CN117957239A - Cancer therapeutic agents - Google Patents

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 agents

Related Applications

This application claims priority to European patent application serial number 21382612.6 filed on July 7, 2021. The contents of the aforementioned application are incorporated herein by reference.

Field of the Invention

The present invention relates generally to cancer therapy and, more particularly, to novel therapeutic compounds, including peptidomimetics 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 papillomavirus infection, Epstein-Barr virus, and human immunodeficiency virus (HIV).

Conventional cancer treatment aims to remove cancerous tissue and prevent it from spreading. Such treatment options include surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and palliative care. Treatment is usually based on the type, location, and grade of the cancer, as well as the patient's health status and preferences. Because cancer cells divide faster than most normal cells, they may be sensitive to chemotherapy drugs.

RAS genes include a family of oncogenes (HRAS, NRAS and KRAS) associated with cell proliferation processes. 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% of pancreatic cancers, 45% of colon cancers, and 25% of lung cancers.

In recent years, due to the large number of protein-protein interactions (PPIs) involved in cellular machinery, the regulation of PPIs has attracted widespread attention in the scientific community. However, due to the nature of these interactions, the regulation of PPIs is very challenging. An alternative to the use of small molecules for the regulation of PPIs is the use of biological products, such as antibodies. PPIs, which are considered to be 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. Therefore, an object of the present invention is to provide a PPI that can recognize and interact with large protein surfaces and has the ability to cross biological barriers.

Because a need exists for treating cancer, including cancers 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 the therapeutic agents.

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 and not limitation.

In aspects of the present invention, therapeutic compounds including peptide mimetics that can be administered to patients are provided. In another aspect of the present invention, therapeutic compounds can be administered individually or in combination with one or more other therapeutic compounds. These additional therapeutic compounds can include antibodies, biologics, small molecules or other therapeutic compounds as listed in Figure 8.

In aspects of the invention, a therapeutic compound that is a peptidomimetic is administered to a patient along 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 peptide mimetic, which is a small protein-like chain designed to mimic a peptide. In another aspect of the invention, the peptide mimetic is suitably engineered to recognize and bind to protein patches (binding sites or sites where a protein interacts with another protein or molecule, including being engineered to trigger an enzymatic pathway) in a specific manner and cross biological barriers. In one aspect of the invention, the therapeutic compound of the invention has a molecular formula of C ₄₇ H ₅₈ N ₆ O _5. It can be named: (S)-N-(3-(((S)-3-amino-1-((S)-4-methyl-1-oxo-1-(pyrrolidin-1-yl)pentan-2-ylamino)-1-oxopropyl-2-yl)(methyl)amino)-3-oxopropyl)-3-(biphenyl-4-yl)-2-(2,2-diphenylacetamido)-N-methylpropanamide.

In aspects of the invention, therapeutic compounds (I) have the ability to effectively inhibit the interaction of RAS (an acronym from rat sarcoma) with its effectors (other proteins in the cascade triggered by RAS) in cells in vitro, 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, therapeutic compounds inhibit the survival of PDAC cell lines while being non-toxic to non-cancerous normal cell lines. In aspects of the invention, therapeutic compounds and therapeutically acceptable salts thereof can be used to treat 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, therapeutic strategies involve finding anticancer drugs that inhibit the binding of RAS to its effectors.

Another aspect of the present invention relates to a pharmaceutical composition (eg, medicine or medicament) comprising a therapeutic compound and a pharmaceutically acceptable salt 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 treating cancer, including human cancers. This aspect may relate to the use of therapeutic compounds and pharmaceutically acceptable salts thereof in the manufacture of medicaments for treating cancer, including human cancers. Alternatively, this aspect may relate to a method of treating cancer comprising administering a therapeutically effective amount of a therapeutic compound and a pharmaceutically acceptable salt thereof.

In embodiments of the aspects mentioned in the previous paragraphs, the cancer comprising human cancer is pancreatic cancer, lung cancer, or colorectal cancer. In other embodiments, the cancer comprising human cancer is pancreatic cancer. And, in other embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

Throughout the specification and claims, the word "comprise" and variations of the word are not intended to exclude other technical features, elements, limitations, additives or components. In addition, the word "comprise" covers the situation of "consisting of". Additional objects, advantages and features of the present invention will become apparent to those skilled in the art after reviewing the specification, or may be understood through practice of the present invention. The following examples and drawings are provided by way of example, and they are not intended to limit the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate aspects of the present invention. In such drawings:

Figure 1 shows the results of immunoprecipitation of HA-KRASG12V with C-RAF or PI3K in starved HeLa cells expressing HA-KRASG12V after incubation with 100 μM of the compound of formula (I) for 2 h and EGF stimulation for 10 min (EGF = epidermal growth factor). Immunoprecipitation was performed with anti-HA antibody, and Western blotting was performed on the bound (BO) fraction and the input (IN) fraction with anti-p110αPI3K and anti-C-RAF. 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 24 h, at which time cell viability was determined by MTS assay. The experiment was repeated 3 times. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test, and differences were considered significant when p≤0.05.

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

Figure 4 shows the GTPase-RAS protein pharmacodynamic site (PDB: 5P21): (A) shows the composite GTPase-RAS, with highly conserved residues in the RAS effector protein underlined in the shaded bar. The RAS protein surface is colored in gray, and residues involved in intermolecular contacts with highly conserved residues in the effector protein are underlined (Asp33, Glu37, Asp38, and Tyr64). (B) shows computational predictions of more relevant residues for designing a new set of peptide mimetics.

Fig. 5 shows the Western blot of different proteins of KRAS signal transduction 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 (GAP120) is used as a load control. Before EGF (50ng/ml) treatment lasts for 10min 2h, the test therapeutic compound is applied to the serum starved cell culture (0.5% FCS lasts for 24h) of hTERT-RPE (hereinafter, also referred to as RPE cell line). DMSO is the solvating agent for diluting peptides, and then after diluting with cell culture medium, the final percentage of DMSO is 0.5%.

Figure 6 shows a protein blot performed under the same conditions as the protein blot of Figure 5, except that 0.5% β-cyclodextrin was used 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).

Fig. 7 shows the Western blot for evaluating 2018/IP-14-01 (P1) and its derived peptide mimics, including IPR-471 (P1.1.), IPR-472 (P1.2), IPR-473 (P1.3) and IPR-474 (P1.4).For this experiment, we follow the same protocol conditions used in the previous WB assay for the first generation peptide mimics described in Fig. 5.Compound is applied to the cell culture of hTERT-RPE cells with 50 μM with 0.5% DMSO for 2h, and then the cells are treated with EGF (50ng/ml) for 10min.We do not determine any solubility problems of these peptide mimics.

FIG8 shows a list of therapeutic compounds useful in treating cancer.

definition

In this specification, reference to "an embodiment/aspect" or "an embodiment/aspect" means that a particular feature, structure or characteristic described in conjunction with the embodiment/aspect is included in at least one embodiment/aspect of the present disclosure. The use of the phrases "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 selectable embodiment/aspect that is mutually exclusive with other embodiments/aspects. In addition, various features are described that may be exhibited by some embodiments/aspects but not by other embodiments/aspects. Similarly, various requirements are described that may be requirements of some embodiments/aspects but not requirements of other embodiments/aspects. Embodiments and aspects may be used interchangeably in some cases.

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

Therefore, optional language and synonyms can be used for any one or more terms discussed herein. Whether a term is elaborated or discussed herein does not have any special meaning. Synonyms for certain terms are provided. The description 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 term discussed herein, is only illustrative and is not intended to further limit the scope and meaning of the present disclosure or any exemplary term. Likewise, the present disclosure is not limited to the multiple embodiments given in this specification.

Without intending to further limit the scope of the present disclosure, examples of instruments, devices, methods and related results thereof according to the embodiments of the present disclosure are given below. Note that, for the convenience of the reader, titles or subtitles may be used in the examples, which should never limit the scope of the present disclosure. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, this document, including definitions, shall prevail.

Where applicable, as used herein in this specification and the appended claims and unless otherwise indicated, the term "about" or "typically" means a margin of +/-20%. Furthermore, where applicable, as used herein in this specification and the appended claims, the term "substantially" means a margin of +/-10% unless otherwise indicated. It should be understood that not all uses of the above terms are quantifiable such that the ranges mentioned can be applied.

The term "subject" or "patient" refers to any single animal in need of treatment, 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). Most preferably, the patient herein is a human. In embodiments, the "subject" of 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 compositions and methods include the listed elements, but do not exclude other unlisted elements. When "consisting essentially of" is used to define compositions and methods, other elements that change the basic properties of the composition and/or method are excluded, but other unlisted elements are not excluded. Therefore, a composition consisting essentially of elements as defined herein will not exclude trace elements, such as pollutants or pharmaceutically acceptable carriers from any separation and purification methods, such as phosphate-buffered saline, preservatives, etc., but will exclude additional unspecified amino acids. "Consisting of" excludes trace elements exceeding other ingredients and substantial method steps for applying the compositions described herein. The 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.

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

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

As used herein, the term "alkyl" refers to a saturated straight or branched hydrocarbon, such as a straight or branched group 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, 2,2-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, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

As used herein, the term "alkynyl" refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-8 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.

As used herein, the term "amide" 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, halogenated alkyl, heteroaryl, heterocyclyl and hydrogen. Amide can be attached to another group through carbon, nitrogen, Rb or Rc. Amide can also be cyclic, for example Rb and Rc can be connected to form a 3-8 ring, such as a 5- or 6-ring. The term "amide" encompasses groups such as sulfonamide, urea, urea groups, carbamates, carbamic acid and cyclic forms thereof. The term "amide" also encompasses amide groups attached to carboxyl groups, for example, -amide-COOH or salts such as -amide-COONa, amino groups attached to carboxyl groups (for example, -amino-COOH or salts such as -amino-COONa).

As used herein, the term "amine" or "amino" refers to the form -NRdRe or -N (Rd) Re -, wherein Rd and Re are independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, carbamate, cycloalkyl, halogenated alkyl, heteroaryl, heterocyclic radical and hydrogen. The amino group can be attached to the parent molecular group through nitrogen. The amino group can also be cyclic, for example, any two of Rd and Re can be connected together or connected to N to form a 3-12 ring (for example, morpholino or piperidinyl). The term amino also includes the corresponding quaternary ammonium salt 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 can each be optionally substituted by hydroxyl, halogen, alkoxy, ester or amino.

The term "aryl" as used herein refers to an aromatic ring system of a monocarbocyclic, dicarbocyclic or other polycarbocyclic rings. The aryl group may optionally be fused with one or more rings selected from aryl, cycloalkyl and heterocyclic groups. The aryl group of the present disclosure may be substituted by a group selected from the following: alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halogenated alkyl, heteroaryl, heterocyclic group, hydroxyl, 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, and 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 wherein the ring contains 6 carbon atoms, referred to herein as "(C6)aryl."

As used herein, the term "arylalkyl" refers to an alkyl group having at least one aryl substituent (e.g., -aryl-alkyl-). 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".

As used herein, the term "carbamate" 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, arylcarbamates or heteroarylcarbamates (e.g., wherein at least one of Rg, Rh and Ri is independently selected from aryl or heteroaryl, such as pyridine, pyridazine, pyrimidine and pyrazine).

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

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

The term "cycloalkyloxy" as used herein refers to a cycloalkyl group attached to an oxygen.

