Peptide derivatives and conjugates thereof for treating cancer

Abstract

To provide luteinizing hormone-releasing hormone (LHRH) peptide derivatives that target the LHRH receptor; LHRH peptide-drug conjugates (LHRH-PDCs); and methods of using the derivatives and / or conjugates thereof to treat an LHRH receptor expressing cancer.SOLUTION: The present invention provides a LHRH peptide-drug conjugate (LHRH-PDC) or a pharmaceutically acceptable salt thereof, comprising the following sequence: X1-His-Trp-Ser-X2-X3(L-D)-X4-X5-Pro-NHR, where X1 is pGlu; X2 is Tyr, Phe, or His; X3 is D-Lys(Ahx); X4 is Leu, Val, Trp, or Met; X5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr, or Lys; R is CH2CH3 or CH3; L is a linker; and D is a cytotoxic agent.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to LHRH peptide derivatives that target luteinizing hormone-releasing hormone (LHRH) receptors. In particular, LHRH peptide derivatives, LHRH peptide-drug conjugates (LHRH-PDCs), and methods of treating LHRH receptor-expressing cancers using such derivatives and / or conjugates are described.

Background Art

[0002] Any discussion of prior art throughout the specification should never be construed as an admission that such prior art is widely known or forms part of the common general knowledge in the art.

[0003] Triple-negative breast cancer (TNBC) constitutes 15% - 20% of all breast cancers and is immunohistochemically defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression (A. Diana et al., Curr. Oncol. Rep., 2008, 20; A.C. Garrido-Castro et al., Cancer Discov., 2019, 9). While TNBC is often more chemosensitive than other types of breast cancer, it is a more aggressive form with a higher prevalence in younger women and a risk of recurrence within the first 3 - 5 years after completion of adjuvant chemotherapy. Over the past few decades, therapeutic progress in treating TNBC has been limited, and chemotherapy remains the standard treatment. Thus, there is an urgent unmet need for the development of targeted therapeutics with increased selectivity and efficacy and reduced toxicity for this patient population.

[0004] Targeted therapy can be achieved through a targeted delivery system centered on antibody-drug conjugates (ADCs) and peptide-drug conjugates (PDCs) (JM Reichert, MAbs, 2011, 3). ADCs are generally associated with many drawbacks, including high production costs, structural heterogeneity, low coupling ratios of antibodies to cytotoxic drugs, and limited tumor penetration (MA Firer & G Gellarman, J Hematol Oncol, 2012, 5). The LHRH receptor is expressed on various tumors, including breast cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, and pancreatic cancer. For example, approximately 74% of TNBC expresses the receptor for LHRH, and a ligand for this receptor potential carrier enables the delivery of a cytotoxic agent directly to these cancerous cells (C Fost, Oncol Rep, 2011, 25). LHRH-conjugated doxorubicin reached clinical trials for prostate cancer, but this PDC failed in phase III clinical trials. Currently, there is still no targeted therapy available for TNBC patients. Thus, a targeted delivery system for treating cancer patients, especially TNBC patients, is needed.

[0005] An object of the present invention is to overcome or improve at least one of the drawbacks of the prior art or to provide a useful alternative.

Summary of the Invention

[0006] The present invention relates to LHRH peptide derivatives and / or LHRH peptide-drug conjugates (LHRH-PDCs). The LHRH peptide derivatives described herein exhibit high affinity for their receptors. The LHRH peptide derivatives described herein have an improved half-life compared to native LHRH peptides and known LHRH agonists such as [w 6 LHRH and [k 6 LHRH.

[0007] In some embodiments, the LHRH peptide derivatives had high enzymatic activity. In certain embodiments, the LHRH peptide derivatives had high efficacy.

[0008] The LHRH peptide derivatives described herein exhibit direct anti-proliferative activity. In certain embodiments, the LHRH peptide derivatives exhibit higher anti-proliferative activity compared to both native LHRH and agonist 6 LHRH in breast cancer cell lines including TNBC cell models. As described herein, the LHRH peptide derivatives of the invention are LHRH receptor agonists.

[0009] Provided is an LHRH peptide derivative that is conjugated to a cytotoxic agent via a linker or is conjugated to a cytotoxic agent.

[0010] Provided is a method for treating LHRH receptor-expressing cancer, comprising administering an LHRH peptide derivative and / or an LHRH peptide-drug conjugate (LHRH-PDC). In particular, a method for treating TNBC comprising administering an LHRH peptide derivative and / or an LHRH-PDC is provided.

[0011] Provided is a method of treating cancer, comprising administering an LHRH peptide derivative and / or an LHRH-PDC. Exemplary cancers that can be treated include, but are not limited to, cancers that target LHRH receptor expression, such as breast cancer, prostate cancer, colon cancer, ovarian cancer, endometrial cancer, and the like. In particular, a method for targeting LHRH receptor-expressing cancer in TNBC is provided.

[0012] In a first aspect, the present invention provides an LHRH peptide-drug conjugate (LHRH-PDC) comprising the following sequence or a pharmaceutically acceptable salt thereof. X1-His-Trp-Ser-X2-X3(L-D)-X4-X5-Pro-NHR (wherein, X1 is pGlu or Gln; X2 is Tyr, Phe or His; X3 is D-Lys or D-Lys(Ahx); X4 is Leu, Val, Trp, or Met; X5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; R is CH2CH3 or CH3; wherein, L is a linker; D is a cytotoxic agent) In certain embodiments, X1 is Gln, X3 is D-Lys, and R is CH2CH3. In some embodiments, X1 is pGlu, X3 is D-Lys, and R is CH2CH3. In some embodiments, X1 is pGlu, X3 is D-Lys(Ahx), and R is CH2CH3. In certain embodiments, X1 is Gln, X2 is Tyr, X3 is D-Lys, X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, X1 is pGlu, X2 is Tyr, X3 is D-Lys, X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, X1 is pGlu, X2 is Tyr, X3 is D-Lys(Ahx), X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, the linker is a cleavable linker, e.g., a dipeptide-based linker with or without PAB (p-aminobenzyl alcohol), or a non-peptide cleavable linker such as a glucuronide linker incorporating a hydrophilic sugar group cleaved by b-glucuronidase. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the cleavable linker is a self-immolative linker. In related embodiments, maleimidocaproyl valine-citrulline-p-aminobenzyl carbamoyl (mc-vc-PABC) serves as a self-immolative linker. In certain embodiments, the cytotoxic agent is an anti-mitotic agent, an alkylating agent, an anti-metabolite, a topoisomerase inhibitor or a protein kinase inhibitor. In some embodiments, the cytotoxic agent is monomethyl auristatin E (MMAE).

[0013] In certain embodiments, the LHRH peptide derivative can be fused to a cytotoxic agent. In related embodiments, the LHRH peptide derivative is fused to the cytotoxic agent via a non-cleavable linker.

[0014] In a second aspect, the present invention provides an anti-proliferative LHRH peptide derivative comprising the following sequence or a pharmaceutically acceptable salt thereof. X1-His-Trp-Ser-X2-X3-X4-X5-Pro-NHR (Wherein, X1 is pGlu or Gln; X2 is Tyr, Phe or His; X3 is D-Lys or D-Lys(Ahx); X4 is Leu, Val, Trp, or Met; X5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys, and R is CH2CH3 or CH3). In certain embodiments, X1 is Gln, X3 is D-Lys, and R is CH2CH3. In some embodiments, X1 is pGlu, X3 is D-Lys, and R is CH2CH3. In some embodiments, X1 is pGlu, X3 is D-Lys(Ahx), and R is CH2CH3. In certain embodiments, X1 is Gln, X2 is Tyr, X3 is D-Lys, X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, X1 is pGlu, X2 is Tyr, X3 is D-Lys, X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, X1 is pGlu, X2 is Tyr, X3 is D-Lys(Ahx), X4 is Leu, X5 is Arg, and R is CH2CH3.

[0015] In a third aspect, the present invention provides an LHRH peptide derivative comprising the sequence: Gln-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH2CH3 or a pharmaceutically acceptable salt thereof.

[0016] In a fourth aspect, the present invention provides an LHRH peptide derivative comprising the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH2CH3 or a pharmaceutically acceptable salt thereof.

[0017] In a fifth aspect, the present invention provides an LHRH peptide derivative comprising the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys(Ahx)-Leu-Arg-Pro-NHCH2CH3 or a pharmaceutically acceptable salt thereof.

