US 2015O1 19330A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0119330 A1 McGee et al. (43) Pub. Date: Apr. 30, 2015

(54) SUBSTRATES AND INHIBITORS OF PROLYL Publication Classification OLGOPEPTIDASE AND FIBROBLAST ACTIVATION PROTEIN AND METHODS OF (51) Int. Cl. USE C07K 7/06 (2006.01) (71) Applicant: The Board of Regents of the University C07K5/II (2006.01) of Oklahoma, Norman, OK (US) C07K5/09 (2006.01) (52) U.S. Cl. (72) Inventors: Patrick A. McGee, Oklahoma City, OK CPC ...... C07K 7/06 (2013.01); C07K5/0817 (US); Kenneth W. Jackson, Edmond, OK (US); Victoria J. Christiansen, (2013.01); C07K5/1019 (2013.01) Oklahoma City, OK (US) (21) Appl. No.: 14/398,886 (57) ABSTRACT (22) PCT Filed: May 3, 2013 Inhibitors of fibroblast activation protein alpha (FAP) and (86). PCT No.: PCT/US2013/0395.43 Prolyl Oligopeptidase (POP) are disclosed, along with their S371 (c)(1), use in various therapies related to conditions, diseases, and (2) Date: Nov. 4, 2014 disorders involving abnormal cell proliferation Such as malig nancies and angiogenesis, and in neural disorders such as Related U.S. Application Data Alzheimer's disease. Stalk portions of the inhibitor mol (60) Provisional application No. 61/643,001, filed on May ecules, and substrates of FAP and POP, are also disclosed and 4, 2012, provisional application No. 61/793,183, filed may be used, for example, in screening methods for identify on Mar. 15, 2013. ing Such inhibitors. US 201S/O119330 A1

Iaun3]+. ;0zzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzz…

Patent Application Publication Apr. 30, 2015 Sheet 2 of 11 US 201S/O119330 A1

§§§§§§§§§§§ §§§§§§§§§§§§)§§§§§3

§§§§§§§§§§¶¡¿$¢$$$$$$§§§§§

$$$

§28,

$$¢£€$£§§§§§§§§

§§§§§§§§§§§§§§§§§§ ? §§?ae,

§ § §§§§§§§§§§§§§§§§§§§§§??$$§§§§§§§§§§§§ $

§§§§§§§§§§§§§§§ a. *.) se §§§§§§§§§§§§§§§§§§§§§§§ {{}} §§§§§§§§§§§§§§§§§§§§§§§§§§§ {{}{} Patent Application Publication Apr. 30, 2015 Sheet 3 of 11 US 201S/O119330 A1

(suu)au?L

-000Z •009 009Z 009|| 000|| Se30 ISU W. Patent Application Publication Apr. 30, 2015 Sheet 4 of 11 US 201S/O119330 A1

SOOH §§§§§§§***

?Odlae------****

O

Seo O WSU CC n Patent Application Publication Apr. 30, 2015 Sheet 5 of 11 US 201S/O119330 A1

a. f ; CD : : 3rt a Sp

e A------1 ico S. s - : dip -Sea & . E Y. r a a s:----. in Sir ; CSS : bes :

N N y y C f 1990. S.L. W. Patent Application Publication Apr. 30, 2015 Sheet 6 of 11 US 201S/O119330 A1

A Cells on plastic 400 -H Control -- M831OuM & J941 OuM : --

POP Activity

0-8-i-us------. & 6 8 10 12 14 16 18 20 Time (hrs) Cells on Matrigel 400 --Control M831OLM “&“J941OLM 300- l r POP - & Activity

s

&: --

^ & FAP & -1 . m m Activity 0 &-i-S$.3s-Sax8------ax&s1 0 2 4 6 8 10 12 14, 16 18 2 Time (hrs) Figure 6 Patent Application Publication Apr. 30, 2015 Sheet 7 of 11 US 201S/O119330 A1

A 1 O O

8 O

6 O 40 o

WI38 MCF 12A HMVECO Cell Type B S. E O s ce S, a a 3 o o SN S N 38 3 & 555 & 3

kDa as s > S S E E 100-8 in in six 75 a

50 Patent Application Publication Apr. 30, 2015 Sheet 8 of 11 US 201S/O119330 A1

OW dod {{SOAO ojoso Ao ? eue CueN eubuqueW eleSA |e. eyesAeo.

OSN dod : : & 9)

x ins * O 2 : &. . .

s y s su

al Patent Application Publication Apr. 30, 2015 Sheet 9 of 11 US 201S/O119330 A1

Rs' ssss w SSS 8. x &xxxxxxxxxxxxxxxxxxxxx : X & SS SNS

NyS. sas s &x swww.www.www.www.www.www.s. Xs &s S.

sSKXs say s SaaS & saxxxxxxxxxxxxxxxxxxssssssssssssssssssssss s sar

a. w Sas SS X NY s SS sar

SS s & S

wsS& SY SSX SS SS wSXSS Sass S ass Sixx Sas * S. s S. & &ss

s w &S s 8xx Sw Sassas s & s xxx xx S X S\s & s ss & S& & & & &ss Sws x SS sy

YS

s a\\\\\\\\\\\SSSSSSSSSSSSSSSSSSSSSSSSSSSSssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss'S W 1. s s & S S y S & SSY &y as S. xx & syas S&Sx - N SS&x w s S$ &\s sr. wers

Patent Application Publication Apr. 30, 2015 Sheet 10 of 11 US 201S/O119330 A1

s:

Figure 10A

Figure 10B Patent Application Publication Apr. 30, 2015 Sheet 11 of 11 US 201S/O119330 A1

HCT116 Colon Cancer Xenografts in Mice-Treatment with J94 and M83 Tumor Volume versus Days of treatment

4000 ^Contro YJ94-50 ^M33-50

3600

3200

O mirretty OD 3: 7D 1OD 18D 2AD 28D Treatment day

FIGURE 11 US 2015/01 19330 A1 Apr. 30, 2015

SUBSTRATES AND INHIBITORS OF PROLYL genetic deletion or pharmacologic inhibition of FAP by OLGOPEPTIDASE AND FIBROBLAST glutamyl-proline boronic acid (Glu-boroPro) decreased stro ACTIVATION PROTEIN AND METHODS OF mal growth in mouse models of lung and colon cancer. How USE ever, Glu-boroPro has an exceptionally short plasma half-life before cyclizing and losing inhibitory activity 22. More CROSS-REFERENCE TO RELATED over, it also inhibits dipeptidyl peptidase IV (DPPIV), which APPLICATIONS/INCORPORATION BY is important in plasma glucose regulation and immune func REFERENCE STATEMENT tion 23. Hence, despite inhibiting FAP and suppressing 0001. The present application claims the benefit under 35 tumor growth, Glu-boroPro is not likely to be therapeutically U.S.C. 119(e) of U.S. Provisional Application Ser. No. useful in cancer 24. 61/643,001, filed May 4, 2012, and U.S. Provisional Appli 0006. The measurement of cellular FAP activity and inhi cation Ser. No. 61/793,183, filed Mar. 15, 2013. The entire bition is confounded by another prolyl endopeptidase: contents of each of the above-referenced applications are namely, Prolyl Oligopeptidase (POP) which is elevated in hereby expressly incorporated herein by reference. many cancers 25. POP is a prolyl-specific serine protein ases, which cleaves peptides of less than about 30 amino acids STATEMENT REGARDING FEDERALLY in length. The enzyme is present in most tissues, but is noted to be more abundant in selected organs, e.g., brain and kidney. SPONSORED RESEARCH ORDEVELOPMENT Recently POP has been indicated as making secondary cleav 0002 This invention was made with government support ages of thymosin-B4 to yield the derivative peptide, acetyl under Contract Number W81XWH-081-0588 awarded by the serine-asparagine-lysine-proline, which appears to be a Department of Defense (DOD) and Contract Number potent stimulator of angiogenesis 26. Both FAP and POP HL072995 awarded by the National Institutes of Health activities are commonly measured using non-specific Sub (NIH). The government has certain rights in the invention. strates such as Z-Gly-Pro-AMC or succinyl-Gly-Pro-AMC, neither of which distinguishes between the two activities BACKGROUND 27. Consequently, total prolyl-specific endopeptidase activ 0003 C-Antiplasmin (CAP) is a glycoprotein in blood ity, which is often attributed to FAP alone, may also include plasma that rapidly and specifically inhibits the enzyme, plas POP activity. This complicates interpretations about the min, which digests blood clots, whether presenting early as effects of inhibiting either enzyme on cancer growth. Pre intravascular platelet-fibrin deposits or as partially or com clinical studies have suggested other promising applications pletely occlusive thrombi. Similarly, plasmin and CAP of POP inhibition for managing memory, learning disorders activities are important to the development and survival of and depression, but development of relatively benign, highly fibrinas occurs in inflammation, wound healing and virtually effective POP inhibitors for in vivo testing have been elusive. all forms of cancer and its metastases. Human a-antiplasmin The results, newly described herein, indicate that POP is (CAP), also known as C-plasmin inhibitor, is the main expressed in significant amounts by a variety of cancer cells inhibitor of plasmin. Plasmin plays a critical role in fibrin grown in culture. proteolysis and tissue remodeling. The inventors discovered 0007 Certain compounds which specifically inhibit either antiplasmin-cleaving enzyme (APCE) in human plasma and one or both of FAP and POP and therefore can be used to treat showed that it is a soluble isoform or derivative of fibroblast various conditions which involve these proteins are desirable. activation protein-alpha (FAP). Like APCE, FAP is also a Thus, the presently disclosed and claimed inventive concept prolyl-specific enzyme that exhibits both endopeptidase and (s) is directed to, but not limited to, substrates and inhibitors dipeptidyl peptidase activities. of FAP and/or POP, and to methods of using FAP and/or POP 0004 FAP significantly over-expresses in >90% of epithe inhibitors for treating conditions such as but not limited to, lial-derived cancers 1-3. FAP is produced transiently by cancers, neural disorders, and angiogenesis, and to Screening activated Stromal fibroblasts during embryogenesis 4 and methods for identifying such inhibitors, that overcome the wound healing 3, but other than an occasional normal fibro disadvantages and defects of the prior art. blast or pancreatic islet C.-cell, it is not expressed by normal adult tissues or benign tumors 2, 3, 5). FAP is prominent on BRIEF DESCRIPTION OF THE DRAWINGS the membranes of proliferating fibroblasts in diseases where 0008 FIG. 1 is a characterization of FAP and POP in fibrous tissue growth is a conspicuous feature, such as pri fibroblasts. Panel A. Confocal images of permeabilized mary pulmonary fibrosis 6; chronic hepatitis 7; certain WI-38 and VA-13 fibroblasts grown on plastic slides and bone-associated malignancies 8, 9; and the arthritides 10. labeled with mouse anti-FAP mab F19 followed by anti Selected parenchymatous cancer cells may also occasionally mouse-AlexaFluor 568 (red) and the DNA stain DAPI express FAP11. (green). Panel B. FAP and POP activities offibroblasts grown 0005 While a biologic substrate for the proteinase activity on plastic wells as measured by cleavage of a fluorescent of FAP has not been definitively established, results indicate substrate designated as “C95” (acetyl-Arg-AEEA-Gly-Pro that FAP helps digest extracellular matrix (ECM) compo AMC), and using a POP specific inhibitor compound desig nents as tissue is remodeled to accommodate cancer expan nated as “J94 (acetyl-Lys-Leu-Arg-(L)boroPro) to separate sion 2, 16, 17. Paradoxically, activated fibroblasts not only the two activities. One FAP unit=A fluorescence/min on digest ECM, but also synthesize ECM components of the cleavage of C95 by one ng APCE. Panel C. Immunostains of stromal scaffolding that Support cell division and motility cell lysates from fibroblast cultures using mouse anti-FAP during neoplastic growth 18. FAP has been considered a mAb 6D2. One ng APCE served as a positive control while potential target in the diagnosis and therapy of cancer, with intracellular contents of C-tubulin (50 kDa) and actin (43 inhibition of FAP proteinase activity posed as possibly thera kDa) were used for standardizing the amount of cell lysate peutically useful 19, 20. Santos et al. 21 have shown that protein applied in each lane. Panel D. Immunostains of cell US 2015/01 19330 A1 Apr. 30, 2015

lysates and membrane and cytosol fractions from WI-38 0013 FIG. 6 shows FAP and POP activities in microvas fibroblast cultures using mouse anti-FAP mab 6D2. One ng cular endothelial cells. Panel A. HMVEC-d cells plated on APCE served as a positive control. Panel E. Immunostains of plastic wells allowed to settle for one hour, then inhibitors and cell lysates from fibroblast cultures using goat anti-POP. One Substrates were added and fluorescence was measured over ng of POP served as a positive control, while C-tubulin (50 time for 18 hours. Panel B. HMVEC-d cells plated on Matri kDa) and actin (43 kDa) were used for standardizing loads. gelTM (BDBiosciences, San Jose, Calif.), allowed to settle for Panel F. Immunostains of cell lysates and membrane and one hour to initiate tubule formation, and assayed for FAP and cytosol fractions from W 138 fibroblast cultures using goat POP activities as in Panel A. Note that POP activity is detect anti-POP. One ng of POP served as a positive control. able from the start in both assay conditions, but FAP activity 0009 FIG.2 shows the amino acid sequence of FAP (SEQ only appears in the MatrigelTM assay at about four hours after ID NO: 1) and corresponding tryptic FAP peptides isolated tubule formation. Each point represents 15 readings over from four human cell types. Protein was purified by immu three separate experiments. noprecipitation and SDS-polyacrylamide gel electrophoresis (0014 FIG. 7 shows FAP activity and FAP protein levels in followed by sequence determination by LC/MS/MS. Corre stressed cells. WI-38 fibroblasts, MCF-12A normal breast spondence to FAP protein is indicated: Human mesenchymal cells, and HMVEC-d endothelial cells were grown in the stem cells (—) 51% (coverage FAP sequence); WI-38 fibro presence of hydrocortisone (hyc) as customarily present in blasts (), 42%; MDA-MB436 cancer cells (OOO growth media, or in its absence. Cells were grown from 7 to O), 41%; and HMVEC-dendothelial cells deprived of hydro 14 days as specified before assessing FAP activity and FAP cortisone (2869626), 6.4%. In each case, the FAP-containing protein levels. Panel A. FAP activity was measured as previ region of the gel was located by reference to a 100kDa protein ously described. FAP activity in the absence of hydrocorti standard band in an adjacent lane. sone was set at 100%, with the level of FAP activity in the 0010 FIG. 3 shows the dose-response inhibition of FAP presence of hydrocortisone expressed as a relative percent. activity on WI-38 fibroblast surfaces by a FAP inhibitor com Panel B. Immunostains of cell lysates from fibroblast, normal pound designated as “M83'' (acetyl-Arg-AEEA-(D)Ala-(L) breast and endothelial cell cultures, using mab 6D2 to FAP. boroPro). WI-38 fibroblasts were grown on plastic, washed APCE 1 ng is used as a positive control; C-tubulin (50 kDa) once with HBSS, after which the M83 inhibitor 10 uM in and actin (43 kDa) were used to standardize cell lysate protein HBSS was added at Zero time to one set, and buffer only to the amounts applied to each electrophoretic lane. MCF 12A nor other four sets. The fluorescent substrate C95 was added to all mal breast cells were grown in the absence of hydrocortisone, sets of wells and A fluorescence/min, reflecting cleavage of (-)hyc, for 7 days, followed by repletion of the normal media C95 over time, was allowed to proceed for two hours when 10 (+) hyc content, as Supplied by Lonza, for 7 days, labeled uM, 1 uM or 100 nM of the M83 inhibitor was added to each (-)hyc/(+)hyc, prior to assessing both FAP activity and FAP of three sets of wells. As shown, each inhibitor concentration protein concentration. instantly, and essentially totally, inhibited the proteolytic 0015 FIG. 8 shows the identification and characterization activity of FAP on the cell surface. of FAP and POP in human mesenchymal stem cells (MSC). 0011 FIG. 4 is a characterization of FAP and POP in Panel A. Confocal image of permeabilized MSCs grown on normal breast and breast cancer cells. Panel A. Confocal glass slides and labeled with mAb F19 to FAP followed by images of permeabilized MCF-12A (normal breast cells), anti-mouse-AlexaFluor 568 (red) and DAPI (green). Panel B. MDA-MB436 or HCC1419 (breast cancer cells) grown on FAP and POP activities found on mesenchymal stem cell glass slides and labeled with mAb F19 to FAP followed by Surfaces grown in plastic wells were measured exactly the anti-mouse-AlexaFluor 568 (red) and DAPI (green). Panel B. same as described in FIGS. 1 and 4. Panel C. Immunostaining FAP and POP activities on surfaces of normal and breast was performed as described in FIGS. 1 and 4. Panel D. Immu cancer cells grown on plastic wells as measured by cleavage nostaining for FAP in cell lysates and membrane and cytoso of fluorescent substrate C95, and using the POP specific lic fractions of mesenchymal stem cells was performed as inhibitor J94 to separate the two activities. One FAP unit=A described in FIG. 1. Panel E. Immunostaining for POP was fluorescence/min on cleavage of C95 by one ng APCE. Panel performed as described in FIGS. 1 and 4. Panel F. Immun C. Immunostains of cell lysates from normal and breast can ostaining for POP in cell lysates and membrane and cytosolic cer cell cultures, using mAb 6D2 to FAP APCE 1 ng was used fractions of mesenchymal stem cells was performed as as a positive control, while intracellular contents of C-tubulin described in FIG. 1. (50 kDa) and actin (43 kDa) were used for standardizing the 0016 FIG. 9 is a graph showing the inhibition of lung amount of cell lysate protein applied in each lane. Panel D. cancer Xenografts in nude mice by compound M83 over a Immunostains of cell lysates from normal and breast cancer period of 28 days. cell cultures using goat anti-POP POP 1 ng was used as a 0017 FIGS. 10A and B are micrographs which demon positive control, while C-tubulin (50 kDa) and actin (43 kDa) strate that application of 50 um of compound J94 inhibited served as load controls. formation of capillary-like tubes in MatrigelTM. 0012 FIG. 5 is a characterization of FAP and POP in 0018 FIG. 11 is a graph showing the inhibitory effects of microvascular endothelial cells. Panel A. Confocal images of M83 and J94 on growth of colon cancer tumors. permeabilized HMVEC-d grown on glass slides and labeled DETAILED DESCRIPTION with mAb F19 to FAP followed by anti-mouse-AlexaFluor 568 (red) and DAPI (green). Panel B. FAP and POP activities 0019. Before explaining the at least one non-limiting of endothelial cells grown on plastic wells measured by embodiment of the inventive concept(s) disclosed herein in exactly the same methods described in FIGS. 1 and 4. Panel detail, it is to be understood that the presently disclosed and C. Immunostaining performed as described in FIGS. 1 and 4. claimed inventive concept(s) is not limited in its application Panel D. Immunostaining for POP performed as described in to the details of examples, experiments, exemplary data, and/ FIGS. 1 and 4. or methods or steps as set forthin the following description, or US 2015/01 19330 A1 Apr. 30, 2015