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

As used herein, the term "dicarboxylic acid" refers to a group 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. Dicarboxylic acids can be substituted by: alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halogenated alkyl, heteroaryl, heterocyclic radical, hydrogen, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Dicarboxylic acids include but are not limited to succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, toxilic acid, phthalic acid, aspartic acid, glutamic acid, malonic acid, fumaric acid, (+)/(-)-malic acid, (+)/(-)-tartaric acid, isophthalic acid and terephthalic acid. The dicarboxylic acid also includes carboxylic acid derivatives thereof such as anhydrides, imides, hydrazides (eg, 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 combined with hydrogen, and Rj and Rk can be independently selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, cycloalkyl, ether, halogenated alkyl, heteroaryl and heterocyclic. Rk can be hydrogen, but Rj cannot be hydrogen. Esters can be cyclic, for example, carbon atoms and Rj, oxygen atoms and Rk, or Rj and Rk can be connected 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 an alkyl, such as -O-C(O)-alkyl-, -C(O)-O-alkyl-, and -alkyl-C(O)-O-alkyl-. Exemplary esters also include aryl esters 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 with structure -RkC(O)O-, wherein oxygen is combined with the parent molecule. Exemplary reverse esters include succinate, D-arginine ester, L-arginine ester, L-lysine ester and D-lysine ester. Esters also include carboxylic anhydrides and acyl halides.

As used herein, the term "halo" or "halogen" refers to F, Cl, Br or I.

As used herein, the term "haloalkyl" refers to an alkyl group substituted by one or more halogen atoms."Haloalkyl" also encompasses an alkenyl group or an alkynyl group substituted by one or more halogen atoms.

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

As used herein, the term "heterocycle", "heterocyclyl" or "heterocyclic" 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 alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thione. Heterocycles also include bicyclic groups, tricyclic groups and tetracyclic groups, wherein any one of the above heterocycles is fused with one or two rings independently selected from aryl, cycloalkyl and heterocycle. Exemplary heterocycles include acridinyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxazolyl, biotinyl, cinnolinyl, dihydrofuranyl, dihydroindolinyl, dihydropyranyl, dihydrothiophenyl, dithiazolyl, furanyl, homopiperidinyl, imidazolidinyl, imidazolinyl, imidazolyl, indolyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isoxazolidinyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazolidinyl, pyrazolyl, oxazinyl, pyrazolyl, pyrazolinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrimidyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, pyrrolyl, quinolinyl, quinoxaloyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, thiadiazolyl, thiazolidinyl, thiazolyl, thienyl, thiomorpholinyl, thiopyranyl, and triazolyl.

As used herein, the terms "hydroxy" and "hydroxyl" refer to -OH.

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

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

As used herein, the term "ketone" refers to the structure -C(O)-Rn (such as acetyl, -C(O)CH3) or -Rn-C(O)-Ro-. The ketone can be attached to another group through Rn or Ro. Rn or Ro can be an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl, or Rn or Ro can be connected to form a 3- to 12-membered ring.

As used herein, the term "monoester" 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 intended to protect an amino group from undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis," 4th Edition (John Wiley & Sons, Hoboken, NJ, 2006), which is incorporated herein by reference. N-protecting groups include: 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 agents such as protected or unprotected D-amino acids, L-amino acids or D,L-amino acids, such as alanine, leucine, phenylalanine, etc.; sulfonyl-containing groups, such as benzenesulfonyl, p-toluenesulfonyl, etc.; carbamate-forming groups, such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxy a cyclopentyloxycarbonyl group, a cyclopentyloxycarbonyl group, a cyclohexyloxycarbonyl group, a cyclohexyloxycarbonyl group, a cyclopent ... Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, tert-butylacetyl, alanyl, phenylsulfonyl, benzyl, tert-butoxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

As used herein, the term "phenyl" refers to a 6-membered carbocyclic aromatic ring. The phenyl group can also be fused with a cyclohexane ring or a cyclopentane ring. The phenyl group can be substituted with one or more substituents, and the one or more substituents include alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, halogenated alkyl, heteroaryl, heterocyclic radical, hydroxyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone.

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

The term "acetylation" or "ethanoylation" in IUPAC nomenclature refers to the reaction that introduces an acetyl functional group into a compound. In contrast, deacetylation refers to the removal of an acetyl group.

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

As used herein, suitable substituents on optionally substituted substituents refer to groups that do not invalidate the synthesis or pharmaceutical utility of the compounds of the present disclosure or the intermediates that can be used to prepare 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 alkoxy; 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; formyl; ketone, such as -CO (C1-8 alkyl), -CO (C6 aryl) ester, such as -CO2 (C1-8 alkyl) and -CO2 (C6 aryl). Those skilled in the art can easily select suitable substituents based on the stability of the compounds of the present disclosure and the pharmacological and synthetic activity.

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 on a subject to which it is administered. In other words, an "active agent" or "active ingredient" refers to one or more components of a composition to which all or part of the effect of the composition is attributed. An active agent can be a primary active agent, or in other words, a component of a composition to which all or part of the effect of the composition is attributed. An active agent can be a secondary agent, or in other words, a component of a composition to which an additional portion of the composition is attributed and/or to which other effects of the composition are attributed.

In an embodiment, a "pharmaceutical composition" is intended to include a combination of an active agent such as a therapeutic compound of the invention and 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 endotoxins or is nontoxic to recipients at the doses or concentrations employed.

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

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

As used herein, the term "pharmaceutically acceptable prodrug" means those prodrugs of the compounds of the present disclosure that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as zwitterionic forms of the compounds of the present disclosure (where possible). A discussion is provided in Higuchi et al., "Prodrugs as Novel Delivery Systems," ACS Symposium Series, Vol. 14, and in Roche, E.B., ed., 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. The acids that may 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 sulfates, citrates, malates, acetates, oxalates, chlorides, bromides, iodides, nitrates, sulfates, bisulfates, phosphates, acid phosphates, isonicotinates, acetates, lactates, salicylates, citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sugar acids, succinates ... The compounds comprising the amino moiety included in the compositions of the present invention can form pharmaceutically acceptable salts with a variety of amino acids in addition to the acids mentioned above. The naturally acidic compounds included in the compositions of the present invention can form basic salts with a variety of pharmacologically acceptable cations. Examples of such salts include alkali metal salts or alkaline earth metal salts, and particularly calcium salts, magnesium salts, sodium salts, lithium salts, zinc salts, potassium salts and iron salts.

The compounds of the present disclosure may contain one or more chiral centers and/or double bonds, and therefore exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. When used in this article, the term "stereoisomer" consists of all geometric isomers, enantiomers or diastereomers. These compounds can be represented by the symbols "R" or "S" according to the configuration of the substituents around the stereoisomer carbon atom. The present disclosure encompasses a variety of stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. A mixture of enantiomers or diastereomers can be designated as "±" in nomenclature, but the skilled person will recognize that the structure can represent chiral centers implicitly.

Individual stereoisomers of the compounds of the present disclosure may be prepared synthetically from commercially available starting materials containing asymmetric centers or stereocenters, or by preparing racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These resolution methods are exemplified by (1) attaching a 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 a mixture of optical enantiomers on a chiral chromatographic column. Stereoisomers may also be resolved into their component stereoisomers by well-known methods such as chiral gas chromatography, chiral high performance liquid chromatography, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent. Stereoisomers may also be obtained from stereopure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

Geometric isomers can also be present in the compounds of the present disclosure. The present disclosure encompasses a variety of geometric isomers and mixtures thereof produced by substituent arrangements around carbon-carbon double bonds or around carbocyclic rings. Substituents around carbon-carbon double bonds are designated as being in "Z" or "E" configurations, wherein the terms "Z" and "E" are used according to IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both E and Z isomers.

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

The compounds disclosed herein may exist as tautomers, and both tautomeric forms are intended to be included within the scope of the present disclosure 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 appropriate amino acid insertions or deletions 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 a cap protein, a rep protein, or a fragment thereof of at least 8 amino acids 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% identity, preferably at least 90% identity and more preferably more than 97% identity. The identity is readily determined by those skilled in the art with the aid 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 outcome. In embodiments, the outcome may be an effective cancer treatment.

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

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

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. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes, as well as a large number of immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ or ε, which in turn define immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen binding region of an antibody will be the most critical in terms of specificity and affinity of binding, and is encoded by the variable domains. The antibody can be a complete antibody, an antigen binding fragment or its single chain.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer comprises two identical pairs of polypeptide chains, each pair having a "light chain" (about 25 kD) and a "heavy chain" (about 50 kD-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 chain and heavy chain, respectively.

Antibodies exist, for example, as complete immunoglobulins or as many well-characterized fragments produced by digestion with a variety of peptidases. Thus, for example, pepsin digests the antibody below the disulfide bonds in the hinge region to produce F(ab)' ₂ , _which is a dimer of Fab, which itself is a light chain VL-CL connected to VH-CH1 by a disulfide bond. Although various antibody fragments are defined in terms of digestion of complete antibodies, it will be appreciated by the skilled artisan that such fragments can be synthesized de novo chemically or by using recombinant DNA methods. Therefore, as used herein, the term antibody also includes antibody fragments produced by modifying the entire antibody, or antibody fragments (e.g., single-chain Fv) synthesized de novo using a recombinant DNA method, or antibody fragments identified using a phage display library (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

Therefore, in any aspect of the present invention, the term antibody also includes miniantibodies, scFv, double antibodies, three antibodies, etc. ScFv and double antibodies are small bivalent biological specific antibody fragments with high affinity and specificity. Their high signal-to-noise ratio is usually better, because better specificity and rapid blood clearance increase their potential for diagnosis and treatment targeting of specific antigens (Sundaresan et al., J Nucl Med 44:1962-9 (2003)). In addition, these antibodies are advantageous because, if necessary, they can be engineered into different types of antibody fragments, ranging from small single-chain Fv (scFv) to complete IgG (Wu & Senter, Nat. Biotechnol. 23:1137-1146 (2005)) with different subtypes. In some embodiments, antibody fragment is a part of scFv-scFv or double antibodies. In some embodiments, in any aspect, the invention provides a high-affinity antibody for use according to the present 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, etc. Regardless of the structure, an 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 that contains an immunoglobulin variable region that acts like an antibody by binding to a specific antigen to form a complex.

The term "antigen binding fragment" or "Fab" refers to the region on an antibody that binds to an antigen. It includes one constant domain and one variable domain (i.e., four domains: VH, CH1, VL, and CL1) in each of the heavy and light chains. The variable domains contain the paratope (antigen binding site) that includes a set of complementary determining regions at the amino terminus of the monomer. Thus, each arm of the Y binds to an epitope on the antigen.

The term "Fc region" or "fragment crystallizable region" refers to the tail region of the antibody CH2-CH3, which interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This "effector function" allows the antibody 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 the binding of Fc to Fc receptors on the surface of cells of the immune system. CDC is mediated by the binding of Fc to proteins of the complement system (e.g., C1q).

In IgG, IgA and IgD antibody isotypes, the Fc region has two identical protein fragments, which are derived from the second constant domain and the third constant domain of the two heavy chains of the antibody. IgM Fc region and IgEFc region have three heavy chain constant domains (CH domains 2-4) in each polypeptide chain, while IgG contains 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 biantennary structures. In addition, a small amount of these N-glycans also carry bisecting GlcNAc and α-2,6-linked sialic acid residues.

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

"Conservatively modified variants" are applicable to both amino acid sequences and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids encoding the same or substantially the same amino acid sequence, or when the nucleic acid does not encode an amino acid sequence, refer to substantially the same sequence. Due to 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. Therefore, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described that do not change the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. The technician will recognize that each codon in the nucleic acid (except AUG and TGG, AUG is usually the only codon for methionine, and TGG is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of the nucleic acid encoding a polypeptide is implicit in each described sequence with respect to the expression product rather than with respect to the actual probe sequence.