[0018] In some embodiments, the half-life of the LHRH peptide derivative is at least about 200 minutes. In certain embodiments, the LHRH peptide derivative has a half-life of from about 200 minutes to about 250 minutes. In some embodiments, the LHRH peptide derivative has a half-life of from about 250 minutes to about 300 minutes. In some embodiments, the LHRH peptide derivative has a half-life of from about 300 minutes to about 350 minutes. In certain embodiments, the LHRH peptide derivative has a half-life of about 365 minutes.

[0019] In some embodiments, the LHRH-PDC comprises an LHRH peptide derivative conjugated to a cytotoxic agent via a self-cleaving linker. In certain embodiments, the LHRH-PDC comprises an LHRH peptide derivative conjugated to MMAE via a self-cleaving linker. In certain embodiments, the LHRH-PDC comprises an LHRH peptide derivative conjugated to MMAE via mc-vc-PABC.

[0020] In a sixth aspect, the present invention provides an LHRH-PDC comprising an LHRH peptide derivative comprising the sequence: Gln-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH2CH3 or a pharmaceutically acceptable salt thereof, wherein the LHRH peptide derivative is conjugated to MMAE via mc-vc-PABC.

[0021] In a seventh aspect, the present invention provides an LHRH-PDC comprising an LHRH peptide derivative having the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH2CH3 or a pharmaceutically acceptable salt thereof, wherein the LHRH peptide derivative is conjugated to MMAE via mc-vc-PABC.

[0022] In an eighth aspect, the present invention provides an LHRH-PDC comprising an LHRH peptide derivative having the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys(Ahx)-Leu-Arg-Pro-NHCH2CH3 or a pharmaceutically acceptable salt thereof, wherein the LHRH peptide derivative is conjugated to MMAE via mc-vc-PABC.

[0023] In a ninth aspect, the present invention provides a pharmaceutical composition comprising an LHRH-PDC or a pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable excipient.

[0024] In a tenth aspect, the present invention provides a pharmaceutical composition comprising an LHRH peptide derivative of the present invention or a pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable excipient.

[0025] In an eleventh aspect, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an LHRH-PDC of the present invention or a pharmaceutically acceptable salt thereof, an LHRH peptide derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, wherein the subject has an LHRH receptor-expressing cancer. In some embodiments, the LHRH receptor-expressing cancer is breast cancer, prostate cancer, colorectal cancer, ovarian cancer, pancreatic cancer or endometrial cancer. In some embodiments, the LHRH receptor-expressing cancer is TNBC. In certain embodiments, the method of treating cancer comprises administering an LHRH-PDC, wherein the cancer is TNBC.

[0026] In a twelfth aspect, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an LHRH-PDC or a pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable excipient having one or more additional cytotoxic agents, wherein the subject has an LHRH receptor-expressing cancer. In some embodiments, the LHRH receptor-expressing cancer is breast cancer, prostate cancer, colorectal cancer, ovarian cancer, pancreatic cancer or endometrial cancer. In some embodiments, the LHRH receptor-expressing cancer is TNBC.

[0027] In a thirteenth aspect, the present invention provides a method of arresting and / or delaying the cell proliferation and / or growth of LHRH receptors expressed by tumors in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an LHRH-PDC of the present invention or a pharmaceutically acceptable salt thereof, an LHRH peptide derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition.

[0028] In a fourteenth aspect, the present invention provides a method of arresting and / or delaying the cell proliferation and / or growth of LHRH receptors expressed by tumors in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an LHRH-PDC or a pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable excipient and one or more additional cytotoxic agents.

[0029] In a fifteenth aspect, the present invention provides a method of treating hormone-sensitive and / or refractory breast cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide conjugate of the present invention, an LHRH peptide derivative or a pharmaceutical composition of the present invention.

[0030] In a 16th aspect, the present invention provides a method of treating hormone-sensitive and / or refractory breast cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an LHRH-PDC or a pharmaceutically acceptable salt, and optionally at least one pharmaceutically acceptable excipient and one or more additional cytotoxic agents.

[0031] In a 17th aspect, the present invention provides the use of an LHRH-PDC of the present invention or a pharmaceutically acceptable salt thereof, an LHRH peptide derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, in the manufacture of a medicament for treating cancer, wherein the cancer is an LHRH receptor-expressing cancer.

[0032] In an 18th aspect, the present invention provides the use of an LHRH-PDC of the present invention or a pharmaceutically acceptable salt thereof, an LHRH peptide derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, in the manufacture of a medicament for arresting or delaying the cell growth and / or proliferation of LHRH receptors expressed by tumors.

[0033] In a 19th aspect, the present invention provides the use of an LHRH-PDC of the present invention or a pharmaceutically acceptable salt thereof, an LHRH peptide derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, in the manufacture of a medicament for treating hormone-sensitive and / or refractory breast cancer.

[0034] In a 20th aspect, the present invention provides the use of an LHRH-PDC of the present invention or a pharmaceutically acceptable salt thereof, an LHRH peptide derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, in the manufacture of a medicament for treating TNBC.

[0035] The method for synthesizing and / or producing the LHRH peptide derivatives and / or LHRH-PDCs disclosed herein is not particularly limited, and any suitable method may be used. <Definition>

[0036] Unless the context clearly requires otherwise, throughout the description and claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

[0037] As used herein, the singular forms "a", "an", and "the" when used in this application mean "one or more", so that, for example, a reference to "a sample" includes a plurality of such samples and the like.

[0038] The term "about" as used herein can be within one or more standard deviations of the practice in the art. Alternatively, "about" should be assumed to be within an acceptable error range for that particular value. For example, in the context of a half-life value, the term "about" can mean a range of up to 10%.

[0039] The term "luteinizing hormone-releasing hormone" or "LHRH" is also known in the art as "gonadotropin-releasing hormone (GnRH)" or "luteinizing hormone-releasing factor (LRF)". LHRH is translated from mRNA as a prohormone and converted to a mature decapeptide with the native sequence pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2. All of the amino acids of the native LHRH peptide are of the L-type.

[0040] The term "peptide" as used herein includes, but is not limited to, two or more amino acids or residues covalently linked by an amide bond or an equivalent. In certain embodiments, the amino acids can be linked by non-natural and non-amide chemical bonds including, but not limited to, D-Lys, Pro-Et, or Ahx.

[0041] The term "amino acid" includes well-known amino acids such as alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); or glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S) or threonine (Thr or T); or tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V). The three-letter or one-letter abbreviations of the above amino acids are known and standard in the art. The amino acids described herein may be in the "L" or "D" stereoisomeric form, except for Gly. Stereoisomeric forms can be designated by including "D" or "L" in the standard three-letter or one-letter abbreviations, such as D-Lys and L-Lys. The one-letter code amino acids in the above cases are of the L form, while the one-letter code amino acids in the following cases are of the D form. When there is no designation of "D" or "L", the amino acid with a three-letter abbreviation is of the "L" form. Non-traditional amino acids are also within the scope of the present invention and include norleucine, ornithine, norvaline, homoserine, and other amino acid analogs. Preferably, non-traditional amino acids are used in place of D-Lys. The term amino acid residue means an amino acid contained in a peptide. It will be understood that the amino acid residue is at the N-terminus or C-terminus of the peptide.

[0042] The term "pGlu" or "pyroGlu" is known in the art and means L-pyroglutamic acid.

[0043] The term "NHEt" is known in the art and refers to N-ethylamide. It is also known as ethylmaleimide or NEM.

[0044] The term "Ahx" is known in the art and refers to 6-aminohexanoic acid.

[0045] The terms "LHRH peptide derivative" or "peptide derivative" are used interchangeably herein and refer to a native LHRH peptide comprising at least two modifications, where the at least two modifications include substitution of the 6th amino acid residue Gly with D-Lys or D-Lys(Ahx) and substitution of the 10th amino acid residue Gly with NHR, where R is defined herein. The LHRH peptide derivative may comprise at least two or more modifications, where the at least two modifications include substitution of the 6th amino acid residue Gly with D-Lys or D-Lys(Ahx), and substitution of the 10th amino acid residue Gly with NHR, where R is defined, and further modifications include substitution of the 1st amino acid residue pGlu with Gln. In some embodiments, the LHRH peptide derivative may comprise additional modifications at amino acid residue positions 5, 7, and / or 8.

[0046] As used herein, the terms "conjugate", "peptide conjugate" or "LHRH peptide-drug-conjugate (LHRH-PDC)" are used interchangeably and mean a molecule comprising an LHRH peptide derivative conjugated to a cytotoxic agent via a linker.

