WO2004098629A1 - GnRH CONJUGATES COMPRISING A PHOTOSENSITIZER FOR PHOTODYNAMIC THERAPY - Google Patents

Abstract

The present invention relates to GnRH conjugates comprising a GnRH analog coupled to a photosensitizer and to pharmaceutical compositions comprising same. The conjugates of the invention bind to GnRH receptors with high affinity and exhibit selective phototoxic effect in GnRH-receptor bearing cells, The GnRH conjugates are thus useful for photodynamic therapy of sex hormone-dependent diseases.

Description

GnRH CONJUGATES COMPRISING A PHOTOSENSITIZER FOR PHOTODYNAMIC THERAPY

FIELD OF THE INVENTION

The present invention relates to gonadotropin-releasing hormone (GnRH) conjugates comprising a GnRH analog and a photosensitizer, to pharmaceutical compositions comprising same, and to methods of treating sex hormone related diseases using the GnRH conjugates.

BACKGROUND OF THE INVENTION

A promising process for the treatment of diseases, especially of tumors, is photodynamic therapy (PDT). In PDT, oxygen radicals such as singlet oxygen (^!O₂), superoxides or peroxides are produced by irradiation of previously applied photosensitizers. Due to differences in microcirculation, distribution and exchange intensity, it has been shown that photosensitizer molecules tend to accumulate in neoplastic tissues. As a result of the formation of the oxygen radicals, and as these radicals are highly cytotoxic, destruction or impairment of the tumor tissue occurs. Recently, porphyrins, chlorins, and bacteriochlorins including their analogs and derivatives have found superior utility as photodynamic compounds for use in diagnosis and treatment of certain cancers. However, the main side effect in their use for PDT resides in the cytotoxicity to normal cells, which is manifested by an enhanced light sensitivity of the skin and eyes of the treated subjects.

Targeted chemotherapy was conceived as a route to differentiate between healthy and afflicted cells, minimizing undesirable toxification and affording high drug concentrations at selected loci. This can be achieved through conjugation of drugs to vectors that possess specific binding sites on the cancerous cells. Vectors such as monoclonal antibodies, proteins, peptides or oligonucleotides have been used as carriers for drug targeting. Therapy of a malignant disease may often exploit receptors that are preferentially expressed by tumor cells. Utilization of these receptors to target tumor cells is a potentially attractive approach, as it may offer the possibility of minimizing non-selective toxic effects. For example, conjugation of Chlorin e6 (Ce6), a well-known porphyrin-based photosensitizer, to insulin or to epidermal growth factor (EGF) enhanced dramatically the corresponding photodynamic activity toward cells that express insulin or EGF receptors as compared to free Chlorin e6 (Akhlynina T.V., et al. Cancer Res., 55: 1014-1019, 1995; Gijsens, A. et al., Cancer Res., 60: 2197-2202, 2000). In addition, multiple drug resistance is expected to be less significant in the case of receptor-mediated active transport of a drug across the cell membrane.

In view of the abundance of gonadotropin-releasing hormone (GnRH) receptors on tumor cells, targeted chemotherapy based on GnRH analogs has gained considerable attention (Janaky, T., et al. Proc. Natl. Acad. Sci. USA, 89: 972-976, 1992; Nagy, A., et al. Proc. Natl. Acad. Sci. USA, 93: 7269-7273, 1996). GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH₂) is a key integrator between the nervous and the endocrine systems and plays a pivotal role in the regulation of the reproductive system. The pulsatile secretion of GnRH prompts the anterior pituitary to release the gonadotropin hormones, i.e., luteinizing hormone (LH) and follicle stimulating hormone (FSH), which, in turn, stimulate gonadal steroidogenesis and gametogenesis. GnRH analogs, both agonists and antagonists, have attracted great interest because of their potential application in the treatment of diseases such as prostate and breast cancer (Emons, G., and Schally, A. V. Human Reprod., 9: 1364-1379, 1994; Schally AN. Peptides 20: 1247-1262, 1999). Their mechanism of action is believed to be related, at least partially, to gonadal steroid deprivation. This phenomenon results from the continuous action of the GnRH analogs that leads to down-regulation of the GnRH receptors and to desensitization of the pituitary gonadotropes. However, GnRH analogs have also been shown to exert direct inhibitory effects on cancer cells of prostate, breast and ovary (Halmos, G., et al. J. Urol. 163: 623-629, 2000; Limonta, P., et al. J. Clin. Endocrinol. Metab. 75: 207-212, 1992; Yano, T, et al. Proc. Νatl. Acad. Sci. USA 91: 1701-1705, 1994).

It is well established that conjugation of bulky moieties, such as tetramethylrhodamine, to the ε-amino group of [D-Lys⁶] GnRH does not significantly affect the bioactivity of GnRH analogs, nor their internalization by gonadotropes (Hazum, E., et al. Mol. Cell. Endocinol. 30: 291-301, 1983). Thus, several cytotoxic compounds were attached to agonists or antagonists of GnRH and the resulting conjugates were evaluated for their anti cancer activity. The conjugates exhibited a wide range of specific binding affinities toward GnRH receptors and were shown to be internalized by the cells. The cytotoxicity of some peptide- drug conjugates was markedly augmented, far beyond that of the drug component. Yet, treatment of GnRH-related tumors by these conjugates caused damage to normal pituitary gonadotropes, probably because of their concomitant binding to healthy cells. Although the damage to pituitary cells was reported to be reversible, the use of these conjugates for treatment of malignancies should be carefully considered.

The use of PDT in the treatment of tumors has been established for many years, beginning with the use of hematoporphyrin derivative (HPD, a mixture of porphyrins) and subsequently with a more potent derivative of HPD, Photofrin® porfimer sodium (see, for example, U.S. Pat. No. 4,932,934). A particularly useful group of photosensitizers, designated "green porphyrins," has been also described (see, for example, U.S. Pat. No. 5,171,749). A preferred form of the green porphyrins is "BPD-MA", a benzoporphyrin derivative in the monoacid form. BPD-MA is currently in clinical trials in photodynamic therapy of various tumors and other conditions.

U.S. Pat. No. 5,258,492 discloses GnRH analogs, which possess high agonistic or antagonistic effect and comprise cytotoxic moieties, such as nitrogen mustard, platinum complexes or complexes of metals derived from the non-platinum-group antitumor agents.

U.S. Pat. No. 6,214,969 discloses GnRH analogs, which contain cytotoxic side chains such as moieties with quinone structure, alkylating agents, antitumor antibodies, and antimetabolites. According to U.S. Pat. No. 6,214,969 the GnRH analogs induce cytotoxic effect on different breast cancer cells.

Aiming at synthesizing GnRH analogs suitable for selective PDT of GnRH-receptor bearing cells, the applicants of the present application have conjugated emodic acid to GnRH analogs (Rahimipour, S., et al., J. Med. Chem. 46: 3965-3974, 2003). According to that disclosure free emodic acid is shown to exert phototoxic activity in pituitary cells, however the emodic acid-GnRH conjugate is found to be devoid of phototoxic effect.

Thus, there is an unmet need for potent GnRH agonists and antagonists that can be conjugated to a photosensitizer to yield GnRH conjugates, which exhibit selective and localized phototoxic activity to GnRH-receptor bearing cells upon irradiation of the cells in an afflicted area. SUMMARY OF THE INVENTION

The present invention relates to the synthesis of GnRH agonists or antagonists conjugated to a photosensitizer and to the biological activity of these conjugates. The GnRH conjugates bind specifically to GnRH receptors and exhibit selectivity in their phototoxic effect. They are, therefore, useful in treating sex hormone-dependent diseases.

The present invention provides GnRH conjugates comprising a GnRH agonist or antagonist coupled to a photosensitizer, which retain the binding specificity of the parent peptide to GnRH-receptor bearing cells. The present invention further provides GnRH conjugates comprising a GnRH agonist or antagonist coupled to a photosensitizer, which exert phototoxic activity in GnRH-receptor bearing cells but not in neighboring healthy cells.

According to one aspect, the present invention provides a GnRH conjugate comprising a GnRH analog coupled to a photosensitizer, or a pharmaceutically acceptable salt or hydrate thereof.

According to particular embodiments, the photosensitizer is selected from the group consisting of porphyrin-based materials, bacteriochlorophylls, purpurins, hypericins, pheophorbides, texaphyrins, phthalocyanines, and derivatives thereof. Non-limiting examples of porphyrin-based materials are hematoporphyrins and hydroporphyrins. In currently preferred embodiments, the photosensitizer is selected from protoporphyrin IX, chlorin e6 (Ce6), and l,6-diaminohexanoyl-3,4,8,13-tetramethyl-hypericin (AmHyp).

According to one embodiment, the GnRH analog is a GnRH agonist of the general formula: pGlu- His-Trp-Y-Tyr-X-Leu-Arg-Pro-Z (SEQ ID NO: 1) wherein Y and X are each independently selected from Lys, Dab, Orn, Ser, hSer, D-

Lys, D-Dab, D-Orn, D-Ser, D-hSer, Lys(photosensitizer), Dab(photosensitizer). Orn(photosensitizer), Ser(photosensitizer), hSer(photosensitizer), D-Lys(photosensitizer), D- Dab(photosensitizer), D-Orn(photosensitizer), D-Ser(photosensitizer), D- hSer(photosensitizer), wherein at least one of Y and X is coupled to photosensitizer; Z is selected from Gly-NH2 or ethylamide; or a pharmaceutically acceptable salt or hydrate thereof. According to the principles of the present invention, coupling of a photosensitizer to an amino acid residue is performed through an available functional group of the amino acid residue with an available functional carboxylic group of the photosensitizer. Examples of functional groups of amino acid residues that may be used for coupling of a photosensitizer are the free amino group of Lys, Orn, Dab, or Cit.

According to some embodiments, the GnRH analog is a GnRH agonist having the amino acid sequence: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH₂ (SEQ ID NO:2) and is designated [D-Lys⁶]GnRH.

According to further embodiments, the GnRH analog is a GnRH antagonist having the formula [D-pGlu¹, D-Phe², D-Trp³, D-Lys⁶]GnRH (SEQ ID NO:3) and is designated [D-

Lys⁶]Antg.