illustrated in the drawings. The presently disclosed and filed Jun. 7, 2006; and 60/836,365, filed Aug. 8, 2006. The claimed inventive concept(s) is capable of other embodiments entire contents of each of these applications are hereby or of being practiced or carried out in various ways. As such, expressly incorporated herein by reference in its entirety. the language used herein is intended to be given the broadest Also, U.S. Ser. Nos. 10/774,242; 1 1/810,997; 11/986,058: possible scope and meaning; and the embodiments are meant and 60/445,774 are hereby expressly incorporated herein by to be exemplary—not exhaustive. Also, it is to be understood reference in their entireties. that the phraseology and terminology employed herein is for 0023 All of the compositions and/or methods disclosed the purpose of description and should not be regarded as and claimed herein can be made and executed without undue limiting. experimentation in light of the present disclosure. While the 0020. In the following detailed description of embodi compositions and methods of the inventive concept(s) have ments of the presently disclosed and claimed inventive con been described in terms of particular embodiments, it will be cept(s), numerous specific details are set forth in order to apparent to those of skill in the art that variations may be provide a more thorough understanding of the inventive con applied to the compositions and/or methods and in the steps or cept(s). However, it will be apparent to one of ordinary skill in in the sequence of steps of the method described herein with the art that the inventive concept(s) within the disclosure may out departing from the concept, spirit and scope of the pres be practiced without these specific details. In other instances, ently disclosed and claimed inventive concept(s). All Such well-known features have not been described in detail to similar Substitutes and modifications apparent to those skilled avoid unnecessarily complicating the present disclosure. in the art are deemed to be within the spirit, scope and concept 0021. Unless otherwise defined herein, scientific and tech of the inventive concept(s) as defined by the appended claims. nical terms used in connection with the presently disclosed 0024. In order that the presently disclosed and claimed and claimed inventive concept(s) shall have the meanings that inventive concept(s) may be more readily understood, certain are commonly understood by those of ordinary skill in the art. term are first defined. Additional definitions are set forth Further, unless otherwise required by context, singular terms throughout the detailed description. Unless defined other shall include pluralities and plural terms shall include the wise, all technical and scientific terms used herein have the singular. Generally, nomenclatures utilized in connection same meanings as commonly understood by one skilled in the with, and techniques of cell and tissue culture, molecular art to which the presently disclosed and claimed inventive biology, and protein and oligo- or polynucleotide chemistry concept(s) pertains (e.g., amino acids may be referred to by and hybridization described herein are those well known and their commonly used abbreviations). commonly used in the art. Standard techniques are used for 0025 APCE is Antiplasmin-Cleaving Enzyme. FAP is recombinant DNA, oligonucleotide synthesis, and tissue cul Fibroblast Activation Protein-alpha. POP is Prolyl Oligopep ture and transformation (e.g., electroporation, lipofection). tidase. DPPIV is Dipeptidyl peptidase IV. CAP is C-Anti Enzymatic reactions and purification techniques are per plasmin. AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid, formed according to manufacturer's specifications or as com also referred to herein as 8-amino-3,6-dioxaoctanoic acid. monly accomplished in the art or as described herein. The AMC is 7-amido-4-methylcoumarin. L-boroPro is L-boronyl foregoing techniques and procedures are generally performed proline. M83 is acetyl-Arg-AEEA-(D)Ala-(L)boroPro. C95 according to conventional methods well known in the art and is acetyl-Arg-AEEA-Gly-Pro-AMC. J94 is acetyl-Lys-Leu as described in various general and more specific references Arg-(L)boroPro. L96 is acetyl-Lys-Leu-Arg-Pro-AMC. Abz that are cited and discussed throughout the present specifica is aminobenzoyl. Ac is acetyl. BZ is benzoyl. Z is benzyloxy tion. See e.g., Sambrook et al. Molecular Cloning: A Labo carbonyl. Boc is t-Butyloxycarbonyl. Fa is Fury lacryloyl. ratory Manual (2nd ed., Cold Spring Harbor Laboratory MeOSuc is methoxysuccinyl. Pyr is pyroglutamate. AFC is Press, Cold Spring Harbor, N.Y. (1989) and Coligan et al. 7-amino-trifluoromethylcoumarin. OEt is ethyl ester. OMe is Current Protocols in Immunology (Current Protocols, Wiley methyl ester (OMe). 2NA is 2-Naphthylamide (2NA). p-NA Interscience (1994)), which are incorporated herein by refer is p-Nitroanilide. ONp is p-Nitrophenyl ester. SBzI is ence. The nomenclatures utilized in connection with, and the Thiobenzyl ester. “Tic' refers to 1,2,3,4 tetrahydro isoquino laboratory procedures and techniques of analytical chemis line-3-carboxylic acid. HCC, HCC1419 breast cancer cells; try, synthetic organic chemistry, and medicinal and pharma HMVEC-d, human microvascular endothelial cells from der ceutical chemistry described herein are those well known and mis; MCF12A, normal breast cells; MDA, MDA-MB436 commonly used in the art. Standard techniques are used for breast cancer cells; MSC, mesenchymal stem cells; VA-13, chemical syntheses, chemical analyses, pharmaceutical WI-38 VA-13 2RA SV40 viral transformed fibroblast cells; preparation, formulation, and delivery, and treatment of WI-38, fibroblast cells. patients. 0026. The use of the word “a” or “an when used in con 0022 All patents, published patent applications, and non junction with the term “comprising in the claims and/or the patent publications mentioned in the specification are indica specification may mean “one but it is also consistent with tive of the level of skill of those skilled in the art to which this the meaning of"one or more.” “at least one.” and “one or more presently disclosed and claimed inventive concept(s) per than one.” The singular forms “a,” “an,” and “the include tains. All patents, published patent applications, and non plural referents unless the context clearly indicates otherwise. patent publications referenced in any portion of this applica Thus, for example, reference to “a compound may refer to 1 tion are herein expressly incorporated by reference in their or more, 2 or more, 3 or more, 4 or more or greater numbers entirety to the same extent as if each individual patent or of compounds. The term “plurality” refers to “two or more.” publication was specifically and individually indicated to be The use of the term 'or' in the claims is used to mean'and/or incorporated by reference. In particular, the present applica unless explicitly indicated to refer to alternatives only or the tion contains subject matter which is related to U.S. Ser. No. alternatives are mutually exclusive, although the disclosure 12/969,161, filed Dec. 15, 2010: 61/286,558, filed Dec. 15, supports a definition that refers to only alternatives and “and/ 2009: Ser. No. 1 1/811002, filed Jun. 6, 2007; 60/811,568, or.” Throughout this application, the term “about is used to US 2015/01 19330 A1 Apr. 30, 2015

indicate that a value includes the inherent variation of error ratio. The phrase “pharmaceutically-acceptable carrier as for the device, the method being employed to determine the used herein means a pharmaceutically-acceptable material, value, or the variation that exists among the study Subjects. composition, or vehicle, such as a liquid or solid filler, diluent, For example but not by way of limitation, when the term excipient, or solvent encapsulating material, involved in car “about is utilized, the designated value may vary by +20% or rying or transporting the Subject compound from one organ, +10%, or +5%, or +1%, or +0.1% from the specified value, as orportion of the body, to another organ, orportion of the body. Such variations are appropriate to perform the disclosed meth Each carrier must be “acceptable' in the sense of being com ods and as understood by persons having ordinary skill in the patible with the other ingredients of the formulation and not art. The use of the term “at least one' will be understood to injurious to the patient. include one as well as any quantity more than one, including 0031. As used herein, a “therapeutically effective amount but not limited to,2,3,4,5,10,15, 20, 30, 40, 50, 100, etc. The the inhibitor or chemotherapeutic agent of the presently dis term “at least one' may extend up to 100 or 1000 or more, closed and claimed inventive concept(s) refers to an amount depending on the term to which it is attached; in addition, the of a compound that is effective, upon single- or multiple-dose quantities of 100/1000 are not to be considered limiting, as administration to the Subject, e.g., a patient, at enhancing the higher limits may also produce satisfactory results. In addi inhibition of the growth or proliferation, or inducing the kill tion, the use of the term “at least one of X, Y and Z' will be ing, of hyperproliferative cells, e.g., cancer cells by a chemo understood to include X alone, Yalone, and Zalone, as well therapeutic compound or by radiation treatment. The term, as any combination of X, Y and Z. The use of ordinal number for example, “therapeutically effective amount” refers to an terminology (i.e., “first”, “second, “third”, “fourth”, etc.) is amount of an inhibitory compound of the presently disclosed solely for the purpose of differentiating between two or more and claimed inventive concept(s) that is administered, e.g., items and is not meant to imply any sequence or order or coadministered, (i.e., sequentially or concomitantly) with importance to one item over another or any order of addition, one or more cytotoxic agents such that the inhibitory com for example. pound and the cytotoxic agent, are effective, upon single- or 0027. As used in this specification and claim(s), the terms multiple-dose administration to the Subject, e.g., a patient, at “comprising (and any form of comprising, Such as "com inhibiting the growth or proliferation, or inducing the killing, prise' and "comprises”), “having (and any form of having, of hyperproliferative orangiogenic cells. Such growth inhi such as “have and “has'), “including (and any form of bition or killing can be reflected as a prolongation of the including, such as “includes” and “include’) or “containing Survival of the Subject, e.g., a patient beyond that expected in (and any form of containing, such as “contains” and “con the absence of such treatment, or any improvement in the tain’) are inclusive or open-ended and do not exclude addi prognosis of the Subject relative to the absence of such treat tional, unrecited elements or method steps. ment. As used herein, the term “carcinomas' refers to lesions 0028. The term “or combinations thereof as used herein that are cancerous. As used herein, the term “neoplasm’ refers to all permutations and combinations of the listed items refers to both precancerous and cancerous lesions. As used preceding the term. For example, "A, B, C, or combinations herein, the terms “inhibit or “inhibiting, mean decreasing thereof is intended to include at least one of A, B, C, AB, FAP, POP or APCE activity, or tumor cell growth rate orother AC, BC, or ABC, and if order is important in a particular targeted activity from the rate that would occur without treat context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. ment of the inhibitor compound and/or causing tumor mass to Continuing with this example, expressly included are combi decrease. Inhibiting also includes causing a complete regres nations that contain repeats of one or more item or term, Such sion of the tumor. Thus the compounds of the presently dis as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, closed and claimed inventive concept(s) can be either cyto CABABB, and so forth. The skilled artisan will understand static or cytotoxic to the tumor cells, when used alone or in that typically there is no limit on the number of items or terms combination with other therapies. in any combination, unless otherwise apparent from the con 0032. As used herein, the terms “cytotoxic agent”, “che text. motherapeutic agent”, “anticancer agent, and “antitumor 0029. As used herein, the term “substantially’ means that agent” are used interchangeably herein and refer to agents the Subsequently described event or circumstance completely that have the property of inhibiting the growth or proliferation occurs or that the Subsequently described event or circum (e.g., a cytostatic agent), or inducing the killing, of hyperpro stance occurs to a great extent or degree. For example, the liferative cells. As used herein, “chemosensitization' and term “substantially’ means that the subsequently described “chemosensitizing effect” are used interchangeably and refer event or circumstance occurs at least 90% of the time, or at to the enhancement of radiation or chemotherapy efficacy by least 95% of the time, or at least 98% of the time. the compound. “Chemosensitizer” refers to the agent that 0030. As used herein, “pharmaceutically acceptable' enhances the efficacy of another agent, such as the cytotoxic refers to those properties and/or Substances, which are accept agent or radiation. able to the patient from a pharmacological/toxicological 0033. The term “a drug or compound for overcoming a point of view including bioavailability and patient acceptance resistance to an anticancer drug or an anticancer-drug-resis or to the manufacturing chemist from a physical-chemical tance overcoming drug or “a pharmaceutical composition point of view regarding composition, formulation, stability for overcoming a resistance to an anticancer drug or an anti and isolatability. The phrase “pharmaceutically acceptable' cancer-drug-resistance overcoming pharmaceutical-compo is employed herein to refer to those compounds, materials, sition” refers to a drug which has no carcinostatic activity compositions, and/or dosage forms which are, within the itself but has a function of reducing a resistance of cancer Scope of sound medical judgment, Suitable for use in contact cells to an anticancer drug. In other words, it means a drug with the tissues of human beings and animals without exces having a function for increasing sensitivity to an anticancer sive toxicity, irritation, allergic response, or other problem or drug of cancer cells having an acquired resistance to the complication, commensurate with a reasonable benefit/risk anticancer drug. In this case, the increase of the sensitivity US 2015/01 19330 A1 Apr. 30, 2015

means not only to increase an effect of an anticancer drug to vided, as are substrates of APCE, FAP or POP. The presently anticancer-drug resistant cells in a higher level than that to disclosed and claimed inventive concept(s) also includes, but anticancer-drug sensitive cells but also to increase the effect is not limited to, methods of using Such inhibitors to treat, of the anticancer drug to the anticancer-drug resistant cells in inhibit, and/or ameliorate conditions and/or diseases involv approximately the same level as that to the anticancer-drug ing FAP and/or POP, such as, but not limited to, epithelial sensitive cells. Further, another term equivalent to “overcom derived cancers, angiogenesis (such as angiogenesis which ing a resistance' may include “restraining or inhibiting a occurs during formation of tumors or in diabetes), Alzhe resistance”, “releasing resistance'. “releasing tolerance' or imer's disease, atherosclerosis, and thrombus disorders and “increasing or enhancing a sensitivity. conditions involving abnormal cell proliferation, as well as 0034. The term “administrating together with in the pres other cancers and disorders identified herein. Substrates of ently disclosed and claimed inventive concept(s) means APCE, FAP, and POP may also be used, for example, in administering two kinds of drugs simultaneously, continu screening methods for identifying inhibitors of FAP and POP. ously or at intervals. The two kinds of drugs may be admin 0039 Thus, the presently disclosed and claimed inventive istered as a mixture or as separate drugs. When administering concept(s) is directed to (but not limited to) methods and as separate drugs, each administering route may be or may be compounds for treating conditions characterized by abnormal not the same. As defined herein, treating cancer (i.e., with an cell proliferation, angiogenesis, and/or neural disorders, anticancer therapy) in a patient includes achieving, partially including, but not limited to, cancer and metastasis, and or Substantially, one or more of the following: arresting the Alzheimer's disease, by using the inhibitors of the present growth or spread of a cancer, reducing the extent of a cancer disclosure to inhibit the enzymatic activity of FAP and POP in (e.g., reducing size of a tumor or reducing the number of vivo. In one embodiment, the presently disclosed and claimed affected sites). Inhibiting the growth rate of a cancer, and inventive concept(s) provides a method for treating a subject ameliorating or improving a clinical symptom or indicator having a condition characterized by abnormal mammalian associated with a cancer (Such as tissue or serum compo cell proliferation and/or angiogenesis. In the method, an nents). The term “treatment as used herein is also intended to agent is administered to a subject in need of such treatment in encompass prophylaxis, therapy and cure. an amount effective to inhibit cell proliferation and/orangio 0035 Also, where used herein, the terms “heterocycle” or genesis. The agent is, or comprises, a compound having at "heterocyclic” refer to ring structures, particularly 4, 5, 6, or least one of Formula I and Formula II as described herein. 7-membered ring structures, and more particularly 5- to 0040. The inventors discovered the circulating antiplas 6-membered rings, whose ring structures include one to four min-cleaving enzyme (APCE) and showed it to be either a heteroatoms such as nitrogen, Sulfur, or oxygen. Heterocyclic derivative of fibroblast activation protein (FAP) or a slight groups include, but are not limited to, thiophene, furan, pyran, variant thereof due to gene splicing differences 1.2. Except pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, for the absence of an about 26-residue amino terminal peptide pyrazine, pyrimidine, and pyridazine. The heterocyclic ring constituting the transmembrane and intracytosolic domains, can be substituted at one or more positions with Such Sub APCE is otherwise identical to FAP in molecular structure stituents as, for example, halogen, alkyl, aralkyl, alkenyl, and function. FAP is over expressed by stromal cells in >90% alkynyl, cycloalkyl, hydroxyl, amino, nitro, Sulfhydryl, of epithelial-derived malignancies, but not by normal tissues imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, or benign tumors, hence causing FAP to be viewed as having silyl ether, alkylthio, Sulfonyl, ketone, aldehyde, ester, a het major potential as a unique diagnostic and therapeutic target erocyclyl, an aromatic or heteroaromatic moiety, CF, CN, or in a wide array of human cancers. the like. Where used herein, the term “carbocycle' or “car 0041. Subsequent research indicated that POP, another bocyclic” refers to an aromatic or non-aromatic ring in which member of the clade of prolyl-specific serine proteinases, had each atom of the ring is carbon and may particularly comprise proteolytic activity which overlapped that of FAP when con 4, 5, 6 or 7 carbons per ring, Such as 5 or 6 carbons per ring. ventionally available synthetic substrates such as Z-Gly-Pro 0036. The term “stalk” or “stalk portion” as used herein AMC were used for FAP activity measurements. Although will be understood to refer to portions of the compounds the principal inhibitor of FAP manifested both high selectiv disclosed herein, such as but not limited to, Formulas I-IV, ity and sensitivity, it also inhibited POP, and this has been a that are missing the Cyc groups thereof. consistent problem in trying to separate and quantitate the 0037 Additionally, the proline analogs and derivatives or activity of each of these enzymes. Very effective and specific other amino acids of the peptidomimetic compounds of the inhibitors of POP have thus been designed and synthesized, presently disclosed and claimed inventive concept(s) may as described herein. It has also been discovered that the POP exist in particular geometric or stereoisomeric forms. The inhibitor very rapidly and effectively blocked capillary-like presently disclosed and claimed inventive concept(s) tube formation by human dermal endothelial cells. Thus, in includes all such compounds, including cis- and trans-iso one aspect of the presently disclosed and claimed inventive mers, R- and S-enantiomers, diastereomers, (D)-isomers, concept(s), the POP inhibitors described hereincan be used as (L)-isomers, the racemic mixtures thereof, and other mixtures anti-angiogenic agents and as effective, high affinity, sensi thereof, as falling within the scope of the presently disclosed tive inhibitors of POP in certain mammalian diseases, par and claimed inventive concept(s). Additional asymmetric car ticularly where therapeutic effectiveness has been limited due bon atoms may be present in a Substituent such as, but not to problems of inhibitor solubility and/or inhibition of other limited to, an alkyl group. All Such isomers, as well as mix enzymes where such inhibition was undesirable and carried tures thereof, are intended to be included in the presently potential for adverse pharmacologic outcomes. disclosed and claimed inventive concept(s). 0042. Further, it has been discovered that POP is exten 0038 Turning now to the presently disclosed and claimed sively expressed by activated fibroblasts that characteristi inventive concept(s), inhibitors of prolyl oligopeptidase cally participate in the development of scaffolding over which (POP) and fibroblast activation protein-alpha (FAP) are pro cancer cells grow as the tumor expands. One embodiment of US 2015/01 19330 A1 Apr. 30, 2015

the presently disclosed and claimed inventive concept(s) is a various cancers and/or other FAP-related conditions or other pseudo-peptide, acetyl-Lys(K)-Leu(L)-Arg(R), which forms conditions involving abnormal cell proliferation, as described a stalk of a very specific, sensitive fluorescent substrate for the in further detail below. With respect to FAP inhibition of this POP enzyme, namely acetyl-KLRP-AMC (designated herein enzyme will limit or obviate the ability of epithelial-derived as L96), which has been shown to be cleaved by POP with a cancers to invade the Surrounding extracellular matrix, K of 30+3 uM and k=2.4+0.1 s'. Neither DPPIV nor thereby limiting the cancer's spread and allowing more effec APCE/FAP cleaved this substrate. The substrate can be used tive use of chemotherapy or radiation therapy. Without wish to assess POP activity from a variety of sources including, but ing to be bound by theory, the FAP inhibitors described herein not limited to, human plasma, tissue, and culture media. In another embodiment, the acetyl-LyS(K)-Leu(L)-Arg(R) pep are effective, highly efficient inhibitors which prevent, tide is used as a stalk of another pseudo-peptide, acetyl-KLR inhibit, and/or reduce the expansion of extracellular space by (L)-boroPro, and alternate versions thereofas represented by digestion of FAP's Substrate proteins, e.g., type I collagen Formula II, which proved to be a very highly sensitive, selec within the extracellular matrix (ECM). This will then abro tive POP inhibitor (K3100 nM), and useful for separating gate Subsequent movementofactivated fibroblasts for remod the activities of POP and ACE/FAP. Using standard tissue eling ECM space with new stromal scaffolding to which culture methods for growing blood vessel capillaries in vitro, malignant cells adhere for migration and mitosis. As a con it is also demonstrated that POP is also expressed by human sequence, the neoplasm will then atrophy and undergo necro endothelial cells as they became confluent and progress to sis, or be arrested to an extent that radiation and/or chemo capillary-like tube formation. It has also been demonstrated therapy measures, desirably at lower than standard doses, will that application of 50 uM POP inhibitor (J94) abrogated the eradicate the malignancy. Previously a number of studies of genesis of capillary-like tubes (FIG. 10). Hence one of the metastatic cancer indicated that Val-boroPro, a dipeptide con potent effects of the inhibitor described here is that it disrupts taining a boronic acid derivative of proline (for example, as angiogenesis by instantaneously inhibiting POP activity. described in U.S. Pat. No. 7,399,869), inhibits FAP, and as a Given the obligatory need of microcirculation to enable tissue consequence, cancer growth. Unfortunately, however, Val growth, the inhibition of POP by the inhibitor indicates its boroPro also non-selectively inhibits most prolylpeptidases ability to abrogate undesirable tissue expansion, i.e., malig such as dipeptidyl peptidases (such as DPPIV) and up-regu nant growth, by blocking the development of a requisite lates cytokine and chemokine-5 activities. Several other pro microcirculation. lylboronic acid derivatives have been developed and reported 0043. To date, many of the POP inhibitors designed and as putative selective inhibitors for FAP, but their instability in invented by others have not been used clinically for inhibiting aqueous environments at physiologic pH and their non-spe angiogenesis. An important advantage of the novel inhibitors cific reactivities with other enzymes due to the electrophilic described herein is the high degree of solubility manifested property of boronic acid has complicated progress in their under aqueous conditions, which is a requirement for use in SC. tissues where water is the overwhelming physiologic solvent, 0047. POP activity has been shown to be elevated in sub and counters the significant negative of previous inhibitors jects with neurological disorders such as, but not limited to, that are incompletely soluble under in vivo conditions. bipolar disorder, autism, Schizophrenia, Various stress-related 0044. In one embodiment, the presently disclosed and disorders, memory loss, and memory deficits (such as those claimed inventive concept(s) is directed to the use of these characteristic of Alzheimer's disease). Without wishing to be POP inhibitors for the treatment of human disorders and bound by theory, POP appears to be involved in cognitive diseases, such as various mood, memory, and behavioral dis functions via the cleavage of neuropeptides. Increased levels orders, as well as learning disorders, and also for blocking of POP may lead to reduced levels of key neuropeptides, angiogenesis by specifically and selectively inhibiting POP. which may be restored by administration of POP inhibitors. In certain embodiments, the presently disclosed and claimed In any event, by whatever mechanism POP has its effect, inventive concept(s) is particularly directed to those human inhibition of POP can be used as a treatment of these diseases, diseases whereformation of new blood vessels is essential for disorders, and conditions (see, for example, references 55-58 disease progression, as in malignancies (specifically epithe and U.S Published Patent Applications 2003/0096392, 2005/ lial-derived malignancies), in retinal blood vessel prolifera 0020677, 2007/0060550, and 2008/0269313, in regard to the tion, in sickle cell disease, and in diabetes mellitus. use of POP inhibitors in the treatment of such neural condi 0045. In one embodiment, inhibition of FAP or POP is tions; the entirety of each of these references being expressly defined herein as at least 50% inhibition of activity of FAP or incorporated herein by reference). POP, respectively, for example, at an inhibitor concentration 0048 Prior to the presently disclosed and claimed inven of 20 uM. Examples of the inhibitors are described below, and tive concept(s), there were no effective inhibitors of FAP or in one embodiment comprise a boroPro linked to the stalk POP that have sufficient specificity to allow exploration of unit. Groups which may substitute for boroPro (boronyl pro effects on the pathogenesis and/or therapy of chronic diseases line), include, but are not limited to, otherboronic acid deriva such as atherosclerosis or a variety of different cancers. For tives, carbocyclic groups, heterocyclic groups, carbonitriles, example, various dipeptide boronyl-proline constructs (e.g., carboxynitriles, or nitrilic-containing compounds, where the “val-boroPro') have been used in efforts to inhibit FAP, but as Arg (or other positively-charged N-terminal amino acid) may noted above, these constructs also inhibited several other or may not be blocked with a protecting group Such as, but not prolyl peptidases, some of the latter being critical for impor limited to. Succinyl, acetyl, benzoyl, benzyloxycarbonyl, or tant metabolic functions. Moreover, the design of these amino other protecting group commonly used for blocking peptides acid boroProline inhibitors did not prevent or slow their from attack by proteases. cyclization and inactivation that occurs within a few minutes 0046. The FAP inhibitors of the presently disclosed and in aqueous environments. The inhibitors of the presently dis claimed inventive concept(s) can be used in treatment of closed and claimed inventive concept(s) avoid these pitfalls. US 2015/01 19330 A1 Apr. 30, 2015

