WO2007060012A2

WO2007060012A2 - Use of l-phenylalanine conjugated to an emitting isotope for therapy of hormone dependent carcinoma

Info

Publication number: WO2007060012A2
Application number: PCT/EP2006/011368
Authority: WO
Inventors: Samuel Samnick
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-11-25
Filing date: 2006-11-27
Publication date: 2007-05-31
Anticipated expiration: 2008-05-25
Also published as: WO2007060012A3

Abstract

The present invention provides a use of a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine-211 for the preparation of a pharmaceutical composition for the treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma. Furthermore, the invention provides an according method for the treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma and a method for the monitoring of the progress of such treatment.

Description

Therapy of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma

The present invention provides a use of a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine- 211 for the preparation of a pharmaceutical composition for the treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma. Furthermore, the invention provides a method for the treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma and a method for the monitoring of the progress of such treatment.

A variety of documents is cited throughout this specification. The disclosure content of said documents including manufacturer's manuals is herewith incorporated by reference in its entirety.

Breast and prostate cancer, which are hormone dependent carcinomas, are the second most common cause of cancer-related death, for women and men in the western world [1-4]. According to [5], the highest cancer incidence and death rates for each racial and ethnic population in the USA continued to be for cancers of the prostate, and lung, and colon among men and for cancers of the breast, lung and colon among women. Moreover, more than 30% of newly diagnosed cancers are breast and prostate cancer, making them a leading cause of death from cancer worldwide [2, 3].

The more current treatments used for prostate cancer (PC) are the androgen- deprivation therapies, radiotherapy and chemotherapy [6 - 8]. However, although these therapies are effective for patients with localized and androgen-responsive PCs, they have demonstrated limited curative effect against advanced metastatic and androgen-resistant states of PCs, and no demonstrable survival benefit has been demonstrated in randomized studies [8, 9]. Various new treatment modalities are currently being developed, including second-line hormonal therapy, immunotherapy, radical prostectomy, gene therapy, dendritic cell vaccination therapy, antisense therapy, growth factor inhibition, vitamin D, and biphosphonates. Unfortunately, in spite of all these efforts the prognosis of metastatic, hormone-refractory prostate cancer remains poor, and no significant improvement in median survival has been demonstrated [9].

For early-stage of breast cancer, two treatment regimens have been used: 1) breast- conserving surgery and radiation, and 2) adjuvant chemotherapy and hormonal therapy [5, 10, 11]. The first treatment regimen has shown to be an appropriate primary therapy for the majority of women with stage I and Il breast cancer, whereas the second one has been used in women with early-stage node-positive disease [10, 11]. Despite advances in screening for breast cancer, improved locoregional treatments, and adjuvant systemic therapy, the management of metastatic breast cancer remains in analogy to metastatic androgen-resistant prostate cancers a major clinical challenge. The main goals of treatment are palliation of symptoms, disease control, and, when possible, prolongation of life [12]. Cytotoxic chemotherapy using anthracyclines or melphalan as well as newer drugs such like paclitaxel or docetaxel, has been shown to be of clinical benefit [13]. However, pilot studies using these chemotherapy regimens to treat advanced stage cancers revealed high rates of congestive cardiac failure [18 % to 20 %], and toxic effects on bone marrow and other organs, limiting clinical application [14]. In summary, none of the treatments currently used has shown a significant survival benefit in patients with metastatic, hormone- refractory prostate cancer or metastatic breast cancer as yet. The median survival time for these patients remains in the range of 1 to 3 years [9, 12]. Accordingly, there is still a lack for a therapy approach which is capable to improve the outcome of patients with hormone-refractory and/or metastatic disease. A cytostatically active drug, with a favorable tolerability profile, overcoming cellular detoxification strategies, and ideally conveying additional anti-tumor activity, therefore, would be desirable as an alternative for maintenance or induction therapy in tumor, to improve tolerability and efficacy of existing chemo- and radiotherapy regimens.

Thus, the technical problem underlying the present invention is to provide means and methods for an improved treatment of hormone dependent carcinoma and hormone- refractory and/or metastatic disease. The solution to this technical problem is achieved by the embodiments characterized in the claims.

Accordingly, the present invention relates to the use of a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine-211 for the preparation of a pharmaceutical composition for the treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma, wherein the hormone dependent carcinoma is selected from the group consisting of prostate carcinoma and mamma carcinoma.

The term "hormone dependent carcinoma" describes in the context of the present invention any human cancer which is histologically derived from tissues of the primary or secondary male or female sexual organs, and the functional regulation of which is controlled primarily by sexual hormones of the steroid class, typically androgens (e.g. testosterone) or gestagens (e.g. progesterone) or estrogens (e.g .estradiol). Typically, the development of the primary tumor depends on hormone availability. Tumors can initially be controlled by anti-hormon agents (e.g. tamoxifen, an anti-estrogen). However, most tumors later progress to more malignant hormone-independent and/or metastatic forms. These forms may be resistant to many current therapies. The term "metastasized carcinoma derived from hormone dependent carcinoma" defines according to the invention such malignant hormone-independent and/or metastatic forms. The carcinoma to be treated by the use or method of the invention are selected from the group consisting of prostate carcinoma and mamma carcinoma. The term "prostate carcinoma" describes a type of hormone dependent cancer of the human prostate gland which is initially characterized by androgen-dependence, and which does typically progress to an androgen-resistant state. Such a carcinoma may demonstrate resistance to many chemotherapeutic agents that are diverse both in structure and mechanism of action.

The term "mamma carcinoma" is understood in the context of the invention as a synonym for "breast cancer". The term describes a further type of hormone dependent cancer of the human mammary glands which is also characterized by progress to therapy resistant metastatic state.

An indication to treat a subject according to the invention can be derived by the diagnosis of minimal residual disease, preferably early solid tumor, advanced solid tumor or metastatic solid tumor, which is characterized by the local and non-local recurrence of the tumor caused by the survival of single cells. According to the invention, it is envisaged that a L-phenylalanine is conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine- 211. Exemplary for the compounds of the invention and in particular as a proof of principle for IPA-131 , orphan status was granted by the EMEA on April 11 , 2006 under the EU designation number EU/3/06/363.

