CN117957239A

CN117957239A - Cancer therapeutic agent

Info

Publication number: CN117957239A
Application number: CN202280060793.9A
Authority: CN
Inventors: 涅韦斯·阿吉尔简; 蒙特塞拉特·若莫特皮霍安; 黛博拉·卡伯特罗梅罗; 索尼亚·布朗洛萨诺; 巴拉亚·阿布阿萨克尔; 弗兰塞斯克·拉瓦纳尔安格拉达; 奇亚拉·帕拉拉; 赫苏斯·塞科莫拉尔; 何塞普·里瓦斯桑托斯; 罗格·普拉德斯科萨诺; 特里萨·塔拉戈克卢瓦
Original assignee: Universitat de Barcelona UB
Current assignee: Universitat de Barcelona UB
Priority date: 2021-07-07
Filing date: 2022-07-06
Publication date: 2024-04-30

Abstract

Therapeutic compounds of the invention include peptidomimetics that have been prepared using solid phase peptide synthesis, including those of formula (I). It can be used for treating cancer; including for the treatment of pancreatic, lung or colorectal cancer; in addition, treatment of pancreatic ductal adenocarcinoma is included.

Description

Cancer therapeutic agent

RELATED APPLICATIONS

The present application claims priority from european patent application serial number 21382612.6 filed 7 at 2021, 7. The contents of the foregoing application are incorporated herein by reference.

Technical Field

The present invention relates generally to cancer therapies, and more particularly, to novel therapeutic compounds, including peptide mimetics thereof, for treating cancer.

Background

Cancer is generally defined as a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancer is associated with several factors including smoking, obesity, poor diet, lack of physical activity, and excessive alcohol consumption. Other factors include certain infections, exposure to ionizing radiation and environmental pollutants. Certain cancers are associated with infections such as helicobacter pylori, hepatitis b, hepatitis c, human papilloma virus infection, epstein-Barr virus (Epstein-Barr virus) and Human Immunodeficiency Virus (HIV).

Conventional cancer treatments aim to remove the cancerous tissue and prevent its spread. Such treatment options include surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy, and palliative care (PALLIATIVE CARE). Treatment is typically based on the type, location and grade of cancer and the health and preference of the patient. Because cancer cells divide faster than most normal cells, they may be sensitive to chemotherapeutic drugs.

RAS genes include the oncogene families (HRAS, NRAS and KRAS) associated with the cell proliferation process. Highly mutated forms of these RAS genes have been found in several cancers, with mutated forms of KRAS found in about 86% of RAS-related cancers, and mutated forms of N-RAS found in about 11%, and finally mutated forms of HRAS found in about 3%. It is common to find mutated RAS genes associated with some of the most deadly cancers. This includes about 90% pancreatic cancer, 45% colon cancer, and 25% lung cancer.

In recent years, the modulation of PPI has attracted considerable attention in the scientific community due to the large number of protein-protein interactions (PPI) involved in cellular machinery (cellular machinery). However, due to the nature of these interactions, the modulation of PPI is very challenging. An alternative to using small molecules for modulation of PPI is to use biological products, such as antibodies. PPIs, which are considered large structures rather than small molecules, have the ability to recognize and interact with large protein surfaces, but do not have the ability to cross biological barriers, a feature that limits their therapeutic use. It is therefore an object of the present invention to provide a PPI capable of recognizing and interacting with large protein surfaces, while having the ability to cross biological barriers.

Because of the need for treatment of cancer, including cancer associated with mutated RAS genes, the present invention provides compositions and methods for treating diseases such as cancer using therapeutic agents, pharmaceutical compositions and articles of manufacture of therapeutic agents (article of manufacture).

Summary of The Invention

The invention described and claimed herein has many attributes and embodiments, including but not limited to those set forth or described or mentioned in this brief summary. The invention described and claimed herein is not limited to or by the features or embodiments identified in this summary, which are included for purposes of illustration only and not limitation.

In aspects of the invention, therapeutic compounds are provided that include peptidomimetics that can be administered to a patient. In another aspect of the invention, the therapeutic compound may be administered alone or in combination with one or more other therapeutic compounds. These additional therapeutic compounds may include antibodies, biologics, small molecules, or other therapeutic compounds as listed in fig. 8.

In aspects of the invention, therapeutic compounds that are peptidomimetics are administered to a patient with 1, 2, 3, 4, 5, 6,7, 8, 9,10, or more different therapeutic compounds (including those listed in fig. 8).

In aspects of the invention, the therapeutic agent is a peptidomimetic, which is a small proteinaceous chain designed to mimic a peptide. In another aspect of the invention, the peptidomimetics are suitably engineered to recognize and bind in a specific manner to a protein region (protein patches) (binding sites or sites at which a protein interacts with another protein or molecule, including being engineered to trigger an enzymatic pathway) and across a biological barrier. In one aspect of the invention, the therapeutic compounds of the invention have the formula C ₄₇H₅₈N₆O₅. It can be named: (S) -N- (3- (((S) -3-amino-1- ((S) -4-methyl-1-oxo-1- (pyrrolidin-1-yl) pent-2-ylamino) -1-oxopropan-2-yl) (methyl) amino) -3-oxopropyl) -3- (biphenyl-4-yl) -2- (2, 2-diphenylacetamido) -N-methylpropanamide.

In aspects of the invention, the therapeutic compound (I) has the ability to effectively inhibit in vitro the interaction of RAS (acronym from rat sarcoma) with its effectors (other proteins in the cascade triggered by RAS) in cells, and it shows high selectivity in reducing the viability of cancer cells, including pancreatic tumor cells expressing oncogenic forms of KRAS. In another aspect of the invention, the therapeutic compound inhibits the survival of a PDAC cell line while being non-toxic to non-cancerous normal cell lines. In aspects of the invention, therapeutic compounds and their therapeutically acceptable salts are useful for treating cancer; wherein the cancer is selected from pancreatic cancer, lung cancer or colorectal cancer; wherein the pancreatic cancer is PDAC.

In aspects of the invention, the therapeutic strategy involves the search for anti-cancer drugs that inhibit the binding of the RAS to its effectors.

Another aspect of the invention relates to a pharmaceutical composition (e.g., a drug (medicine) or medicament) comprising a therapeutic compound and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable excipient, diluent or carrier.

Another aspect of the invention relates to therapeutic compounds and pharmaceutically acceptable salts thereof for use in the treatment of cancer, including human cancer. The present aspects may relate to the use of therapeutic compounds and pharmaceutically acceptable salts thereof in the manufacture of medicaments for the treatment of cancer, including human cancer. Alternatively, the present aspects may relate to a method of treating cancer comprising administering a therapeutically effective amount of a therapeutic compound and pharmaceutically acceptable salts thereof.

In an embodiment of the aspect mentioned in the preceding paragraph, the cancer comprising human cancer is pancreatic cancer, lung cancer or colorectal cancer. In other embodiments, the cancer, including human cancer, is pancreatic cancer. Also, in other embodiments, the pancreatic cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

Throughout the description and claims, the word "comprise" and variations of the word are not intended to exclude other technical features, elements, limitations, additives or components. Furthermore, the word "comprising" encompasses the case of "consisting of. Additional objects, advantages, and features of the invention will become apparent to those skilled in the art upon examination of the specification or may be learned by practice of the invention. The following examples and figures are provided by way of example and are not intended to limit the invention.

Brief Description of Drawings

The figures illustrate aspects of the invention. In such figures:

fig. 1 shows the results of co-immunoprecipitation of HA-KRASG12V with C-RAF or PI3K after incubation with 100 μm of compound of formula (I) for 2h and EGF stimulation for 10min (EGF = epidermal growth factor) in starved HeLa cells expressing HA-KRASG 12V. Immunoprecipitation was performed with anti-HA antibodies and western blotting was performed with anti-p110αpi3k and anti-C-RAF Binding (BO) fractions and Input (IN) fractions. The experiment was repeated at least three times.

Figure 2 shows the effect of compounds of formula (I) on Cell Viability (CV) of six pancreatic adenocarcinoma human cell lines (all carrying oncogenic KRAS mutations) and non-transformed cell lines (hTERT-RPE, shown as RPE in figure 2). Cells cultured in 10% FCS were treated with compound (I) in a dose range from 0 μm to 25 μm and incubated for 24h, at which time cell viability was determined by MTS assay. Experiments were repeated 3 times. The differences were assessed using a one-way ANOVA and Tukey multiple comparison test, and when p.ltoreq.0.05, the differences were considered significant.

FIG. 3 shows that RAS is activated into RAS GTP-binding conformation by a guanine nucleotide exchange factor (GEF) protein state that allows RAS to associate with its protein effectors and initiate downstream protein cascades. In addition, the activated RAS may be recruited by Farnesyl Transferase (FT) and transported through the cytosol until it reaches the cell membrane.

FIG. 4 shows the GTPase-RAS protein pharmacodynamic site (PDB: 5P 21): (A) Highly conserved residues in the complex gtpase-RAS, RAS effector proteins are underlined in the negative bars. The RAS protein surface is grey colored, residues involved in intermolecular contacts with highly conserved residues in effector proteins are underlined (Asp 33, glu37, asp38 and Tyr 64). (B) Computational predictions of more relevant residues for designing a new set of peptidomimetics are shown.

FIG. 5 shows Western blots of different proteins of the KRAS signaling pathway to evaluate the synthetic therapeutic compounds IP-14-01 (P1), IP-14-02 (P2), IP-14-03 (P3), IP-14-04 (P4), IP-14-07 (P7), IP-14-08 (P8) and IP-14-09 (P9). Gtpase activating protein (GAP 120) was used as a load control. The test therapeutic compound was applied to serum starved cell cultures (0.5% FCS for 24 h) of hTERT-RPE (hereinafter also referred to as RPE cell line) 2h before EGF (50 ng/ml) treatment was continued for 10 min. DMSO is a solvating agent to dilute the peptide, and then after dilution with cell culture medium, the final percentage of DMSO is 0.5%.

FIG. 6 shows Western blotting performed under the same conditions as Western blotting of FIG. 5, except that 0.5% of beta-cyclodextrin was used instead of DMSO to evaluate the compounds IP-14-01 (P1), IP-14-02 (P2), IP-14-03 (P3), IP-14-04 (P4), IP-14-07 (P7), IP-14-08 (P8) and IP-14-09 (P9).

FIG. 7 shows Western blots for evaluation of 2018/IP-14-01 (P1) and its derived peptidomimetics, including IPR-471 (P1.1.), IPR-472 (P1.2), IPR-473 (P1.3) and IPR-474 (P1.4). For this experiment, we followed the same protocol conditions used in the previous WB assay for the first generation of peptide mimetics as set forth for fig. 5. Compounds were applied to cell cultures of hTERT-RPE cells at 50. Mu.M in 0.5% DMSO for 2h, and then the cells were treated with EGF (50 ng/ml) for 10min. We did not determine any solubility problems for these peptidomimetics.

Fig. 8 shows a list of therapeutic compounds for treating cancer.

Definition of the definition

In this specification, reference to "one embodiment/aspect" or "an embodiment/aspect" means that a particular feature, structure, or characteristic described in connection with the embodiment/aspect is included in at least one embodiment/aspect of the present disclosure. The use of the phrase "in one embodiment/in one aspect" or "in another embodiment/in another aspect" throughout this specification does not necessarily refer to the same embodiment/aspect, nor is it a separate or alternative embodiment/aspect mutually exclusive of other embodiments/aspects. Furthermore, various features are described which may be exhibited by some embodiments/aspects and not by others. Similarly, various requirements are described which may be requirements for some embodiments/aspects but not other embodiments/aspects. Embodiments and aspects may be used interchangeably in certain circumstances.

The terms used in this specification generally have their ordinary meaning in the art, in the context of this disclosure, and in the specific context in which each term is used. Certain terms used to describe the disclosure are discussed below or elsewhere in this specification to provide additional guidance to the practitioner regarding the description of the disclosure. It should be understood that the same thing may be stated in more than one way.

Thus, alternative languages and synonyms may be used for any one or more of the terms discussed herein. Nor is it of any particular significance to be construed or discussed herein. Synonyms for certain terms are provided. The recitation of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and is not intended to further limit the scope and meaning of the disclosure or any exemplary terms. Also, the present disclosure is not limited to the various embodiments set forth in the specification.

Without further limiting the scope of the present disclosure, examples of apparatus, devices, methods, and related results according to embodiments of the present disclosure are given below. Note that headings or sub-headings may be used in the examples for the convenience of the reader and should not in any way limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present document, including definitions, will control.

As used herein in this specification and the appended claims and unless otherwise indicated, the term "about" or "generally" means a margin of +/-20%. Furthermore, as applicable, the term "substantially" as used herein in this specification and the appended claims, unless indicated otherwise, means a margin of +/-10%. It is to be understood that not all uses of the above terms are quantifiable so that the noted ranges can be applied.

The term "subject" or "patient" refers to any individual animal, more preferably a mammal (including non-human animals such as, for example, dogs, cats, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates) in need of treatment. Most preferably, the patient herein is a human. In embodiments, a "subject" for diagnosis or treatment is a prokaryotic or eukaryotic cell, tissue culture, tissue, or animal, such as a mammal, including a human.

As used herein, the term "comprising" is intended to mean that the compositions and methods comprise the listed elements, but do not exclude other non-listed elements. When the compositions and methods are defined using "consisting essentially of the composition (consisting essentially of), other elements that alter the basic properties of the compositions and/or methods are excluded, but other elements not listed are not excluded. Thus, a composition consisting essentially of the elements as defined herein will not exclude trace elements, such as contaminants from any isolation and purification method or pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, etc., but will exclude additional unspecified amino acids. "consists of (consisting of)". Removing trace amounts exceeding other components elements and essential method steps for administering the compositions described herein. Embodiments defined by each of these transitional terms are within the scope of the present disclosure and the invention embodied therein.

As used herein, the term "hydrate" refers to a crystalline form having a stoichiometric or non-stoichiometric amount of water incorporated into the crystal structure.

The term "alkenyl" as used herein refers to an unsaturated, straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2 to 8 carbon atoms, referred to herein as a (C2-C8) alkenyl group. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexyl alkenyl, 2-propyl-2-butenyl, and 4- (2-methyl-3-butenyl) -pentenyl.

The term "hydrocarbyloxy" (alkoxy) as used herein refers to an alkyl group (-O-alkyl-) attached to oxygen. "hydrocarbyloxy" groups also include alkenyl groups attached to oxygen ("alkenyloxy") or alkynyl groups attached to oxygen ("alkynyloxy" groups). Exemplary hydrocarbyloxy groups include, but are not limited to, groups of alkyl, alkenyl, or alkynyl groups having 1-8 carbon atoms, referred to herein as (C1-C8) hydrocarbyloxy. Exemplary hydrocarbyloxy groups include, but are not limited to, methoxy and ethoxy.

The term "alkyl" as used herein refers to saturated straight or branched chain hydrocarbons, such as straight or branched chain groups of 1 to 8 carbon atoms, referred to herein as (C1-C8) alkyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2-dimethyl-1-butyl, 3-dimethyl-1-butyl, 2-ethyl-1-butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

The term "alkynyl" as used herein refers to an unsaturated, straight or branched chain hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched chain group of 2 to 8 carbon atoms, referred to herein as (C2-C8) alkynyl. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl and 4-butyl-2-hexynyl.

The term "amide" as used herein refers to the form-NRaC (O) (Rb) -or-C (O) NRbRc, wherein Ra, rb and Rc are each independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl and hydrogen. The amide may be attached to another group through carbon, nitrogen, rb or Rc. The amide may also be cyclic, e.g., rb and Rc may be linked to form a 3-to 8-membered ring, such as a 5-or 6-membered ring. The term "amide" encompasses groups such as sulfonamide, urea, ureido, urethane, carbamic acid and cyclic forms thereof. The term "amide" also encompasses amide groups attached to a carboxyl group, e.g., -amide-COOH or salts such as-amide-COONa, amino groups attached to a carboxyl group (e.g., -amino-COOH or salts such as-amino-COONa).

The term "amine" or "amino" as used herein refers to the form-NRdRe or-N (Rd) Re-, wherein Rd and Re are independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, carbamate, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen. Amino groups may be attached to parent molecular groups through nitrogen. The amino group may also be cyclic, e.g., any two of Rd and Re may be linked together or with N to form a 3-to 12-membered ring (e.g., morpholino or piperidinyl). The term amino also includes the corresponding quaternary ammonium salts of any amino group. Exemplary amino groups include alkylamino groups wherein at least one of Rd or Re is an alkyl group. In some embodiments, rd and Re may each be optionally substituted with hydroxy, halo, hydrocarbyloxy, ester, or amino.

The term "aryl" as used herein refers to a monocyclic, bicyclic, or other multicyclic aromatic ring system. The aryl group may optionally be fused to one or more rings selected from aryl, cycloalkyl and heterocyclyl. The aryl groups of the present disclosure may be substituted with a group selected from the group consisting of: hydrocarbyloxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halohydrocarbon, heteroaryl, heterocyclyl, hydroxy, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione. Exemplary aryl groups include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7, 8-tetrahydronaphthyl. Exemplary aryl groups also include, but are not limited to, monocyclic aromatic ring systems in which the ring contains 6 carbon atoms, referred to herein as "(C6) aryl.

The term "arylalkyl" as used herein refers to an alkyl group (e.g., -aryl-alkyl-) having at least one aryl substituent. Exemplary arylalkyl groups include, but are not limited to, arylalkyl groups having a monocyclic aromatic ring system, wherein the ring contains 6 carbon atoms, referred to herein as "(C6) arylalkyl.

