>2 through a solution of tetramethyl-ammonium hydroxide.

EXAMPLE K Using the procedure of Example J, a 10 weight percent aqueous solution of tetraethyl-ammonium-bi-sulphite was prepared from tetraethyl-ammonium hydroxide. 30 EXAMPLE L Using the procedure of Example J, a 10 weight percent aqueous solution of trimethyl-ethyl-ammonium-bisulphite was prepared from trimethyl-ethyl-ammonium hydroxide. 2497 - 19 - EXAMPLE H Using the procedure of Example J, a 5 weight percent solution of tetrabutyl-ammonium bisulphite was prepared from tetrabutyl-ammonium hydroxide.

EXAMPLE N A sample of the tetramethyl-ammonium bisulphite of Example J was evaporated under vacuum to dryness in a Buchi rotary evaporator. When all the water had been removed, an oil bath heated to 130°C was applied 10 to the flask containing the solid and heating under vacuum was continued for another 4 hours, so as to remove water from the bisulphite and produce the metabisulphite.

EXAMPLE 0 15 A. 5 weight percent aqueous solution of tetra- methyX-ammonium sulphite was prepared by bubbling S02 through a solution of tetramethyl-ammonium hydroxide.

EXAMPLE P A solution of sodium formaldehyde bisulphite 20 was prepared by dissolving commercially-available solid sodium formaldehyde bisulphite in distilled water at room temperature to form a 3 percent by weight aqueous solution.

EXAMPLE Q Example P was repeated except that a 10 percent by weight aqueous solution was formed.

EXAMPLE R Capsules of sodium formaldehyde bisulphite were prepared by charging standard gelatin capsules 30 with sufficient sodium formaldehyde bisulphite to provide 25 mg of active agent per capsule.

EXAMPLE S The procedure of Example R was repeated, except that capsules containing 50 mg of active agent were 35 prepared. Γ 52497 - 20 -EXAMPLE 1 A. variety of agents were coded and evaluated under a code designation. Among these compounds, thiouracil, mercaptosuccinic acid, thiosemicarbazide, 5 phenothiazine and sodium thiosulphate were included, so as to compare sodium bisulphite with certain compounds mentioned in USP 4,148,855.

After determination of PT and PTT values, the coded compounds were decoded. The bisulphites and 10 sulphites showed an effect in vitro and only one other compound (sodium formaldehyde bisulphite) was effective in increasing PT and PTT values.

The results are shown in Table I where mean values of 5 replications in each case are reported.

The findings demonstrate the reliability of the test method. This procedure was repeated with some additional compounds on two different days using a different control on each day. The results are shown in Table II below. These data indicate that the active moieties 20 are probably the bisulphite and sulphite groups. - 21 - 58487 i · 22Ϊ a * I 2 c «ίί. a Ο X ψ4 « 4 · i 5 a -*4 ο ϋ 2 u 3 N i 2 3 a 3- 3 3 143 3 3 .3ss 33 oS||S| « flg Isas-i || isUitsi go 43U11 sS 1 i 8! 3 1 s 1 J β - s 33343 a" Hs!Ss33 S§Hi^i5 | 12.5.2.5 g < g f | 3 5 i 3 * 03,-332533 21322¾¾¾¾ . ΪΙ1ΙΠ11 iiilsllll 0 0 " 38S33S22S « -«•«'•’βΜ* t ή ·* « * - » « * v * j;;;:;;;; i ϊ I m .a utinaiininit.. n «»«««"«"" 0 s| is » is - S S 2 * S 2 S S S n ? ~ ° « *§ S iissisisas : -qsanssss ° I W tJ I .", (Η Π ««PIWNPiNN t si U c g| Ϊ, i jl I . X 9 • UJ J5 5 2 4 9 7 - 22 - «··> S -. ° · * • n · et tft «» O 2 ~ w -5 ior»nSSSnS rt^wenw^HH g 3 E= g · i 5 a S 5 iSiJSS! ΐ β ο -*4 • XI α« χ ο Cl] «Η «•4 e® »*»****«» I » ZS 3 g «η ο 2 ο Η | 5 5 S 3 2 s S S S 5 * , .. « β · · - | ί ε 3 a a a » s s s ssssaasss is Μ cs § ς o I uj · *i E £8 g “3 y 0® U1lAlAlAiAlA«nin ZB , j v e «μ - Ϊ s | * · S . “ «•a u 4i « (I «4 f P «4 9 3- 5 c 2 -3 a >» j? · «4 ·* « λ a λ • U M O M 4J « • Solution A was found to prolong PT and PTT in a dose-related fashion. When added to human and to rabbit plasma in vitro, the agent triethyl-ammonium 5 bisulphite was found to prolong PT and PTT significantly at a concentration of 0.5 mg/ml and the effects are directly proportional to dose. The agent is active in inhibiting various coagulation, factors, including factors VII, IX, X, XI, and XII.

