EP0809518A1 - PROCEDE DE MESURE IN VITRO DE L'ELIMINATION D'hCG MARQUEE CHEZ L'HOMME, AGENT A UTILISER AVEC CE PROCEDE ET EMPLOI DE CE DERNIER POUR DETERMINER L'ELIMINATION D'hCG PRODUITE DE FA ON PATHOPHYSIOLOGIQUE, OU POUR DETERMINER LE FONCTIONNEMENT D'ORGANES - Google Patents

PROCEDE DE MESURE IN VITRO DE L'ELIMINATION D'hCG MARQUEE CHEZ L'HOMME, AGENT A UTILISER AVEC CE PROCEDE ET EMPLOI DE CE DERNIER POUR DETERMINER L'ELIMINATION D'hCG PRODUITE DE FA ON PATHOPHYSIOLOGIQUE, OU POUR DETERMINER LE FONCTIONNEMENT D'ORGANES

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Publication number
EP0809518A1
EP0809518A1 EP96902902A EP96902902A EP0809518A1 EP 0809518 A1 EP0809518 A1 EP 0809518A1 EP 96902902 A EP96902902 A EP 96902902A EP 96902902 A EP96902902 A EP 96902902A EP 0809518 A1 EP0809518 A1 EP 0809518A1
Authority
EP
European Patent Office
Prior art keywords
hcg
elimination
establishment
labelled
well
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96902902A
Other languages
German (de)
English (en)
Inventor
Thomas Broe Christensen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0809518A1 publication Critical patent/EP0809518A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Definitions

  • 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 human Chorion Gonadotropin
  • 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.
  • 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 .
  • 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.
  • hCG level is observed in connection with extra-uterine pregnancies as part of the normal pregnancy physiology.
  • 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.
  • 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?.
  • 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.
  • the invention consequently relates to a method for the in vitro measurement of the elimination of labelled hCG in humans, which is characterised by
  • 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.
  • I-hCG is well-known from laboratory analyses, where it is used as a reagent in radi ⁇ -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).
  • 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.
  • 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.
  • 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. 5 hCG measurements of both 125 I-hCG as well as the total amount of hCG in a healthy normal subject.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 example concerns a woman with ola who, in spite of chemotherapy, continues to have an increased hCG level.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.

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  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
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  • Urology & Nephrology (AREA)
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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

La présente invention concerne un agent destiné à l'analyse de l'élimination d'hCG (hormone chorionique gonadotrophique) produite physiologiquement par un patient, le procédé de mesure in vitro de l'élimination de l'hCG chez les patients qui présentent un niveau augmenté de cette hormone et l'emploi d'un tel agent. Cela permet de déterminer l'effet d'un traitement sur des patients ayant des niveaux anormaux d'hCG, comme les patients atteints de cancer des testicules, et de déterminer le fonctionnement de différents organes.
EP96902902A 1995-02-15 1996-02-14 PROCEDE DE MESURE IN VITRO DE L'ELIMINATION D'hCG MARQUEE CHEZ L'HOMME, AGENT A UTILISER AVEC CE PROCEDE ET EMPLOI DE CE DERNIER POUR DETERMINER L'ELIMINATION D'hCG PRODUITE DE FA ON PATHOPHYSIOLOGIQUE, OU POUR DETERMINER LE FONCTIONNEMENT D'ORGANES Withdrawn EP0809518A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK16995A DK171745B1 (da) 1995-02-15 1995-02-15 Fremgangsmåde til fastlæggelse af eliminationen af mærket hCG i mennesker, middel til anvendelse ved fremgangsmåden samt anvendelse heraf til fastlæggelse af patofysiologisk dannet hCG's elimination eller til fastlæggelse af organfunktion
DK169/95 1995-02-15
PCT/DK1996/000070 WO1996025180A1 (fr) 1995-02-15 1996-02-14 PROCEDE DE MESURE IN VITRO DE L'ELIMINATION D'hCG MARQUEE CHEZ L'HOMME, AGENT A UTILISER AVEC CE PROCEDE ET EMPLOI DE CE DERNIER POUR DETERMINER L'ELIMINATION D'hCG PRODUITE DE FAÇON PATHOPHYSIOLOGIQUE, OU POUR DETERMINER LE FONCTIONNEMENT D'ORGANES

Publications (1)

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EP0809518A1 true EP0809518A1 (fr) 1997-12-03

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EP96902902A Withdrawn EP0809518A1 (fr) 1995-02-15 1996-02-14 PROCEDE DE MESURE IN VITRO DE L'ELIMINATION D'hCG MARQUEE CHEZ L'HOMME, AGENT A UTILISER AVEC CE PROCEDE ET EMPLOI DE CE DERNIER POUR DETERMINER L'ELIMINATION D'hCG PRODUITE DE FA ON PATHOPHYSIOLOGIQUE, OU POUR DETERMINER LE FONCTIONNEMENT D'ORGANES

Country Status (4)

Country Link
EP (1) EP0809518A1 (fr)
AU (1) AU4712796A (fr)
DK (1) DK171745B1 (fr)
WO (1) WO1996025180A1 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4874693A (en) * 1986-10-10 1989-10-17 Mark Bogart Method for assessing placental dysfunction

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9625180A1 *

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Publication number Publication date
DK171745B1 (da) 1997-04-28
WO1996025180A1 (fr) 1996-08-22
AU4712796A (en) 1996-09-04
DK16995A (da) 1996-08-16

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EP0809518A1 (fr) PROCEDE DE MESURE IN VITRO DE L'ELIMINATION D'hCG MARQUEE CHEZ L'HOMME, AGENT A UTILISER AVEC CE PROCEDE ET EMPLOI DE CE DERNIER POUR DETERMINER L'ELIMINATION D'hCG PRODUITE DE FA ON PATHOPHYSIOLOGIQUE, OU POUR DETERMINER LE FONCTIONNEMENT D'ORGANES
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