The use of various steroids to increase the intensity of the heartbeat is known. A number of steroid glycosides have been found useful in cardiotherapy, but the corresponding aglycons (genins), which share the characteristic steroid structure of the glycosides are convulsive poisons.
For purposes of definition, a cardiotonic glycoside is one which increases the intensity of the heart beat but may decrease the rate of heartbeat. These glycosides may be designated by the following general formula: ##SPC1##
The corresponding aglycons have a hydroxy group at position 3 rather than a glycoside conjugate.
A wide number of sugars may be employed to form cardiotonic glycosides, as long as the steroid nucleus is a cardenolide which contains a five membered lactone ring at the C.sub.17 position. Particular cardiotonic glycosides useful in the treating of heart disceases and which are readily available include digoxin, digitoxin and gitoxin. These compounds differ at the C.sub.12 and C.sub.16 position of the steroid (R.sub.1 and R.sub.2), in that digitoxin contains a hydrogen substituent at each position, digoxin contains a hydroxyl at C.sub.12 and a hydrogen at C.sub.16 while gitoxin contains a hydrogen at C.sub.12 and hydroxyl at C.sub.16. The glycoside portion of each of these compounds comprises three digitoxose sugars. The 3', 3", 3'" and 4'" positions bear hydroxyl groups while the 5', 5" and 5'" bear methyl substituents. Other sugars which may be found in cardiotonic glycosides include the lanatoside series which differs from the digitoxose in that the 3'" position bears an acetyl while the 4'" position contains an additional glucose sugar. When this glycoside is conjugated to digoxigenin it is designated lanatoside A, while if it is conjugated with gitoxigenin it is designated lanatoside B, and if it is conjugated with digitoxigenin it is designated lanatoside C.
Other sugar units which may give a compound which has cardiotonic activity, when they are conjugated to a cardenolide steroid, include rhamnose, antiarose, digitalose, thevetose, talomethylose, cymarose, oleandrose, sarmentose, boivinose and diginose. By combining three of these units with an aglycon, various cardiotonic glycosides may be formed. The aglycons alone are usually convulsive poisons not useful in medicine. For a more thorough discussion of cardiotonic glycosides and aglycons, see "Rodd's Chemistry of Carbon Compounds", Second Edition, Vol. 2, part D, chapter 17, and "Chemistry and Metabolism of Digitalis", Digitalis, edited by Charles Fisch and Borys Furawicz, Grune & Stratton, Inc., New York, 1969.
As previously mentioned, although the glycosides have been found to exhibit much less toxicity than the corresponding aglycon, in many instances the difference between a therapeutic and a toxic dose of a glycoside is only in the order of a few micrograms per kilogram of blood. Digitalis intoxication occurs frequently due to the small differences between therapeutic and toxic quantities. The therapeutic quantity of any cardiotonic glycoside cannot be predicted in advance of medication since each patient may react differently to the dosage, and retain different quantities of the drug in the blood stream.
Digitalis intoxication occurs with increasing frequency in elderly heart patients, since diseased renal function in the aged can result in higher and sometimes toxic levels of cardiotonic glycoside residue in the blood. Also, there may be some overlapping between therapeutic and toxic blood levels due to susceptability of the myocardium to digitalis. For example, the patient whose myocardium is diseased may show signs of toxicity despite therapeutic blood levels. Other clinical conditions contributing to digitalis intoxication are coronary ischemia and potassium depletion. The problems of digitalis intoxication are compounded in that its symptoms may often be mistaken for fatigue or restlessness.
Generally, accepted therapeutic levels for digoxin range from 0.8 to 2.4 ng/ml while toxic levels range from 2.1 to 8.7 ng/ml Toxic versus non-toxic levels of digitoxin also overlap, i.e., 3.0 to 39.0 ng/ml may be therapeutic while 26.0 to 43.0 ng/ml may be toxic. Higher levels of digoxin and digitoxin are generally lethal.
Radioimmunoassay for cardiotonic glycosides has proved extremely effective in detecting undermedicated, adequately medicated and over-medicated or toxic levels of the drug in blood serum. That sampling technique comprises the use of a radioactive steroid glycoside or aglycon and an antibody which will bind both the radioactive steroid and the cardiotonic glycoside in the blood on a competitive basis. By counting the amount of radioactive material reacting with the antibody, the amount of cardiotonic glycoside in the blood may be calculated. From the results, it can be quickly determined whether the patient has been undermedicated, properly medicated or over-medicated and is suffering digoxin toxicity.