0049 Further, the presently disclosed inhibitor com linker or filler) between the P group and the positively pounds with high specificity for FAP and/or POP can be used charged amino acid in one or more of the P. Ps, P or P, in in vitro assays based on cancer cell lines to determine the positions may for example comprise one or more neutral, role of FAP at various stages of pathogenesis of FAP-related non-charged amino acids, e.g., glycine, alanine, leucine, iso cancers. The exact mode of how FAP operates in specific leucine, Valine, proline, methionine, tryptophan, tyrosine, cancer etiologies is still under study, and an inhibitor mol threonine, serine, B-alanine, y-amino butyric acid, epsilon ecule for selectively inhibiting FAP can be most useful. amino caproic acid; or PEG, (n=1-6), PPG, (n=1-6), amino 0050 FAP and POP Inhibitors PEG-carboxy group (n=1-6), including for example, 0051 Certain embodiments of the presently disclosed and 8-amino-3,6-dioxaoctanoic acid, 11-amino-3,6,9-trioxaun claimed inventive concept(s) include APCE-inhibitory, FAP decanoic acid, and 14-amino-3,6,9,12-tetraoxatetradecanoic inhibitory, and/or POP-inhibitory peptidomimetics. The pep acid, and amino-PPG-carboxy oligomers (e.g., n-1-6). tidomimetics may have up to 28 amino acids or more, as well These individual spacer molecules may be the same (e.g., all as less than 28 amino acids. The peptidomimetics comprise glycine, alanine, etc., or other singleamino acid or molecule) proline analogs and derivatives thereof for use as a P proline or different (e.g., more than one type of amino acid, ethylene Substitute, including, but not limited to, the proline analogs glycol/propylene glycol, or a hybrid amino acid/amino and derivatives shown in Table 1 or discussed or described PEG-carboxy or amino-PPG-carboxy where n=1-6), and elsewhere herein. The peptidomimetics (inhibitors) particu may be in a range of 3.0-21 A (0.3 nm-2.1 nm, or 1 to 7amino larly may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, acids) in length. The spacer may be comprised of neutral 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids, monomers comprising ethylene glycol for example, or other and the peptidomimetics may further comprise a spacer com similar monomer units (e.g., propylene glycol) each having a pound (which may be an amino acid) for separating certain length of about 0.3 nm, which together have a length of 3.0-21 amino acids of the compound, or compounds which may be A (0.3 nm-2.1 nm) such that the spacer places the positively bound or complexed thereto, for extending the serum life of charged amino acid (or, in the case of a POP substrate an/dor the peptide. Such spacers are discussed further below. inhibitor, a negatively- or positively-charged amino acid) 0052 Inhibitors of the presently disclosed and claimed within about 5-25A (0.5 nm-2.5 nm) of the proline or proline inventive concept(s) may possess a charged residue (posi substitute, analog, or derivative at the P. position. The inhibi tively-charged in the case of FAP inhibitors) at a distance tors optionally comprise a blocking group on the N-terminal corresponding to the length of two to seven residues upstream end for inhibiting protease degradation of the inhibitor. of the proline analog (0.3 nm-2.4 nm) and can be used as a 0055 FAP Inhibitors rapid, tightly binding and effective inhibitor of APCE, FAP, 0056. In one embodiment, FAP inhibitors of the presently and/or POP. Such inhibitors can be useful for the treatment of disclosed and claimed inventive concept(s) comprise com disorders relating to FAP and POP, for example, as described pounds having Formula I as shown below: elsewhere herein. B-Xaa1-Sp-Xaa-Cyc (Formula I). 0053. In certain embodiments, the inhibitors and/or sub In Formula I, Xaa, may be a positively-charged amino acid, strates include a sequence having 2,3,4, 5, 6, or 7 amino acids including but not limited to, C.f3-diaminopropionic acid, C.Y- in the N-terminal direction from the proline or proline sub diaminobutyric acid, ornithine, B-homoornithine, arginine, stitute. In the case of FAP inhibitors and/or substrates, these 2, B-homoarginine, homoarginine, lysine, homolysine, B-ho 3, 4, 5, 6, or 7 amino acids include a positively-charged amino acid Such as, but not limited to, C.f3-diaminopropionic acid, molysine, or histidine, and Xaa, is glycine, D-alanine, C.Y-diaminobutyric acid, ornithine, B-homoornithine, argin D-serine, or D-threonine. Alternatively, Xaa, may be absent ine, B-homoarginine, homoarginine, lysine, homolysine, Such that the compound comprises the Formula Ia: B-homolysine, and histidine, and a glycine, D-alanine, (Formula Ia). D-serine, or D-threonine at the P. position. In the case of POP 0057 B is a blocking (protecting) group. Examples of inhibitors and/or substrates, these 2, 3, 4, 5, 6, or 7 amino Such protecting groups include, but are not limited to, ami acids include at least one positively-charged or negatively nobenzoyl (Abz), acetyl (Ac), benzoyl (BZ), carbobenzoxy, charged amino acid, such as but not limited to, C.f3-diamino benzyloxycarbonyl (Z), t-Butyloxycarbonyl (Boc), Fury propionic acid, C.Y-diaminobutyric acid, ornithine, B-ho lacryloyl (Fa), Methoxysuccinyl (MeOSuc), Pyroglutamate moornithine, arginine, B-homoarginine, homoarginine, (Pyr), Pyrazine, Phenylalanine, peptides comprising any lysine, homolysine, 3-homolysine, histidine, aspartic acid, or combination of 1-3 natural amino acids, and Succinyl (Suc). glutamic acid, and a positively-charged amino acid at P. Such Where present, the blocking group B may have a molecular as but not limited to, C.f3-diaminopropionic acid, C.Y-diami weight<400 Da, such as a molecular weight<300 Da. In other nobutyric acid, ornithine, B-homoornithine, arginine, B-ho embodiments, B may be absent such that the compound com moarginine, homoarginine, lysine, homolysine, B-homol prises the Formula Ib: ysine, or histidine. The inhibitors and/or substrates may also comprise a negatively-charged or aromatic amino acid (e.g., (Formula Ib). asn, gln, asp, glu, trp, tyr, and phe) at a position downstream 0.058 Sp is a spacer molecule, which has a length in a (C-terminal direction) from the proline or proline substitute. range of 0.3 nm to 0.6nm to 0.9 nm to 1.2 nm to 1.5 nm to 1.8 0054. In one embodiment of the presently disclosed and nm to 2.1 nm to 2.5 nm (including any Subrange therein, Such claimed inventive concept(s), the inhibitor and/or substrate as 0.3 nm to 1.5 nm). Examples of Such spacer molecules compounds comprise a spacer (linker or filler) group between include, but are not limited to, Y-aminobutyric acid, e-ami the P group and the positively-charged amino acid (e.g., nocaproic acid, 8-amino-3,6-dioxaoctanoic acid, 11-amino arginine) on the N-terminal side. The positively-charged 3,6,9-trioxaundecanoic acid, 14-amino-3,6,9,12-tetraoxatet amino acid, e.g., arg, his, or lys or other listed herein, may be radecanoic acid; C.-aminobutyric acid; 5-aminopentanoic in a position equivalent to P7, P., Ps, or P. The spacer (i.e., acid; 6-aminohexanoic acid; 7-aminoheptanoic acid; 8-ami US 2015/01 19330 A1 Apr. 30, 2015

nooctanoic acid; 3-(aminooxy)acetic acid, B-alanine, gly, ala, Such as 0.3 nm to 1.5 nm). Examples of such spacer molecules thr, trp, tyr, met, leu, ile, val, ser, proline, a PEG.; PPG, an include, but are not limited to, Y-aminobutyric acid; E-ami aminocarboxy PEG, or PPG, or a combination of any of the nocaproic acid; 8-amino-3,6-dioxaoctanoic acid; 11-amino above wherein Sp has a length of 0.3 nm to 2.5 nm, and 3,6,9-trioxaundecanoic acid; 14-amino-3,6,9,12-tetraoxatet wherein n=1-6. radecanoic acid; C.-aminobutyric acid; 5-aminopentanoic 0059 Cyc is a carbocyclic or heterocyclic ring. The car acid; 6-aminohexanoic acid; 7-aminoheptanoic acid; 8-ami bocyclic ring may comprise 4, 5, 6, or 7 carbon atoms, for nooctanoic acid; 3-(aminooxy)acetic acid; B-alanine, gly example. The heterocyclic ring may comprise 4, 5, 6, or 7 cine, alanine, threonine, tryptophan, tyrosine, methionine, atoms, for example, wherein at least one atom is a heteroatom leucine, isoleucine, valine, serine, or proline; PEG (wherein Such as nitrogen or other atom as discussed elsewhere herein. n=1-6); PPG, (wherein n=1-6); an aminocarboxy PEG, or Alternatively, Cyc may be absent such that the compound PPG (wherein n=1-6); or a combination of any of the above, comprises the Formula Ic: So long as Sp has a length of 0.3 nm to 2.5 nm, and. Particular versions of Spare leucine, isoleucine, Valine, and alanine. (Formula Ic). 0.066 Cyc is a carbocyclic or heterocyclic ring. The car 0060. The compound may further comprise one or more bocyclic ring may comprise 4, 5, 6, or 7 carbon atoms, for amino acids extending upstream from the Cyc group, includ example. The heterocyclic ring may comprise 4, 5, 6, or 7 ing, but not limited to, aspartic acid, glutamic acid, glutamine, atoms, for example wherein at least one atom is a heteroatom aspargine, serine, threonine, histidine, tyrosine, alanine, phe Such as nitrogen or other atom as discussed elsewhere herein. nylalanine, glycine, Valine, leucine, isoleucine, methionine, The compound may further comprise one or more amino tyrosine, tryptophan, or any negatively-charged or aromatic acids extending upstream from the Cyc group, including, but amino acid. Examples of Cyc include, but are not limited to, not limited to aspartic acid, glutamic acid, glutamine, those shown in Table 1 and desirably comprise boronyl pro aspargine, serine, threonine, histidine, tyrosine, alanine, phe lines (L, D, or D/L), carbonitrile prolines, or nitrile pyrro nylalanine, glycine, Valine, leucine, isoleucine, methionine, lidines. Non-limiting examples of various FAP inhibitor.com tyrosine, tryptophan, or any negatively-charged or aromatic pounds of the presently disclosed and claimed inventive amino acid. Examples of Cyc include, but are not limited to, concept(s) having the structure of Formula I are shown in those shown in Table 1 and desirably comprise boronyl pro Table 2. These FAP inhibitors are generally also inhibitors of lines (L, D, or D/L), carbonitrile prolines or nitrile pyrro POP. lidines. Non-limiting examples of various POP inhibitor 0061 POP Inhibitors compounds of the presently disclosed and claimed inventive 0062. In one embodiment, POP inhibitors of the presently concept(s) having the structure of Formula II are shown in disclosed and claimed inventive concept(s) comprise com Tables 3-5. pounds having Formula II as shown below: 0067. In a particular embodiment, the inhibitor com B-Xaa1-Sp-Xaa-Cyc (Formula II). pounds of the presently disclosed and claimed inventive con cept(s) are non-immunogenic (i.e., induce no antibody In this embodiment Xaa, is a negatively- or positively response) and have Zero cell membrane permeability, as well charged amino acid, including but not limited to, C.B-diami as a solubility in water of at least 5 mg/ml. nopropionic acid, C.Y-diaminobutyric acid, ornithine, B-ho 0068. The inhibitors described herein may comprise isos moornithine, arginine, B-homoarginine, homoarginine, tere bonds. For example but not by way of limitation, in a lysine, homolysine, 3-homolysine, histidine, aspartic acid, or compound comprising acetyl-arginyl-amino-PEG-carboxy glutamic acid. Alternatively, Xaa, may be absent such that D-alanyl-L-boroproline, the carbonyl of the carboxyl group the compound comprises the Formula IIc: of the arginine moiety may be reduced to a methylene group (Formula IIc). to form a reduced isostere bond between the residual arginine 0063 Xaa, is a positively-charged amino acid, including group and the amino-PEG-carboxy spacer group. Any of the but not limited to, C.B-diaminopropionic acid, C.Y-diami inhibitor compounds of the presently disclosed and claimed nobutyric acid, ornithine, B-homoornithine, arginine, B-ho inventive concept(s) may comprise Such a reduced isostere moarginine, homoarginine, lysine, homolysine, B-homol bond, or may be formed with the regular peptide bond, ysine, or histidine. Xaa, and Xaa, may be the same when between the arginine (or other charged amino acid) and the both are positively-charged. spacer group, i.e., between Xaa (or Xaa) and Sp. The 0064 B is a blocking (protecting) group. Examples of reduced isostere bond is effective in further reducing pepti Such protecting groups include, but are not limited to, ami dase activity upon the compound. nobenzoyl (Abz), acetyl (Ac), benzoyl (BZ), carbobenzoxy, 0069. The compound may further comprise, with, or in benzyloxycarbonyl (Z), t-Butyloxycarbonyl (Boc), Fury place of B, an N-terminal oligopeptide having 1 to 10 amino lacryloyl (Fa), Methoxysuccinyl (MeOSuc), Pyroglutamate acids extending in an N-terminal direction from Xaa, or (Pyr), Pyrazine, Phenylalanine, peptides comprising any Xaa, and/or a C-terminal oligopeptide having 1-10 amino combination of 1-3 natural amino acids, and Succinyl (Suc). acids extending in a C-terminal direction from Cyc, wherein Where present, the blocking group B may have a molecular the N-terminal oligopeptide and C-terminal oligopeptide weight <400 Da, such as a molecular weight <300 Da. In may comprise one or more of the 20 naturally-occurring other embodiments, B may be absent such that the compound amino acids in any combination. comprises the Formula IIa. 0070. In certain embodiments of a FAP inhibitor com pound of the presently disclosed and claimed inventive con (Formula IIa). cept(s), D-ala replaces gly, because it was observed in a 0065 Sp is a spacer molecule which may have a length in Surprising result that this unnatural amino acid significantly a range of 0.3 nm to 0.6nm to 0.9 nm to 1.2 nm to 1.5 nm to amplified inhibitory selectivity of the compound for FAP or 1.8 nm to 2.1 nm to 2.5 nm (including any Subrange therein, APCE versus DPPIV. D-ser and D-thr may also substitute for US 2015/01 19330 A1 Apr. 30, 2015 gly. For example, as shown in Table 7, an inhibitor compound compound may be disposed in a pharmaceutically-acceptable comprising D-ala-L-boroPro had an APCE K, of 5.7 nMand carrier as described elsewhere herein. DPPIV Ki of 6130 nM, while a compound comprising gly 0073 FAP and POP Substrates boroPro had an APCEK, of 1.8 nM and DPPIV K, of 440 nM. 0074 Embodiments of the presently disclosed and Unlike FAP, DPPIV is expressed by normal tissues and is claimed inventive concept(s) include substrates for APCE, involved in normal physiologic reactions; therefore, a high K, FAP, and/or POP, and particularly substrates which are spe (low affinity) for DPPIV is greatly desired. The D-amino cific for POP acid-containing inhibitors may inhibit APCE with a K in the 0075 FAP Substrates low nM range (e.g., such as <100 nM, <50 nM, <20 nM, <15 0076. In one embodiment, the presently disclosed and nM, or <10 nM) and do not significantly inhibit DPPIV (e.g., claimed inventive concept(s) includes substrates of FAP (and >5,000 nM), unless used at unacceptable and unusually high APCE) having Formula III as shown below: concentrations. In a particular version of the presently dis B-Xaa1-Sp-Xaa-Pro-Rep closed and claimed inventive concept(s), the inhibitor com (Formula III). pounds are highly selective for APCE and FAP versus DPPIV. 0077 B is a blocking (protecting) group. Examples of For example, the ratio of K, (DPPIV): K, (APCE/FAP) may be Such protecting groups include, but are not limited to, ami >200, such as >500, or >600, or >700, or >800, or >900, or nobenzoyl (Abz), acetyl (Ac), benzoyl (BZ), carbobenzoxy, >1,000. The K, (DPPIV) may be >200 nM, such as >500 nM, benzyloxycarbonyl (Z), t-Butyloxycarbonyl (Boc), Fury >1,000 nM, >2,500 nM, >4,000 nM, >5,000 nM, >6,000 nM, lacryloyl (Fa), Methoxysuccinyl (MeOSuc), Pyroglutamate >7,500 nM, or >10,000 nM. The molecular weight of the (Pyr), Pyrazine, Phenylalanine, peptides comprising any inhibitor compound of the presently disclosed and claimed combination of 1-3 natural amino acids, and Succinyl (Suc). inventive concept(s) may be <1000 Da, such as <800 Da, or Where present, the blocking group B may have a molecular <600 Da. The inhibitors which are specific for POP may have weight <400 Da, such as a molecular weight <300 Da. In a K (POP)<100 nM while the K, (FAP) is >200 nM or >500 other embodiments, B may be absent such that the compound uM. The inhibitors of POP may have a Ki (DPPIV)>200 nM, comprises the Formula IIIa: such as >500 nM, >1,000 nM, >2,500 nM, >4,000 nM, Xaa-Sp-Xaa-Pro-Rep (Formula IIIa). >5,000 nM, >6,000 nM, >7,500 nM, or >10,000 nM. 0078. In Formula III, Xaa is a positively-charged amino 0071. The inhibitors described herein exhibit very good to acid Such as, but not limited to, C.f3-diaminopropionic acid, excellent selectivity and are highly water soluble; in addition, C.Y-diaminobutyric acid, ornithine, B-homoornithine, argin given this length and the lack of exposed amino-terminal ine, B-homoarginine, homoarginine, lysine, homolysine, amino group, the inhibitors are not prone to cyclization with f-homolysine, or histidine. Xaa, is glycine, D-alanine, resultant loss of activity. In certain embodiments, a chemo D-serine, or D-threonine. therapeutic agent, such as described below, can be linked to 0079 Sp is a spacer molecule comprising one or more of the inhibitor molecule, directly via an exposed amino or car y-aminobutyric acid; e-aminocaproic acid; 8-amino-3,6-di boxy group or via a linking group. In an alternative embodi oxaoctanoic acid; 11-amino-3,6,9-trioxaundecanoic acid; ment, the presently disclosed and claimed inventive concept 14-amino-3,6,9,12-tetraoxatetradecanoic acid; C.-aminobu (s) includes a compound which comprises the 'stalk portion tyric acid; 5-aminopentanoic acid; 6-aminohexanoic acid; of the compound of Formula I, i.e., B-Xaa-Sp-Xaa, (For 7-aminoheptanoic acid; 8-aminooctanoic acid; 3-(aminooxy) mula Ic), wherein Xaa is a positively-charged amino acid, acetic acid; (3-alanine; alanine, threonine, tryptophan, Such as but not limited to, C.f3-diaminopropionic acid, C.Y- tyrosine, methionine, leucine, isoleucine, Valine, serine, pro diaminobutyric acid, ornithine, B-homoornithine, arginine, line; PEG, (n=1-6); PPG, (n=1-6); aminocarboxy PEG, B-homoarginine, homoarginine, lysine, homolysine, B-ho (n=1-6); aminocarboxy PPG, (n=1-6); or a combination of molysine, or histidine (or is absent), and Xaa is glycine, any of the above, so long as Sp has a length in a range of 0.3 D-alanine, D-serine, or D-threonine. The presently disclosed nm to 0.6nm to 0.9 nm to 1.2 nm to 1.5 nm to 1.8 nm to 2.1 and claimed inventive concept(s) also include the stalk por nm to 2.5 nm (including any Subrange therein, Such as 0.3 nm tion of Formula II, i.e., B-Xaa-Sp-Xaa, (Formula IIb). to 1.5 nm). wherein Xaa, is a negatively- or positively-charged amino 0080 Pro is proline or a proline analog which can form a acid, such as but not limited to, C.f3-diaminopropionic acid, Pi—P' bond which is cleavable by FAP. Rep may be absent, C.Y-diaminobutyric acid, ornithine, B-homoornithine, argin or is a reporter group Such as, but not limited to, at least one of ine, B-homoarginine, homoarginine, lysine, homolysine, 7-amido-4-methylcoumarin (AMC), 7-amino-trifluorometh B-homolysine, histidine, aspartic acid, or glutamic acid, and ylcoumarin (AFC), ethyl ester (OEt), methyl ester (OMe), Xaa- is a positively-charged amino acid, such as but not 2-Naphthylamide (2NA), p-Nitroanilide (p-NA), p-Nitrophe limited to, C.f3-diaminopropionic acid, C.Y-diaminobutyric nyl ester (ONp), or Thiobenzyl ester (SBZI). The substrate acid, ornithine, B-homoornithine, arginine, B-homoarginine, may further comprise peptide(s) (n=1-10) extending from the homoarginine, lysine, homolysine, B-homolysine, or histi N-terminal amino acid and/or from the C-terminal amino acid dine (or is absent). Xaa, and Xaa, may be the same when thereof. The compounds of Formula III generally are also both are positively-charged. The “stalks” may be used as Substrates of POP. precursors of the inhibitor compounds, or linked to a targeting 0081 POP Substrates agent or delivery agent, to deliver a “warhead' (i.e., Cyc, or I0082 In another embodiment, the presently disclosed and other compound) to FAP or POP, respectively, for example. claimed inventive concept(s) includes substrates of POP hav 0072 The presently disclosed and claimed inventive con ing Formula IV as shown below: cept(s) is thus directed to compounds comprising or other wise based on these “stalks as a component, and to the use of B-Xaa1-Sp-Xaa-Pro-Rep (Formula IV). these compounds in any therapeutic, diagnostic, or assay B is a blocking (protecting) group. Examples of such protect method described, contemplated, or enabled herein. The ing groups include, but are not limited to, aminobenzoyl US 2015/01 19330 A1 Apr. 30, 2015

