WO2004098652A1

WO2004098652A1 - Radiolabelled thyroid stimulating hormone (tsh) and use thereof for the diagnosis and therapy of the differentiated thyroid tumors

Info

Publication number: WO2004098652A1
Application number: PCT/IT2004/000234
Authority: WO
Inventors: Alberto Signore; Marco Chianelli
Original assignee: Universita' Degli Studi Di Roma La Sapienza
Priority date: 2003-05-05
Filing date: 2004-04-27
Publication date: 2004-11-18
Also published as: ITRM20030214A1; ITRM20030214A0

Abstract

The invention relates to the use of the human recombinant thyroid stimulating hormone (TSH) labelled with radioisotopes suitable for the administration in vivo, having a good stability and specific activity, as scintigraphic tracer and/or as radiomedicament respectively for the diagnosis and therapy of differentiated thyroid tumors and the respective metastases.

Description

RADIOLABELLED THYROID STIMULATING HORMONE (TSH) AND USE THEREOF FOR THE DIAGNOSIS AND THERAPY OF THE DIFFERENTIATED THYROID TUMORS.

The present invention relates to the radiolabelled thyroid stimulating hormone (TSH) and use thereof for the diagnosis and therapy of the differentiated thyroid tumors. More particularly the invention relates to the TSH labelled with suitable radioisotopes for the administration in vivo, having a good stability and specific activity, in order to use it advantageously as scintigraphic tracer and/or as radiomedicament, for the diagnosis and therapy of differentiated thyroid tumors and metastasis, respectively.

It is known that the papillary and follicular thyroid tumors are differentiated thyroid tumors (DTC) that preserve the functional characteristics of the normal thyroid cells from which they originate. In fact, the transformed thyroid cells maintain the ability to catch iodine, to synthesize and release both thyroid hormones or thyroglobulin. Furthermore, the above mentioned cells are able to synthesize the enzyme thyroperoxidase (TPO) and express on the outer cellular membrane the receptor for TSH.

The iodine uptake, synthesis of thyroglobulin and other functions of the thyroid cells are stimulated by TSH which acts through bond with its own receptor. Such functions are expressed to a significantly lesser extent in the transformed cells compared with the normal thyroid tissue.

The therapy of the DTCs is firstly surgical and it includes the removal of the thyroid and contingent metastasis evident at time of the surgical operation. In order to avoid the onset of hypothyroidism following the thyroidectomy, the patient must take exogenous thyroid hormones, like levothyroxine, at such doses to inhibit the TSH production in order to prevent that the latter can stimulate the growth of neoplastic thyroid tissue.

Currently different techniques are used, also in combination, for the diagnosis of the possible topical-regional relapses and/or the metastases at a distance following the surgical operation. Among these techniques it is very diffused, as diagnostic tool, the total body scintigraphy with ¹³¹l (WBS), carried out in absence of thyroid residues following the surgical operation. In fact under such conditions, the ¹³¹l uptake indicates the presence of thyroid tissue compatible with topical-regional relapses and/or metastases at distance.

To increase the efficiency of test in order to detect thyroid tissue catching ¹³¹l, the examination is performed under conditions of elevated plasmatic values of TSH. The increase of haematic levels of TSH can be achieved through induction of severe hypothyroidism, this condition stimulating the production of endogenous TSH. To induce hypothyroidism, the patient must suspend the intake of levσthyroxine for a period of about 40 days. At the end of such period, ¹³¹l (2-5 mCi) is administered and the scintigraphy is performed after 24/48 hours.

Alternatively the use of the recombinant human TSH has been recently introduced in the clinical practice, according to that described, particularly, in the US patents No. 6284491 and No. 6117991 (both to Department of Health and Human Services U.S.A.). The recombinant human TSH, subsequently to intramuscular administration (0,9 mg/day for two days), provokes elevation of TSH plasmatic values. Then iodine is administered and the scintigraphy is carried out after 24/48 hours.

Despite its effectiveness, the scintigraphic technique with ¹³¹1 above described shows some limits and/or disadvantages.

The most substantial limit is represented by the impossibility to carry out a diagnosis or to adopt a therapy in absence of uptake of the radioisotope by DTC cells. In fact, such ability of uptake can be lost in the late phases of the tumors following the loss of differentiation and, therefore, the scintigraphic detection of the thyroid tissue is impaired.