According to another embodiment, the GnRH analog is a GnRH antagonist of SEQ ID NO:4 of the general formula:

Ac-D-Nal-D-Cpa-D-Pal-X-Y-Z-Leu-W-Pro-D-Ala-NH2 wherein X is selected from Ser, hSer, Lys, Dab, Orn, NicLys, Ser (photosensitizer), hSer (photosensitizer), Lys(photosensitizer), Dab(photosensitizer), Orn(photosensitizer), or NycLys (photosensitizer); Y is selected from Tyr, NicLys, Lys, D-Lys, Dab, Orn, NicLys(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), Dab(photosensitizer), or Orn(photosensitizer); Z is selected from D-Ser, D-NicLys, D-iPrLys, D-Lys, Ser(photosensitizer), D-Ser(photosensitizer), D-hSer(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), D-NicLys(photosensitizer), Dab(photosensitizer), D-

Dab(photosensitizer), Orn(photosensitizer), D-Orn(photosensitizer), or iPrLys(photosensitizer); W is selected from Arg, iPrLys, Ser(photosensitizer), D- Ser(photosensitizer), hSer(photosensitizer), D-hSer(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), NicLys(photosensitizer), D-NicLys(photosensitizer),

Dab(photosensitizer), Orn(photosensitizer), or iPrLys(photosensitizer), wherein at least one of X, Y, Z, and W is coupled to a photosensitizer; or a pharmaceutically acceptable salt or hydrate thereof.

According to some embodiments, the GnRH antagonist is [Acetyl-D-3-(2'-naphtyl)- Ala, D-4-chlorophenylalanine, D-3-(3'-pyridyi)-Ala, Ser, Tyr, D-Lys, Leu, Arg, Pro, D-Ala- amidejGnRH (SEQ ID NO: 5) designated herein [D-Lys⁶] SB75. According to yet another embodiment, the GnRH analog is a GnRH antagonist of SEQ ID NO:6 of the general formula:

Ac-Nal-D-Cpa-D-Pal-X-Tyr-Y-Leu-Arg-Pro-D-Ala-NH2 wherein X is selected from Ser, Serφhotosensitizer), hSer, hSer(photosensitizer); Y is selected from D-Lys, D-Ser, D-hSer, D-Orn, D-Cit, D-Dab, D-Lys(photosensitizer), D-

Ser(photosensitizer), D-hSer(photosensitizer), D-Orn(photosensitizer), D-

Cit(photosensitizer), D-Dab(photosensitizer), wherein at least one of X and Y is coupled to a photosensitizer; or a pharmaceutically acceptable salt or hydrate thereof.

Ac denotes acetyl; Dab denotes diaminobutyric acid; D-Cit denotes D-Citrulline; D- Cpa denotes 4-chloro-D-Phe; D-Nal denotes D-3-(2'-naphtyl)-Ala; D-Pal denotes D-3-(3'- pyridyl)alanine; hSer denotes homoserine; iPr denotes isopropyl; Nic denotes nicotinyl; Orn denotes ornithine; and pGlu denotes pyroglutamic acid.

It will be appreciated that the present invention encompasses GnRH agonists and antagonists as are known in the art to which a photosensitizer being coupled according to the principles of the invention so long as the GnRH conjugates comprising a GnRH analog and a photosensitizer retain GnRH receptor binding specificity.

According to another aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient a GnRH conjugate comprising a GnRH analog coupled to a photosensitizer, or a pharmaceutically acceptable salt or hydrate thereof and a pharmaceutically acceptable carrier or diluent.

According to a further aspect, the present invention provides a method of treating a sex hormone-related disease or condition in a subject comprising administering to the subject suffering from the disease or condition a therapeutical ly effective amount of a pharmaceutical composition comprising as an active ingredient a GnRH conjugate comprising a GnRH analog coupled to a photosensitizer, or a pharmaceutically acceptable salt or hydrate thereof and a pharmaceutically acceptable carrier or diluent, the GnRH conjugate binds to cells having GnRH receptors.

According to one embodiment, the method of treating a sex hormone-related disease or condition in a subject further comprises irradiating the cells in an afflicted area with light or X-ray radiation having a wavelength within the photoactivating action spectrum of the photosensitizer to produce a cytotoxic effect. According to another embodiment, the sex hormone-dependent disease or condition that may be treated with the pharmaceutical composition of the invention is selected from the group consisting of malignant and non-malignant sex hormone-dependent diseases. Malignant sex hormone-dependent diseases include prostate cancer, breast cancer, ovarian cancer, cervical cancer, a tumor of the pituitary, testicular cancer, uterine cancer, and the like. Non-malignant sex hormone-dependent diseases include benign prostatic hyperplasia, precocious puberty, aberrant sexual behavior (treatment by chemical castration), late luteal phase dysphoric disorder (premenstrual syndrome), fibroids, endometriosis, myoma, hirsutism, cyclic auditory dysfunction, porphyria, polycystic ovarian syndrome, and the like. It is now disclosed, for the first time, that a GnRH conjugate comprising a GnRH agonist, [D-Lys⁶]GnRH having the formula (pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro- Gly-NH ), coupled to a photosensitizer such as protoporphyrin IX (PplX), hypericin derivative (AmHyp), or chlorin e6 (Ce6) binds GnRH receptors with high affinity, induces LH release in vitro, exhibits long-lasting activity of LH release in vivo, and exerts selective phototoxic activity in pituitary, breast, and prostate cell lines.

It is further disclosed that a GnRH conjugate comprising a GnRH antagonist, [D-Lys⁶]Antg having the formula [D-pGlu¹, D-Phe², D-Trp³, D-Lys⁶]GnRH or [D-Lys⁶]SB75 having the formula [Acetyl-D-3-(2'-naphtyl)-Ala-D-4-chlorophenylalanine-D-3-(3'-pyridyl)-Ala-Ser- Tyr-D-Lys-Leu-Arg-Pro-D-Ala-amide]GnRH, coupled to a photosensitizer such as PplX or Ce6 binds GnRH receptors with high affinity, inhibits GnRH-induced LH release in vitro, and exerts selective phototoxic activity in pituitary, breast, and prostate cell lines.

These and other embodiments of the present invention will be better understood in relation to the figures, description, examples, and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a scheme of the synthesis of the GnRH analog-PpIX conjugates.

FIG. 2 shows the chemical structures of chlorin e6 (Ce6), hypericin, and various potential derivatives of hypericin.

FIGS. 3 A-B show the ultraviolet- visible spectra of PplX and its GnRH conjugates. FIG. 3A shows the spectrum of PplX and its GnRH conjugates (10 μM) in ethanol as compared to their spectra when dissolved in PBS (FIG. 3B). FIGS. 4A-B show the displacement (%) of the specific binding of ^I25I[D-Lys⁶]GnRH from pituitary membranes of pro-estrous rats by increasing concentrations of unlabeled GnRH analogs and conjugates. FIG. 4A, [D-Lys⁶]GnRH (O), [D-Lys⁶(PpIX)]GnRH (•); FIG. 4B, [D-Lys⁶]Antg (D) and [D-Lys⁶(PpIX)]Antg (■). Membranes were incubated for 90 min at 4°C with ¹²⁵I[D-Lys⁶]GnRH (40,000 - 50,000 cpm, 23.5 pM) either with or without the unlabeled peptides. Non-specific binding was defined as the binding of the labeled ligand in the presence of 1 μM of [D-Lys⁶]GnRH (FIG. 4A) or of 10 μM of [D-Lys⁶]Antg (FIG. 4B). These values were subtracted from the total binding for the calculation of specific binding. FIGS. 5A-B show the displacement (%) of the specific binding of ¹²⁵I[D-Lys⁶]GnRH from pituitary membranes of pro-estrous rats by increasing concentrations of additional unlabeled GnRH analogs and conjugates. FIG. 5A, [D-Lys⁶]GnRH (•), [D-Lys⁶(Ce6)]GnRH (Δ), and [D-Lys⁶(AmHyp)]GnRH (D); FIG. 5B, [D-Lys⁶]SB75 (Δ) and [D-Lys⁶(Ce6)]SB75 (■)• FIGS. 6A-B show the effect of GnRH analogs and conjugates on the secretion of LH from primary cultures of rat pituitary cells. FIG. 6A, LH releasing potency of [D-Lys⁶]GnRH and of [D-Lys⁶(PpIX)]GnRH. Primary cultures of rat pituitary cells were incubated in M-199 containing the indicated concentrations of [D-Lys⁶]GnRH (O) or [D-Lys⁶(PpIX)]GnRH (•). FIG. 6B, The inhibitory effect of a GnRH antagonist conjugate on GnRH-induced LH secretion from primary cultures of rat pituitary cells. Cells were incubated in the dark in M- 199 containing GnRH (1 nM) in the absence (white bar) or presence of increasing concentrations of [D-Lys⁶]Antg (D) or [D-Lys⁶(PpIX)]Antg (■). At the end of the incubation period (4 h at 37°C) media were collected and LH concentration was determined by RIA. FIGS. 7A-B show the effect of additional GnRH analogs and conjugates on the secretion of LH from primary cultures of rat pituitary cells. FIG. 7A shows the LH releasing potency of [D-Lys⁶]GnRH and of [D-Lys⁶]GnRH-photosensitizer conjugate. Primary cultures of rat pituitary cells were incubated in M-199 containing the indicated concentrations of [D-Lys⁶]GnRH (•), [D-Lys⁶(Ce6)]GnRH (Δ), or [D-Lys⁶(AmHyp)]GnRH (D). FIG. 7B shows the inhibitory effect of a GnRH antagonist conjugate on the GnRH- induced LH secretion from primary cultures of rat pituitary cells. Cells were incubated in the dark in M-199 containing GnRH (1 nM) in the presence of increasing concentrations of [D- Lys⁶]SB75 (Δ) or [D-Lys⁶(Ce6)]SB75 (■).

FIG. 8 shows the induction of LH secretion in rats following intraperitoneal administration of [D-Lys⁶(PpIX)]GnRH (•) or of the parent peptide, [D-Lys⁶]GnRH (O). Rats were injected with 2 nmol/rat of [D-Lys⁶]GnRH or with [D-Lys⁶(PpIX)]GnRH. Blood samples were taken from each rat at the indicated time intervals and serum LH levels were determined by RIA.

FIGS. 9 A-B show the time dependent phototoxicity of GnRH analog-PpIX conjugates in mouse pituitary gonadotrope cells (αT3-l)s. Cells were incubated in the dark with [D- Lys⁶(PpIX)]GnRH (FIG. 9A), [D-Lys⁶(PpIX)]Antg (FIG. 9B), or with PplX (C) in phenol- and serum- free DMEM for 45 (Δ) or 180 min (♦). Cells were then washed and illuminated (λ_max > 400 nm) with an irradiance of 14.3 mW cm^"2 and a total fluent of 17 J cm^"2. Incubation was then continued for 24 h in a fresh medium and cell survival was determined by the XTT method. Values are expressed as % survival. Complete survival (100%) is defined as the survival rate of cells in the control group that were incubated without any PplX derivative and were either illuminated or kept in the dark.