[0047] The term "linker" means a moiety intended to covalently bond or join a cell targeting enhancing moiety, such as an LHRH peptide derivative and a cytotoxic agent. The linker used herein may be a cleavable linker or a non-cleavable linker. In the context of the LHRH peptide derivatives described herein, the linker is covalently bonded to the LHRH peptide derivative via the 6th amino acid residue (D-Lys or D-Lys(Ahx)) or the C- or N-terminus.

[0048] As used herein, the term "cytotoxic agent" includes, but is not limited to, anti-mitotic agents, alkylating agents, anti-metabolites, topoisomerase inhibitors or protein kinase inhibitors. Cytotoxic agents can be vinca alkaloids, cryptophycins, bortezomib, thio-bortezomib, tubulysins, aminopterin, rapamycin, paclitaxel, docetaxel, daunorubicin, everolimus, a-amanitin, bemcarlit, didemnin B, geldanamycin, purvalanol A, ispinesib, budesonide, dasatinib, epothilones, maytansines, doxorubicin, camptothecin, methotrexate (MTX) or monomethyl auristatin E (MMAE). In certain embodiments, the cytotoxic agent is MMAE.

[0049] The method for preparing the LHRH peptide derivative and / or LHRH-PDC is not particularly limited. Exemplary methods include Fmoc solid-phase chemistry and click chemistry. It will be recognized that LHRH peptide derivatives may be commercially available. Further, it is understood that commercially available kits may be available for conjugating a cytotoxic agent, such as MMAE, to the LHRH peptide derivatives described herein.

[0050] Also contemplated are pharmaceutically acceptable salts of the LHRH peptide derivative and / or LHRH-PDC. The term "pharmaceutically acceptable salts" includes both acid and base addition salts, and means salts that retain the biological effectiveness and properties of the free base or acid and that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts can be formed with inorganic or organic acids or bases and can be prepared by reacting the purified compound in its free base or acid form separately with a suitable inorganic or organic acid or base and isolating the salt thus formed, during the final isolation and purification of the compound.

[0051] As used herein, the term "pharmaceutical composition" or "composition" means a mixture of at least one LHRH peptide derivative or LHRH-PDC, or a pharmaceutically acceptable salt, solvate, hydrate thereof, and other chemical components such as pharmaceutically acceptable excipients. Pharmaceutical compositions suitable for the delivery of the peptide derivatives or conjugates described herein and methods for their preparation will be apparent to those skilled in the art.

[0052] Also contemplated are pharmaceutical compositions comprising at least one LHRH peptide derivative and / or LHRH-PDC, and optionally at least one pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" means any pharmaceutically acceptable inert component of the composition. As is known in the art, excipients include diluents, buffers, binders, lubricants, disintegrants, colorants, antioxidants / preservatives, pH adjusters, and the like. The excipient is selected based on the desired physical form of the final product: for example, a parenteral formulation for injection, a tablet having the desired hardness and brittleness, which disperses rapidly and is easy to swallow. Suitable forms of the pharmaceutical composition include, but are not limited to, tablets, capsules, elixirs, liquid formulations, sustained release or extended release agents, etc. The intended physical form and / or contents of the pharmaceutical composition are conventional formulations that can be formulated by those skilled in the pharmaceutical formulation art.

[0053] The cancers described herein that express the LHRH receptor include neoplastic cancer cell populations, including benign and malignant tumors, or non-neoplastic cancer cell populations. Methods for determining LHRH receptor expression in cancer are not particularly limited. Exemplary methods include Western blotting, immunocytochemistry, flow cytometry, and PCR (polymerase chain reaction). Exemplary cancers include, but are not limited to, carcinomas, sarcomas, lymphomas, melanomas, mesotheliomas, nasopharyngeal cancers, leukemias, adenocarcinomas, and myelomas. In certain embodiments, the cancer is lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, uveal melanoma, uterine cancer, ovarian cancer, endometrial cancer, leiomyosarcoma, rectal cancer, gastric cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, small cell lung cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer, Burkitt lymphoma, ureteral cancer, kidney cancer, renal cell cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, spinal cord tumors, brainstem glioma, pituitary adenocarcinoma, cholangiocarcinoma, Hurthle cell thyroid cancer, or gastroesophageal junction adenocarcinoma.

[0054] It is also contemplated that LHRH peptide derivatives and / or LHRH-PDCs can be delivered to cancer cells in vitro or in vivo. In some embodiments, the LHRH-PDC is administered to cancer cells in vitro or in vivo. In certain embodiments, the LHRH peptide derivative is administered to cancer cells in vitro or in vivo and exhibits anti-proliferative activity. The LHRH peptide derivative and / or LHRH-PDC can be administered to cells together with a pharmaceutically acceptable carrier in a composition, as described herein.

[0055] As used herein, the term "tumor" means neoplastic cell growth and proliferation, and premalignant and malignant cells and tissues, whether malignant or benign. For example, certain cancers can be characterized by a solid mass tumor. The term "tumor" includes solid tumors and non-solid tumors. The LHRH peptide derivatives, LHRH-PDCs, or compositions described herein can be administered to a subject locally (e.g., by direct injection) or remotely (e.g., by systemic administration) at the site of a tumor. In some embodiments, the LHRH peptide derivatives, LHRH-PDCs, or compositions described herein can be administered systemically to a subject, e.g., intravascularly such as by intravenous administration.

[0056] The term "antiproliferative activity" means that a compound can stop the proliferation of cells.

[0057] A "subject" to be treated by the methods described herein includes mammals, including humans ("patients") or non-human subjects (e.g., cats, dogs, etc.). The LHRH peptide derivatives, LHRH-PDCs, or compositions described herein can be administered to a human or non-human subject. The LHRH peptide derivatives, LHRH-PDCs, or compositions described herein can be administered to human cancer cells or non-human cancer cells in vitro or in vivo. In certain embodiments, the cells are mammalian cells.

[0058] As used herein, a "therapeutically effective amount" of an LHRH peptide derivative, LHRH-PDC, or composition, when administered (either as a single dose or over the course of multiple treatments), includes an amount that promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or a prevention of disability or impairment due to disease pain. A therapeutically effective amount of an LHRH peptide derivative, LHRH-PDC, or composition described herein, when administered to a subject at risk of developing a disease or at risk of suffering a recurrence of a disease, includes a "prophylactically effective amount" which is any amount of the LHRH peptide derivative, LHRH-PDC, or composition that inhibits the onset or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the occurrence or recurrence of a disease can be evaluated by assaying the activity of the agent in in vitro assays, or using various methods known to those of skill in the art, such as animal model systems that predict efficacy in humans. As an example for the treatment of cancer, a therapeutically effective amount of an LHRH peptide derivative, LHRH-PDC, or composition described herein can inhibit the growth of cancer cells by at least about 20%, at least about 40%, at least about 60%, or at least about 80% relative to untreated cancer cells. In some embodiments, for the treatment of cancer, a therapeutically effective amount of an LHRH peptide derivative as described herein can inhibit the growth of cancer cells by up to about 60%. In some embodiments, for the treatment of cancer, a therapeutically effective amount of an LHRH-PDC as described herein can inhibit the growth of cancer cells by up to about 80%. A therapeutically effective amount of an LHRH peptide derivative, LHRH-PDC, or composition described herein can completely inhibit cell growth or tumor growth. A therapeutically effective amount of an LHRH peptide derivative, LHRH-PDC, or composition described herein can inhibit or decrease cell growth or tumor growth to a statistically significant extent as compared to a control. "Statistical significance" means significance at the p<0.05 level, or other measures of statistical significance as used by those of skill in biomedical statistics in the context of a particular type of treatment or prevention.

[0059] Depending on the cancer type described in this specification, a wide range of acceptable dosages are contemplated depending on whether the route of administration and / or the LHRH peptide derivatives, LHRH-PDCs, or compositions described herein are administered locally or systemically. Suitable dosages include dosages falling within the range of from about 0.5 mg / kg to about 5 mg / kg. The dosage may be single or divided and may be administered according to a variety of protocols including q.d., b.i.d., t.i.d., or every other day, every other week (b.i.w.), once a week, once a month, once a quarter, etc. In each of these cases, the therapeutically effective amount described herein is understood to correspond to the example of administration as determined by the administration protocol or, alternatively, to the total daily dosage, total weekly dosage, total monthly dosage, or total quarterly dosage.

