A METHOD FOR THE IN VITRO MEASUREMENT OF THE ELIMINATION OF LABELLED hCG IN HUMANS, AN AGENT TO BE USED IN THE METHOD AS WELL AS THE USE THEREOF FOR THE ESTABLISHMENT OF THE ELIMINATION OF PATHO-PHYSIOLOGICALLY PRODUCED hCG OR FOR THE ESTABLISHMENT OF ORGAN FUNCTION

Abstract: An agent for the analysis of the elimination of physiologically produced hCG in a human patient, the method for the in vitro measurement of hCG disappearance in patients with an increased hCG level as well as the use of such an agent. It is hereby made possible to establish the effect of treatment in patients with abnormal hCG levels, such as patients with testicular cancer, and to establish the function of various organs.

Description: Title: A method for the in vitro measurement of the elimination of labelled hCG in humans, an agent to be used in the method as well as the use thereof for the establishment of the elimination of patho-physiologically produced hCG or for the establishment of organ function. Technical field The present invention relates to a method for the in vitro measurement of the elimination of labelled hCG (human Chorion Gonadotropin) in humans, an agent to be used in the method as well as the use thereof for the establishment of the elimination of patho-physiologically produced hCG or for the establishment of organ function. hCG is a glycoprotein with a molecular weight of about 36,700 Daltons. The protein consists of an alpha and a beta chain. hCG has a carbohydrate content of about 30%. Because of its many sial acid side chains hCG is negatively charged, which is probably also the reason why hCG in connection with size chromatography is eluted at an apparent molecular weight of about 60,000 Daltons 1 . hCG is applied as a medicine in connection with ovulation stimulation and is secreted, to some degree unaltered, in the urine. No other elimination or metabolisation organs other than the kidneys 2 have been established in humans. In women, hCG is observed under normal physiological conditions during pregnancy. hCG is secreted from the syncytiotrophoblast cell layer during pregnancy and has a predominantly luteinizing effect. Just a few days after conception, hCG may be measured in blood and urine. The hCG level rises until about the 10th to 12th week of pregnancy, after which the level remains constant until birth. Following birth the hCG disappears rapidly from the blood and urine 3"6 . Under normal physiological conditions hCG does not occur in men in quantities measurable by usual measuring methods. By its presence in measurable quantities in men hCG is thus a sign of disease activity. Examples of diseases in which an increase of the hCG level in plasma is observed , are: Men: testicular cancer patients, in which about 30% -50% of these patients have a raised hCG level, Women: mola, choriocarcinoma. Further, an increase of the hCG level is observed in connection with extra-uterine pregnancies as part of the normal pregnancy physiology. Finally, an increase of the hCG level is observed sporadically in connection with a long range of other malignant conditions, such as cancer of the lung, pancreas, ventricle, liver, prostate, uterus, breast and bladder. BaςkgTQμπd art When patients are treated for a disease which is accompanied by an abnormal hCG production, repeated measurements of the hCG level have hitherto been taken in order to assess the effect of the treatment. hCG is produced by cancer patients in tumour cells, and a declining hCG level has thus been interpreted as a reduced or ceased hCG production and, thereby, a positive effect of the treatment undertaken?. At present only measurements of the hCG level by means of immunological techniques are employed to assess the fall in the hCG level in patients with a disease accompanied by an abnormal production of hCG 8"15 . The ascertained fall in the hCG plasma level is compared with normal values obtained by examination of hCG disappearance in healthy persons following the injection of hCG and the measurement of hCG in plasma by means of immunological techniques 16,17 . The disappearance or elimination rate for hCG, measured as the half-life, is for healthy persons found to be an average of about 1.5 days. By means of several clinical measurements of patients with testicular cancer after orchiectomy, and later during chemotherapy, it has, however, not been possible to reproduce these half-lives 9 , 1 8-2 0 τhus half-lives of between 1.4 and 3.2 days after orchiectomy are found in patients with testicular cancer, and half-lives of right up to 34 days have been described. Today half-lives for hCG of more than 3 days are considered as prolonged. Since the half-lives for hCG, determined in healty men and women following the injection of purified hCG from pregnant women as well as from women