The term "alpha-, beta- or Auger-electron emitting isotope" defines in the context of the present invention radioactive istopes, characterised by the emission of different particles (rays) formed during radioactive decay or by nuclear transition processes. An alpha emitting isotope is defined as a radioactive nuclide emitting alpha particles, corresponding to a helium nucleus consisting of two protons and two neutrons. A beta emitting isotope is defined as a nuclide emitting fast nuclear electrons (negatrons) formed during radioactive decay. An Auger-electron emitting isotope is defined as a nuclide emitting low energy nuclear electrons, formed by nuclear electron capture or internal transition processes. The maximum path lengths of these particles are in a range from 10 nm to 12 mm.

L-phenylalanine derivatives in the forms applicable in accordance with the present invention are presented in by general formula I:

General formula I in which,

X is bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 or astatine-211 linked to L-phenylalanine at the 3- (meta-) or 4- (para-) position within the aromatic ring.

Ri is H, alkyl group, amino acid, peptide, protein or an other residue known to facilitate or improve tumor targeting. R₂ is OH, amino acid, or an other residue known to facilitate or improve tumor targeting. Preferred conjugates according to the formula I are those in which X is a bromine-77, bromine-82, iodine-125, iodine-131 , or astatine-211 linked to L-phenylalanine at the para- position of the aryl group, while Ri is H and R₂ is OH.

According to the physical half life of the radionuclide conjugated to the L- phenylalanine, also the conjugates have a corresponding half life of 16.2 h for bromine-76, 57.04 h for bromine-77, 35.3 h for bromine-82, 4.17 d for iodine-124, 59.41 d for iodine-125, 8.02 d for iodine-131 or 7.21 h astatine-211 labelled L- phenylalanine, respectively. As described in more detail in the appended examples, the halogen isotope may e.g. be conjugated following a protocol for a "non-carrier- added" (n.c.a.) conjugation as well as following a protocol for a "carrier-added" (c.a.) conjugation; see e.g. appended example 1.

Due to the presence of a radionuclide, which is an alpha-, beta- or Auger-electron emitting isotope, in the described conjugate the effective compound is an endoradiotherapeutic agent. The term "endoradiotherapeutic agent" defines in the context of the present invention an agent which comprises at least one type of radioactive isotopes. Such agent is to be administered to a subject in the need thereof and is effective in the therapy of the above described carcinoma with the ability to metastasize due to an endogenic irradiation, i.e. an irradiation with the radioactive compound within the body of the subject to be treated by the endoradiotherapy. The L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope may be administered to the subject in the need thereof via a parenteral, transdermal, intraluminal, intra-arterial, intrathecal or intravenous route or by direct injection into malignant tissue. Also within the scope of the invention is an administration wherein the L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope is bound to a matrix of a medical device. The conjugate may be bound, for example, to a suitable matrix via an amino binding between the amino group of the phenylalanine and a matrix coated with polypeptides. The medical device may be e.g. a wound plating such as a gauze, a metal plating or different, preferably biological, carrier material.

It is preferred that the effective compound is formulated in form of a pharmaceutical composition to the subject in the need thereof. In accordance with this invention, the term "pharmaceutical composition" relates to a composition for administration to a subject, preferably a human patient. As generally noted above the pharmaceutical composition is preferably administered parenterally, transdermal^, intraluminally, intra-arterially, intrathecally or intravenously. Also preferred is a direct injection of the pharmaceutical composition into malignant tissue. It is in particular envisaged that said pharmaceutical composition is administered to a patient via infusion or injection. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, subcutaneous, intraperitoneal, intramuscular, topical or intradermal administration. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Preferred dosages for the administration of the L- phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope are described herein below. The compositions may be administered locally or systematically. Administration will generally be parenteral, e.g., intravenous. In an preferred embodiment, the pharmaceutical composition is administered subcutaneously and in an even more preferred embodiment intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition might comprise proteinaceous carriers, like, e.g., serum albumine or immunoglobuline, preferably of human origin. It is envisaged that the pharmaceutical composition might comprise, in addition to L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope, further biologically active agents, depending on the intended use of the pharmaceutical composition in a treatment comprising the administration of additional agents for a concomitant therapy. Examples for such further biologically active agents are described herein below in the context of uses and methods comprising a concomitant therapy.

Generally, the L-phenyalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope is to be administered in doses of 10^"5 to 10^~18 g / kg body weight. More preferably, the L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope is administered in doses of 10^"7 to 10^~15 g / kg body weight and more preferably, in doses of 10^"8 to 10^"10 g / kg body weight. It is preferred that such a dose is formulated contained in 1 to 10, preferably 2 to 5 ml of sterile solution, such as phosphate buffered saline solutions, water for injection, etc.

It has been surprisingly found that the above described conjugates are capable to accumulate specifically and with marked retention in a limited group of hormone dependent carcinoma and hormone-refractory or metastasized carcinomas derived from hormone dependent carcinomas. As demonstrated in the appended examples, the L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope specifically accumulates in prostate carcinoma cells, whereas other carcinoma with the ability to metastasize do not or do not specifically accumulate the conjugate. Examples for carcinomas, respectively for metastasized carcinomas which do not or do not specifically accumulate the conjugate are known in the art and include lung cancer, pancreatic cancer, colorectal cancer, metastases of malignant melanoma, myeloma, lymphoma and metastases of cancers with unknown primary localization.

Due to the specific accumulation of the L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope this conjugate is useful in the treatment of such specific hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma by specifically targeting the cells of this carcinoma and to destroy it by the irradiation activity of the emitting isotope.