The term "carbamate" as used herein refers to the form-RgOC (O) N (Rh) -, -RgOC (O) N (Rh) Ri-or-OC (O) NRhRi, wherein Rg, rh and Ri are each independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl and hydrogen. Exemplary carbamates include, but are not limited to, aryl carbamates or heteroaryl carbamates (e.g., wherein at least one of Rg, rh, and Ri is independently selected from aryl or heteroaryl groups such as pyridine, pyridazine, pyrimidine, and pyrazine).

The term "carboxy" as used herein refers to-COON or its corresponding carboxylate salt (e.g., -COONa). The term carboxy also includes "carboxycarbonyl", e.g., a carboxyl group attached to a carbonyl group, e.g., -C (O) -COOH or a salt, such as-C (O) -COONa.

The term "cyano" as used herein refers to-CN.

The term "cycloalkoxy" as used herein refers to a cycloalkoxy group attached to oxygen.

The term "cyclic hydrocarbon group" as used herein refers to a saturated or unsaturated cyclic hydrocarbon group of 3-12 carbons or 3-8 carbons derived from a cycloalkane, a bicyclic hydrocarbon group, or a bridged bicyclic hydrocarbon group, referred to herein as a "(C3-C8) cyclic hydrocarbon group). Exemplary cycloalkyl groups include, but are not limited to, cyclohexane, cyclohexene, cyclopentane and cyclopentene. The cycloalkyl group may be substituted as follows: hydrocarbyloxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halohydrocarbon, heteroaryl, heterocyclyl, hydroxy, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione. The cycloalkyl group may be fused to other saturated or unsaturated cycloalkyl groups, aryl groups or heterocyclyl groups.

The term "dicarboxylic acid" as used herein refers to groups comprising at least two carboxylic acid groups, such as saturated and unsaturated hydrocarbon dicarboxylic acids and salts thereof. Exemplary dicarboxylic acids include alkyl dicarboxylic acids. The dicarboxylic acid may be substituted as follows: hydrocarbyloxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halohydrocarbon, heteroaryl, heterocyclyl, hydrogen, hydroxy, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione. Dicarboxylic acids include, but are not limited to, succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, maleic acid, phthalic acid, aspartic acid, glutamic acid, malonic acid, fumaric acid, (+)/(-) -malic acid, (+)/(-) -tartaric acid, isophthalic acid, and terephthalic acid. Dicarboxylic acids also include carboxylic acid derivatives thereof, such as anhydrides, imides, hydrazides (e.g., succinic anhydride and succinimide).

The term "ester" refers to the structure-C (O) O-, -C (O) O-Rj-, rkC (O) O-Rj-or-RkC (O) O-, wherein O is not bonded to hydrogen, and Rj and Rk may be independently selected from hydrocarbyloxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, cycloalkyl, ether, halohydrocarbon, heteroaryl, and heterocyclyl. Rk may be hydrogen, but Rj may not be hydrogen. The esters may be cyclic, for example, carbon atoms and Rj, oxygen atoms and Rk, or Rj and Rk may be linked to form a 3-to 12-membered ring. Exemplary esters include, but are not limited to, alkyl esters wherein at least one of Rj or Rk is alkyl, such as-O-C (O) -alkyl-, -C (O) -O-alkyl-and-alkyl-C (O) -O-alkyl-. Exemplary esters also include aryl or heteroaryl esters, for example, wherein at least one of Rj or Rk is a heteroaryl group, such as pyridine, pyridazine, pyrimidine, and pyrazine, such as nicotinate. Exemplary esters also include reverse esters having the structure-RkC (O) O-, wherein oxygen is bound to the parent molecule. Exemplary reverse esters include succinic acid esters, D-arginine esters, L-lysine esters, and D-lysine esters. Esters also include carboxylic acid anhydrides and acid halides.

The term "halo" or "halogen" as used herein refers to F, cl, br or I.

The term "haloalkyl" as used herein refers to an alkyl group substituted with one or more halogen atoms. "halo-substituted hydrocarbyl" also encompasses alkenyl or alkynyl groups substituted with one or more halogen atoms.

The term "heteroaryl" as used herein refers to a monocyclic, bicyclic or polycyclic aromatic ring system containing one or more heteroatoms, e.g., 1-3 heteroatoms, such as nitrogen, oxygen and sulfur. Heteroaryl groups may be substituted with one or more substituents including hydrocarbyloxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halohydrocarbon, heteroaryl, heterocyclyl, hydroxy, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione. Heteroaryl groups may also be fused to non-aromatic rings. Illustrative examples of heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, pyrimidinyl (pyrimidyl), pyrazinyl (pyrazyl), triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1, 2, 3) -triazolyl and (1, 2, 4) -triazolyl, pyrazinyl (pyrazinyl), pyrimidinyl (pyrimidinyl), tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, furyl, phenyl, isoxazolyl and oxazolyl. Exemplary heteroaryl groups include, but are not limited to, monocyclic aromatic rings, wherein the rings contain 2-5 carbon atoms and 1-3 heteroatoms, referred to herein as "(C2-C5) heteroaryl.

The term "heterocycle (heterocycle)", "heterocyclyl" or "heterocyclics" as used herein refers to a saturated or unsaturated 3-, 4-, 5-, 6-, or 7-membered ring containing one, two, or three heteroatoms independently selected from nitrogen, oxygen, and sulfur. The heterocycle may be aromatic (heteroaryl) or non-aromatic. The heterocycle may be substituted with one or more substituents including hydrocarbyloxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halohydrocarbon, heteroaryl, heterocyclyl, hydroxy, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione. The heterocyclic ring also includes bicyclic, tricyclic, and tetracyclic groups, wherein any one of the above heterocyclic rings is fused to one or two rings independently selected from aryl, cycloalkyl, and heterocyclic. Exemplary heterocycles include acridinyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benzothienyl, benzoxazolyl, biotinyl, cinnolinyl, dihydrofuranyl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, furanyl, homopiperidinyl, imidazolidinyl, imidazolinyl, imidazolyl, indolyl, isoquinolyl, isothiazolidinyl, isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazinyl, pyrazolyl, pyrazolinyl, pyridazinyl, pyridinyl, pyrimidinyl (pyrimidinyl), pyrimidinyl (pyrimidyl), pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, pyrrolyl, quinolinyl, quinoxalinyl formyl (quinoxaloyl), tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, thiadiazolyl, thiazolidinyl, thiazolyl, thiothienyl, triazolyl (thiomorpholinyl), and thiomorpholinyl.

The terms "hydroxyl" and "hydroxyl" as used herein refer to-OH.

The term "hydroxyalkyl" as used herein refers to a hydroxyl group attached to an alkyl group.

The term "hydroxyaryl" as used herein refers to a hydroxyl group attached to an aryl group.

The term "ketone" as used herein refers to the structure-C (O) -Rn (such as acetyl, -C (O) CH 3) or-Rn-C (O) -Ro-. The ketone may be attached to another group by Rn or Ro. Rn or Ro may be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl, or Rn or Ro may be linked to form a 3-to 12-membered ring.

The term "monoester" as used herein refers to an analog of a dicarboxylic acid in which one of the carboxylic acids is functionalized as an ester and the other carboxylic acid is a free carboxylic acid or a salt of a carboxylic acid. Examples of monoesters include, but are not limited to, monoesters of succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, oxalic acid, and maleic acid.

The term "N-protecting group" refers to a group that is intended to protect an amino group from undesired reactions during the synthetic procedure. Commonly used N-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis," 4 th edition (John Wiley & Sons, hoboken, N.J., 2006), incorporated herein by reference. The N-protecting group includes: acyl groups, aroyl groups or carbamoyl groups, such as formyl, acetyl, propionyl, pivaloyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, o-nitrophenoxyacetyl, α -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl; and chiral auxiliary such as protected or unprotected D-amino acid, L-amino acid or D, L-amino acid, such as alanine, leucine, phenylalanine, etc.; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; urethane forming groups such as benzyloxycarbonyl, p-chlorobenzoxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3, 4-dimethoxybenzyloxycarbonyl, 3, 5-dimethoxybenzyloxycarbonyl, 2, 4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4, 5-dimethoxybenzyloxycarbonyl, 3,4, 5-trimethoxybenzyloxycarbonyl, 1- (p-biphenyl) -1-methylethoxycarbonyl, α -dimethyl-3, 5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenyloxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, benzyloxycarbonyl, such as benzyl, trimethoxymethyl, etc.; and silyl groups such as trimethylsilyl and the like. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butoxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

The term "phenyl" as used herein refers to a 6 membered carbocyclic aromatic ring. The phenyl group may also be fused to a cyclohexane ring or a cyclopentane ring. The phenyl group may be substituted with one or more substituents including hydrocarbyloxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halohydrocarbon, heteroaryl, heterocyclyl, hydroxy, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione.

The term "thioalkyl" as used herein refers to an alkyl group (-S-alkyl-) attached to sulfur.

The term "acetylation (acetylation)" or "acetylation (ethanoylation)" in IUPAC nomenclature refers to a reaction in which an acetyl functional group is introduced into a compound. In contrast, deacetylation refers to the removal of acetyl groups.

The "alkyl", "alkenyl", "alkynyl", "hydrocarbyloxy", "amino" and "amide" groups may be optionally substituted or interrupted or branched with at least one group selected from hydrocarbyloxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carbonyl, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halohydrocarbon, heteroaryl, heterocyclyl, hydroxy, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, thione, ureido and N. The substituents may be branched to form a substituted or unsubstituted heterocyclic or cyclic hydrocarbon group.

As used herein, suitable substituents on optionally substituted substituents refer to groups that do not negate the synthetic or pharmaceutical utility of the compounds of the present disclosure or of intermediates useful in preparing them. Examples of suitable substituents include, but are not limited to: c1-8 alkyl, alkenyl or alkynyl; c1-6 aryl, C7-5 heteroaryl; c3-7 cycloalkyl; c1-8 hydrocarbyloxy; c6 aryloxy; -CN; -OH; oxo; halo, carboxyl; amino, such as-NH (C1-8 alkyl), -N (C1-8 alkyl) 2, -NH ((C6) aryl) or-N ((C6) aryl) 2; a formyl group; ketones such as-CO (C1-8 alkyl), -CO (C6 aryl) esters such as-CO 2 (C1-8 alkyl) and-CO 2 (C6 aryl). Suitable substituents can be readily selected by those skilled in the art based on the stability, pharmacological and synthetic activity of the compounds of the present disclosure.

The term "active agent" or "active ingredient" refers to a substance, compound, or molecule that is biologically active or otherwise induces a biological or physiological effect in a subject to which it is administered. In other words, "active agent" or "active ingredient" refers to one or more components of a composition, all or part of the effect of which is attributed to the one or more components. The active agent may be the primary active agent, or in other words, a component of the composition, the full or partial effect of which is attributed to that component. The active agent may be a secondary agent, or in other words, a component of the composition to which additional portions of the composition are attributed and/or to which other effects of the composition are attributed.

In embodiments, "pharmaceutical compositions" are intended to include combinations of an active agent such as a therapeutic compound of the invention with an inert or active carrier in a sterile composition suitable for in vitro, in vivo, or ex vivo diagnostic or therapeutic use. In one aspect, the pharmaceutical composition is substantially free of endotoxin, or is non-toxic to the recipient at the dosage or concentration employed.

The term "pharmaceutically acceptable carrier" as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds that provide supplemental, additional, or enhanced therapeutic functions.

The term "pharmaceutically acceptable composition" as used herein refers to a composition comprising at least one compound as disclosed herein formulated with one or more pharmaceutically acceptable carriers.

The term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds of the present disclosure, and zwitterionic forms (where possible) of the compounds of the present disclosure, which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and commensurate with a reasonable benefit/risk ratio, and are effective for their intended use. Discussion is provided in Higuchi et al, "Prodrugs as Novel DELIVERY SYSTEMS," ACS Symposium Series, volume 14 and in Roche, e.b. editors Bioreversible CARRIERS IN Drug Design, american Pharmaceutical Association and Pergamon Press,1987, both of which are incorporated herein by reference.

The term "pharmaceutically acceptable salt" refers to salts of acidic or basic groups that may be present in the compounds used in the compositions of the present invention. The naturally basic compounds included in the compositions of the present invention are capable of forming various salts with a variety of inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts (i.e., salts containing pharmacologically acceptable anions including, but not limited to, sulfate, citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoate)), the amino moiety-containing compounds included in the compositions of the present invention can form pharmaceutically acceptable salts with a variety of amino acids other than the above-mentioned acids.

The compounds of the present disclosure may contain one or more chiral centers and/or double bonds, and thus exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. As used herein, the term "stereoisomer" consists of all geometric isomers, enantiomers or diastereomers. These compounds may be represented by the symbols "R" or "S" depending on the configuration of the substituents around the carbon atom of the stereoisomer. The present disclosure encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated "±" in nomenclature, but the skilled artisan will recognize that the structure may represent a chiral center intemally.

Individual stereoisomers of the compounds of the present disclosure may be prepared synthetically from commercially available starting materials containing asymmetric or stereogenic centers or by preparing racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These splitting methods are exemplified by: (1) attaching the mixture of enantiomers to a chiral auxiliary, separating the resulting mixture of diastereomers by recrystallization or chromatography, and releasing the optically pure product from the auxiliary, (2) salt formation with an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on a chiral chromatographic column. The stereoisomer mixture may also be resolved into their constituent stereoisomers by well known methods such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallization of compounds into chiral salt complexes, or crystallization of compounds in chiral solvents. Stereoisomers may also be obtained from stereopure intermediates, reagents and catalysts by well known asymmetric synthetic methods.

Geometric isomers may also be present in the compounds of the present disclosure. The present disclosure encompasses a variety of geometric isomers arising from the placement of substituents around a carbon-carbon double bond or the placement of substituents around a carbocyclic ring, and mixtures thereof. Substituents around a carbon-carbon double bond are designated as being in the "Z" or "E" configuration, wherein the terms "Z" and "E" are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the E isomer and the Z isomer.

Substituents around a carbon-carbon double bond may alternatively be referred to as "cis" or "trans", where "cis" means that the substituent is on the same side of the double bond and "trans" means that the substituent is on the opposite side of the double bond. The arrangement of substituents around a carbocycle is designated "cis" or "trans". The term "cis" means that the substituents are on the same side of the ring plane, and the term "trans" means that the substituents are on opposite sides of the ring plane. A mixture of compounds in which substituents are disposed on both the same side and opposite sides of the ring plane is designated "cis/trans".

The compounds disclosed herein may exist as tautomers, and the scope of the disclosure is intended to include both tautomeric forms, even though only one tautomeric structure is depicted.

When referring to amino acids or fragments thereof, the term "substantial homology" or "substantial similarity" indicates that when optimally aligned with the appropriate amino acid insertion or deletion of another amino acid (or its complementary strand), there is amino acid sequence identity in at least about 95% to 99% of the aligned sequences. Preferably, the homology is over the full length sequence or a protein thereof, such as cap protein, rep protein or a fragment thereof of at least 8 amino acids in length or more desirably at least 15 amino acids in length. Examples of suitable fragments are described herein.

The term "highly conserved" means at least 80% identical, preferably at least 90% identical and more preferably more than 97% identical. Identity is readily determined by those skilled in the art by means of algorithms and computer programs known to those skilled in the art.

In embodiments, an "effective amount" refers to, but is not limited to, an amount of a defined compound sufficient to achieve a desired therapeutic result. In embodiments, the result may be an effective cancer treatment.

In embodiments, as used herein, the terms "treatment", "treatment" and the like are used herein, but are not limited to, meaning obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disorder or a sign or symptom thereof, and/or may be therapeutic in terms of alleviating a symptom of the disease or infection or partially or completely curing the disorder and/or side effects attributable to the disorder.

As used herein, the term "recombinant" refers to a polypeptide or polynucleotide that does not occur in nature, and which may be produced by combining polynucleotides or polypeptides in an arrangement that does not normally occur together. The term may refer to a polypeptide produced by a biological host selected from the group consisting of mammalian expression systems, insect cell expression systems, yeast expression systems, and bacterial expression systems.

As used herein, the term "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen through one or more immunoglobulin variable regions. Putative immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a number of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin classes IgG, igM, igA, igD and IgE, respectively. Typically, the antigen binding region of an antibody will be most critical in terms of binding specificity and affinity, and is encoded by the variable domain. The antibody may be an intact antibody, an antigen-binding fragment, or a single chain thereof.

Exemplary immunoglobulin (antibody) structural units comprise tetramers. Each tetramer comprises identical two pairs of polypeptide chains, each pair having one "light chain" (about 25 kD) and one "heavy chain" (about 50kD-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to the variable domains of the light and heavy chains, respectively.

Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bond in the hinge region to produce F (ab)' ₂,F(ab)'₂, which is a dimer of Fab, which itself is the light chain VL-CL linked to VH-CH1 via a disulfide bond. While various antibody fragments are defined in terms of digestion of intact antibodies, the skilled artisan will appreciate that such fragments may be synthesized de novo, either chemically or by using recombinant DNA methods. Thus, as used herein, the term antibody also includes antibody fragments produced by modification of the entire antibody, or antibody fragments synthesized de novo using recombinant DNA methods (e.g., single chain Fv), or antibody fragments identified using phage display libraries (see, e.g., mcCafferty et al, nature 348:552-554 (1990)).