post partum, may thus not be used immediately as a reference for half-lives found in patients with testicular cancer in chemotherapy, there is a need for an agent by means of which it can reliably and with certainty be established whether an hCG production is still taking place, or whether the maintenance of a high hCG level is due to elimination phenomena. Until today a prolonged half-life (longer than 3 days) has thus been interpreted as an expression of continued production of hCG and, thereby, the presence of vital tumour cells. Brief description of the invention However, it has now surprisingly been shown that by employing labelled hCG as a marker it is possible to determine with great certainty whether a continued hCG production is taking place in patients with initially abnormal plasma hCG levels. The invention consequently relates to a method for the in vitro measurement of the elimination of labelled hCG in humans, which is characterised by 1) measuring the disappearance rate of labelled hCG in samples from said human, 2) comparing the disappearance rate obtained under 1) with a) the disappearance rate of the total amount of hCG in samples from said human for the establishment of a possible presence of patho- physiological hCG production, or b) the disappearance rate of labelled hCG in samples from a healthy subject for the establishment of organ function. The invention further relates to an agent for use in the method, said agent being characterised in that it as active ingredient contains sterilely produced, labelled hCG, preferably 125 I-hCG. 125 I-hCG is well-known from laboratory analyses, where it is used as a reagent in radiό-immuno-assays. Iodization techniques and sterile production of medicines are well-known techniques. The sample may be a plasma sample, a whole-blood sample or another tissue sample, preferably a plasma sample. 0.5-50 Mbq is administered, preferably about 5 MBq 125 I-hCG with a specific activity of 1-100 Mbq/ g, preferably about 50 MBq/mg (lmg ~ about 3000 IU hCG). Thereafter the plasma disappearance curve for 125 I-hCG is followed by taking blood samples of 0.5-100 ml, preferably about 5 ml 1 to 20 times a week, preferably 2 to 5 times weekly. These blood samples are counted for activity in the range of 0 to 1 MeV, preferably 15-75 KeV in a well counter. The background activity, calculated as an average of the activity found by counting an empty glass, a glass of plasma and a glass of water, is subtracted from these counts. If the patient has previously received an 125 I-hCG injection, the counting of the initial plasma sample cannot be used for background correction. An average of the counts from empty samples and samples with H 2 O is then employed. By measurement of the disappearance rate of labelled hCG it is possible to determine the pure elimination rate of labelled hCG, primarily 125 I-hCG, since labelled hCG is not produced in the human organism. In addition an immunological determination is made of the total amount of hCG in samples, preferably taken at the same time as the samples for the determination of the activity. As the injected amount of the labelled hCG is negligible compared to the total amount of hCG, this measurement will thus reflect the production as well as the elimination phenomena for hCG. By comparing the plasma disappearance curve obtained by the immunological determination of the total amount of hCG in plasma with the plasma disappearance curve for 125 I-hCG, it is possible to establish whether a continued hCG production is taking place. A parallel course of the two curves illustrates that, irrespective of a prolonged half-life, an hCG production is no longer taking place, and the prolonged half-life is therefore due to elimination phenomena and not hCG production. If an hCG production is no longer taking place, the treatment with, for example, chemotherapy, may thus be terminated. The invention further relates to the use of an agent according to the invention for the establishment of the elimination of patho-physiologically produced hCG in humans. The use of labelled hCG as an elimination marker has thus afforded significantly improved opportunities for choosing therapy for patients with diseases which are accompanied by an abnormal hCG production. The new decision basis thus rests on the patient's own metabolism of labelled hCG as an expression of hCG elimination, seen in the light of the patient's own physiology and patho-physiology, rather than normal values for hCG elimination approximated and extrapolated from scientific examinations of other patients or animal models. In the light of the above technique it is also possible, when the elimination organs for hCG are finally established, to establish the decay rates of 125 I-hCG, which correlate with the relevant organ function. In this way a significantly improved possibility of establishing the function of the organ in question is made possible. The invention thus also relates to the use of an agent according to the invention for the establishment of organ function. The scope of applicability of the invention will appear from the following with reference to the drawings and the examples. It should, however, be understood that the detailed description and the specific examples are merely included to illustrate preferred embodiments, and that various alterations and modifications within the scope of protection will be obvious to persons skilled in the art on the basis of the detailed description. Brief description of the drawing The invention is further illustrated with reference to the drawings, in which Fig. 1 shows hCG measurements during and after chemotherapy in a patient with testicular cancer, as well as how the half-life for hCG is assessed at a given moment, Fig. 2 hCG measurements of both 125 I-hCG as well as total amount of hCG during and after chemotherapy in the same patient, Fig. 3 hCG measurements of both 125 I-hCG as well as the total amount of hCG during and after chemotherapy in a patient with mola, Fig. 4 hCG measurements of both 125 I-hCG as well as the total amount of hCG in a patient without kidney function, and Fig. 5 hCG measurements of both 125 I-hCG as well as the total amount of hCG in a healthy normal subject. Detailed description of the invention. Fig. 1 illustrates a sequence for hCG fall in a patient with increased hCG level. The arrows indicate the commencement day of a 5 days chemotherapy. The broken line indicates the detection limit for hCG measured by means of an immunological technique. hCG measured by means of an immunological technique is indicated by circles. Initially a slow fall is noted (day 0 to 5). Thereafter the half-life (T 1/4 ) decreases to about 1.8 days (day 8 to 20) and increases upon the second treatment (day 20) to about 16 days. During the last phase of the sequence (day 60 to 132) the half-life is seen to be prolonged to about 40 days. The prolonged half-lives (longer than 3 days) after day 20 would, according to the prior art, have been taken as an expression of continued hCG production and thereby the necessity of a continued treatment. Fig. 2 illustrates the same patient, but with a drop in the plasma activity after 125 I- hCG injection plotted in the same coordinate system (indicated by asterisks). If the two curves are compared it will be seen that 125 I-hCG (injected in the patient) disappears in the same manner as the total amount of hCG (produced in the patient). From this it will be seen that there are no reasons for assuming a continued hCG production. The prolonged half-life is therefore due to an elimination problem, and - in the light of this - no further treatment should be given. The method is thus a valuable supplement to the monitoring of the course of treatment. Example 1 A patient with testicular cancer and increased hCG level has twice received 5 days of chemotherapy at an interval of 21 days. Resistance to chemotherapy is suspected, as the half-life for hCG is longer than 3 days (Fig .1 at approx. day 34 to 43 assessed graphically as indicated). The patient's thyroid gland is iodine blocked with 400 mg of potassium iodide administered orally at least 1 hour before the commencement of the examination in order to avoid the uptake of free 125 I. In this way the radiation dose to the thyroid gland is reduced significantly. A well-functioning intravenous insert (e.g. venflon) is made in the patient's arm vein. 10 ml of blood is taken for a background plasma sample as well as 5 ml of blood for the determination of the total amount of hCG in the plasma at time 0. 5 3.3215 MBq of 125 I-hCG supplied by the Institutt for Energiteknikk, Kjeller, Norway is then injected. Two blood samples are taken of 10 ml and 5 ml respectively, for the determination of the activity of 125 I-hCG and the total amount of hCG in plasma at time 2 hours. Thereafter corresponding samples are taken on day: 1, 2, 4, 5, 6, 7, 8, 10, 11, 12, 10 21, 28, 34, 35, 36, 37, 42, 49, 57, 62, 75, 83, 89, 96 and 104, until the patient is hCG-negative, i.e. the total amount of hCG is below detection limit. All blood samples are centrifuged at 3000 rpm for 10 minutes, and plasma is pipetted. 