The therapeutic peptides of the present invention may have amino acid additions, deletions or substitutions. The modified amino acid sequence is a sequence that is different from the natural amino acid sequence due to deletions, insertions, non-conservative or conservative substitutions or combinations thereof of one or more amino acid residues. 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 can be conservative or non-conservative. As used herein, "conservative amino acid substitutions" are substitutions in which one of the amino acid residues is replaced by another amino acid residue with 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The most common exchanges are bidirectional 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 generally do not change 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 a functional side group. 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 tert-butoxycarbonyl group, a chloroacetyl group, or a formyl group. The free carboxyl group can be derivatized to form a salt, a methyl ester and an ethyl ester or other types of esters or hydrazides. The free hydroxyl group can be derivatized to form an O-acyl derivative or an O-alkyl derivative. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. Also included as derivatives are peptides containing one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine can replace lysine.

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

The term unnatural amino acid or unusual amino acid includes those amino acids that can be built into synthetic peptides. These amino acids include D-amino acids, homoamino acids, β-homoamino acids, N-methyl amino acids, α-methyl amino acids, unnatural side chain variant amino acids and other unusual amino acids. D-amino acids refer to the mirror image of the naturally occurring L-isomer. Homoamino acids are amino acids that include a methylene (CH ₂ ) group added to the α-carbon of the amino acid. β-homoamino acids are analogs of standard amino acids in which the carbon skeleton has been extended by inserting a carbon atom immediately after the acid group. N-methyl amino acids are amino acids that carry a methyl group instead of a proton at the nitrogen. α-methyl amino acids are natural amino acid variants in which the proton on the α-carbon atom of the natural original amino acid (between the amino group and the carboxyl group) has been replaced by a methyl group. Unusual amino acids are most commonly found in microbial peptides and proteins and are formed after translation. Unusual amino acids often contribute to the special biological activities of these peptides. In addition, amino acids can be synthetic and non-natural.

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

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form, and their complements. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. 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 specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by 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 "prevention" refers to all actions that inhibit or delay the occurrence of a disease.

As used herein, the term "treatment" means all actions that have alleviated, improved or alleviated the symptoms of a disease. In this specification, "treatment" means alleviating, improving or alleviating the symptoms of cancer, neurodegenerative or infectious diseases by administering the antibodies disclosed herein.

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

The term "subject" refers to those suspected of having or diagnosed with cancer, neurodegenerative disease, or infectious disease. However, it includes, but is not limited to, any subject to be treated with a pharmaceutical composition disclosed herein. A pharmaceutical composition comprising an anti-DLL3 antibody disclosed herein is administered to a subject suspected of having cancer, neurodegenerative disease, or infectious disease.

The term "cancer" refers to human cancers and malignancies, sarcomas, adenocarcinomas, etc., including solid tumors, kidney cancer, breast cancer, lung cancer, renal cancer, bladder cancer, urinary tract cancer, urethral cancer, penis cancer, vulvar cancer, vaginal cancer, cervical cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer and liver cancer. Additional cancers include, for example, Hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, small cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer and adrenocortical carcinoma.

In any of the above embodiments, one or more cancer therapies, such as chemotherapy, radiotherapy, immunotherapy, surgery, or hormone therapy, can 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-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin. Tetrazines include dacarbazine, mitozolomide and temozolomide. Aziridines include thiotepa, mitomycin and diaziquone (AZQ). Cisplatin and derivatives include cisplatin, carboplatin and oxaliplatin. In one embodiment, the chemotherapeutic agent is an antimetabolite: antifolate (e.g., methotrexate), fluoropyrimidine (e.g., fluorouracil and capecitabine), deoxynucleoside analogs, and thiopurines. In another embodiment, the chemotherapeutic agent is an antimicrotubule 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 .

Contacting the patient with the therapeutic compound can 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 agent.

As used herein, the term "formulation" refers to the therapeutic compounds disclosed herein and excipients combined together, which 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 including ranges, e.g., pH, temperature, time, concentration, and molecular weight, will be understood as approximate values according to common practice in the art. When used herein, the term "about", as appropriate in view of the context, may represent a variation of (+) or (-) 1%, 5%, or 10% of the stated amount. It should be understood that, although not always explicitly stated, the agents described herein are exemplary only, and 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 (13th ed.), Mack Publishing Company, Easton, PA, a standard reference for many types of administration. As used herein, the term "preparation" means a combination of at least one active ingredient with one or more other ingredients, also commonly referred to as excipients, which may be independently active or inactive. The term "preparation" may or may not refer to a pharmaceutically acceptable composition for administration to humans or animals, and may include compositions that are useful intermediates for storage or research purposes.

Since the patients and subjects of the methods of the present invention are veterinary subjects in addition to humans, formulations suitable for these subjects are also suitable. These subjects include livestock and pets as well as sports animals such as horses, spirits, wait.

For use as a treatment for human and animal subjects, the therapeutic compounds of the invention can be formulated as pharmaceutical compositions or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or treatment), the therapeutic compound is formulated in a manner consistent with these parameters. An overview of such technology is found in: Remington: The Science and Practice of Pharmacy, 21 "ed., Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

The therapeutic compounds described herein can be present in an amount of 1%-95% by weight of the total weight of the pharmaceutical composition. The pharmaceutical composition can be provided in a dosage form suitable for intra-articular, oral, parenteral (e.g., intravenous, intramuscular), rectal, skin, subcutaneous, local, transdermal, sublingual, nasal, vaginal, intracapsular, intraurethral, intrathecal, epidural, ear or eye administration, or provided by injection, inhalation or direct contact with nasal, urogenital, gastrointestinal, reproductive or oral mucosa. Therefore, the pharmaceutical composition can be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels (including hydrogels), pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, products suitable for iontophoresis delivery or aerosols. The composition can be prepared according to conventional pharmaceutical practices.

Details

Embodiments of the invention include methods of using therapeutic compounds, pharmaceutical compositions and preparations thereof, to treat, monitor and prevent cancer, including refractory cancers.

Treatment

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

Any suitable route of administration or mode of administration can be used to provide a therapeutic compound of a therapeutically effective dose or a prophylactic effective dose to a patient. Exemplary routes of administration or modes of administration include parenteral (e.g., intravenous, intraarterial, 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, intestinal, intrapulmonary, intralymphatic, intracavitary, intraorbital, intracapsular and transurethral, as well as local delivery by catheter or stent.

The pharmaceutical composition comprising the therapeutic compound according to the present disclosure can 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 agents and absorption delay agents and physiologically compatible analogs. The pharmaceutical composition may include a suitable solid phase or gel phase carrier or excipient. Exemplary carriers or excipients include calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin and polymers such as polyethylene glycol. Exemplary pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and combinations thereof. In many cases, it will be preferred to include an isotonic agent in the composition, such as sugar, polyols such as mannitol, sorbitol or sodium chloride. Pharmaceutically acceptable carriers may also include a small amount of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, which enhance the shelf life or effectiveness of the therapeutic agent.

In embodiments, therapeutic compounds can be incorporated into pharmaceutical compositions suitable for parenteral administration. Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to change the toxicity of solutions having a concentration of 0mM-300mM (optimally 150mM for liquid dosage forms). Lyophilized dosage forms can include cryoprotectants, mainly 0%-10% sucrose (optimally 0.5%-1.0%). Other suitable cryoprotectants include trehalose and lactose. Lyophilized dosage forms can include bulking agents, mainly 1%-10% mannitol (optimally 2%-4%). Stabilizers can be used for both liquid dosage forms and lyophilized dosage forms, mainly 1mM-50mM L-methionine (optimally 5mM-10mM). Other suitable bulking agents include glycine, arginine, which can 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 surfactant.

Pharmaceutical compositions comprising therapeutic compounds can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol as a preservative) or in sterile water prior to injection. Pharmaceutical compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.

The therapeutic compound in the pharmaceutical composition can be formulated in a "therapeutically effective amount" or a "prophylactically effective amount." A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the dosage and time period necessary. The therapeutically effective amount of the recombinant vector can 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 preventive or therapeutic purposes, the bioavailability of the particular agent, the ability of the trispecific antibody to elicit a desired response in an individual, previous therapy, the patient's age, weight and sex, the patient's clinical history and response to the antibody, the type of trispecific antibody used, the judgment of the attending physician, etc. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the recombinant vector are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the dosage and time period necessary.

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

In an embodiment, the therapeutic compound of the invention comprises the peptide mimetic P1.3 (hereinafter also referred to as IPR-473) or a derivative of P1.3 having the following structure:

Wherein, _R1 includes one of the following:

CH3, CH2-CH3, CH2-CH2-CH3, CH2-CH2-CH2-CH3 or CH2-CH2-CH2-hexyl;

And _R2 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 _R4 includes one of the following:

H, CH3 or alkyl group

And R ₅ includes one of the following:

H, CH3 or alkyl group

In an embodiment, the therapeutic compound of the invention includes one of the following peptide mimetics, wherein P1.3 is the structure listed above, wherein the specific _R1 , _R2 and _R3 of each peptide mimetic are listed in Table 1 below, and the relevant KRAS-GTPase docking scores of the specific peptide mimetics 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:

R ₁ ＝CH ₃

R ₂ ＝CH ₂ -NH ₂ (Dap side chain)

R ₃ ＝CH ₂ CH(CH ₃ ) ₂ (L-Leu side chain)

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

wherein R ₁ is CH ₃ , R ₂ is (CH ₂ ) ₄ NH ₂ (Lys), R ₃ is CH(CH ₃ ) ₂ (Val), and R ₅ is NCH ₃ . And,

R ₁ ＝H

R ₂ ＝CH ₂ -NH ₂ (Dap side chain)

R ₃ ＝CH ₂ CH(CH ₃ ) ₂ (L-Leu side chain)

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

R ₁ = -CH ₂ CH ₂ - (intercalated in pyrrolidine)

R ₂ ＝CH ₂ -NH ₂ (Dap side chain)

R ₃ ＝CH ₂ CH(CH ₃ ) ₂ (L-Leu side chain)

R ₁ = -CH ₂ CH ₂ CH ₂ -(intercalated in piperidine)

R ₂ ＝CH ₂ -NH ₂ (Dap side chain)

R ₃ ＝CH ₂ CH(CH ₃ ) ₂ (L-Leu side chain)

R ₁ ＝-H

R ₂ ＝CH ₂ -NH ₂ (Dap side chain)

R ₃ ＝CH ₂ CH(CH ₃ ) ₂ (L-Leu side chain).

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

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

In other embodiments, the therapeutic compound is administered in a range of about 10 ng to about 100 ng per single administration, about 10 ng to about 1 g per single administration, about 10 ng to about 10 g per single administration, about 10 ng to about 100 mg per single administration, about 10 ng to about 1 mg per single administration, about 10 ng to about 10 mg per single administration, about 10 ng to about 100 mg per single administration, about 10 ng to about 1000 mg per injection, about 10 ng to about 10,000 mg per single administration, about 100 ng to about 1 mg per single administration, about 100 ng per single administration, to about 10 mg, about 100 ng to about 100 mg per single administration, about 100 ng to about 1 mg per single administration, about 100 ng to about 10 mg per single administration, about 100 ng to about 100 mg per single administration, about 100 ng to about 1000 mg per single administration, about 100 ng to about 10,000 mg per single administration, about 1 mg to about 10 mg per single administration, about 1 mg to about 100 mg per single administration, about 1 mg to about 100 mg per single administration, about 1 mg to about 100 mg per single administration, about 1 mg to about 100 mg per single administration, about 1 m per injection g to about 1000 mg, about 1 mg to about 10,000 mg per separate administration, about 10 mg to about 100 mg per separate administration, about 10 mg to about 1 mg per separate administration, about 10 mg to about 10 mg per separate administration, about 10 mg to about 100 mg per separate administration, about 10 mg to about 1000 mg per injection, about 10 mg to about 10,000 mg per separate administration, about 100 mg to about 1 mg per separate administration, about 100 mg to about 100 mg per separate administration, about 100 mg to about 1000 mg per injection, Each single administration is about 100 mg to about 10,000 mg, each single administration is about 1 mg to about 10 mg, each single administration is about 1 mg to about 100 mg, each injection is about 1 mg to about 1000 mg, each single administration is about 1 mg to about 10,000 mg, each single administration is about 10 mg to about 100 mg, each injection is about 10 mg to about 1000 mg, each single administration is about 10 mg to about 10,000 mg, each injection is about 100 mg to about 1000 mg, each single administration is about 100 mg to about 10,000 mg, each injection is about 100 mg to about 1000 mg, each single administration is about 100 mg to about 10,000 mg, and each single administration is about 1000 mg to about 10,000 mg. The trispecific antibody can be administered every day, 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 specific embodiments, an amount of the therapeutic compound can be administered at a dosage 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 dosage will depend on the patient's condition, size, age, and condition.