Thus, in any aspect of the invention, the term antibody also includes minibodies, scFv, diabodies, triabodies, and the like. ScFv and diabodies are small bivalent biospecific antibody fragments with high affinity and specificity. Their high signal-to-noise ratio is generally better because better specificity and rapid blood clearance increase their potential for diagnostic and therapeutic targeting of specific antigens (Sundaresan et al, J nucleic Med 44:1962-9 (2003)). Furthermore, these antibodies are advantageous because they can be engineered into different types of antibody fragments, if necessary, ranging from small single chain Fv (scFv) to complete IgG with different subtypes (Wu & Senter, nat. Biotechnol.23:1137-1146 (2005)). In some embodiments, the antibody fragment is part of an scFv-scFv or diabody. In some embodiments, in any aspect, the invention provides a high affinity antibody for use according to the invention.

The term "antibody fragment" or "antigen binding fragment" is used to refer to a portion of an antibody, such as Fab', fab, fv, scFv, and the like. Regardless of structure, the antibody fragment binds to the same antigen that is recognized by the intact antibody. The term "antibody fragment" also includes diabodies and any synthetic or genetically engineered protein comprising immunoglobulin variable regions that act like antibodies by binding to a specific antigen to form a complex.

The term "antigen binding fragment" or "Fab" refers to the region of an antibody that binds to an antigen. It comprises one constant domain and one variable domain (i.e., four domains: VH, CH1, VL, and CL 1) for each of the heavy and light chains. The variable domain comprises a paratope (antigen binding site) comprising a set of complementarity determining regions at the amino terminus of the monomer. Thus, each arm of Y binds an epitope on the antigen.

The term "Fc region" or "fragment crystallizable region" refers to the tail region of an antibody CH2-CH3, which interacts with cell surface receptors known as Fc receptors and some proteins of the complement system. This "effector function" allows antibodies to activate the immune system, resulting in cytotoxicity (ADCC), antibody Dependent Cellular Phagocytosis (ADCP), and/or Complement Dependent Cytotoxicity (CDC). ADCC and ADCP are mediated by Fc binding to Fc receptors on the cell surface of the immune system. CDC is mediated by the binding of Fc to proteins of the complement system (e.g., C1 q).

In IgG, igA and IgD antibody isotypes, the Fc region has two identical protein fragments derived from the second constant domain and the third constant domain of the two heavy chains of the antibody. The IgM Fc region and IgE Fc region have three heavy chain constant domains (CH domains 2-4) in each polypeptide chain, whereas IgG comprises 2 CH domains 2 and 3. The Fc region of IgG has a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor mediated activity. The N-glycans attached to this site are mainly complex types of core fucosylated double antenna structures. In addition, small amounts of these N-glycans also carry bisecting GlcNAc and alpha-2, 6 linked sialic acid residues.

The term "scFv" or "scFv fragment antibody" refers to a small molecule antibody consisting of a VH domain and a VL domain in the configuration of a VL-VH or VH-VL with a linker region between them. scFv fragment antibodies can penetrate more easily blood vessel walls and solid tumors, making them preferred carriers for targeted drugs.

"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acids do not encode an amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one of the conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. The skilled artisan will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce functionally identical molecules. Thus, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, not with respect to the actual probe sequence.

The therapeutic peptides of the invention may have amino acid additions, deletions or substitutions. The modified amino acid sequence is a sequence that differs from the native amino acid sequence by the deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof. In one embodiment, the modification is a point mutation. In one aspect, the modified therapeutic peptide does not have a naturally occurring sequence.

Amino acid substitutions may be conservative or non-conservative. As used herein, a "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The most common exchanges are bi-directional Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/Glu and Asp/Gly. Amino acid exchanges in Proteins and peptides that do not normally alter The activity of The protein or peptide are known in The art (H.Neurath, R.L.Hill, the Proteins, ACADEMIC PRESS, new York, 1979).

The term "derivative of a peptide" refers to a peptide having one or more residues chemically derivatized by reaction of functional side groups. Such derivatized molecules include, for example, those in which the free amino group has been derivatized to form an amine hydrochloride, a p-toluenesulfonyl group, a benzyloxycarbonyl group, a t-butoxycarbonyl group, a chloroacetyl group, or a formyl group. The free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. The free hydroxyl groups may be derivatized to form O-acyl derivatives or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzyl histidine. Also included as derivatives are those peptides containing one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may replace proline; the 5-hydroxy lysine may be substituted for lysine; 3-methylhistidine may replace histidine; homoserine may be substituted for serine; ornithine may be substituted for lysine.

Alternatively, the amino acid may be a modified amino acid residue and/or may be an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation). The non-naturally occurring amino acid may be a "non-natural" amino acid that may be used in the therapeutic compounds of the invention.

The term unnatural amino acid or unusual amino acid (unusual amino acid) includes those that can be constructed as synthetic peptides. These amino acids include D-amino acids, homoamino acids, beta-homoamino acids, N-methyl amino acids, alpha-methyl amino acids, unnatural side chain variant amino acids, and other unusual amino acids. D-amino acids involve the mirror image of the naturally occurring L-isomer. A high amino acid is an amino acid that includes an alpha-carbon that adds a methylene (CH ₂) group to the amino acid. Beta-homoamino acids are analogues of standard amino acids in which the carbon skeleton has been extended by insertion of one carbon atom immediately after the acid group. An N-methyl amino acid is an amino acid that carries a methyl group at the nitrogen rather than a proton. An alpha-methyl amino acid is a natural amino acid variant in which a proton on the alpha-carbon atom of the natural original amino acid (between the amino group and the carboxyl group) has been substituted with a methyl group. Unusual amino acids are most commonly found in microbial peptides and proteins and are formed post-translationally. Unusual amino acids tend to contribute to the specific biological activity of these peptides. In addition, the amino acids may be synthetic non-natural.

In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of identical amino acid residues or nucleotides that are about 60% identical, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity, over a specified region when compared and aligned for maximum correspondence over a comparison window or specified region, as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection. Such sequences are thus said to be "substantially identical". The definition also refers to the complement of the test sequence or complement that can be applied to the test sequence. The definition also includes sequences with deletions and/or additions, as well as those with substitutions. As described below, a preferred algorithm may interpret the interval (gap) and the like. Preferably, identity exists over a region of at least about 25 amino acids or nucleotides in length, or more preferably over a region of 50-100 amino acids or nucleotides in length.

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof as well as their complements in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which one or more of the third positions of the selected (or all) codons are substituted with mixed bases and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J. Biol. Chem.260:2605-2608 (1985); rossolini et al, mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

As used herein, the term "preventing" means inhibiting or delaying all actions of the occurrence of a disease.

As used herein, the term "treatment" means all actions that have been taken to alleviate, ameliorate or mitigate the symptoms of a disease. In this specification, "treatment" means alleviation, amelioration, or alleviation of symptoms of cancer, neurodegenerative or infectious disease by administration of antibodies disclosed herein.

The term "administering" refers to introducing an amount of a predetermined substance into a patient by some suitable method. The composition disclosed herein may be administered via any common route so long as it is capable of reaching the desired tissue, such as, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or intrarectal administration. However, since the peptide is digested after oral administration, the active ingredients of the composition for oral administration should be coated or formulated for protection from degradation in the stomach.

The term "subject" refers to those persons suspected of having or diagnosed with cancer, neurodegenerative disease, or infectious disease. However, including but not limited to any subject to be treated with the pharmaceutical compositions disclosed herein. A pharmaceutical composition comprising an anti-DLL 3 antibody disclosed herein is administered to a subject suspected of having cancer, a neurodegenerative disease, or an infectious disease.

The term "cancer" refers to human cancers and malignancies, sarcomas, adenocarcinomas, etc., including solid tumors, kidney cancers, breast cancers, lung cancers, kidney cancers, bladder cancers, urinary tract cancers, penis cancers, vulva cancers, vagina cancers, cervical cancers, colon cancers, ovarian cancers, prostate cancers, pancreatic cancers, stomach cancers, brain cancers, head and neck cancers, skin cancers, uterus cancers, testicular cancers, esophagus cancers and liver cancers. Additional cancers include, for example, hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung tumor, primary brain tumor, stomach cancer, colon cancer, malignant pancreatic islet tumor, malignant carcinoid, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenocortical cancer.

In any of the above embodiments, one or more cancer therapies, such as chemotherapy, radiation therapy, immunotherapy, surgery or hormonal therapy, may be further co-administered with the antibodies of the invention.

In one embodiment, the therapeutic compound is an alkylating agent: nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatin and derivatives, and non-classical alkylating agents. Nitrogen mustards include dichloromethyl diethylamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan. Nitrosoureas include N-nitroso-N-methyl urea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin. Tetrazines include dacarbazine, mitozolomide and temozolomide. Aziridines include thiotepa, mitomycin and deaquinone (diaziquone, AZQ). Cisplatin and its derivatives include cisplatin, carboplatin and oxaliplatin. In one embodiment, the chemotherapeutic agent is an antimetabolite: antifolates (e.g., methotrexate), fluoropyrimidines (e.g., fluorouracil and capecitabine), deoxynucleoside analogues, and thiopurines. In another embodiment, the chemotherapeutic agent is an anti-microtubule agent, such as vinca alkaloids (e.g., vincristine and vinblastine) and taxanes (e.g., paclitaxel and docetaxel). In another embodiment, the chemotherapeutic agent is a topoisomerase inhibitor or a cytotoxic antibiotic, such as doxorubicin, mitoxantrone, bleomycin, actinomycin, and mitomycin.

In another embodiment, the therapeutic compound is a compound identified in fig. 8.

The contacting of the patient with the therapeutic compound may be performed by administering the antibody to the patient intravenously, intraperitoneally, intramuscularly, intratumorally, or intradermally. In some embodiments, the therapeutic compound is co-administered with a cancer therapeutic.

The term "formulation" as used herein refers to the therapeutic compounds and excipients disclosed herein in combination that can be administered and have the ability to bind to the corresponding receptor and initiate a signal transduction pathway leading to the desired activity. The formulation may optionally contain other agents.

All numerical designations of the inclusive range, e.g., pH, temperature, time, concentration, and molecular weight, will be understood to be approximations according to the practices common in the art. As used herein, the term "about," considering the context, may represent a variation (+) or (-) 1%, 5% or 10% of the stated amount, as the case may be. It is to be understood that the agents described herein are merely exemplary, although not always explicitly stated, and that equivalents of such agents are known in the art.

Many known and useful compounds and the like can be found in Remington's Pharmaceutical Sciences (13 th edition), mack Publishing Company, easton, PA-standard references for multiple types of administration. As used herein, the term "formulation" means a combination of at least one active ingredient with one or more other ingredients (which may be independently active or inactive), also commonly referred to as excipients. The term "formulation" may or may not refer to a pharmaceutically acceptable composition for administration to humans or animals, and may include a composition that is useful as an intermediate for storage or research purposes.

Since the patients and subjects of the methods of the invention are veterinary subjects in addition to humans, formulations suitable for these subjects are also suitable. These subjects include domestic and companion animals and sports animals such as horses, troopsEtc.

For use as a treatment for human and animal subjects, the therapeutic compounds of the invention may be formulated as pharmaceutical or veterinary compositions. Therapeutic compounds are formulated in a manner that meets these parameters, depending on the subject to be treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or treatment). An overview of such techniques is found in the following: remington, THE SCIENCE AND PRACTICE of Pharmacy, 21 "edition, lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, incorporated by reference herein, J.Swarbrick and J.C.Boylan,1988-1999,Marcel Dekker,New York.

The therapeutic compounds described herein may be present in an amount of 1% -95% by weight of the total weight of the pharmaceutical composition. The pharmaceutical composition may be provided in a dosage form suitable for intra-articular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intracapsular, intraurethral, intrathecal, epidural, otic or ocular administration, or by injection, inhalation or direct contact with nasal, genitourinary, gastrointestinal, reproductive or oral mucosa. Thus, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, pill, powder, granule, suspension, emulsion, solution, gel (including hydrogels), paste, ointment, cream, plaster, drenching agent (drench), osmotic delivery device, suppository, enema, injectable (injectable), implant, spray, article of manufacture suitable for iontophoretic delivery, or aerosol. The composition may be formulated according to conventional pharmaceutical practice.

Detailed description of the preferred embodiments

Embodiments of the invention include methods of treating, monitoring and preventing cancer, including refractory cancer, using therapeutic compounds, pharmaceutical compositions and articles of manufacture thereof.

Therapeutic method

Another aspect of the application relates to a method for treating a cell proliferative disorder. The method comprises administering to a subject in need thereof an effective amount of a therapeutic compound according to the present disclosure. In another aspect, a method for treating a cell proliferative disorder comprises administering to a subject in need thereof an effective amount of a therapeutic compound according to the present disclosure.

Any suitable route or mode of administration may be used to provide a therapeutically effective dose or a prophylactically effective dose of the therapeutic compound to the patient. Exemplary routes or modes of administration include parenteral (e.g., intravenous, intra-arterial, intramuscular, subcutaneous, intratumoral), oral, topical (nasal, transdermal, intradermal, or intraocular), mucosal (e.g., nasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, enteral, intrapulmonary, intralymphatic, intracavity, intraorbital, intracapsular, and transurethral, and local delivery through a catheter or stent.

Pharmaceutical compositions comprising therapeutic compounds according to the present disclosure may be formulated in any pharmaceutically acceptable carrier or excipient. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and physiologically compatible analogs. The pharmaceutical composition may comprise a suitable solid or gel phase carrier or excipient. Exemplary carriers or excipients include calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol. Exemplary pharmaceutically acceptable carriers include one or more of the following: water, brine, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include an isotonic agent, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride in the composition. The pharmaceutically acceptable carrier may also contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers which enhance the shelf life or effectiveness of the therapeutic agent.

In embodiments, the therapeutic compound may be incorporated into a pharmaceutical composition suitable for parenteral administration. Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride may be used to alter the toxicity of solutions having concentrations of 0mM to 300mM (150 mM being optimal for liquid dosage forms). The lyophilized dosage form may comprise a cryoprotectant, primarily 0% -10% sucrose (optimally 0.5% -1.0%). Other suitable cryoprotectants include trehalose and lactose. The lyophilized dosage form may include a bulking agent (bulking agent), mainly 1% -10% mannitol (optimally 2% -4%). Stabilizers can be used in both liquid and lyophilized dosage forms, mainly 1mM-50mM L-methionine (most preferably 5mM-10 mM). Other suitable fillers include glycine, arginine, which may be included as 0% -0.05% > polysorbate-80 (optimally 0.005% -0.01%). Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.

The pharmaceutical composition comprising the therapeutic compound may be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol preservative) or in sterile water prior to injection. The pharmaceutical composition may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion.

The therapeutic compounds in the pharmaceutical compositions may be formulated in "therapeutically effective amounts" or "prophylactically effective amounts". "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result over the necessary dosage and period of time. The therapeutically effective amount of the recombinant vector may vary depending on the condition to be treated, the severity and course of the condition, the mode of administration, whether the antibody or agent is administered for prophylactic or therapeutic purposes, the bioavailability of the particular agent, the ability of the trispecific antibody to elicit a desired response in the individual, previous therapies, the age, weight and sex of the patient, the clinical history of the patient and the response to the antibody, the type of trispecific antibody used, the discretion of the attending physician, and the like. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the recombinant vector are exceeded by the therapeutic benefit. "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result over the necessary dosage and period of time.

The therapeutic compound is suitably administered to the patient at one time or in a series of treatments, and may be administered to the patient at any time from diagnosis. The therapeutic compounds may be administered as a sole treatment or in combination with other drugs or therapies useful in treating the condition in question.

In embodiments, the therapeutic compounds of the present invention include peptide mimetics P1.3 (also referred to hereinafter as IPR-473) or derivatives of P1.3 having the structure:

Wherein R ₁ comprises one of the following:

CH3, CH2-CH2-CH2-CH3 or CH2-CH2-CH 2-hexyl;

and R ₂ includes one of the following:

side chains corresponding to Dab, cyclohexylglycine, asn, cys, gly, ile, thr or Lys

And R ₃ includes one of the following:

Side chains corresponding to Leu, ala, gly, phe, ile, tyr or Val

And R ₄ includes one of the following:

H. CH3 or alkyl groups

And R ₅ includes one of the following:

H. CH3 or alkyl groups

In an embodiment, the therapeutic compounds of the invention include one of the following peptidomimetics, wherein P1.3 is the structure listed above, wherein the specific R ₁、R₂ and R ₃ of each peptidomimetic are listed in Table 1 below, and the relevant KRAS-GTPase docking scores for the specific peptidomimetics are listed in Table 1 below (including 140 variants of P1.3):

TABLE 1

Other analogs or derivatives of P1.3 include Fmoc-P1.3 as disclosed below:

R₁＝CH₃

R ₂＝CH₂-NH₂ (Dap side chain)

R ₃＝CH₂CH(CH₃)₂ (L-Leu side chain)

Additional analogues or derivatives of P1.3 include the following:

Wherein R ₁ is CH ₃,R₂ (CH ₂)₄NH₂(Lys),R₃ is CH (CH ₃)₂ (Val), and R ₅ is NCH ₃, and

R₁＝H

R ₂＝CH₂-NH₂ (Dap side chain)

R ₃＝CH₂CH(CH₃)₂ (L-Leu side chain)

Another analogue or derivative of P1.3 has the following structure:

R ₁＝-CH₂CH₂ - (embedded in pyrrolidine)

R ₂＝CH₂-NH₂ (Dap side chain)

R ₃＝CH₂CH(CH₃)₂ (L-Leu side chain)

R ₁＝-CH₂CH₂CH₂ - (embedded in piperidine)

R ₂＝CH₂-NH₂ (Dap side chain)

R ₃＝CH₂CH(CH₃)₂ (L-Leu side chain)

R₁＝-H

R ₂＝CH₂-NH₂ (Dap side chain)

R ₃＝CH₂CH(CH₃)₂ (L-Leu side chain).