As above mentioned, in order to carry out the scintigraphic examination it is necessary to induce the elevation of endogenous TSH levels. If this last is achieved by administration of recombinant exogenous TSH, the patient remains in a state of euthyroidism throughout the examination. Though, such practice is still poorly diffused and in addition the recombinant human TSH is expensive and not always it is refunded by the national sanitary service. Where it is not possible to use the recombinant human TSH, the scintigraphy can not be immediately performed, but it is necessary to suspend therapy with levothyroxine and to await at least 40 days for the preparation of the examination. Such a preparation induces a severe hypothyroidism, that involves important changes in the life quality of the patient due to reduction of the renal function, reduced cardiac functionality, deep asthenia, reduced working ability. Further sometimes the clinical conditions of the patient don't allow the suspension of therapy and accordingly the execution of the scintigraphic examination.

It is also to underline that the pre-operating ability of the total body scintigraphy with ¹³¹l to notice DTC metastases is very scarce. In fact under such conditions the thyroid iodine uptake is of higher level, for which the uptake by possible metastases is reduced. Another not inconsiderable factor is to be recognized in the elevated effective dose of radiations absorbed by the patient, equal to about 20 mSv, for the WBS execution with ³¹i, that is repeated several times in the patient life depending on the clinical picture. This can contribute to increase the incidence frequency of potentially carcinogenic genetic mutations, and can induce a reduction of the salivary glands function through which the iodine is excreted.

Finally, a reduction of the therapeutic effectiveness of the ¹³¹l doses can be found on possible metastasis, because of reduced functionality in terms of uptake by the cells previously treated with diagnostic doses of ¹³¹l, this is a phenomenon known as thyroid "stunning".

A further method of diagnosis for possible relapses and/or metastases, in addition to that above described regarding the total body scintigraphy, is represented by the measurement of the thyroglobulin (TG) plasmatic levels. In fact it is known that the increase of the TSH values, achieved in patients like in the preceding case, stimulates thyroglobulin production in the potentially present tumorai cells, provoking the increase of the plasmatic values thereof. Also when the DTCs lose the capability to catch the iodine, in most cases they continue to possess the ability to synthesize thyroglobulin. Tough characterized by elevated diagnostic accuracy, the determination of thyroglobulin plasmatic levels, besides to be subject to the same inconveniences of the total body scintigraphy with regard to induction of increased levels of TSH, shows some further limitations described below.

First of all it is not possible to use thyroglobulin also for therapeutic purposes, as it is carried out with ¹³¹l. Therefore, in case of a discovered pathological increase of the protein plasmatic values, however the therapy depends on the ¹³¹l uptake. As well the location of the neoplastic foci is impossible through the detection of thyroglobulin elevated levels. Moreover, such values are not predictive of the presence of metastases from DTC in the preoperation stage, when the same gland contributes to produce thyroglobulin physiologically. From the viewpoint of possible therapeutic interventions in the eventuality of metastases, if these last show ability to catch the iodine it is possible to resort to radiometabolic therapy through ¹³¹l administered in therapeutic doses (100-200 mCi). Such therapeutic approach is effective in most cases. However, also radiometabolic therapy with ³¹l shows some limitations which reflect those already discussed for diagnostic use of ¹³¹|, and which consist substantially in the impossibility to achieve a therapeutic effect in the DTCs in case of unsuccessful uptake of ³¹1. In the light of the above described, it is therefore clear the need to have new means, either for the diagnosis and for therapy of the differentiated thyroid tumors (DTC), that doesn't show the disadvantages of the heretofore known techniques.

As noticed previously, DTCs preserve the TSH-receptor expression (Namba H et al.; Overexpression of the Intact Thyrotropin Receptor in a Human Thyroid Carcinoma Cell Line. Endocrinology 1993; 132:2). This ability is also preserved in most cases also when the ¹³¹l uptake is impaired. In fact unsuccessful iodine uptake doesn't depend on TSH-receptor unsuccessful expression but depends on the defective signal transduction following the TSH binding to its own receptor. The authors of the present invention have found that the radiolabelled recombinant TSH retains the ability to bind its own receptor and accordingly it can be advantageously employed as scintigraphic tracer and as radiomedicament, respectively in the diagnosis and therapy of differentiated thyroid tumors and related metastases, bypassing disadvantages of currently available techniques in this field. Particularly, radiolabelled TSH, intravenously injected, allows the imaging by scintigraphy with γ room of the tumor and normal thyroid tissue, consequently to tissue build-up of the thyroid-stimulating hormone due to the binding with the receptor thereof.

As the practical realization of the diagnostic and therapeutic means of the invention, several techniques for labelling peptides and proteins with radioisotopes are already known, particularly with the metastable radioisotope gamma emitting ^99mTc (technetium), which is particularly useful for diagnostic applications for imaging. Along these, for instance, US patents No. 5206370 and No. 5420285 disclose methods which use several pyridil-hydrazines, pyridil-hydrazides and derivatives thereof, such as metallic ion bifunctional chelating agents, including technetium and rhenium. The same subject has been more recently investigated with specific reference to 6-hydrazinonicotinamide (HYNIC) (Rennen HJJ et al..; Labelling proteins with ^99mTC via Hydrazinonicotinamide (HYNIC): Optimization of the Conjugation Reaction. Nuclear Medicine & Biology 2000; 27:599-604).

Specifically considering the thyrotropin (TSH), the authors have developed a protocol for efficient preparation of the scintigraphic tracer according to the invention, which allows to maintain an elevated specific activity and stability of the protein even subsequently to the delicate labelling process with ^99mTC. The same labelling method can be used, with slight changes, for the proteinlabelling with Rhenium radioisotopes (¹⁸⁶Re and ¹⁸⁸Re) that, even emitting beta-type radiations, have chemical characteristic similar to those of ^99mTc. As previously noticed, the recombinant human TSH is already employed in humans in a non-radioactive form and its systemic administration is safe and does not produce side effects. TSH can be advantageously used for diagnostic purposes, when it is labelled with gamma emitting isotopes, which are characterized by elevated ability of penetration, whi le it can be used for therapeutic purposes when is labelled with sotopes emitting primarily beta particles, which, having a low abili ty of penetration assure a more selective dose deposition on short-range with respect to that obtainable with use of gamma-emitting isotopes.

The use of radiolabelled human recombinant TSH both for the diagnosis and DTCs therapy is characterized by several advantages with respect to above described techniques currently employed in this field.

First of all the scintigraphic examination performed with radiolabelled TSH does not need suspension of the therapy with levothyroxine and the hypothyoidism induction in patients. On the contrary, the chronic administration of levothyroxine in suppressive doses involves an over-regulation of the TSH-receptor expression, and this facilitates DTCs individualization through the receptor binding with the tracer of the invention. Another not inconsiderable positive aspect of the fact that it is not necessary to induce a endogenous TSH increase, it is that the examination does not involve long waiting times for the preparation of the patient.

In addition, the DTC metastasis which not catch iodine, but still expressing TSH-receptor can be both diagnosed and treated by radiolabelled TSH according to the invention, unlike with conventional methods based on the iodine. As already noticed, for the diagnosis and therapy, the TSH according to the invention is labelled with different isotopes, respectively, γ-emitting and β- emitting.

The employment of radiolabelled TSH should be suitable to allow a pre-operating stadiation of the disease by detecting possible metastases. Currently ¹³¹I-WBS it does not allow the pre- operating stadiation because, before the intervention, the thyroid uptakes an elevated quantity of the administered iodine (from 20% to 60%). The iodine uptake by metastases, under these conditions, would be very low due to ¹³¹l reduced availability and also due to reduced ability to uptake iodine normally observable in neoplastic thyroid tissue in comparison to the normal one (at least 10 times lower). The thyroid uptake intensity, therefore, makes impossible the simultaneous imaging of the metastases.

Based on recent experiences with other receptorial-type medicaments, such as radiolabelled somatostatin, it is presupposed that the radiolabelled recombinant TSH uptake by the normal thyroid does not represent an impediment to the systemic location of tumor thyroid tissue.

It is also interesting to notice that the effective dose, resulting from a scintigraphy performed employing the tracer according to the invention labelled with γ-emitting radioisotopes such as ^99mTc or ¹²³l, is assessed below 5 mSv. This means, in fact, that the patients are exposed to lower doses of radiation with respect to the total body scintigraphy (WBS) with ¹³¹l, which radiates both γ- radiations or β- radiations (and in fact it is used both for diagnostic and therapeutic purposes), and which, as already noticed, provokes the absorption by the patient of an about 20 mSv effective dose.

Finally, an further advantage, which arises from diagnostic use of labelled recombinant TSH with radioisotopes emitting only gamma rays, is represented by the absence of the phenomenon of thyroid "stunning" previously described, because of the scarce irradiation of the target tissue.

Therefore it is specific object of the present invention the use of the human recombinant thyroid stimulating hormone (TSH) labelled with a radioactive isotope suitable for the administration in vivo, for the preparation of a radiomedicament for the diagnosis and therapy of differentiated thyroid tumors (DTC) and the respective metastases.

More particularly, if the radiomedicament according to the invention is for the diagnosis of differentiated thyroid tumors and respective metastases, the radioactive isotope is substantially a γ- emitting radioisotope, which can be chosen from the group consisting of ^99mTc, ¹²³l, ¹²⁵l, ^{1 1}ln. Alternatively positron (β+) emitting radioisotopes, such as ⁶²Cu, ¹¹C and ¹⁸F, can be employed. The radiated positrons result in gamma rays which can be opportunely used for diagnostic purposes using opportune machineries allowing the positron emission tomography (PET). Where the radiomedicament according to the invention is directed to the therapy of differentiated thyroid tumors and respective metastases, the radioactive isotope, whereby the TSH is labelled, is substantially a β-emitting radioisotope, and can be chosen from the group consisting of ¹³¹l, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ¹⁶⁶Ho, ³²P, ⁷⁶As, ⁸⁶Rb and ⁸⁹Sr.