References for PT, PTT and assays of all the coagulation factors can be found in the above-cited textbook, "Human Blood Coagulation, Haemostasis and Thrombosis".

EXAMPLES 3 and 4 15 When solution A and Solution I were each further similarly tested as in Example 2, increases in PT and PTT values at similar respective concentrations are observed, although somewhat different values are obtained.

Data evaluated by the procedure above described are provided in Tables III and IV below.

EXAMPLE 5 To demonstrate that triethylamine sulphite and triethylamine bisulphite have an effect upon blood 25 coagulation factors, the data in Table III (below) for fibrinogen, Factor IX and Factor X using Biggs procedure were obtained. The results show a substantial effect on these factors, with the bisulphite being the more active.

EXAMPLE 6 To demonstrate reproducibility of the Biggs procedure, the work of Example 5 was repeated 5 times and the deviation is summarized and shown in Table IV below. 5249? ι, , - 24 -EXAMPLE 7 Λ rabbit was injected with about 72 mg/kg of body weight of triethylamine bisulphite. After a period of 5 hours, a blood sample (220 g) was withdrawn 5 and centrifuged at 1000 r.p.m. for 10 minutes to give a platelet-rich plasma. The material was evaluated for platelet aggregation by the method described by Born (cited above) and it was found that aggregation of platelets was markedly decreased in comparison with an 10 untreated rabbit. It is concluded that triethylamine bisulphite is an agent which inhibits thrombotic condition formation in vivo.

Other amine bisulphites, such as n-octylamine bisulphite and tri-n-butylamine bisulphite, also demon-15 strate similar inhibition of platelet aggregation when similarly tested.

EXAMPLE 8 Platelet-rich plasma from rabbits and men was prepared by the centrifugation technique mentioned in 20 Example 7. .Using ADP (adenosine diphosphate) as a stimulus, rapid aggregation of platelets occurred. When the stimulated blood had been treated with each of the agents Of Example 7, there was a strong inhibition of platelet aggregation. This procedure is described in 25 the article by Born in Nature, Yol. 194, pgs. 927-929 (1962). The results demonstrate that these agents cause inhibition of platelet aggregation in vitro.

Other stimuli, collagen and arachidonic acid, yielded similar results. 52497 - 25 - m * * Ο ♦ Μ fit ο *+ C* X « §5 = 2 <ρ« cl α -ψ * 3ο c * ·* ~ ο2 § " 8 2 £ β: ί 85 I α χ 5 - 2 3 25 - 3 5 55 = -3 . *•52-· ! S S a S · 5J 3 ο 1 ££ £ 2 £ S ' ί g ° 5 ««-·- 28.1 a* * S ϊ 5 Zr. α S h μ * η 5 * S 5S ' 2 · 1 Ζζ ί 9 8 S " C · ϊ fig 8 5-5 I · I ιΙ ι - as >6 £ : * * κ) | ϋ §s | 5 s c U «I Ζ UM Ν3 δ ·2 <2 a 38 1 g“ £“ 1 3 5 2 S ίϊ «η μ ** ** w Μ S 5 Μ Γ4 Λ Μ ά ·τ ν « a 2 8 •ζτ- S3 49 7 .1, - 26 - *i 1 tj r - E S* f2 STi 5 nto me tnm , *s | dii ή it *i\ > 3 s si 1 n I a ft _ m { * * o · z 2 ~ gi V 2 3 >ng a 3 3 g * 3 T “ S m | a a a i p I δ s' — u x i .n s i i i i · , , 5 s S » « a 2 P a j . U rt H rn M U 3 > 6 i I e> i«S I i i i i s A is J5 ac -i vj * 5j «. μ «e 2Ϊ » ® * 3 J α a? 3* ° 8 Ϊ - - - * 8 s s ε s | - i s &. w «a id in ^ 3 0 *< £ λ S e M § ·Ε x os 1 i« , n » ε | £ w ri ui oa z κ S B - « " «» X I-s $ i if ^|j Λ «· Λ SO. i 2 3 5 ί ΐ S'4" | | ** i f ft h! 3 n η n U tA tO ΙΛ 52497 - 27 -EXAMPLE 9 The procedure of Biggs was repeated for PT and PTT determination using various compounds of the formula in (A), with multiple replications for accuracy and consistency 5 reasons. These compounds are generally effective in .increasing PT and PTT. In general, the bisulphites of the Formula ih (A) are somewhat more effective in increasing PT and PTT values than are the sulphites; the bisulphites of diamines (as compared with monoamines) are 10 effective. Such an increase indicates an anticoagulation effectiveness in vitro. The diallyl-amine bisulphite is unexpectedly of low activity in increasing PT and PTT values.