Doses can be tested in any of several art-accepted animal models applicable to any particular cell proliferative disorder.

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

Pharmaceutical compositions disclosed herein can include solvents, emulsions or other diluents that are sufficient to dissolve the amount of therapeutic compounds disclosed herein. In other aspects of this embodiment, pharmaceutical compositions disclosed herein can be included in solvents, emulsions or diluents in the following amount: 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 can include a solvent, emulsion, or other diluent in an amount 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 40% (v/v), about 4% (v/v) to 30% (v/v), about 4% (v/v) to 20% (v/v), about 4% (v/v) to 10% (v/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 desired concentration. 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 0.00001 mg/mL, at least 0.0001 mg/mL, at least 0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100 mg/mL, at least 200 mg/mL or at least 500 mg/mL. In other aspects of this embodiment, the final concentration of the therapeutic compound in the pharmaceutical composition can be in the range of, for example, about 0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mL to about 3,000 mg/mL, about 1,000 mg/mL to about 3,000 mg/mL, about 1 The dosage range is from about 100 mg/mL to about 2,000 mg/mL, from about 250 mg/mL to about 2,000 mg/mL, from about 500 mg/mL to about 2,000 mg/mL, from about 750 mg/mL to about 2,000 mg/mL, from about 1,000 mg/mL to about 2,000 mg/mL, from about 100 mg/mL to about 1,500 mg/mL, from about 250 mg/mL to about 1,500 mg/mL, from about 500 mg/mL to about 1,500 mg/mL, from about 750 mg/mL to about 1,500 mg/mL, from about 1,000 mg/mL to about 1,500 mg/mL, from about 100 mg/mL to about 1,200 mg/mL, from about 250 mg/mL to about 1,500 mg/mL mL to about 1,200 mg/mL, about 500 mg/mL to about 1,200 mg/mL, about 750 mg/mL to about 1,200 mg/mL, about 1,000 mg/mL to about 1,200 mg/mL, about 100 mg/mL to about 1,000 mg/mL, about 250 mg/mL to about 1,000 mg/mL, about 500 mg/mL to about 1,000 mg/mL, about 750 mg/mL to about 1,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL to about 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100 mg/mL to about 500 mg/mL, About 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mL to about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about 0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.1 mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or about 0.001 mg/mL to about 100 mg/mL.

Aspects of the present specification disclose, in part, treating an individual suffering from cancer. As used herein, the term "treat" refers to reducing or eliminating clinical symptoms of cancer in an individual; or delaying or preventing the onset of clinical symptoms of cancer in an individual. For example, the term "treat" can 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 a person 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 tissues or organs affected by the cancer. Those skilled in the art will understand the appropriate symptoms or 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 the symptoms of a disorder associated with cancer. In aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the severity of the symptoms of a disorder 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% or at least 95%. In other aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the severity of symptoms of a disorder associated with 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 100%. 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 symptoms associated with cancer by, e.g., 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 symptoms associated with cancer by, e.g., 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 symptoms 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 yet other aspects of this embodiment, the therapeutically effective amount of the pharmaceutical composition disclosed herein is generally in the range of about 0.001 mg/kg to about 100 mg/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, the effective amount of the pharmaceutical composition disclosed herein can be, for example, at least 0.001 mg/kg, at least 0.01 mg/kg, at least 0.1 mg/kg, at least 1.0 mg/kg, at least 5.0 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, or at least 50 mg/kg, and is 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 in the range of, for example, about 0.001 mg/kg to about 10 mg/kg, about 0.001 mg/kg/day to about 15 mg/kg, about 0.001 mg/kg to about 20 mg/kg, about 0.001 mg/kg to about 25 mg/kg, about 0.001 mg/kg to about 30 mg/kg, about 0.001 mg/kg to about 35 mg/kg, about 0.001 mg/kg to about 40 mg/kg, about 0.001 mg/kg to about 45 mg/kg, about 0.001 mg/kg to about 50 mg/kg, about 0.001 mg/kg to about 75 mg/kg, or about 0.001 mg/kg to about 100 mg/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 in the range of, for example, 0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 15 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about 0.01 mg/kg to about 25 mg/kg, about 0.01 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 35 mg/kg, about 0.01 mg/kg to about 40 mg/kg, about 0.01 mg/kg to about 45 mg/kg, about 0.01 mg/kg to about 50 mg/kg, about 0.01 mg/kg to about 75 mg/kg, or about 0.01 mg/kg to about 100 mg/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 in the range of, for example, about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 15 mg/kg, about 0.1 mg/kg to about 20 mg/kg, about 0.1 mg/kg to about 25 mg/kg, about 0.1 mg/kg to about 30 mg/kg, about 0.1 mg/kg to about 35 mg/kg, about 0.1 mg/kg to about 40 mg/kg, about 0.1 mg/kg to about 45 mg/kg, about 0.1 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 75 mg/kg, or about 0.1 mg/kg to about 100 mg/kg, and administered, for example, every 3 days, every 5 days, every 7 days, every 10 days, or every 14 days.

In liquid and semisolid formulations, the concentration of the therapeutic compounds disclosed herein can generally be between about 50 mg/mL and about 1,000 mg/mL. In aspects of this embodiment, the therapeutically effective amount of the therapeutic compounds disclosed herein can be, for example, from about 50 mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 300 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50 mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50 mg/mL to about 900 mg/mL, about 50 mg/mL to about 1,000 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 400 mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50 mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50 mg/mL to about 900 mg/mL, about 50 mg/mL to about 1,000 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about L to about 300 mg/mL, about 100 mg/mL to about 400 mg/mL, about 100 mg/mL to about 500 mg/mL, about 100 mg/mL to about 600 mg/mL, about 100 mg/mL to about 700 mg/mL, about 100 mg/mL to about 800 mg/mL, about 100 mg/mL to about 900 mg/mL, about 100 mg/mL to about 1,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about 200 mg/mL to about 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about 200 mg/mL to about 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about 200 mg/mL to about 800 mg/mL, about 200 mg/mL to about 900 mg/mL, about 200 mg/mL to about 1,000 mg/mL, about 300 mg/mL to about 400 mg/mL, about 300 mg/mL to about 500 mg/mL, about 300 mg/mL to about 600 mg/mL, about 300 mg/mL to about 700 mg/mL, about 300 mg/mL to about 800 mg/mL, about 300 mg/mL to about 900 mg/mL, about 300 mg/mL to about 1,000 mg/mL, about 400 mg/mL to about 500 mg/mL, about 400 mg/mL to about 600 mg/mL, about 400 mg/mL to about In some embodiments, the present invention relates to an intravenous in vitro medicated drug, wherein the intravenous in vitro medicated drug is administrated in an amount of 100 mg/mL to 200 mg/mL and/or 400 mg/mL to 600 mg/mL. In some embodiments, the intravenous in vitro medicated drug is administrated in an amount of 100 mg/mL to 200 mg/mL and/or 400 mg/mL to 600 mg/mL. In some embodiments, the intravenous in vitro medicated drug is administrated in an amount of 100 mg/mL to 200 mg/mL and/or 400 mg/mL to 600 mg/mL.

Administration can be a single dose or cumulative (continuous administration), and can be easily determined by those skilled in the art. For example, treating cancer can include a one-time administration of an effective dose of a pharmaceutical composition disclosed herein. Alternatively, treating cancer can include multiple administrations of an effective dose of a 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 can vary from person to person, depending on factors such as the severity of individual symptoms. For example, an effective dose of a pharmaceutical composition disclosed herein can be administered once a day to an individual for an indefinite period of time, or until the individual no longer needs treatment. Those of ordinary skill in the art will recognize that the state of the individual can be monitored throughout the treatment process, and the effective amount of the pharmaceutical composition disclosed herein administered can be adjusted accordingly.

In one embodiment, the therapeutic compounds disclosed herein are capable of reducing the number of cancer cells or tumor size in an individual suffering from 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% compared to a patient not receiving the same treatment.

In another embodiment, the therapeutic compounds disclosed herein are capable of reducing the number of cancer cells or tumor size in an individual suffering from cancer by, e.g., 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%, compared to a patient not receiving 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 suffering from 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% compared to a patient not receiving 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 suffering from 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%, or about 10% to about 100%. 0%, 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 other embodiments, the therapeutic compounds and derivatives thereof have a half-life of 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 therapeutic compound is administered for a period of 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 during which administration is ceased 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 the size of cancer cell populations and/or tumor cells in an individual by, 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%.

In other aspects of this embodiment, a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains the size of cancer cell populations and/or tumor cells in a subject 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 other aspects of this embodiment, a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains the size of cancer cell populations and/or tumor cells in an individual 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%, about 95%, or about 100%.

In yet other aspects of this embodiment, a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains the size of cancer cell populations and/or tumor cells in an individual 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%.

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

In embodiments, pharmaceutical compositions or therapeutic compounds are administered to treat sarcomas. In embodiments, sarcomas are one or more of the following: angiosarcomas, chondrosarcomas, dermatofibrosarcomas protuberans, desmoplastic small round cell tumors, epithelioid sarcomas, Ewing sarcomas, gastrointestinal stromal tumors (GIST), Kaposi's sarcomas, leiomyosarcomas, liposarcoma, malignant peripheral nerve sheath tumors, myxofibrosarcomas, osteosarcomas, rhabdomyosarcomas, soft tissue sarcomas, solitary fibrous tumors, synovial sarcomas, and undifferentiated pleomorphic sarcomas. In another embodiment, the sarcoma to be treated is a uterine sarcoma. In further embodiments, pharmaceutical compositions or therapeutic compounds are administered to treat uterine cancer. Uterine cancer is endometrial cancer or uterine sarcoma.

In one aspect, the pharmaceutical compositions disclosed herein reduce the symptoms of a disorder associated with cancer. In aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the symptoms of a disorder 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%, or at least 95%. In other aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the symptoms of a disorder associated with 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%, or about 10% to about 100%. 0%, 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 80%, or about 50% to about 70%.

In another aspect, the pharmaceutical compositions disclosed herein reduce the frequency of the symptoms of the disorder associated with cancer that occur within a given time period. In aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the frequency of the symptoms of the disorder associated with cancer that occur within a given time period 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 time period 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 10% to about 100%, about 15 ... From about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 10% to about 80%, from about 20% to about 80%, from about 30% to about 80%, from about 40% to about 80%, from about 50% to about 80%, or from about 60% to about 80%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, from about 40% to about 70%, or from about 50% to about 70%.