Generally, a therapeutically effective amount or a prophylactically effective amount of a therapeutic compound will be administered in a range from about 1ng/kg body weight/day to about 100mg/kg body weight/day, whether by one or more administrations. In particular embodiments, the therapeutic compound is administered in the following ranges: from about 1ng/kg body weight to about 10mg/kg body weight/day, about 1ng/kg body weight/day to about 1mg/kg body weight/day, about 1ng/kg body weight/day to about 100g/kg body weight/day, about 1ng/kg body weight/day to about 10 g/body weight/day, about 1ng/kg body weight/day to about 1g/kg body weight/day, about 1ng/kg body weight/day to about 100ng/kg body weight/day, about 1ng/kg body weight/day to about 10ng/kg body weight/day, about 10ng/kg body weight/day to about 100mg/kg body weight/day, about 10ng/kg body weight/day to about 10mg/kg body weight/day, about 10ng/kg body weight/day to about 100g/kg body weight/day, about 10ng/kg body weight/day to about 10mg/kg body weight/day, about 10ng/kg body weight/day to about 1 ng/day, about 10ng/kg body weight/day to about 100 ng/day, about 100ng/kg body weight/day to about 100 mg/day, about 100ng/kg body weight/day to about 100mg/kg body weight/day, about 100 ng/day to about 100mg/kg body weight/day, about 1mg/kg body weight/day to about 100mg/kg body weight/day, about 1mg/kg body weight/day to about 10mg/kg body weight/day, about 1mg/kg body weight/day to about 1mg/kg body weight/day, about 1mg/kg body weight/day to about 100mg/kg body weight/day, about 1mg/kg body weight/day to about 10mg/kg body weight/day, about 10mg/kg body weight/day to about 100mg/kg body weight/day, about 10mg/kg body weight/day to about 10mg/kg body weight/day, about 10mg/kg body weight/day to about 1mg/kg body weight/day about 10mg/kg body weight/day to about 100mg/kg body weight/day, about 100mg/kg body weight/day to about 10mg/kg body weight/day, about 100mg/kg body weight/day to about 1mg/kg body weight/day, about 1mg/kg body weight/day to about 100mg/kg body weight/day, about 1mg/kg body weight/day to about 10mg/kg body weight/day, about 10mg/kg body weight/day to about 100mg/kg body weight/day.

In other embodiments, the therapeutic compound is administered at a dose of 500g to 20g every three days or 25mg/kg body weight every three days.

In other embodiments, the therapeutic compound is administered in the following ranges: about 10ng to about 100ng each alone, about 10ng to about 1g each alone, about 10ng to about 10g each alone, about 10ng to about 100mg each alone, about 10ng to about 1mg each alone, about 10ng to about 10mg each alone, about 10ng to about 100mg each alone, about 10ng to about 1000mg each alone, about 10ng to about 10,000mg each alone, about 100ng to about 1mg each alone, about 100ng to about 10mg each alone, about 100ng to about 100mg each alone, about 100ng to about 1000mg each alone, about 100ng to about 10,000mg each alone, about 1mg to about 10mg each alone, about 1mg to about 1mg each alone about 1mg to about 100mg per individual administration, about 1mg to about 1000mg per individual administration, about 1mg to about 10,000mg per individual administration, about 10mg to about 100mg per individual administration, about 10mg to about 1mg per individual administration, about 10mg to about 10mg per individual administration, about 10mg to about 100mg per individual administration, about 10mg to about 1000mg per individual administration, about 10mg to about 10,000mg per individual administration, about 100mg to about 1mg per individual administration, about 100mg to about 10mg per individual administration, about 100mg to about 100mg per individual administration, about 100mg to about 1000mg per individual administration, about 100mg to about 10,000mg per individual administration, about 1mg to about 10mg per individual administration, about 1mg to about 100mg per individual administration, about 1mg to about 1000mg per individual administration, about 1mg to about 10,000mg per individual administration, about 10mg to about 10mg per individual administration, about 10mg to about 10,000mg per individual administration, about 100mg to about 1000mg per injection, about 100mg to about 10,000mg per individual administration, and about 1000mg to about 10,000mg per individual administration. The trispecific antibodies may be administered daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days or every 7 days or every 1 week, every 2 weeks, every 3 weeks or every 4 weeks.

In other particular embodiments, an amount of therapeutic compound may be administered at a dose of about 0.0006 mg/day, 0.001 mg/day, 0.003 mg/day, 0.006 mg/day, 0.01 mg/day, 0.03 mg/day, 0.06 mg/day, 0.1 mg/day, 0.3 mg/day, 0.6 mg/day, 1 mg/day, 3 mg/day, 6 mg/day, 10 mg/day, 30 mg/day, 60 mg/day, 100 mg/day, 300 mg/day, 600 mg/day, 1000 mg/day, 2000 mg/day, 5000 mg/day, or 10,000 mg/day. As expected, the dose will depend on the condition, size, age and condition (condition) of the patient.

Dosages may be tested in several prior art accepted animal models suitable for any particular cell proliferative disorder.

In other aspects of this embodiment, a pharmaceutical composition disclosed herein reduces the size of a tumor by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

In other aspects of this embodiment, a pharmaceutical composition disclosed herein reduces the size of a tumor by, for example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

In other aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the size of a tumor by, for example, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, no more than 50%, no more than 55%, no more than 60%, no more than 65%, no more than 70%, no more than 75%, no more than 80%, no more than 85%, no more than 90%, or no more than 95%.

In still other aspects of this embodiment, a pharmaceutical composition disclosed herein reduces the size of a tumor from, for example, about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

The amount of the pharmaceutical composition disclosed herein is sufficient to allow for routine administration to an individual. In aspects of this embodiment, a pharmaceutical composition disclosed herein can be, for example, a pharmaceutical composition of at least 5mg, at least 10mg, at least 15mg, at least 20mg, at least 25mg, at least 30mg, at least 35mg, at least 40mg, at least 45mg, at least 50mg, at least 55mg, at least 60mg, at least 65mg, at least 70mg, at least 75mg, at least 80mg, at least 85mg, at least 90mg, at least 95mg, or at least 100 mg. In other aspects of this embodiment, a pharmaceutical composition disclosed herein can be, for example, a pharmaceutical composition of at least 5mg, at least 10mg, at least 20mg, at least 25mg, at least 50mg, at least 75mg, at least 100mg, at least 200mg, at least 300mg, at least 400mg, at least 500mg, at least 600mg, at least 700mg, at least 800mg, at least 900mg, at least 1,000mg, at least 1,100mg, at least 1,200mg, at least 1,300mg, at least 1,400mg, or at least 1,500mg. In yet other aspects of this embodiment, the pharmaceutical compositions disclosed herein can be within the following ranges: for example, about 5mg to about 100mg, about 10mg to about 100mg, about 50mg to about 150mg, about 100mg to about 250mg, about 150mg to about 350mg, about 250mg to about 500mg, about 350mg to about 600mg, about 500mg to about 750mg, about 600mg to about 900mg, about 750mg to about 1,000mg, about 850mg to about 1,200mg, or about 1,000mg to about 1,500mg. In still other aspects of this embodiment, the pharmaceutical compositions disclosed herein may be within the following ranges: for example, about 10mg to about 250mg, about 10mg to about 500mg, about 10mg to about 750mg, about 10mg to about 1,000mg, about 10mg to about 1,500mg, about 50mg to about 250mg, about 50mg to about 500mg, about 50mg to about 750mg, about 50mg to about 1,000mg, about 50mg to about 1,500mg, about 100mg to about 250mg, about 100mg to about 500mg, about 100mg to about 750mg, about 100mg to about 1,000mg, about 100mg to about 1,500mg, about 200mg to about 500mg, about 200mg to about 750mg, about 200mg to about 1,000mg, about 200mg to about 1,500mg, about 5mg to about 1,000mg, or about 5mg to about 250mg.

The pharmaceutical compositions disclosed herein may comprise an amount of solvent, emulsion, or other diluent sufficient to dissolve the therapeutic compounds disclosed herein. In other aspects of this embodiment, the pharmaceutical compositions disclosed herein may comprise solvents, emulsions, or diluents in the following amounts: for example, less than about 90% (v/v), less than about 80% (v/v), less than about 70% (v/v), less than about 65% (v/v), less than about 60% (v/v), less than about 55% (v/v), less than about 50% (v/v), less than about 45% (v/v), less than about 40% (v/v), less than about 35% (v/v), less than about 30% (v/v), less than about 25% (v/v), less than about 20% (v/v), less than about 15% (v/v), less than about 10% (v/v), less than about 5% (v/v), or less than about 1% (v/v). In other aspects of this embodiment, the pharmaceutical compositions disclosed herein may comprise solvents, emulsions, or other diluents in amounts within the following ranges: for example, about 1% (v/v) to 90% (v/v), about 1% (v/v) to 70% (v/v), about 1% (v/v) to 60% (v/v), about 1% (v/v) to 50% (v/v), about 1% (v/v) to 40% (v/v), about 1% (v/v) to 30% (v/v), about 1% (v/v) to 20% (v/v), about 1% (v/v) to 10% (v/v), about 2% (v/v) to 50% (v/v), about 2% (v/v) to 40% (v/v), about 2% (v/v) to 30% (v/v), about 2% (v/v) to 20% (v/v), about 2% (v/v) to 10% (v/v), about 4% (v/v) to 50% (v/v), about 4% (v/v) to 30% (v), about 20% (v/v) to 20% (v/v), about 4% (v/v) to 10% (v), about 6% (v/v) to 50% (v/v), about 6% (v/v) to 40% (v/v), about 6% (v/v) to 30% (v/v), about 6% (v/v) to 20% (v/v), about 6% (v/v) to 10% (v/v), about 8% (v/v) to 50% (v/v), about 8% (v/v) to 40% (v/v), about 8% (v/v) to 30% (v/v), about 8% (v/v) to 20% (v/v), about 8% (v/v) to 15% (v/v), or about 8% (v/v) to 12% (v/v).

The final concentration of the therapeutic compound disclosed herein in the pharmaceutical composition disclosed herein can be any concentration desired. In aspects of this embodiment, the final concentration of the therapeutic compound in the pharmaceutical composition can be a therapeutically effective amount. In other aspects of this embodiment, the final concentration of the therapeutic compound in the pharmaceutical composition can be, for example, at least 0.00001mg/mL, at least 0.0001mg/mL, at least 0.001mg/mL, at least 0.01mg/mL, at least 0.1mg/mL, at least 1mg/mL, at least 10mg/mL, at least 25mg/mL, at least 50mg/mL, at least 100mg/mL, at least 200mg/mL, or at least 500mg/mL. In other aspects of this embodiment, the final concentration of the therapeutic compound in the pharmaceutical composition can be within the following range: for example, the number of the cells to be processed, about 0.00001mg/mL to about 3,000mg/mL, about 0.0001mg/mL to about 3,000mg/mL, about 0.01mg/mL to about 3,000mg/mL, about 0.1mg/mL to about 3,000mg/mL, about 1mg/mL to about 3,000mg/mL, about 250mg/mL to about 3,000mg/mL, about 500mg/mL to about 3,000mg/mL, about 750mg/mL to about 3,000mg/mL, about 1,000mg/mL to about 3,000mg/mL, about 100mg/mL to about 2,000mg/mL about 250mg/mL to about 2,000mg/mL, about 500mg/mL to about 2,000mg/mL, about 750mg/mL to about 2,000mg/mL, about 1,000mg/mL to about 2,000mg/mL, about 100mg/mL to about 1,500mg/mL, about 250mg/mL to about 1,500mg/mL, about 500mg/mL to about 1,500mg/mL, about 750mg/mL to about 1,500mg/mL, about 1,000mg/mL to about 1,500mg/mL about 100mg/mL to about 1,200mg/mL, about 250mg/mL to about 1,200mg/mL, about 500mg/mL to about 1,200mg/mL, about 750mg/mL to about 1,200mg/mL, about 1,000mg/mL to about 1,200mg/mL, about 100mg/mL to about 1,000mg/mL, about 250mg/mL to about 1,000mg/mL, about 500mg/mL to about 1,000mg/mL, about 750mg/mL to about 1,000mg/mL, about 100mg/mL to about 750mg/mL, about 250mg/mL to about 750mg/mL, about 500mg/mL to about 750mg/mL, about 100mg/mL to about 500mg/mL, about 250mg/mL to about 500mg/mL, about 0.00001mg/mL to about 0.0001mg/mL, about 0.00001mg/mL to about 0.001mg/mL, about 0.00001mg/mL to about 01mg/mL, about 0.00001mg/mL, about 0.01mg to about 0.1mg/mL, about 0.00001mg/mL to about 1mg/mL, about 0.001mg/mL to about 0.01mg/mL, about 0.001mg/mL to about 0.1mg/mL, about 0.001mg/mL to about 1mg/mL, about 0.001mg/mL to about 10mg/mL, or about 0.001mg/mL to about 100mg/mL.

Aspects of the present specification disclose, in part, treating a subject afflicted with cancer. As used herein, the term "treating" refers to reducing or eliminating clinical symptoms of cancer in an individual; or delay or prevent the onset of clinical symptoms of cancer in an individual. For example, the term "treating" may mean reducing the symptoms of a condition characterized by cancer, including but not limited to tumor size, by, for example, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. The actual symptoms associated with cancer are well known and can be determined by one of ordinary skill in the art by considering a variety of factors including, but not limited to, the location of the cancer, the cause of the cancer, the severity of the cancer, and/or the tissue or organ affected by the cancer. Those skilled in the art will know the appropriate symptoms or signs (indicators) associated with a particular type of cancer and will know how to determine whether an individual is a candidate for treatment as disclosed herein.

In another aspect, the pharmaceutical compositions disclosed herein reduce the severity of symptoms of a cancer-related disorder. In aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the severity of a symptom of a cancer-related disorder by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In other aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the severity of a symptom of a cancer-related disorder by, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

In aspects of this embodiment, a therapeutically effective amount of a pharmaceutical composition disclosed herein reduces a symptom associated with cancer by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In other aspects of this embodiment, a therapeutically effective amount of a pharmaceutical composition disclosed herein reduces a symptom associated with cancer by, for example, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 100%. In yet other aspects of this embodiment, a therapeutically effective amount of a pharmaceutical composition disclosed herein reduces a symptom associated with cancer by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

In still other aspects of this embodiment, a therapeutically effective amount of the pharmaceutical composition disclosed herein is generally in the range of about 0.001mg/kg to about 100mg/kg and is administered, for example, every 3 days, every 5 days, every 7 days, every 10 days, or every 14 days. In aspects of this embodiment, an effective amount of a pharmaceutical composition disclosed herein can be, for example, at least 0.001mg/kg, at least 0.01mg/kg, at least 0.1mg/kg, at least 1.0mg/kg, at least 5.0mg/kg, at least 10mg/kg, at least 15mg/kg, at least 20mg/kg, at least 25mg/kg, at least 30mg/kg, at least 35mg/kg, at least 40mg/kg, at least 45mg/kg, or at least 50mg/kg, and administered, for example, every 3 days, every 5 days, every 7 days, every 10 days, or every 14 days. In other aspects of this embodiment, an effective amount of a pharmaceutical composition disclosed herein can be within the following range: for example, about 0.001mg/kg to about 10mg/kg, about 0.001mg/kg to about 15mg/kg, about 0.001mg/kg to about 20mg/kg, about 0.001mg/kg to about 25mg/kg, about 0.001mg/kg to about 30mg/kg, about 0.001mg/kg to about 35mg/kg, about 0.001mg/kg to about 40mg/kg, about 0.001mg/kg to about 45mg/kg, about 0.001mg/kg to about 50mg/kg, about 0.001mg/kg to about 75mg/kg, or about 0.001mg/kg to about 100mg/kg, and is administered, for example, every 3 days, every 5 days, every 7 days, every 10 days, or every 14 days. In yet other aspects of this embodiment, an effective amount of a pharmaceutical composition disclosed herein can be within the following range: for example, 0.01mg/kg to about 10mg/kg, about 0.01mg/kg to about 15mg/kg, about 0.01mg/kg to about 20mg/kg, about 0.01mg/kg to about 25mg/kg, about 0.01mg/kg to about 30mg/kg, about 0.01mg/kg to about 35mg/kg, about 0.01mg/kg to about 40mg/kg, about 0.01mg/kg to about 45mg/kg, about 0.01mg/kg to about 50mg/kg, about 0.01mg/kg to about 75mg/kg, or about 0.01mg/kg to about 100mg/kg, and administered, for example, every 3 days, every 5 days, every 7 days, every 10 days, or every 14 days. In still other aspects of this embodiment, an effective amount of a pharmaceutical composition disclosed herein can be within the following range: for example, about 0.1mg/kg to about 10mg/kg, about 0.1mg/kg to about 15mg/kg, about 0.1mg/kg to about 20mg/kg, about 0.1mg/kg to about 25mg/kg, about 0.1mg/kg to about 30mg/kg, about 0.1mg/kg to about 35mg/kg, about 0.1mg/kg to about 40mg/kg, about 0.1mg/kg to about 45mg/kg, about 0.1mg/kg to about 50mg/kg, about 0.1mg/kg to about 75mg/kg, or about 0.1mg/kg to about 100mg/kg, and is administered, for example, every 3 days, every 5 days, every 7 days, every 10 days, or every 14 days.