The effectiveness and safety of single photon emission tomography (SPET) with 4- [¹²³l]iodo-L-phenylalanine (IPA-123) for brain tumor imaging not only in the experimental C6 glioma model but also in patients with malignant gliomas has been previously demonstrated [15 - 17]. This data demonstrates that this group of compounds is also capable to cross the blood-brain barrier after the intravenous administration. Moreover, and advantageous for the safety of the administration according to the present invention, it was demonstrated that the accumulation in the normal tissue and also in cerebral parenchyma is moderate and decreases rapidly with time. However, due to the complete difference of the origin and the stages of development of hormone dependent carcinoma and malignant gliomas it could not be expected that an L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope might be effective in the treatment of hormone dependent carcinoma. On the one hand development and upgrading of gliomas appear to be etiologically hormone independent [18, 19]. In addition, malignant gliomas lack the ability to form metastases. It is understood that the high accumulation of IPA-123 in gliomas is primarily associated with the increased amino acid transport into glioma cells. This has been shown to be specific for many tumors [20, 21]. On the other hand hormone dependent carcinomas are characterized by a progress to a hormone-independent and therapy resistant metastatic state, which has been associated with the activation of other signaling cascades by growth factors that control the strict balance between the growth rate and apoptosis [7, 22]. Among them, the EGF and transforming growth factor alpha (TGF-α) play a major role during carcinogenesis. In particular, the stimulation of EGFR, insulin-like growth factor-l (IGF-I)₁ protein Kinase A cascades and members of the erbB family is understood to lead to recruitment of proteins that interact and activate androgen or estrogen receptors in the absence of hormones [7, 22, 23]. It is further understood that the expression levels of EGFR and its ligands EGF and TGF-α in prostate carcinoma cells is generally enhanced during the disease progression to more malignant hormone-independent and metastatic PC forms, where it regulates angiogenesis, tumor growth and progression, and metastasis [7, 22]. Because the uptake of IPA-123 was previously known only as moderate in hormone dependent PC cells and in brain metastases of small cell lung cancers and adenocarcinomas [17], the surprising feature of the L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope is the unexpected high accumulation in androgen-independent PC3 and DU 145 PC cells, and in metastatic breast tumor cells. This provides the rationale for an attractive novel tool with a clinical impact on targeting of relapsed carcinomas derived from hormone dependent tissues. The surprisingly high accumulation of the para-iodinated L-phenylalanine in androgen- independent PC3 and DU145 PC cells, and in hormon-independent or metastatic breast tumor cells is understood as associated with the modified transcriptional activity or inactivation of the hormonal receptor pathway in disease, which leads to enhanced erbB transduction and, therefore, to enhanced protein demand in malignant cells or metastases [24, 25]. There is no known correlation between cellular overexpression of EGFR and enhanced amino acid transport in the tumor cell, although overexpression of EGFR, and increased erbB oncogene has been demonstrated in high grade gliomas [19, 26].

Accordingly, the invention teaches that the L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope specifically accumulates in hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma as well as such metastasized carcinoma which migrated into the brain. As described herein above it is preferred that the conjugated L-phenylalanine is to be administered intravenously. The intravenous administration may be effected by administering the conjugate in form of an above described pharmaceutical composition for parenteral administration.

It is also preferred that the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope conjugated to the L-phenylalanine is in the range of 0.1 to 1000 MBq/kg body weight. Generally, but not exclusively, this dose range overlaps with the above indicated dose range calculated in terms of g / kg body weight. More preferably, the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope is in the range of 10 to 400 MBq/kg body weight, more preferably the dose is in the range of 20 to 120 MBq/kg body weight. The administered dose can be determined using an appropriate dose meter, calibrated to quantitatively measure alpha, beta or gamma radiation.

It is also preferred that the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope conjugated to the L-phenylalanine is to be administered as a single dose once or as fractionated doses in 2 to 60 fraction doses. More preferably, the conjugate is administered fractionized in 2 to 10 fraction doses. Dose fractionation is an established procedure in radiation therapy. By fractionating a total administered dose, improved tolerability for healthy non-target tissue, as well as an increased cytotoxic effect to tumor tissue is achieved. Repeated fractionated irradiation allows to therapeutically impact a higher percentage of cells in radiation sensitive stages of the cell cycle, compared to a one time single high dose irradiation. Therapeutic irradiation induces single and double strand breaks of DNA, which is counteracted by nuclear repair mechanisms upregulated following irradiation. It is believed that cells undergoing DNA repair, are more susceptible to a renewed irradiation than radiation- naive cells.

In a further preferred embodiment of the use of the invention the conjugated L- phenylalanine is 4-[¹³¹l]iodo-L-phenylalanine (IPA-131), 4-[¹²⁴l]iodo-L-phenylalanine (IPA-124) and 4-[²¹¹At]astatine-L-phenylalanine (AtPA-211). lodine-131 is widely available, has a favourable half life and can be handled by most institutions licensed to apply open radionuclides, lodine-131 allows for convenient extracorporal therapy monitoring using a gamma camera owing to a gamma ray component, emitted in a fixed ratio relative to the therapeutic beta particle emission, which is itself not detectable extracorporally. Another preferred embodiment of the method of the invention makes use of 4-[¹²⁴l]iodo-L-phenylalanine. lodine-124 has a positron emission component, allowing for PET imaging, in addition to the therapeutic beta- emission. Using quantitative PET imaging, internal dosimetry measurements at an ongoing basis can be conducted for therapy planning and therapy monitoring for a period of up to 15 days following a single injection. Astatine-211 is also preferred, as it emits high energy (6.8 MeV) alpha particles, with a short path length in tissue (65μm), allowing to administer a highly cytotoxic radiation to targeted tissue, while minimising undesirable radiation effects to non-target tissue.

Moreover, it is preferred that the pharmaceutical composition further comprises a chemo therapeutic agent, an immunotherapeutic agent, a gene therapeutic agent, a vaccine, an antisense nucleotide therapeutic agent, an siRNA therapeutic agent, a further endoradiotherapeutic agent and/or a further radiosensitising agent. The administration of a chemo therapeutic agent, an immunotherapeutic agent, a gene therapeutic agent, a vaccine, an antisense nucleotide therapeutic agent, an siRNA therapeutic agent, a further endoradiotherapeutic agent and/or a further radiosensitising agent is understood as a concomitant therapy. Methods and means for concomitant therapies are well known in the art.

The conjugated L-phenylalanine and the additional therapeutic may be formulated as a single pharmaceutical composition for simultaneous administration of the effective compounds or in separate pharmaceutical composition for sequential administration. Accordingly, an administration of a composition comprising the conjugated L- phenylalanine prior to the administration of a composition comprising one or more therapeutics selected from the group of a chemo therapeutic, an immunotherapeutic, a gene therapeutic, a vaccine, an antisense nucleotide therapeutic, an siRNA therapeutic, a further endoradiotherapeutic agent and/or a further radiosensitising agent is envisaged as well as simultaneous or subsequent administration. An example for a chemo therapeutic agent comprises bioactive agents known to be effective in retarding or arresting the malignant growth or to be effective in the regression or elimination of malignant tissues or cells. Such agents might be e.g. drugs acting as cytostatics. Accordingly, a chemotherapy comprises in line with the medical standards in any systemic or local treatment the administration of cytostatic or cytotoxic agents. Chemotherapeutic agents used in oncology include among others, nitroso urea compunds (ACNU [nimustin], BCNU [carmustin], CCNU [lomustin]), temozolomid, procarbacin, metothrexate, cytarabin, gemcitabine, fluorouracil, cyclophosphamide, mitoxantron, anthracyclins, estramustin, or taxanes. The chemotherapeutic agents are intended to be administered in appropriate dosing regimens according to medical practise. In line with the invention nitroso urea compounds, temozolomide, procarbacin, and methotrexate are preferred chemotherpeutic agents.