The treatment method of this 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 a physician through appropriate medical judgment and administered once or several times. The specific therapeutic effective dose level of any particular patient can vary according to a variety of factors well known in the medical field, including the type and degree of response to be achieved, according to whether the pharmaceutical composition is used with other agents, the patient's age, weight, health status, sex and diet, the time and route of administration, the secretion rate of the pharmaceutical composition, the time period of treatment, other drugs combined with the pharmaceutical composition disclosed herein or used simultaneously, and similar factors well known in the medical field.

In still another aspect, the present specification provides use of a therapeutic compound and a pharmaceutical composition comprising the therapeutic compound in the preparation of a medicament for preventing or treating cancer, a neurodegenerative disease, or an infectious disease.

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

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

In one embodiment, the weekly dose can 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 can be in the range of from 5mg/kg to 20mg/kg. In selectable aspects, the weekly dose can be in the range of from 10mg/kg to 15mg/kg.

This specification also provides a pharmaceutical composition for administration to a subject. The pharmaceutical composition disclosed herein may also include 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 harmful side effects, and can be easily determined based on the type of disease, the patient's age, weight, health status, sex and drug sensitivity, route of administration, mode of administration, frequency of administration, duration of treatment, drugs used in combination or simultaneously with the composition disclosed herein, and other factors known in medicine.

The pharmaceutical composition comprising the therapeutic compounds disclosed herein may also comprise a pharmaceutically acceptable carrier. For oral administration, the carrier may include, but is not limited to, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, and a flavoring agent. For injectable products, the carrier may include a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, and a stabilizer. For products for topical administration, the carrier may include a matrix, an excipient, a lubricant, and a preservative.

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

On the other hand, the examples of carriers, excipients and diluents suitable for pharmaceutical preparations include but are not limited to lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate and mineral oil. In addition, pharmaceutical preparations can also include fillers, anticoagulants, lubricants, humectants, flavoring agents and antiseptics.

Furthermore, the pharmaceutical composition disclosed herein may have any formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

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

The composition can be used by blending with various pharmaceutically acceptable carriers such as physiological saline or organic solvents. In order to increase stability or absorbability, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used.

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

The total effective dose of the pharmaceutical composition disclosed herein can be administered to the patient in a single dose, or can be administered in multiple doses for a long period of time according to a fractionated treatment regimen. In the pharmaceutical composition disclosed herein, the content of the active ingredient can vary according to the severity of the disease. Preferably, the total daily dose of the peptide mimetic disclosed herein can be about 0.0001 μg to 500 mg per 1 kg patient body weight. However, in addition to the route of administration and treatment frequency of the pharmaceutical composition, various factors are considered to determine the effective dose of the peptide mimetic, including the patient's age, weight, health status, sex, severity of disease, diet and secretion rate. In view of this, those skilled in the art can easily determine the effective dose suitable for the specific use of the pharmaceutical composition disclosed herein. The pharmaceutical composition disclosed herein is not particularly limited to preparations, and routes of administration and modes, as long as it shows a suitable effect.

Furthermore, the pharmaceutical composition may be administered alone, or in combination or simultaneously with other pharmaceutical agents having or within active agents showing preventive or therapeutic efficacy.

In still another aspect, the present specification provides a method for preventing or treating cancer, infectious diseases or neurodegenerative diseases, 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, it will be understood by those skilled in the art that the formulations described herein can be equally applicable to many types of peptide mimetics and other therapeutic compounds, including those exemplified, as well as other compounds known in the art. In view of the teachings and guidance provided herein, it will also be understood by those skilled in the art that, for example, the selection of the type and/or amount of one or more excipients, surfactants and/or optional components can be based on the chemical and functional compatibility with the biopharmaceutical to be formulated and/or the mode of administration and other chemical, functional, physiological and/or medical factors well known in the art. For example, compared to reducing sugars, non-reducing sugars exhibit favorable excipient properties when used with polypeptide biopharmaceuticals. Therefore, exemplary formulations are further illustrated herein with reference to different peptide mimetics. However, the scope of applicability, chemical and physical properties, considerations and methodology applied to polypeptide biopharmaceuticals can be similarly applied to biopharmaceuticals other than polypeptide biopharmaceuticals.

In various embodiments, the pharmaceutical composition may include, 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 include a single therapeutic compound, such as a peptide mimetic, for treating one or more conditions (including but not limited to diseases). In an embodiment, a pharmaceutical composition as described herein may also include, 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 a formulation to treat, for example, a pathological condition and one or more side effects caused by the primary treatment. In another embodiment, a variety of therapeutic compounds may also be included, but not limited to, in a pharmaceutical composition as described herein to achieve different medical purposes, including, for example, simultaneous treatment and monitoring of the progression of a pathological condition. In another embodiment, multiple simultaneous therapies, such as those exemplified herein and other combinations well known in the art, are particularly useful for patient compliance because a single pharmaceutical composition may be sufficient for some or all of the proposed treatments and/or diagnoses. Those skilled in the art will know those therapeutic compounds that can be mixed for a wide range of combination therapies. Similarly, in various embodiments, a first therapeutic compound can be used with a second therapeutic compound or more therapeutic compounds and a combination of one or more therapeutic compounds together 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 include one or more preservatives and/or additives known in the art. Similarly, the pharmaceutical composition may also be formulated into but not limited to any of a variety of known delivery formulations. For example, in embodiments, the pharmaceutical composition may include surfactants, adjuvants, biodegradable polymers, hydrogels, etc., such optional components, their chemical properties and functional properties are known in the art. Similar known in the art is a pharmaceutical composition that promotes rapid release, sustained release or delayed release of bioactive agents after administration. Preparations as described can be produced to include these formulation components or other formulation components known in the art.

Therefore, the pharmaceutical composition can be used as a single dose or as two or more doses (which may or may not contain the same amount of desired molecules) over time, or as a continuous infusion via an implant device or catheter. The further refinement of appropriate dosage is routinely performed by those of ordinary skill in the art, and within the scope of their routinely performed tasks. Appropriate dosage can be determined by using appropriate dose-response data. In various embodiments, the therapeutic compound in the pharmaceutical composition described herein can be, but is not limited to, administered to the patient over an extended period of time, such as for long-term administration of chronic conditions. The composition can be solid, semisolid or aerosol, and the pharmaceutical composition is formulated as a tablet, gel tab, lozenge, oral dissolvable strip, capsule, syrup, oral suspension, emulsion, granule, sprinkle or pellet.

In embodiments, for oral, rectal, vaginal, parenteral, pulmonary, sublingual and/or intranasal delivery formulations, tablets may be prepared by compression or molding, optionally 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., but not limited to, povidone, gelatin, hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., but not limited to, sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), and/or a surfactant or dispersant.

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

In an embodiment, the capsule pharmaceutical composition can utilize hard capsules or soft capsules, including but not limited to gelatin capsules or vegetarian capsules, such as capsules made of hydroxymethyl propyl cellulose (HMPC). In an embodiment, the type of capsule is a gelatin capsule. In an embodiment, as described in detail in Pharmaceutical Capules, Second Supplement, F.Podczeck and B.Jones, 2004, a capsule filling machine such as but not limited to a capsule filling machine available from a commercial supplier such as Miranda International can be used, or capsules are filled using capsule manufacturing techniques known in the industry. In an embodiment, the capsule pharmaceutical composition can be prepared using, but not limited to, a processing center such as Chao Center for Industrial Pharmacy & Contract Manufacturing located at Purdue Research Park.

The packaging and equipment 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 regimen, geopolitical environment (e.g., exposure to extreme temperature conditions in developing countries), and other practical considerations.

Injection devices include pen syringes, automatic syringes, safety syringes, syringe pumps, infusion pumps, glass pre-filled syringes, plastic pre-filled syringes and needleless syringes. The syringe can be pre-filled with a liquid, or can be a dual chamber, such as for use with a lyophilized material. An example of a syringe for such a purpose is Lyo-Ject ^TM , Lyo-Ject ^TM is a dual chamber pre-filled lyophilized syringe available from Vetter GmbH, Ravensburg, Germany. Another example is LyoTip, which is a pre-filled syringe designed to conveniently deliver a lyophilized formulation available from LyoTip, Inc., Camarillo, California, USA. Administration by injection can be, but is not limited to, intravenous, intramuscular, intraperitoneal or subcutaneous, as appropriate. Administration by non-injection routes can be, but is not limited to, nasal, oral, ocular, skin or pulmonary, as appropriate.

In certain embodiments, kit can include but is not limited to one or more single chamber or multi-chamber syringes (for example, liquid syringe and lyophilizing syringe), for applying one or more pharmaceutical compositions described herein.In various embodiments, kit can include the pharmaceutical composition for parenteral, subcutaneous, intramuscular or IV administration, the pharmaceutical composition is sealed in the bottle under partial vacuum in the form of being ready to be loaded into syringe and administered to experimenter.In this regard, pharmaceutical composition can be arranged therein under partial vacuum.In all these embodiments and other embodiments, kit can include one or more bottles according to any embodiment in the aforementioned, wherein each bottle includes a single unit dose for being administered to experimenter.

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

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 dosage of the pharmaceutical composition formulation will depend on the subject's disease or condition and the actual clinical setting.

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

The pharmaceutical compositions as described herein can be used in the manufacture of medicaments and in the treatment of humans and other animals by administration according to conventional procedures.

Also provided herein are combinatorial methods for developing suitable pharmaceutical compositions using combinations 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 the 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 an embodiment, 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.

Example

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 to be examples of a single aspect only. Any functionally equivalent method is within the scope of the present 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 based on the foregoing description and accompanying drawings. Such modifications fall within the scope of the present invention.

Example 1

Treatment with compounds of formula (I) selectively kills pancreatic tumor cells expressing oncogenic KRAS, while having negligible effects in normal cells.

The results shown in Figure 1 illustrate that the compound of formula (I) reduces the interaction of PI3K and c-RAF1 with KRAS. The results shown in Figure 2 illustrate that the compound of formula (I) is able to reduce cell viability with an IC ₅₀ of approximately 20 μM in all tumor cell lines, but only affects less than 5% of normal cells at these concentrations. Therefore, the compound of formula (I) reduces pancreatic tumor cells, but not normal cells.

General preparation process of compounds of formula (I) and similar compounds thereof

Compounds of formula (I) and some analogs thereof (the latter are not disclosed herein) were prepared by solid phase peptide synthesis (SPPS) following the Fmoc/t-Bu strategy. Synthesis was performed at a scale of 100 μmol/time using 2-chlorotrityl chloride resin as a solid polymer support. Synthesis was performed manually in a polypropylene syringe with a porous disk at the bottom. While growing the peptide mimetic chain, intermittent manual stirring was performed to ensure proper mixing of the reagents. Solvents and soluble reagents were removed by aspiration. The extent of the amino acid coupling reaction was monitored using the Kaiser test (primary amines) (see E. Kaiser et al.; "Color test for detection of freeterminal amino groups in the solid-phase synthesis of peptides"; Anal. Biochem. 1970; Vol. 34; pp. 595-598) or the tetrachlorobenzoquinone test (secondary amines) (see T. Vojkovsky; "Detection of secondary amines on solid phase"; Pept. Res. 1995; Vol. 8; pp. 236-237). In those cases where the coupling was not completely completed, a re-coupling step was performed using standard coupling conditions. Selective N-alkylation of the backbone of the compounds 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; Vol. 119; pp. 2301-2302).

2-Chlorotrityl chloride resin was placed in a syringe (reaction vessel) equipped with a polyethylene fritted disk. The resin was swelled by washing with dichloromethane (DCM) and dimethylformamide (DMF).