The results are shown in Table V below.

Even though aromatic amines, such as aniline, form active agents, they are considered to be excessively toxic for mammalian use and so are not included within the scope of the formula in (A).

The alkanolamine bisulphites are of approximately 20 the same activity era the lower alkyl amine bisulphites.

EXAMPLE 10 To evaluate whether or not an amine moiety has an effect on blood factors, some amines were evaluated as their hydrochlorides (as opposed to the 25 corresponding sulphites and bisulphites).

After their respective preparations the amine salts were evaluated by the Biggs method for their effect on PT and PTT. The data shown in Table VI below show that these hydrochlorides have no effect 30 upon FT and PTT in vitro. 52497 - 28: - ^ ''J ® λ 2222 ta C4 Μ f% Μ r>t *4 Μ *m ** ·* Ν Η Ν " 3 ί^οαβΓ"*0Β»β<Λ«7<» ^*^2 0ΒΛθβ»-ί*»ρΙθ·«4«βΜ 2 2 2 2 Η I «ΗΜΜΝΡίΠΗΝ^ r* W ΡΊ ** β! - β» U « L, ιΑ*4|%4*(η*Ρ*0<0· W « Λ «·< -> μ ο « *ί π β! a β! « 5 « 2222 u ΝΜΝρ4ΠΗ*4ΗΗΗ Ν W « ** 4 8 C Ο ζ C c. < 3 ί ««o^aeicet^cB 'β « « ^ J r· J»e»e\o>e^N-4> 2222 β 0] «f«nmiAiAv a a c a: < ft. 3 t 2222 2 Of«9eBQO«>A>nr* W N f B S 4 4 P* ^ III z 5 g s 5 S 5 j 2 x > i < κ 2b w *, M J 5 2 5 3 2 u u o J u S Z 9» O U Z ^ S * o < = z m < x ta tax ο. m x x o S z «3 z « 53*9 » u m > 2 5 S*,i «5ίί S g is “jo* g § I * £ ^ ? 3 5.3 § 5 I S 2 S f= § sssssAsssi i s«i z £3 « Μ

Claims (19)

1. 52497 - 39 -

2. 1. A pharmaceutical composition for use in treating an actual or incipient thrombotic condition in a mammal, which contains at least 5 one carrier substance and, as the active treating agent, at least one compound selected from: (A) compounds of theformula: Rl\ R,-N# SO-H (2~n^ 2 r 3 n _ 3 m wherein:

3. 10 R1 is an alkyl radical containing less than 11 carbon atoms, a cycloalkyl radical containing 6 to 10 carbon atoms or a monohydroxyalkyl radical containing less than 11 carbon atoms, R2 is a hydrogen atom, an alkyl radical 15 containing less than 11 carbon atoms or a monohydroxy alkyl radical containing less than 11 carbon atoms, or Rj and R2 together form an unsubstituted morpholine, piperidine or hexamethyleneimine ring or - 40 -- 5249 7 such a ring carrying on one of its carbon atoms an alkyl radical containing less than 11 carbon atoms, R^ is a hydrogen atom, an alkyl radical containing less than 11 carbon atoms, a monohydroxyalkyl 5 radical containing less than 11 carbon atoms or a radical of the formulas I' SO H (2_n^ Hni@-- j n R2 J m wherein and Rj have the meanings defined above, R^ is a divalent saturated aliphatic radical 10 containing less than 11 carbon atoms, and m is 1 or 2, n is 0 or 1 and the sum of m and n is 2; (B) compounds -of either of the formulae: RS Ί 'R5 vX r6 N® S0„H (2_η)θ ε_°ς 20 R7^ 3” r7^/ V > “d [v J2 15 wherein: R_, R., R and R_ are the same or different DO/ O and each is an alkyl radical, containing less than 11 carbon atoms, and m and n have the meanings defined above; 20 (C) compounds of the formula: R-CHOH-S03M 52497 - 41 - wherein: R is a hydrogen atom or a hydroxymethyl radical and M is an alkali metal or an ammonium group, 5 but excluding: (a) 0.6M injectable aqueous solutions of ethanolamine bisulphite, and (b) compositions containing a formaldehyde bisulphite and a protamine. 10 2. A pharmaceutical composition as claimed in Claim 1, wherein the above treating agent is a compound of type (A) in which Rj, Rg and Rg each represent an alkyl group containing 1 to 4 carbon atoms.