In liquid and semi-solid formulations, the concentration of the therapeutic compounds disclosed herein may generally be between about 50mg/mL to about 1,000mg/mL. In an aspect of this embodiment, A therapeutically effective amount of a therapeutic compound disclosed herein can be, for example, from about 50mg/mL to about 100mg/mL, about 50mg/mL to about 200mg/mL, about 50mg/mL to about 300mg/mL, about 50mg/mL to about 400mg/mL, about 50mg/mL to about 500mg/mL, about 50mg/mL to about 600mg/mL, about 50mg/mL to about 700mg/mL, about 50mg/mL to about 800mg/mL, about 50mg/mL to about 900mg/mL, about 50mg/mL to about 1,000mg/mL, about 100mg/mL to about 200mg/mL, about 100mg/mL to about 300mg/mL, about 100mg/mL to about 400mg/mL, about 100mg/mL to about 500mg/mL, about 100mg/mL to about 600mg/mL, about 100mg/mL to about 700mg/mL, about 100mg/mL to about 800mg/mL about 100mg/mL to about 900mg/mL, about 100mg/mL to about 1,000mg/mL, about 200mg/mL to about 300mg/mL, about 200mg/mL to about 400mg/mL, about 200mg/mL to about 500mg/mL, about 200mg/mL to about 600mg/mL, about 200mg/mL to about 700mg/mL, about 200mg/mL to about 800mg/mL, about 200mg/mL to about 900mg/mL, about 200mg/mL to about 1,000mg/mL, about 300mg/mL to about 400mg/mL, about 300mg/mL to about 500mg/mL, about 300mg/mL to about 600mg/mL, about 300mg/mL to about 700mg/mL, about 300mg/mL to about 800mg/mL, about 300mg/mL to about 900mg/mL, about 300mg/mL to about 1,000mg/mL, about 400mg/mL, about 500mg/mL, about 400mg/mL to about 600mg/mL, about 400mg/mL to about 700mg/mL, about 400mg/mL to about 800mg/mL, about 400mg/mL to about 900mg/mL, about 400mg/mL to about 1,000mg/mL, about 500mg/mL to about 600mg/mL, about 500mg/mL to about 700mg/mL, about 500mg/mL to about 800mg/mL, about 500mg/mL to about 900mg/mL, about 500mg/mL to about 1,000mg/mL, about 600mg/mL to about 700mg/mL, about 600mg/mL to about 800mg/mL, about 600mg/mL to about 900mg/mL, or about 600mg/mL to about 1,000mg/mL.

Administration may be single dose or cumulative (continuous administration) and can be readily determined by one skilled in the art. For example, treating cancer may comprise administering an effective dose of the pharmaceutical composition disclosed herein at one time. Alternatively, treating cancer may include multiple administrations of an effective dose of the pharmaceutical composition over a series of time periods, such as, for example, once a day, twice a day, three times a day, once every few days, or once a week. The time of administration may vary from person to person, depending on factors such as the severity of the individual's symptoms. For example, an effective dose of a pharmaceutical composition disclosed herein may be administered to an individual once daily for an indefinite period of time, or until the individual no longer requires treatment. One of ordinary skill in the art will recognize that the status of an individual may be monitored throughout the course of treatment and that the effective amount of the pharmaceutical composition disclosed herein administered may be adjusted accordingly.

In one embodiment, a therapeutic compound disclosed herein is capable of reducing the number of cancer cells or tumor size in an individual afflicted with cancer by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% as compared to a patient who has not received the same treatment.

In another embodiment, a therapeutic compound disclosed herein is capable of reducing the number of cancer cells or tumor size in an individual afflicted with a cancer by, for example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% as compared to a patient who has not received the same treatment.

In embodiments, the therapeutic compounds disclosed herein are capable of reducing the number of cancer cells or tumor size in an individual afflicted with cancer by, for example, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, no more than 50%, no more than 55%, no more than 60%, no more than 65%, no more than 70%, no more than 75%, no more than 80%, no more than 85%, no more than 90%, or no more than 95% as compared to a patient who has not received the same treatment. In other aspects of this embodiment, the therapeutic compound is capable of reducing the number of cancer cells or tumor size in an individual afflicted with a cancer by, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a patient who has not been treated with the same.

In further embodiments, the therapeutic compounds and derivatives thereof have the following half-lives: 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months or more.

In embodiments, the period of administration of the therapeutic compound lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer. In further embodiments, the period of time that administration is stopped lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer.

In aspects of this embodiment, a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains, for example, 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the size of a cancer cell population and/or tumor cells in an individual.

In other aspects of this embodiment, a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains, for example, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 100% of the cancer cell population and/or tumor cell size in an individual.

In other aspects of this embodiment, a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains, for example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% the size of a cancer cell population and/or tumor cells in an individual.

In still other aspects of this embodiment, a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50% of the cancer cell population and/or tumor cell size in an individual.

The pharmaceutical composition or therapeutic compound is administered to the subject. The individual is typically a human, but may be an animal, including but not limited to dogs, cats, birds, cattle, horses, sheep, goats, reptiles, and other animals, whether or not domesticated. Typically, any individual that is a candidate for treatment is a candidate for having some form of cancer, whether the cancer is benign or malignant, a solid tumor or other tumor, cancer cells that are not located in a tumor, or some other form of cancer. Among the most common types of cancer include, but are not limited to, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, leukemia, lung cancer, melanoma, non-hodgkin's lymphoma, pancreatic cancer, prostate cancer, gastric cancer, and thyroid cancer. In addition to full informed consent disclosing all relevant risks and benefits of the procedure, pre-operative evaluation typically includes routine medical history and physical examination.

In embodiments, the pharmaceutical composition or therapeutic compound is administered to treat a sarcoma. In embodiments, the sarcoma is one or more of the following: hemangiosarcoma, chondrosarcoma, dermatofibrosarcoma carinii, desmoplasia small round cell tumors, epithelioid sarcoma, ewing's sarcoma, gastrointestinal stromal tumor (GIST), kaposi's sarcoma, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, soft tissue sarcoma, isolated fibrotumor, synovial sarcoma, and undifferentiated polymorphous sarcoma. In another embodiment, the sarcoma to be treated is uterine sarcoma. In further embodiments, the pharmaceutical composition or therapeutic compound is administered to treat uterine cancer. The uterine cancer is endometrial cancer or uterine sarcoma.

In one aspect, the pharmaceutical compositions disclosed herein reduce the symptoms of a cancer-related disorder. In aspects of this embodiment, a pharmaceutical composition disclosed herein reduces a symptom of a cancer-related disorder by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In other aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce symptoms of a cancer-related disorder by, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

In another aspect, the pharmaceutical compositions disclosed herein reduce the frequency of symptoms of cancer-related disorders that occur over a given period of time. In aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the frequency of symptoms of a cancer-related disorder occurring over a given period of time by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In other aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the frequency of symptoms of a cancer-related disorder occurring within a given period of time by, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

The methods of treatment of the present specification may include the step of administering a pharmaceutical composition comprising a therapeutic compound in a pharmaceutically effective amount. The total daily dose should be determined by the physician with appropriate medical judgment and administered once or several times. The specific therapeutically effective dosage level for any particular patient may vary depending on a variety of factors well known in the medical arts, including the type and extent of response to be achieved, the pharmaceutical composition with which the other agents are to be used, the age, weight, health, sex and diet of the patient, the time and route of administration, the secretion rate of the pharmaceutical composition, the period of treatment, other drugs in combination or concurrent with the pharmaceutical composition disclosed herein, and similar factors well known in the medical arts.

In yet another aspect, the present specification provides the use of a therapeutic compound and a pharmaceutical composition comprising the therapeutic compound in the manufacture of a medicament for the prevention or treatment of cancer, neurodegenerative disease or infectious disease.

In one embodiment, the dosage of the pharmaceutical composition may be administered daily, half-week, weekly, biweekly, or monthly. The treatment period may last one week, two weeks, one month, two months, four months, six months, eight months, one year or more. The initial dose may be greater than the maintenance dose.

In one embodiment, the dosage is in the range of weekly dosages of at least 0.01mg/kg, at least 0.25mg/kg, at least 0.3mg/kg, at least 0.5mg/kg, at least 0.75mg/kg, at least 1mg/kg, at least 2mg/kg, at least 3mg/kg, at least 4mg/kg, at least 5mg/kg, at least 6mg/kg, at least 7mg/kg, at least 8mg/kg, at least 9mg/kg, at least 10mg/kg, at least 15mg/kg, at least 20mg/kg, at least 25mg/kg, or at least 30 mg/kg.

In one embodiment, the weekly dose may be at most 1.5mg/kg, at most 2mg/kg, at most 2.5mg/kg, at most 3mg/kg, at most 4mg/kg, at most 5mg/kg, at most 6mg/kg, at most 7mg/kg, at most 8mg/kg, at most 9mg/kg, at most 10mg/kg, at most 15mg/kg, at most 20mg/kg, at most 25mg/kg, or at most 30mg/kg. In a particular aspect, the weekly dose may be in the range from 5mg/kg to 20 mg/kg. In alternative aspects, the weekly dose may be in the range of from 10mg/kg to 15 mg/kg.

The present specification also provides a pharmaceutical composition for administration to a subject. The pharmaceutical compositions disclosed herein may also comprise a pharmaceutically acceptable carrier, excipient or diluent. As used herein, the term "pharmaceutically acceptable" means that the composition is sufficient to achieve a therapeutic effect without deleterious side effects and can be readily determined based on the type of disease, age, weight, health, sex and drug sensitivity of the patient, route of administration, mode of administration, frequency of administration, duration of treatment, drugs combined or used concurrently with the compositions disclosed herein, and other factors known in medicine.

Pharmaceutical compositions comprising the therapeutic compounds disclosed herein may also comprise a pharmaceutically acceptable carrier. For oral administration, carriers can include, but are not limited to, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants and flavorants. For injectable preparations, the carriers may include buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers. For articles for topical application, the carrier may include a matrix, an excipient, a lubricant, and a preservative.

The disclosed pharmaceutical compositions can be formulated into a variety of dosage forms in combination with the aforementioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical compositions may be formulated as tablets, troches, capsules, elixirs, suspensions, syrups or wafers (wafer). For injectable preparations, the pharmaceutical compositions may be formulated as ampoules as single dosage forms or as multi-dose containers. The pharmaceutical compositions may also be formulated as solutions, suspensions, tablets, pills, capsules and long-acting formulations.

On the other hand, examples of carriers, excipients and diluents suitable for pharmaceutical formulations include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical formulation may also contain fillers, anticoagulants, lubricants, moisturizers, flavors, and antibacterial agents (antiseptic).

Furthermore, the pharmaceutical compositions disclosed herein may have any formulation selected from the group consisting of: tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, nonaqueous solvents, lyophilized formulations and suppositories.

The pharmaceutical composition may be formulated into a single dosage form suitable for the patient's body and is preferably formulated into a preparation useful for peptide mimetic medicaments according to typical methods in the pharmaceutical arts for administration by oral or parenteral routes, such as by cutaneous, intravenous, intramuscular, intraarterial, intramedullary, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intracolonic, topical, sublingual, vaginal or rectal administration, but is not limited thereto.

The composition may be used by blending with various pharmaceutically acceptable carriers such as physiological saline or an organic solvent. For increased stability or absorption, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used.

The dosage and frequency of administration of the pharmaceutical compositions disclosed herein is determined by the type of active ingredient and a variety of factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.

The total effective dose of the pharmaceutical compositions disclosed herein may be administered to a patient in a single dose, or may be administered in multiple doses for a prolonged period of time according to a fractionated treatment regimen. In the pharmaceutical compositions disclosed herein, the amount of active ingredient may vary depending on the severity of the disease. Preferably, the total daily dose of the peptide mimetic disclosed herein can be about 0.0001 μg to 500mg per 1kg patient body weight. However, in addition to the route of administration and frequency of treatment of the pharmaceutical composition, a variety of factors are considered to determine the effective dose of the peptidomimetic, including the age, weight, health, sex, severity of the disease, diet and secretion rate of the patient. In view of this, one of ordinary skill in the art can readily determine an effective dose suitable for the particular use of the pharmaceutical compositions disclosed herein. The pharmaceutical composition disclosed herein is not particularly limited to the formulation, and the route and mode of administration, as long as it shows a suitable effect.

Furthermore, the pharmaceutical composition may be administered alone or in combination or simultaneously with other pharmaceutical formulations of the active agent showing a prophylactic or therapeutic efficacy or within the active agent showing a prophylactic or therapeutic efficacy.

In yet another aspect, the present specification provides a method for preventing or treating cancer, an infectious disease, or a neurodegenerative disease, the method comprising the step of administering a therapeutic compound or a pharmaceutical composition comprising the therapeutic compound to a subject.

In view of the teachings and guidance provided herein, those skilled in the art will appreciate that the formulations described herein may be equally applicable to many types of peptidomimetics and other therapeutic compounds, including those illustrated, as well as other compounds known in the art. In view of the teachings and guidance provided herein, those skilled in the art will also appreciate that the selection of, for example, the type and/or amount of one or more excipients, surfactants, and/or optional components may be based on chemical and functional compatibility with the biopharmaceutical to be formulated and/or mode of administration, as well as other chemical, functional, physiological, and/or medical factors well known in the art. For example, non-reducing sugars exhibit advantageous excipient properties when used with polypeptide biopharmaceuticals as compared to reducing sugars. Thus, exemplary formulations are further illustrated herein with reference to different peptidomimetics. However, the applicability ranges, chemical and physical properties, considerations and methodologies applied to polypeptide biopharmaceuticals may similarly apply to biopharmaceuticals other than polypeptide biopharmaceuticals.

In various embodiments, the pharmaceutical composition may comprise, but is not limited to, a combination of therapeutic compounds (such as viruses, proteins, antibodies, peptides, etc., as described herein) in the pharmaceutical composition. For example, a pharmaceutical composition as described herein may comprise a single therapeutic compound, such as a peptidomimetic, for treating one or more conditions, including but not limited to diseases. In embodiments, a pharmaceutical composition as described herein may also comprise, but is not limited to, two or more different therapeutic compounds for a single condition or multiple conditions. The use of multiple therapeutic compounds in a formulation may be directed to, for example, the same or different indications. Similarly, in another embodiment, a variety of therapeutic compounds may be used in the formulation to treat, for example, both a pathological condition and one or more side effects caused by primary treatment. In further embodiments, a plurality of therapeutic compounds may also be included in, but are not limited to, pharmaceutical compositions as described herein to achieve different medical objectives, including, for example, simultaneous treatment and monitoring of the progression of a pathological condition. In further embodiments, multiple concurrent therapies, such as those exemplified herein, as well as other combinations well known in the art, are particularly useful for patient compliance, as a single pharmaceutical composition may be sufficient for some or all of the suggested treatments and/or diagnoses. Those skilled in the art will recognize that therapeutic compounds may be mixed for a wide range of combination therapies. Similarly, in various embodiments, a first therapeutic compound may be used in combination with a second therapeutic compound or more therapeutic compounds and one or more therapeutic compounds along with one or more other therapeutic compounds (including small molecules or antibody drugs). Thus, in various embodiments, formulations comprising 1,2,3,4, 5, or 6 or more different therapeutic compounds are provided, as well as formulations comprising one or more therapeutic compounds in combination with one or more other therapeutic compounds.

In various embodiments, the pharmaceutical composition may comprise one or more preservatives and/or additives known in the art. Similarly, the pharmaceutical composition may also be formulated into, but is not limited to, any of a variety of known delivery formulations. For example, in embodiments, the pharmaceutical compositions may comprise surfactants, adjuvants, biodegradable polymers, hydrogels, and the like, such optional components, their chemical and functional properties being known in the art. Similarly known in the art are pharmaceutical compositions that facilitate rapid, sustained or delayed release of the bioactive agent following administration. Formulations as described may be produced to contain these formulation components or other formulation components known in the art.

Thus, the pharmaceutical composition may be administered over time as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implant device or catheter. Further refinement of the appropriate dose is routinely performed by those of ordinary skill in the art and is within the scope of the tasks they routinely perform. The appropriate dose may be determined by using appropriate dose-response data. In various embodiments, the therapeutic compounds in the pharmaceutical compositions described herein may be administered to a patient over an extended period of time, such as for chronic administration of a chronic condition, but are not limited to. The composition may be solid, semi-solid or aerosol, and the pharmaceutical composition is formulated as a tablet, gel sheet (geltab), lozenge, orally dissolving strip, capsule, syrup, oral suspension, emulsion, granule, spray (sprinkle), or pellet (pellet).

In embodiments, for oral, rectal, vaginal, parenteral, pulmonary, sublingual and/or intranasal delivery formulations, tablets may be prepared by compression or molding, optionally together with one or more auxiliary ingredients or additives. In embodiments, compressed tablets are prepared, for example, by compressing in a suitable tablet press a therapeutic compound in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., without limitation, povidone, gelatin, hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., without limitation, sodium starch glycolate, crospovidone, croscarmellose sodium), and/or a surfactant or dispersant.

In embodiments, molded tablets are prepared, for example, but not limited to, by molding in a suitable tablet press a mixture of the powdered compound moistened with an inert liquid diluent. In embodiments, the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient using, for example, but not limited to, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. In embodiments, the tablets may optionally be provided with a coating, but are not limited thereto, such as a film, sugar coating, or enteric coating, to provide release in portions of the intestine other than the stomach. In embodiments, processes, equipment and processors for tablet and capsule preparation are well known in the art.