After swelling and preparing the resin, the protected form of the amino acid of the first Fmoc protection of the peptide mimetic to be synthesized is attached to the resin through its carboxylic acid moiety using N,N-diisopropylethylamine (DIEA) in DMF as a coupling agent. In order to couple, 0.6 equivalents of protected amino acid are mixed with a few drops of DCM and added to the resin. Subsequently, 5 equivalents of DIEA are added twice, first adding 1/3 part, and the remaining 2/3 part is added after 10min. The reaction is allowed to continue for 50min. Afterwards, the unreacted active sites of the polymer support are capped by pouring methanol (1mL/g polymer support) into the reaction mixture. Ten minutes later, the solvent and unreacted reagent are removed by suction. Next, the Fmoc group is removed by treating the resin with 20% piperidine in DMF. Washing liquid is collected and measured by UV spectroscopy to quantify the load of the first amino acid in the polymer support.

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

Amino acid N-alkylation was performed on-resin. The on-resin process used for N-methylation of amino acids was the following 3-step method (these steps were 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 using β-mercaptoethanol and 1,8-diazabicyclo[5.4.0]undec-7-ene.

After the peptide sequence was completed, the resulting peptide (which still remained anchored to the polymer support) was washed with DCM (5×1 min) and dried by suction. The peptide was then cleaved from the resin using 5% trifluoroacetic acid (TFA) in DCM. The process washes and DCM washes (5×1 min) were collected and combined to obtain the cleaved peptide 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 a mixture of DCM, pyrrolidine, 1-hydroxy-7-azabenzotriazole (HOAt) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·Cl) in the smallest possible volume. After the C-terminal capping was completed, the solvent was removed under vacuum and then it was diluted again with a solution of ACN:H ₂ O 50:50 and lyophilized.

The side chain protecting groups were removed by applying a mixture of TFA 95%: triisopropylsilane (TIS) 2.5%: H ₂ O 2.5% for 1.5 h. All filtrates were pooled and TFA was evaporated under a N ₂ flow stream. The crude 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. The 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 were: Fmoc-Bip-OH, Fmoc-Nme-β-Ala-OH, Fmoc-Dap(Boc)-OH and Fmoc-Leu-OH.

Anchoring of the first amino acid : Before adding the first amino acid, the 2-chlorotrityl chloride resin was first washed (5×1 min DCM, 5×1 min DMF and 1×5 min DMC). Then, 0.6 equivalents of the amino acid were mixed with a few drops of DCM and added to the resin. Subsequently, 5 equivalents of DIEA were added in 2 portions, first 1/3 portion, which was allowed to react for 10 min, and then the same steps were repeated with the remaining 2/3 portion for a reaction time of 50 min. The unreacted active sites of the resin were capped using methanol (1 mL/g polymer support). The reaction mixture was then removed by suction and the polymer support was washed with DMF (5×1 min). Next, the Fmoc group was removed by treating the resin with 20% piperidine in DMF (4 mL/g resin, 2×1 min and 1×10 min). The washings were collected and measured by UV spectroscopy to determine the loading capacity of the first amino acid.

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

Selective N-methylation of the diaminopropionic acid moiety : The selective N-methylation was performed as follows:

a) Protection and activation of the amino group with o-NBS: 4 equivalents of o-NBS, 3 equivalents of 2,3,5-trimethylpyridine and a few drops of DMF, performed twice (30 min and 20 min).

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

c) o-NBS removal: 10 equivalents of β-mercaptoethanol, 5 equivalents of 1,8-diazabicyclo[5.4.0]undec-7-ene and a few drops of DMF, performed twice (10 min and 40 min).

Cleavage of peptide from polymer support : The peptide-resin was treated with 5% TFA in DCM (3×15 min, 6 mL). The cleavage mixture and subsequent DCM washes (5×1 min) were collected and combined to obtain the cleaved peptide from the resin. Then, the solvent from the collected mixture was 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 allowed the retention of the protective side chain groups of the peptide.

C-terminal capping : The crude lyophilizate 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 for 3 h at room temperature under constant stirring. The mixture was then washed with saturated solutions of NaHCO ₃ , NH ₄ Cl and NaCl (three times each). The organic layer was collected and dried under vacuum. It was then diluted with a mixture of ACN:H ₂ O (50:50) and lyophilized.

Removal of side chain protecting groups : Side chain protecting groups were removed by acidic mixture treatment (1.5 h) using TFA 95%:TIS 2.5%:H ₂ O 2.5%. The cleavage mixture was evaporated using a stream of N _2. The crude was then diluted with a mixture of ACN:H ₂ O (50:50) and lyophilized.

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

Characterization of peptides : The identity of the peptides 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 (2.1×100 mm×3.5 μm, Waters) and Masslynx software. Flow rate: 0.3 ml/min, mobile phase: H ₂ O (0.1% formic acid) and ACN (0.1% formic acid). UV detection: 220 nm. 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 (100×4.6 mm×5 μm, Waters) and Empower software. Flow rate: 1.6 mL/min, mobile phase: H ₂ O (0.1% TFA) and ACN (0.1% TFA). Detection was performed at 220 nm.

-Molecular formula: C ₄₇ H ₅₈ N ₆ O ₅

- Calculated mass: 787.00 g/mol

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

- Gradient and retention time (min): gradient 0-100% B in 3 min (A = 0.1% TFA in _H2O , B = 0.1% TFA in ACN), 3.1 min; gradient 40-100% B in 20 min (A = 0.1% TFA in _H2O , B = 0.1% TFA in ACN), 1.9 min.

-Purity: 95%.

Cell lines and culture

hTERT-RPE (immortalized retinal pigment epithelial human cells) and HeLa (epithelioid cervical carcinoma cells) both express RAS wild type and were obtained from the American Tissue and Cell Collection (ATCC). MPanc-96, HPAF-II, PA-TU-8902, SW1990, PA-TU 8988T and PANC-1 PDAC cell lines (obtained as described in: C. Barcelo et al.: "Ribonucleoprotein HNRNPA2B1 interacts with and regulates oncogenic KRAS in pancreatic ductaladenocarcinoma cells"; Gastroenterology 2014 Oct;147(4):882-892.e8.doi:10.1053/j.gastro.2014.06.041. Epub 2014 Jul 3. PMID:24998203), all express oncogenic mutant KRASG12V.

HeLa cells and PDAC cells were grown in Dulbecco's modified Eagle's medium (DMEM), and hTERT-RPE were grown in DMEM-HAM's F12 (1:1) medium, both of which were supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel), penicillin, streptomycin, and non-essential amino acids.

Drug treatment and EGF-dependent signaling and activation

Cells were seeded in medium containing 10% FBS for 24 h and serum starved for the next 24 h. Then, they were incubated with different concentrations of compounds for 2 h. Continuous treatment with EGF (50 ng/mL; Sigma-Aldrich) was performed for 10 min to activate cell signaling.

Cell transfection and plasmids

HeLa cells were transfected with pEF-HA-KRASG12V plasmid obtained 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 Apr 18; 283(16): 10621-31. doi: 10.1074/jbc.M706238200. Epub 2008 Jan 8. PMID: 18182391). 2000 transfection reagent (Invitrogen) was used as the transfection method.

SDS-PAGE, Western blotting, and antibodies

Proteins were resolved by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore). Non-specific binding of antibodies was assessed by incubating the membranes with a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20 and 5% bovine serum albumin for 1 h at room temperature. Protein expression was determined by probing the blots overnight at 4°C with the following specific antibodies: anti-c-RAF (BD Transduction 610151, 1:500); anti-phospho-c-RAF S338 (Cell Signaling 9427, 1:500); anti-PI3K p110α (Cell Signaling 4249, 1:1000); anti-AKT (Cell Signaling 9272, 1:1000); anti-phospho-AKT S473 (Cell Signaling 4060, 1:1000); anti-phospho-AKT Thr308 (Cell Signaling 4056, 1:1000); anti-p44/42 MAPK (ERK1/2) (Cell Signaling 9102, 1:2000); anti-phospho-p44/42 MAPK (ERK1/2) T202/Y204 (Cell Signaling 4370, 1:2000); anti-GAP120 (Santa Cruz SC-63, 1:200); anti-HA (Sigma-Aldrich H6908, 1:1000); or anti-α-tubulin (Sigma-Aldrich T9026, 1:2000). Next, after washing the membranes, they were incubated with the corresponding HRP-conjugated secondary antibodies (goat anti-rabbit BioRad 170-6515 or goat anti-mouse BioRad 170-6516, 1:3000) at room temperature for 60 min and washed again. Protein detection was performed by enhanced chemiluminescence (EZ-ECL, Biological Industries). The emitted light was titled and quantified (ChemiDoc, BioRad).

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

Co-immunoprecipitation (Co-IP)

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

Cell viability assay

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

Example 2

We conducted a preliminary evaluation of the effector binding sites on the surface of 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 sites (effectors after RAS activation) (see Figure 3). Effectors include RAF, RAL and PI3K. This was done using computational methods as described below.

Computational hit identification

Computational methods were applied to provide potent and permeable peptide mimetics as drug candidates. Our approach was to design peptide mimetics that would bind to the RAS effector binding site, blocking the possibility of RAS to interact with the effector protein, therefore reducing its tumor activity.

Identification of RAS-effector binding sites was performed by structural comparison of GTPase-RAS in complex with several effector proteins such as phosphoinositide 3-kinase (PI3K), Bry2RBD, RalGDS, phospholipase C, NORE1A and RAF and applying computational standard protocols.

A total of one aromatic residue and three negatively charged residues (i.e., Asp33, Glu37, Asp38, and Tyr64) were identified on the RAS interface with RAS effector proteins. We identified several residues on the surface of RAS proteins, namely, Asp33, Glu37, Asp38, and Tyr64, that 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 (Figure 4, Table 4), which facilitates interactions with possible binders.

Based on the results obtained from both the analysis of the RAS protein crystal structure and the MD simulation, Asp33 residue and Asp38 residue were selected as substrate binding sites because they were identified by both analyses. These data were then used for virtual screening of putative RAS inhibitors to adjust the composition of the peptide mimetic library used as a ligand (such as in the hit identification step) and to set the position and size of the docking box (docking box) (in both the hit identification step and the hit optimization step).

We created an initial library with more than 80,000 tripeptides and tetrapeptides, which were formed by both natural and unnatural amino acids. The tripeptides and tetrapeptides produced each contained at least one positively charged residue. We used the SMINA docking program to screen for peptide mimetics. This was achieved by using the K-RAS GTPase crystal structure as a receptor (PDB 5P21) and setting the binding site around negatively charged hotspot residues on the surface of the K-RAS GTPase.

Materials and methods

Synthesis of peptide mimetics

All compounds were synthesized by solid phase peptide synthesis (SPPS) following the Fmoc/tBu strategy. Syntheses were performed at 100 μmol/time using 2-chlorotrityl chloride resin (except P1.1, which used H-Rink amide chemmatrix). Syntheses were performed manually in polypropylene syringes equipped with a porous disk at the bottom. Intermittent manual stirring was performed while the peptide chain was growing to ensure proper mixing of the reagents. Solvents and soluble reagents were removed by aspiration. Amino acid coupling (L-, D- or non-natural) was performed using 4 equivalents of Fmoc-protected amino acids, 4 equivalents of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU) and 8 equivalents of N,N-diisopropylethylamine (DIEA) in dimethylformamide (DMF) (1×75 min). The extent of the reaction was monitored using the Kaiser test (primary amines) or the tetrachlorobenzoquinone test (secondary amines). In those cases where coupling was not fully completed, a re-coupling step was performed using standard coupling conditions.The Fmoc group was removed from the amino acid (after successful completion of the coupling reaction) using a mixture of 20% piperidine in DMF (2 x 1 min and 1 x 10 min).