4. 3. A pharmaceutical composition as claimed in 15 Claim 2, wherein R^, Rg and Rg each represent ethyl.

5. 4. A pharmaceutical composition as claimed in Claim 1, wherein the active treating agent is a compound of type (A) in which Rj represents an alkyl group containing 5 to 10 carbon atoms and Rg and Rg both 2o represent hydrogen.

6. 5. A pharmaceutical composition as claimed in Claim 4, wherein Rj^ represents n-octyl.

7. 6. A pharmaceutical composition as claimed in Claim 1,wherein the active treating agent is a compound 25 of type (B) in which Rg, Rg, R7 and Rg each represent an alkyl group containing 1 to 4 carbon atoms.

8. 7. A pharmaceutical composition as claimed in Claim 6, wherein R_, R,, R_ and R0 each represent DO/ O butyl. 30 8. A pharmaceutical composition as claimed in Claim 1, wherein the active treating agent is sodium formaldehyde bisulphite.

9. A pharmaceutical composition as claimed in any one of the preceding Claims, wherein the active 35 treating agent is a bisulphite. - 42 - 53497

10. A pharmaceutical composition as claimed in any one of the preceding Claims, wherein the composition is in an orally administerable form.

11. A pharmaceutical composition as claimed in 5 Claim 10, wherein the composition is an aqueous solution containing 1 to 10% by weight of the active treating agent.

12. A pharmaceutical composition as claimed in any one of Claims 1 to 9, wherein the composition is in 10 an injectable form.

13. A pharmaceutical composition as claimed in Claim 12, wherein the composition is an injectable aqueous solution containing 1 to 11% by weight of the active treating agent. 15 14. A pharmaceutical composition as claimed in any one of the preceding Claims, wherein the active treating agent is the only pharmacologically active agent in the composition.

14. 15. A pharmaceutical composition as claimed in 20 any one of Claims 1 to 13^ wherein the composition is in the form of an aqueous solution containing dissolved sulphur dioxide.

15. 16. A composition - as claimed in any one of Claims i to 15 for use in a method of treatment of the human or 25 other mammal body by therapy or in a diagnostic method practised on the human or other mammal body.

16. 17. A process for the preparation of a pharmaceutical composition as claimed in any one of claims 1 to 16, which comprises combining the carrier substance, the 30 active treating agent and any other components in the sterile state and under sterile conditions and optionally packaging portions of the resultant composition in dosage unit form. - 43 - 52487

17. 18. A method for modifying blood coagulation and platelet aggregation factors in vitro which comprises adding to blood and/or to plasma derived from blood a compound as defined in any one of claims 1 to 9. 5 19. A method for treating a thrombotic condition in a non-human mammal, which comprises administering to such mammal an antithrombotically-effective amount of a composition according to any one of Claims 1 to 15.

18. 20. A method for treating a thrombotic condition in a 10 non-human mammal, which comprises! (A) determining from a sample of blood removed from the mammal (1) one or both of the blood coagulation factors consisting of prothrombin time and partial thromboplastin 15 time and (2) the platelet aggregation factor, (B) when such factors fall within normal ranges for such mammal, administering to such mammal a pharmaceutical composition according to any one of claims 1 to 15 at a 20 dosage rate and for a time sufficient to inhibit the blood coagulation and platelet aggregation factors to an extent which is at least sufficient to inhibit thrombus development in the mammal but insufficient to cause spontaneous internal bleeding, 25 (C) at intervals during administration, removing further samples of blood from the mammal and determining the lengthened blood coagulation factor(s) and the inhibited platelet aggregation factor, (D) adjusting administration of the composition so 30 as to achieve and maintain predetermined desired values for the lengthened blood coagulation factor(s) and the inhibited platelet aggregation factor in the mammal, and (E) continuing steps (B), (C) and (D) until the mammal has recovered from the thrombotic condition. 52497 - 44 -

19. 21. A pharmaceutical composition according to Claim I, substantially as hereinbefore described with particular reference to Examples A - S of the accompanying Examples. 5 22. A method according to Claim 18, substantially as hereinbefore described and exemplified. F. R. KELLY & CO. AGENTS FOR THE APPLICANTS.