Examples for an immunotherapeutic agent comprise but are not limited to compounds such as antibodies, antibody fragments and/or derivatives thereof which specifically detect malignant tissue and/or cellular therapeutics, including those consisting of adoptively transferred autologous, heterologous, xenogenous or endogenous cells, which have the ability to eliminate malignant cells or tissues. The term "antibody fragment or derivative thereof relates to single chain antibodies, or fragments thereof, synthetic antibodies, antibody fragments, such as Fab, a F(ab2)', Fv or scFv fragments, single domain antibodies etc., or a chemically modified derivative of any of these. Antibodies to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified outside the motifs using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) (e.g. posttranslational and chemical modifications, such as glycosylation and phosphorylation) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition 1989 and 3rd edition 2001. The specific detection of malignant tissue or cells may be effected via the detection of tumor specific markers by the antibodies, antibody fragments and/or derivatives thereof. A tumor-specific marker is a tumor-associated cell surface antigen which is either found exclusively on tumor cells or is overexpressed on tumor cells as compared to non- malignant cells. Tumor-associated cell surface antigens can be expressed not only on tumor cells but also on cells/tissue which are/is not essential for survival or which can be replenished by stem cells not expressing tumor-associated cell surface antigen. Furthermore, a tumor-associated cell surface antigen can be expressed on malignant cells and non-malignant cells but is better accessible by a therapeutic agent of interest on malignant cells. Examples of over-expressed tumor-associated cell surface antigens are Her2/neu, EGF-Receptor, Her-3 and Her-4. An example of a tumor- associated cell surface antigen which is tumor specific is EGFRV-III. An example of a tumor-associated cell surface antigen which is presented on a cell which is non- essential for survival is PSMA. Examples of tumor-associated cell surface antigens which are presented on cells which are replenished are CD19, CD20 and CD33. An example of a tumor-associated cell surface antigen which is better accessible in a malignant state than in a non-malignant state is EpCAM. Moreover, the definition of "immunotherapeutics" may comprise agents such as T-cell co-stimulatory molecules or cytokines, agents activating B-cells, NK-cells or other cells of the immune system as well as drugs inhibiting immune reactions (e.g. corticosteroids).

The term "gene therapeutic agent" defines in the context of the invention means for a therapy comprising the administration of one or more nucleic acid constructs functionally encoding e.g. one or more antigens which are characteristic for malignant cells. Such antigens comprise tumor specific markers. The sequence encoding such antigen is operably linked to a nucleic acid sequence which is a regulatory sequence. Thus, a gene therapy comprises the functional expression of a heterologous gene in a patient according to standard medical protocols using appropriate vector systems known in the art; see e.g. Haberkorn et al., Curr Med Chem. 2005; 12(7)779-94. The term "regulatory sequence" refers to DNA sequences which are necessary to effect the expression of coding sequences to which they are ligated. Control sequences in the context of the described gene therapy generally include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors. The term "control sequence" is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components. The term "operably linked" refers to a arrangement/configuration wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

The administration of a vaccine aims in the context of the present invention at activating the innate or adaptive immune system of the patient to act against the tumor tissue or the malignant cells. Such therapy comprises e.g. administering one or more antigen preparations containing tumor substances, or cells selected to react against tumor tissue or the malignant cells.

An antisense therapeutic agent is e.g. a nucleotide sequence being complementary to tumor-specific gene sequences, aiming at functionally neutralising tumor gene expression, and consequently inducing tumor cell death.

An siRNA therapeutic agent according to the invention is e.g. a small interfering RNA capable of sequence-specifically silencing the expression and activity of various tumor-specific target genes by triggering cleavage of specific unique sequences in the mRNA transcript of the target gene and disrupting translation of the target mRNA, consequently inducing tumor cell death.

A concomitant therapy which requires the administration of additional bioactive agents which are effective in the treatment of the carcinoma with the ability to metastasize may be accompanied by the administration of additional compounds which minimize potential side effects of said bioactive agents such as drugs acting on the gastro- intestinal system, drugs preventing hyperuricemia, and/or drugs acting on the circulatory system, e.g. on the blood pressure, known in the art. Such additional bioactive agents may be formulated in the form of the same or a separate pharmaceutical composition.

The term "endoradiotherapeutic agent" has been described herein above.

Examples for radiosensitizing agents are 3-iodo-L-phenylalanine or 4-iodo- phenylalanine, which are characterized by a stable, non-radioactive iodine of the [¹²⁷I]- isotope. The term "radiosensitizing effect" describes in the context of the present invention the capacity of a compound to enhance the therapeutic response to concomitantly administered radiation therapy. Such radiation therapy includes external and internal radiation therapies. This is understood as the ability to induce an increased response to a given radiation dose administered in the presence of the radiosensitizing compound, compared to the response induced by the same radiation dose in the absence of the radiosensitizing compound, or, alternatively, to induce selectively the sensitivity of neoplastic cells for a radiotherapy, not present in the absence of the compound.

It is preferred for the use of the invention that the pharmaceutical composition is to be administered to a patient who is subsequently to be irradiated percutaneously (percutaneous radiotherapy). Such percutaneous exoradiotherapy is understood in the context of the invention as a concomitant therapy. Percutaneous radiotherapy is typically administered as an external beam radiation stemming from among others, radioactive cobalt-60 sources, linear accelerators, proton, neutron, or hadron beam sources. Preferably, the irradiation is started in a period of 0 to 7 days subsequent to the administration of the conjugated L- phenylalanine. More preferably, the irradiation is started in a period of 0.5 to 24 hours subsequent to the administration of the conjugated L-phenylalanine.

The concomitant radiotherapy may comprise a cumulative external irradiation of a patient in a dose of 1 to 100 Gy. A preferred range of the irradiation dose is 1 to 60 Gy. It is preferred that the external irradiation dose is administered in 1 to 60 fractional doses, more preferably in 5 to 30 fractional doses. Preferably, the fractionized doses are administered over a period of 1 to 26 weeks, more preferably over a period of 6 to 12 weeks.

