US 3035974 A
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The present invention relates to injectable pharmaceutical compositions, more particularly for the parenteral therapeutic use of thyroidal substances.
Compositions according to the present invention have broad utility in human and veterinary medicine and are particularly useful in the treatment of hypercholesterolemia by the parenteral administration of thyroidal substances such as thyroxine.
However, the parenteral use of thyroxine and other thyro-mimetic substances in humans as therapeutic agents for prolonged administration has not proven satisfactory. As a matter of fact, it is impossible to continue parenteral administration in this manner, since these substances, when administered alone, induce symptoms attributable to the [temperature-regulating system of the body and to the autonomic nervous system, whereby the administration of thyroidal substances in large doses or in frequently repeated normal doses, in most human subjects, or even in relatively infrequent usual doses in occasional human subjects, causes palpitation, extreme nervousness and tremulousness and an increase in body heat production. The increase in body heat production is known as the specific dynamic action of thyroidal substances and may give rise to dangerous effects, since the augmented heat production of the body may not be rapidly dissipated and hence may cause heat accumulation so as to cause the temperature of the thyroid-medicated subject to rise to dangerous levels. Thus, the benefit to be derived from medication with appropriate doses of thyroidal substances can be more than ofiset by the incursion of these untoward effects, which can be so severe as to prescribe the rational use of thyroidal substances in subjects whose illness would otherwise warrant such administration. See, for example, the discussion of this problem in connection with atherosclerosis in British Medical Journal, 1958, volume I, page 509. A valuable therapeutic tool has thus heretofore been placed beyond the reach of medical science.
It has now been discovered that thyroidal substances are rendered therapeutic-ally acceptable for parenteral administration even when large doses are administered or when usual doses are given in rapid sequence, if in association with those thyroidal substances there is parenterally administered at least a certain quantity of macromolecular substance of a certain electrical charge.
Accordingly, it is an object of the present invention to provide therapeutic agents containing thyroidal substances rendered therapeutically acceptable.
Still another object of the present invention is the provision of new and improved parenteral dosage forms of thyroidal substances.
The components, proportions and procedural steps of the present invention will be separately set forth as follows:
THE THY ROIDAL SUBSTANCES The thyroidal substances according to the present in- States Patent ice vention are members of the class consisting of watersoluble salts of I BOOB and mixtures thereof, where R is a monobasic carboxylic group in which the chain carrying the carboxyl is short chain aliphatic having 1-4 carbon atoms including the carbon atom of the carboxyl group. These thyroidal substances are in water-soluble form as used in the present invention, and are rendered water-soluble by' formation of their corresponding water-soluble salts as the sodium or potassium salts. Examples of thyroidal substances according to the present invention are thyroxine, tri-iodothyronine, 3,5-diiodo-L-tyrosine, N-acetyl-3,5-diiodo-L- tyrosine, 3,5-diiodo-4-hydroxybenzoic acid, 3,5-diiodo-4- hydroxylphenylacetic acid, 3,5-diiodo-4-hydroxyphenylpropionic acid, 4-(4'-hydroxy-3',5'-diiodophenoxy)-3,5- diiodobenzoic acid, tri-iodothyroacetic acid, diiodothyronine, 3,S-diiodo-4-hydroxyphenylbutyric acid, and thyroglobulin. Particularly preferred is thyroxine. In addition, there have been reports that decarboxylated thyroniue amine is thyro-mimetic and that cellular metabolism has been stimulated by simple iodonated benzene derivatives. However, in the absence of evidence of their ability in humans, it cannot be known at the present time whether such substances are equivalent to those of the present invention.
THE MACROMOLECULAR SUBSTANCES The macromolecular substances of the present invention are therapeutically acceptable electronegative colloids in aqueous dispersion. They are of very high molecular weight, at least about 100,000, and are properly referred to briefly as macroanions. Upon solution or dispersion in a menstruum or solvent, macroanions exhibit an electronegative charge due to the ionization of functional acidic groups which they contain. Thus, heparin is a macroanion because of the dissociation of sulfamido and sulfate groups in its molecular structure, while carboxymethylcellulose is a macroanion because it is a massive, colloidally dispersable molecule which in aqueous dispersion yields an electronegative charge because of ionization of its carboxyl group. The same is true of other macroanions because of the presence in the molecule of other acidic or potentially acidic groups such as nitrite, nitro, sulfone, free tertiary phosphate or imino groups.