[0060] Also, it is contemplated that the LHRH peptide derivatives, LHRH-PDCs, or compositions described herein may be administered together with one or more cytotoxic agents. Administration as an LHRH peptide derivative, LHRH-PDC, or as a composition described herein with one or more additional cytotoxic agents may include co-administration or sequential administration. Sequential administration includes an administration schedule in which the administration of the LHRH peptide derivative, LHRH-PDC, or composition described herein and one or more cytotoxic agents are separated by one hour or more, or one day or more.

[0061] As described herein, in vitro metabolic stability in plasma is defined as the sensitivity of the compound to in vivo conversion in plasma and is represented as the in vitro half-life (T 1 / 2 ).

Brief Description of the Drawings

[0062] Embodiments of the present invention are described herein by way of example only, with reference to the following accompanying drawings.

[0063]

FIG. 1A-1B

[0064]

FIG. 2

[0065]

FIG. 3A-3B

[0066]

FIG. 4A-4E

[0067]

FIG. 5A-5B

[0068]

FIG. 6

[0069]

FIG. 7A-7B

[0070]

FIG. 8A-8B

[0071]

FIG. 9A-9B

[0072]

FIG. 10A-10B

[0073]

FIG. 11

[0074] In one embodiment, there is provided an LHRH-PDC or a pharmaceutically acceptable salt thereof comprising the following sequence. X1-His-Trp-Ser-X2-X3(L-D)-X4-X5-Pro-NHR In the formula, X1 is pGlu or Gln; X2 is Tyr, Phe or His; X3 is D-Lys or D-Lys(Ahx); X4 is Leu, Val, Trp, or Met; X5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; R is CH2CH3 or CH3; In the formula, L is a linker; D is a cytotoxic agent. In certain embodiments, X1 is Gln, X3 is D-Lys, and R is CH2CH3. In some embodiments, X1 is pGlu, X3 is D-Lys, and R is CH2CH3. In some embodiments, X1 is pGlu, X3 is D-Lys(Ahx), and R is CH2CH3. In certain embodiments, X1 is Gln, X2 is Tyr, X3 is D-Lys, X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, X1 is pGlu, X2 is Tyr, X3 is D-Lys, X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, X1 is pGlu, X2 is Tyr, X3 is D-Lys(Ahx), X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the cleavable linker is a self-immolative linker. In related embodiments, maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamoyl (mc-vc-PABC) serves as the self-immolative linker. In certain embodiments, the cytotoxic agent is an anti-mitotic agent, an alkylating agent, an anti-metabolite, a topoisomerase inhibitor, or a protein kinase inhibitor. In some embodiments, the cytotoxic agent is selected from the group consisting of vinca alkaloids, cryptophycins, bortezomib, thio-bortezomib, tubulysin, aminopterin, rapamycin, paclitaxel, docetaxel, daunorubicin, everolimus, a -amanitin, bemcentinib, didemnin B, geldanamycin, purvalanol A, ispinesib, budesonide, dasatinib, epothilone, maytansine, doxorubicin, camptothecin, methotrexate (MTX), or monomethyl auristatin E (MMAE). In certain embodiments, the cytotoxic agent is MMAE.

[0075] In some embodiments, the linker comprises at least one amino acid. In certain embodiments, the linker comprises one or more amino acid residues where the amino acid is Lys, Asn, Thr, Ser, He, Met, Pro, His, Gin, Arg, Gly, Asp, Glu, Ala, Vai, Phe, Leu, Tyr, Cys, and / or Trp. In some embodiments, the linker comprises a carbon chain, an amide bond or an ether bond. In some embodiments, the linker comprises a hydrazone bond, a vinyl ether bond, an acetal bond, a ketal bond or a disulfide bond. In certain embodiments, the linker comprises Gly-Phe-Leu-Gly. In some embodiments, the linker consists of Val-Cit (Cit = citrulline). In some embodiments, the linker comprises Phe-Lys. In some embodiments, the linker is a polyethylene glycol (PEG) chain, an acetate linker, an ester linker, a lectin, buSS (disulfirubutyrate) or a maleimide.

[0076] In a further embodiment, there is provided an LHRH-PDC or a pharmaceutically acceptable salt thereof comprising the following sequence. X1-His-Trp-Ser-X2-X3(L-D)-X4-X5-Pro-NHR Wherein, X1 is pGlu or Gln; X2 is Tyr, Phe or His; X3 is D-Lys or D-Lys(Ahx); X4 is Leu, Val, Trp, or Met; X5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; R is CH2CH3 or CH3; Wherein, L is a linker; D is a cytotoxic agent. In some embodiments, X1 is Gln. In some embodiments, X1 is pGlu. In some embodiments, X1 is Gln and X2 is Tyr. In some embodiments, X1 is Gln and X2 is Phe. In some embodiments, X1 is Gln and X2 is His. In some embodiments, X3 is D-Lys. In some embodiments, X3 is D-Lys(Ahx). In some embodiments, X1 is Gln and X4 is Leu. In some embodiments, X1 is Gln and X4 is Val. In some embodiments, X1 is Gln and X4 is Trp. In some embodiments, X1 is Gln and X4 is Met. In some embodiments, X1 is Gln and X5 is Arg. In some embodiments, X1 is Gln and X5 is Gln. In some embodiments, X1 is Gln and X5 is Trp. In some embodiments, X1 is Gln and X5 is Ser. In some embodiments, X1 is Gln and X5 is Leu. In some embodiments, X1 is Gln and X5 is Asn. In some embodiments, X1 is Gln and X5 is Phe. In some embodiments, X1 is Gln and X5 is Tyr. In some embodiments, X1 is Gln and X5 is Lys. In certain embodiments, R is CH2CH3. In certain embodiments, R is CH3.

[0077] In certain embodiments, the LHRH peptide derivative can be fused to a cytotoxic agent. In related embodiments, the LHRH peptide derivative is fused to the cytotoxic agent via a non-cleavable linker.

[0078] In a further embodiment, there is provided an antiproliferative LHRH peptide derivative comprising the following sequence or a pharmaceutically acceptable salt thereof: X1-His-Trp-Ser-X2-X3-X4-X5-Pro-NHR wherein, X1 is pGlu or Gln; X2 is Tyr, Phe or His; X3 is D-Lys or D-Lys(Ahx); X4 is Leu, Val, Trp, or Met; X5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys R is CH2CH3 or CH3. In certain embodiments, X1 is Gln, X3 is D-Lys, and R is CH2CH3. In some embodiments, X1 is pGlu, X3 is D-Lys, and R is CH2CH3. In some embodiments, X1 is pGlu, X3 is D-Lys(Ahx), and R is CH2CH3. In certain embodiments, X1 is Gln, X2 is Tyr, X3 is D-Lys, X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, X1 is pGlu, X2 is Tyr, X3 is D-Lys, X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, X1 is pGlu, X2 is Tyr, X3 is D-Lys(Ahx), X4 is Leu, X5 is Arg, and R is CH2CH3.

[0079] In a further embodiment, there is provided an LHRH peptide derivative comprising the following sequence or a pharmaceutically acceptable salt thereof: X1-His-Trp-Ser-X2-X3-X4-X5-Pro-NHR wherein, X1 is Gln; X2 is Tyr, Phe or His; X3 is D-Lys or D-Lys(Ahx); X4 is Leu, Val, Trp, or Met; X5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys R is CH2CH3 or CH3. In certain embodiments, X3 is D-Lys and R is CH2CH3. In certain embodiments, X2 is Tyr, X3 is D-Lys, X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, X2 is Tyr, X3 is D-Lys(Ahx), X4 is Leu, X5 is Arg, and R is CH2CH3. In certain embodiments, R is CH2CH3. In certain embodiments, R is CH3. In some embodiments, the present invention includes an LHRH peptide derivative comprising the sequence: Gln-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH2CH3, wherein the LHRH peptide derivative provides an LHRH peptide conjugate that is linked to a cytotoxic agent via a self-cleaving linker.

[0080] In some embodiments, the present invention includes an LHRH peptide derivative comprising the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH2CH3, wherein the LHRH peptide derivative provides an LHRH peptide conjugate that is linked to a cytotoxic agent via a self-cleaving linker.

[0081] In some embodiments, the present invention includes an LHRH peptide derivative comprising the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys(Ahx)-Leu-Arg-Pro-NHCH2CH3, wherein the LHRH peptide derivative provides an LHRH peptide conjugate that is linked to a cytotoxic agent via a self-cleaving linker.