3 ml of plasma is used for the determination of 125 I-hCG and 1 ml of plasma for determining the total amount of hCG . The activity in the individual plasma samples 15 is counted in a well counter in the spectrum of 15 to 75 KeV. All samples are counted simultaneously in order to avoid correction for physical decay of 125 I, since the counting period is < < T 14 physical for 125 I. The activity of 125 I-hCG in the individual plasma samples is plotted in a semi- logarithmic coordinate system with the point in time for the taking of the sample as 20 the abscissa (Fig. 2). In the same coordinate system the total amount of hCG measured by means of an immunological technique is plotted as a function of the point in time for the sample taking. The curves are compared: In spite of a prolonged half-life for hCG (Tι Λ > 3 days) a parallel course of the two curves is seen. This is interpreted as ceased production 25 of hCG and thus no living hCG-producing cancer cells. In the light of this further chemotherapy ceases after the patient has received the standard treatment of 4 times 5 days of chemotherapy. The patient has thus been cured by means of less chemotherapy than would have been employed before the present invention. 5 Example 2 The example concerns a woman with ola who, in spite of chemotherapy, continues to have an increased hCG level. Resistance to chemotherapy is suspected, as the patient has an increasing hCG level (Fig. 3). 10 The patient's thyroid gland is iodine blocked with 400 mg of potassium iodide administered orally at least 1 hour before the commencement of the examination in order to avoid the uptake of free I25 I. In this way the radiation dose to the thyroid gland is reduced significantly. A well-functioning intravenous insert (e.g. venflon) is made in the patient's arm vein. 15 10 ml of blood is taken for a background plasma sample as well as 5 ml of blood for the determination of the total amount of hCG in the plasma at time 0. 3.1415 MBq 125 I-hCG supplied by Institutt for Energiteknikk, Kjeller, Norway, is injected. Two blood samples of 10 ml and 5 ml, respectively, are taken for the determination of the activity of 125 I-hCG and the total amount hCG in the plasma, respectively, at 0 time 2 hours. Thereafter corresponding samples are taken on day 4, 7, 14, 17, 21, 24, 52, 55, 58, 62, 77, 84, 94, 98, 107 and 121 (Fig. 3). The blood samples are examined in the same manner as in example 1 and the results plotted in a semi-logarithmic coordinate system. The curves are compared: the patient is hCG-negative on day 20 (Fig. 3), and a uniform course of the curves is seen. This is interpreted as cessation of the production of hCG , since the fall in the hCG level has proceeded at the same rate as the fall in the activity from 125 I-hCG. There are thus no living hCG-producing cells, and the suspicion regarding the presence of chemotherapy-resistant hCG-producing cells has been shown to be unfounded. The patient is thus cured and may continue with normal checks. Example 3. This example illustrates the use of the agent according to the invention for the assessment of the function of the conversion organs for small proteins, where hCG is employed as a model substance. In a patient where an assessment of the disappearance of small proteins, i.e. proteins with a molecular weight of less than 50,000 Daltons, such as the proteins b 2 - microglobulin and Retinol binding protein is desired, the patient's thyroid gland is iodine blocked by the administration of 400 mg of potassium iodide at least 1 hour before the commencement of the examination in order to avoid the uptake of free 125 I. In this way the radiation dose to the thyroid gland is reduced significantly. A well-functioning intravenous insert (e.g. venflon) is made in the patient's arm vein. 10 ml of blood is taken as background plasma sample as well as 5 ml of blood for the determination of the total amount of hCG in the plasma at time 0. 2.9415 Mbq 125 I-hCG, supplied from Institutt for Energiteknikk, Kjeller, Norway, is then injected. Two blood samples of 10 ml and 5 ml respectively are taken for the determination of the activity of 125 I-hCG and the total amount of hCG in plasma, respectively, at time 2 hours. Corresponding samples are then taken on day 1, 2, 3, 4, 5, 6, 7, 8, 10, 12 and 14 (Fig. 4). The blood samples are examined in the same manner as in example 1 and the results plotted in a semi-logarithmic coordinate system. The course of the curve is compared with that found by the examination of a normal person, said examination being undertaken analogously with the above indicated examination. The curve courses are compared (cf. Fig. 4 and Fig. 5). It is seen that the patient has a lower disappearance rate of the administered 125 I-hCG, and there is thus no doubt that the patient's conversion organs for small proteins function more poorly than for a healthy subject. References 1. Birken S, Canfield RE. Structural and immunochemical properties of human choriogonadotropin. pp. 47-80. [Review]. In: McKerns KW, ed 1978; Structure and function. 2. Nisula BC, Blithe DL, Akar A, Lefort G, Wehmann RE. Metabolic fate of human choriogonadotropin. [Review]. J Steroid Biochem 1989; 33: 733-737. 