FIGS. 10 A-B show the phototoxicity of GnRH analog-Ce6 conjugates in mouse pituitary gonadotrope cells ( T3-l) (FIG. 10A) and in human prostate cell line(LNCap) (FIG. 10B). Cells were incubated in the dark with Ce6 (♦), [D-Lys⁶(Ce6)]GnRH (Δ), or with [D-Lys⁶(Ce6)]SB75 (■) in phenol- and serum- free DMEM for 4 h. Cells were then washed and illuminated (λ_max> 550 nm) with an irradiance of 14.3 mW cm^"2 and a total fluent of 17 J cm^"2. Incubation was then continued for 24 h in a fresh medium and cell survival was determined by the XTT method. Values are expressed as % survival. Complete survival (100%) is defined as the survival rate of cells in the control group that were incubated without any Ce6 derivative and were either illuminated or kept in the dark.

FIGS. 11 A-B show the reduction of the phototoxicity of [D-Lys⁶(PpIX)]GnRH as a result of competition for receptor binding sites with [D-Lys⁶]GnRH. The αT3-l pituitary cells (FIG. 11 A) or MCF-7 cells transfected with GnRH receptors (FIG. 1 IB) were incubated for 45 min with various concentrations of [D-Lys⁶]GnRH, followed by the addition of 1 μM of [D-Lys⁶(PpIX)]GnRH (black) or PplX (white bars in the control and in the 0.1 μM groups) and incubation was continued for an additional 3 h. The cells were washed, illuminated and incubated for an additional 24 h in a fresh medium. Cell survival was determined by the XTT method.

FIG. 12 shows the selective phototoxicity of [D-Lys⁶(PpIX)]Antg and of PplX in primary rat pituitary cells. Cells were incubated with lμM [D-Lys⁶(PpIX)]Antg (black bars) or with lμM PplX (white bars) for the indicated time and irradiated (λ> 350 nm) with an irradiance of 14.3 mW cm^"2 and with a total fluence of 17 J cm^"2. The media were then changed and the incubation continued for an additional 24 h, after which the media were removed for the evaluation of luteinizing hormone (LH) and growth hormone (GH) concentrations by RIA. Data represent the multiple of the basal LH/GH release.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides GnRH agonists or antagonists coupled to a photosensitizer, pharmaceutical compositions comprising same, and methods for treating sex hormone-dependent diseases in subjects. According to the present invention the amino acid residues used for synthesis of GnRH analogs are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, e.g., sequential, divergent and convergent synthetic approaches. The amino acid residues and derivatives thereof are represented throughout the specification and claims by three-letter codes according to IUPAC conventions. When there is no indication, the amino acid residue occurs in L isomer configuration. Amino acid residues present in D isomer configuration are indicated by "D" before the residue abbreviation.

The GnRH analogs of the present invention contain natural and non-natural amino acid residues. The non-natural amino acid residues include: Cit which denotes citrulline, Cpa which denotes 4-chloro-Phe, Dab which denotes diaminobutyric acid, hSer which denotes homoserine, iPrLys which denotes isopropyl Lys, Nal which denotes 3-(2'-naphtyl)-Ala, NicLys which denotes nicotinyl Lys, Orn which denotes ornithine, Pal which denotes 3-(3'- pyridyl-Ala), and pGlu which denotes pyroglutamic acid.

The term "analog" means any variant of GnRH peptide and includes both GnRH agonists and antagonists. As defined herein a GnRH "agonist" means a GnRH analog, which binds cell membrane GnRH receptors and activates them. A GnRH "antagonist" means a GnRH analog, which binds cell membrane GnRH receptors and inactivates them or inhibit the binding and activity of a GnRH agonist. According to one aspect, the present invention provides a GnRH conjugate comprising a GnRH analog coupled to a photosensitizer, or a pharmaceutically acceptable salt or hydrate thereof.

According to one embodiment, the GnRH analog is a GnRH agonist having the general formula: pGlu- His-Trp-Y-Tyr-X-Leu-Arg-Pro-Z (SEQ ID NO : 1) wherein Y and X are each independently selected from Lys, Dab, Orn, Ser, hSer, D- Lys, D-Dab, D-Orn, D-Ser, D-hSer, Lys(photosensitizer), Dab(photosensitizer), Orn(photosensitizer), Ser(photosensitizer), hSer(photosensitizer), D-Lys(photosensitizer), D- Dab(photosensitizer), D-Orn(photosensitizer), D-Ser(photosensitizer), D- hSer(photosensitizer), wherein at least one of Y and X is coupled to photosensitizer; Z is selected from Gly-NH2 or ethylamide; or a pharmaceutically acceptable salt or hydrate thereof.

According to some embodiments, the GnRH analog is a GnRH agonist having the amino acid sequence of SEQ ID NO:2 and is designated [D-Lys⁶]GnRH. According to further embodiments, the GnRH analog is a GnRH antagonist having the amino acid sequence of SEQ ID NO:3 and is designated [D-Lys ]Antg.

According to another embodiment, the GnRH analog is a GnRH antagonist having the general formula:

Ac-D-Nal-D-Cpa-D-Pal-X-Y-Z-Leu-W-Pro-D-Ala-NH2 (SEQ ID NO:4) wherein X is selected from Ser, hSer, Lys, Dab, Orn, NicLys, Ser (photosensitizer), hSer (photosensitizer), Lys(photosensitizer), Dab(photosensitizer). Orn(photosensitizer), or NycLys(photosensitizer); Y is selected from Tyr, NicLys, Lys, D-Lys, Dab, Orn, NicLys(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), Dab(photosensitizer), or Orn(photosensitizer); Z is selected from D-Ser, D-NicLys, D-iPrLys, D-Lys, Ser(photosensitizer), D-Ser(photosensitizer), D-hSer(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), D-NicLys(photosensitizer), Dab(photosensitizer), D- Dab(photosensitizer), Orn(photosensitizer), D-Orn(photosensitizer), or iPrLys(photosensitizer); W is selected from Arg, iPrLys, Ser(photosensitizer), D- Ser(photosensitizer), hSer(photosensitizer), D-hSer(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), NicLys(photosensitizer), D-NicLys(photosensitizer), Dab(photosensitizer), Orn(photosensitizer), or iPrLys(photosensitizer), wherein at least one of X, Y, Z, and W is coupled to a photosensitizer; or a pharmaceutically acceptable salt or hydrate thereof.

According to some embodiments, the GnRH antagonist is [Acetyl-D-3-(2'-naphtyι)- Ala, D-4-chlorophenylalanine, D-3-(3'-pyridyl)-Ala, Ser, Tyr, D-Lys, Leu, Arg, Pro, D-Ala- amide]GιιRH (SEQ ID NO:5) designated [D-Lys⁶]SB75.

According to yet another embodiment, the GnRH analog is a GnRH antagonist having the general formula:

Ac-Nal-D-Cpa-D-Pal-X-Tyr-Y-Leu-Arg-Pro-D-Ala-NH2 (SEQ ID NO:6) wherein X is selected from Ser, Ser(photosensitizer), hSer, hSer(photosensitizer); Y is selected from D-Lys, D-Ser, D-hSer, D-Orn, D-Cit, D-Dab, D-Lys(photosensitizer), D-

Ser(photosensitizer), D-hSer(photosensitizer), D-Orn(photosensitizer), D-

Cit(photosensitizer), D-Dab(photosensitizer), wherein at least one of X and Y is coupled to a photosensitizer; or a pharmaceutically acceptable salt or hydrate thereof.

It will be appreciated that the present invention encompasses GnRH agonists and antagonists as are known in the art to which a photosensitizer is coupled according to the principles of the invention so long as the GnRH conjugates comprising a GnRH analog and a photosensitizer retain GnRH receptor binding specificity. Examples of GnRH agonists and antagonists are listed, for example, in U.S. Pat. No. 5,508,383, 6,214,798 and 6,384,017 and references therein, the content of which is incorporated herein by reference as if fully set forth (see also U.S. Pat. Nos. 5,248,492, and 6,184,374, the content of which is incorporated herein by reference as if fully set forth, which disclose GnRH analogs coupled to a cytotoxic moiety, the cytotoxic moiety may be substituted by a photosensitizer disclosed in the present invention). For review of GnRH agonists and antagonists see also Schaison, G., J. Steroid Biochem. 33: 745, 1989. GnRH analog synthesis may be performed by solid phase peptide synthesis method of

Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). Alternatively, a peptide of the present invention can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer- Verlag, 1984) or by any other method known in the art for peptide synthesis.

In general, these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain bound to a suitable resin. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support (resin) or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions conductive for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups are removed sequentially or concurrently, and the peptide chain, if synthesized by the solid phase method, is cleaved from the solid support to afford the final polypeptide. A particularly preferred method of preparing peptides involves solid phase peptide synthesis. In this method of preparing peptides, the alpha-amino group of the amino acid is protected by an acid or base sensitive group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain. Suitable protecting groups are t- butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t- amyloxycarbonyl, isobornyloxycarbonyl, (alpha,alpha)-dimethyl-

3,5dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, 9- fluorenylmethyloxycarbonyl (FMOC) and the like. The FMOC protecting group is preferred.

In the solid phase peptide synthesis method, the C-terminal amino acid is attached to a suitable solid support. Suitable solid supports useful for the above synthesis are those materials, which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the solvent media used. Suitable solid supports are chloromethylpolystyrene-divinylbenzene polymer, hydroxymethyl-polystyrene-divinylbenzene polymer, and the like. The coupling reaction is accomplished in a solvent such as ethanol, acetonitrile, N,N-dimethylformamide (DMF), and the like. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as is well known in the art. A preferred procedure is described, for example, in Rahimipour, S., at al., J. Med. Chem. 44: 3645-3652, 2001.

The present invention provides GnRH conjugates comprising GnRH analogs. The term "analog" includes, but is not limited to, GnRH amide derivatives, GnRH ester derivatives, and the like. In addition, this invention further includes hydrates of the GnRH analogs described herein. The term "hydrate" includes, but is not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, and the like.