[0082] Further preferred embodiments of the present invention are described herein by way of example only, with reference to the accompanying drawings.

[0083] (Example)

Example

[0084] Example 1: Synthesis of Various LHRH Peptide Derivatives

[0085] The LHRH peptide derivatives of the present invention were designed and synthesized on Rink amide resin according to the in situ neutralization protocol (P. Varaminietal., J Med Chem., 2017, 60; P. Varaminietal., Int J Pharm, 2017, 521) for Fmoc solid-phase chemistry (the structure of the LHRH peptide derivative is shown in Fig. 1A). The purity of each of the peptides (LD4, LD5 and LD6) was over 98%. The peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a Shimadzu system using a Vydac C18 column (5 mm, 22 250 mm) running a gradient of two solvents: A: H2O, 0.1% TFA, and B: acetonitrile / H2O 9:1, 0.1% TFA. A gradient of 20% - 60% B over 60 minutes (peptides 1 - 3, 7 and 9 - 10) or a gradient of 10% - 60% B over 70 minutes (peptides 4 - 6 and 8) was used at a flow rate of 10 mL / min. The collected fractions were analyzed by high-resolution MS and ESI-MS and analytical RP-HPLC using Vydac C4 and C18 columns (5 mm, 4.6 250 mm) with a gradient of 0% - 100% B over 30 minutes at a flow rate of 1 mL / min. The pure fractions were combined and lyophilized.

[0086] Example 2: Synthesis of LD5-mc-vc-PABC-MMAE conjugate

[0087] The LD5-mc-vc-PABC-MMAE conjugate was synthesized by Fmoc solid-phase chemistry technology (P. Varamini et al., J Med Chem., 2017, 60; P. Varamini et al., Int J Pharm, 2017, 521).

[0088] Example 3: Metabolic stability of LHRH peptide derivatives

[0089] The inventors have surprisingly found that the LHRH peptide derivatives improve metabolic stability. The metabolic stabilities of native LHRH peptide, LHRH peptide derivatives LD4, LD5 and LD6, and the known LHRH agonists, tryptorelin ([w6]LHRH) and [k6]LHRH, were examined in human plasma (Figure 1B). The LHRH peptide derivatives LD4, LD5 and LD6 have significantly improved half-lives (T of about 257 minutes, 365 minutes and 309 minutes for LD-4, LD-5 and LD-6, respectively) compared to the native LHRH peptide (T of about 10 minutes 1 / 2 ) and LHRH agonists, namely tryptorelin ([w 6 LHRH) and [k 6 LHRH (T of about 19 minutes and 39 minutes, respectively 1 / 2 ). 1 / 2 )

[0090] LD4, LD5 and LD6 show unique high stability, and among all, LD5 has the highest half-life of 365 minutes and was selected for conjugation to MMAE via the self-cleavable linker, mc-vc-PABC. The binding affinity of the LD5-mc-vc-PABC-MMAE conjugate to the LHRH receptor was examined by the DuoLink assay. As shown in Figure 6, this assay confirmed the high binding affinity of the peptide ligand to the LHRH receptor.

[0091] Example 4: Antiproliferative activity

[0092] The inventors have further discovered that LD4, LD5 and LD6 have significantly higher antiproliferative activity in three different breast cancer cell lines compared to both native LHRH and the agonist [w6]LHRH used clinically in hormone-dependent gynecological cancers (see Table 1).

[0093] Example 5: The LHRH receptor (LHRH-R) is expressed in human breast cancer cell lines

[0094] Culture media were collected from breast cancer cells MDA-MB-231, SK-BR-3, and MCF-7, lysed in lysis buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM Tris HCl, pH 8.0, and protease inhibitor mixture), sonicated 15 times for 1 second on ice, and then centrifuged at 16,100×g for 30 minutes at 4°C. 50 micrograms of total protein extract was loaded onto an 8% SDS / PAGE gel. After transferring overnight at 4°C, polyvinylidene fluoride (PVDF) membranes were blocked in 5% bovine serum albumin (BSA) for 1 hour and incubated with anti-LHRH receptor primary antibody (SolarBio Life Sciences) overnight at 4°C. After washing with Tween in Tris-buffered saline (TBS-T), the membranes were incubated with horseradish peroxidase-conjugated (HRP-conjugated) secondary antibody for 1 hour. After washing, proteins were detected using an ECL-Plus chemiluminescence detection system (GE Healthcare). Densities were measured using the ImageJ program. β-Actin was used as a control in Western blotting to measure LHRH-R expression in MDA-MB-231, SK-BR-3, and MCF-7 cancer cell lines. Western blot analysis showed LHRH receptor expression in three breast cancer cell lines with different characteristics (MDA-MB-231: ER-, HER-2-, PR-; MCF-7, ER+, HER-2+, PR+; and SK-BR-3, ER-, HER-2+, PR-). These cell lines were used for anti-proliferation assays of LHRH peptide derivatives. This study was performed using an antibody specifically produced against LHRH-R. In MCF-7, MDA-MB-231, and SK-BR-3, a major protein band of approximately 64 kDa molecular weight, known for the human pituitary LHRH receptor, was identified (lanes 1, 2, and 3 of Figure 2, respectively). The levels of LHRH-R expression relative to β-actin in MCF-7, MDA-MB-231, and SK-BR-3 indicated that these breast cancer cells could be actively targeted via the LHRH receptor.

[0095] Using immunohistochemistry, the expression of LHRH-R in MDA-MB-231 (TNBC cell model), SKOV-3 (LHRH-R low-expression cancer cell model), as well as HMEC and MCF-10A (i.e., normal breast cells) was confirmed (Figure 3A). As a result of quantitative analysis (Figure 3B), in MDA-MB-231 (TNBC cell model), the LHRH-R signal intensity was significantly higher compared to SKOV-3 (LHRH-R low-expression cancer cell model) and normal breast cells (p<0.05).

Table 1

[0096] Example 6: Cytotoxicity of LD5-mv-vc-PABC-MMAE

[0097] The cytotoxicity of the LHRH peptide derivative pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH2CH3 (LD5) conjugated with MMAE by the self-cleaving linker, mv-vc-PABC (i.e., LD5-mv-vc-PABC-MMAE), was measured by the MTT assay. Using this assay, it was investigated whether LD5-mv-vc-PABC-MMAE affects the proliferation of TNBC cells expressing LHRH-R. Using the TNBC cell model, MDA-MB-231, the relative cytotoxicity of LD5-mc-vc-PABC-MMAE compared to MMAE, as well as normal breast cells, HMEC and MCF-10A, were screened as in the case of SKOV-3 (LHRH-R negative control). Cells were incubated with each compound for 72 hours, and the relative cell viability was measured using the colorimetric MTT assay. The growth inhibitory effects of LD5-mc-vc-PABC-MMAE and MMAE were determined for normal breast cells, MDA-MB-231 and SKOV-3 IC50 reported as (nM) (Figure 4A).

[0098] Normal breast cells (HMEC and MCF-10A) were sensitive to MMAE at 0.11 nM and 0.83 nM, respectively. However, LD5-mc-vc-PABC-MMAE showed no significant cytotoxic effect on normal breast cells (IC 50 value > 1000 nM). In contrast, the TNBC cell line (MDA-MB-231) showed significant sensitivity to LD5-mc-vc-PABC-MMAE at an IC 50 value of 1.26 nM. The SKOV-3 cell line was sensitive to MMAE at an IC 50 value of 0.03 nM, but this cell line was resistant to LD5-mc-vc-PABC-MMAE with an IC 50 value > 1000 nM).

[0099] The cytotoxicity of LD5-mc-vc-PABC-MMAE was evaluated by comparing it with MMAE for all cells. The potency of LD5-mc-vc-PABC-MMAE was significantly lower than that of MMAE at specific concentrations in normal breast cells (HMEC and MCF-10A), TNBC cells (MDA-MB-231), and the LHRH-R negative control (SKOV-3) (Figures 4B, 4C, 4D, and 4E). This decrease in the cytotoxicity of MMAE when conjugated with LD5 indicates the selectivity of the LHRH uptake pathway via the LHRH receptor compared to the simple diffusion of free MMAE in cell lines.