3. van der Lugt B, Drogendijk AC. The disappearance of human chorionic gonadotro- pin from plasma and urine following induced abortion. Disappearance of HCG after induced abortion. Acta Obstet Gynecol Scand 1985; 64: 547-552. 4. Steier JA, Bergsjo P, Myking OL. Human chorionic gonadotropin in maternal plas¬ ma after induced abortion, spontaneous abortion, and removed ectopic pregnancy. Obstet Gynecol 1984; 64: 391-394. 5. Thyssen HH, Christensen H, Schebye O, Berget A, Arends J, Larsen SO. Elimina¬ tion of human chorionic gonadotropin in serum and urine after uncomplicated induced abortion during the first trimester. [Danish]. Ugeskr Laeger 1992; 154: 2071-2072. 6. Vejerslev LO, Arends J, Larsen SO. Spontaneous regression of serum HCG in 18 patients following evacuation of uncomplicated androgenic moles. [Danish]. Ugeskr Laeger 1988; 150: 2081-2083. 7. Mann K, Sailer B, Hoermann R. Clinical use of HCG and hCG beta determinati- ons. [Review]. Scand J Clin Lab Invest Suppl 1993; 216: 97-104. 8. Paus E, Fossa SD, Risberg T, Nustad K. The diagnostic value of human chorionic gonadotrophin in patients with testicular seminoma. Br J Urol 1987; 59: 572-577. 9. Toner GC, Geller NL, Tan C, Nisselbaum J, Bosl GJ. Serum tumor marker half- life during chemotherapy allows early prediction of complete response and survival in nonseminomatous germ cell tumors. Cancer Res 1990; 50: 5904-5910. 10. Motzer RJ, Gulati SC, Crown JP, et al. High-dose chemotherapy and autologous bone marrow rescue for patients with refractory germ cell tumors. Early intervention 5 is better tolerated. Cancer 1992; 69: 550-556. 11. Gerl A, Clemm C, Lamerz R, Mann K, Wilmanns W. Prognostic implications of tumour marker analysis in non-seminomatous germ cell tumours with poor prognosis metastatic disease. Eur J Cancer 1993; 29A: 961-965. 12. Droz JP, Kramar A, Ghosn M, et al. Prognostic factors in advanced nonsemino- ιo matous testicular cancer. A multivariate logistic regression analysis. Cancer 1988; 62: 564-568. 13. Cassels JW, Jr. , Mann K, Blithe DL, Nisula BC, Wehmann RE. Reduced metabolic clearance of acidic variants of human choriogonadotropin from patients with testicular cancer. Cancer 1989; 64: 2313-2318. 15 14. Kuhbock J, Aiginger P, Kuzmits R, Spona J. Prognostic value of tumor marker determinations in testicular cancer patients. Cancer Detect Prev 1987; 10: 389-392. 15. Klein EA. Tumor markers in testis cancer. Urol Clin North Am 1993; 20: 67-73. 16. Wehmann RE, Nisula BC. Metabolic and renal clearance rates of purified human chorionic gonadotropin. J Clin Invest 1981 ; 68: 184-194. 20 17. Saal W, Glowania HJ, Hengst W, Happ J. Pharmacodynamics and pharmacokine- tics after subcutaneous and intramuscular injection of human chorionic gonadotropin. Fertil Steril 1991; 56: 225-229. 18. Horwich A, Peckham MJ. Serum tumour marker regression rate following chemotherapy for malignant teratoma. Eur J Cancer Clin Oncol 1984; 20: 1463-1470. 19. Pedersen BN. Tumour markers in testicular germ cell tumours. Acta Oncol 1984; 24: 287-294. 20. van der Gaast A, Hoekstra JW, Croles JJ, Splinter TA. Elevated serum tumor markers in patients with testicular cancer after induction chemotherapy due to a reser¬ voir of markers in cystic differentiated mature teratoma. J 1991; 145: 829-831. While the invention has been described with reference to specific embodiments thereof, it is obvious that it can be varied in many ways. Such variations are not be considered a deviation from the scope of the invention, and all such modifications which are obvious to persons skilled in the art are also to be considered comprised by the scope of the accompanying claims.

Claims: Claims: 1. A method for the in vitro measurement of the elimination of labelled hCG in humans, characterised by 1) measuring the disappearance rate of labelled hCG in samples from said 5 human, 2) comparing the disappearance rate obtained under 1) with a) the disappearance rate of the total amount of hCG in samples from said human for the establishment of a possible presence of patho- physiological hCG production, or 10 b) the disappearance rate of labelled hCG in samples from a healthy subject for the establishment of organ function. 2. An agent for use in the method according to claim 1, characterised by containing as active ingredient labelled hCG. 3. An agent according to claim 2, characterised by the labelled hCG being 15 125 I-hCG. 4. The use of an agent according to claim 2 or 3 for the establishment of patho- physiologically produced hCG in humans. 5. The use of an agent according to claim 2 or 3 for the establishment of organ function.

Inventors: CHRISTENSEN THOMAS BROE, CHRISTENSEN, THOMAS, BROE
Applicants: CHRISTENSEN THOMAS BROE, CHRISTENSEN, THOMAS, BROE
Application year: 1996
Main classification: A61K49
IPC codes: A61K51, G01N33