It is evident that the choice from the radionuclides above mentioned depends on several factors, in the first place among them, the commercial availability, cost and intrinsic characteristics such as maximum energy and the biological half-life. Particularly, energy determines the penetration into tissues, therefore the greater the energy, the greater the penetration into tissues, while the decay time determines the irradiation duration of the target tissue.

According to another aspect thereof, the present invention has as a further object, the human recombinant thyroid stimulating hormone (TSH) labelled with ^99mTc, as used in medicine like a tracer for the scintigraphic diagnosis of differentiated thyroid tumors (DTC) and the respective metastases. For such use, as already noticed, the TSH is conjugated preferably with the bifunctional chelating agent 6- hydrazinonicotinamide (HYNIC), and preferably the radiolabelled product includes also tricine and/or EDDA as co-ligand.

In addition it is a further object of the invention a radiopharmaceutical composition for the diagnosis and therapy of differentiated thyroid tumors and respective metastases comprising like active ingredient the recombinant human TSH labelled with a radioactive isotope suitable for the administration in vivo, such as a γ-emitting or β⁺-emitting isotope for the diagnosis, or β^"-emitting for therapy, along with one or more adjuvants and/or pharmacologically acceptable excipients. Similarly, the object of the invention can take form of a radiopharmaceutical kit for the diagnosis or therapy of differentiated thyroid tumors (DTC) and the respective metastases comprising the following components: a) a reducing agent, a bifunctional chelating agent conjugated to recombinant human TSH, a coligand, an antioxidant agent; and b) a radioactive isotope suitable for the administration in vivo.

For the diagnostic use the kit to be used for the labelling could be formulated with ^99mTc and should comprise a glass vial vacuum closed with hermetic rubber plug for medical use that contains the following constituents in a freeze dried, sterile and pyrogen free form, prepared according to the principles of Buona Pratica Farmaceutica: a reducing agent (stannous chloride), a bifunctional chelating agent conjugated to hrTSH (HYNIC-hrTSH), a coligand (tricine and/or EDDA), an anti-oxidant agent (gentisic acid). If necessary the kit can be reconstituted with ^99mTc.

For the therapeutic use, the labelling can be performed with ¹⁸⁶Re or ¹⁸⁸Re according to the same procedure and, because of the similar chemical characteristics of ^99mTc and ¹⁸⁶Re or ¹⁸⁸Re, contain the same constituents with doses of the relative constituent slightly different in the two cases.

Some experimental results achieved within the present invention, including the data relative to the characteristics and the performances of radiolabelled TSH for the DTC diagnosis and therapy, are set forth by a way of example, and they are illustrated in the enclosed figures, in which: figure 1 shows the time variation of the relation between the radioactivity present in the target organ and the background radioactivity (background) within the results of biodistribution studies in vivo on mice implanted with tumors not expressing THS-receptor (ARO) or with tumors expressing such receptor (TPC-1), studied in both cases with radioiodinated TSH according to the invention. figure 2 shows, within the same studies of the preceding figure, the scintigraphic images at two hours of tumor accumulation of ¹²³I-TSH, in mice implanted with ARO (A and B) or with TPC-1 (C and D); the arrows indicate the point in which the tumors have been implanted; figure 3 shows, within the labelling experimentation of TSH-HYNIC with ^99mTc, the percentage variation of the labelling efficiency of the tricine, used as coligand, as a function of the labelling pH, employing a tricine concentration equal to 27,98 mg/ml; figure 4 shows, within the same experimentation illustrated in figure 3, the percentage variation of the labelling efficiency of the tricine as a function of the same tricine concentration; figure 5 shows, within the same experimentation illustrated in figure 3, the percentage course of the labelling efficiency of the tricine versus of the SnCI₂-2H₂0 concentration; figure 6 shows, within the same experimentation illustrated in figure 3, the percentage variation of the labelling efficiency of the tricine over time (stability), for a tricine concentration equal to 102,94 mg/ml and at pH 5; figure 7 shows, within the same experimentation illustrated in figure 3, the percentage variation of the labelling efficiency of the tricine over time (stability), for a tricine concentration equal to 102,94 mg/ml and at pH 7; figure 8 shows, within the same experimentation illustrated in figure 3, the percentage course of the labelling efficiency of the TSH-HYNIC versus the incubation time; figure 9 shows, within the same experimentation illustrated in figure 3, the percentage variation of the labelling efficiency of the TSH-HYNIC versus the radioactivity concentration (mCi/ml).