[0100] Example 7: Role of the LHRH Receptor in the Cytotoxicity of LD5-mc-vc-PABC-MMAE

[0101] A receptor binding competition assay was performed to examine the relationship between cytotoxicity and binding to LHRH-R. TNBC cells (MDA-MB-231) were pretreated with 100 μM tryptophan (TRN) for 2 hours to block LHRH-R. The significant cytotoxic effect of LD5-mc-vc-PABC-MMAE (LM) was reversed after pretreatment with TRN (Figures 5A and 5B). The MTT assay showed that blocking LHRH-R through pretreatment with TRN significantly increased the cell viability of LD5-mc-vc-PABC-MMAE-treated cells. This indicates that LHRH-R plays an important role in the anti-cancer activity of LD5-mc-vc-PABC-MMAE (Figure 5).

[0102] Example 8: Interaction between LHRH-R and LHRH in an LD5-mc-vc-PABC-MMAE-treated environment

[0103] The proximity ligation assay (PLA) was used to measure the interaction between LHRH-R and LHRH in LD5-mc-vc-PABC-MMAE-treated cells. The Duolink® proximity ligation assay (PLA) enables the detection of endogenous proteins, protein modifications, and protein interactions in situ with high specificity and high sensitivity. Protein targets can be easily detected and localized at the single-molecule resolution in unmodified cells and tissues. Typically, two unique protein targets are detected using two primary antibodies raised in different species. The PLA reagent was added to fixed MDA-MB-231 cells after incubating these cells with primary antibodies specific for LHRH-R and LHRH. In cells treated with LD5-mc-vc-PABC-MMAE, it was revealed from the signal localization that the protein interaction was in the intracellular compartment as well as in the cell membrane (Figure 6). This qualitative result confirmed the interaction between LHRH-R and its ligand during the uptake of LD5-mc-vc-PABC-MMAE by MDA-MB-231 cells.

[0104] Example 9: In vitro uptake of LD5-mc-vc-PABC-MMAE in TNBC cells

[0105] Using a TNBC cell line (MDA-MB-231) that overexpresses LHRH-R, the uptake of LD5-mc-vc-PABC-MMAE by TNBC cells was examined. The uptake was compared with normal breast cells (LHRH-R negative control). LD5-mc-vc-PABC-MMAE was incubated with MDA-MD-231 normal breast cells for 18 hours, and the intracellular uptake of the conjugate was monitored using confocal LSM (Figure 7A).

[0106] As shown in Figure 7A, significantly higher uptake of LD5-mc-vc-PABC-MMAE was observed in LHRH-R positive cells (MDA-MB-231) compared with normal breast cells (MCF-10A and HMEC). This was supported by a quantitative comparison of the intracellular uptake of LD5-mc-vc-PABC-MMAE in LHRH-R positive and negative cells (Figure 7B). These data support the active targeting delivery of the compound via LHRH-R in TNBC cells (p<0.05).

[0107] Example 10: Effects of LD5-mc-vc-PABC-MMAE on α-tubulin polymerization in normal and cancer cells

[0108] The intracellular effect of MMAE on α-tubulin polymerization was examined using α-tubulin immunostaining in TNBC cells (MDA-MB-231) and normal breast cells (MCF-10A and HMEC) treated with MMAE conjugated with LD5 (LD5-mc-vc-PABC-MMAE). TNBC cells and normal breast cells were fixed after incubation with 1 μM MMAE and LD5-mc-vc-PABC-MMAE with PBS as a control for 18 hours. α-Tubulin was stained by immunostaining and observed by confocal LSM. As shown in Figure 8A, TNBC cells and normal breast cells treated with MMAE showed a dramatic decrease in α-tubulin formation compared to the PBS control, indicating its non-selective activity against normal breast cells and cancer cells (i.e., TNBC cell model). The α-tubulin signal of normal breast cells treated with PBS and LD5-mc-vc-PABC-MMAE (LM) was significantly higher than that of the MMAE treatment group (Figure 8B), indicating that the effect of the molecular target drug on α-tubulin polymerization as a factor for cell survival was low (Figure 4).

[0109] Example 11: Effect of silencing of the LHRH-R gene on the uptake of LD5-mc-vc-PABC-MMAE by TNBC cells

[0110] To further investigate the role of LHRH-R in the uptake of LD5-mc-vc-PABC-MMAE, LHRH-R expression was blocked by silencing of those genes in MDA-MB-231 (TNBC cell model). This study was performed by co-transfection of siRNA (RNAi-Mate transfection reagent and siRNA, GNRHR-homo-2242, GNRHR-homo-2701, scrambled RNA, manufactured by GenePharma) and fluorescently labeled negative control siRNA (FAM transfection efficiency control manufactured by GenePharma). As shown in Figure 9, silencing resulted in a decrease of more than 83% in LHRH-R expression in transfected MDA-MB-231 cells compared to the negative control (Figure 9, p<0.05). The uptake of LD5-mc-vc-PABC-MMAE was significantly decreased after silencing of LHRH-R gene expression, indicating that this construct is actively targeted to be taken up by cancer cells via overexpressed LHRH-R (Figure 10, p<0.05).

[0111] Example 12: In vitro metabolic stability of LD5-(mc-vc-PABC)-MMAE

[0112] The metabolic stability of LD5-(mc-vc-PABC)-MMAE was investigated in cell culture medium (c-medium), human and mouse sera. An LC / MS method was developed to detect both the presence of free MMAE and any degradation species from the overall construct, LD5-(mc-vc-PABC)-MMAE. To evaluate the stability of the valine-citrulline linkage, LD5-(mc-vc-PABC)-MMAE was incubated at 1 μM for 10 days at 37 °C in c-medium, human and mouse sera. Aliquots were taken at pre-determined time intervals (t = 0, 1, 2, 4, 7, 10 days) and analyzed by LC / MS for the release of free MMAE.

[0113] LD5-(mc-vc-PABC)-MMAE was stable over the test period and was shown to be 10 days (Figure 11). This study revealed that the drug released as MMAE in human plasma and c-medium after 10 days was less than 3% of the total drug. In mouse plasma, less than 5% of the total drug was released after 10 days. Furthermore, qualitative full-scan LC / MS analysis including UV detection of the same samples did not reveal the presence of other molecular species that could be identified as drugs or drug-linker degradation products.

[0114] The use of highly stable peptide derivatives and intracellular linkers in the design of LHRHD-(mc-vc-PABC)-MMAE (i.e., LD-5-(mc-vc-PABC)-MMAE) resulted in a stable conjugate in human and mouse plasma and cell culture medium (Example 12). This drug was stably attached to the peptide, and the release of MMAE after 10 days of incubation in human plasma was only about 3%, but it was shown to be cleaved by lysosomal proteases once taken into cells via the LHRH receptor in vitro. These stability data are comparable to those of clinically relevant ADCs containing MMAE such as brentuximab vedotin (cAC10-vcMMAE) or trastuzumab emtansine (T-DM1) (JA Francisco et al., Blood, 2003, 102; B Bender et al., The AAPS Journal, 2014, 16). These conjugates regarding these stability data have been successful in clinical trials and are currently on the market, but there is no report on the in vitro stability of zoptarelin doxorubicin (AEZS108, a PDC targeting the LHRH receptor that reached clinical trials). The reason for the failure of this conjugate in phase III clinical trials was the lack of stability and release of doxorubicin in plasma before reaching the tumor site. From this, it was concluded that PDC has a safety profile similar to that of free doxorubicin. No superiority was recognized in terms of toxicity or efficacy over free doxorubicin.