In embodiments, the capsule pharmaceutical composition may utilize hard or soft capsules, including but not limited to gelatin or vegetarian capsules, such as those made of hydroxymethyl propyl cellulose (HMPC). In embodiments, the type of capsule is a gelatin capsule. In embodiments, as described in detail in Pharmaceutical Capules, second supplement, f.podczeck and b.jones,2004, the capsules may be filled using a capsule filling machine such as, but not limited to, a capsule filling machine available from commercial suppliers such as Miranda International, or using capsule manufacturing techniques well known in the industry. In embodiments, the encapsulated pharmaceutical compositions may be prepared, but are not limited to, using a processing center such as Chao Center for Industrial Pharmacy & Contract Manufacturing located in Purdue RESEARCH PARK.

The packaging and instrumentation used for administration may be determined by a variety of considerations, such as, but not limited to, the volume of material to be administered, storage conditions, whether a skilled medical practitioner will administer or patient self-compliance, dosage regimens, geopolitical environments (e.g., exposure to extreme temperature conditions in developing countries), and other practical considerations.

Injection devices include pen-type injectors, automatic injectors, safety injectors, syringe pumps, infusion pumps, glass pre-filled injectors, plastic pre-filled injectors, and needleless injectors. The syringe may be prefilled with a liquid, or may be dual-chambered, for example for use with lyophilized materials. An example of a syringe for such use is Lyo-select ^TM,Lyo-Ject^TM which is a dual chamber prefilled freeze-dried syringe available from Vetter GmbH, ravensburg, germany. Another example is LyoTip, which is a prefilled syringe available from LyoTip, inc., camarillo, california, u.s.a. designed to conveniently deliver lyophilized formulations. Administration by injection may be intravenous, intramuscular, intraperitoneal or subcutaneous, as the case may be. Administration by the non-injection route may be, but is not limited to, nasal, oral, ocular, dermal or pulmonary, as the case may be.

In certain embodiments, the kits may include, but are not limited to, one or more single or multi-chamber syringes (e.g., liquid syringes and lyophilized syringes) for administering one or more pharmaceutical compositions described herein. In various embodiments, the kit may include a pharmaceutical composition for parenteral, subcutaneous, intramuscular, or IV administration, sealed in a vial under partial vacuum in a form ready to be loaded into a syringe and administered to a subject. In this regard, the pharmaceutical composition may be disposed therein under a partial vacuum. In all of these and other embodiments, the kit may comprise one or more vials according to any of the preceding embodiments, wherein each vial comprises a single unit dose for administration to the subject.

The kit may comprise a lyophilisate as treated herein, which when reconstituted provides a pharmaceutical composition corresponding thereto. In various embodiments, the kit may comprise a lyophilizate and a sterile diluent for reconstitution of the lyophilizate.

Also described herein are methods for treating a subject in need of treatment, the method comprising administering to the subject an effective amount of a pharmaceutical composition as described herein. The therapeutically effective amount or dose of the pharmaceutical composition formulation will depend on the disease or condition of the subject and the actual clinical setting.

In embodiments, the pharmaceutical compositions as described herein may be administered by any suitable route, particularly by parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient and the disease being treated. Methods of determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with, but are not limited to, the pharmaceutical composition used for the treatment, the purpose of the treatment, and the subject being treated. Single administration or multiple administrations may be performed, but are not limited thereto, with the dosage level and mode being selected by the treating physician. Suitable dosages of pharmaceutical compositions and methods of administering agents are known in the art.

Pharmaceutical compositions as described herein may be used in the manufacture of medicaments and for the treatment of humans and other animals by administration according to conventional procedures.

Also provided herein are combination methods for developing suitable pharmaceutical compositions using the combination of amino acids as excipients. These methods are effective for developing stable liquid pharmaceutical compositions or lyophilized pharmaceutical compositions, and in particular pharmaceutical compositions comprising one or more therapeutic compounds.

The compositions according to embodiments described herein have desirable properties such as desirable solubility, viscosity, injectability, and stability. The lyophilisates according to the embodiments described herein also have desirable properties, such as desirable recovery, stability and reconstitution.

In embodiments, the pH of the pharmaceutical composition is at least about 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, or 9.

In embodiments, the pH of the pharmaceutical composition is from about 3 to about 9, about 4 to about 19, about 5 to about 9, about 6 to about 8, about 6 to about 7, about 6 to about 9, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 7 to about 8, about 7 to about 9, about 7 to about 10.

Examples

The compositions and methods described herein will be further understood by reference to the following examples, which are intended to be purely exemplary. The compositions and methods described herein are not limited in scope by the exemplary embodiments, which are intended as illustrations of a single aspect only. Any functionally equivalent method is within the scope of the invention. In addition to the compositions and methods explicitly described herein, various modifications of the compositions and methods described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are within the scope of the invention.

Example 1

Treatment with the compound of formula (I) selectively kills pancreatic tumor cells expressing oncogenic KRAS, while the effect in normal cells is negligible.

The results shown in figure 1 demonstrate that the compounds of formula (I) reduce PI3K and c-RAF1 interactions with KRAS. The results shown in fig. 2 demonstrate that the compound of formula (I) is able to reduce cell viability in all tumor cell lines with an IC ₅₀ of about 20 μm, but only affects less than 5% of normal cells at these concentrations. Thus, the compounds of formula (I) reduce pancreatic tumor cells, but not normal cells.

General Process for the preparation of Compounds of formula (I) and their analogues

The compound of formula (I) and some of its analogues (the latter not disclosed herein) are prepared by means of Solid Phase Peptide Synthesis (SPPS) following the Fmoc/t-Bu strategy. The synthesis was carried out on a 100. Mu. Mol scale/time using 2-chlorotrityl chloride resin as solid polymer support. The synthesis was performed manually in a polypropylene syringe with a porous disc at the bottom. Intermittent manual agitation was performed while growing the peptidomimetic chain to ensure proper mixing of the reagents. The solvent and soluble reagents were removed by aspiration. The extent of the amino acid coupling reaction was monitored using the Kaiser test (primary amine) (see E.Kaiser et al ;"Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides";Anal.Biochem.1970;, volume 34; pages 595-598) or the tetrachloroquinone test (secondary amine) (see T.Vojkovsky; "Detection of secondary amines on solid phase"; pept. Res.1995; volume 8; pages 236-237). In those cases where the coupling is not fully completed, the re-coupling step is performed using standard coupling conditions. Selective N-alkylation of the compound backbone was performed using the method described by S.C.Miller et al (see "Site-SELECTIVE N-methylation of peptides on solid support"; J.Am.chem.Soc.1997; volume 119; pages 2301-2302).

The 2-chlorotrityl chloride resin was placed in a syringe (reaction vessel) equipped with a polyethylene porous plate. The resin was swollen by washing with Dichloromethane (DCM) and Dimethylformamide (DMF).

After swelling and preparation of the resin, the protected form of the first Fmoc protected amino acid of the peptidomimetic to be synthesized is attached to the resin via its carboxylic acid moiety using N, N-Diisopropylethylamine (DIEA) in DMF as a coupling agent. For coupling, 0.6 equivalent of protected amino acid was mixed with a few drops of DCM and added to the resin. Subsequently, 5 equivalents of DIEA were added in two portions, 1/3 of the parts first being added and the remaining 2/3 of the parts being added after 10 min. The reaction was allowed to proceed for 50min. Thereafter, unreacted active sites of the polymer support were capped by pouring methanol (1 mL/g polymer support) into the reaction mixture. After ten minutes, the solvent and unreacted reagents were removed by aspiration. Next, fmoc groups were removed by treating the resin with 20% piperidine in DMF. The wash solution was collected and measured by UV spectroscopy to quantify the loading of the first amino acid in the polymer support.

The subsequent amino acids of the peptidomimetic were coupled using 4 equivalents of Fmoc-protected amino acid, 4 equivalents of 2- (1H-benzotriazol-1-yl) -1, 3-tetramethyluronium tetrafluoroborate (TBTU), 8 equivalents of DIEA and a few drops of DMF poured into a reaction vessel containing the polymeric support. The mixture was allowed to react for 75min. The degree of coupling reaction was monitored using the Kaiser test or the tetrachloroquinone test. In the case of incomplete coupling, the reaction was repeated using the same conditions. Washing with DMF (5X 1 min) and DCM (5X 1 min) was performed during the coupling step. After the coupling was completed, fmoc groups were removed using a mixture of 20% piperidine in DMF (4 mL/g resin, 2X 1min and 1X 10 min). Fmoc group removal was monitored using the Kaiser test or the Tetrachloranil test, which were performed after washing the polymer support with DMF (5X 1 min) and DCM (5X 1 min). Subsequent amino acids are coupled using the same reaction conditions until the sequence of the target compound is completed.

The amino acid N-alkylation is carried out on the resin. The on-resin process used for N-methylation of amino acids is the following 3-step method (these steps are performed after Fmoc removal of the last coupled amino acid on the peptide sequence anchored to the polymer support):

a) Protection and activation of amino groups with o-N-bromosuccinimide (o-NBS).

B) Deprotonation and N-methylation with diazabicyclo [5.4.0] undec-7-ene and dimethyl sulfate.

C) o-NBS removal with beta-mercaptoethanol and 1, 8-diazabicyclo [5.4.0] undec-7-ene.

After completion of the peptide sequence, the resulting peptide (which remains anchored to the polymer support) was washed with DCM (5×1 min) and dried by aspiration. The peptide was then cleaved from the resin using 5% trifluoroacetic acid (TFA) in DCM. The treatment washes and DCM washes (5X 1 min) were collected and combined to obtain cleaved peptides from the resin. The collected solvent was then evaporated under vacuum until dryness. The crude peptide was diluted with Acetonitrile (ACN): H ₂ O solution (50:50) and lyophilized.

Next, C-terminal capping was added by diluting the peptide powder in the smallest possible volume of a mixture of DCM, pyrrolidine, 1-hydroxy-7-azabenzotriazole (HOAt) and N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC. Cl). After the C-terminal capping was completed, the solvent was removed under vacuum, then it was diluted again with a solution of ACN: H ₂ O50:50 and lyophilized.

The side chain protecting groups were removed by applying a mixture of TFA 95%: triisopropylsilane (TIS) 2.5%: H ₂ O2.5% for 1.5H. All filtrates were pooled and TFA was evaporated under a flow of N ₂. The crude product was then diluted with a mixture of ACN: H ₂ O (50:50) and lyophilized. Finally, the compound was purified using semi-preparative RP-HPLC. Fractions of interest were collected and lyophilized to yield the desired compound.

Specific preparation of Compounds of formula (I)

Support and Fmoc protected amino acids: 2-chlorotrityl chloride resin was used as a polymer support. The amino acids used are: fmoc-Bip-OH, fmoc-Nme-beta-Ala-OH, fmoc-Dap (Boc) -OH and Fmoc-Leu-OH.

Anchoring of the first amino acid: before the addition of the first amino acid, the 2-chlorotrityl chloride resin (5X 1min DCM, 5X 1min DMF and 1X 5min DMC) was washed first. Then, 0.6 equivalent of amino acid was mixed with a few drops of DCM and added to the resin. Subsequently, 5 equivalents of DIEA, first 1/3 of the amount, were added in 2 portions, the reaction was allowed to continue for 10min, and then the same procedure was repeated with the remaining 2/3 portions for a reaction time of 50min. Unreacted active sites of the resin were capped with methanol (1 mL/g polymer support). Then, the reaction mixture was removed by suction, and the polymer support was washed with DMF (5×1 min). Next, fmoc groups were removed by treating the resin (4 mL/g resin, 2X 1min and 1X 10 min) with 20% piperidine in DMF. The wash solution was collected and measured by UV spectroscopy to determine the loading capacity of the first amino acid.

Peptide mimetic chain extension: after coupling the first amino acid into the polymer support, the peptidomimetic chain was extended by pouring a pre-activated (3 min) mixture of 4 equivalents of Fmoc-protected amino acid, 4 equivalents of TBTU, 8 equivalents of DIEA, and a few drops of DMF, and pouring into a reaction vessel containing the polymer support. The mixture was allowed to react for 75min with intermittent manual stirring. After that, the reagents and solvents were removed by aspiration and the polymer support was washed with DMF (5 x 1 min) and DCM (5 x 1 min). The extension of the coupling was monitored by means of the Kaiser test (coupling on primary amine) or the tetrachloroquinone test (coupling on secondary amine). In those cases where the reaction was not complete, the coupling step was repeated using the same coupling conditions (4 equivalents of Fmoc protected amino acid, 4 equivalents of TBTU and 8 equivalents of DIEA and a few drops of DMF,75min reaction). After evaluating the integrity of the coupling, fmoc groups were removed by treatment with 20% piperidine in DMF (4 mL/g resin, 2X 1min and 1X 10 min). Fmoc group removal was monitored using the Kaiser test or the Tetrachloranil test, which were performed after washing the polymer support with DMF (5X 1 min) and DCM (5X 1 min). Subsequent amino acids are coupled using the same reaction conditions until the sequence of the target compound is completed.

Selective N-methylation of diaminopropionic acid moieties: the selective N-methylation proceeds as follows:

a) Protection and activation of amino groups with o-NBS: 4 equivalents of o-NBS, 3 equivalents of 2,3, 5-trimethylpyridine and a few drops of DMF are carried out 2 times (30 min and 20 min).

B) Deprotonation and N-alkylation: 3 equivalents of 1, 8-diazabicyclo [5.4.0] undec-7-ene in DMF was added to the resin (5 min), and after 5min 10 equivalents of dimethyl sulfate (10 min) were added. This process was repeated twice.

C) o-NBS removal: 10 equivalents of beta-mercaptoethanol, 5 equivalents of 1, 8-diazabicyclo [5.4.0] undec-7-ene and a few drops of DMF are carried out 2 times (10 min and 40 min).

Cleavage of peptides from polymeric support: the peptide-resin was treated with 5% TFA (3X 15min,6 mL) in DCM. The cleavage mixture and the subsequent DCM washes (5×1 min) were collected and combined to obtain cleaved peptides from the resin. The solvent from the collected mixture was then evaporated under vacuum until dryness. Finally, the crude peptide obtained was diluted with ACN/H ₂ O solution (50:50) and lyophilized. This acidic treatment allows retention of the protective side chain groups of the peptide.

C-terminal capping: the lyophilizate crude was diluted in the smallest possible volume of DCM and 3 equivalents of pyrrolidine, 3 equivalents of HOAt and 3 equivalents of EDC. Cl were added. The mixture was allowed to react at room temperature with continuous stirring for 3h. The mixture was then washed with saturated solution of NaHCO ₃、NH₄ Cl and NaCl (three times each). The organic layer was collected and dried under vacuum. Then, it was diluted with a mixture of ACN: H ₂ O (50:50) and lyophilized.

Removal of side chain protecting groups: the side chain protecting groups were removed by treatment with an acidic mixture (1.5H) using TFA 95%: TIS2.5%: H ₂ O2.5%. The cleavage mixture was evaporated using a stream of N ₂. The crude product was then diluted with a mixture of ACN: H ₂ O (50:50) and lyophilized.

Peptide purification: the peptide was purified using semi-preparative RP-HPLC. The crude product was dissolved in ACN: H ₂ O (the smallest possible amount of ACN: H ₂ O was used). The column used was a C18 (100 mm. Times.30 mm,5 μm,) A gradient of 0-100% B over 20min was used (a=0.1% TFA in H ₂ O, b=0.1% TFA in ACN). Flow = 16mL/min. Detection = 220nm. The fractions of interest were analyzed by combined analytical HPLC and HPLC/MS and lyophilized.

Characterization of the peptides: the identity of the peptide was confirmed using HPLC-MS. HPLC-MS chromatograms were recorded on a WATERS ALLIANCE 2796 separation module system equipped with a Waters 2996 photodiode array detector, a quadruple 3100 mass detector and a Sunfire C18 column (2.1 x 100mm x 3.5 μm,Waters) and Masslynx software. Flow rate: 0.3ml/min, mobile phase: h ₂ O (0.1% formic acid) and ACN (0.1% formic acid). UV detection: 220nm. Purity was quantified by analytical HPLC. HPLC chromatograms were recorded on a WATERS ALLIANCE 2695 separation module equipped with a 2996 photodiode array detector (PDA) and a Sunfire C18 column (100×4.6mm×5 μm,Waters) and Empower software. Flow rate: 1.6mL/min, mobile phase: h ₂ O (0.1% TFA) and ACN (0.1% TFA). Detection was performed at 220 nm.

-A molecular formula: c ₄₇H₅₈N₆O₅

-Calculating mass: 787.00g/mol

-Quality identification: [ M+H ] ⁺ =787.4 Da

Gradient and retention time (min): a gradient of 0-100% B over 3 minutes (a=0.1% TFA in H ₂ O, b=0.1% TFA in ACN) for 3.1min; a gradient of 40-100% B over 20 min (a=0.1% TFA in H ₂ O, b=0.1% TFA in ACN) for 1.9min.

Purity-of: 95%.

Cell lines and cultures

Both hTERT-RPE (immortalized retinal pigment epithelial human cells) and HeLa (epithelial-like cervical cancer cells) express RAS wild type and are obtained from the american tissue and cell collection (American Tissue and Cell Collection, ATCC). MPanc-96, HPAF-II, PA-TU-8902, SW1990, PA-TU 8988T and PANC-1PDAC cell lines (obtained as described in C.Barcelo et al, 10 months in :"Ribonucleoprotein HNRNPA2B1 interacts with and regulates oncogenic KRAS in pancreatic ductal adenocarcinoma cells";Gastroenterology2014; 147 (4): 882-892.e8.doi:10.1053/j. Gastro.2014.06.041.Epub 2014, 7 months 3. PMID: 24998203) all expressed oncogenic mutated KRASG12V.

HeLa cells and PDAC cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) and hTERT-RPE was grown in DMEM-HAM's F (1:1) medium, both supplemented with 10% fetal bovine serum (FBS; biological Industries, israel), penicillin, streptomycin and nonessential amino acids.