The invention further includes pharmaceutically acceptable salts of the GnRH analogs. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and the like. Thus, organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropylamine, methylamine, dimethylamine and the like) and optionally substituted ethanolamines (e.g. ethanolamine, diethanolamine and the like) may be used according to the principles of the invention. When the GnRH analog of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, oxalic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

According to the principles of the present invention coupling of a photosensitizer to a GnRH agonist, exemplified herein by [D-Lys⁶] GnRH, or to a GnRH antagonists, exemplified herein by [D-Lys⁶]Antg and [D-Lys⁶]SB75, is performed by reacting the ε-amino group of D-Lys of the GnRH analog with one of the carboxylic functional groups of a photosensitizer. Previous studies have shown that coupling of bulky moieties, such as tetramethylrhodamine, to the ε-amino group of the [D-Lys⁶] GnRH does not significantly affect the bioactivity of the GnRH analogs nor their internalization by gonadotropes (Hazum, E., et al. Mol. Cell. Endocinol. 30: 291-301, 1983). According to the Examples disclosed herein below, coupling of a photosensitizer to a GnRH agonist or antagonist preserved GnRH receptor binding specificity of the parent peptide (see Example 2 herein below). Thus, according to the principles of the present invention coupling of a photosensitizer to a GnRH agonist or antagonist preserves GnRH receptor binding specificity of the parent peptide.

It will be appreciated that although the coupling of a photosensitizer to the GnRH analogs is exemplified herein below by reacting one of the free carboxylic functional groups of PplX with the free ε-amino group of D-Lys, coupling of a photosensitizer to other amino acid residues, such as, for example Ser, hSer, Dab, Orn, Cit, NicLys, iPrLys, or derivatives thereof, is also encompassed in the present invention. Coupling of a photosensitizer to other amino acid residues involves available functional groups of the amino acid residues, for example, coupling of a photosensitizer may be performed through the free functional amino group of Orn, Dab, or Cit, thus forming an amide bond. Alternatively, coupling of a photosensitizer to Ser or hSer may be performed through the available hydroxyl group, thus forming an ester bond. Additionally, coupling of a photosensitizer to a GnRH analog may be performed to at least one amino acid residue of the GnRH analog, the amino acid residue being located at position 4, 5, 6 or 8 according to the principles of the invention. According to currently preferred embodiments, a photosensitizer is coupled to one amino acid residue of a GnRH analog.

Coupling of a photosensitizer to a GnRH analog may be performed by using a carbodiimide coupling reagent such as dicyclohexyl-carbodiimide (DCC) in the presence of a catalytic amount of 4-dimethylaminopyridine (DMAP) or via N-hydroxysuccinimide (NHS) and DCC. The coupling may also be performed by benzotriazole-1-yl-oxy-tris-pyrrolidino- phosphonium hexafluoro phosphate (PyBOP) as a coupling reagent and 4-methylmorpholine (NMM) as a base. Thus, coupling of a photosensitizer to an available functional amino group of an amino acid residue such as Lys, Orn, Dab, or Cit, to from an amide bond may be performed in the presence of a coupling reagent such as PyBoP, HBTU, or carbodiimides such as DCC in the presence of NHS or N-hydroxybenzotriazole (HOBT). Also, coupling of a photosensitizer to an available functional hydroxyl group of an amino acid residue such as Ser or hSer to form an ester bond may be performed by the anhydride coupling reaction in the presence of DMAP and DCC. Coupling may be performed in solution, for example, in DMF, or may be performed when the peptide is still bound to the resin. Alternatively, other coupling methods well known in the art, for example, by alkylation reactions with iodinated or brominated photosensitizer, may be used for the practice of the invention. The terms "conjugation" and "coupling" are used interchangeably throughout the specification of the present invention and refer to the chemical reaction, which results in covalent attachment of a photosensitizer to a GnRH analog to yield a GnRH conjugate.

The term "photosensitizer" refers to a light or an X-ray activatable compound. The localized exposure of photosensitizer-containing tissues to light or X-rays at a wavelength appropriate for activating the photosensitizer ordinarily does not induce a chemical reaction between cell components and the photosensitizer molecules. Instead, the photosensitizer molecules act as catalysts by trapping the energy of the photoactivating light or X-ray and then passing it on to molecules of oxygen, which in turn are raised to an excited state that is capable of oxidizing adjacent molecules or structures. The resulting cell death is not caused primarily by damage to the DNA, but rather by damage to essential cellular structures. It has been stated that the biochemical effects of such photoactivation include cross-linking of membrane proteins, peroxidation of lipids, inhibition of transport of some essential metabolites, lysis of lysosomal and mitochondrial membranes, and inactivation of several enzymes, which lead to cell death. Thus, without wishing to be bound to any mechanism of action, it is apparent that a photosensitizer produces a phototoxic or cytotoxic effect when irradiated with light or X-ray having a wavelength within the photoactivating action spectrum of the photosensitizer. Ideally, a photosensitizer should exhibit low background toxicity, i.e., it should not be toxic in the absence of irradiation with energy of the appropriate wavelength.

Suitable photosensitizers that may be coupled to the GnRH analogs according to the present invention include porphyrin-based materials including, but not limited to, hematoporphyrins, such as hematoporphyrin HCl and hematoporphyrin esters; dihematoporphyrin ester (Wilson et al., Oral Microbiol. Immunol. 8:182-187, 1993); hematoporphyrin IX and its derivatives; hydroporphyrins such as chlorin and bacteriochlorin, and synthetic diporphyrins and dichlorins; o-substituted tetraphenyl porphyrins; chlorin e6 monoethylendiamine monamide (CMA; available from Porphyrin Products, Logan, Utah); mono-1-aspartyl derivative of chlorin e6, and mono- and diaspartyl derivatives of chlorin e6; the hematoporphyrin mixture Photofrin II (quardra Logic Technologies, Inc., Vancouver, BC, Canada); benzoporphyrin derivatives (BPD), including benzoporphyrin monoacid Ring A (BPD-MA), purpurins; bacteriochlorophylls; hypericins; texaphyrins; phthalocyanines; analogs and derivatives thereof. Other potential photosensitizers include, but are not limited to, pheophorbides such as pyropheophorbide compounds, anthracenediones; anthrapyrazoles; and aminoanthraquinone. In currently preferred embodiments, protoporphyrin IX (PplX), chlorin e6 (Ce6) and a Hypericin derivative designated AmHyp, are selected. It will be appreciated that the present invention also encompasses GnRH conjugates, which comprise a GnRH analog coupled to different photosensitizers.

The present invention discloses the synthesis of the GnRH conjugate [D- Lys6(PpIX)]GnRH . This GnRH conjugate was shown to bind specifically to GnRH receptors, to induce LH release in vitro, to exhibit long-lasting activity of LH release in vivo, and to exert phototoxic effect in pituitary cells higher than the phototoxic effect obtained by the non-conjugated PplX. It should be emphasized that [D-Lys6(PpIX)]GnRH did not possess any toxicity under dark conditions.

The present invention further discloses two additional GnRH agonist conjugates, i.e., the GnRH agonist [D-Lys6]GnRH was conjugated to Ce6 and to AmHyp. The conjugates thus formed were shown to preserve GnRH receptor binding specificity and GnRH activity and to induce phototoxic effect in pituitary and prostate cell lines.

The present invention discloses the synthesis of the GnRH antagonist conjugate, [D- pGlu¹, D-Phe², D-Trp³, D-Lys⁶ (PpIX)]GnRH designated [D-Lys⁶(PpIX)]Antg. This conjugate was found to bind specifically to GnRH receptors, to inhibit LH release in vitro, to exhibit long-lasting inhibitory activity on LH release in vivo, to exert phototoxic effect in pituitary cells higher than that obtained with the non-conjugated PplX, and to be highly selective in its phototoxic activity to GnRH receptor bearing cells. Notably, it did not possess any toxicity under dark conditions.

The present invention further discloses another GnRH antagonist [D-Lys⁶] SB75 conjugated to Ce6. The conjugate thus formed was found to bind specifically to GnRH receptors, to inhibit LH release from pituitary cells, and to induce phototoxic effect in pituitary and prostate cell lines at a lower concentration than Ce6 alone.

Various assays may be performed to determine whether GnRH agonists or antagonists of the present invention preserve their GnRH activity after coupling to a photosensitizer. These assays include, but are not limited to, competition binding experiments of a labeled

GnRH analog to GnRH receptor preparations in the presence of an unlabeled GnRH conjugate, induction or inhibition of LH release by pituitary cells in vitro, and induction of LH release in vivo. Assays to establish the biological activity of GnRH conjugates are disclosed in Examples 2-4 of the present invention. However, other methods well known in the art may be used to determine whether a GnRH conjugate according to the principles of the present invention preserve its biological activity. The present invention also provides pharmaceutical compositions comprising as an active ingredient a GnRH conjugate comprising a GnRH analog coupled to a photosensitizer or a pharmaceutically acceptable salt or hydrate thereof and a pharmaceutically acceptable carrier or diluent.

The pharmaceutical compositions comprising the GnRH conjugates of the invention may be prepared as injectables, either as liquid solutions or suspensions or as solid forms, which can be suspended or solubilized prior to injection. The preparation can also be emulsified. The active therapeutic ingredient is mixed with inorganic and/or organic carriers, which are pharmaceutically acceptable and compatible with the active ingredient.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Carriers are more or less inert substances when added to a pharmaceutical composition to confer suitable consistency or form to the composition. As used herein a "pharmaceutically acceptable carrier" may be a solid carrier for solid formulations, a liquid carrier for liquid formulations, or mixtures thereof. Solid carriers include, but are not limited to, a gum, a starch (e.g. corn starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a ceUulosic material (e.g. microcrystalline cellulose), an acrylate (e.g. polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. For liquid formulations, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene gly col, 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 (for subcutaneous, intravenous, intraarterial, intraperitoneal or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

In addition, the compositions may further comprise binders (e.g. cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCl, acetate, phosphate) of various pH and ionic strength, additives such as gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

The pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, gels, ointments, creams, lotions, and the like. The compositions may be formulated in depot, implant or sustained-release formulations, in which the GnRH conjugate is released over a period of time after administration. For example, the composition may include a slow release polymer. Particularly preferred formulations include controlled-release compositions such as are known in the art for the administration of leuprolide (trade name: Lupron®), injectable formulations (U.S. Pat. No. 4,849,228), and sustained-release compositions for water-soluble polypeptides (U.S. Pat. No. 4,675,189). The compositions may be formulated in immediate-release compositions in which the GnRH conjugate is released immediately after administration. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the GnRH conjugate, together with a suitable amount of a carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration.