[0115] Example 13: In Vivo Minimum Tolerable Dose and Toxicity Test

[0116] A. Phase I: MTD Single Dose

[0117] LD5-(mc-vc-PABC)-MMAE was intravenously administered to the base of 3 female NOD / SCID mice (23±3 g). The animals were administered an initial dose of 3 mg / kg. If the animals survived for 72 hours, the dose of the next cohort was increased. If one or more animals died, the dose of the next cohort was decreased. The test was terminated when all individuals survived at the upper limit, or when 2 - 3 dosing levels were tested, or when the upper or lower limit was reached. At each dose level, the presence of acute toxicity symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and the effects on the autonomic nervous system (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) were observed during the first 15 minutes, and then again at 1 and 2 hours later. Body weights were recorded before dosing and 72 hours after dosing. After 3 days of compound administration, the animals were observed and the mortality was recorded daily. Gross necropsy was performed on all animals without tissue collection. At all monitoring time points (15 minutes, 1 and 2 hours), no significant adverse effects were observed with intravenous injection at 3 and 10 mg / kg. No mortality and body weight changes were observed, indicating the tolerance of the dosing level (Tables 2 and 3). Subsequently, a dosing amount of 10 mg / kg of this drug was determined for the following MTD repeated dosing test (Phase II). [Table 2] Maximum Tolerable Dose and Autonomic Signs in Mice (Phase I: Mortality) JPEG2025090582000002.jpg65127[Table 3] Maximum Tolerable Dose and Autonomic Signs in Mice (Phase I: Body Weight) JPEG2025090582000003.jpg91127

[0118] B. Phase II: MTD Repeated Dosing

[0119] LD-5-(mc-vc-PABC)-MMAE (10 mg / kg; determined by the results of the Phase I trial) was intravenously administered once a week on days 1 and 8 to a group consisting of 3 female NOD / SCID mice (23 ± 3 g). At 1 and 2 hours after each administration on days 1 and 8, the presence of acute toxic symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) was observed for the first 15 minutes. Body weights were recorded before administration and on days 1, 4, 8, 12, and 15. The animals were observed, and mortality was recorded daily after the first compound was administered for 15 days. Gross necropsy was performed on all animals without tissue collection. No significant adverse effects were observed at 10 mg / kg IV after the first and second administrations of LD-5(mc-vc-PABC)-MMAE on days 1 and 8 (15 minutes, 1 hour, and 2 hours). Furthermore, since all the test animals survived at the end of the test period, it was suggested that the dosing level was tolerated after repeated administrations on days 1 and 8 (Tables 3 and 4). No abnormalities were observed after gross necropsy in both phases. [Table 4] Maximum Tolerated Dose, Autonomic Signs - Phase 2 Mortality in Mice JPEG2025090582000004.jpg132170[Table 5] Maximum Tolerated Dose, Autonomic Signs - Phase 2 Body Weight in Mice JPEG2025090582000005.jpg70170

[0120] The MTD and toxicity tests of LHRHD-(mc-vc-PABC)-MMAE (i.e., LD-5-(mc-vc-PABC)-MMAE) were conducted in female NOD / SCID mice, and no toxicity was observed in any of the mice that received a dose of 10 mg / kg. Therefore, this dose did not reach the MTD. For the corresponding ADC with MMAE, the MTD was considerably lower. For example, the MTD of brentuximab vedotin in mice was achieved at 30 - 40 mg / kg, which corresponds to approximately 70 mg / kg of LHRHD-(mc-vc-PABC)-MMAE. Considering the equivalent selectivity and stability to the corresponding ADC of LHRHD-(mc-vc-PABC)-MMAE and the significantly lower MTD, an excellent safety profile is predicted for this PDC. Another advantage of this PDC over current ADCs is that the production cost is significantly lower and the manufacturing process is simple.

Claims

1. An LHRH peptide-drug conjugate (LHRH-PDC) comprising the sequence: X 1 -His-Trp-Ser-X 2 -X 3 (L-D)-X 4 -X 5 -Pro-NHR During the ceremony, X 1 is pGlu or Gln; X 2 is Tyr, Phe or His; X 3 is D-Lys or D-Lys(Ahx); X 4 is Leu, Val, Trp, or Met; X 5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; R is CH 2 CH 3 or CH 3 and During the ceremony, L is a linker; D is a cytotoxic agent.

2. X 1 is Gln, and X 3 is D-Lys and R is CH 2 CH 3 The LHRH-PDC of claim 1,

3. X 1 is pGlu, and X 3 is D-Lys and R is CH 2 CH 3 The LHRH-PDC of claim 1,

4. X 1 is pGlu, and X 3 is D-Lys(Ahx) and R is CH 2 CH 3 The LHRH-PDC of claim 1,

5. X 1 is Gln, and X 2 is Tyr, and X 3 is D-Lys, and X 4 is Leu, and X 5 is Arg and R is CH 2 CH 3 The LHRH-PDC of claim 1,

6. X 1 is pGlu, and X 2 is Tyr, and X 3 is D-Lys, and X 4 is Leu, and X 5 is Arg and R is CH 2 CH 3 The LHRH-PDC of claim 1,

7. X 1 is pGlu, and X 2 is Tyr, and X 3 is D-Lys(Ahx), and X 4 is Leu, and X 5 is Arg and R is CH 2 CH 3 The LHRH-PDC of claim 1,

8. X 1 The LHRH-PDC of claim 1, wherein is Gln.

9. X 1 The LHRH-PDC of claim 1, wherein is pGlu.

10. X 2 The LHRH-PDC of claim 8 or claim 9, wherein is Tyr.

11. X 2 The LHRH-PDC of claim 8 or claim 9, wherein is Phe.

12. X 2 The LHRH-PDC of claim 8 or claim 9, wherein is His.

13. X 3 The LHRH-PDC of any one of claims 8 to 12, wherein is D-Lys.

14. X 3 The LHRH-PDC of any one of claims 8 to 12, wherein is D-Lys(Ahx).

15. X 4 The LHRH-PDC of any one of claims 8 to 14, wherein is Leu.

16. X 4 The LHRH-PDC of any one of claims 8 to 14, wherein is Val, Trp or Met.

17. X 5 The LHRH-PDC of any one of claims 8 to 16, wherein is Arg.

18. X 5 The LHRH-PDC of any one of claims 8 to 16, wherein is Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys.

19. R is CH 2 CH 3 The LHRH-PDC of any one of claims 8 to 18,

20. R is CH 3 The LHRH-PDC of any one of claims 8 to 18,

21. The LHRH-PDC of any one of the preceding claims, wherein the linker is a self-immolating linker.

22. 22. The LHRH-PDC of claim 21, wherein the self-immolating linker is maleimidocaproylvaline-citrulline-p-aminobenzylcarbamoyl (mc-vc-PABC).

23. 2. The LHRH-PDC of any one of the preceding claims, wherein the cytotoxic agent is an antimitotic agent, an alkylating agent, an antimetabolite, a topoisomerase inhibitor or a protein kinase inhibitor.

24. 2. The LHRH-PDC of any one of the preceding claims, wherein the cytotoxic agent is a vinca alkaloid, cryptophycin, bortezomib, thiobortezomib, tublysin, aminopterin, rapamycin, paclitaxel, docetaxel, daunorubicin, everolimus, a-amanate, vemcarin, didemnin B, geldanomycin, purvalanol A, ispinesib, budesonide, dasatinib, epothilone, maytansine, doxorubicin, camptothecin, methotrexate (MTX) or monomethylauristatin E (MMAE).

25. The LHRH-PDC of any one of claims 1 to 24, wherein the cytotoxic agent is MMAE.

26. An antiproliferative LHRH peptide derivative comprising the sequence: or a pharma- ceutically acceptable salt thereof. X 1 -His-Trp-Ser-X 2 -X 3 -X 4 -X 5 -Pro-NHR During the ceremony, X 1 is pGlu or Gln; X 2 is Tyr, Phe or His; X 3 is D-Lys or D-Lys(Ahx); X 4 is Leu, Val, Trp, or Met; X 5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys R is CH 2 CH 3 or CH 3 It is.

27. X 1 is Gln, and X 3 is D-Lys and R is CH 2 CH 3 27. The LHRH peptide derivative of claim 26, wherein:

28. X 1 is pGlu, and X 3 is D-Lys and R is CH 2 CH 3 27. The LHRH peptide derivative of claim 26, wherein:

29. X 1 is pGlu, and X 3 is D-Lys(Ahx) and R is CH 2 CH 3 27. The LHRH peptide derivative of claim 26, wherein:

30. X 1 is Gln, and X 2 is Tyr, and X 3 is D-Lys, and X 4 is Leu, and X 5 is Arg and R is CH 2 CH 3 27. The LHRH peptide derivative of claim 26, wherein:

31. X 1 is pGlu, and X 2 is Tyr, and X 3 is D-Lys, and X 4 is Leu, and X 5 is Arg and R is CH 2 CH 3 27. The LHRH peptide derivative of claim 26, wherein:

32. X 1 is pGlu, and X 2 is Tyr, and X 3 is D-Lys(Ahx), and X 4 is Leu, and X 5 is Arg and R is CH 2 CH 3 27. The LHRH peptide derivative of claim 26, wherein:

33. X 1 27. The LHRH peptide derivative of claim 26, wherein is Gln.

34. X 1 27. The LHRH peptide derivative of claim 26, wherein is pGlu.

35. X 2 35. The LHRH peptide derivative of claim 33 or claim 34, wherein is Tyr.

36. X 2 35. The LHRH peptide derivative of claim 33 or claim 34, wherein is Phe.

37. X 2 35. The LHRH peptide derivative of claim 33 or claim 34, wherein is His.

38. X 3 The LHRH peptide derivative according to any one of claims 33 to 37, wherein is D-Lys.

39. X 3 38. The LHRH peptide derivative according to any one of claims 33 to 37, wherein is D-Lys(Ahx).

40. X 4 40. The LHRH peptide derivative according to any one of claims 33 to 39, wherein is Leu.

41. X 4 40. The LHRH peptide derivative of any one of claims 33 to 39, wherein is Val, Trp or Met.

42. X 5 42. The LHRH peptide derivative according to any one of claims 33 to 41, wherein is Arg.

43. X 5 42. The LHRH peptide derivative of any one of claims 33 to 41, wherein is selected from the group consisting of Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys.