In an alternative embodiment, the invention provides a method for the monitoring of the progress of a treatment of carcinoma with the ability to metastasize (in a human subject) selected from the group consisting of prostate carcinoma and mamma carcinoma (breast cancer), the method comprising the step of localizing and/or dosimetrically measuring a L-phenylalanine conjugated to an alpha-, beta- or Auger- electron emitting isotope selected from the group consisting of bromine-76, bromine- 77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine-211 in the subject by using a γ-camera subsequent to the administration of a pharmaceutical composition comprising said conjugate to a subject in the need thereof. The detection of an interception of tumor progression, a decrease of the tumor size or an elimination of a tumor is indicative of a therapeutic success.

An example for the localization and/or dosimetric measurement of conjugated L- phenylalanine in the subject by using a γ-camera is exemplified e.g. in [16, 17]. It is preferred that the conjugated L-phenylalanine is localized and/or dosimetrically measured at least 0 to 7 days, preferably 0.5 to 48 h subsequent to the administration of the pharmaceutical composition.

In a further alternative embodiment, the invention provides a method for the treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma, wherein the hormone dependent carcinoma is selected from the group consisting of prostate carcinoma and mamma carcinoma, the method comprising the steps of administering a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine-211 to a subject in the need thereof. It is preferred that the effective compound, which is the conjugated L-phenylalanine, is formulated in form of a pharmaceutical composition. The term "pharmaceutical composition" has been defined herein above. The route of administration of the effective compound depends inter alia from its formulation. Different routes for differentially formulated compositions have been described herein above. It is preferred for the method of the invention that the conjugated L-phenylalanine is administered intravenously.

It is further preferred that the conjugated L-phenylalanine labeled with alpha-, beta- or Auger-electron emitting isotope is administered to the subject in a doses of 10^"5 to 10^" ¹⁸ g / kg body weight. More preferably, the L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope is administered in doses of 10^"7 to 10^"15 g / kg body weight of the subject and more preferably in doses of 10^"8 to 10^"10 g / kg body weight of the subject. It is preferred that the radioactive dose of the conjugated L- phenylalanine labeled with alpha-, beta- or Auger-electron emitting isotope is in the range of 0.1 to 1000 MBq/kg body weight. Generally, but not exclusively, this dose range overlaps with the above indicated dose range calculated in terms of g / kg body weight. More preferably, the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope is in the range of 10 to 400 MBq/kg body weight and more preferably in the range of 20 to 120MBq/kg body weight. As described herein above, it is also preferred that such dose is formulated contained in 1 to 10, preferably 2 to 5 ml of sterile solution, such as phosphate buffered saline solutions, water for injection, etc.

Also preferred for the method for the treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma of the invention is the administration of the conjugated L-phenylalanine to a subject in the need thereof, wherein the conjugated L-phenylalanine is 4 -[¹³¹l]iodo-L- phenylalanine (IPA-131), 4-[¹²⁴l]iodo-L-phenylalanine (IPA-124) or 4-[²¹¹At]astatine-L- phenylalanine (AtPA-211).

In an also preferred embodiment of the invention it is envisaged that the method further comprises the step of a treatment of the subject by a concomitant therapy. Said concomitant therapy may be selected from the group consisting of a surgical therapy, a chemotherapy, an endo- or exoradiotherapy, an immunotherapy, a gene therapy, a vaccine therapy, an antisense nucleotide therapy, an siRNA therapy, an intracavitary therapy, a radiosensitizer therapy, or a device-based treatment. The step of the concomitant therapy may be effected prior, simultaneous or subsequent to the step of administering a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope.

Definitions for a chemotherapy, an endo- or exoradiotherapy, an immunotherapy, a gene therapy, a vaccine therapy, an antisense nucleotide therapy, an siRNA therapy and radiosensitizer therapy have been provided herein above. Methods and means for concomitant therapies are well known in the art. An example for a surgical therapy comprises a resection of a solid tumour or of malignant tissue. A further concomitant therapy in line with the invention comprises the surgical implantation of a radioactive device such as a radioactive seed. Such seed may be implanted locally to the tumor site. The technique of implanting radioactive devices is known in the art and described herein above in the discussion of the state of the art.

In a preferred embodiment of the method of the invention the concomitant therapy is an exoradiotherapy (also called external beam radiation therapy) and wherein the irradiation is a percutaneous radiotherapy. Preferably, the irradiation is started in a period of 0 to 7 days subsequent to the administration of the conjugated L- phenylalanine. More preferably, the irradiation is started in a period of 0.5 to 24 hours subsequent to the administration of the conjugated L-phenylalanine.

The above described concomitant exoradiotherapy may comprise a cumulative external irradiation of a patient in a dose of 1 to 100 Gy. A preferred range of the irradiation dose is 1 to 60 Gy. It is preferred that the external irradiation dose is administered in 1 to 60 fractional doses, more preferably in 5 to 30 fractional doses. Preferably, the fractionized doses are administered over a period of 1 to 26 weeks, more preferably over a period of 6 to 12 weeks. As described herein above, external field radiation therapy is typically administered as an external beam radiation stemming from among others, radioactive cobalt-60 sources, linear accelerators, proton, neutron, or hadron beam sources. In accordance with the present invention, the term 'fractional dose' is to be understood to mean that the overall activity of the fractional dose adds up or essentially adds up to the cumulative external irradiation otherwise also achievable by administering one single dose.

It is also preferred that the subject to be treated by all embodiments of the method of the invention is a human subject.

In an alternative embodiment the invention relates to a method for the monitoring of the progress of a treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma (in a human subject), wherein the hormone dependent carcinoma is selected from the group consisting of prostate carcinoma and mamma carcinoma, the method comprising the steps: (a) administering a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine-211 to a subject in the need thereof; and (b) localizing and/or dosimethcally measuring the conjugated L-phenylalanine in the subject by using a γ-camera.

The detection of an interception of tumor progression, a decrease of the tumor size or an elimination of a tumor is indicative of a therapeutic success.

A method for the administration of a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope is described herein above. As also described herein above, an example for the localization and/or dosimetric measurement of conjugated L-phenylalanine in the subject by using a γ-camera is exemplified e.g. in [16, 17]. The method for the monitoring of the progress of a treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma may also comprise the step of a treatment of the subject by an above described concomitant therapy. As described herein above, the step of the concomitant therapy may be effected prior, simultaneous or subsequent to the step of administering a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope.

Preferably, the conjugated L-phenylalanine is localized and/or dosimetrically measured at least 0 to 7 days, more preferably 0.5 to 48 h subsequent to its administration.