The preparation of pharmaceutical compositions, which contain an active component is well understood in the art, for example by mixing, granulating, or tablet-forming processes. The active therapeutic ingredient is often mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. For parenteral administration, the pharmaceutical composition is converted into a solution, suspension, or emulsion, if desired with the substances customary and suitable for this purpose, for example, solubilizers or other. Formulations for inhalation administration may be solid and contain as excipients, for example, lactose, or may be aqueous or oily solutions for administration in the form of nasal drops. The invention further provides a method of treating a sex hormone-dependent disease in a subject, the method comprises administering to a subject suffering from the disease a therapeutically effective amount of pharmaceutical composition comprising as an active ingredient a GnRH conjugate comprising a GnRH analog coupled to a photosensitizer and a pharmaceutically acceptable carrier, the GnRH conjugate binds to cells having cell membrane GnRH receptors. According to one embodiment, the method of treating a sex hormone-dependent disease further comprises irradiating the cells in an afflicted area with light or X-ray radiation having a wavelength within the photoactivating action spectrum of the photosensitizer to produce a cytotoxic effect.

As used herein, the term "administering" refers to bringing a subject in contact with a pharmaceutical composition comprising a GnRH conjugate of the present invention. As used herein, administration can be accomplished in vitro, i.e., in a test tube, or in vivo, i.e., in cells or tissues of living organisms, for example humans. As used herein, the term "treating" means remedial treatment, and encompasses the terms "reducing", "suppressing", "ameliorating" and "inhibiting", which have their commonly understood meaning of lessening or decreasing.

The administration of the pharmaceutical composition may be performed by a variety of routes including oral, intranasal, rectal, and topical. Alternatively, the pharmaceutical composition is intravenously, intraarterially, intraperitoneaUy, subcutaneously, intramuscularly injected in liquid form. It may also be injected into other regions, such as into synovial fluids. Delivery of the composition transdermally is also contemplated, such as by diffusion via a transdermal patch. The pharmaceutical compositions can be applied on conventional intravaginal rings or other intravaginal devices. The pharmaceutical compositions of the invention may also be administered to the patient by standard procedures used in PDT.

The amount of the pharmaceutical composition to be administered and the route of administration will be determined according to the kind of disease, for example the kind of tumor, stage of the disease, age and health conditions of the patient. Acting as a kind of medicinal peptide, the GnRH agonists and antagonists described herein are not likely to be administrated orally. However, these peptides can be easily made into a lyophilized powder, which can be readily dissolved in a saline solution for injection intravenously, subcutaneously, or intramuscularly. The preferable routes of administration are intravenous or direct injection into the afflicted site, for example into the solid tumor, or intranassaly by inhalation.

As defined herein, the term "sex-hormone related disease or condition" encompasses diseases or conditions involving the reproductive system, and/or which are dependent upon a sex hormone, i.e., a male hormone or female hormone. In one embodiment, these include diseases or conditions occurring due to an excess of such hormones in mammals or non- mammalian vertebrates (e.g. human, monkey, bovine, horse, dog, cat, sheep, rabbit, rat, mouse, fish etc.).

In a further embodiment, sex hormone-dependent diseases include malignant sex hormone-dependent diseases exemplified, but are not limited to, prostate cancer, breast cancer, ovarian cancer, cervical cancer, tumor of the pituitary, testicular cancer, and uterine cancer. In another embodiment, sex hormone-dependent diseases include non-malignant

(benign) sex hormone-dependent diseases. Non-limiting examples include benign prostatic hyperplasia, precocious puberty, aberrant sexual behavior (treatment by chemical castration), late luteal phase dysphoric disorder (premenstrual syndrome), fibroids, endometriosis, myoma, hirsutism, cyclic auditory dysfunction, porphyria, or polycystic ovarian syndrome.

The term "therapeutically effective amount" of the conjugate of the invention refers to that amount that of the conjugate that when administered to a subject is capable of treating a sex hormone-dependent disease. A non-limiting range for a therapeutically effective amount of a GnRH conjugate is 0.01 μg/kg-10 mg/kg of body weight per day, preferably between 0.01 and 5 mg/kg of body weight per day. The administration may be accomplished by a single daily administration, by administration over several applications, or by slow release in order to achieve the most effective results.

It will be appreciated that the pharmaceutical compositions comprising the GnRH conjugates of the invention may be used in combination therapy with standard medicaments for the diseases listed herein above. Standard medicaments for treating cancer include, for example, cytotoxic drugs such as adriamycin, daunomycin, taxol, taxotere and vincristine. Alternatively, the pharmaceutical compositions may be administered with anti-estrogens, anti-androgens, or inhibitors of sex steroid biosynthesis (see, for example, U.S. Pat. Nos. 6,384,017 and 6,211,153).

According to the principles of the present invention, the conjugate binds to cells having cell membrane receptors and accumulates therein. However, while in normal tissues these cells are separated from each other, in a tumor the cells are usually clustered and thus the local concentration of the conjugate is higher. As a result, in photodynamic therapy, the phototoxic or cytotoxic effect of the GnRH conjugate in a tumor can be higher by several orders of magnitude than its effect in the normal tissue. Consequently the threshold of irradiation that is destructive for the tumor is expected to be reduced to a level that is non- destructive for the normal tissue. Under these circumstances, the phototoxic or cytotoxic effect will be essentially limited to the tumor site. This application is of a particular importance for tumors that are inaccessible to conventional surgery.

The radiation may be supplied through any convenient means appropriate to the performance of PDT. In the model system, the radiation is conveniently supplied simply by a standard light source such as a halogen lamp or a laser source. Alternatively, an X-ray source may be used. For treatment of a subject, fiber optics or other more specialized means of delivery are preferred. The wavelength chosen will depend on the choice of the photosensitizer; it will be apparent from the absorption spectrum of the photosensitizer what range of light will be suitable. The choice may also depend on the availability of a convenient light source.

The intensity and energy levels supplied are also dependent on the nature of the condition being treated. Typical levels of light energy are in the range of at least 1 J/cm^"2. Photodynamic therapy (PDT) may be conducted at traditional energy levels, which may result in erythema as a side-effect, or may be conducted at "low dose" levels, which are considered those equivalent to ambient light. For "ordinary" PDT, the light fluent rate generally varies between more than 50-200 J/cm^" . However, at "low-dose" levels, the fluent rate is typically less than that. Suitable low-dose levels may be determined by experimentally determining the level, which first elicits the symptoms of erythema and then cutting the energy to one-fourth to one-sixth of that level. Low-dose PDT usually involves energies of less than 10 J/cm^"2, preferably less than 5 J/cm^"2, and more preferably 1 J/cm^"2 or less. In the present application, however, somewhat higher energies may be desirable~for example, in the range of 10-20 J/cm^"2. The energy can be supplied at a suitable rate with, in some instances, very low irradiation levels. For sub lethal PDT, somewhat higher intensities slightly less than 50 mW/cm^"2 may be employed. In order to maintain viability, the optimum balance between drug concentration, irradiation and time may be determined using a suitable model system. Typically, PDT employs higher total energies and higher fluent rates— energies on the order of 100-200 J/cm^"2 and irradiances on the order of 200 mW/cm^"2. This PDT can be used to prevent neovascularization —for example, in treating tumors. Thus, a subject is irradiated with light at appropriate wavelength that causes the photosensitizer to produce a cytotoxic effect. Preferably, the cytotoxic effect is substantial enough to kill at least 50%, more preferably at least 70%, and most preferably at least 90% of the pathogenic cells.

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. EXAMPLE 1

Preparation of the GnRH analog-Photosensitizer conjugates

Parent peptides, GnRH agonist (pGlu¹-His²-Trp³-Ser⁴-Tyr⁵-D-Lys⁶-Leu⁷-Arg⁸-Pro⁹- Gly¹⁰-NH₂) [(D-Lys⁶) GnRH] and (D-pGlu¹, D-Phe²-D-Trp³-Ser⁴-Tyr⁵-D-Lys⁶-Leu⁷-Arg⁸- Pro⁹-Gly¹⁰-NH₂) [(D-Lys⁶)Antg], were synthesized on a multiple solid-phase peptide synthesizer as described (Rahimipour, S., et al. Lett. Pept. Sci. 5: 421-427, 1998), cleaved from the resin, precipitated, dissolved in H O and then the solution was lyophilized. The purity of the crude peptides was usually over 90%, and therefore they were used for the next stage without further purification.

Conjugation of protoporphyrin IX (PplX) to the peptides (FIG. 1) was performed by reacting the free ε-amino group of the D-Lys residue of the GnRH analogs with one of the carboxylic functional groups of the PplX in a homogeneous solution of DMF as follows: to a DMF solution (1 ml) of crude [D-Lys⁶]GnRH (31 mg, 25 μmol) and PplX (15.5 mg, 27.5 μmol; Sigma (St. Louis, MO)), a DMF solution (0.5 ml) containing benzotriazole-1-yl-oxy- tris-pyrrolidino-phosphonium hexafluoro phosphate (PyBOP; 13 mg, 27.5 μmol) as a coupling reagent and 4-methylmorpholine (NMM; 8.2 μl, 75 μmol) as a base was added. The mixture was stirred for 2 h at room temperature. The completion of the reaction was determined by the disappearance of [D-Lys ]GnRH as revealed by analytical reverse phase (RP) HPLC using an HPLC system (Spectra Series P200 liquid chromatography system equipped with a Spectra Series UV100 variable wavelength absorbance detector; Thermo Separation Products, Riviera Beach, FL) equipped with a prepacked column of Vydac RP-4 (250 x 3.2 mm; 5 μm, Vydac, Hesperia, CA). The crude conjugate was then precipitated with ice-cold tert-butyl methyl ether (10 ml), dried, and purified to homogeneity (>95%) by preparative RP-HPLC using a prepacked Vydac RP-4 column (250 x 22 mm; 10 μm, Vydac, Hesperia, CA) to yield 27 mg (15 μmol; 60%) of [D-Lys⁶(PpIX)]GnRH. All HPLC purification and analyses were achieved by using 0.1% trifluoroacetic acid (TFA) in water as buffer A and 0.1% TFA in 75% acetonitrile in water (v:v) as buffer B. Eluent composition was 10-100 % B over 40 min. Mass spectrometry of [D-Lys⁶(PpIX)]GnRH was carried out on a Micromass Platform

LCZ 4000 (Manchester, UK) using an electron spray ionization technique (ESI) and resulted in a m/z [M + H]⁺ value of 1797.8; the calculated m/z value for C₉₃H_n6N₂₂Oi₆ [M + H]⁺ is 1798.0. In order to determine the amino acid composition, [D-Lys⁶(PpIX)]GnRH was first hydro lyzed with 6 N HCl at 110 °C for 22 h and then the hydrolysate was analyzed by an AccQ.Tag amino acid analysis method using a pre-column derivatization technique (Waters 2690, Milford, MA). Amino acid analysis of [D-Lys⁶ (PplX)] GnRH resulted in the following yields: Glu 1.02, His 0.99, Ser 0.90, Tyr 0.96, Lys 1.02, Leu 0.98, Arg 0.99, Pro 0.77, Gly 1.13. Trp could not be detected because of its destruction under the acidic conditions of hydrolysis. This 'one-pot synthesis' method resulted in a better yield and purity than other methods that use carboidiimides as coupling reagents. Conjugation of [(D-Lys )GnRH] with other photosensitizers such as, for example, chlorin e6 (Ce6) and a derivative of Hypericin (AmHyp) was performed as described above for the conjugation of [(D-Lys⁶)GnRH] with PplX. The structures of Ce6, AmHyp, and of additional derivatives of AmHyp are presented in FIG. 2.