44. R is CH 2 CH 3 44. The LHRH peptide derivative according to any one of claims 33 to 43, wherein

45. R is CH 3 44. The LHRH peptide derivative according to any one of claims 33 to 43, wherein

46. LHRH peptide derivative containing the sequence: Gln-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH 2 CH 3 or a pharma- ceutically acceptable salt thereof.

47. LHRH peptide derivative containing the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH 2 CH 3 or a pharma- ceutically acceptable salt thereof.

48. LHRH peptide derivative containing the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys(Ahx)-Leu-Arg-Pro-NHCH 2 CH 3 or a pharma- ceutically acceptable salt thereof.

49. An LHRH peptide derivative comprising the following sequence: or a pharma- ceutically acceptable salt thereof. X 1 -His-Trp-Ser-X 2 -X 3 -X 4 -X 5 -Pro-NHR During the ceremony, X 1 is Gln; X 2 is Tyr, Phe or His; X 3 is D-Lys or D-Lys(Ahx); X 4 is Leu, Val, Trp, or Met; X 5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; and R is CH 2 CH 3 or CH 3 It is.

50. An LHRH peptide derivative comprising the following sequence: or a pharma- ceutically acceptable salt thereof. X 1 -His-Trp-Ser-X 2 -X 3 -X 4 -X 5 -Pro-NHR During the ceremony, X 1 is pGlu; X 2 is Tyr, Phe or His; X 3 is D-Lys or D-Lys(Ahx); X 4 is Leu, Val, Trp, or Met; X 5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; and R is CH 2 CH 3 or CH 3 It is.

51. D-Lys and R is CH 2 CH 3 51. The LHRH peptide derivative of claim 49 or claim 50, which is

52. D-Lys(Ahx) and R is CH 2 CH 3 51. The LHRH peptide derivative of claim 49 or claim 50, which is

53. X 2 is Tyr, and X 3 is D-Lys, and X 4 is Leu, and X 5 is Arg and R is CH 2 CH 3 51. The LHRH peptide derivative of claim 49 or claim 50, which is

54. X 2 is Tyr, and X 3 is D-Lys(Ahx), and X 4 is Leu, and X 5 is Arg and R is CH 2 CH 3 51. The LHRH peptide derivative of claim 49 or claim 50, which is

55. 55. An LHRH-PDC comprising the LHRH peptide derivative of any one of claims 26 to 54, wherein the LHRH peptide derivative is linked to a cytotoxic agent via a self-destructing linker.

56. 56. The LHRH-PDC of claim 55, wherein the cytotoxic agent is MMAE.

57. The LHRH-PDC of claim 55 or claim 66, wherein the self-disintegrating linker is mc-vc-PABC.

58. Sequence: Gln-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH 2 CH 3 or a pharma- ceutically acceptable salt thereof, wherein the LHRH peptide derivative is conjugated to MMAE via mc-vc-PABC.

59. Sequence: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH 2 CH 3 or a pharma- ceutically acceptable salt thereof, wherein the LHRH peptide derivative is conjugated to MMAE via mc-vc-PABC.

60. Sequence: pGlu-His-Trp-Ser-Tyr-D-Lys(Ahx)-Leu-Arg-Pro-NHCH 2 CH 3 or a pharma- ceutically acceptable salt thereof, wherein the LHRH peptide derivative is conjugated to MMAE via mc-vc-PABC.

61. A pharmaceutical composition comprising an LHRH-PDC of any one of claims 1 to 25 or claims 55 to 60, or a pharma- ceutically acceptable salt thereof, or an LHRH peptide of any one of claims 26 to 54, or a pharma- ceutically acceptable salt thereof, and optionally at least one pharma- ceutically acceptable excipient.

62. A method of treating cancer comprising administering to a subject an LHRH-PDC or a pharma- ceutically acceptable salt thereof described in any one of claims 1 to 25 or claims 55 to 60, an LHRH peptide derivative or a pharma- ceutically acceptable salt thereof described in any one of claims 26 to 54, or a pharmaceutical composition described in claim 61, wherein the subject has an LHRH receptor-expressing cancer.

63. 63. The method of claim 62, wherein the LHRH receptor-expressing cancer is breast cancer, prostate cancer, colon cancer, ovarian cancer, pancreatic cancer or endometrial cancer.

64. 64. The method of claim 62 or claim 63, wherein the LHRH receptor-expressing cancer is triple-negative breast cancer (TNBC).

65. A method of arresting or slowing cell proliferation and / or growth of an LHRH receptor expressing tumor in a subject, comprising administering to the subject an LHRH-PDC or a pharma- ceutically acceptable salt thereof of any one of claims 1 to 25 or claims 55 to 60, an LHRH peptide derivative or a pharma- ceutically acceptable salt thereof of any one of claims 26 to 54, or a pharmaceutical composition of claim 61.

66. A method for treating hormone-sensitive and / or refractory breast cancer in a subject, comprising administering to the subject an LHRH-PDC or a pharma- ceutical acceptable salt thereof of any one of claims 1 to 25 or claims 55 to 60, an LHRH peptide derivative or a pharma- ceutical acceptable salt thereof of any one of claims 26 to 54, or a pharmaceutical composition of claim 61.

67. Use of an LHRH-PDC or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 25 or claims 55 to 60, an LHRH peptide derivative or a pharma- ceutically acceptable salt thereof according to any one of claims 26 to 54, or a pharmaceutical composition according to claim 61, in the manufacture of a medicament for treating cancer, wherein the cancer is an LHRH receptor-expressing cancer.

68. Use of an LHRH-PDC as defined in any one of claims 1 to 25 or claims 55 to 60, or a pharma- ceutically acceptable salt thereof, or an LHRH peptide derivative as defined in any one of claims 26 to 54, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition as defined in claim 61, in the manufacture of a medicament for arresting or slowing cell proliferation and / or growth of an LHRH receptor expressing tumor.

69. Use of an LHRH-PDC as defined in any one of claims 1 to 25 or claims 55 to 60, or a pharma- ceutically acceptable salt thereof, or an LHRH peptide derivative as defined in any one of claims 26 to 54, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition as defined in claim 61, in the manufacture of a medicament for treating hormone-sensitive and / or refractory breast cancer.

70. Use of an LHRH-PDC as claimed in any one of claims 1 to 25 or claims 55 to 60, or a pharma- ceutically acceptable salt thereof, or an LHRH peptide derivative as claimed in any one of claims 26 to 54, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 61, in the manufacture of a medicament for treating triple-negative breast cancer (TNBC).

71. An LHRH-PDC according to any one of claims 1 to 25 or claims 55 to 60, or a pharma- ceutically acceptable salt thereof, an LHRH peptide derivative according to any one of claims 26 to 54, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to claim 61, for use in a method for treating an LHRH-expressing cancer.

72. An LHRH-PDC or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 25 or claims 55 to 60, an LHRH peptide derivative or a pharma- ceutical acceptable salt thereof according to any one of claims 26 to 54, or a pharmaceutical composition according to claim 61, for use in the treatment of an LHRH-expressing cancer, wherein the cancer is triple-negative breast cancer (TNBC).

73. 74. The method of claim 72 or 73, comprising administering LHRH-PDC or a pharma- ceutically acceptable salt thereof, an LHRH peptide derivative or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, wherein the LHRH-PDC or a pharma- ceutically acceptable salt thereof, an LHRH peptide derivative or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in the method of claim 72 or 73, wherein the LHRH-PDC or a pharma- ceutically acceptable salt thereof, an LHRH peptide derivative or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof is administered in combination with one or more additional cytotoxic agents.

Images