(Abz), acetyl (Ac), benzoyl (BZ), carbobenzoxy, benzyloxy compound of Formula IV which is cleavable by POP, and carbonyl (Z), t-Butyloxycarbonyl (Boc), Fury lacryloyl (Fa), which has signaling activity when cleaved by the POP, is Methoxysuccinyl (MeOSuc), Pyroglutamate (Pyr), Pyrazine, obtained along with quantity of POP. The POP is then Phenylalanine, peptides comprising any combination of 1-3 exposed to a POP inhibitor candidate to form a test mixture, natural amino acids, and Succinyl (Suc). Where present, the and the test mixture is combined with the substrate com blocking group B may have a molecular weight <400 Da. pound. The signal emitted from the test mixture/substrate such as <300 Da. In other embodiments, B may be absent compound mixture is measured to identify a POP inhibitor such that the compound comprises the Formula IVa: which inhibits the activity of the POP Xaa1-Sp-Xaa-Pro-Rep (Formula IV). I0089. In one particular embodiment, the presently dis closed and claimed inventive concept(s) is directed to a com 0083. In Formula IV. Xaa, is a negatively- or positively pound (and to a composition containing the compound) hav charged amino acid, such as but not limited to, C.f3-diamino ing the formula: propionic acid, C.Y-diaminobutyric acid, ornithine, B-ho moornithine, arginine, B-homoarginine, homoarginine, B-Xaa1-Sp-Xaa-Cyc (Formula II). lysine, homolysine, 3-homolysine, histidine, aspartic acid, or B is defined as a protecting group or is absent. Xaa, is a glutamic acid. positively-charged or negatively-charged amino-acid, or is 0084. Xaa, is a positively-charged amino acid, Such as absent. Sp is a spacer molecule having a length in the range of but not limited to, C.f3-diaminopropionic acid; C.Y-diami 0.3 nm to 2.5 nm. Xaa, is a positively-charged amino acid. nobutyric acid; ornithine; 3-homoornithine; arginine; (3-ho Cyc is a carbocyclic or heterocyclic compound. moarginine; homoarginine; lysine; homolysine; B-homol 0090 Xaa, may be, for example, C.B-diaminopropionic ysine; or histidine. Xaa, and Xaa, may be the same when acid, C.Y-diaminobutyric acid, ornithine, B-homoornithine, both are positively-charged. arginine, B-homoarginine, homoarginine, lysine, homol 0085 Sp is a spacer molecule comprising one or more of ysine, B-homolysine, histidine, aspartic acid or glutamic acid. y-aminobutyric acid; e-aminocaproic acid; 8-amino-3,6-di Xaa, may be, for example, C.f3-diaminopropionic acid, C.Y- oXaoctanoic acid; 11-amino-3,6,9-trioxaundecanoic acid; diaminobutyric acid, ornithine, B-homoornithine, arginine, 14-amino-3,6,9,12-tetraoxatetradecanoic acid; C.-aminobu B-homoarginine, homoarginine, lysine, homolysine, B-ho tyric acid; 5-aminopentanoic acid; 6-aminohexanoic acid; molysine, or histidine. Sp may be selected from the group 7-aminoheptanoic acid; 8-aminooctanoic acid; 3-(aminooxy) consisting of for example, Y-aminobutyric acid; e-aminoca acetic acid; (3-alanine; alanine, threonine, tryptophan, proic acid; 8-amino-3,6-dioxaoctanoic acid; 11-amino-3,6,9- tyrosine, methionine, leucine, isoleucine, Valine, serine, pro trioxaundecanoic acid; 14-amino-3,6,9,12-tetraoxatetrade line; PEG, (n=1-6); PPG, (n=1-6); aminocarboxy PEG, canoic acid; C.-aminobutyric acid; 5-aminopentanoic acid; (n=1-6); aminocarboxy PPG, (n=1-6); or a combination of 6-aminohexanoic acid; 7-aminoheptanoic acid; 8-aminooc any of the above, so long as Sp has a length in a range of 0.3 tanoic acid; 3-(aminooxy)acetic acid; B-alanine:glycine; ala nm to 0.6nm to 0.9 nm to 1.2 nm to 1.5 nm to 1.8 nm to 2.1 nine; threonine, tryptophan; tyrosine; methionine; leucine; nm to 2.5 nm (including any Subrange therein, such as 0.3 nm isoleucine; Valine; serine; proline; ethylene glycol; PEG, to 1.5 nm). (wherein n=1-6); propylene glycol; PPG, (wherein n=1-6): I0086 Pro is proline or a proline analog which can form a amino-PEG-carboxy (wherein n=1-6); amino-PPG-car P-P bond which is cleavable by POP Rep may be absent, boxy (wherein n=1-6); and combinations thereof. B may be, or is a reporter group Such as, but not limited to, at least one of for example, at least one of aminobenzoyl (Abz), acetyl (Ac), 7-amido-4-methylcoumarin (AMC), 7-amino-trifluorometh benzoyl (BZ), benzyloxycarbonyl (Z), t-Butyloxycarbonyl ylcoumarin (AFC), ethyl ester (OEt), methyl ester (OMe), (Boc), Furylacryloyl (Fa), Methoxysuccinyl (MeOSuc), 2-Naphthylamide (2NA), p-Nitroanilide (p-NA), p-Nitrophe Pyroglutamate (Pyr), Pyrazine, Phenylalanine, a 1-3 merpep nyl ester (ONp), or Thiobenzyl ester (SBZI). The substrate tide, and Succinyl (Suc). may further comprise peptides (n=1-10) extending from the 0091 Cyc may be, for example, a 4, 5, 6, or 7-member N-terminal amino acid and/or from the C-terminal amino carbon carbocycle. Cyc may be, for example, a 4, 5, 6, or acid. 7-member carbon heterocycle, and may comprise a nitrogen I0087 Screening for APCE, FAP and POP Inhibitors heteroatom. Cyc may comprise, for example, a boronylpro 0088 Certain embodiments of the presently disclosed and line, proline carbonitrile, nitrile pyrrolidone, or cyanopyrro claimed inventive concept(s) also include methods of screen lidine. Sp may be selected from the group consisting of for ing for inhibitors of APCE, FAP, and/or POP. In the method, example, ethylene glycol, PEG, (wherein n=1-6), propylene at least one of an APCE, FAP or POP enzyme substrate such glycol, 8-amino-3,6-dioxaoctanoic acid, PPG, (wherein n=1- as is contemplated herein is provided; the Substrate may par 6)), amino-PEG-carboxy group (wherein n=1-6), an amino ticularly comprise a reporter group. A quantity of at least one PPG-carboxy group (wherein n=1-6), and combinations of APCE, FAP, and POP if provided, and the enzyme is thereof. The compound may comprise an isostere bond exposed to an inhibitor candidate to form a test mixture. The between Xaa, and Sp. Xaa, may comprise, for example, a test mixture is combined with the substrate, and the fluores methylene group in Substitution for the carbonyl group adja cence emission from the test mixture is measured to identify cent Sp. Sp may have a length in a range of 0.6nm to 1.75 nm. when the activity of the enzyme is inhibited by the enzyme Sp may be, for example, leucine, isoleucine, Valine, or ala inhibitor candidate. Any cleavable substrate of APCE, FAP, nine. The compound may further comprise a 1-10mer peptide and/or POP which produces an observable signal (fluores or oligopeptide extending from Cyc in the C-terminal direc cence or other signal). Such as discussed elsewhere herein, tion. may be used. In one embodiment, the presently disclosed and 0092. In one embodiment, the compound is capable of claimed inventive concept(s) is directed to a method of binding to the active site of POPata K.<100 nM, <50 nM, or screening for inhibitors of POP. In the method, a substrate <20 nM, for example, and capable of binding to DPPIV at a US 2015/01 19330 A1 Apr. 30, 2015

Ki>500 nM, or >1000 nMorgreater and/or has a Ki (DPPIV): absent. Sp is a spacer molecule having a length in the range of Ki (POP) ratio >500. In one embodiment B is an acetyl or 0.3 nm to 2.5 nm. Xaa, is a positively-charged amino acid. pyrazine; Xaa, is lysine; Xaa, is arginine; and Cyc is 0098. The presently disclosed and claimed inventive con L-boroproline. In one embodiment the compound is com cept(s) is further directed to a method of inhibiting activity of bined with a pharmaceutically-acceptable carrier or vehicle prolyl oligopeptidase (POP) in a subject suffering from a to form a pharmaceutical composition. disorder for which inhibition of POP provides a therapeuti 0093. In another particular embodiment of the presently cally-effective benefit. In the method, any of the POP inhibi disclosed and claimed inventive concept(s), a pharmaceutical tors described herein is administered to a subject in need of composition is provided that includes any of the compounds Such therapy. described herein disposed within or otherwise combined with 0099. In one embodiment, the therapeutically-effective a pharmaceutically-acceptable carrier or vehicle. In one benefit is the inhibition of angiogenesis. In one embodiment, embodiment, the compound is the compound of Formula II as the compound is the compound of Formula II or Formula IIb described herein above. In particular embodiments, Xaa, of as described herein above. the compound is lysine, and the Sp of the compound is leu 0100 Certain embodiments of the presently disclosed and cine, isoleucine, Valine, or alanine. claimed inventive concept(s) include FAP and POP inhibitor 0094. Yet another particular embodiment of the presently compounds described herein which are conjugated to carrier disclosed and claimed inventive concept(s) is directed to a compounds which are able to pass through the cell mem method of inhibiting activity of prolyl oligopeptidase (POP) brane, including, but not limited to, protein transduction in a POP-expressing cell or tissue, comprising administering domains (PTDs). PTDs are positively charged peptides or to the POP-expressing cell or tissue any of the compounds peptide-like molecules that permeate cell membrane lipid described herein. In one embodiment, the compound is the bilayers. Typically PTDs contain several arginine residues compound of Formula II as described herein. The POP-ex and can be used to deliver other agents, such as peptides, pressing cells or tissues to which the compound is adminis proteins, oligonucleotides or Small molecules through a cell tered may be cancer cells and/or activated fibroblast cells. The membrane and into the cytosol. One PTD is a highly efficient compound may be administered to cells and/or tissues in vitro molecular transporter formed by synthesizing an oligomer of or in vivo. For example, the compound may be administered arginines alternating with EACAS. PTDs are well-known in to a patient, such as but not limited to, a mammalian patient. the art. Examples of PTDs which may be used herein are In one embodiment of the method, formation of acetyl-ser shown, for example, in U.S. Pat. Nos. 7,166.692; 7,217,539; asp-lys-pro is inhibited upon administration of the compound 7,053,200; 6,835,810; 6,645,501; and Published U.S. Patent to the cells or tissues. Applications 2002/0009491; 2003/0032593; 2003/01627 19: 2006/0159719, 2006/0293234; and 2007/0105775, each of 0095. Another particular embodiment of the presently dis which is expressly incorporated herein in its entirety by ref closed and claimed inventive concept(s) is directed to a method of inhibiting angiogenesis in a tissue. In the method, CCC. a pharmaceutically acceptable amount of any of the com 0101 Similarly, based on the results showing that the pounds described herein is administered to a tissue exhibiting inhibitors likewise inhibit FAP with high sensitivity and angiogenesis and/or having the potential to exhibit angiogen specificity, the inhibitors can be used to selectively inhibit the esis, thereby inhibiting angiogenesis in the tissue. In one proteolytic activity of FAP on cell surfaces offibroblasts and embodiment, the compound is the compound of Formula II as cancer cells towards collagen within the extracellular matrix, described herein above. The tissue exhibiting angiogenesis and without impacting other prolyl-specific proteinases (e.g., and/or having the potential to exhibit angiogenesis to which DPPIV) for which it has no specificity thus enabling analysis the compound is administered may comprise cancer cells of various characteristics of the sample with other prolyl and/or activated fibroblast cells. The compound may be specific proteinases present while FAP is inhibited. Further, administered to cells and/or tissues in vitro or in vivo. For the high aqueous solubility of the particular inhibitors (>500 example, the compound may be administered to a patient, ug/ml) indicates they will not permeabilize the universal Such as but not limited to, a mammalian patient. In one highly lipophilic, hydrophobic nature of cell membranes. embodiment of the method, formation of acetyl-ser-asp-lys 01.02 Utility pro is inhibited upon administration of the compound to the 0103) Further to, and in addition to the utilities already cells or tissues. described hereinabove, in one embodiment, a subject may be treated with a compound described herein in a manner and in 0096. Yet another particular embodiment of the presently an amount So as to treat any of the conditions, diseases or disclosed and claimed inventive concept(s) is directed to a disorders described herein. For example, the compounds can method of treating cancer in a Subject. In the method, a be used to inhibit proliferation of a primary tumor, or to therapeutically-effective amount of any of the compounds inhibit metastatic spread or growth while minimizing the described herein is administered to a subject in need of such potential for systemic toxicity. In certain embodiments, the therapy. In one embodiment, the compound is the compound abnormal mammalian cell proliferation is manifested as a of Formula II as described herein above. tumor. Some conditions intended to be treated by the present 0097 Another particular embodiment of the presently dis methods using the present compounds include benign (i.e., closed and claimed inventive concept(s) is directed to a com non-cancerous), pre-malignant and malignant (i.e., cancer pound (and to a composition containing the compound) hav ous) tumors especially those characterized by angiogenesis. ing the formula: In some embodiments, the condition characterized by abnor B-Xaa-Sp-Xaa- (Formula IIb). mal mammalian cell proliferation is further characterized by the presence of reactive stromal fibroblasts. Inhibitors of POP B is defined as a protecting group or is absent. Xaa, is a described herein are intended to inhibitangiogenesis, particu positively-charged or negatively-charged amino-acid, or is larly angiogenesis which is related to Such malignancies. US 2015/01 19330 A1 Apr. 30, 2015