EXAMPLE 1: Studies of biodistribution in vivo and of the binding of recombinant human TSH labelled with radioiodine to its own receptor in experimental animal models.

For these studies recombinant human TSH has been used (rhTSH; Thyrogen, G-enzyme).

TSH labelling with radioiodine

The labelling has been performed through enzymatic method (Burrin JM; Comparison between human ¹²⁵l-labelled TSH labelled with lactoperoxidase or chloramine-T advantages in the use of enzymically labelled antigen in a RIA for human TSH. Clinica Chimica Acta 1976; 70:153-159), because comparative studies of several iodination methods (Kermode JC, Thompson BD; lodination of TSH for receptor-binding studies with human thyroid membranes: effects of specific activity and method of iodination. J Endocr 1980; 84:439-447) have shown that the protein does not suffer damages, maintaining affinity for its receptor. TSH has been incubated in 0,01 M phosphate buffer at pH 7, with lactoperoxidase, glucose oxididase, D-glucose (reagents from Sigma-Aldrich), ¹²⁵l (IMS30, Amersham) for in vitro studies or ¹²³l for in vivo studies in the animal, according to the labelling protocol of the recombinant human TSH with ¹²⁵i or ¹²³l set forth in table 1. The incubation time for labelling was 30 minutes.

For the labelling with ¹²⁵l has been used a stoichiometric ratio ¹²⁵I:TSH 1 :1 , because it is known from literature that the hormone biological activity is not altered.

As a result, rhTSH has been labelled with ¹²³l at an elevated specific activity, equal to 132,2 mCi/mg and efficiency of 55%. Labelling with ¹²⁵l a specific activity equal to 94,3 mCi/mg and an efficiency of 98% has been achieved.

The stability tests in vitro have shown a not remarkable release of the radioiodine, equal to 4%, after 4 hours, both in plasma and in saline.

Experiments in vivo with radioiodinated TSH

Cell lines - For the targeting experiments in vivo at tumor level in the animal model the following two cellular lines have been used: TPC-1 , deriving from the papillary carcinoma of the thyroid, which expresses the receptor for TSH and ARO, deriving from the anaplastic carcinoma of the thyroid, that does not express the TSH receptor.

The cells of the TPC-1 line has been cultured in DMEM, supplemented with 10% bovine fetal serum, 10 ml/I penicillin/streptomycin (10000 U of G penicillin and 10 mg of streptomycin), 10 ml/l B amphotericin (250 μg/ml), 1 % L-glutamine. The cells of the ARO line have been cultured in RPMI 1640, supplemented with 10% bovine fetal serum, 10 ml/I penicillin/streptomycin (10000 U of G penicillin and 10 mg of streptomycin), 10 ml/l B amphotericin (250 μg/ml), 1% L-glutamine. In both cases, at the confluence, the cells are detached with a 0,25 % solution of trypsin-EDTA after washing with HBSS without Ca²⁺ and Mg²⁺.

In vivo experiments - For in vivo experiments mice without thymus have been used. In the animals thyroid carcinoma tumors have been induced by injecting in the right thigh T10⁶ cells suspended in 100 μl of PBS. In three mice, cells of the TPC-1 line have been injected which constitute the positive control to detect the specific binding of TSH to its own receptor. The group of negative control is constituted of others three mice in which cells of the ARO line have been injected which do not express the TSH receptor.

Tumors are developed in the animals, and after 25 biodistribution experiments with 10 μg of radioiodinated TSH have been performed. To avoid the uptake by the thyroid of potentially present free iodine, before the radiolabelled TSH injection, 100 μl of a saturated solution of KCI0₄ for 20 minutes have been injected. The animals have been preventively sedated with 100 μl of Domitek.

Both dynamic for 30 min and static at 1 h, 2 h, 3 h scintigraphic images have been acquired. The images have been quantitatively analyzed tracing "regions of interest" (ROI) on whole body and following organs: blood, head, brain, heart, lungs, stomach, spleen, liver, kidneys, small intestine, large intestine, bladder, fat tissue, carcass, thyroid, thigh with the tumor, contralateral thigh.

After several acquisitions, a mouse for any line has been sacrificed, the organs have been collected, weighed and counted at the gamma counter after 2 days, in order to assess the radioactivity accumulation over time. Activity for every organ has been normalized for the weight of the organ and for the total activity, the tumor/contralateral thigh ratio, percentage variations of the same at 2 and 3 h in comparison to the measured value at 1 h have been calculated. The contralateral thigh radioactivity has been considered background and subtracted to the value found for the tumor. From the achieved values it is possible to find the value of the percentage of injected dose per gram of tissue (% ID/g). Results

It has been found that rhTSH labelled with iodine, as already noticed with an high specific activity, was stable for the use in vivo.