GnRH antagonist conjugate was synthesized and characterized by the same methods used for the GnRH agonist conjugate synthesis and the yield of [D-Lys⁶(PpIX)]Antg was 21 mg (13.3 μmol, 53%). Mass spectrometry resulted in a m/z [M + H]⁺ value of 1807.5; the calculated m/z value for C₉₆Hn₈N₂oOι₆ [M + H]⁺ is 1807.9. Amino acid analysis performed after hydrolysis with 6 M HCl at 110 °C for 22 h resulted in the following yields: Glu 0.97, Phe 1.19, Ser 0.8, Tyr 0.76, Lys 0.98, Leu 0.96, Arg 1.02, Pro 0.98, Gly 1.04. Another GnRH antagonist, [Acetyl-D-3-(2'-naphtyl)-Ala, D-4-chlorophenylalanine, D-

3-(3'-pyridyl)-Ala, L-Ser, L-Tyr, D-Lys, L-Leu, L-Arg, L-Pro, D-Ala-amide]GnRH ([D- Lys⁶]SB75) was conjugated to Ce6. The conjugation was performed as described above for PplX conjugation to [D-Lys⁶]Antg.

FIGS. 3A-B show the UV-visible spectra of PplX and its GnRH conjugates. As shown in FIG. 3, PplX was found to be more aggregated in PBS (FIG. 3A) than in ethanol (FIG. 3B), as evidenced by the flattening of its characteristic peak at 400 nm. Covalent binding of PplX to the GnRH analogs significantly reduced its aggregation state in PBS as indicated by the sharper peaks of the conjugates compared to that of PplX alone (FIG. 3B). It should be noted that the efficiency of a photosensitizer in producing singlet oxygen is believed to be at least partly related to the degree of its aggregation in aqueous solution. When aggregated, the photoactivity of a photosensitizer is reduced since its photo-excited state is rapidly deactivated to its ground state by internal energy transfer. EXAMPLE 2

Receptor binding assay

The ability of the GnRH analog-photosensitizer conjugates to bind GnRH receptors was evaluated in vitro by displacement assays using I[D-Lys ]GnRH.

[D-Lys⁶] GnRH was iodinated by the chloramine T method and purified by analytical HPLC. The binding affinities of the GnRH analogs to rat pituitary receptor preparations were compared through competitive binding experiments (Yahalom, D., et al. Life Sci. 64: 1543- 1552, 1999) as follows: rat pituitary membranes containing approximately 0.1 pituitary equivalent (25 μg protein/tube, prepared from proestrous rats) in 0.4 ml of assay buffer (0.1% BSA in 10 mM Tris-HCl; pH 7.4) were incubated in triplicates with ¹²⁵I[D- Lys⁶]GnRH (40,000-50,000 cpm, 23.5 pM, 50 μl) in the presence (0.1 nM-10 μM) or absence of a GnRH conjugate (50 μl) in a total volume of 0.5 ml assay buffer for 90 min at 4°C. The reaction was terminated by rapid filtration through Whatman GF/C filters, presoaked in 2-3% polyethylenimine solution in 10 mM Tris-HCl to minimize filter absorption. The filters were washed three times with cold 10 mM Tris-HCl (3 ml each) and were counted in a Packard Auto-Gamma Counting System (Packard, Meriden, CT). Nonspecific binding was defined as the residual radioactivity in the presence of an excess of the parent peptide: [D-Lys⁶]GnRH (1 μM), [D-Lys⁶]Antg (10 μM), or SB75 (1 μM). Specific binding was calculated by subtracting the non-specific binding from the maximal binding determined in the absence of any competing peptide.

FIGS. 4A-B show the ability of the GnRH conjugates to compete with ¹²⁵I[D-Lys⁶] GnRH for its binding to GnRH receptors. As shown in FIG. 4A, the agonist-based conjugate, [D-Lys⁶(PpIX)]GnRH, was shown to bind specifically to the GnRH receptors, although its binding affinity was lower than that of the parent agonist (IC₅₀ = 22 and 1.1 nM, respectively). Incorporation of PplX to the GnRH antagonist resulted in an even larger decrease in binding affinity, with an IC₅₀ of 1.8 μM for [D-Lys⁶(PpIX)]Antg versus 6 nM for the parent antagonist (FIG. 4B).

FIGS. 5A-B show the ability of additional GnRH conjugates to compete with ¹²⁵I[D- Lys⁶] GnRH for its binding to GnRH receptors. As shown in FIG. 5A, [D-Lys⁶(Ce6)]GnRH and [D-Lys⁶(AmHyp)]GnRH were shown to bind specifically to the GnRH receptors, although their binding affinity was lower than that of the parent agonist (IC₅₀ = 60, 2.7, and 0.2 nM for [D-Lys⁶(Ce6)]GnRH, [D-Lys⁶(AmHyp)]GnRH, and [D-Lys⁶]GnRH, respectively). Similarly, conjugation of Ce6 to the GnRH antagonist, [D-Lys⁶]SB75 reduced its binding affinity compared to the parent peptide (IC₅₀ = 40 and 0.4 nM, respectively).

EXAMPLE 3

Biological activity of the GnRH conjugates in vitro

To evaluate the correlation between GnRH binding affinity and GnRH bioactivity, the effect of each of the GnRH conjugates on LH release from primary rat pituitary cell cultures was compared to that of the parent peptides.

Primary pituitary cell cultures prepared from 21 -day-old Wistar-derived female rats as described (Yahalom, D., et al. J. Med. Chem. 43: 2824-2830, 2000) were maintained in M- 199 supplemented with 10% horse serum and antibiotics, and were plated (50,000 cells/well) in 96-multiwell tissue culture dishes. After 48 h, the medium was replaced by phenol red- and serum-free M-199 containing the desired concentration of either the GnRH agonist or its conjugates (4 wells/experimental group) and the cells were incubated (4 h at 37°C) in the dark to avoid photo-activation of the photosensitizer. To evaluate the activity of the antagonistic conjugate, primary cultures of rat pituitary cells were incubated with native GnRH (1 nM) alone or in the presence of increasing concentrations of either the conjugate or the parent antagonist peptide. The GnRH peptides were dissolved in dimethylsulfoxide (DMSO) to obtain stock solutions of 1 mM that were then diluted in the medium to obtain the desired concentrations. The DMSO concentration was always less than 1%. As a control, cells were incubated with identical concentrations of DMSO only, and the results indicated that DMSO at these concentrations did not have a significant effect either on receptor binding nor on hormone secretion. At the end of the incubation period, supernatants (0.1 ml) were diluted with 1% BS A/PBS (0.9 ml) and analyzed for LH and GH content by double- antibody radioimmunoassay (RIA) (Daane, T. A., and Parlow, A.F. Endocrinology 88: 653- 667, 1971; Desbuquois, B., and Aurbach, G.D. J. Clin. Endocrinol. Metab. 33: 732-738, 1971) using kits kindly supplied by the National Institute of Arthritis, Metabolism and Digestive Diseases (NIMDD). LH and GH levels are expressed in terms of the LH-RP-3 and GH-RP-2 rat reference preparation, respectively. FIGS. 6A-B show the effect of [D-Lys⁶]GnRH, [D-Lys⁶]Antg, and their PplX conjugates on LH release from primary rat pituitary cell cultures. The bioactivity of [D- Lys⁶(PpIX)]GnRH and of the parent peptide [D-Lys⁶]GnRH (FIG. 6A) demonstrated that both peptides stimulated LH release, although the agonist conjugate exhibited somehow lower LH releasing activity than its parent peptide. In the case of GnRH antagonist, both Lys⁶(PpIX)]Antg and [D-Lys⁶]Antg inhibited LH release, however, the bioactivity of [D- Lys⁶(PpIX)]Antg (FIG. 6B) was lower than that of its parent antagonist (ED₅₀=1.8 and 0.08 μM, respectively).

FIG. 7A shows the effect of [D-Lys⁶]GnRH, [D-Lys⁶(Ce6)]GnRH, and [D- Lys⁶ (AmHyp)] GnRH on LH release from primary rat pituitary cell cultures. As shown in Fig. 7A, the two GnRH conjugates retained their biological activity and stimulated LH release.

FIG. 7B shows the effect of GnRH antagonist, [D-Lys⁶] SB75, and its conjugate [D- Lys⁶(Ce6)]SB75 on LH release from primary rat pituitary cell cultures. The inhibitory effect of [D-Lys⁶ (Ce6)]SB75 on LH release was not significantly reduced compared to that of the parent peptide.

EXAMPLE 4

Biological activity of the GnRH conjugate in vivo The agonistic activity of GnRH in vivo was evaluated by assessing the induction of LH release following intraperitoneal injection of [D-Lys⁶ (PplX)] GnRH or its parent peptide into rats.

Fifteen week-old female rats (220-250 g) were injected intraperitoneaUy with 2 nmol of [D-Lys⁶(PpIX)]GnRH or [D-Lys⁶]GnRH in 0.5 ml of PBS. Blood samples were withdrawn by cardiac puncture under light ether anesthesia at the indicated time intervals, and serum LH levels were determined by RIA as described in Example 3.

FIG. 8 shows the effect of [D-Lys⁶(PpIX)]GnRH and of [D-Lys⁶]GnRH on serum LH concentration in female rats. As shown in FIG. 8, [D-Lys⁶(PpIX)]GnRH was found to be less active at shorter time periods than [D-Lys⁶]GnRH. However, at longer time periods, the conjugate's bioactivity was higher (FIG. 8). For example, the level of LH release induced 24 h after administration of 2 nmol of the conjugate was significantly (p<0.05) higher than that induced by an equimolar amount of the parent peptide (1.7 vs. 1 folds of basal release). These results indicate the long-lasting bioactivity of [D-Lys⁶ (PplX)] GnRH. Long-lasting in vivo bioactivity was also evident when PplX was conjugated to [D-Lys⁶]Antg.