The mechanism by which macroanions according to the present invention vitiate the undesirable effects of thyroidal substances in the human body is believed to be as follows:
Macroanions of the type contemplated by the present invention have a tendency to inhibit certain enzymes of the body known as depoly-merases. Among the latter enzymes are those which cause a reduction in molecular weight of highly-polymerized compounds in tissues, such as proteins, mucopolysaccharides and nucleic acids. Macroani'ons offer substrate competition to such enzymes and thus act as competitive inhibitors to all of them. Thy-roidal substances, on the other hand, have just the opposite efiect on the depolymerization of body tissues and ground-substances, either through activation of the enzymes concerned in this depoly-merization or by direct interaction with the polymerized materials or both. The exact mode by which thyroidal substances enhance depolymerization, however, is not known at present; but it is clear that they do so, and in so doing are responsible for the exaggerated catabolisnr which constitutes the undesirable or toxic effects of thyroidal substances. Thus, by opposing this catabolic depolymerization, macroanions furnish an antidote or vitiator of undesirable efiects of parenterally administered thy-roidal substances as set forth above.
In view of the above mechanism, it is obvious that the macroanions of the present invention are characterized by certain physical and electrical properties held in common among them and are not necessarily chemically related to each other at all. The very size of the molecule is such that its behavior for purposes of the present invention is virtually independent of its exact chemical nature. As stated above, the molecular weight of these macroanions is at least about 100,000, and may range up into the millions dependent only on the condition that they remain colloidal in aqueous dispersion, that is, that they do not settle out.
Their size, however, is not their only operative characteristic, but also their electronegative charge. The macroanions of the present invention are strongly electronegative. Weakly electronegative colloids are not operative for purposes of the present invention, as evidenced by the fact that their natural occurrence in the human organism is not suflicient to cover the toxic efiects of thyroidal substances. In this sense, the macro'anions of the present invention are heparinoids in that they are pronounced anticoagulants for the blood for the same reason as heparin. Hence, the measure of their electronegative charge can be stated in terms of antithrombic effect, not that the present invention operates through an antithrombic mechanism, but rather because with respect to the charge of the molecules the measure of suitability of the substance and the measure of its anti-thrombic effect are interrelated parameters.
Stated another Way, the macroanions of the present invention are operative according to their antidepolymerase activity. Therefore, their operativeness for purposes'of the present invention can be determined by observing their eifect on the course of a specific depolymerization. The coagulation of fibrinogen, which occurs during the clotting of blood, is just such a depolymeriza- 3 tion. Blood clotting can be initiated by any proteolytic enzyme that is, any enzymethat depolymerizes, proteins. One such enzyme that is highly convenient is the physiologically-occurring enzyme thrombase. The clotting of fibrinogen upon the addition of thrombase can be delayed if there is also present an antidepolymerase, e.g., an antithrombin. Thus, the retardation of clot-ting under standardized conditions can serve as a measurement of the suitability for purposes of the present invention of macroanions with regard to their antidepolyrnerase activity.
The determination of antithrombic and hence depolymerase activity can be conducted for this purpose by i a given quantity of the plasma to clot in 3 seconds 20.2 seconds. A 0.3% aqueous solution of purified human fibrinogen is equivalent and may be used in place of the the B12.
plasma. The thrombase is used in highly concentrated solution so that it will not add poportionately greatly to the volume of the test solution; otherwise the end point would be difficult to determine with precision. Preferably, the volume of thrombase solution is about one quarter the volume of the plasma. For example, it is highly convenient to use 0.2 ml. of plasma and so to standardize the thrombase solution that a single drop (1 minim) effects clotting in 3 seconds. The samples are kept at uniform temperature, preferably 3639 C.
Plasma samples are then established which contain the macroanion in question in a concentration of 0.5% by weight. The same quantity of thrombase solution is then added that would cause the sample to clot in 3 seconds in the absence of the colloid. If the clotting time is extended to 5 seconds or more, then the colloid possesses antithrombin, i.e., antidepolymerase activity according to the present invention such that the undesirable effects of parenterally administered thy-ro-active substances will be abrogated.
The preferred macroanion of the present invention is an aggregate of purified or crystalline vitamin B and gelatin or other non-allergenic protein, with the gelatin present in substantially greater quantity than the B In the presence of thyroxine as the thyroidail substance, this B -gelatin aggregate has the particular advantage of providing a medium for the dissolution of a watersoluble thyroxine salt in which thyroxine will dissolve in substantial quantities at neutral pH. Ordinarily, the pH of an aqueous solvent for thyroxine must be at least about 10 in order to dissolve "an appreciable amount of thyroxine. But at such high pH, the B is unstable and rapidly loses its activity, probably decomposing. Moreover, B does not aggregate with itself in aqueous media, but remains lmonomolecular. Hence, the aggregation of B with gelatin not only provides a medium in which thyroxine will remain in solution without precipitation at neutral pH, but also provides the required macromolecu lar structure. It is not the gelatin that gives the electro negative characteristic to this macromolecule, but rather The gelatin molecules are at least fifteen times larger than the B molecules; and hence, the gelatin serves in eifect as an integral excipient for the B by which the B is given the character of a micromolecule but at the same time retains its characteristic electro negative charge. This aggregation of B and gelatin is confirmed by electrophoretic studies, in which the B is seen to migrate with the gelatin.