Drug therapy and EGF dependent signalling and activation

Cells were inoculated in medium containing 10% FBS for 24h and serum starved for the next 24h. They were then incubated with different concentrations of compound for 2h. Continuous treatment with EGF (50 ng/mL; sigma-Aldrich) was performed for 10min in order to activate cell signaling.

Cell transfection and plasmid

HeLa cells were treated as described in C.Lopez-Alcala et al ("Identification of essential interacting elements in K-Ras/calmodulin binding and its role in K-Ras localization.";J.Biol.Chem.2008, 18 days 4; 283 (16) transfection of the pEF-HA-KRASG12V plasmid obtained as described in 10621-31.Doi:10.1074/jbc.M706238200. Epub.2008, 8. PMID: 18182391). According to the instructions of the manufacturer, use2000 Transfection reagent (Invitrogen) as a transfection method.

SDS-PAGE, western blotting and antibodies

Proteins were resolved by SDS-PAGE and transferred to PVDF membrane (Immobilon-P, millipore). Nonspecific binding of antibodies was assessed by incubating the membranes with a buffer containing 20mM Tris-HCl pH 7.5, 150mM NaCl, 0.05% Tween 20 and 5% bovine serum albumin at room temperature for 1 h. Protein expression was determined by probing the blots overnight at 4 ℃ with the specific antibodies below: anti-c-RAF (BD transmission 610151, 1:500); anti-phospho-c-RAF S338 (CELL SIGNALING 9427, 1:500); anti-PI 3Kp110α (CELL SIGNALING 4249, 1:1000); anti-AKT (CELL SIGNALING9272, 1:1000); anti-phospho-AKT S473 (CELL SIGNALING:4060, 1:1000); anti-phospho-AKT Thr308 (CELL SIGNALING 4056, 1:1000); anti-p 44/42MAPK (ERK 1/2) (CELL SIGNALING 9102, 1:2000); anti-phospho-p 44/42MAPK (ERK 1/2) T202/Y204 (CELL SIGNALING 4370, 1:2000); GAP120 (Santa Cruz SC-63, 1:200); anti-HA (Sigma-Aldrich H6908, 1:1000); or anti-alpha-tubulin (Sigma-Aldrich T9026, 1:2000). Next, after washing the membranes, they were incubated with the corresponding HRP conjugated secondary antibodies (goat anti-rabbit BioRad170-6515 or goat anti-mouse BioRad 170-6516, 1:3000) for 60min at room temperature and washed again. Protein detection was performed by enhanced chemiluminescence (EZ-ECL, biological Industries). The emitted light is titled and quantified (ChemiDoc, bioRad).

To analyze RAS signaling, cells were lysed in a buffer containing 67mM Tris-HCl pH 6.8 and 2% SDS, and then the samples were heated at 97 ℃ for 15min. After this, the protein concentration of the lysate was determined using the Lowry method. An aliquot of 15 μg of protein per sample was loaded onto the gel.

Co-immunoprecipitation (Co-IP)

Cells were transfected with pEF-HA-KRASG12V plasmid for 24h and starved for the next 24h before treatment with peptide and EGF. Next, IP using an anti-HA antibody crosslinked with agarose beads was performed. Briefly, cells were lysed on ice for 10min with a buffer containing 20mM Tris-HCl pH 7.5, 100mM NaCl, 2mM EDTA, 5mM MgCl ₂, 1% (v/v) Triton X-100, 10% glycerol (v/v), 1mM Dithiothreitol (DTT) plus protease and phosphatase inhibitors (150 nM aprotinin, 20. Mu.M leupeptin, 1mM phenylmethylsulfonyl fluoride, 5mM sodium fluoride and 0.2mM sodium orthovanadate). After clarification by centrifugation, the supernatant (500. Mu.g-2000. Mu.g) was incubated with 40. Mu.L-50. Mu.L of anti-HA-tag antibody (clone HA-7, sigma-Aldrich A20956) cross-linked to agarose beads at 4℃for 3h under rotation. Next, the immune complex obtained after 2min of rotation at 10000g at 4 ℃ was washed and subjected to immunoblotting with the corresponding antibody.

Cell viability assay

10,000 Cells in 50 μl of 10% FBS-containing medium were cultured for 24h and then treated with drug (50 μl final volume) in each well (100 μl final volume) of the 96-well plate for another 24h. MTS viability assay (CellTiter)Aqueous One Solution Cell Proliferation Assay, promega G3580) was performed according to manufacturer's specifications. The absorbance of each well was measured at 490nm using a multi-mode plate reader (Spark, tecan). The percent cell viability was calculated by dividing the absorbance of each well by the average absorbance of the control wells (control wells have no significant deviation when tested using Student's t).

Example 2

We have performed a preliminary evaluation of the effector binding sites on the surface of the RAS proteins, which led us to determine the use of peptide mimetics to modulate the interaction of RAS proteins with those proteins that interact at the RAS-effector binding site (effectors after RAS activation) (see figure 3). Effectors include RAF, RAL and PI3K. This is accomplished using a calculation method as described below.

Computational hit authentication

Computational methods are applied to provide efficient and permeable peptidomimetics as drug candidates. Our approach is to design a peptidomimetic that will bind to the RAS effector binding site, blocking the possibility of RAS interaction with effector proteins, thus reducing its tumor activity.

Identification of RAS-effector binding sites is performed by comparing the structure of gtpase-RAS complexed with several effector proteins such as phosphoinositide 3-kinase (PI 3K), bry2RBD, ralGDS, phospholipase C, NORE a and RAF and applying a computational standard protocol.

A total of one aromatic residue and three negatively charged residues (i.e., asp33, glu37, asp38, and Tyr 64) were identified at the RAS interface with the RAS effector protein. We identified several residues on the RAS protein surface, asp33, glu37, asp38 and Tyr64, which are involved in intermolecular contacts with specific residues that are highly conserved in almost all effector proteins. We determined that these residues are conserved in interactions with almost all RAS binding site effector proteins. We also determined that these RAS binding sites are generally highly positively charged between Asp33 and Asp38 (fig. 4, table 4), which facilitates interactions with possible binders.

Based on the results obtained from both analysis of RAS protein crystal structure and MD simulation, asp33 and Asp38 residues were selected as substrate binding sites, as they were identified by both assays. These data are then used for virtual screening of putative RAS inhibitors to tailor the composition of the peptide mimetic library used as ligand, such as in the hit identification step, and to set the position and size of the docking box (docking box), both in the hit identification step and in the hit optimization step.

We created an initial library with more than 80,000 tripeptides and tetrapeptides formed from both natural and unnatural amino acids. The tripeptides and tetrapeptides produced each contain at least one positively charged residue. We performed peptide mimetic screening using SMINA docking procedure. This is achieved by using the K-RAS GTPase crystal structure as the receptor (PDB 5P 21) and locating the binding site around negatively charged hot spot residues on the surface of the K-RAS GTPase.

Materials and methods

Synthesis of peptidomimetics

All compounds were synthesized by means of Solid Phase Peptide Synthesis (SPPS) following the Fmoc/tBu strategy. The synthesis was carried out on a 100. Mu. Mol scale/time using 2-chlorotrityl chloride resin (which used H-Rink amide chemmatrix in addition to P1.1). The synthesis was performed manually in a polypropylene syringe with a porous disc at the bottom. Intermittent manual agitation was performed while growing the peptide chain to ensure proper mixing of the reagents. The solvent and soluble reagents were removed by aspiration. Amino acid coupling (L-, D-or unnatural) (1X 75 min) was performed using 4 equivalents of Fmoc-protected amino acid, 4 equivalents of 2- (1H-benzotriazol-1-yl) -1, 3-tetramethylammonium tetrafluoroborate (TBTU) and 8 equivalents of N, N-Diisopropylethylamine (DIEA) in Dimethylformamide (DMF). The extent of the reaction was monitored using the Kaiser test (primary amine) or the tetrachloroquinone test (secondary amine). In those cases where the coupling is not fully completed, the re-coupling step is performed using standard coupling conditions. The Fmoc group was removed from the amino acid (after successful completion of the coupling reaction) using a 20% piperidine mixture in DMF (2X 1min and 1X 10 min).

The selective N-alkylation of the backbone of the compound was carried out using the method described by Miller et al, which was divided into three steps (these steps were carried out after Fmoc removal of the amino acid anchored to the resin to be N-alkylated).

Protection and activation of amino groups with o-N-bromosuccinimide (o-NBS): 4 equivalents of o-NBS in DMF, 3 equivalents of 2,3, 5-trimethylpyridine (1X 30min and 2X 20 min), 2) deprotonation and N-alkylation: 3 equivalents of 1, 8-diazabicyclo [5.4.0] undec-7-ene in DMF was added to the resin (5 min), followed by 10 equivalents of the desired alkyl sulfate to the resin (10 min). This process was repeated twice. 3) o-NBS removal: two treatments (1X 10min and 1X 40 min) were performed with a mixture of 10 equivalents of beta-mercaptoethanol and 5 equivalents of 1, 8-diazabicyclo [5.4.0] undec-7-ene in DMF.

Mixed solvent molecular dynamics (MixMD) simulation of RAS GTPase proteins

The crystal structure of RAS GTPase protein (PDB code 5P 21) is downloaded from the RCSB PDB database (https:// www.rcsb.org /) and used as an input structure to perform 50-ns long explicit mixed solvent molecular dynamics (MixMD) simulations.

In a first preliminary step, the co-crystallized GppNHp molecules are replaced by the GTP raw cofactor, and the parameter file of the GTP raw cofactor is downloaded from the AMBER parameter database (http:// research. Bmh. Manchester. Ac. Uk/bryce/AMBER /). Thus, the entire system was properly protonated and placed in a periodic cubic mixed solvent cartridge containing benzene, propane, ethanol, propionic acid and ethylamine organic probes in proper combination with TIP3P water molecules (minimum distance between protein and cartridge edge set to)。

The entire protonation, solvation and parameterization of the system was performed using the Leap module of AmberTool and using the ff12 AMBER force field.

As detailed below, a two-step conjugate gradient based minimization was run followed by a four-step equilibrium and 50-ns long MD simulation ⁵⁷ using NAMD simulation package. The SHAKE algorithm and PARTICLE MESH EWALD (PMD) method (used to suppress all bonds to hydrogen atoms and calculate long range coulomb interactions, respectively) were applied in all simulations. A time step of 2fs andCut-off distance of long-range interactions of (c).

Thus, the solvating system is relaxed first with an unlimited minimization step of 500 cycles long, followed by another 5000 cycles, during which harmonic suppression is only usedIs applied to the backbone atoms of the protein. Subsequently, the proteins equilibrate in a four-step scheme, in which the system is gradually heated from 0K to 300K using the Langmuir dynamics (LANGEVIN DYNAMIC) model, and the initial position limits are regularly relaxed. Thus, NVT conditions (i.e., constant number of molecules (N), volume (V), and temperature (T)) are used toThe harmonic potential of (2) suppresses the main chain atoms and increases the temperature from 100K to 300K, and MD simulations of 100-ps length were performed. Then, NVT conditions are used and expressed asThe harmonic potential of (2) suppresses the main chain atoms, running a 120-ps long heating phase from 300K to 600K. NVT is then used andThe harmonic potential of (c) suppresses the backbone atoms from cooling the system from 600K to 300K for 120ps. Finally, NPT conditions (i.e., constant molecular number (N), pressure (P) and temperature (T)) are used and are expressed asThe harmonic potential of (2) suppresses the backbone atoms and is modeled at 300K as 100-ps long.

After equilibration, use is made of the atoms applied only to the main chainIs run in 50-ns long simulations at 300K under NVT conditions.

Finally, the trajectory is used to identify protein surface regions with a high propensity for ligand binding based on the distribution of organic probes during the trajectory, since the frequency of occupation of probes in a given region should be proportional to their binding affinity for that particular region. Thus, using the cpptraj module of AmberTools, the last 25ns of the simulation along each organic probe is integrated into the probe-occupancy map and ultimately visualized as an isosurface corresponding to the most frequently sampled region of each organic probe.

Virtual screening of putative RAS inhibitors

Using extensive computational investigation of RAS interfaces, we were able to generate a set of peptidomimetics that could bind to target binding sites with theoretical potency greater than-8.0 kcal/mol (μM-nM scale of expected experimental inhibition potency range) while exhibiting good in silico permeability profiles with average Polar Accessible Surface Area (PASA) belowThese parameters represent the potential activity and permeability of the designed peptidomimetics. /(I)

To exclude the possibility of identifying false negatives from the docking, we performed short implicit peptide binding site MD simulations to identify peptide mimics that exhibit stable binding patterns (RMSD simulations less than). The method enriches the choice of compounds capable of binding KRAS-gtpase based on thermodynamic points while preserving the computational properties and peptide structure of the binding target region.

In screening two data sets of putative RAS protein inhibitors, two separate and subsequent in silico molecular docking experiments were applied with the aim of identifying and ultimately optimizing hit compounds.

In the first molecular docking experiment (hit identification step), data sets of tripeptide and tetrapeptide mimics were constructed using natural and unnatural amino acids. The N-terminal and C-terminal portions of the compounds are enriched by different capping moieties having different size and polarity profiles, such as diphenylacetic acid, 2- (4-t-butylphenoxy) acetic acid, phenylacetic acid, 9-anthroic acid or benzoic acid for the N-terminal portion, and formamide, pyrrolidine, pyridine or 3-azaspiro [5.5] undecane for the C-terminal portion. Based on the results of the substrate-binding site analysis, only positively singly charged compounds were selected and thus a library of 80,000 compounds was generated.

Molecular docking

In both the hit identification step and the optimization step, the same protocol was used to perform the molecular docking experiments. All putative three-dimensional structures of RAS protein inhibitors were created starting from the first order, parameterized using AmberTool Leap modules and ff12 AMBER force field, and finally minimized with NAMD analog package. A parametric library of non-natural peptide building blocks (if any) and capping residues was written using the AmberTool modules Antechamber and Leap.

RAS GTPase protein Structure (PDB code 5P 21)The resolution crystal structure is used as a receptor for interfacing to all putative RAS inhibitors. In a first preliminary step, the co-crystallized GppNHp and water molecules are removed. Thus, hydrogen atoms are added and the entire system is properly protonated using an H++ web server (http:// biophysics. Cs. Vt. Edu/H++) and default parameters.

All docking calculations were performed using SMINA, SMINA is the energy minimization optimization fork (fork) ⁶¹ of the AutoDock Vina docking procedure. All ligand and receptor structures were converted to input files appropriate for SMINA using the preparation_ligand 4.Py script and the preparation_receptor 4.Py script provided by AutoDock Tools. Modulated in RAS effector binding regions the size is 60 multiplied by 60 and the grid space isIs nearly cubic and centered at the Asp33 residue and the Asp38 residue. The exhaustive, number of modes and energy ranges are set to 32, 100 and 50, respectively.

After all docking simulations have been completed, the phi/psi dihedral Ramachandran distribution, geometry of the corresponding peptide bonds, and intermolecular contacts of all building blocks are calculated for the 20 top-ranked docking poses. Thus, the docking conformation, which is incompatible with phi/psi dihedral Ramachandran distribution, with non-planar or cis peptide bonds or with any intramolecular contact, is filtered out. Thus, the top-ranked docking poses (if any) of each compound are combined together and sorted according to SMINA docking energy, filtering out all compounds with docking energy below-8 kcal/mol. Then, the binding stability and predicted membrane permeability filter (see below for details) are applied sequentially, and after that, the most promising compound is selected and subjected to visual inspection.

For the final selection of peptide candidates, additional factors are considered (e.g., peptide binding patterns consistent with predicted substrate binding site positions and chemistry, lack of tight orientation of positively charged residues to hydrogen bond donors, tight orientation of negatively charged residues to hydrogen bond acceptors, and insertion of polar residues into highly hydrophobic clefts).

RAS binding stability assessment

Using NAMD simulation packages, the docking model for each compound complexed with RAS proteins underwent conjugate gradient minimization, equilibration, and implicit solvent MD simulation of 3-ns length. Thus, the first preliminary step, namely the overall protonation and parameterization of the system, is performed using the Leap module of AmberTool and the ff12 AMBER force field. The system then uses a 1000 cycle long minimization toThe force constant of (a) applies harmonic suppression to all backbone atoms (both acceptor and ligand) of the system to relax. Then, an equilibration step of 200-ps length was performed by gradually heating the system to 300K and at 5/>, respectivelyAndIs performed by applying harmonic suppression to the backbone atoms of the protein and ligand. Finally, 3-ns long MD simulation passesThe force constant of (2) applies harmonic suppression only to the backbone atoms of the receptor protein. In all simulations, SHAKE ⁵⁸ was applied to suppress all bonds to hydrogen atoms, while setting the simulation time step of 2-fs andCut-off distance of long-range interactions of (c).

Finally, a binding stability assessment was performed using a MolSoft ICM browser (www.molsoft.com) to calculate the average root mean square deviation (LIGRMSDAVG) ⁶³ of each compound along the last 1.5 ns of the trace. LIGRMSDAVG values belowThe compounds of (2) are predicted to be stable binders and are therefore selected for cell membrane permeability evaluation.

Computer permeability prediction

In a first preliminary step, the three-dimensional structure of each compound is created from scratch and parameterized using AmberTool Leap modules and ff12 AMBER force fields.