The figures show:

Figure 1 :

Induction of primary necrosis and apoptosis by IPA-131 vs. External irradiation (15 Gy) in a EGFR-positive human model cell line Figure 2:

Effect of IPA-131 and external irradiation on survival of EGFR-positive cells (human model cell line in vitro) Figure 3:

Cellular uptake kinetics of IPA (IPA-123) in hormone independent human prostate cancer cell lines in vitro

The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of scope of the present invention.

Example 1 : 4-Bromo-L-phenylalanine (4-BrPA), 3-bromo-L-phenylalanine (3-BrPA), 4-iodo-L- phenylalanine (4-IPA), 4-ter.butyltinn-L-phenylalanine (4-TBSnPA), 3-ter.butyltinn-L- phenylalanine (3-TBSnPA), 4-methylsilyl-L-phenylalanine (4-Me₃SiPA) and 3- methylsilyl-L-phenylalanine (3-Me₃SiPA) used as starting materials (precursor) for radiolabeling were either purchased commercially or prior synthesized in analogy to the literature. Unless stated otherwise, all other chemicals and solvents were of analytical grade and obtained commercially or via our local hospital pharmacy. Sodium [¹²⁴l]iodide, sodium [¹²⁵l]iodide, sodium [¹³¹l]iodide, sodium [⁷⁷Br]bromide, sodium [⁸²Br]bromide, and sodium [²¹¹At]astate for radiolabeling was obtained in the highest obtainable radiochemical purity, generally in 0.01 N NaOH or in phosphate buffered saline (PBS) from different suppliers. HPLC purification was performed on a Hewlett Packard HPLC system consisting of a binary gradient pump (HP 1100), a Valco 6-port valve with 2500 μl loop, a variable wavelength detector (HP 1100) with a UV detection at 254 nm and a sodium iodide scintillation detector (Berthold, Wildbad, Germany), using reversed-phased column (250 x 4 mm, Nucleosil-100). The column was eluted at different flow rates in with water/ethanol/acetic acid (89:10:1 ; v/v) or PBS / ethanol (90:10; v/v).

The proposed radiolabeled phenylalanines were obtained either by non-isotopic halogen exchange (carrier-added/c.a.) or by radio-demetalation of the corresponding precursor as described in the general scheme 1 , resulting to no-carrier-added (n. c. a) products after HPLC separation.

R = 124/125/131| _or 77/82g_r

X = m- , p-Br or m-, p-l for 77/82Br n.c.a. (m, p)-IPA-124, -IPA-125, -IPA-131 or n.c.a. (m, p)-BrPA-77 and -BrPA-82

X = t-Bu₃Sn or R = (124/125/131)^ 77/82_Br

(CH₃)₃Si Or ^{21 1}At n.c.a. (m, p)-BrPA-77, -BrPA-82, -IPA-124, -IPA-125, -IPA-131 and n.c.a. (m, p)-AtPA-211

Scheme 1 : scheme of the radiosyntheses of n.c.a. IPA-124, IPA-125, IPA-131 , BrPA- 77, BrPA-82 and AtPA-211

Example 2:

phenylalanine (m,p-IPA-125) and 3,4-r¹³¹lliodo-L-phenylalanine (m,p-IPA-131) by non- isotopic radioiodo-debromination A solution of carrier free sodium [¹²⁴l]iodide, sodium [¹²⁵l]iodide or sodium [¹³¹l]iodide (up to 5 GBq) and 5 μl aqueous Na₂S₂O₅ (4.0 mg Na₂S₂O₅ZmI) was evaporated to dryness by passing a stream of nitrogen through a reaction vessel at 100⁰C, followed by addition of 200 μl of the corresponding L-bromophenylalanine (0.25 - 0.5 mg/ ml 0.1 N H₃PO₄), 20 μl aqueous L-ascorbic acid (10 mg/ml) and 20 μl aqueous Cu(II) sulphate (0.10 mol/l). The reaction vessel was heated for 30 min at 170⁰C, cooled and the mixture diluted with up to 500 μl water. The radioiodinated product was separated from unreacted starting materials and radioactive impurities by HPLC. Generally, 3/4- IPA-124, 3/4-IPA-125 and m/p-IPA-131 were obtained in 88 ± 10% radiochemical yield, with a specific activity > 500 GBq / μmol. The fraction containing the radioiodinated products was collected into a sterile tube, buffered with 0.5 M phosphate buffered saline (pH 7.0; Braun, Melsungen, Germany), and sterile filtered through a 0.22 μm sterile membrane (Millex GS, Millipore, Molsheim, France) to an isotonic and injectable radiopharmaceutical for in vitro and in vivo investigations.

Example 3:

Synthesis of 3,4-r¹²⁴niodo-L-phenylalanine (m, p-IPA-124), 3,4-r¹²⁵πiodo-L- phenylalanine (m,p-IPA-125). 3.4-r¹³¹πiodo-L-phenylalanine (m.p-IPA-13P). and 3.4- f²¹¹llastatine-L-phenylalanine (m, p-AtPA-211) by iodo-demetalation Alternatively (m, p)-IPA-124, (m, p)-IPA-125, (m, p)-IPA-131 , and (m, p)-AtPA-211 were prepared by iodo-demetalation in acidic condition in the presence of chloramine- T for in situ oxidation of the corresponding radioiodide and astatine-211 , using the protected tributyltin- or trialkylsilyl-L-phenylalanine as starting material. In details: Sodium [^124/125/131l]iodide (up to 2 GBq in 50 μl PBS) was added to a mixture consisting of protected (m,p)-tert. tributyltin- or trialkylsilyl-L-phenylalanine (50 - 80 μg) in 50 μl of methanol and 10 μl of 1 N HCI in a 1 ml-vial, followed by 5 μl aqueous chloramine-T (CAT) from a solution of 1 mg CAT / ml water, while shaking. After a 2 min reaction time at room temperature, 20 μl aqueous Na₂S₂O₅ (4.0 mg Na₂S₂O₅ZmI) was added to the reaction vessel to bind the unreacted free radioisotop, followed by deprotection of the amino and carbonyl groups under acidic condition at 100 ⁰C. (m, p)-IPA-124, (m, p)-IPA-125, (m, p)-IPA-131 , and (m, p)-AtPA-211 were isolated by HPLC and formulated as described above for in vitro and in vivo studies. The radiochemical yields were > 90 %, with a specific activity > 1000 GBq / μmol.