EXAMPLE 5

Phototoxicity of GnRH conjugates in cultured cells

The phototoxicity of the GnRH analog-photosensitizer conjugates and of the unconjugated photosensitizer were evaluated in mouse pituitary gonadotrope cell line (ccT3- 1), in a human breast cancer cell line (MCF-7), and in human prostate cell line (LNCap). αT3-l cells (50,000 cells/well) were plated in 96-well tissue culture plates in 0.1 ml of

DMEM supplemented with 10% FCS and antibiotics. After 24 h, the medium was replaced with fresh medium (but without phenol red and usually without 10% FCS, unless otherwise indicated) containing various concentrations of the tested compounds and cells were incubated for the indicated time periods at 37° C in the dark. The medium was then removed, cells were washed with PBS and irradiated in PBS to activate the photosensitizer moiety. The intensity of the light source in all experiments was 14.3 mW cm^'2 with a total fluent of either 8.5 or 17 J cm^"2. Following illumination, the medium was replaced by DMEM supplemented with 10% FCS and plates were incubated for an additional 24 h at 37°C. Cell survival was then determined using the XTT kit (Biological Industries, Beit-Haemek, Israel) following the manufacturer's protocol. The results indicated that when PplX was used as a photosensitizer, increasing the illumination dose from 8.5 to 17 (J/cm²) in the presence of the conjugates significantly decreased cell survival but did not affect the phototoxicity of PplX. Therefore, the irradiation dose was set at 17 (Jem^" ) for all the photosensitizers used. It should be noted that the presence of GnRH receptors in the αT3-l is well established (Anderson, L., et al. J. Endocrinol. 136: 51-58, 1993) and was also confirmed by receptor- binding experiments. In addition, the conjugates and PplX did not possess any toxicity under dark conditions.

Both agonist and antagonist-derived conjugates were more phototoxic to αT3-l cells than the unconjugated PplX. The LD₅₀ values for [D-Lys⁶(PpIX)]GnRH and [D- Lys⁶(PpIX)]Antg were 95 and 100 nM, respectively, compared to 150 nM for the free PplX. FIGS. 9A-B show the phototoxicity effect of [D-Lys⁶(PpIX)]GnRH and of [D- Lys⁶(PpIX)]Antg on pituitary gonadotrope cell line. Increasing the incubation time from 45 min to 180 min significantly increased the phototoxicity of the conjugates in αT3-l cell line (FIGS. 9A and 9B), whereas it did not affect the phototoxicity of unconjugated PplX (FIG. 9C). The highest change was observed for [D-Lys⁶(PpIX)]Antg, of which the phototoxicity increased by an order of magnitude, from LD₅₀=1.01 μM at 45 min to LD₅₀=0.1 μM at 180 min (FIG. 9B). The optimal period of incubation needed for a GnRH analog to reach binding equilibrium to GnRH receptors was demonstrated to be about 90 min. Therefore, an analog with lower binding affinity, such as [D-Lys⁶(PpIX)]Antg, may require a longer period of incubation in order to reach binding equilibrium. This is consistent with the conjugates that their phototoxicity is being receptor-mediated. Since PplX is known to accumulate in cells through mechanisms that are not receptor-mediated, its phototoxicity was not dependent on incubation time FIG. 9C). It should be noted that the phototoxicity of both [D- Lys⁶ (PplX)] GnRH and PplX was similar and significant in MCF-7 cells at a concentration of O.lμM, reaching maximum at lμM. No toxicity was observed under dark conditions even at a 10 μM concentrations.

FIGS. 10A-B show the phototoxicity effect of [D-Lys⁶(Ce6)]GnRH, [D- Lys⁶(Ce6)]SB75, and Ce6 on pituitary gonadotrope cell line (FIG. 10 A) and on prostate cell line (FIG. 10B). As shown in FIG. 10, the agonistic conjugate, [D-Lys⁶(Ce6)]GnRH, showed lower phototoxicity than that of the antagonistic conjugate, [D-Lys⁶(Ce6)]SB75, and free Ce6 in αT3-l and to LnCaP as compared to. Conjugation of Ce6 to the antagonist increased significantly its phototoxicity to both cell lines.

In order to determine whether the phototoxicity of the PplX conjugates toward cells that express GnRH receptors is receptor-mediated, αT3-l cells were treated for 45 min in the dark with increasing concentrations of the parent peptide [D-Lys⁶]GnRH (1-100 nM) followed by incubation with [D-Lys⁶(PpIX)]GnRH (lμM, 3 h). If phototoxicity is receptor mediated then [D-Lys⁶] GnRH would be expected to compete with [D-Lys⁶(PpIX)]GnRH for the same binding sites on the cell membrane and reduce the binding and the internalization of the latter, and consequently its light-induced phototoxicity. FIG. 11A shows the protective effect of [D-Lys⁶]GnRH against the phototoxicity of

[D-Lys⁶(PpIX)]GnRH. As shown in FIG. 11 A, pre-treatment of αT3-l cells with 0.1 μM of [D-Lys⁶] GnRH resulted in an approximately 40 times increase in the survival rate of the pituitary cell line as compared to cells treated with [D-Lys (PplX)] GnRH alone. On the other hhaanndd,, pprree--ttreatment of αT3-l cells with [D-Lys⁶]GnRH did not affect the phototoxicity of free PplX.

Treatment of wild type MCF-7 cells with [D-Lys⁶(PpIX)]GnRH in the presence of [D- Lys⁶] GnRH did not affect phototoxicity. However, when MCF-7 cells were transfected with human GiiRH-I receptor plasmid DNA (kindly provided by Dr. Z. Naor, Tel Aviv University, Israel) using the calcium phosphate method (Sambrook, J., et al. Molecular cloning: A laboratory Manual, 2nd edition. New York, Cold Spring Harbor Laboratory Press, 1989), similar results to those obtained for αT3-l were observed (FIG. 11B). The results indicated that the phototoxicity induced by the GnRH agonist-PpIX conjugate is mediated through GnRH receptors. It should be noted that each experiment using MCF-7 cells transfected with GnRH-receptor plasmid DNA included MCF-7 cells transfected with a positive control reporter plasmid containing the cytomegalovirus (CMV) promoter to enable estimation of transfection efficiencies and comparison with results obtained from other experiments.

The selectivity of phototoxicity of [D-Lys⁶(PpIX)]Antg to cells that contain GnRH receptors was further evaluated using primary rat pituitary cell cultures. The primary pituitary cell culture, unlike the T3-l cell line, is a heterogeneous cell culture which contains cells that release a variety of hormones, including luteinizing hormone, growth hormone, prolactin, and others. Therefore, a comparison of the release of LH to GH may indicate whether [D-Lys⁶(PpIX)]Antg is preferentially toxic to gonadotropes that express GnRH receptors.

In order to test the selective phototoxicity of [D-Lys⁶(PpIX)]Antg, rat pituitary cells were prepared as described in Example 3 and maintained in DMEM containing 10% horse serum and antibiotics. Cells were then incubated for the indicated time periods in the dark with the tested compounds (lμM) in phenol free and serum free DMEM at 37° C. Following irradiation at an intensity of 14.3 mW cm^" and with a total fluent of 17 J cm^" , the medium was replaced with fresh DMEM containing serum and antibiotics, and the cells were incubated in a humidified incubator at 37° C for an additional 24 h. The supernatants (0.1 ml) were diluted with 1% BSA/PBS (0.9 ml), and LH and GH concentrations were determined by RIA. The fold of selectivity (LH/GH) was calculated using the formula: LH

'GH„

Selectivity =

'-'"^■basal/

'GH basal .

where LΗ_X and GH_X refer to net amount of LH and GH poured out from pituitary cells following irradiation of either [D-Lys⁶ (PplX)] Antg or unconjugated PplX. When primary pituitary cells were treated with PplX or [D-Lys⁶(PpIX)]Antg in the dark, only basal release of LH and GH was detected. Treatment of the cells with PplX followed by illumination brought about an increase in the release of both LH and GH into the medium, indicating that cell death is associated with illumination.

The selective phototoxicity of [D-Lys⁶(PpIX)]Antg in rat pituitary cells is presented in FIG. 12. As shown in FIG. 12, when primary rat pituitary cells were incubated with [D- Lys⁶(PpIX)]Antg for various time intervals prior to illumination, the LH/GH ratios in the medium were significantly higher than those induced by PplX (FIG. 12). For example, the LH/GH ratio in media obtained from pituitary cells incubated for 60 min with 1 μM of [D- Lys⁶(PpIX)]Antg was found to be approximately nine fold higher than the LH/GH ratio in media obtained from cells incubated with unconjugated PplX (FIG. 12). However, when the time of incubation was increased to 3 h the selectivity of the conjugate decreased and became comparable to that of PplX.

It will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described herein above. Rather the scope of the invention is defined by the claims that follow.

Claims

CLAIMS:

1. A GnRH conjugate comprising a GnRH analog coupled to a photosensitizer, or a pharmaceutically acceptable salt or hydrate thereof. 2. The GnRH conjugate according to claim 1, wherein the photosensitizer is selected from the group consisting of porphyrin-based materials, bacteriochlorophylls, purpurins, hypericins, pheophorbides, texaphyrins, phthalocyanines, and derivatives thereof.

3. The GnRH conjugate according to claim 2, wherein the photosensitizer is selected from protoporhyrin IX (PplX), chlorin e6, and AmHypericin. 4. The GnRH conjugate according to claim 1, wherein the GnRH analog is a GnRH agonist having the general formula: pGlu- His-Trp-Y-Tyr-X-Leu-Arg-Pro-Z wherein Y and X are each independently selected from Lys, Dab, Orn, Ser, hSer, D- Lys, D-Dab, D-Orn, D-Ser, D-hSer, Lys(photosensitizer), Dab(photosensitizer), Orn(photosensitizer), Ser(photosensitizer), hSer(photosensitizer), D-

Lys(photosensitizer), D-Dab(photosensitizer), D-Orn(photosensitizer), D- Ser(photosensitizer), D-hSer(photosensitizer), wherein at least one of Y and X is coupled to photosensitizer; Z is selected from Gly-NH2 or ethylamide; or a pharmaceutically acceptable salt or hydrate thereof. 5. The GnRH conjugate according to claim 4, wherein the GnRH analog is a GnRH agonist having the amino acid sequence: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH₂ and designated [D-Lys⁶] GnRH.