Inhibitors of POP and FAP described herein may also be used ment, an agent in an amount effective to inhibit angiogenesis to treat conditions which exhibit excessive or undesirable in an abnormal proliferative cell mass, wherein the agent is an stromal growth Such as idiopathic pulmonary fibrosis, rheu inhibitor as described herein. matoid arthritis, and peritoneal and pleural adhesions. Effec 0109. In one embodiment, an inhibitor having Formula I tive treatment is defined in at least one embodiment as result or II, or a combination thereof, is provided along with at least ing in reduced tumor growth and/or tumor shrinkage, defined, one other anti-cancer compound (i.e., an anti-cancer com for example, as a reduction in tumor volume of at least a 10%, pound other than a compound having Formula I or Formula II or at least 25%, or at least 50% after a predetermined course or as otherwise contemplated herein), and a pharmaceutically of treatment. acceptable carrier. In another aspect, a pharmaceutical prepa 0104. In other embodiments, the abnormal mammalian ration is provided which comprises a compound having For cell proliferation treated with the presently described inhibi mula I or Formula II, or a combination thereof, at least one tors is a carcinoma, a sarcoma, or a melanoma or others other anti-angiogenic compound (i.e., an anti-angiogenic described elsewhere herein. More particularly, the condition compound other than a compound of Formula I or Formula II, may be, but is not limited to, a breast cancer, colorectal or as otherwise contemplated herein), and a pharmaceutically cancer, ovarian cancer, prostate cancer, pancreatic cancer, acceptable carrier. kidney cancer, lung cancer, melanoma, or fibrosarcoma, or bone and connective tissue sarcomas, including, but not lim 0110. In other embodiments, anti-cancer cocktails con ited to, osteosarcoma and fibrosarcoma. The abnormal mam taining an inhibitor compound of the presently disclosed and malian cell proliferation may be epithelial cell-derived, claimed inventive concept(s) and other anti-proliferative meaning that it is epithelial cells which are abnormally pro compounds and/or other anti-angiogenic compounds as liferating. Some conditions characterized by abnormal mam described herein are also provided. In still other embodi malian epithelial cell proliferation include adenomas of epi ments, compounds having the Formula I or Formula II, or thelial tissues such as the breast, colon, pancreas, lung, and combinations thereof, are used in the preparation of a medi prostate, as well as malignant tumors identified above. cament for treating Subjects having conditions characterized According to other embodiments of the presently disclosed by abnormal mammalian cell proliferation. and claimed inventive concept(s), a method is provided for 0111. In still other embodiments, the inhibitory compound treating a Subject having a metastasis of epithelial origin. may be targeted to a cell mass (e.g., a tumor) through the use 0105. According to some embodiments of the presently of a targeting compound specific for a particular tissue or disclosed and claimed inventive concept(s), the inhibitor tumor type. In some embodiments, the inhibitors may be (agent) is administered locally. In some embodiments, the targeted to primary or in some instances, secondary (i.e., agent is targeted to a tumor. This can be achieved by the metastatic) lesions through the use of targeting compounds particular mode of administration. For example, certain more which preferentially recognize a cell Surface marker. easily accessible tumors such as breast or prostate tumors 0.112. As described herein, the inhibitors and substrates of may be targeted by direct needle injection to the site of the the presently disclosed and claimed inventive concept(s) lesion. Lung tumors may be targeted, for example, by the use comprise peptides or peptidomimetics which may include of inhalation as a route of administration. non-amino acid residues such as saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or 0106. In some embodiments, the agents may be adminis structural analogs of the above, or combinations thereof and tered in a systemic manner, via administration routes such as, the like. In particular, as described herein, it is possible to but not limited to, oral, intravenous, intramuscular and intra Substitute non-naturally occurring amino acids as described peritoneal administration. Systemic administration routes hereinforthe "P" proline residue. In one embodiment, the P. may be desired, for example, if the Subject has metastatic group is an analog of proline in which the carboxylate group lesions. In other embodiments, the agent is administered in a (COOH) is replaced with a boronyl group (BOH). Alterna Sustained release formulation. tive compounds of the presently disclosed and claimed inven 0107. In administering the present compounds to subjects, tive concept(s) have an analogous structure in which the dosing amounts, dosing schedules, routes of administration boronyl group is replaced by, for example, a nitrile, a carbo and the like may be selected so as to affect the other known nitrile, carboxynitrile, a phosphonate or a fluoroalkylketone, activities of these compounds. For example, amounts, dosing alphaketos, N-peptiolyl-3-(acylhydroxylamines), azapep schedules and routes of administration can be selected as tides, azetidines, fluoroolefins dipeptide isoesteres, peptidyl described herein, whereby therapeutically effective levels for (alpha-aminoalkyl) phosphonate esters, aminoacyl pyrroli inhibiting proliferation are provided, yet are provided at lev dine-2-nitriles and 4-cyanothiazolidides, or other structures els which do not affect other proteins (e.g., enzymes neces for example as shown in Table 1. sary for healthy function such as DPPIV) in the subject. In 0113. As noted herein, certain embodiments of the pres Some embodiments of the presently disclosed and claimed ently disclosed and claimed inventive concept(s) include inventive concept(s), a method is provided in which the methods for treating a Subject having a condition character inhibitor is administered in combination with surgery (before, ized by an abnormal cell proliferation or other conditions during, or after) to remove an abnormal proliferative cell described herein. As used herein, the term subject is intended a SS. to refer to a mammal including, but not limited to, humans, 0108. In another aspect, the FAP and/or POP inhibitors as apes, monkeys, other nonhuman primates, dogs, cats, sheep, described herein may be used in treatment for inhibiting llamas, goats, horses, cattle, Zoo animals, pigs, and rodents. angiogenesis in a Subject having a condition characterized by As used herein, the terms Subject and patient are used inter abnormal mammalian cell proliferation, such as a cancer, changeably. An abnormal mammalian cell proliferation dis comprising administering to a Subject in need of Such treat order or condition, as used herein, refers to a localized region US 2015/01 19330 A1 Apr. 30, 2015 13 of cells (e.g., a tumor) which exhibit an abnormal (e.g., tumors, also referred to as carcinomas, and secondary tumors, increased) rate of division as compared to their normal tissue also referred to as metastases of epithelial origin. Carcinomas counterparts. intended for treatment with the methods of the presently 0114. In one aspect, as noted, the presently disclosed and disclosed and claimed inventive concept(s) include, but are claimed inventive concept(s) includes methods for treating a not limited to, acinar carcinoma, acinous carcinoma, alveolar Subject having a condition characterized by an abnormal epi adenocarcinoma (also called adenocystic carcinoma, thelial cell proliferation. Epithelial cells are cells occurring in adenomyoepithelioma, cribriform carcinoma and cylin one or more layers which cover the entire surface of the body droma), carcinoma adenomatosum, adenocarcinoma, carci and which line most of the hollow structures of the body, noma of adrenal cortex, alveolar carcinoma, alveolar cell excluding the blood vessels, lymph vessels, and the heart carcinoma (also called bronchiolar carcinoma, alveolar cell interior which are lined with endothelium, and the chest and tumor and pulmonary adenomatosis), basal cell carcinoma, abdominal cavities which are lined with mesothelium. carcinoma basocellulare (also called basaloma, or basiloma, Examples of such epithelial cells include, but are not limited and hair matrix carcinoma), basaloid carcinoma, basosqua to, cells of the anterius corneae, anteriorepithelium of cornea, mous cell carcinoma, breast carcinoma, bronchioalveolar Barrett's epithelium, capsular epithelium, ciliated epithe carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, lium, columnar epithelium, corneal epithelium, cubical epi cerebriform carcinoma, cholangiocellular carcinoma (also thelium, cuboidal epithelium, epithelium eductus semicircu called cholangioma and cholangiocarcinoma), chorionic car laris, enamel epithelium, false epithelium, germinal cinoma, colloid carcinoma, comedo carcinoma, corpus car epithelium, gingival epithelium, glandular epithelium, glom cinoma, cribriform carcinoma, carcinoma en cuirasse, carci erular epithelium, laminated epithelium, epithelium of lens, noma cutaneum, cylindrical carcinoma, cylindrical cell epithelium lentis, mesenchymal epithelium, olfactory epithe carcinoma, duct carcinoma, carcinoma durum, embryonal lium, pavement epithelium, pigmentary epithelium, pig carcinoma, encephaloid carcinoma, epibulbar carcinoma, mented epithelium, protective epithelium, pseudostratified epidermoid carcinoma, carcinoma epitheliale adenoides, car epithelium, pyramidal epithelium, respiratory epithelium, cinoma exulcere, carcinoma fibrosum, gelatiniform carci rod epithelium, seminiferous epithelium, sense epithelium, noma, gelatinous carcinoma, giant cell carcinoma, giganto sensory epithelium, simple epithelium, squamous epithe cellulare, glandular carcinoma, granulosa cell carcinoma, lium, Stratified epithelium, Subcapsular epithelium, Sulcular hair-matrix carcinoma, hematoid carcinoma, hepatocellular epithelium, tessellated epithelium, and transitional epithe carcinoma (also called hepatoma, malignant hepatoma and lium. hepatocarcinoma), Hurthle cell carcinoma, hyaline carci 0115 One category of conditions characterized by abnor noma, hypernephroid carcinoma, infantile embryonal carci mal epithelial cell proliferation is proliferative dermatologic noma, carcinoma in situ, intraepidermal carcinoma, intraepi disorders. These include, but are not limited to, conditions thelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell Such as keloids, seborrheic keratosis, papilloma virus infec carcinoma, lenticular carcinoma, carcinoma lenticulare, tion (e.g., producing Verruca Vulbaris, Verruca plantaris, Ver lipomatous carcinoma, lymphoepithelial carcinoma, carci ruca plana, condylomata, etc.) and eczema. An epithelial noma mastitoides, carcinoma medullare, medullary carci precancerous lesion is a skin lesion which has a propensity to noma, carcinoma melanodes, melanotic carcinoma, muci develop into a cancerous condition. Epithelial precancerous nous carcinoma, carcinoma muciparum, carcinoma skin lesions also arise from other proliferative skin disorders mucocellulare, mucoepidermoid carcinoma, carcinoma Such as hemangiomas, keloids, eczema, and papilloma virus mucosum, mucous carcinoma, carcinoma myxomatodes, infections producing verruca Vulbaris, Verruca plantaris and nasopharyngeal carcinoma, carcinoma nigrum, oat cell car Verruca planar. The symptoms of the epithelial precancerous cinoma, carcinoma ossificans, osteoid carcinoma, ovarian lesions include skin-colored or red-brown macule or papule carcinoma, papillary carcinoma, periportal carcinoma, prein with dry adherent scales. Actinic keratosis is the most com vasive carcinoma, prostate carcinoma, renal cell carcinoma of mon epithelial precancerous lesion among fair skinned indi kidney (also called adenocarcinoma of kidney and hypeme viduals. It is usually present as lesions on the skin which may phoroid carcinoma), reserve cell carcinoma, carcinoma Sar or may not be visually detectable. The size and shape of the comatodes, Scheinderian carcinoma, Scirrhous carcinoma, lesions varies. This is a photosensitive disorder and may be carcinoma Scroti, signet-ring cell carcinoma, carcinoma sim aggravated by exposure to Sunlight. Bowenoid actinic kera plex, Small-cell carcinoma, Solanoid carcinoma, spheroidal tosis is another form of an epithelial precancerous lesion. In cell carcinoma, spindle cell carcinoma, carcinoma Spongio Some cases, the lesions may develop into an invasive form of Sum, Squarnous carcinoma, squamous cell carcinoma, string squamous cell carcinoma and may pose a significant threat of carcinoma, carcinoma telangiectaticum, carcinoma telang metastasis. Other types of epithelial precancerous lesions iectodes, transitional cell carcinoma, carcinoma tuberosum, include, but are not limited to, hypertrophic actinic keratosis, tuberous carcinoma, Verrucous carcinoma, carcinoma vilo arsenical keratosis, hydrocarbon keratosis, thermal keratosis, sum. In certain embodiments, the methods of the inventive radiation keratosis, viral keratosis, Bowen's disease, eryth concept(s) are used to treat Subjects having cancer in at least roplaquia of queyrat, oral erythroplaquia, leukoplakia, and one or more of the breast, cervix, ovary, prostate, lung, colon, intraepidermal epithelialoma. rectum, pancreas, stomach and kidney. 0116. As noted above, another category of conditions 0117. Other conditions characterized by an abnormal characterized by abnormal epithelial cell proliferation is mammalian cell proliferation to be treated by the methods tumors of epithelial origin. Epithelial tumors are known to described herein include, but are not limited to, sarcomas. those of ordinary skill in the art and include, but are not Sarcomas are rare mesenchymal neoplasms that arise in bone limited to, benign and premalignant epithelial tumors, such as and soft tissues. Different types of sarcomas are recognized breast fibroadenoma and colon adenoma, and malignant epi and these include: liposarcomas (including myxoid liposar thelial tumors. Malignant epithelial tumors include primary comas and pleiomorphic liposarcomas), leiomyosarcomas, US 2015/01 19330 A1 Apr. 30, 2015 rhabdomyosarcomas, malignant peripheral nerve sheath require angiogenesis particularly for oxygen and nutrient tumors (also called malignant Schwannomas, neurofibrosar Supply. It has been previously shown that inhibition of angio comas, or neurogenic sarcomas), Ewings tumors (including genesis in Solid tumor can cause tumor regression in animal Ewing's sarcoma of bone, extraskeletal Ewing's sarcoma, models. Thus in one aspect, the presently disclosed and and primitive neuroectodermal tumor), synovial sarcoma, claimed inventive concept(s) relates to methods for inhibiting angiosarcomas, hemangiosarcomas, lymphangiosarcomas, angiogenesis by inhibiting the proliferation, migration or Kaposi's sarcoma, hemangioendothelioma, fibrosarcoma, activation of endothelial cells and fibroblasts, wherein this desmoid tumor (also called aggressive fibromatosis), der angiogenesis is unrelated to wound healing in response to matofibrosarcoma protuberans, malignant fibrous histiocy injury, infection or inflammation. toma, hemangiopericytoma, malignant mesenchymoma. 0.122 Thus, the present methods are intended for the treat alveolar soft-part sarcoma, epithelioid sarcoma, clear cell ment of diseases and processes that involve or are mediated sarcoma, desmoplastic Small cell tumor, gastrointestinal stro by angiogenesis including, but not limited to, hemangioma, mal tumor (also known as GI stromal sarcoma), osteosarcoma Solid tumors, tumor metastasis, benign tumors, for example (also known as osteogenic sarcoma)-skeletal and extraskel hemangiomas, acoustic neuromas, neurofibromas and tra etal, and chondrosarcoma. chomas, Osler-Webber Syndrome, telangiectasia, myocar 0118. The methods of the presently disclosed and claimed dial angiogenesis, angiofibroma, plaque neovascularization, inventive concept(s) also include the treatment of Subjects coronary collaterals, ischemic limb angiogenesis, corneal with melanoma. Melanomas are tumors arising from the mel diseases, rubiosis, neovascular glaucoma, diabetic retinopa anocytic system of the skin and other organs. Examples of thy, retrolental fibroplasia, diabetic neovascularization, melanoma include lentigo maligna melanoma, Superficial macular degeneration, keloids, ovulation, menstruation, pla spreading melanoma, nodular melanoma, and acral lentigi centation, and any cancer involving angiogenesis. nous melanoma. I0123. The compositions and methods of the presently dis 0119 Conditions characterized by an abnormal mamma closed and claimed inventive concept(s) in certain instances lian cell proliferation as noted are cancers including, but not may be useful for replacing existing Surgical procedures or limited to, biliary tract cancer, endometrial cancer, esoph drug therapies, although in most instances the methods are ageal cancer, gastric cancer, pancreatic cancer, intraepithelial useful in improving the efficacy of existing therapies for neoplasms, including Bowen's disease and Paget’s disease, treating Such conditions. Accordingly combination therapy liver cancer, oral cancer, including squamous cell carcinoma, may be used to treat the subjects. For example, the inhibitors sarcomas, including fibrosarcoma and osteosarcoma, skin may be administered to a subject in combination with another cancer, including melanoma, Kaposi's sarcoma, testicular anti-proliferative (e.g., an anti-cancer) therapy. Suitable anti cancer, including germinal tumors (seminoma, non-semi cancer therapies include, but are not limited to, Surgical pro noma (teratomas, choriocarcinomas)), stromal tumors and cedures to remove the tumor mass, chemotherapy or local germ cell tumors, thyroid cancer, including thyroid adenocar ization radiation. The other anti-proliferative therapy may be cinoma and medullar carcinoma, and renal cancer including administered before, concurrent with, or after treatment with adenocarcinoma and Wilms tumor. the inhibitors of the presently disclosed and claimed inventive 0120) Further, certain embodiments of the presently dis concept(s). There may also be a delay of several hours, days closed and claimed inventive concept(s) include a method of and in some instances weeks between the administration of treating a subject having an abnormal proliferation originat the different treatments, such that the inhibitor may be admin ing in bone, muscle or connective tissue. Exemplary condi istered before or after the other treatment. As an example, the tions intended for treatment by the present methods include inhibitor may be administered in combination with Surgery to primary tumors (i.e., sarcomas) of bone and connective tis remove an abnormal proliferative cell mass. As used herein, sue. The methods also include treatment of subjects with “in combination with Surgery' means that the agent may be metastatic tumors, for example metastatic tumors of epithe administered prior to, during or after the Surgical procedure. lial origin. Carcinomas may metastasize to bone, as has been 0.124. The subjects treated with the presently disclosed observed with breast cancer, and liver, as is sometimes the FAP and/or POP inhibitors may be treated in combination case with colon cancer. The methods of the presently dis with other non-Surgical anti-proliferative (e.g., anti-cancer) closed and claimed inventive concept(s) are intended to treat drug therapy. In one embodiment, the inhibitor may be metastatic tumors regardless of the site of the metastasis administered in combination with an anti-cancer compound and/or the site of the primary tumor. Such as a cytostatic compound. A cytostatic compound is a 0121 The presently disclosed and claimed inventive con compound (e.g., a nucleic acid, or a protein) that Suppresses cept(s) includes methods for inhibiting POP and/or FAP in a cell growth and/or proliferation. In some embodiments, the Subject having a pathology which involves angiogenesis. cytostatic compound is directed towards the malignant cells Angiogenesis is defined as the formation of new blood ves of a tumor. In yet other embodiments, the cytostatic com sels. These disorders include conditions characterized by pound is one which inhibits the growth and/or proliferation of abnormal mammalian cell proliferation, such as cancerous vascular smooth muscle cells or fibroblasts. conditions wherein overexpression of FAP and/or POP asso 0.125. The presently disclosed and claimed inventive con ciated with the tumors stimulates angiogenesis and rapid cept(s) in one embodiment is directed to a method of treating tumor growth, as well as non-cancer conditions including cancer comprising administering to a patient in need thereof diabetes, diabetic retinopathy, neovascular glaucoma and a cancer treatment comprising radiation and/or an effective psoriasis. Thus, in particular embodiments, the present meth amount of a chemotherapeutic composition, and administer ods are aimed using the disclosed FAP and/or POP inhibitors ing at least one compound of the presently disclosed and to inhibit tumor and/or non-tumor angiogenesis. Tumor claimed inventive concept(s), with pharmaceutical accept angiogenesis refers to the formation of new blood vessels in able additives, diluents, carriers and excipients, and pharma the vicinity or within a tumor mass. Solid tumor cancers ceutically acceptable salts thereof. US 2015/01 19330 A1 Apr. 30, 2015