A rapid haematic clearance of the radiomedication has been observed. After the first hour from the intravenous injection the circulating activity linked to the radiomedicament disappears almost completely from the circulatory stream allowing a clear imaging of the organs and tissues wherein the radiomedicament is accumulated in a specific way.

As it is shown in figures 1 and 2, during 30 initial minutes ¹²³I-TSH is localized in the stomach, liver, kidneys and in the bladder, while accumulation has not been noticed in the thyroid. During first 30 minutes an accumulation is also verifiable in the area of implant of the tumors, both of type ARO and of type TPC-1. However for the TPC-1 type tumors it is noticed an increase of the ratio T/B from 1 ,35 to 1 ,81 after three hours, while for the ARO tumors the T/B ratio is decreased from 1 ,26 to 1 , 18.

From the biodistribution studies on the organs, it results that the uptake by the tumor, expressed like % ID/g of tissue at first hour, increased from 100 to 277 in the TPC-1 tumors and decreased from 100 to -10 in the ARO tumors. The quite low and decreasing accumulation in mice with ARO tumors is due to a non-specific accumulation caused by the increased vascularizzation.

In fact only the TPC-1 tumors show significant uptake of the tracer, as can be noticed in figure 2. The greater and time increasing accumulation in the TPC-1 tumors denotes a specific uptake by the tumor, due to the interaction of the TSH-receptor with TSH. This allows to conclude that the labelled TSH has a notable potentia in the diagnosis and follow up of the DTCs.

EXAMPLE 2: Study of the recombinant human TSH labelled with ^99mTc Also for this study recombinant human TSH has been used

(rhTSH; Thyrogen, G-enzyme). Preparation of the conjugate HYNIC-TSH

The radioisotope ^99mTc has been complexed with the bifunctional chelating agent 6-hydrazinonicotinamide (HYNIC). In order to prepare the conjugate HYNIC-TSH, rhTSH has been incubated with HYNIC at room temperature for 1 hour to a stoichiometric ratio HYNIC:TSH 3:1(200 μl TSH at the concentration of 1 mg/ml and 6,62 μl HYNIC at the concentration of 10 mg/ml in DMSO). In fact from a recent work it results that such stoichiometric ratio has revealed to be optimal for labelling biologically active peptides with retention of biological activity (Rennen HJJM et al..; Labelling proteins with ^99mTC via Hydrazinonicotinamide (HYNIC): Optimization of the Conjugation Reaction. Nuclear Medicine & Biology 2000; 27:599-604). The employed conjugation conditions between HYNIC and TSH are set forth in table 2. Table 2

In order to remove the excess of unbound HYNIC it has been performed a purification through size exclusion chromatography using a Sephadex G 25 PD10 column (Amersham Pharmacia Biotech), eluting with PBS. Subsequently in order to increase the protein concentration, the conjugated protein has been concentrated in the same purification solvent (PBS) at 400 μl (final concentration 0,5 mg/ml) and subsequently at 200 μl (final concentration 1 mg/ml) with Speed-vac. TSH labelling with ⁹⁹ TC The TSH-HYNIC has been labelled with ^99mTc0₄ ^" using tricine as coligand and SnCI₂-2H₂0 as reducing agent, like set forth in the following table 3. In order to optimize the labelling procedure have been studied the effects of the concentration of HYNIC-TSH, tricine, SnCI₂-2H₂0, pH, concentration and volume of Tc0₄ ^" on the labelling efficiency of TSH and tricine, as it will be illustrated after the table.

Table 3

As tricine the labelling efficiency has been assessed on the base of pH, which has been varied during the experiment from 7 to 9, maintaining the concentration at 27,98 mg/ml. The resultant plot is shown in figure 3. The effects of the tricine concentration on the labelling efficiency of the same tricine have been assessed with dilutions 1 :2 from 27,28 mg/ml to 416 mg/ml values, as can be observed by the profile of the plot set forth in figure 4.

Furthermore, in order to optimize the labelling efficiency of tricine the concentration of SnCI₂-2H₂0 has been varied from 0,075 mg/ml to 0,3 mg/ml; the profile of the resulting plot is set forth in figure 5.

Studies have been also carried out in order to test stability over time of the labelled tricine (at concentration of 102,94 mg/ml), at pH 5 and pH 7, respectively. The profile of the two plot is set forth in figures 6 and 7.

The labelling efficiency of HYNIC-TSH has been assessed both versus the time of incubation and concentration of radioactivity from 4 mCi/ml to 143,65 mCi/ml. The concentration of HYNIC-TSH has been varied between 0,033 mg/ml and 0,13 mg/ml. The profile of the respective plots resulting from the experiments is shown in figures 8 and 9.