6. The GnRH conjugate according to claim 1, wherein the GnRH analog is a GnRH antagonist having the formula of [D-pGlu¹, D-Phe², D-Trp³, D-Lys⁶]GnRH and designated [D-Lys⁶]Antg.

7. The GnRH conjugate according to claim 1, wherein the GnRH analog is a GnRH antagonist having the general formula:

Ac-D-Nal-D-Cpa-D-Pal-X-Y-Z-Leu-W-Pro-D-Ala-NH2 wherein X is selected from Ser, hSer, Lys, Dab, Orn, NicLys, Ser (photosensitizer), hSer (photosensitizer), Lys(photosensitizer), Dab(photosensitizer), Orn(photosensitizer) or NycLys9photosensitizer); Y is selected from Tyr, NicLys, Lys, D-Lys, Dab, Orn, NicLys(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer),

Dab(photosensitizer), or Orn(photosensitizer); Z is selected from D-Ser, D-NicLys, D- iPrLys, D-Lys, Ser(photosensitizer), D-Ser(photosensitizer), D-hSer(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), D-NicLys(photosensitizer),

Dab(photosensitizer), D-Dab(photosensitizer), Orn(photosensitizer), D-

Orn(photosensitizer), or iPrLys(photosensitizer); W is selected from Arg, iPrLys, Ser(photosensitizer), D-Ser(photosensitizer), hSer(photosensitizer), D- hSer(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), NicLys(photosensitizer), D-NicLys(photosensitizer), Dab(photosensitizer),

Orn(photosensitizer), or iPrLys(photosensitizer), wherein at least one of X, Y, Z, and W is coupled to a photosensitizer; or a pharmaceutically acceptable salt or hydrate thereof.

8. The GnRH conjugate according to claim 7, wherein the GnRH analog is a GnRH antagonist having the formula [Acetyl-D-3-(2'-naphtyl)-Ala, D-4-chlorophenylalanine, D-3-(3'-pyridyl)-Ala, Ser, Tyr, D-Lys, Leu, Arg, Pro, D-Ala-amide]GnRH designated

[D-Lys⁶] SB75, or a pharmaceutically acceptable salt or hydrate thereof.

9. The GnRH conjugate according to claim 1, wherein the GnRH analog is a GnRH antagonist having the general formula:

Ac-Nal-D-Cpa-D-Pal-X-Tyr-Y-Leu-Arg-Pro-D-Ala-NH2 wherein X is selected from Ser, Ser(photosensitizer), hSer(photosensitizer); Y is selected from D-Lys, D-Ser, D-Cit, D-Lys(photosensitizer), D-Ser(photosensitizer), D- hSer(photosensitizer), D-Orn(photosensitizer), D-Cit(photosensitizer), D- Dab(photosensitizer), wherein at least one of X and Y is coupled to a photosensitizer; or a pharmaceutically acceptable salt or hydrate thereof. 10. The GnRH conjugate according to claim 1 having the formula [D-Lys⁶(PpIX)]GnRH or a pharmaceutically acceptable salt or hydrate thereof.

11. The GnRH conjugate according to claim 1 having the formula [D-Lys⁶(Ce6)]GnRH or a pharmaceutically acceptable salt or hydrate thereof.

12. The GnRH conjugate according to claim 1 having the formula [D-Lys⁶(AmHyp)]GnRH or a pharmaceutically acceptable salt or hydrate thereof.

13. The GnRH conjugate according to claim 1 having the formula [D-pGlu¹, D-Phe², D-Trp³, D-Lys⁶ (PpIX)]GnRH or a pharmaceutically acceptable salt or hydrate thereof.

14. The GnRH conjugate according to claim 1 having the formula [D-Lys⁶(Ce6)]SB75 or a pharmaceutically acceptable salt or hydrate thereof. 15. A pharmaceutical composition comprising as an active ingredient a GnRH conjugate comprising a GnRH analog coupled to a photosensitizer or a salt or hydrate thereof and a pharmaceutically acceptable carrier or diluent.

16. The pharmaceutical composition according to claim 15, wherein the photosensitizer is selected from the group consisting of porphyrin-based materials, bacteriochlorophylls, purpurins, hypericins, pheophorbides, texaphyrins, phthalocyanines, and derivatives thereof.

17. The pharmaceutical composition according to claim 16, wherein the photosensitizer is selected from protoporhyrin IX (PplX), chlorin e6, and AmHypericin.

18. The pharmaceutical composition according to claim 15, wherein the GnRH analog is a GnRH agonist having the general formula: pGlu- His-Trp-Y-Tyr-X-Leu-Arg-Pro-Z wherein Y and X are each independently selected from Lys, Dab, Orn, Ser, hSer, D- Lys, D-Dab, D-Orn, D-Ser, D-hSer, Lys(photosensitizer), Dab(photosensitizer), Orn(photosensitizer), Ser(photosensitizer), hSer(photosensitizer), D- Lys(photosensitizer), D-Dab(photosensitizer), D-Orn(photosensitizer), D-

Ser(photosensitizer), D-hSer(photosensitizer), wherein at least one of Y and X is coupled to photosensitizer; Z is selected from Gly-NH2 or ethylamide; or a pharmaceutically acceptable salt or hydrate thereof.

19. The pharmaceutical composition according to claim 18, wherein the GnRH analog is a GnRH agonist having the amino acid sequence: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-

Pro-Gly-NH₂ designated [D-Lys⁶]GnRH.

20. The pharmaceutical composition according to claim 15, wherein the GnRH analog is a GnRH antagonist having the formula [D-pGlu¹, D-Phe², D-Trp³, D-Lys⁶]GnRH designated [D-Lys⁶]Antg.

21. The pharmaceutical composition according to claim 15, wherein the GnRH analog is a GnRH antagonist having the general formula:

Ac-D-Nal-D-Cpa-D-Pal-X-Y-Z-Leu-W-Pro-D-Ala-NH2 wherein X is selected from Ser, hSer, Lys, Dab, Orn, NicLys, Ser (photosensitizer), hSer (photosensitizer), Lys(photosensitizer), Dab(photosensitizer), Orn(photosensitizer), or NycLys(photosensitizer); Y is selected from Tyr, NicLys, Lys, D-Lys, Dab, Orn, NicLys(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer),

Dab(photosensitizer), or Orn(photosensitizer); Z is selected from D-Ser, D-NicLys, D- iPrLys, D-Lys, Ser(photosensitizer), D-Ser(photosensitizer), D-hSer(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), D-NicLys(photosensitizer),

Dab(photosensitizer), D-Dab(photosensitizer), Orn(photosensitizer), D-

Orn(photosensitizer), or iPrLys(photosensitizer); W is selected from Arg, iPrLys, Ser(photosensitizer), D-Ser(photosensitizer), hSer(photosensitizer), D- hSer(photosensitizer), Lys(photosensitizer), D-Lys(photosensitizer), NicLys(photosensitizer), D-NicLys(photosensitizer), Dab(photosensitizer),

Orn(photosensitizer), or iPrLys(photosensitizer), wherein at least one of X, Y, Z, and W is coupled to a photosensitizer; or a pharmaceutically acceptable salt or hydrate thereof.

22. The pharmaceutical composition according to claim 21, wherein the GnRH analog is a GnRH antagonist having the formula [Acetyl-D-3-(2'-naphtyl)-Ala, D-4- chlorophenylalanine, D-3-(3'-pyridyl)-Ala, Ser, Tyr, D-Lys, Leu, Arg, Pro, D-Ala- amide]GnRH designated [D-Lys⁶]SB75, or a pharmaceutically acceptable salt or hydrate thereof.

23. The pharmaceutical composition according to claim 15, wherein the GnRH analog is a GnRH antagonist having the general formula: Ac-Nal-D-Cpa-D-Pal-X-Tyr-Y-Leu-Arg-Pro-D-Ala-NH2 wherein X is selected from Ser, Ser(photosensitizer), hSer(photosensitizer); Y is selected from D-Lys, D-Ser, D-Cit, D-Lys(photosensitizer), D-Ser(photosensitizer), D- hSer(photosensitizer), D-Orn(photosensitizer), D-Cit(photosensitizer), D-

Dab(photosensitizer), wherein at least one of X and Y is coupled to a photosensitizer; or a pharmaceutically acceptable salt or hydrate thereof.

24. The pharmaceutical composition according to claim 15, wherein the GnRH conjugate having the formula [D-Lys⁶(PpIX)]GnRH or a pharmaceutically acceptable salt or hydrate thereof.

25. The pharmaceutical composition according to claim 15, wherein the GnRH conjugate having the formula [D-Lys⁶(Ce6)]GnRH or a pharmaceutically acceptable salt or hydrate thereof.

26. The pharmaceutical composition according to claim 15, wherein the GnRH conjugate having the formula [D-Lys⁶ (AmHyp)] GnRH or a pharmaceutically acceptable salt or hydrate thereof. 27. The pharmaceutical composition according to claim 15, wherein the GnRH conjugate having the formula [D-pGlu¹, D-Phe², D-Trp³, D-Lys⁶ (PpIX)]GnRH or a pharmaceutically acceptable salt or hydrate thereof.

28. The pharmaceutical composition according to claim 15, wherein the GnRH conjugate having the formula of [D-Lys⁶(Ce6)]SB75 or a pharmaceutically acceptable salt or hydrate thereof.

29. A method for treating a sex hormone-dependent disease or condition comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising as an active ingredient a GnRH conjugate comprising a GnRH analog coupled to a photosensitizer or a pharmaceutically acceptable salt or hydrate thereof and a pharmaceutically acceptable carrier, the GnRH conjugate binds to cells having cell membrane GnRH receptors.

30. The method according to claim 29, further comprising irradiating the cells in an afflicted area with light or X-ray radiation having a wavelength within a photoactivating action spectrum of the photosensitizer to produce a phototoxic or cytotoxic effect. 31. The method according to any one of claims 29 and 30, wherein the sex hormone- dependent disease is selected from the group consisting of malignant and non-malignant sex hormone-dependent diseases.

32. The method according to claim 31, wherein the malignant sex hormone-dependent disease is selected from the group consisting of prostate cancer, breast cancer, ovarian cancer, cervical cancer, tumor of the pituitary, testicular cancer, and uterine cancer.

3. The method according to claim 31, wherein the non-malignant sex hormone-dependent disease is selected from the group consisting of benign prostatic hyperplasia, precocious puberty, aberrant sexual behavior, late luteal phase dysphoric disorder, fibroids, endometriosis, myoma, hirsutism, cyclic auditory dysfunction, porphyria, and polycystic ovarian syndrome.