0126 The presently disclosed and claimed inventive con chloride; Elsamitrucin; Enloplatin; Enpromate: Epipropi cept(s) also provides the use of compositions which comprise dine: Epirubicin Hydrochloride; Erbulozole; Esorubicin of one or more of the compounds of the presently disclosed Hydrochloride; Estramustine; Estramustine Phosphate and claimed inventive concept(s), their derivatives, metabo Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Eto lites, analogues and/or mimic molecules with pharmaceutical prine: Fadrozole Hydrochloride; Fazarabine: Fenretinide; acceptable additives, diluents, carriers and excipients and Floxuridine: Fludarabine Phosphate: Fluorouracil; Fluoroc pharmaceutically acceptable salts thereof, for the manufac itabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gem ture of a medicament for a cancerous condition. As noted citabine Hydrochloride; Hydroxyurea; Idarubicin Hydro elsewhere herein, the pharmaceutical formulations may be chloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a: administered in combination (before or simultaneously) with Interferon Alfa-2b: Interferon Alfa-n1; Interferon Alfa-n3; other therapeutic treatments, such as radiation treatment or Interferon Beta-Ia, Interferon Gamma-Ib; I proplatin; Irino chemotherapeutic drugs. tecan Hydrochloride; Lanreotide Acetate; Letrozole; Leupro 0127. The presently disclosed and claimed inventive con lide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; cept(s) is exemplified in terms of in vitro and in vivo activity Lomustine; Losoxantrone Hydrochloride; Masoprocol; May against various neoplastic cell lines. The test cell lines tansine; Mechlorethamine Hydrochloride; Megestrol employed in the in vitro assays are well recognized and Acetate; Melengestrol Acetate; Melphalan: Menogaril; Mer accepted as models for anti-tumor activity in animals. The captopurine; Methotrexate; Methotrexate Sodium; Meto term animals as used herein includes, but is not limited to, prine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin: mice, rats, domesticated animals such as but is not limited to, Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane: cats, dogs, and other animals but is not limited to, cattle, Mitoxantrone Hydrochloride: Mycophenolic Acid; Nocoda sheep, pigs, horses, and primates Such as but not limited to, Zole; Nogalamycin; Ormaplatin: Oxisuran: Paclitaxel; Pegas monkeys, humans and more generally mammals. pargase; Peliomycin; Pentamustine: Peplomycin Sulfate; 0128. In one aspect, certain embodiments of the presently Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydro disclosed and claimed inventive concept(s) feature the use of chloride; Plicamycin; Plomestane: Porfimer Sodium; Porfiro an inhibitory compound of the presently disclosed and mycin; Prednimustine; Procarbazine Hydrochloride; Puro claimed inventive concept(s) as a chemosensitizer, in combi mycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; nation with at least one other chemotherapeutic agent or Rogletimide: Safingol: Safingol Hydrochloride; Semustine: radiation dosage. In a particular embodiment, the compound SimtraZene, Sparfosate Sodium; Sparsomycin; Spirogerma is co-administered with the chemotherapeutic agent, to a sub nium Hydrochloride; Spiromustine; Spiroplatin: Streptoni ject. In a particular embodiment, the compound is co-admin grin: Streptozocin; Sulofenur; Talisomycin; Taxol; Taxotere: istered with repeated dosages of the same, or a different Tecogalan Sodium; Tegafur, Teloxantrone Hydrochloride: chemotherapeutic agent, to a Subject. In a particular embodi Temoporfin; Teniposide; Teroxirone; Testolactone: Thiami ment, the inhibitory compound of the presently disclosed and prine; Thioguanine: Thiotepa; Tiazofurin; Tirapazamine; claimed inventive concept(s) enhances the efficacy of the Topotecan Hydrochloride; Toremifene Citrate; Trestolone chemotherapeutic agent, e.g., a cytotoxic agent or radiation Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate dosage, relative to the effect of the cytotoxic agent or radia Glucuronate; Tubulozole Hydrochloride; Uracil Mustard; tion dosage in the absence of the compound. The inhibitory Uredepa; Vapreotide: Verteporfin; Vinblastine Sulfate; Vinc compound may be used in combination therapy with conven ristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sul tional cancer chemotherapeutics or treatments. Conventional fate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine treatment regimens for tumors include radiation, antitumor Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; agents, interferons, interleukins, tumor necrosis factors, or a Zeniplatin: Zinostatin; and Zorubicin Hydrochloride. combination of two or more of these agents, as well as other 0.130. Other anti-cancer drugs include, but are not limited chemotherapeutic (cytotoxic) agents described herein. to: 20-epi-1.25 dihydroxyvitamin D3; 5-ethynyluracil; abi 0129 Suitable anti-proliferative drugs or cytostatic com raterone; acylfulvene; adecypenol; adoZelesin; ALL-TK pounds to be used in combination with the presently disclosed antagonists; ambamustine; amidox; amifostine; aminole and claimed inhibitors include anti-cancer drugs. Numerous Vulinic acid; amrubicin; anagrelide; andrographolide; angio anti-cancer drugs which may be used are well known and genesis inhibitors; antagonist D; antagonist G, antarelix; anti include, but are not limited to: Acivicin; Aclarubicin; Acoda dorsalizing morphogenetic protein-1, antiestrogen; Zole Hydrochloride: Acronine; Adozelesin; Aldesleukin; antineoplaston; antisense oligonucleotides; aphidicolin gly Altretamine; Ambomycin; Ametantrone Acetate; Aminoglu cinate; apoptosis gene modulators; apoptosis regulators; apu tethimide; Amsacrine; AnastroZole; Anthramycin; Asparagi rinic acid; ara-CDP-DL-PTBA; arginine deaminase; asula nase; Asperlin; AZacitidine, AZetepa, Azotomycin; Batimas crine; atamestane; atrimustine; axinastatin 1; axinastatin 2: tat; Benzodepa; Bicalutamide: Bisantrene Hydrochloride; axinastatin 3; aZasetron; azatoxin; aZatyrosine; baccatin III Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequi derivatives: balanol; batimastat; BCR/ABL antagonists; ben nar Sodium; Bropirimine; Busulfan; Cactinomycin; Caluster Zochlorins; benzoylstaurosporine; beta lactam derivatives; one; Caracemide: Carbetimer; Carboplatin: Carmustine: beta-alethine; betaclamycin B; betulinic acid; bFGF inhibi Carubicin Hydrochloride; Carzelesin; Cedefingol; Chloram tor; bisaziridinylspermine; bisnafide; bistratene A: breflate: bucil; Cirolemycin; Cisplatin: Cladribine; Crisinatol Mesy budotitane; buthionine Sulfoximine; calcipotriol; callphostin late: Cyclophosphamide; Cytarabine; Dacarbazine; Dactino C; camptothecin derivatives; canarypox IL-2; capecitabine; mycin; Daunorubicin Hydrochloride; Decitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest Dexormaplatin, DeZaguanine; DeZaguanine Mesylate; M3; CARN 700; cartilage derived inhibitor; casein kinase Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydro inhibitors (ICOS); castanospermine; cecropin B; cetrorelix: chloride; Droloxifene; Droloxifene Citrate; Dromostanolone chlorins; chloroquinoxaline Sulfonamide; cicaprost; cis-por Propionate; Duazomycin; Edatrexate. Eflornithine Hydro phyrin, clomifene analogues; clotrimazole; collismycin A; US 2015/01 19330 A1 Apr. 30, 2015 collismycin B; combretastatin A4, combretastatin analogue; phosphatase inhibitors; purine nucleoside phosphorylase conagenin, crambescidin 816; crisinatol; cryptophycin 8: inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemo cryptophycin A derivatives; curacin A; cyclopentan globin polyoxyethylene conjugate; raf antagonists; raltitr thraquinones; cycloplatam, cypemycin; cytarabine ocfosfate; exed; ramosetron; ras farnesyl protein transferase inhibitors; cytolytic factor; cytostatin: dacliximab; dehydrodidemnin B; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated: deslorelin; dexifosfamide; dexraZOxane; dexVerapamil; rhenium Re 186 etidronate; rhizoxin: ribozymes: RII retina didemnin B; didox; diethylnorspermine; dihydro-5-azacyti mide; rohitukine; romurtide; roquinimex: rubiginone B1; dine; dihydrotaxol. 9-; dioxamycin; diphenyl spiromustine; ruboxyl; Saintopin; SarCNU; sarcophytol A. SargramoStim; Sdi 1 mimetics; senescence derived inhibitor 1; sense oligo docosanol; dolasetron: doxifluridine; dronabinol; duocarmy nucleotides; signal transduction inhibitors; signal transduc cin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflo tion modulators; single chain antigen binding protein; sizofi rnithine; elemene; emitefur, epirubicin; epristeride; estra ran; Sobuzoxane: Sodium borocaptate; sodium phenylacetate; mustine analogue; estrogen agonists; estrogen antagonists; Solverol; somatomedin binding protein; Sonermin; Sparfosic etanidazole; etoposide phosphate; exemestane; filgrastim; acid; spicamycin D; spiromustine; splenopentin; spongistatin finasteride; flavopiridol; flezelastine; fluasterone; fludara 1; squalamine; stem cell inhibitor; stem-cell division inhibi bine; fluorodaunorunicin hydrochloride; forfenimex; formes tors; stipiamide: Stromelysin inhibitors; Sulfinosine; Superac tane; fotemustine; gadolinium texaphyrin, gallium nitrate; tive vasoactive intestinal peptide antagonist; Suradista; galocitabine; ganirelix; gelatinase inhibitors; glutathione Suramin; Swainsonine; synthetic glycosaminoglycans; talli inhibitors; hepsulfam; heregulin; hexamethylene bisaceta mustine; tamoxifen methiodide; tauromustine, tazarotene; mide; hypericin; ibandronic acid; idoxifene; idramantone; tecogalan Sodium, tegafur, tellurapyrylium; telomerase ilmofosine; illomastat; imidazoacridones; imiquimod; immu inhibitors; temozolomide; tetrachlorodecaoxide; tetraZom nostimulant peptides; insulin-like growth factor-I receptor ine; thaliblastine; thalidomide; thiocoraline; thrombopoietin: inhibitor, interferon agonists; interferons; interleukins; thrombopoietin mimetic; thymalfasin; thymopoietin receptor iobenguane; iododoxorubicin; ipomeanol, 4-, irinotecan; agonist; thymotrinan; thyroid stimulating hormone; tin ethyl iroplact; irsogladine; isobengaZole; isohomohalicondrin B; etiopurpurin; titanocene dichloride; topsentin; toremifene; itasetron; jasplakinolide; kahalalide F. lamellarin-N triac totipotent stem cell factor; translation inhibitors; tretinoin; etate; lanreotide; leinamycin; lenograstim; lentinan Sulfate; triacetyluridine; triciribine; tropisetron; turosteride; tyrosine leptolstatin; leukemia inhibiting factor, leukocyte alpha inter kinase inhibitors; tyrphostins; UBC inhibitors: ubenimex: feron; leuprolide-estrogen-progesterone; leuprorelin; urogenital sinus-derived growth inhibitory factor, urokinase levamisole; liarozole; linear polyamine analogue; lipophilic receptor antagonists; variolin B; vector System, erythrocyte disaccharide peptide; lipophilic platinum compounds; lisso gene therapy; Velaresol; Veramine; Verdins; Vinorelbine; clinamide 7: lobaplatin; lombricine; lometrexol; lonidamine: Vinxaltine; vitaxin, Zanoterone; Zilascorb; and Zinostatin Sti losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannosta malamer. tin A; marimastat; masoprocol; maspin; matrilysin inhibitors; 0131 Anti-cancer Supplementary potentiating com matrix metalloproteinase inhibitors; merbarone; meterelin; pounds include, but are not limited to: Tricyclic anti-depres methioninase; metoclopramide: MIF inhibitor; mifepristone; sant drugs (e.g., imipramine, desipramine, amitryptyline, clo miltefosine; mirimostim; mismatched double stranded RNA; mipramine, trimipramine, doxepin, nortriptyline, mitoguaZone, mitolactol; mitomycin analogues; mitonafide; protriptyline, amoxapine and maprotiline); non-tricyclic anti mitotoxin fibroblast growth factor-Saporin; mofarotene; mol depressant drugs (e.g., Sertraline, traZodone and citalopram); gramostim; monoclonal antibody, human chorionic gonadot Ca" antagonists (e.g., Verapamil, nifedipine, nitrendipine rophin, monophosphoryl lipid A+myobacterium cell wall sk; and caroverine); Calmodulin inhibitors (e.g., prenylamine, mopidamol; multiple drug resistance gene inhibitor, multiple trifluoroperazine and clomipramine); Amphotericin B; Tri tumor Suppressor 1-based therapy; mustard anti cancer com paranol analogues (e.g., tamoxifen); antiarrhythmic drugs pound; mycaperoxide B: mycobacterial cell wall extract; (e.g., quinidine); antihypertensive drugs (e.g., reserpine); myriaporone; N-acetyldinaline; N-substituted benzamides: Thiol depleters (e.g., buthionine and Sulfoximine) and mul nafarelin; nagrestip; naloxone-pentazocine; napavin; naph tiple drug resistance reducing compounds such as Cremaphor terpin, nartograstim; nedaplatin; memorubicin; neridronic EL. acid; neutral endopeptidase; nilutamide; nisamycin; nitric 0.132. Other compounds which are useful in combination oxide modulators; nitroxide antioxidant; nitrullyn; 06-ben therapy for the purposes of the presently disclosed and Zylguanine; octreotide; okicenone; oligonucleotides; claimed inventive concept(s) include, but are not limited to, onapristone; ondansetron; ondansetron; oracin; oral cytokine the antiproliferation compound Piritrexim Isethionate; the inducer, osaterone; Oxaliplatin, oxaunomycin; paclitaxel antiprostatic hypertrophy compound Sitogluside; the benign analogues; paclitaxel derivatives; palauamine; palmitoyl prostatic hyperplasia therapy compound Tamsulosin Hydro rhizoxin; pamidronic acid; panaxytriol; panomifene; para chloride; the prostate growth inhibitor Pentomone; radioac bactin; paZelliptine; pegaspargase; peldesine; pentosan tive compounds such as Fibrinogen I 125, Fludeoxyglucose F polysulfate Sodium; pentostatin: pentroZole; perflubron; per 18, Fluorodopa F 18, Insulin I 125, Insulin I 131, lobenguane fosfamide; perillyl alcohol; phenazinomycin; phenylacetate; I 123, Iodipamide Sodium I 131, Iodoantipyrine I 131, Iodo phosphatase inhibitors; picibanil; pilocarpine hydrochloride; cholesterol I 131, Iodohippurate Sodium I 123, Iodohippurate pirarubicin; piritrexim; placetin A; placetin B; plasminogen Sodium I 125, Iodohippurate Sodium I 131, Iodopyriacet I activator inhibitor, platinum complex; platinum compounds; 125, Iodopyracet I 131, Iofetamine Hydrochloride I 123, platinum-triamine complex; porfimer Sodium; porfiromycin; Iomethin I 125, Iomethin I 131, Iothalamate Sodium I125, propyl bis-acridone; prostaglandin J2, proteasome inhibitors; Iothalamate Sodium I 131, Iotyrosine I 131, Liothyronine I protein A-based immune modulator, protein kinase C inhibi 125, Liothyronine I 131, Merisoprol Acetate Hg 197, Mer tor, protein kinase C inhibitors, microalgal; protein tyrosine isoprol Acetate Hg 203, Merisoprol Hg 197, Selenomethion US 2015/01 19330 A1 Apr. 30, 2015

ine Se 75, Technetium Tc 99m Antimony Trisulfide Colloid, cal condition of the subject being treated, the severity of the Technetium Tc 99m Bicisate, Technetium Tc 99m Disofenin, condition, the duration of the treatment, the nature of the Technetium Tc 99m Etidronate, Technetium Tc 99m Exam concurrent or combination therapy (if any), the specific route etazine, Technetium Tc 99m Furifosmin, Technetium Tc 99m of administration and like factors within the knowledge and Gluceptate, Technetium Tc 99m Lidofenin, Technetium Tc expertise of the health practitioner. For example, in connec 99m Mebrofenin, Technetium Tc 99m Medronate, Techne tion with methods directed towards treating Subjects having a tium Tc 99m Medronate Disodium, Technetium Tc 99m Mer condition characterized by abnormal cell proliferation, an tiatide, Technetium Tc 99m Oxidronate, Technetium Tc 99m effective amount to inhibit proliferation would be an amount Pentetate, Technetium Tc 99m Pentetate Calcium Trisodium, sufficient to reduce or halt altogether the abnormal cell pro Technetium Tc 99m Sestamibi, Technetium Tc 99m Siboroxime, Technetium Tc 99m Succimer, Technetium Tc liferation so as to slow or halt the development of or the 99m Sulfur Colloid, Technetium Tc 99m Teboroxime, Tech progression of a cell mass such as, for example, a tumor. As netium Tc 99m Tetrofosmin, Technetium Tc 99m Tiatide, used in the embodiments, “inhibit embraces all of the fore Thyroxine 1125, Thyroxine 1131, Tolpovidone 1131, Tri going. In other embodiments, a therapeutically effective olein 1125 and Triolein 1131. amount will be an amount necessary to extend the dormancy 0.133 According to the methods of the presently disclosed of micrometastases or to stabilize any residual primary tumor and claimed inventive concept(s), the inhibitors of FAP and/ cells following Surgical or drug therapy. or POP may be administered prior to, concurrent with, or 0.137 Generally, a therapeutically effective amount will following the other anti-cancer compounds described herein vary with the Subjects age, condition, and sex, as well as the (or others not listed). The administration schedule may nature and extent of the disease in the subject, all of which can involve administering the different agents in an alternating be determined by one of ordinary skill in the art. The dosage fashion. In other embodiments, the inhibitor may be delivered may be adjusted by the individual physician or veterinarian, before and during, or during and after, or before and after particularly in the event of any complication. A therapeuti treatment with other therapies. In some cases, the inhibitor is cally effective amount is typically, but not limited to, an administered more than 24 hours before the administration of amount in a range from 0.1 ug/kg to about 2000 mg/kg, or the other anti-proliferative treatment. In other embodiments, from 1.0 g/kg to about 1000 mg/kg, or from about 0.1 mg/kg more than one anti-proliferative therapy may be administered to about 500 mg/kg, or from about 1.0 mg/kg to about 100 to a Subject. For example, the Subject may receive the present mg/kg, in one or more dose administrations daily, for one or inhibitors, in combination with both surgery and at least one more days. If desired, the effective daily dose of the active other anti-proliferative compound. Alternatively, the inhibi compound may be administered as two, three, four, five, six or tor may be administered in combination with more than one more Sub-doses for example administered separately at anti-cancer drug. appropriate intervals throughout the day, optionally, in unit 0134. Other compounds useful in combination therapies dosage forms. In some embodiments, the inhibitors are with the inhibitor compounds of the presently disclosed and administered for more than 7 days, more than 10 days, more claimed inventive concept(s) include, but are not limited to, than 14 days and more than 20 days. In still other embodi anti-angiogenic compounds such as angiostatin, endostatin, ments, the inhibitoris administered over a period of weeks, or fumagillin, non-glucocorticoid steroids and heparin or hep months. In still other embodiments, the inhibitor is delivered arin fragments and antibodies to one or more angiogenic on alternate days. For example, the agent is delivered every peptides such as C-FGF, B-FGF, VEGF, IL-8 and GM-CSF. two days, or every three days, or every four days, or every five These latter anti-angiogenic compounds may be administered days, or every six days, or every week, or every month. along with the inhibitor compounds of the presently disclosed and claimed inventive concept(s) for the purpose of inhibiting 0.138. The inhibitor compounds of the presently disclosed proliferation or inhibiting angiogenesis in all of the afore and claimed inventive concept(s) can also be administered in mentioned conditions as described herein. In certain embodi prophylactically effective amounts, particularly in Subjects ments, the inhibitors may be administered in combination diagnosed with benign or pre-malignant tumors or conditions with an anti-angiogenic compound and at least one of the likely to present pathogenic angiogenesis, such as diabetes. In anti-proliferative therapies described above including sur these instances, the inhibitors are administered in an amount gery or anti-proliferative drug therapy. effective to prevent the development of an abnormal mam 0135) In yet other examples, the inhibitors according to the malian cell proliferative mass or to prevent angiogenesis in presently disclosed and claimed inventive concept(s) can be the Solid tumor mass, depending on the embodiment. The used to treat CNS maladies such as strokes, tumors, ischemia, inhibitors may also be administered in an amount effective to Parkinson's disease, memory deficits, memory loss, eating prevent metastasis of cells from a tumor to other tissues in the disorders, senile dementia, hearing loss, vision loss, body. In these latter embodiments, the presently disclosed migraines, depression, brain injury, bipolar disorder, spinal and claimed inventive concept(s) includes methods of pre cord injury, Alzheimer's disease, and amyotrophic lateral venting the metastatic spread of a primary tumor or angio sclerosis (which has a CNS component) and can be used to genesis related to pathogenic conditions. improve learning and memory function. 0.139. According to another aspect of the presently dis 0136. The inhibitor compounds of the presently disclosed closed and claimed inventive concept(s), a kit is provided. and claimed inventive concept(s) are administered in thera The kit is a package which houses a container which contains peutically effective amounts. An effective amount is a dosage an inhibitor of the presently disclosed and claimed inventive of the inhibitor sufficient to provide atherapeutically or medi concept(s) and also includes instructions for administering cally desirable result or effect in the subject to which the the inhibitor to a subject having a condition characterized by compound is administered. The effective amount will vary an abnormal mammalian cell proliferation or other condition with the particular condition being treated, the age and physi described herein. The kit may optionally also contain one or US 2015/01 19330 A1 Apr. 30, 2015