For radio labelling of steroids, 125-Iodine or 131-Iodine are preferred, since they exhibit high specific activity, while .sup.14 C and .sup.3 H are also available. The use of compounds exhibiting a high specific activity permits the use of considerably less material for the same counting efficiency. Counting with such isotopes can be achieved by liquid scintillation or gamma ray spectroscopy counting procedures.
The sampling procedure preferably consists of the use of an antibody polymer in tablet form, which antibody has been raised in an animal host by the administration of an appropriate antigen. The radioactive steroid and the cardiotonic glycoside isolated in the blood serum then complete with each other for a limited number of binding sites on the antibody to form an insoluble complex. The antigen/antibody complex is then separated via centrifuge techniques and the radioactive portion is measured, as for example, by gamma counter. From the difference in radioactivity in the test specimen, compared to standards of known cardiotonic glycoside concentrations in blood, the level of cardiotonic glycoside in the test sample may be accurately computed.
For a more complete discussion of radioimmunoassay principles, see A. R. Midgday, Jr. and G. D. Niswender, in "Karolinska Symposia on Research Methods in Endocrinology, 2nd Symposium: Steroid Assay by Protein Binding," The Reproductive Endocrinology Research Unit, Stockholm (1970), 320-333 and W. D. Odell and W. H. Daughadey, "Principles of Competitive Protein-Binding Assays," J. B. Lippincott Company, Philadelphia (1971). For a complete discussion on the preparation of protein-digoxin conjugates necessary for the production of antibodies, see "Digoxin-Specific Antibodies", Butler et al., Proc. Natl. Acad. Sci. U.S. 57:71.
The method most commonly used in conjugating a radioactive isotope, such as 125-Iodine, to a steroid is via an electrophilic substitution reaction. Because of the high susceptability of phenol derivatives to electrophilic substitution, since the hydroxyl is an activating group, radioactive steroids have been synthesized by conjugating tyrosine or tyrosine methyl ester (TME) onto the steroid, after which the compound may be reacted with the radioactive isotope (.sup.125 I).
Oliver et al., Journal of Clinical Investigation, 47, 1035 (1968) teach the synthesis of a digitoxigenin-3-0-succinyl-iodine-125 tyrosine methyl ester for use in cardiotonic glycoside radioimmunoassay procedures. Such a compound, although suitable for measuring the level of the drug in blood serum, suffers from the disadvantage that it is extremely difficult and expensive to produce. The succinylation step of the C.sub.3 -hydroxyl group of the genin, which is used at the binding site for the TME and iodine-125, proceeds very slowly and may require reaction time on the order of months. It is believed that the slow reaction time associated with the succinylation of the C.sub.3 -hydroxyl, is due to the fact the C.sub.3 -hydroxyl group is fixed in an axial position and offers a large amount of steric hindrance. Additionally, although such a reaction procedure is suitable for the synthesis of digitoxigenin derivatives, where the C.sub.12 substituent is a hydrogen, it is not per se suitable for the synthesis of radioactive digoxigenin derivatives. Digoxigenin has at the C.sub.12 position and equatorial hydroxyl substituent which will more readily be succinylated than the axial-3-hydroxyl group, since it offers less steric hindrance. Thus succinylation of the 3-hydroxyl of the genin not only proceeds slowly but for many compounds produces little or no yield of a useful immunoreactive derivative unless other positions are blocked. In addition, Stall et al., "The Specificity of the Digoxin Radioimmunoassay Procedure", Res. Commun. Chem. Pathol Pharmacol, 4, 503 (1972) shows that digoxigenin has a lower affinity for antibody than digoxin itself. They show a crossreactivity of about 34% wherein the optimum ratio of binding ability of radiolabelled to unlabelled antigen should be 1:1. Thus it would be expected that a radioactive glycoside will bind on a more competitive basis with an antibody than the corresponding aglycon.
Although Draws et al., "Faster and Easier Radioimmunoassay of Digoxin," Clinical Chemistry, Vol. 20, No. 3, 343-347 (1974) discusses the use of a .sup.125 I-tyrosine methyl ester of digoxin, there is no indication that such a compound should be a sugar hydroxyl substituted material or any indication of how such a compound might be synthesized.
It is an object of this invention to provide a process of synthesizing a radioactive steroid which will complete on a predictable basis for sites on an antibody with cardiotonic glycosides to be measured.
It is another object of this invention to provide a process of synthesizing such radioimmunoassay compounds via an improved and simplified procedure.
It is yet another object of this invention to provide a radioimmunoassay compound having a high specific activity and improved stability upon storage.