For the purification of the reaction mixture two techniques have been employed: an elution of Sephadex G 25 column by using PBS and a solid phase extraction with tC2 Sep-pak (Waters).

The evaluation of the labelling efficiency has been carried out by a flash thin-layer chromatography (ITLC) with ITLC-SG plates (Gelman Laboratories) using 0,9% NaCI as mobile phase to measure both the colloids and protein labelled at the beginning. In order to detect the presence of free ^99mTc, acetone as mobile phase has been used. In this system ^99mTc migrates with the front of solvent.

Stability of the native TSH and the TSH conjugated to HYNIC

The TSH stability has been tested at different values of pH: 3, 5, 7, 8 and 10, using size exclusion chromatography (SEC), with PBS as eluent at a flow rate of 1 ml/min, after incubation with the solvents used for the labelling. Then sodium dodecil sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the native TSH and conjugated TSH has been performed. The concentrations of the stacking and running gel were 12 % and 10%, respectively. Electrophoresis has been carried out in pH 6,8 buffer. The proteins have been boiled for 2 minutes with loading buffer. 10 μg of conjugated TSH, and volumes of labelled TSH equal to 4, 8, 10 ml, have been used. To do this labelled TSH both in not purified and Sep-pak or PD10 purified form has been employed.

TSH had been labelled with a labelling efficiency equal to 44,1 % according to the conditions of the labelling protocol set forth in table 3. 20 μg of every markers of the table 4 have been used. Table 4

A potential difference of 200 V has been applied to the electrodes and a current of 25 mA for 90 min has been used. Purification and quality controls of TSH

The non-conjugated, native protein, conjugated HYNIC- TSH and labelled TSH have been analyzed with HPLC, size exclusion chromatography (SEC), using the TSK-GEL G 3000 SWXL column and PBS as eluent. Reverse phase chromatography has been performed on C8 column (Inertsil, GL Science Inc.) using a two solvent gradient: - A, composition: H₂0, CH₃CN (ACN) 5%, CF₃COOH

(TFA) 0,1%;

- B, composition : CH₃CN, CF₃COOH 0,1%; and using the elution schedule set forth in table 5.

Table 5

HPLC chromatograph was equipped with an UV spectrophotometer and a gamma radiation detector (photomultiplier tube).

The radiochemical purity has been evaluated by flash thin- layer chromatography (ITLC) with - ITLC-SG plates (Gelman Laboratories) and with 80% methanol as mobile phase. For the in vivo experiments the purification has been performed on Sephadex G 25 PD10 column (Amersham Pharmacia Biotech) eluting with PBS.

In vitro stability of TSH labelled with ¹²⁵l has been tested by incubation of 125 μl of the reaction solution in 1 ml of plasma and saline. Several ITLCs have been performed in consecutive times, after 1 , 3, 4 h, respectively, in order to detect the possible release of free iodine.

Results The purpose of experiments has been to provide a labelling protocol for TSH with a satisfactory labelling efficiency, and therefore the conjugation and labelling reactions of TSH have been optimized considering every parameter that can affect the yield of the conjugation and labelling reactions. Increasing the initial concentrations of native and conjugated TSH, an increase of the labelling efficiency has been noticed and the best results have been achieved concentrating the conjugated at 0,13 mg/ml. It has been found that the labelling of the tricine is carried out in more efficient manner increasing the value of the labelling pH. Tricine, at concentration of 27,98 mg/ml, has been labelled at values of pH 7, 8 and 9, obtained by adding 1M NaOH, at concentration of SnCI₂-2H₂0 of 0,15 mg/ml. The profile of the plot set forth in figure 3 allows to detect such correspondence.

It has been shown that the TSH is stable at values of pH 5 and 7 and it is unstable at pH 9. Although tricine has been labelled with a very elevated efficiency just at the value of pH 9, as shown in figure 3, the value of pH 7 has been chosen for the labelling, as compromise.

The labelling efficiency of tricine also results to increase versus the increase of its own concentration (figure 4), until a maximum value, for a concentration equal to 205,9 mg/ml. A further increase of the concentration does not result in better results. Furthermore it has been shown that the increase of the concentration of SnCI₂-2H₂0 provokes a decrease of the labelling efficiency of the tricine. A concentration equal to 0,08 mg/ml of SnCI₂-2H₂0 has provided the highest value of labelling efficiency, but also the highest concentration of colloids. Therefore it has been selected as optimal a concentration of SnCI₂-2H₂0 equal to 0,15 mg/ml.

The labelled tricine does not release significantly free ^99mTc within 2 hours from the labelling carried out both at pH 5 and 7, showing a good stability. The achieved results are represented by the plots set forth in figure 6 and 7, respectively.