more other anti-proliferative compounds or one or more anti lactated Ringer's or fixed oils. Intravenous vehicles include angiogenic compounds for use in combination therapies as fluid and nutrient replenishers, electrolyte replenishers (such described herein. as those based on Ringer's dextrose), and the like. Preserva 0140. The compounds of the presently disclosed and tives and other additives may also be present Such as, for claimed inventive concept(s) may be administered alone or in example, antimicrobials, anti-oxidants, chelating com combination with the above-described drug therapies by a pounds, and inert gases and the like. The pharmaceutical variety of administration routes available. The particular compositions may conveniently be presented in unit dosage mode selected will depend of course, upon the compound form and may be prepared by any of the methods well-known selected, the condition being treated, the severity of the con in the art of pharmacy. dition, whether the treatment is therapeutic or prophylactic, 0145 Compositions suitable for oral administration may and the dosage required for efficacy. The methods of the particularly comprise discrete units, such as capsules, tablets, presently disclosed and claimed inventive concept(s), gener lozenges, each containing a predetermined amount of the ally speaking, may be practiced using any mode of adminis inhibitor. Other compositions include Suspensions in aqueous tration that is medically acceptable, meaning any mode that liquids or non-aqueous liquids such as a syrup, elixir, or an produces effective levels of the active compounds without emulsion. In yet other embodiments, the particular vehicle is causing clinically unacceptable adverse effects. The admin a biocompatible microparticle or implant that is suitable for istration may, for example, be oral, intraperitoneal, intra implantation into the mammalian recipient. cavity Such as rectal or vaginal, transdermal, topical, nasal, 0146 In other embodiments of the methods and com inhalation, mucosal, interdermal, or parenteral routes. The pounds of the presently disclosed and claimed inventive con term "parenteral' includes Subcutaneous, intravenous, intra cept(s), the compound is targeted to a site of abnormal cell muscular, or infusion. proliferation, such as, a tumor, through the use of a targeting 0141 Intravenous or intramuscular routes may not par compound specific for a particular tissue or tumor type. The ticularly Suitable for long term therapy and prophylaxis. In compounds of the presently disclosed and claimed inventive certain embodiments, however, it may be appropriate to concept(s) may be targeted to primary or in Some instances, administer the compound in a continuous infusion every sev secondary (i.e., metastatic) lesions through the use of target eral days, or once a week, or every several weeks, or once a ing compounds which preferentially recognize a cell Surface month. Intravenous or intramuscular routes may be desired in marker. The targeting compound may be directly conjugated emergency situations. Oral administration may be used for to the compounds of the presently disclosed and claimed prophylactic treatment because of the convenience to the inventive concept(s) via a covalent linkage. The compound patient as well as the dosing schedule. Likewise, Sustained may be indirectly conjugated to a targeting compound via a release devices as described herein may be useful in certain linker. Alternatively, the targeting compound may be conju embodiments for prophylactic or post Surgery treatment, for gated or associated with an intermediary compound such as, example. for example, a liposome within which the inhibitor is encap 0142. When using the compounds of the presently dis Sulated. Liposomes are artificial membrane vessels which are closed and claimed inventive concept(s) in Subjects in whom useful as a delivery vector in vivo or in vitro. It has been the primary site of abnormal proliferation is well delineated shown that large unilamellar vessels (LUV), which range in and easily accessible, direct administration to the site may be size from 0.2-4.0 um can encapsulate large macromolecules. desired, provided the tumor has not already metastasized. For Liposomes may be targeted to a particular tissue. Such as the example, administration by inhalation for lung tumors or by vascular cell wall, by coupling the liposome to a specific Suppositories in the treatment of cervical, ovarian or rectal ligand Such as a monoclonal antibody, Sugar, glycolipid, or tumors may be desired. Likewise, melanoma, for example, protein. In still other embodiments, the targeting compound may be treated with the compound via topical administration may be loosely associated with the compounds of the pres in and around the area of the lesion. In still other embodi ently disclosed and claimed inventive concept(s). Such as ments aimed at the treatment of Subjects with breast, lung, within a microparticle comprising a polymer, the compound pancreatic, or prostate cancer, for example, the compounds of the presently disclosed and claimed inventive concept(s) may be delivered by injection directly into the tissue with, for and the targeting compound. example, a biopsy needle and Syringe. 0147 Targeting compounds useful according to the meth 0143 Systemic administration may be desired in some ods of the presently disclosed and claimed inventive concept instances such as, for example, if the Subject is known to have (s) are those which direct the compound to a site of abnormal or is suspected of having metastases. In this way, all tumor proliferation Such as a tumor site. The targeting compound of sites, whether primary or secondary, may receive the com choice will depend upon the nature of the tumor or the tissue pound. Systemic delivery may be accomplished through for origin of the metastasis. In some instances it may be desirable example, oral or parenteral administration. Inhalation may be to target the compound to the tissue in which the tumor is used in either systemic or local delivery, as described herein. located. For example, the compounds can be delivered to 0144 Compositions of the compound for parenteral breast epithelium by using a targeting compound specific for administration particularly include, but are not limited to, breast tissue. In particular embodiments, the target is specific sterile aqueous or non-aqueous solutions, Suspensions, and for malignant breast epithelium. Examples of compounds emulsions. Examples of non-aqueous solvents are propylene which may localize to malignant breast epithelium include, glycol, polyethylene glycol, vegetable oils such as olive oil, but are not limited to, estrogen and progesterone, epithelial and injectable organic esters such as ethyl oleate. Aqueous growth factor (EGF) and HER-2/neu ligand, among others. carriers include water, alcoholic/aqueous Solutions, emul The HER-2/neu ligand may also be used to target compounds sions or Suspensions, including saline and buffered media. to ovarian cancers. Ovarian cancers are also known to express Parenteral vehicles include, for example, sodium chloride EGFR and c-fms, and thus could be targeted through the use Solution, Ringer's dextrose, dextrose and Sodium chloride, of ligands for either receptor. In the case of c-fms which is US 2015/01 19330 A1 Apr. 30, 2015 also expressed by macrophages and monocytes, targeted glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) delivery to an ovarian cancer may require a combination of agar, (14) buffering agents, such as magnesium hydroxide local administration Such as a vaginal Suppository as well as and aluminum hydroxide; (15) alginic acid, (16) pyrogen a targeting compound. Prostate cancers can be targeted using free water; (17) isotonic saline; (18) Ringer's solution; (19) compounds such as peptides (e.g., antibodies or antibody ethyl alcohol; (20) pH buffered solutions; (21) polyesters, fragments) which bind to prostate specific antigen (PSA) or polycarbonates and/or polyanhydrides; and (22) other non prostate specific membrane antigen (PSMA). Other markers toxic compatible Substances employed in pharmaceutical for which may be used for targeting of the agent to specific mulations. tissues include, for example, in liver: HGF, insulin-like growth factor I, II, insulin, OV-6, HEA-125, hyaluronic acid, 0151. As set forth above, in certain embodiments, the collagen, N-terminal propeptide of collagen type III, man compounds of the presently disclosed and claimed inventive nose/N-acetylglucosamine, asialoglycoprotein, tissue plas concept(s) contain a basic functional group. Such as amino or minogen activator, low density lipoprotein, carcinoembry alkylamino, and are, thus, capable of forming pharmaceuti onic antigen; in kidney cells: angiotensin II, vasopressin, cally-acceptable salts with pharmaceutically-acceptable antibodies to CD44v6; in keratinocytes and skin fibroblasts: acids. The term “pharmaceutically-acceptable salts' in this KGF, very low density lipoprotein, RGD-containing pep respect, refers to the relatively non-toxic, inorganic and tides, collagen, laminin; in melanocytes: kit ligand; in gut: organic acid addition salts of the compounds. These salts can cobalamin-intrinsic factor, heat stable enterotoxin of E. Coli: be prepared in situ in the administration vehicle or the dosage in breast epithelium: heregulin, prolactin, transferrin, cad form manufacturing process, or by separately reacting a puri fied compound of the presently disclosed and claimed inven herin-11. Other markers specific to particular tissues are tive concept(s) in its free base form with a suitable organic or available and would be known to one of ordinary skill in the inorganic acid, and isolating the salt thus formed during Sub art sequent purification. Representative salts include, but are not 0148. In still other embodiments, the compounds of the limited to, the hydrobromide, hydrochloride, sulfate, bisul presently disclosed and claimed inventive concept(s) may be fate, phosphate, nitrate, acetate, Valerate, oleate, palmitate, targeted to fibroblasts specifically, via ligands or binding Stearate, laurate, benzoate, lactate, phosphate, tosylate, cit partners for fibroblast specific markers. Examples of these rate, maleate, fumarate. Succinate, tartrate, napthylate, mesy markers include, but are not limited to fibroblast growth fac late, glucoheptonate, lactobionate, and laurylsulphonate salts tors (FGF) and platelet derived growth factor (PDGF). In and the like. some embodiments, it is desirable to target the compound to FAP specifically through the use of binding peptides for FAP 0152 The pharmaceutically acceptable salts of the com which do not interfere with inhibition by the compound of the pounds of the presently disclosed and claimed inventive con presently disclosed and claimed inventive concept(s). cept(s) include, but are not limited to, the conventional non 0149 Other embodiments of the presently disclosed and toxic salts or quaternary ammonium salts of the compounds, claimed inventive concept(s) include pharmaceutically e.g., from non-toxic organic or inorganic acids. For example, acceptable compositions which comprise a therapeutically Such conventional nontoxic salts include those derived from effective amount of one or more of the compounds described inorganic acids such as hydrochloride, hydrobromic, Sulfuric, herein, formulated together with one or more pharmaceuti Sulfamic, phosphoric, nitric, and the like; and the salts pre cally acceptable carriers (additives) and/or diluents. As pared from organic acids such as acetic, propionic, succinic, described in detail below, the pharmaceutically acceptable glycolic, Stearic, lactic, malic, tartaric, citric, ascorbic, palm compositions may be specially formulated for administration itic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, in Solid or liquid form, including, but not limited to, those salicyclic, Sulfanilic, 2-acetoxybenzoic, fumaric, toluene adapted for the following: (1) oral administration, for Sulfonic, methanesulfonic, ethane disulfonic, oxalic, example, aqueous or non-aqueous Solutions or Suspensions, isothionic, and the like. tablets, e.g., those targeted for buccal, Sublingual, and sys 0153. In other cases, the compounds of the presently dis temic absorption, boluses, powders, granules, pastes for closed and claimed inventive concept(s) may contain one or application to the tongue; (2) parenteral administration, for more acidic functional groups and, thus, are capable of form example, by Subcutaneous, intramuscular, intravenous or epi ing pharmaceutically-acceptable salts with pharmaceuti dural injection as, for example, a sterile Solution or Suspen cally-acceptable bases. The term “pharmaceutically-accept Sion, or Sustained-release formulation; (3) topical applica able salts' in these instances refers to the relatively non-toxic, tion, for example, as a cream, ointment, or a controlled inorganic and organic base addition salts of compounds of the release patch or spray applied to the skin; (4) intravaginally or presently disclosed and claimed inventive concept(s). These intrarectally, for example, as a cream or foam; (5) Sublin salts can likewise be prepared in situ in the administration gually; (6) ocularly; (7) transdermally, or (8) nasally. vehicle or the dosage form manufacturing process, or by 0150. Some examples of materials which can serve as separately reacting the purified compound in its free acid pharmaceutically-acceptable carriers include, but are not lim form with a suitable base, such as the hydroxide, carbonate or ited to: (1) Sugars, such as lactose, glucose and Sucrose; (2) bicarbonate of a pharmaceutically-acceptable metal cation, starches. Such as corn Starch and potato starch; (3) cellulose, with ammonia, or with a pharmaceutically-acceptable and its derivatives, such as Sodium carboxymethyl cellulose, organic primary, secondary or tertiary amine. Representative ethyl cellulose and cellulose acetate; (4) powdered traga alkali or alkaline earth salts include, but are not limited to, the canth; (5) malt, (6) gelatin; (7) talc.; (8) excipients, such as lithium, Sodium, potassium, calcium, magnesium, and alumi cocoa butter and Suppository waxes; (9) oils, such as peanut num salts and the like. Representative organic amines useful oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil for the formation of base addition salts include, but are not and Soybean oil: (10) glycols, such as propylene glycol; (11) limited to, ethylamine, diethylamine, ethylenediamine, etha polyols. Such as glycerin, Sorbitol, mannitol and polyethylene nolamine, diethanolamine, piperazine and the like. US 2015/01 19330 A1 Apr. 30, 2015 20

0154 Wetting agents, emulsifiers and lubricants, includ compound or compounds are mixed with one or more phar ing, but not limited to, sodium lauryl Sulfate and magnesium maceutically-acceptable carriers, including, but not limited Stearate, as well as coloring agents, release agents, coating to, sodium citrate or dicalcium phosphate, and/or any of the agents, Sweetening, flavoring and perfuming agents, preser following: (1) fillers or extenders, such as starches, lactose, Vatives and antioxidants can also be present in the composi Sucrose, glucose, mannitol, and/or silicic acid; (2) binders, tions. Examples of pharmaceutically-acceptable antioxidants Such as, for example, carboxymethylcellulose, alginates, include, but are not limited to: (1) water soluble antioxidants, gelatin, polyvinyl pyrrolidone. Sucrose and/or acacia; (3) Such as ascorbic acid, cysteine hydrochloride, sodium bisul humectants, such as glycerol; (4) disintegrating agents, such fate, sodium metabisulfite, sodium sulfite and the like; (2) as agar-agar, calcium carbonate, potato or tapioca starch, oil-soluble antioxidants, such as ascorbyl palmitate, buty alginic acid, certain silicates, and sodium carbonate; (5) solu lated hydroxyanisole (BHA), butylated hydroxytoluene tion retarding agents. Such as paraffin; (6) absorption accel (BHT), lecithin, propyl gallate, alpha-tocopherol, and the erators, such as quaternary ammonium compounds; (7) wet like; and (3) metal chelating agents, such as citric acid, eth ting agents, such as, for example, cetyl alcohol, glycerol ylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, monostearate, and non-ionic Surfactants; (8) absorbents. Such phosphoric acid, and the like. as kaolin and bentonite clay; (9) lubricants, such a talc, cal 0155 As noted above, formulations of the compounds of cium Stearate, magnesium Stearate, Solid polyethylene gly the presently disclosed and claimed inventive concept(s) cols, sodium lauryl sulfate, and mixtures thereof; and (10) include those Suitable for oral, nasal, topical (including buc coloring agents. In the case of capsules, tablets and pills, the cal and Sublingual), rectal, vaginal and/or parenteral admin pharmaceutical compositions may also comprise buffering istration. The formulations may conveniently be presented in agents. Solid compositions of a similar type may also be unit dosage form and may be prepared by any methods well employed as fillers in Soft and hard-shelled gelatin capsules known in the art of pharmacy. The amount of active ingredient using Such excipients as lactose or milk Sugars, as well as high which can be combined with a carrier material to produce a molecular weight polyethylene glycols and the like. single dosage form will vary depending upon the host being 0160 A tablet may be made by compression or molding, treated, the particular mode of administration. The amount of optionally with one or more accessory ingredients. Com active ingredient which can be combined with a carrier mate pressed tablets may be prepared using binder (for example, rial to produce a single dosage form will generally be that gelatin or hydroxypropylmethyl cellulose), lubricant, inert amount of the compound which produces atherapeutic effect. diluent, preservative, disintegrant (for example, sodium Generally, out of one hundred percent, this amount will range starch glycolate or cross-linked sodium carboxymethyl cel from about 1 percent to about ninety-nine percent of active lulose), Surface-active or dispersing agent. Molded tablets ingredient, such as from about 5 percent to about 70 percent, may be made by molding in a Suitable machine a mixture of or from about 10 percent to about 30 percent. the powdered compound moistened with an inert liquid dilu 0156. In certain embodiments, a formulation of the com ent pounds of the presently disclosed and claimed inventive con 0.161 The tablets, and other solid dosage forms of the cept(s) comprises an excipient selected from the group con pharmaceutical compositions of the presently disclosed and sisting of cyclodextrins, liposomes, micelle forming agents, claimed inventive concept(s). Such as dragees, capsules, pills e.g., bile acids, and polymeric carriers, e.g., polyesters and and granules, may optionally be scored or prepared with polyanhydrides. coatings and shells, such as enteric coatings and other coat 0157 Methods of preparing these formulations or compo ings well known in the pharmaceutical-formulating art. They sitions may include the step of bringing into association a may also be formulated so as to provide slow or controlled compound of the presently disclosed and claimed inventive release of the compound or compounds therein using, for concept(s) with the carrier and, optionally, one or more acces example, hydroxypropylmethyl cellulose in varying propor sory ingredients. In general, the formulations are prepared by tions to provide the desired release profile, other polymer uniformly and intimately bringing into association a com matrices, liposomes and/or microspheres. They may be for pound of the presently disclosed and claimed inventive con mulated for rapid release, e.g., freeze-dried. They may be cept(s) with liquid carriers, or finely divided solid carriers, or sterilized by, for example, filtration through a bacteria-retain both, and then, if necessary, shaping the product. ing filter, or by incorporating sterilizing agents in the form of 0158 Formulations of the compounds of the presently sterile solid compositions which can be dissolved in sterile disclosed and claimed inventive concept(s) suitable for oral water, or some other sterile injectable medium immediately administration may be, but are not limited to, the form of before use. These compositions may also optionally contain capsules, cachets, pills, tablets, lozenges (using a flavored opacifying agents and may be of a composition that they basis, usually Sucrose and acacia or tragacanth), powders, release the active ingredient(s) only, or preferentially, in a granules, or as a solution or a Suspension in an aqueous or certain portion of the gastrointestinal tract, optionally, in a non-aqueous liquid, or as an oil-in-water or water-in-oil liq delayed manner. Examples of embedding compositions uid emulsion, or as an elixir or syrup, or as pastilles (using an which can be used include polymeric Substances and waxes. inert base, such as gelatin and glycerin, or Sucrose and acacia) The active ingredient can also be in micro-encapsulated form, and/or as mouth washes and the like, each containing a pre if appropriate, with one or more of the above-described determined amount of a compound of the presently disclosed excipients. and claimed inventive concept(s) as an active ingredient. A 0162 Liquid dosage forms for oral administration of the compound of the presently disclosed and claimed inventive compounds of the presently disclosed and claimed inventive concept(s) may also be administered as a bolus, or paste. concept(s) include, but are not limited to, pharmaceutically 0159. In solid dosage forms of the compounds of the pres acceptable emulsions, microemulsions, solutions, Suspen ently disclosed inventive concept(s) for oral administration sions, syrups and elixirs. In addition to the active ingredient, (capsules, tablets, pills, powders, granules and the like), the the liquid dosage forms may contain inert diluents commonly US 2015/01 19330 A1 Apr. 30, 2015

used in the art, such as, for example, water or other solvents, ointments, powders, solutions and the like, are also contem solubilizing agents and emulsifiers, such as ethyl alcohol, plated as being within the scope of the presently disclosed and isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alco claimed inventive concept(s). hol, benzyl benzoate, propylene glycol. 1,3-butylene glycol, 0166 Pharmaceutical compositions of the compounds of oils (in particular, cottonseed, groundnut, corn, germ, olive, the presently disclosed and claimed inventive concept(s) Suit castor and sesame oils), glycerol, tetrahydrofuryl alcohol, able for parenteral administration comprise one or more com polyethylene glycols and fatty acid esters of Sorbitan, and pounds of the presently disclosed and claimed inventive con mixtures thereof. Besides inert diluents, the oral composi cept(s) in combination with one or more pharmaceutically tions can also include adjuvants such as wetting agents, emul acceptable sterile isotonic aqueous or nonaqueous Solutions, Sifying and Suspending agents, Sweetening, flavoring, color dispersions, Suspensions or emulsions, or sterile powders ing, perfuming and preservative agents. Suspensions, in which may be reconstituted into sterile injectable solutions or addition to the active compounds, may contain Suspending dispersions just prior to use, which may contain Sugars, alco agents as, for example, ethoxylated isostearyl alcohols, poly hols, antioxidants, buffers, bacteriostats, Solutes which ren oxyethylene Sorbitol and Sorbitan esters, microcrystalline der the formulation isotonic with the blood of the intended cellulose, aluminum metahydroxide, bentonite, agar-agar recipient or Suspending or thickening agents. and tragacanth, and mixtures thereof. 0.167 Examples of Suitable aqueous and nonaqueous car riers which may be employed in the pharmaceutical compo 0163 Formulations of the compounds of the pharmaceu sitions of the presently disclosed and claimed inventive con tical compositions of the presently disclosed and claimed cept(s) include, but are not limited to, water, ethanol, polyols inventive concept(s) for rectal or vaginal administration may (such as glycerol, propylene glycol, polyethylene glycol, and be presented as a Suppository, which may be prepared by the like), and Suitable mixtures thereof, vegetable oils, such as mixing one or more compounds of the presently disclosed olive oil, and injectable organic esters, such as ethyl oleate. and claimed inventive concept(s) with one or more Suitable Proper fluidity can be maintained, for example, by the use of nonirritating excipients or carriers comprising, for example, coating materials, such as lecithin, by the maintenance of the cocoa butter, polyethylene glycol, a Suppository wax or a required particle size in the case of dispersions, and by the use salicylate, and which is solid at room temperature, but liquid of Surfactants. at body temperature and, therefore, will melt in the rectum or 0.168. These compositions may also contain adjuvants vaginal cavity and release the active compound. Such as preservatives, wetting agents, emulsifying agents and 0164. Formulations of the presently disclosed and claimed dispersing agents. Prevention of the action of microorgan inventive concept(s) which are suitable for vaginal adminis isms upon the Subject compounds may be ensured by the tration also include, but are not limited to, tampons, creams, inclusion of various antibacterial and antifungal agents, for gels, pastes, foams or spray formulations containing Such example, paraben, chlorobutanol, phenol Sorbic acid, and the carriers as are known in the art to be appropriate. Dosage like. It may also be desirable to include isotonic agents. Such forms for the topical or transdermal administration of a com as Sugars, sodium chloride, and the like into the compositions. pound of the presently disclosed and claimed inventive con In addition, prolonged absorption of the injectable pharma cept(s) include, but are not limited to, powders, sprays, oint ceutical form may be brought about by the inclusion of agents ments, pastes, creams, lotions, gels, Solutions, patches and which delay absorption Such as aluminum monostearate and inhalants. The active compound may be mixed under sterile gelatin. conditions with a pharmaceutically-acceptable carrier, and 0169. In some cases, in order to prolong the effect of a with any preservatives, buffers, or propellants which may be drug, it is desirable to slow the absorption of the drug from required. The ointments, pastes, creams and gels may con Subcutaneous or intramuscular injection. This may be accom tain, for example, in addition to an active compound of the plished by the use of a liquid Suspension of crystalline or presently disclosed and claimed inventive concept(s), excipi amorphous material having poor water solubility. The rate of ents, such as animal and vegetable fats, oils, waxes, paraffins, absorption of the drug then depends upon its rate of dissolu starch, tragacanth, cellulose derivatives, polyethylene gly tion which, in turn, may depend upon crystal size and crys cols, silicones, bentonites, silicic acid, talc and Zinc oxide, or talline form. Alternatively, delayed absorption of a parenter mixtures thereof. Powders and sprays can contain, for ally-administered drug form is accomplished by dissolving or example, in addition to a compound of the presently disclosed Suspending the drug in an oil vehicle. and claimed inventive concept(s), excipients such as lactose, 0170 Injectable depot forms are made by forming talc, silicic acid, aluminum hydroxide, calcium silicates and microencapsule matrices of the Subject compounds in biode polyamide powder, or mixtures of these Substances. Sprays gradable polymers such as polylactide-polyglycolide. can additionally contain customary propellants. Such as chlo Depending on the ratio of drug to polymer, and the nature of rofluorohydrocarbons and volatile unsubstituted hydrocar the particular polymer employed, the rate of drug release can bons, such as butane and propane. be controlled. Examples of other biodegradable polymers 0.165 Transdermal patches have the added advantage of include poly(orthoesters) and poly(anhydrides). Depot providing controlled delivery of a compound of the presently injectable formulations are also prepared by entrapping the disclosed and claimed inventive concept(s) to the body. Such drug in liposomes or microemulsions which are compatible dosage forms can be made by dissolving or dispersing the with body tissue. compound in the proper medium. Absorption enhancers can 0171 When the compounds of the presently disclosed and also be used to increase the flux of the compound across the claimed inventive concept(s) are administered as pharmaceu skin. The rate of such flux can be controlled by either provid ticals, to human or animal Subjects, they are generally given ing a rate controlling membrane or dispersing the compound as a pharmaceutical composition containing, for example, in a polymer matrix or gel. Ophthalmic formulations, eye 0.01% to 99.5% (such as 0.5 to 90%) of the compound (with US 2015/01 19330 A1 Apr. 30, 2015 22 or without other compounds given adjunctively in combina One skilled in the art of preparing formulations can readily tion with a pharmaceutically acceptable carrier). select the proper form and mode of administration depending 0172. The preparations of the presently disclosed and upon the particular characteristics of the compound selected, claimed inventive concept(s) may be given, for example, the disease state to be treated, the stage of the disease, and orally, parenterally, topically, or rectally as explained above. other relevant circumstances using formulation technology They are of course given in forms suitable for each adminis known in the art, described, for example, in Remington's tration route. For example, they are administered in tablets or Pharmaceutical Sciences, latest edition, Mack Publishing capsule form, by injection, inhalation, eye lotion, ointment, Co. Suppository, by injection, infusion or inhalation, topical by 0176 Pharmaceutical compositions can be manufactured lotion or ointment, and rectal by Suppositories. The phrases utilizing techniques known in the art. Typically the therapeu “parenteral administration' and “administered parenterally' tically effective amount of the compound will be admixed as used herein means modes of administration other than with a pharmaceutically acceptable carrier as described else enteral and topical administration, usually by injection, and where herein. In one embodiment, the half-life of the com includes, but is not limited to, intravenous, intramuscular, pounds described herein can be extended by their being con intraarterial, intrathecal, intracapsular, intraorbital, intracar jugated to other molecules such as polymers using methods diac, intradermal, intraperitoneal, transtracheal, Subcutane known in the art to form drug-polymer conjugates. For ous, Subeuticular, intraarticulare, Subcapsular, Subarachnoid, example, the molecules can be bound to molecules of inert intraspinal and intrasternal injection and infusion. The polymers known in the art, such as a molecule of polyethylene phrases “systemic administration.” “administered systemi glycol (PEG) in a method known as "PEGylation’. Pegyla cally.” “peripheral administration' and “administered periph tion can therefore extend the in vivo lifetime and thus thera erally as used herein mean the administration of a com peutic effectiveness of the molecule. pound, drug or other material other than directly into the (0177 PEG molecules can be modified by functional central nervous system, such that it enters the patient’s system groups, for example as shown in Harris et al., “Pegylation, A and, thus, is Subject to metabolism and other like processes, Novel Process for Modifying Phararmacokinetics’. Clin for example, Subcutaneous administration. Pharmacokinet, 2001:40(7): 539-551, and the amino termi 0173 Regardless of the route of administration selected, nal end of the molecule, or cysteine residue if present, or other the compounds of the presently disclosed and claimed inven linking amino acid therein can be linked thereto, wherein the tive concept(s), which may be used in a suitable hydrated PEG molecule can carry one or a plurality of one or more form, and/or the pharmaceutical compositions of the pres types of molecules. ently disclosed and claimed inventive concept(s), are formu (0178. By “polyethylene glycol” or “PEG” is meanta poly lated into pharmaceutically-acceptable dosage forms by con alkylene glycol compound or a derivative thereof, with or ventional methods known to those of skill in the art. Actual without coupling agents or derviatization with coupling or dosage levels of the active ingredients in the pharmaceutical activating moeities (e.g., with thiol, triflate, tresylate, azird compositions of the presently disclosed and claimed inven ine, oxirane, or particularly with a maleimide moiety). Com tive concept(s) may be varied so as to obtain an amount of the pounds Such as maleimido monomethoxy PEG are exemplary active ingredient which is effective to achieve the desired or activated PEG compounds of the inventive concept(s). therapeutic response for a particular patient, composition, Other polyalkylene glycol compounds, such as polypropy and mode of administration, without being toxic to the lene glycol, may be used in the presently disclosed and patient. claimed inventive concept(s). Other appropriate polymer 0.174. The selected dosage level will depend upon a variety conjugates include, but are not limited to, non-polypeptide of factors including the activity of the particular compound of polymers, charged or neutral polymers of the following types: the presently disclosed and claimed inventive concept(s) dextran, colominic acids or other carbohydrate based poly employed, or the ester, salt or amide thereof, the route of mers, biotin deriviatives and dendrimers, for example. The administration, the time of administration, the rate of excre term PEG is also meant to include other polymers of the class tion or metabolism of the particular compound being polyalkylene oxides. employed, the duration of the treatment, other drugs, com (0179 The PEG can be linked to any N-terminal amino pounds and/or materials used in combination with the par acid of the molecule described herein and/or can be linked to ticular compound employed, the age, sex, weight, condition, an amino acid residue downstream of the N-terminal amino general health and prior medical history of the patient being acid, such as lysine, histidine, tryptophan, aspartic acid, treated, and like factors well known in the medical arts. A glutamic acid, and cysteine, for example or other such amino physician or veterinarian having ordinary skill in the art can acids known to those of skill in the art. The PEG carrier readily determine and prescribe the effective amount of the moiety attached to the peptide may range in molecular weight pharmaceutical composition required. For example, the phy from about 200 to 20,000 MW. Particularly, the PEG moiety sician or veterinarian could start doses of the compounds of may be from about 1,000 to 8,000 MW, such as from about the presently disclosed and claimed inventive concept(s) 3,250 to 5,000 MW, or about 5,000 MW. The actual number employed in the pharmaceutical composition at levels lower of PEG molecules covalently bound per molecule of the than that required in order to achieve the desired therapeutic inventive concept(s) may vary widely depending upon the effect and gradually increase the dosage until the desired desired stability (i.e. serum half-life). Molecules contem effect is achieved. While it is possible for compounds of the plated herein can be linked to PEG molecules using tech presently disclosed and claimed inventive concept(s) to be niques shown, for example (but not limited to), in U.S. Pat. administered alone, it may be desired to administer the com Nos., 4,179,337; 5,382,657; 5,972,885; 6,177,087; pound as a pharmaceutical formulation (composition). 6,165,509; 5,766,897; and 6,217,869; the specifications and 0.175. As noted, particular amounts and modes of admin drawings each of which are hereby expressly incorporated istration are able to be determined by one skilled in the art. herein by reference. US 2015/01 19330 A1 Apr. 30, 2015