Example 4: Synthesis of 3.4-r⁸²Br1bromo-L-phenylalanine (m. p-BrPA-82) and 3.4-f⁷⁷Br1bromo-L- phenylalanine (m,p-BrPA-77)

(m, p)-BrPA-82 and (m, p)-BrPA-77 were prepared either by non-isotopic [⁷⁷ ' ⁸²Br]bromo-deiodination at 160 ⁰C or by [⁷⁷ ' ⁸²Br]bromo-demetalation, using (m,p)- iodo-L-phenylalanine or the corresponding (m,p)-tributyltin- or (m, p)-trialkylsilyl-L- phenylalanine, as starting materials in analogy to the procedure described above, (m, p)-BrPA-82 and (m, p)-BrPA-77 were isolated by means of HPLC as no carrier-added products with a specific activity > 500 GBq / μmol. The fractions containing the radiobrominated products were buffered with phosphate buffered saline (PBS), and sterile filtered through a 0.22 μm sterile membrane to an isotonic and injectable radiopharmaceuticals for in vitro and in vivo investigations.

Example 5: Cell lines and cell cultures

Two human prostate cancer cell lines, two human breast cancer cell lines, and two melanoma cell line were investigated. The human prostate cancer cells PC3 and DU145 (American Type Culture Collection, Rockville, MD), the human breast SK-BR-3 and BT-474 cancer cell lines, as well as the melanoma cell lines SK-MEL25 and A101 D were purchased commercially or provided by the oncological research laboratory of the University Medical Center of Saarland (Homburg, Germany). Cells were cultivated in RPMI-1640 medium or in Dulbecco's modified Eagle medium (sodium pyruvate-free, supplemented with L-glucose and pyridoxine), respectively, supplemented with 10 % (v/v) heat-inactivated foetal calf serum (FCS), penicillin (50 U/ml), streptomycin (50 μg/ml), and insulin (50 μg/ml; PromoCell, Heidelberg, Germany). All cells lines were maintained in appropriate flasks in a humidified incubator (5% CO₂) at 37°C. Before the experiment, subconfluent cell cultures were trypsinized with a solution of 0.05% trypsin containing 0.02% EDTA but without Ca²⁺ and Mg²⁺, and resuspended in fresh medium to various cell concentrations after counting by vital staining on a hemocytometer, depending upon the study. Cells were free of mycoplasms. Viability of the cells was > 95 %.

Example 6:

Example of internalisation experiments Uptake experiments were undertaken to evaluate the affinity of the proposed L- phenylalanine derivatives for the proposed human tumors, and to assess their therapeutic activity in vitro.

All experiments were performed fourfold, simultaneously with 250000, 500000 and 10⁶ freshly prepared human tumor cells, including human malignant glioma cells, pancreatic prostatic and breast cancer cells. Before experiments, subconfluent cells were trypsinized as described above. The suspension was mixed thoroughly, transferred to a 50-ml centrifuge tube (Falcon^®, Becton Dickinson, USA). Cells were centrifuged for 5 min at 200 x g; the resulting supernatant was removed and the pellet resuspended in serum-free Dulbecco^'s Modified Eagle medium and then transferred to Eppendorf tubes at concentrations of 10⁶ cells/ml for the uptake investigations. Before incubation with the corresponding radiolabeled phenylalanine, the tumor cells were preincubated for 5 min in 500 μL medium at 37°C in 1.5-ml Eppendorf centrifuge tubes. Aliquots of 30-50 μL (10⁶ - 1.5 x 10⁶ cpm) freshly prepared radiopharmaceutical were added and cells incubated at 37°C / 5% CO₂ for 1 , 2, 5, 15, 30, 60, 90 and 120 min while shaking. Uptake was stopped with 500 μL ice-cold PBS (pH 7.4) and an additional 3-min in an ice bath, the cells were centrifuged for 2 min at 300 x g, the supernatant removed and the pellet washed three time with ice-cold PBS. Cell pellets were counted for radioactivity together with 3 aliquots of standards on a Berthold LB951 counter. The percentage of binding of the radiopharmaceutical was calculated by the formula: (cpm cell pellet/mean cpm radioactive standards) x 100. The results were expressed either as percent of the applied dose per 10⁶ cells or as cpm/1000 cells for better comparison.

Example 7:

Evaluation of the cell survival rate after treatment with 3/4-bromo-L-phenylalanine and

3/4-iodo-L-phenylalanine

After development of a confluent lawn of cells, the cultures were exposed to 0.1 to 5 μmol/ml of the corresponding pharmaceutical for up to 48 hours at 37°C/5% CO₂. In a parallel experiment, cells were irradiated using a 6-MeV linear accelerator with doses from 2 to 15 Gy or treated with IPA-131 or BrPA-79/81 for comparison of cell survival rate. In order to be able to observe the morphology of the glioma cells, the cells were grown on standard glass slides or in standard culture dishes. Then the medium was removed and the cells were fixed either in 70% ethanol for at least 30 min on ice for flow-cytometric analyses after staining or in 4 % neutral buffered formalin for immunohistopathological analysis. Example 8:

Results and discussion

In vitro studies

The cytostatic effect and radiosensitizing effect of IPA in EGFR positive human tumor cells is demonstrated in figures 1 and 2. As shown, the cytostatic effect of IPA-131 on EGFR positiv tumor cells was more pronounced as compared to external irradiation up to 15 Gy (Fig. 1). Combining IPA-131 with external irradiation led to a dramatic reduction of the cell survival rate (Fig. 2). Flow-cytometric analyses of stained cells show dose dependent induction of primary necrosis and apoptosis, which was more significant than that caused by external irradiation, even with 15 Gy, and more pronounced with increasing IPA-concentration. This result attests the high radiosensitizing effect of IPA-131 on EGFR-positive cells. The surviving cells contained only sparse cytoplasm, the nuclei were shrunken and contained condensed or clumped chromatin. Cytologically, the mode of cell death was apoptosis as the remaining tumour cells contained only sparsely cytoplasm and apoptotic bodies, in other cells the nuclei were shrunken and contained condensed chromatin. Identical results were obtained with BrPA-77 in prostate and breast cells in vitro. This results suggests the high therapeutic potential of 3/4-halogenated L-phenylalanine hormone-independent or metastatic human tumors, especially for hormone- independent prostate and metastatic breast cancer.

The results demonstrate that meta- and para-halogenated-L-phenylalanins represent a new class of therapeutic agents for tumour therapy in these tumor entities.