The labelling efficiency of TSH has shown an increasing linear profile with incubation time increases (figure 8) and the maximum value has been observed in correspondence of the maximum tested incubation time, 40 minutes. A further increase has been achieved by increasing the radioactivity concentration of ^99mTc0₄ ^~ up to 50,9 mCi/ml (figure 9), while it has been observed that radioactivity concentrations higher than 50 mCi/ml result in lowering the labelling efficiency, probably because of the saturation of the coordination sites of Tc in the HYNIC molecule.

Following monitoring the influence of all parameters, the final protocol set forth in tables 2 and 3 previously shown has been formulated, concerning respectively the preparation conditions of the conjugate HYNIC-TSH and labelling conditions of HYNIC-TSH with ^99mTc.

By using this protocol as values of labelling efficiency have been achieved 91 ,04% for tricine and 55,66% for TSH, respectively.

SDS-PAGE analysis has detected the presence of a small fraction (<10%) TSH as dimer

The retention time of non-conjugated TSH has been respectively equal to 11 minutes in exclusion chromatography and equal to 21 minutes in reverse phase chromatography.

In table 6 are reported the results of stability studies of TSH carried out at different pH values. As already reported, TSH is stable at pH 5 and 7; while at pH 3, 8 and 9 has been found the presence of aggregate demonstrating the instability of the molecule in solution.

Table 6

Then a protocol for the production of a stable radiomedicament and labelled with a good specific activity has been developed, showing that ¹²³1-TSH and ^99mTc-HYNIC-TSH products constitute a valid solution for the diagnosis of the DTCs and respective metastases, allowing to detect the tumoural cells by binding to the TSH receptors expressed from such cells and following detection of such tracers by scintigraphic examination. It is apparent that analogous optimized solutions can be developed by those skilled in the art in order to delivery in optimal manner beta- emitting radioisotopes, with therapeutic functions, to the DTC cells.

Claims

1. Use of the human recombinant thyroid stimulating hormone (TSH) labelled with a radioactive isotope suitable for the administration in vivo, for the preparation of a radiomedicament for diagnosis and therapy of differentiated thyroid tumors and relevant metastases.

2. Use of the thyroid stimulating hormone (TSH) according to the claim 1 , wherein the radiomedicament is for the diagnosis of differentiated thyroid tumors and relevant metastases and the radioactive isotope is substantially a γ-emitting or positron (β⁺) emitting radioisotope.

3. Use according to the claim 2, wherein the substantially γ-emitting radioisotope is chosen from the group consisting of ^99mTc, ¹²³l, ¹²⁵l, ¹¹¹ln.

4. Use according to the claim 2, wherein the substantially positron (β⁺) emitting radioisotope is chosen from the group consisting of ⁶²Cu, ¹¹C and ¹⁸F.

5. Use of the thyroid stimulating hormone (TSH) according to the claim 1, wherein the radiomedicament is for the therapy of differentiated thyroid tumors and relevant metastases and the radioactive isotope is substantially a β^" emitting radioisotope.

6. Use according to the claim 5, wherein the substantially β^'emitting radioisotope is chosen from the group consisting of ¹³¹l, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ¹⁶⁶Ho, ³²P, ⁷⁶As, ⁸⁶Rb and ⁸⁹Sr.

7. Human recombinant thyroid stimulating hormone (TSH) labelled with ^99mTc as tracer for the scintigraphic diagnosis of differentiated thyroid tumors and related metastases.

8. Radiolabelled thyroid stimulating hormone (TSH) according to the claim 7, wherein the TSH is conjugated to the bifunctional chelating agent 6-idrazinonicotinamide (HYNIC).

9. Radiolabelled thyroid stimulating hormone (TSH) according to the claim 8, wherein tricine and/or EDDA as coligands are also present.

10. Radiopharmaceutical composition for the diagnosis and therapy of differentiated thyroid tumors and related metastases comprising as active ingredient the human recombinant thyroid stimulating hormone (TSH) labelled with a radioactive isotope suitable for the administration in vivo, along with one or more adjuvants and/or pharmacologically acceptable excipients.

11. Radiopharmaceutical composition according to the claim 10 for the diagnosis of differentiated thyroid tumors and related metastases wherein the radioactive isotope is a γ-emitting or positron emitting radioisotope.

12. Radiopharmaceutical composition according to the claim 10 for the therapy of differentiated thyroid tumors and related metastases wherein the radioactive isotope is a β^"-βmitting radioisotope.

13. Radiopharmaceutical kit for the diagnosis or therapy of differentiated thyroid tumors and related metastases comprising the following components: a) a reducing agent, a bifunctional chelating agent conjugated to recombinant human TSH, a coligand, an antioxidant agent; and b) a radioactive isotope suitable for the administration in vivo.