It is believed that the stoichiometric mixture for purposes of forming this aggregate is equimolar, that is, fifteen parts by weight of gelatin to one part by weight of B However, as the B is considerably more expensive than the gelatin, and as the gelatin is innocuous in any event, it is preferred to provide an excess of gelatin over that theoretically required, in order to assure complete utilization of the B Therefore, the preferred weight ratio of gelatin to B is twenty to one.
In addition to the number of electronegative charges on the macromolecule, an important factor contributing to the effectiveness of the colloids within the scope of the present invention is spaciality, specifically, the distribution of those charges in or on the macromolecule. Where proper spacing exists, these charges reinforce each other to enhance the activity of the macromolecule as a whole. In the specific B -gelatin aggregate, for example, the spacing of the unesterified phosphate valence of the B from its neighbors causes the aggregate to display a more marked macroanionic effect than do certain colloidal substances having even more acidic groups per unit molecular weight.
In the B -gelatin aggregate, the gelatin molecules appear to be aggregated by a polymerization reaction with the B which can attach to the gelatin molecule in two manners, both of which are operative for purposes of this invention: (1) by reaction of the acid amides with weakly acidic carboxyls of the gelatin in a salt-type linkage, and (2.) by coordination of the histidyl iminazolyl groups with a residual valence of the cobalt. By both of these mechanisms the B molecules can provide bridges between the gelatin molecules, thereby producing an aggregate in which the free tertiary phosphate radicals of the B are spaced apart by the gelatin substituent groups of the aggregate. The phosphate radicals are thus exposed and reinforce each other. Thus, although both B and gelatin are amphoteric substances possessing both acid and basic groups, the net result of their interaction is to effect a reduction in overall basicity, by neutralizing linkages of the iminazolyl radicals and the salt formation of the acid amides, so that the acidic groups are allowed to exert their maximum electronegativity.
When tested for antidepolymerase activity by the method described above, this B -gelatin aggregate when present in a concentration of 0.5% by weight lengthens the clotting time of human plasma under the influence of thrombase from 3 seconds in the absence of the aggregate to 6 seconds in the presence of the aggregate.
The antithyrotoxic eflect of macroanions containing vitamin B as a minor substituent thereof in aggregation with gelatin should not be confused with the previously known antithyrotoxic effect of vitamin B as reported for example by Monroe, Missouri Agricultural Experiment Station, Research Bulletin No. 446, 1949, pages 5669, as Well as by others. The antithyrotoxic effect reported by Monroe was that when fed orally, the B hastened the removal of thyroxine from the animal organism. According to this teaching, the toxic effects of thyroxine were destroyed because the thyroxine itself was destroyed. Normal or slightly above normal doses of thyroxine could be thus fed orally at short intervals with no accumulation of thyroxine from feeding to feeding and accordingly with none of the effects characteristic of thyroxine accumulation in the animal organism. Obviously, the destruction of t ryroxine would have no benefit and indeed would be detrimental to the present invention, as it would become impossible to conduct massive thyroxine therapy. Instead, the present invention functions according to entirely different mechanisms, as described above, to produce the entirely ditferent result of preserving the thyroxine but abating its toxic effects, within the limits of the compositions of the present invention as set forth herein and in the appended claims.
PROPORTIONS The thyroactive substance is the active ingredient of the pharmaceutical composition of the present invention, and its minimum efiective quantity per dose, according to the present invention, is about 0.3 mg. Its preferred dosage is 0.5 mg. As to thyrotoxicity, no upper limit is imposed on the quantity of thyroidal substance by thyrotoxicity, as the macroanion is efiective to cover all therapeutically acceptable doses; however, limits on the quantity of thyroactive substance are imposed by considerations other than thyrotoxic effects.
The quantity of macroanion and the quantity of thyroidal substance must be related to each other so that the macroanion is present in much greater quantity than the thyroidal substance. Specifically, the macroanion must not be present in amounts less than about five times the weight of the thyroidal substance. Preferably, it is present in quantities at least ten times the amount of the thyroidal substance, and the particularly preferred concentration ratio is twenty to one.