Reference Example Figure 3 shows an example of uptake kinetic of 4-iodo-L-phenylalanine in primary human androgen-independent tumor cells. The radiolabeled derivative 4-[¹²³l]iodo-L- phenylalanine was used to facilitate quantification, using a gamma counter. As shown, IPA exhibit high uptake in human tumor cells with a continuous increase over the investigation time. This result provides evidence of the high affinity of the proposed radiopharmaceuticals for human tumors, including the human malignant gliomas, pancreatic carcinomas, prostate and breast cancer. References:

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Claims

1. Use of a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine-211 for the preparation of a pharmaceutical composition for the treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma, wherein the hormone dependent carcinoma is selected from the group consisting of prostate carcinoma and mamma carcinoma.

2. The use according to claim 1 , wherein the conjugated L-phenylalanine is to be administered intravenously.

3. The use according to claim 1 or 2, wherein the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope conjugated to the L-phenylalanine is in the range of 0.1 to 1000 MBq/kg body weight.

4. The use according to anyone of claims 1 to 3, wherein the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope conjugated to the L- phenylalanine is to be administered as a single dose once or as a fractionated dose in 2 to 60 fraction doses.

5 The use according to anyone of claims 1 to 4, wherein the conjugated L- phenylalanine is 4-[¹³¹l]iodo-L-phenylalanine (IPA-131), 4-[¹²⁴l]iodo-L- phenylalanine (IPA-124) or 4-[²¹¹At]astatine-L-phenylalanine (AtPA-211).

6. The use according to anyone of claims 1 to 5, wherein the pharmaceutical composition further comprises a chemo therapeutic, an immunotherapeutic, a gene therapeutic, a vaccine, an antisense nucleotide therapeutic, an siRNA therapeutic agent, a further endoradiotherapeutic agent and/or a further radiosensitising agent.

7. The use according to anyone of claims 1 to 6, wherein the pharmaceutical composition is to be administered to a patient who is subsequently to be irradiated percutaneously.

8. A method for the monitoring of the progress of a treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma, wherein the hormone dependent carcinoma is selected from the group consisting of prostate carcinoma and mamma carcinoma, the method comprising the step of localizing and/or dosimetrically measuring a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine- 76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine- 211 in the subject by using a γ-camera subsequent to the administration of a pharmaceutical composition comprising said conjugate.

9. The method according to claim 8, wherein the conjugated L-phenylalanine is localized and/or dosimetrically measured at least 0 to 7 days, preferably 0.5 to 48 h subsequent to the administration of the pharmaceutical composition.

10. A method for the treatment of hormone dependent carcinoma and hormone- refractory or metastasized carcinoma derived from hormone dependent carcinoma, wherein the hormone dependent carcinoma is selected from the group consisting of prostate carcinoma and mamma carcinoma, the method comprising the step of administering a L-phenylalanine conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine-211 to a subject in the need thereof.

11. The method according to claim 10, wherein the conjugated L-phenylalanine is administered intravenously.

12. The method according to claim 10, wherein the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope conjugated to the L-phenylalanine is in the range of 0.1 to 1000 MBq/kg body weight.

13. The method according to claim 10, wherein the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope conjugated to the L-phenylalanine is administered as a single dose once or as a fractionated dose in 2 to 60 fraction doses.

12. The method according to claim 10, wherein the conjugated L-phenylalanine is 4-[¹³¹l]iodo-L-phenylalanine (IPA-131), 4-[¹²⁴l]iodo-L-phenylalanine (IPA-124) or 4-[²¹¹At]astatine-L-phenylalanine (AtPA-211).

13. The method according to claim 10, further comprising the step of a treatment of the subject by a concomitant therapy selected from the group consisting of a surgical therapy, a chemotherapy, an endo- or exoradiotherapy, an immunotherapy, a gene therapy, a vaccine therapy, an antisense nucleotide therapy, an siRNA therapy, an intracavitary therapy, a radiosensitizer therapy or a device-based treatment.

14. The method according to claim 13, wherein the concomitant therapy is an exoradiotherapy wherein the irradiation is started in a period of 0 to 7 days subsequent to the administration of the conjugated L-phenylalanine.

15. The method according to claim 13, wherein the concomitant therapy is an endoradiotherapy comprising the implantation of a radioactive device.

16. The method according to claim 10, wherein the subject is a human subject.

17. A method for the monitoring of the progress of a treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma, wherein the hormone dependent carcinoma is selected from the group consisting of prostate carcinoma and mamma carcinoma, the method comprising the steps:

(a) administering a L-phenylalanine conjugated to an alpha-, beta- or Auger- electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine- 211 to a subject in the need thereof; and

(b) localizing and/or dosimetrically measuring the conjugated L-phenylalanine in the subject by using a γ-camera.

18. The method according to claim 17, wherein the conjugated L-phenylalanine is localized and/or dosimetrically measured at least 0 to 7 days, preferably 0.5 to 48 h subsequent to its administration

14. The method according to claim 10, wherein the conjugated L-phenylalanine is 4-[¹³¹l]iodo-L-phenylalanine (IPA-131), 4-[¹²⁴l]iodo-L-phenylalanine (IPA-124) or 4-[²¹¹At]astatine-L-phenylalanine (AtPA-211).

15. The method according to claim 10, further comprising the step of a treatment of the subject by a concomitant therapy selected from the group consisting of a surgical therapy, a chemotherapy, an endo- or exoradiotherapy, an immunotherapy, a gene therapy, a vaccine therapy, an antisense nucleotide therapy, an siRNA therapy, an intracavitary therapy, a radiosensitizer therapy or a device-based treatment.

16. The method according to claim 15, wherein the concomitant therapy is an exoradiotherapy wherein the irradiation is started in a period of 0 to 7 days subsequent to the administration of the conjugated L-phenylalanine.

17. The method according to claim 15, wherein the concomitant therapy is an endoradiotherapy comprising the implantation of a radioactive device.

18. The method according to claim 10, wherein the subject is a human subject.

19. A method for the monitoring of the progress of a treatment of hormone dependent carcinoma and hormone-refractory or metastasized carcinoma derived from hormone dependent carcinoma, wherein the hormone dependent carcinoma is selected from the group consisting of prostate carcinoma and mamma carcinoma, the method comprising the steps:

(a) administering a L-phenylalanine conjugated to an alpha-, beta- or Auger- electron emitting isotope selected from the group consisting of bromine-76, bromiπe-77, bromine-82, iodine-124, iodine-125, iodine-131 and astatine- 211 to a subject in the need thereof; and

20. The method according to claim 19, wherein the conjugated L-phenylalanine is localized and/or dosimetrically measured at least 0 to 7 days, preferably 0.5 to 48 h subsequent to its administration

RECTIFIED SHEET (RULE 91) ISA/EP