Selective N-alkylation of the backbone of the compounds was performed using the method described by Miller et al., which is divided into the following three steps (these steps are performed 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, 3 equivalents of 2,3,5-trimethylpyridine in DMF (1×30 min and 2×20 min), 2) Deprotonation and N-alkylation: 3 equivalents of 1,8-diazabicyclo[5.4.0]undec-7-ene in DMF were added to the resin (5 min), followed by 10 equivalents of the desired alkyl sulfate (10 min). This treatment was repeated twice. 3) o-NBS removal: Two treatments were performed with a mixture of 10 equivalents of β-mercaptoethanol and 5 equivalents of 1,8-diazabicyclo[5.4.0]undec-7-ene in DMF (1×10 min and 1×40 min).

Mixed-solvent molecular dynamics (MixMD) simulations of RAS GTPase proteins

The crystal structure of the RAS GTPase protein (PDB code 5P21) was downloaded from the RCSB PDB database (https://www.rcsb.org/) and used as the input structure to perform a 50-ns long explicit mixed solvent molecular dynamics (MixMD) simulation.

In a first preliminary step, the cocrystallized GppNHp molecules were replaced by the GTP native cofactor, whose parameter file was downloaded from the AMBER parameter database (http://research.bmh.manchester.ac.uk/bryce/amber/). Thus, the entire system was appropriately protonated and placed in a periodic cubic mixed solvent box containing benzene, propane, ethanol, propionic acid and ethylamine organic probes in appropriate combinations with TIP3P water molecules (the minimum distance between the protein and the box edge was set to ).

The Leap module of AmberTool16 was used and the overall protonation, solvation and parameterization of the system was performed using the ff12 AMBER force field.

As detailed below, two-step conjugate gradient-based minimizations were run, followed by four steps of equilibration and 50-ns-long MD simulations using the NAMD simulation package. ⁵⁷ The SHAKE algorithm and the Particle Mesh Ewald (PMD) method were applied in all simulations (to suppress all bonds to hydrogen atoms and calculate long-range Coulomb interactions, respectively). A time step of 2 fs and The cut-off distance for long-range interactions.

Therefore, the solvated system was first relaxed using a 500-cycle long unrestricted minimization step, followed by another 5000 cycles, during which the harmonic suppression was only The protein was then equilibrated in a four-step protocol, where the system was gradually heated from 0 K to 300 K using the Langevin dynamic model and the initial position constraints were regularly relaxed. A 100-ps long MD simulation was performed with the main-chain atoms suppressed by a harmonic potential of 200 K and the temperature increased from 100 K to 300 K. Then, NVT conditions were used and The backbone atoms were suppressed by a harmonic potential and a 120-ps long heating phase was run from 300 K to 600 K. NVT was then used and The system was cooled from 600 K to 300 K for 120 ps using the harmonic potential of . Finally, the NPT conditions (i.e., constant number of molecules (N), pressure (P), and temperature (T)) were used and the The harmonic potentials were used to suppress the backbone atoms in 100-ps long simulations at 300 K.

After equilibration, the The minor harmonic suppression is performed by running a 50-ns long simulation at 300 K under NVT conditions.

Finally, based on the distribution of organic probes during the trajectory, the trajectory was used to identify protein surface regions with high ligand binding propensity, since the frequency with which probes occupy a given region should be proportional to their binding affinity to that particular region. Therefore, using the cpptraj module of AmberTools16, the positions along the last 25ns of the simulation for each organic probe were integrated into the probe-occupancy map and finally visualized as isosurfaces corresponding to the regions most frequently sampled by each organic probe.

Virtual screening of putative RAS inhibitors

Using an extended computational investigation of the RAS interface, we were able to generate a set of peptide mimetics that can bind to the target binding site with a theoretical potency greater than -8.0 kcal/mol (μM-nM expected experimental inhibitory potency range scale) while exhibiting a good in silico permeability profile with an average polar accessible surface area (PASA) below These parameters represent the potential activity and permeability of the designed peptide mimetics.

To exclude the possibility of identifying false negatives for docking, we performed short implicit peptide binding site MD simulations to identify peptide mimetics that exhibited stable binding modes (simulation RMSD less than ). This approach enriched the selection of compounds capable of binding to KRAS-GTPase based on thermodynamic points while retaining the in silico properties and peptide structure of the binding target region.

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

In the first molecular docking experiment (hit identification step), a data set of tripeptide mimetics and tetrapeptide mimetics was constructed using natural and unnatural amino acids. The N-terminal and C-terminal parts of the compounds were enriched with different capping parts with different sizes and polarity profiles (such as diphenylacetic acid, 2-(4-tert-butylphenoxy)acetic acid, phenylacetic acid, 9-anthracene acid or benzoic acid for the N-terminal part; and formamide, pyrrolidine, pyridine or 3-azaspiro [5.5] undecane for the C-terminal part). Based on the results of the substrate-binding site analysis, only compounds with a positive single charge 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 molecular docking experiments. The three-dimensional structures of all putative RAS protein inhibitors were created from scratch starting from the primary sequence, parameterized using the AmberTool16Leap module and the ff12 AMBER force field, and finally minimized using the NAMD simulation package. The parameter libraries of non-natural peptide building blocks (if any) and capping residues were compiled using the AmberTool16 modules Antechamber and Leap.

The protein structure of RAS GTPase (PDB code 5P21) The resolution crystal structure was used as the receptor for docking all putative RAS inhibitors. In the first preliminary step, the co-crystallized GppNHp and water molecules were removed. Therefore, hydrogen atoms were added and the entire system was appropriately protonated using the H++web server ( http://biophysics.cs.vt.edu/H++ ) and default parameters.

All docking calculations were performed using SMINA, an energy minimization optimization fork of the AutoDock Vina docking program ⁶¹ . All ligand and receptor structures were converted into input files suitable for SMINA using the prepare_ligand4.py script and prepare_receptor4.py script provided by AutoDock Tools. A grid of 60 × 60 × 60 with a grid spacing of 100 was adjusted in the RAS effector binding region. The almost cubic grid boxes were centered on residues Asp33 and Asp38. The exhaustiveness, number of patterns, and energy range were set to 32, 100, and 50, respectively.

After all docking simulations have been completed, for the 20 top-ranked docking poses, the phi/psi dihedral angle Ramachandran distribution of all building blocks, the geometry of the corresponding peptide bond and intermolecular contact are calculated. Therefore, docking conformations that are incompatible with the phi/psi dihedral angle Ramachandran distribution, with non-planar or cis-peptide bonds or with any intramolecular contact are filtered out. Therefore, the top-ranked docking poses (if any) of each compound are merged together and sorted according to the SMINA docking energy, filtering out all compounds with docking energies lower than -8kcal/mol. Then, apply binding stability and predicted cell membrane permeability filters (see below for details), and then, select the most promising compounds and undergo visual inspection.

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

RAS binding stability assessment

The docking model of each compound in complex with the RAS protein was subjected to conjugate gradient minimization, equilibration, and 3-ns long implicit solvent MD simulations using the NAMD simulation package. Therefore, the first preliminary step, namely the global protonation and parameterization of the system, was performed using the Leap module of AmberTool16 and the ff12 AMBER force field. Then, the system was minimized with a 1000-cycle length to The harmonic suppression is applied to all backbone atoms (both receptor and ligand) of the system by a force constant of 1.5 Å. Then, a 200-ps long equilibration step is performed by gradually heating the system to 300 K and heating it at 5 and Harmonic restraint was applied to the backbone atoms of the protein and ligand using force constants of . Finally, the 3-ns long MD simulation was performed by Harmonic suppression was applied only to the backbone atoms of the receptor protein with a force constant of 1.5 Å. In all simulations, SHAKE ⁵⁸ was applied to suppress all bonds to hydrogen atoms, and a 2-fs simulation time step and The cut-off distance for long-range interactions.

Finally, binding stability assessment was performed using MolSoft ICM Browser ( www.molsoft.com ) to calculate the average root-mean-square deviation (LigRMSDavg) for each compound along the last 1.5 ns of the trajectory ⁶³ . LigRMSDavg values below The compounds were predicted to be stable binders and were therefore selected for cell membrane permeability evaluation.

Computer permeability prediction

In a first preliminary step, the 3D structure of each compound was created from scratch and parameterized using the AmberTool16 Leap module and the ff12 AMBER force field.

For each compound, a set of 25 2-ns long implicit chloroform solvation unrestricted molecular dynamics (MD) simulations were performed using the ff12 AMBER force field and NAMD simulation packages. Each of the previously generated 3D structures was first used to obtain a small conformational ensemble containing a total of 25 conformers. Therefore, each compound was relaxed by a short 1000-step unrestricted energy minimization, and then a short MD simulation lasting 100 ps was performed, with the system temperature set to 300 K. Finally, 25 conformers were obtained by extracting a trajectory snapshot every 4 ps and used as input for the final chloroform solvation MD simulation.

Therefore, in each simulation, the system was first energy minimized by 5000 conjugate gradient steps. Then, the system underwent an equilibrium process divided into four steps, gradually heating from 0 K to 300 K for 100 ps, and A force constant of was used to apply harmonic restraint to all heavy atoms in order to preserve the initial molecular geometry during heating. During equilibration, the integration time step was set to 2 fs and the nonbonding cutoff distance was set to Finally, a production step consisting of conducting unrestrained MD simulations lasting 2 ns and setting the system temperature to 300 K was run. For each compound, all 25 2-ns-long MD trajectories were combined together, and a total of 12,500 MD frames were extracted using the cpptraj module of AmberTools16.

Finally, for each compound, the average exposure of polar atoms to the solvent during the overall simulation was extracted based on the average polar accessible solvent area value (polASA _avg ) over the overall MD frame using MolSoft ICM Browser. polASA _avg values below The peptides were predicted to be permeable by passive diffusion and were therefore selected for visual inspection.

Cell lines and culture

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

hTERT-RPE were cultured in DMEM-HAM's F12 (1:1) medium, both of which were supplemented with 10% fetal bovine serum (Biological Industries, Israel), penicillin, streptomycin, and non-essential amino acids.

Drug treatment and EGF-dependent signaling activation

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

SDS-PAGE, Western blotting, and antibodies

Proteins were resolved by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore). Nonspecific binding of antibodies was assessed by incubating the membranes with a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM 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°C with the following specific antibodies: anti-c-RAF (BD Transduction 610151, 1:500); anti-phospho-c-RAF S338 (Cell Signaling 9427, 1:500); anti-PI3K p110α (Cell Signaling 4249, 1:1000); anti-AKT (Cell Signaling 9272, 1:1000); anti-phospho-AKT S473 (Cell Signaling 4060, 1:1000); anti-phospho-AKT Thr308 (Cell Signaling 4056, 1:1000); anti-p44/42 MAPK (ERK1/2) (Cell Signaling 9102, 1:2000); anti-phospho-p44/42 MAPK (ERK1/2) T202/Y204 (Cell Signaling 4370, 1:2000); anti-GAP120 (Santa Cruz SC-63, 1:200); anti-HA (Sigma-Aldrich H6908, 1:1000); or anti-α-tubulin (Sigma-Aldrich T9026, 1:2000). Next, after washing the membranes, they were incubated with the corresponding HRP-conjugated secondary antibodies (goat anti-rabbit BioRad 170-6515 or goat anti-mouse BioRad 170-6516, 1:3000) at room temperature for 60 minutes and washed again. Protein detection was performed by enhanced chemiluminescence (EZ-ECL, Biological Industries). The emitted light was titled and quantified (ChemiDoc, BioRad).