Another limitation on the proportions is imposed by the quantity of liquid that may be administered parenterally in a single dosage form. The minimum from a standpoint of the quantity of liquid is 0.1 ml., for less than this cannot accurately be measured in clinical procedures; while the maximum is ml., for obvious reasons. The preferred dosage is 1-2 ml. Thus, considering the maximum volume of liquid to be 5 ml. and the minimum quan- 6 tity of thyroidal substance to be 0.3 mg'., it will be realized that the medium must contain at least about 0.06 mg./ml. of thyroactive substance and at least about 0.3 mg./ml. of macroanion. The maximum quantity of macroanion is conditioned only by the concentration at which the aqueous medium tends to form a gel. By the same token, the minimum quantity of macroanion per dose is 1.5 mg. Thus, for the preferred B -gelatin aggregate, the minimum B is about 0.1 mg., for a maximum ratio of thyroidal substance to B of three to one. But this extreme is not the preferred proportion. Instead, for the B -gelatin system, the preferred ratio of thyroactive substance such as thyroxine to B is one to one, and a particularly desirable single dosage form consists essentially of 0.5
.mg. of B 0.5 mg. of sodium thyroxine, and 10 mg. of
gelatin, in l-2 ml. of physiologic saline (0.9% aqueous solution of sodium chloride). It is intended that these liquid dosage quantities be segregated in sealed ampoules such that one ampoule is substantially consumed for each parenteral administration. Of course, for beginning dosages, it is well to start at fractions of a normal dose until the dosage regimen is established.
The media in which these materials are contained are aqueous, that is, at least about 50% water. Suitable media are water, physiologic saline, solutions of glycols, for example up to about 50% polyethyleneglycol, and various ethanol-water media. They may vary in pH between weakly acidic and strongly alkaline. Above about pH 11, the strong alkalinity renders the injeotant injurious to the tissues or to the blood stream into which they are introduced. Below about pH 6, the macroanions precipitate protein in acid medium, except the phthalocyanines which are stabilized sulfonic acid derivatives. The preferred pH range is neutral to 10, preferably nearer the bottom of the range than the top. Hence, another advantage of the B -gelatin macroanion is that pHs between 6 and 9 are easily maintained despite the presence of the strongly alkaline thyroxine salts.
In order to enable those skilled in this art to practice the invention, the following illustrative example is given:
Example One hundred milliliters of a 1% aqueous solution of gelatin is produced by autoclaving an aqueous dispersion of 1 gram of parenteral grade of gelatin in that quantity of water. Fifty milligrams each of crystalline vitamin B and sodium d-l-thyroxine are added with stirring. The temperature remains at room temperature and the pH remains at 7 throughout, and the mixture is vigorously agitated to effect solution. After sterilization by Seitz filtration, it is sealed in amber ampoules and may be administered parenterally in 1 ml. doses. Prolonged therapy may be conducted with this composition with no evidence of thyrotoxicity.
It has been found that the repeated parenteral administration of the composition of the above example is effective in maintaining human blood cholesterol levels at lower and narrower ranges for long periods of time without relapse.
It is to be understood that the present invention and the appended claims are to be accorded a range of equivalents commensurate in scope with the advance made over the prior art.
What is claimed is:
1. An injectable pharmaceutical composition for the parenteral therapeutic use of thyroxine, said composition being therapeutically acceptacle for such therapy at a protracted dosage regimen under which its thyroxine content would provoke toxic manifestations if administered alone, consisting essentially of an aqueous medium containing not less than about 0.06 mg./ml. of a water-soluble salt of thyroxine, and also vitamin B and gelatin, the gelatin being present in an amount greater than the B and the B and gelatin together having at least about five times the weight of the thyroxine, the B and gelatin being associated with each other in the formof a macroanionic substance in dispersed phase and having a molecular weight of at least about 100,000.
2. A single dose of injectable pharmaceutical composi tion for the parenteral therapeutic use of thyroxine, said dose being therapeutically acceptable for such therapy at a protracted dosage regimen under which its thyroxine content would provoke toxic manifestations if administered alone, consisting essentially of not more than about 5 ml. of an aqueous medium containing not less than about 0.3 mg. of a Water-soluble salt of thyroxine, and also vitamin B and gelatin, the gelatin being present in an amount greater than the B and the B and gelatin together having at least about five times the weight of the thyroxine, the B and gelatin being associated with each other in the form of a macroanionic substance in dispersed phase and having a molecular weight of at least about 100,000. v
3. A sealed ampoule containing about 1-2 ml. of an aqueous medium containing about 0.5 mg. thyroxine, about 0.5 mg. vitamin B and about 10 mg. gelatin, balance essentially water, the B and gelatin being associated with each other in the form of a macroanionic substance 8 in dispersed phase and having a molecular Weight of at least about 100,000;
4. The method of'treating hypercholesterolemia in humans Without substantially increasing metabolic rates, which comprises administering parenterally to said humans a compound consisting essentially of a Water-soluble salt of thyroxine, and vitamin B and gelatin, the gelatin being present in an amount greater than the B and the gelatin and the B together having at least about five times the Weight of the thyroxine salt, the B and gelatin being associated with each other in the form of a macroanionic substance in dispersed phase and having a molecular weight of at least about 100,000.
Monroe: Missouri Agr. Exp. Station Res. Bull. 446 (1949), pp. 56-69.
British Med. 1., March 1, 1958, page 509.