For each compound, a set of 25 unrestricted Molecular Dynamics (MD) simulations of implicit chloroform solvation of 2-ns length were performed using the ff12 AMBER force field and NAMD simulation package. Each of the previously generated 3D structures is first used to obtain a small conformational ensemble comprising a total of 25 conformational isomers. Thus, each compound was relaxed by a short 1000 step unlimited energy minimization and then subjected to a short MD simulation for 100 ps, setting the system temperature to 300 f K f. Finally, 25 conformational isomers were obtained by extracting one track snapshot per 4 ps and used as input to the MD simulation of the final chloroform solvation.

Thus, in each simulation, the system first performs energy minimization through 5000 conjugate gradient steps. The system then undergoes an equilibration process divided into four steps, gradually heating from 0K to 300K for 100 ps to Harmonic suppression is applied to all heavy atoms to maintain the original molecular geometry during heating. During balancing, the integration time step is set to 2fs and the non-bonding cutoff distance is set toFinally, a production step consisting of performing an unrestricted MD simulation for 2ns and setting the system temperature to 300K was run. For each compound, all 25 2-ns long MD tracks were combined together and a total of 12,500 MD frames were extracted using the cpptraj module of AmberTools.

Finally, for each compound, the average exposure of polar atoms to solvent during the overall simulation was extracted from the average polar accessible solvent area values (polASA _avg) on the overall MD frame using MolSoft ICM browser. polASA _avg values belowIs predicted to be permeable by passive diffusion and is therefore selected for visual inspection.

Cell lines and cultures

Expression of RAS wild-type hTERT-RPE (immortalized retinal pigment epithelial human cells) was obtained from the American Tissue and Cell Collection (ATCC).

HTERT-RPE was supplemented with 10% fetal bovine serum (Biological Industries, israel), penicillin, streptomycin, and nonessential amino acids in DMEM-HAM's F (1:1) medium.

Drug therapy and EGF dependent signaling activation

Cells were inoculated in medium containing 10% FBS for 24 hours and serum starved (0.5%) was performed for the next 24 hours. They were then incubated with different concentrations of compound for 2 hours. Continuous treatment with EGF (50 ng/mL) (Sigma-Aldrich) was performed for 10 minutes in order to activate cell signaling.

SDS-PAGE, western blotting and antibodies

Proteins were resolved by SDS-PAGE and transferred to PVDF membrane (Immobilon-P, millipore). Nonspecific binding of antibodies was assessed by incubating the membranes with a buffer containing 20mM Tris-HCl pH 7.5, 150mM NaCl, 0.05% Tween 20 and 5% bovine serum albumin for 1 hour at room temperature. Protein expression was determined by probing the blots overnight at 4 ℃ with the specific antibodies below: anti-c-RAF (BD transmission 610151, 1:500); anti-phospho-c-RAF S338 (CELL SIGNALING 9427, 1:500); anti-PI 3Kp110α (CELL SIGNALING 4249, 1:1000); anti-AKT (CELL SIGNALING9272, 1:1000); anti-phospho-AKT S473 (CELL SIGNALING:4060, 1:1000); anti-phospho-AKT Thr308 (CELL SIGNALING 4056, 1:1000); anti-p 44/42MAPK (ERK 1/2) (CELL SIGNALING 9102, 1:2000); anti-phospho-p 44/42MAPK (ERK 1/2) T202/Y204 (CELL SIGNALING 4370, 1:2000); GAP120 (Santa Cruz SC-63, 1:200); anti-HA (Sigma-Aldrich H6908, 1:1000); or anti-alpha-tubulin (Sigma-Aldrich T9026, 1:2000). Next, after washing the membranes, they were incubated with the corresponding HRP conjugated secondary antibodies (goat anti-rabbit BioRad170-6515 or goat anti-mouse BioRad 170-6516, 1:3000) for 60 minutes at room temperature and washed again. Protein detection was performed by enhanced chemiluminescence (EZ-ECL, biological Industries). The emitted light is titled and quantified (ChemiDoc, bioRad).

To analyze RAS signaling, cells were lysed in a buffer containing 67mM Tris-HCl pH 6.8 and 2% SDS, and then the samples were heated at 97 ℃ for 15 minutes. After this, the protein concentration of the lysate was determined using the Lowry method. An aliquot of 15 μg of protein per sample was loaded onto the gel.

Analysis of peptide mimetics

After final visual inspection, nine peptide mimetic sequences were selected for synthesis as listed in table 2. Nine sequences include tripeptide and tetrapeptide mimics. All of these sequences are capped with a secondary amine at the C-terminus and a hydrophobic group at the N-terminus. The permeability threshold is set toWherein all peptidomimetics below the threshold are predicted to be permeable.

TABLE 2

Experimental evaluation of chemical structures of table 2

We evaluated the tripeptide and tetrapeptide mimics of Table 2 by testing the ability of the peptide mimics to inhibit RAS-GTP signaling proteins in hTERT-RPE cells. To do this we inoculated the cells in culture plates for 48 hours. Cells were serum starved (0.5% FCS) for 24 hours. After this period, we added EGF to cells at a concentration of 50ng/mL for a period of 10 minutes. EGF is added to the culture to activate RAS signaling pathways. To evaluate the activity of the peptidomimetics, we added one tripeptide mimetic or tetrapeptide mimetic to the culture plate at a concentration of (50 μm) 2 hours prior to the addition of EGF, so that each peptidomimetic was tested in the culture plate. We run Western Blotting (WB) to detect the activation levels of both Ras signaling pathways (Raf/ERK and PI 3K/AKT). GAP120 detection was used as a control.

Figure 5 shows western blots where we evaluated the efficacy of the 9 peptide mimetics of table 2 in inhibiting RAS effectors. Gtpase activating protein (GAP 120) was used as a control.

In order for a peptidomimetic to be considered a positive hit, it should inhibit the activity of both protein cascades, and thus phosphorylation of RAF (P-RAF), AKT (P-AKT) and ERK (P-ERK) should not be observed.

When analyzed, we found that compounds IP-14-05 and IP-14-06 were insoluble when diluted in cell culture medium. We have also found that peptide IP-14-04 forms aggregates and precipitates onto cells, causing cell death. Therefore, we did not incorporate IP-14-05 and IP-14-06 into WB (FIG. 5).

As a result, we finally evaluated only 6 of the first 9 peptidomimetics in RPE cells. From the 6 peptidomimetics we evaluated, we determined that 3 of them, in particular, IP-14-01, IP-14-03 and IP-14-08, were able to inhibit two RAS-effector protein cascades. Of these 3 peptidomimetics, we found that IP-14-01 was the most potent inhibitor when analyzed by WB (fig. 5).

As part of our analysis, we evaluated IP-14-01, IP-14-03 and IP-14-08 as well as their biophysical properties by studying them using a solution of 5% DMSO in water. We performed permeability through biological barriers (PAMPA assay) and its internalization in SH-SY5Y cells.

TABLE 3 Table 3

Table 3 shows the results of PAMPA assays (Pe,% transport and% retention) of IP-14-01, IP-14-03 and IP-14-08, as well as the percentage of cellular internalization used to evaluate the permeability of the peptidomimetics. The methods used are well known and will be known to those skilled in the art. As a control, we evaluated the solubility in water containing 5% DMSO to determine any possible problems associated with low solubility. Data are expressed as mean ± SD.

Solubility was measured in 5% DMSO in water and the three compounds (IP-14-01, IP-14-03 and IP-14-08) showed good solubility, providing assurance that the experiment could be performed at a concentration of >100 μm without the risk of any of the three therapeutic compounds precipitating from solution. The results of the PAMPA assay showed high retention, negligible transport and zero permeability Pe for each of the three peptidomimetics. We found that when IP-14-01 was incubated with SH-SY5Y cells at 60. Mu.M for 2h, 13.5% of the peptidomimetic was taken up by the cells. When IP-14-03 and IP-14-08 were incubated with the same cells, we did not find similar results for IP-14-03 and IP-14-08.

In addition, additional efforts were made to reevaluate those peptide mimetics from the first generation that were insoluble under in vitro assay conditions or formed aggregates as described above when incubated with RPE cells. For these peptidomimetics, their solubility in PBS with 15% beta-cyclodextrin was studied (table 5).

TABLE 4 Table 4

Compound code	Concentration in PBS beta-cyclodextrin 15% (μM)
		IP-14-04	780±30
IP-14-05	849±43
		IP-14-06	844±4
IP-14-07	766±19
		IP-14-09	949±8

Table 4 shows that all of these peptidomimetics are diluted and soluble at 1mM in PBS (phosphate-saline buffer) with 15% beta-cyclodextrin. Then, we placed these peptidomimetics under continuous stirring for 24 hours. At the end of this time we centrifuged each solution and then run the supernatant from each solution by HPLC and compare the results with 1mM solution of each compound in ACN/H ₂ O. This control was used to determine the actual solubility in PBS of 15% beta-cyclodextrin. Data are expressed as mean ± SD.

FIG. 6 shows RAS Western blotting we performed under the same conditions as described above, except that in this case we used β -cyclodextrin at a concentration of 0.5% instead of DMSO to evaluate compounds IP-14-01 (P1), IP-14-02 (P2), IP-14-03 (P3), IP-14-04 (P4), IP-14-07 (P7), IP-14-08 (P8) and IP-14-09 (P9). We used DMSO to solubilize gtpase activating protein (GAP 120) in cell culture medium at a diluted concentration of 0.5%.

Because of the inhibition of the RAS effector signaling cascade by IP-14-01, along with its high intracellular internalization value, we selected the therapeutic compound IP-14-01 for optimization and the second round of calculation methods.

Example 3

Evaluation of chemical Structure derivative of IP-14-01

The second generation peptidomimetics derived from IP-14-01 are designed according to the same computer protocol as the previous application. In this regard, we prepared four new peptidomimetics, which we eluted after the completion of the computational study.

In a second molecular docking experiment (hit optimization step), a new peptidomimetic dataset is generated starting from the P1 primary sequence by combining the original building block with carefully selected specific and ad hoc selectable moieties in order to increase the membrane permeability and/or receptor binding affinity of the original compound.

We designed IPR-471 to increase the number of amino acids in the backbone of a peptidomimetic. On this basis, the C-terminus is extended by removal of secondary amines, which have been used to cap proline-carboxamide.

IPR-472 has almost the same structure as IP-14-01, but the N-methylalkylation of beta-alanine is replaced by a longer carbon chain attached to the aromatic group propylbenzene. We make this change to increase the overall compound hydrophobicity along with an increase in n-alkyl shielding ability. We believe that a four carbon chain rather than methyl will provide a degree of flexibility that will allow six carbon aromatic rings to encapsulate the molecule. This will reduce the polarity of IPR-472 in aqueous environments. We expect this to increase the number of contacts with the protein surface.

IPR-473 was designed to be the most conserved among IP-14-01 derived peptidomimetics because the only change is the substitution of isoleucine for alanine and the addition of N-methylation to one of the amide linkages of the sequence backbone. This new molecule is expected to fully maintain the binding pattern of the parent compound, but with the addition of more contacts in order to slightly optimize its potency.

In contrast to IP-14-01, IPR-474 has two substitutions. These are alanine substituted with cyclohexylglycine and another amino acid having a polar group substituted with an amino acid having a polar group. In this case, the new amino acid is threonine.

Finally, we designed these peptidomimetics to maintain the same sequence end by maintaining three identical amino acids in the same order and the diphenyl N-terminus (table 6). These novel peptide mimetics are identified in table 5.

TABLE 5

Table 4 shows that the second generation of four new peptidomimetics was generated using the same docking method previously used to screen the first round of peptidomimetics.

FIG. 7 is a RAS signaling Western blot from our evaluation of IP-14-01 (P1) and its derived peptidomimetics IPR-471 (P1.1.), IPR-472 (P1.2), IPR-473 (P1.3) and IPR-474 (P1.4). For this experiment we used the same protocol and conditions as the WB assay previously described disclosed herein (fig. 5). More specifically, we incubated each peptidomimetic compound with serum-starved hTERT-RPE cells at a concentration of 50 μm in 0.5% DMSO for 2 hours in culture and then treated with EGF (50 ng/ml) for 10min. None of these therapeutic compounds showed any solubility problems.

As shown in FIG. 7, we found that IPR-471 (P1.1) and IPR-474 (P1.4) are not better than IP-14-01 (P1) from which they were derived. In contrast, we found that IP-14-02 (P1.2) was able to inhibit RAF and AKT, but not ERK. We also determined that IPR-473 (P1.3) is able to inhibit even more effectively than IP-14-01 both different protein cascades (RAF/ERK and PI 3K/AKT).

Of the peptidomimetic compounds evaluated in fig. 7, we determined that IPR-473 showed the highest degree of inhibition of RAS effectors.

Next, we incubated IP-14-01 at several concentrations with different cancer and normal cell lines (hTERT-RPE cells). Cell viability was measured by the MTS cell proliferation assay. The same experiment was performed on the parent peptidomimetic IP-14-01. At any incubation concentration, IP-14-01 did not differ in its ability to kill normal versus cancer cells, i.e., the parental peptide mimetic did not show cell line specificity (data not shown).

Next, we incubated several concentrations of IPR-473 with different cancer and normal cell lines (-hTERT-RPE cells). Cell viability was measured by the MTS cell proliferation assay. Fig. 8 shows the results of an MTS viability assay for measuring cell viability of seven different cell lines. Cells were placed in 96-well plate cultures containing medium containing 10% FBS (Bilogical Industries). (10000 cells per well). These cells were then cultured for 24 hours and then incubated with IPR-473 at concentrations of 10. Mu.M, 15. Mu.M, 20. Mu.M, and 25. Mu.M for an additional 24 hours.

Different cell lines used included human pancreatic cancer cells MPANC-96, human pancreatic adenocarcinoma cells HPAF-II, human pancreatic grade II adenocarcinoma PA-TU, human pancreatic ductal adenocarcinoma SW1990, human pancreatic adenocarcinoma 8988-T, and human pancreatic ductal carcinoma PANC-1. Each of these comprises a pancreatic tumor cell line. The control stated is hTERT-RPE cells and is non-cancerous.

The results obtained in the cell viability assay as shown in fig. 8 confirm the potential therapeutic activity of IPR-473. The therapeutic compound is cytotoxic to cancerous cells at a concentration of greater than 15 μm. Meanwhile, it has no effect on hTERT-RPE control cells. Thus, we found that IPR-473 exhibits high specificity for pancreatic cancer cell lines in cell proliferation assays.

Certain embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on the embodiments of these descriptions will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

The grouping of alternative embodiments, elements or steps of the present invention should not be construed as limiting. Each group member disclosed herein may be referred to and claimed either alone or in combination with other group members. It is contemplated that one or more members of a group may be included in or deleted from the group for convenience and/or patentability reasons. When any such inclusion or deletion occurs, the specification is considered to contain the group as modified so as to achieve a written description of all Markush groups (Markush groups) used in the appended claims.

Unless otherwise indicated, all numbers expressing features, items, quantities, parameters, properties, terms, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". As used herein, the term "about" means that a feature, item, quantity, parameter, property, or term so qualified includes a range of plus or minus 10% above and below the stated value of the feature, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the specification as if it were individually set forth herein.

The terms "a," "an," "the," and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The specific embodiments disclosed herein may be further limited in the claims using language consisting or consisting essentially of. The transitional term "consisting of" when used in a claim, whether added as a submitted or per modification, does not include any element, step, or component not specified in the claim. The transitional term "consisting essentially of" limits the scope of the claims to those materials or steps specified and that do not materially affect the basic and novel characteristics. Embodiments of the claimed invention are described and enabled herein, either inherently or explicitly.

The grouping of alternative embodiments, elements or steps of the present invention should not be construed as limiting. Each group member disclosed herein may be referred to and claimed either alone or in combination with other group members. It is contemplated that one or more members of a group may be included in or deleted from the group for convenience and/or patentability reasons. When any such inclusion or deletion occurs, the specification is considered to contain the group as modified so as to achieve a written description of all markush groups used in the appended claims.

All patents, patent publications, and other publications cited and identified in this specification are individually and clearly incorporated by reference herein in their entirety for the purpose of description and disclosure, e.g., the compositions and methodologies described in such publications that may be used in connection with the present application. These publications are provided solely for their disclosure prior to the filing date of the present application. In this regard, nothing is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior application or for any other reason. With respect to dates or statements, all statements concerning the contents of these documents are based on the information available to the present inventors and do not constitute any admission as to the correctness of the dates or contents of these documents.

Finally, it should be understood that although aspects of the present description are emphasized by reference to specific embodiments, those skilled in the art will readily appreciate that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. It is to be understood, therefore, that the disclosed subject matter is in no way limited to the particular methodologies, protocols, and/or reagents, etc. described herein. Accordingly, various modifications or adaptations of the disclosed subject matter, or alternative configurations of the disclosed subject matter, may be made in accordance with the teachings herein without departing from the spirit of the specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. The invention is therefore not limited to the exact details as shown and described.

Claims

1. A compound of formula (I) and pharmaceutically acceptable salts thereof,

2. A compound of formula (I) according to claim 1, having the chemical name: (S) -N- (3- (((S) -3-amino-1- ((S) -4-methyl-1-oxo-1- (pyrrolidin-1-yl) pent-2-ylamino) -1-oxopropan-2-yl) (methyl) amino) -3-oxopropyl) -3- (biphenyl-4-yl) -2- (2, 2-diphenylacetamido) -N-methylpropanamide.

3. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, diluent or carrier.

4. A compound of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of human cancer.

5. The compound for use according to claim 4, wherein the human cancer is selected from the group consisting of pancreatic cancer, lung cancer and colorectal cancer.

6. The compound for use according to claim 5, wherein the human cancer is pancreatic cancer.

7. The compound for use according to claim 6, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma PDAC.