To analyze RAS signaling, cells were lysed in a buffer containing 67 mM Tris-HCl pH 6.8 and 2% SDS, and the samples were then heated at 97°C 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 a final visual inspection, nine peptide mimetic sequences were selected for synthesis as listed in Table 2. The nine sequences included tripeptide mimetics and tetrapeptide mimetics. All of these sequences had a secondary amine cap at the C-terminus and a hydrophobic group cap at the N-terminus. The permeability threshold was set to All peptide mimetics below this threshold were predicted to be permeable.

Table 2

Table 2 Experimental evaluation of the chemical structures

We evaluate the tripeptide mimetics and tetrapeptide mimetics of Table 2 by testing the ability of peptide mimetics to inhibit RAS-GTP signaling proteins in hTERT-RPE cells. To complete this work, we seeded cells in culture plates for 48 hours. Cell serum starvation (0.5% FCS) lasts for 24 hours. After this period, we added EGF to the cells at a concentration of 50ng/mL for a period of 10 minutes. EGF is added to the culture to activate the RAS signaling pathway. In order to evaluate the activity of peptide mimetics, 2 hours before adding EGF, we added a tripeptide mimetics or tetrapeptide mimetics to the culture plates at a concentration of (50 μM) so that each peptide mimetics was tested in the culture plates. We ran Western blot (WB) to detect the activation levels of two Ras signaling pathways (Raf/ERK and PI3K/AKT). GAP120 detection was used as a control.

Figure 5 shows a Western blot in which we evaluated the potency of the 9 peptide mimetics of Table 2 to inhibit RAS effectors. GTPase activating protein (GAP120) was used as a control.

In order for a peptide mimetic 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 performing the analysis, we found that compounds IP-14-05 and IP-14-06 were insoluble when diluted in cell culture medium. We also found that peptide IP-14-04 formed aggregates and precipitated on cells, causing cell death. Therefore, we did not include IP-14-05 and IP-14-06 in WB (Figure 5).

The result is that we have only evaluated 6 kinds of peptide mimics in the first 9 kinds of peptide mimics in RPE cells in the end.From the 6 kinds of peptide mimics we evaluated, we determined that 3 kinds of peptide mimics, especially, IP-14-01, IP-14-03 and IP-14-08 can suppress two kinds of RAS-effector protein cascades.In these 3 kinds of peptide mimics, we found that when analyzed by WB, IP-14-01 was the most effective inhibitor (Fig. 5).

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

Table 3

Table 3 shows the results of PAMPA assays (Pe, % transport and % retention) for 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 peptide mimics. 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 water, and three compounds (IP-14-01, IP-14-03 and IP-14-08) showed good solubility, providing that the experiment can be carried out at a concentration of>100 μM without the guarantee of the risk of precipitation from solution of any of the three therapeutic compounds. The result of PAMPA determination shows the high retention %, negligible transport and zero permeability Pe of each peptide mimetic in the three peptide mimics. We found that when IP-14-01 was incubated with SH-SY5Y cells for 2h at 60 μM, 13.5% of the peptide mimics were absorbed 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 re-evaluate those peptide mimics from the first generation, which were insoluble under in vitro assay conditions, or formed aggregates as described above when incubated with RPE cells. For these peptide mimics, their solubility in PBS with 15% beta-cyclodextrin was studied (Table 5).

Table 4

Compound Code Concentration in PBS β-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 peptide mimetics were diluted and soluble at 1 mM in PBS (phosphate-saline buffer) with 15% β-cyclodextrin. We then placed these peptide mimetics under constant stirring for 24 hours. At the end of this time, we centrifuged each solution and then ran the supernatant from each solution by HPLC and compared the results to a 1 mM solution of each compound in ACN/H ₂ O. This control was used to determine the actual solubility in PBS with 15% β-cyclodextrin. The data are expressed as mean ± SD.

Figure 6 shows a RAS Western blot that 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 dissolve the GTPase activating protein (GAP120) in the cell culture medium at a diluted concentration of 0.5%.

Due to its inhibitory effect on the RAS effector signaling cascade together with its high cellular internalization value, we selected the therapeutic compound IP-14-01 for optimization and the second round of computational approaches.

Example 3

Evaluation of chemical structure derivatives of IP-14-01

The second generation of peptidomimetics derived from IP-14-01 were designed following the same in silico protocol applied previously. In this regard, we prepared four new peptidomimetics, which we eluted after the completion of the computational studies.

In the second molecular docking experiment (hit optimization step), new peptide mimetic data sets were generated starting from the P1 primary sequence by combining the original building blocks with specific and ad hoc alternative moieties carefully selected in order to increase the membrane permeability and/or receptor binding affinity of the original compounds.

We designed IPR-471 to increase the number of amino acids on the backbone of the peptidomimetic. Based on this, the C-terminus was extended by removing the secondary amine that had been used to cap the proline with carboxamide.

IPR-472 has a nearly identical structure compared to IP-14-01, but the N-methyl alkylation of the β-alanine is replaced with a longer carbon chain attached to an aromatic group, propylbenzene. We made this change in order to increase the overall compound hydrophobicity along with the increase in the shielding ability of the n-alkyl group. We believe that the four-carbon chain instead of the methyl group will provide a degree of flexibility that will allow the six-carbon aromatic ring to wrap around the molecule. This will reduce the polarity of IPR-472 in an aqueous environment. We anticipate that this will increase the number of contacts with the protein surface.

IPR-473 was designed to be the most conservative of the IP-14-01-derived peptidomimetics, as the only changes were the substitution of isoleucine for alanine and the addition of an N-methylation to one of the amide bonds in the backbone of the sequence. This new molecule was expected to retain the binding mode of the parent compound exactly, but add a few more contacts in order to slightly optimize its potency.

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

Finally, we designed these peptide mimetics to maintain the same sequence ends by maintaining the same three amino acids in the same order as well as the diphenyl N-terminus (Table 6). These new peptide mimetics are identified in Table 5.

Table 5

Table 4 shows that applying the same docking approach previously used to screen the first round of peptide mimetics, four new peptide mimetics of the second generation were generated.

Fig. 7 is the RAS signal transduction protein blotting from our evaluation of IP-14-01 (P1) and its derived peptide mimics IPR-471 (P1.1.), IPR-472 (P1.2), IPR-473 (P1.3) and IPR-474 (P1.4).For this experiment, we used the same scheme and conditions (Fig. 5) as previously described WB assay disclosed herein.More particularly, we incubated each peptide mimetic compound in culture with 50 μM concentration dissolved in 0.5% DMSO together with serum-starved hTERT-RPE cells for 2 hours, and then treated with EGF (50 ng/ml) for 10 min.None of these therapeutic compounds showed any solubility problems.

As shown in Figure 7, we found that IPR-471 (P1.1) and IPR-474 (P1.4) were no 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) was able to inhibit all two different protein cascades (RAF/ERK and PI3K/AKT) even more effectively than IP-14-01.

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

Next, we incubated the IP-14-01 of several concentrations with different cancer cell lines and normal cell lines (hTERT-RPE cells). Cell viability was measured by MTS cell proliferation assay. Parental peptide mimics IP-14-01 was subjected to the same experiment. At any incubation concentration, there was no difference in the ability of IP-14-01 to kill normal cells and cancer cells, i.e., parental peptide mimics did not show cell line specificity (data not shown).

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

The different cell lines used include human pancreatic cancer cell MPANC-96, human pancreatic adenocarcinoma cell HPAF-II, human pancreatic II grade 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 cell, and is non-cancerous.

The results obtained in the cell viability assay as shown in Figure 8 confirm the potential therapeutic activity of IPR-473. The therapeutic compound is cytotoxic to cancerous cells at concentrations above 15 μM. At the same time, it has no effect on hTERT-RPE control cells. Therefore, we found that IPR-473 exhibits high specificity for pancreatic cancer cell lines in cell proliferation assays.

Certain embodiments of the present invention are described herein, including the best modes known to the inventor for carrying out the present invention. Of course, after reading the foregoing description, the variation of the embodiments described will become apparent to those of ordinary skill in the art. The inventor anticipates that the technician will adopt such variation as appropriate, and the inventor contemplates that the present invention is implemented in a manner different from that clearly described herein in addition. Therefore, the present invention includes all modifications and equivalents of the subject matter set forth in the appended claims herein permitted by applicable law. In addition, the above-mentioned embodiments are encompassed by the present invention in any combination of all possible variations thereof, unless otherwise specified herein or otherwise clearly contradictory to the context.

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

Unless otherwise indicated, all numerals used in this specification and claims to represent features, items, quantities, parameters, properties, terms, etc. should be understood to be modified by the term "about" in all cases. As used herein, the term "about" means that such qualified features, items, quantities, parameters, properties, or terms include the range of 10% above and below the value of the stated features, items, quantities, parameters, properties, or terms. Therefore, unless otherwise indicated, the numerical parameters listed in this specification and the attached claims are variable approximate values. At least, rather than attempting to limit the application of the equivalent principle to the scope of the claims, each digital indication should be interpreted at least according to the reported significant digits and by applying the usual approximation technique. Although the numerical ranges and values of the wide range of the present invention are listed as approximate values, the numerical ranges and values listed in the specific embodiments are reported as accurately as possible. However, any numerical range or value inherently contains a certain error, which is necessarily generated by the standard deviation present in its respective test measurement value. The enumeration of the numerical range of values herein is intended to be used as a shorthand method for individually referring to each individual numerical value falling into the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the specification as if it were individually recited herein.

The terms "a", "an", "the" and similar referents used in the context of describing the present invention (especially in the context of the following claims) should be interpreted as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as") are intended only to better illustrate the present invention rather than to impose limitations on the scope of the invention otherwise claimed. The language in this specification should not be interpreted as indicating that any unclaimed element is essential to the practice of the present invention.

Specific embodiments disclosed herein may be further limited in the claims using the language consisting of or consisting essentially of. When used in the claims, whether as filed or added per amendment, the transition term "consisting of" excludes any elements, steps, or ingredients not specified in the claim. The transition term "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the claimed invention are inherently or explicitly described and enabled herein.

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 individually or in combination with other group members. It is expected that, for convenience and/or patentability reasons, one or more members of a group may be included in the group or deleted from the group. When any such inclusion or deletion occurs, this specification is deemed to include the group as modified, thereby achieving the written description of all Markush groups used in the appended claims.

All patents, patent publications and other publications that quote and identify in this specification are individually and clearly incorporated herein by reference with their entirety for the purpose of description and disclosure, for example, compositions and methodology described in such publications that can be used in connection with the present invention. These publications are provided only for the disclosures prior to the filing date of the present application. In this respect, any content should not be interpreted as the inventor being unqualified by prior invention or for any other reason prior to the recognition of such disclosures. About date or statement, all representations about the content of these files are based on information available to the applicant, and do not constitute any recognition about the date of these files or the correctness of the content.

Finally, it should be understood that although aspects of this specification are emphasized by reference to specific embodiments, those skilled in the art will readily appreciate that these disclosed embodiments are merely illustrations of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is by no means limited to the specific methodology, schemes and/or reagents, etc. described herein. Therefore, various modifications or changes to the disclosed subject matter, or alternative configurations of the disclosed subject matter may be made according to the teachings of this article without departing from the spirit of this specification. Finally, the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the invention, which is solely defined by the claims. Therefore, the present invention is not limited to the precise content as shown and described.

Claims (7)

1. A compound of formula (I) and a pharmaceutically acceptable salt thereof, 2. The compound of formula (I) according to claim 1 has the following chemical name: (S)-N-(3-(((S)-3-amino-1-((S)-4-methyl-1-oxo-1-(pyrrolidin-1-yl)pentan-2-ylamino)-1-oxopropyl-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 treating 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).