0180 Alternatively, it is possible to entrap the molecules EXAMPLES in microcapsules prepared, for example, by coacervation 0185. While the presently disclosed and claimed inventive techniques or by interfacial polymerization (for example, concept(s) will now be described in connection with certain hydroxymethylcellulose or gelatine-microcapsules and poly embodiments in the following examples so that aspects (methylmethacylate) microcapsules, respectively), in colloi thereof may be more fully understood and appreciated, it is dal drug delivery systems (for example, liposomes, albumin not intended to limit the inventive concept(s) to these particu microspheres, microemulsions, nano-particles, and nanocap lar examples. On the contrary, it is intended to cover all Sules), or in macroemulsions. Such techniques are disclosed alternatives, modifications and equivalents as may be in the latest edition of Remington's Pharmaceutical Sciences. included within the scope of the presently disclosed and 0181 U.S. Pat. No. 4,789,734 describes methods for claimed inventive concept(s) as defined herein and in the encapsulating biochemicals in liposomes and is hereby appended claims. Thus, the following examples, which include particular embodiments will serve to illustrate the expressly incorporated by reference herein. Essentially, the practice of the presently disclosed and claimed inventive con material is dissolved in an aqueous solution, the appropriate cept(s), it being understood that the particulars shown are by phospholipids and lipids added, along with Surfactants if way of example and for purposes of illustrative discussion of required, and the material dialyzed or Sonicated, as necessary. particular embodiments of the presently disclosed and A review of known methods is by G. Gregoriadis, Chapter 14. claimed inventive concept(s) only and are presented in the "Liposomes. Drug Carriers in Biology and Medicine, pp. cause of providing what is believed to be the most useful and 287-341 (Academic Press, 1979). Microspheres formed of readily understood description of compounds, methods of polymers or proteins are well knownto those skilled in the art, use, and formulation procedures as well as of the principles and can be tailored for passage through the gastrointestinal and conceptual aspects of the presently disclosed and claimed tract directly into the blood stream. Alternatively, the agents inventive concept(s). can be incorporated and the microspheres, or composite of 0186 Experimental microspheres, implanted for slow release over a period of 0187. The following describes various experimental pro time, ranging from days to months. See, for example, U.S. cedures used to synthesize examples of Substrates and inhibi Pat. Nos. 4,906,474; 4925,673; and 3,625,214 which are tors and further experimentation based on these substrates incorporated by reference herein. and inhibitors. 0182. When the composition of the presently disclosed 0188 Materials and Methods and claimed inventive concept(s) is to be used as an injectable (0189 Cell Culture: material, it can be formulated into a conventional injectable (0190. Fibroblasts, WI-38 and WI-38 VA132RA (VA-13); carrier. Suitable carriers include biocompatible and pharma breast cancer cells, MDA-MB436 (MDA) and HCC1419 ceutically acceptable phosphate buffered saline Solutions, (HCC); and normal breast cells, MCF-12A, were all pur which may be isotonic. chased from American Type Culture Collection (ATCC). Human dermal microvascular endothelial cells (HMVEC-d) 0183 For reconstitution of a lyophilized product in accor and mesenchymal stem cells (MSC) were purchased from dance with the presently disclosed and claimed inventive Lonza. All cells were authenticated by the companies and concept(s), one may employ a sterile diluent, which may used within six months of purchase or recovery from cryo contain materials generally recognized for approximating preservation. WI-38 and VA-13 were grown in MEM (Medi physiological conditions and/or as required by governmental atech Inc.) supplemented with 10% FCS (fetal calf serum, regulation. In this respect, the Sterile diluent may contain a Gibco), 2 mMGlutaMAX-1 (Gibco) and 1 mM sodium pyru buffering agent to obtain a physiologically acceptable pH, vate (Gibco). MDA and HCC were grown in DMEM (Medi Such as sodium chloride, saline, phosphate-buffered saline, atech Inc.) supplemented with 10% FCS. MCF-12A and and/or other substances which are physiologically acceptable HMVEC-d were grown in EGM2-MV (Lonza) with all pro and/or safe for use. In general, the material for intravenous vided supplements, unless otherwise specified. MSC were injection in humans should conform to regulations estab grown in MSCGM (Lonza). lished by the Food and Drug Administration, which are avail (0191 Monoclonal Antibody Production: able to those in the field. The pharmaceutical composition 0.192 Human antiplasmin-cleaving enzyme (APCE) was may also be in the form of an aqueous solution containing purified as previously described 28 and used as the antigen many of the same substances as described above for the for monoclonal antibody (mAb) production. The hybridoma reconstitution of a lyophilized product. cells were produced at the Hybridoma Centerfor Agricultural 0184 The compounds of the presently disclosed and and Biological Sciences at Oklahoma State University. After claimed inventive concept(s) can also be administered as a initial screening against pure APCE by direct ELISA, the pharmaceutically acceptable acid- or base-addition salt, positive cells were cloned three times by limited dilution. formed by reaction with inorganic acids such as, but not Selected antibodies were produced in serum free media limited to, hydrochloric acid, hydrobromic acid, perchloric (SFM, Gibco) in roller bottles, then purified using MEP acid, nitric acid, thiocyanic acid, Sulfuric acid, and phospho Hypercel (Pall) chromatography and isotyped using the ric acid, and organic acids Such as formic acid, acetic acid, Pierce Rapid Isotyping kit (Thermo Scientific). Out of 24 propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic mAbs identified, m.Ab 6D2, an IgG1 K antibody, which rec acid, malonic acid, Succinic acid, maleic acid, and fumaric ognized both APCE and FAP by ELISA and Western blotting acid, or by reaction with an inorganic base Such as sodium with the greatest sensitivity, was used in these studies. Mouse hydroxide, ammonium hydroxide, potassium hydroxide, and F19 mAb to FAP was produced and purified from cultures of organic bases such as mono-, di-, trialkyl and arylamines and hybridoma cells purchased from ATCC, and used for confocal Substituted ethanolamines. imaging and immunoprecipitation (IP). US 2015/01 19330 A1 Apr. 30, 2015 24

0193 Immunostaining: connected to an AB-Sciex QSTAR Elite mass spectrometer. 0194 Selected normal or neoplastic cells were grown to The peptide molecular weights and MS/MS fragment ion confluency in 10 cm tissue culture dishes, rinsed in phosphate spectra observed for each peptide were used to query an buffered saline (PBS) and lysed on the plate in 1 ml of 2x NCBI comprehensive non-identical human protein database Laemmli denaturing sample buffer with DTT for whole cell (updated Jan. 31, 2011) loaded on an in-house MASCOT lysate. Membrane and cytosolic fractions were prepared from database server (version 2.3). confluent tissue culture dishes using the Mem-PER kit 0198 Inhibitors and Substrates: (Pierce) per manufacturer instructions. Whole cell lysates 0199 The pseudo-peptide inhibitor, M83 (acetyl-Arg were electrophoresed under reducing conditions on 4-12% AEEA-(D)Ala-(L)boroPro), has dual FAP (K, 5.7 nM) and Bis-tris SDS-PAGEgels (Invitrogen) and transferred to nitro POP (K–7.4 nM) inhibition. The specific POP inhibitor, J94 cellulose for Western blotting. After blocking with 3% bovine (acetyl-Lys-Leu-Arg-(L)boroPro (K,<100 nM)); and the sub serum albumin (BSA)/TBS-Tween (TBST), blots were incu strate. C95 (acetyl-Arg-AEEA-Gly-Pro-AMC), were bated with a combination of 0.5 g/ml mab 6D2, 0.1 lug/ml designed, synthesized and characterized by us as previously anti-C. tubulin (Sigma #6199) and 0.1 lug/ml anti-actin (Ab reported 27. All are soluble in aqueous buffers. The inhibi cam 2G1055) all in 1% BSA/TBST, and then washed and tors (J94 and M83) were dissolved in Hanks Balanced Salt incubated with 1:50,000 goat anti-mouse-HRP (Thermo Solution (HBSS: Gibco) at 1 mM and stored at -20°C. The Fisher). Blots for POP were blocked with 3% BSA/TBST, substrate (C95) was dissolved in HBSS at 2.5 mM and stored then incubated with a combination of 0.1 ug/ml goatanti-POP at 4° C. FAP and POP at equimolar concentrations cleaved (R& D Systems #AF4308), 0.1 g/ml anti-C. tubulin (Sigma C95 substrate at equivalent rates (data not shown). Tic-Pro #6199) and 0.1 ug/ml anti-actin (Abcam 2CR 1055) in 1.5% AFC, a prolyl-specific fluorescent substrate, was supplied by BSA/0.5M NaC1/TBST. After washing in the same buffer, Vantia (Southampton, England). blots were incubated with 1:18,000 rabbit anti-goat-HRP (R (0200 APCE (4 ug), DPPIV (2 ug) or POP (2 ug) was & D Systems #HAF017) in 1.5% BSA/0.5M NaC1/TBST. incubated in 25 mM sodium phosphate buffer, pH 7.5, con ECL-Plus (Thermo-Fisher) was added and blots were visual taining 1.0 mM EDTA and 2% methanol in a total volume of ized on RPI blue radiographic film (Amersham). 200 ul for 20 min at 22°C. Using Tic-Pro-AFC (10-300 uM 0.195 Confocal analysis of FAP protein in cells was for APCE, 5-200 uM for DPPIV, and 40-800 uM for POP), accomplished first by growing cells to confluence on four fluorescence was monitored with time at excitation/emission well glass chamber slides (Lab-Tek). The cells were fixed wavelengths of 400/508 nm, using a black-sided 96-well with 0.75% paraformaldehyde, blocked with 1% BSA/PBS, plate in a BIO-TEK FL600 fluorescence plate reader. For and then incubated with mouse F19 mAb 2 ug/ml or isotype accurate assessment ofkinetic parameters, saturating concen control antibody, MOPC 21 (Sigma), 2 ug/ml in 0.1% BSA/ trations of substrate were used. For standard curves, dilutions PBS, with 0.1% saponin. After washing, the slides were incu of AFC (7-amino-4-trifluoromethylcoumarin) were prepared bated with goat anti-mouse-Alexa Fluor 568 (Invitrogen) at in the same assay buffer and corresponding fluorescence was 1:2000 in 0.1% BSA/PBS and then mounted with Prolong measured. The substrate in five different concentrations (20. Gold/DAPI (Molecular Probes). Cells were visualized using 40, 60, 80 & 160 uM) was mixed with four different concen a Leica TCSNTMicroscope fitted with a 40x Plan Fluotar 1.0 trations of inhibitors around the preliminary apparent (app) NA oil immersion objective. Images were analyzed using K, values. Leica TCS and Volocity software. 0201 Whole Cell Activity Assays: 0196. Protein Characterization: 0202 Cells were cultured in normal growth media unless 0.197 Cells of each type were grown to confluence in 10 otherwise stated. For cells grown in media with selected cm tissue culture dishes, rinsed in PBS and then lysed in 1 ml components either withheld or added, and depending on each ice-cold IP buffer containing 1% Triton/150 mM. NaCl/10 experimental design as indicated in the Results section, mM Tris, pH 7.5/1 mM EDTA/1 mM EGTA/0.5% NP-40/ growth times were chosen from 7 to 14 days in specific media 10% sucrose with Complete Ultra protease inhibitor cocktail before plating for activity assays. For activity assays, cells (Roche) added. Whole cell lysates were centrifuged to were plated into 96-well black-sided, clear bottom tissue remove detergent-insoluble proteins. Five ug/ml of mAb F19 culture plates (CoStar) at densities selected for achieving con were added and allowed to bind overnight at 4°C. Then 25ul fluency in three days as indicated in the Results section or of 75% slurry of Protein G beads (Amersham) in TBS were each such experiment. Cell densities varied with cell type and added and incubated for 1 hour at 4°C. Protein G beads were the selected growth media; the number of cells necessary for spun down, washed three times with IP buffer, resuspended in plating was estimated from pilot experiments. After three loading buffer and boiled for five minutes, after which the days growth, cultures were washed with HBSS. The wash beads were removed by centrifugation and a portion of the solution was replaced with 188 ul fresh buffer (HBSS) and Supernatant was electrophoresed under reducing conditions either buffer or two ul of M83 or J94 inhibitor (10 uM final) on 4-12% Bis-tris SDS-PAGE gels. To confirm the presence was added. Tenul offluorescent substrate. C95 were added to of FAP, the regions of each lane corresponding to the molecu give a final concentration of 125 uM, and fluorescence was lar weight of FAP were excised and the proteins within each measured with time at 360/460 nm excitation/emission wave gel slice were reduced with Tris(2-carboxyethylphosphine, lengths using an FL600 microplate fluorescence reader (Bio then alkylated with iodoacetamide, and digested with trypsin tek Instruments). Fluorescence units were converted to FAP as described by the In-gel Tryptic Digestion Kit protocol units/100,000 cells by using a conversion factor determined (Thermo-Fisher). Each trypsin digest sample was analyzed from an APCE standard curve of prolyl-specific endopepti by high performance liquid chromatography-tandem mass dase activity, such that 1 FAP unit is equivalent to the fluo spectrometry (LC/MS/MS) on a nanoscale Dionex UltiMate rescence produced by 1 ng of APCE/min. Since FAP and POP 3000 HPLC equipped with an Acclaim PepMap C18 column cleave the C95 substrate at an equivalent rate, both FAP and (75 um internal diameterx15 cm length with 3 um particles) POP activities are expressed as FAP units in all figures. US 2015/01 19330 A1 Apr. 30, 2015

0203 For tube formation assays, MatrigelTM (BD Bio FAP or POP, and the high affinity POP inhibitor J94 which has sciences, San Jose, Calif.) 80 ul was added to black-sided, strict specificity for POP, but none towards FAP, it was pos clear bottom 96-well plates as above and allowed to gel at 37° sible to measure each enzyme’s activity 27. For each cell C. for 30 minutes. Then 15,000 HMVEC-d in EC media type, total prolyl-specific endopeptidase activity on intact cell without serum were added and allowed to settle for 1 hour. membranes was determined using C95 substrate, which is Either buffer, the M83 inhibitor or the J94 inhibitor (10 uM cleaved at equal rates by FAP or POP; on parallel cultures J94 final) was added and the assay then started by adding the dual inhibitor was added to block solely that activity due to POP, FAP/POP fluorescent C95 Substrate. The reader was main thereby defining the remaining endopeptidase activity as tained at 37° C. and fluorescence was measured with time for uniquely attributable to membrane-associated FAP (FIGS. 18 hours. 1B,3,4B,5B, 6 & 8B). As expected, the dipeptidase, DPPIV. 0204 Results neither cleaved C95 substrate nor was it inhibited by M83 or 0205 FAP/APCE and POP are prolyl-specific serine J94. The previous studies showed that APCE and FAP have endopeptidases, with the proteolytic activity of POP gener essentially identical activities 12, thereby allowing the use ally restricted to peptides having less than about 30 residues of an APCE standard curve to determine moles of active FAP 31. FAP appears to be membrane-inserted and POP mem on the cell surface and hence, the number of FAP proteinase brane-associated 32, 33, while APCE circulates in blood, molecules/cell. Therefore, knowing the FAP activity and possibly as a shed soluble derivative or splice-variant of FAP number of WI-38 fibroblasts in a confluent well, each cell was 12. Non-specific single amino acid and dipeptide prolyl estimated to have 117,000 FAP molecules on its membrane boronic acid compounds or mutations of the serine active-site surface. FIG. 1C demonstrates FAP by Western blot and FIG. in FAP inhibit FAP proteolytic activity on cancer associated 1D indicates that the majority of FAP is membrane-associ fibroblasts and diminishes cancer growth 17, 21. POP, com ated, with a lesser amount in the cytosol, which agrees with monly expressed by neoplasms 7, 25, 33-35, manifests our confocal results. FIG. 1E shows POP by Western blot in overlapping proteolytic activity with FAP/APCE when mea both WI-38 and VA-13 fibroblasts, although the activity assay Sured as usual with non-specific fluorescent peptide Sub did not detect any surface accessible POP activity in VA-13 strates such as Z-Gly-Pro-AMC or succinyl-Gly-Pro-AMC cells. The majority of POP is cytosolic with a significant 27. Efforts to use inhibitors to separate the two enzymatic fraction being membrane-associated, which agrees with our activities have likewise been compromised by significant assay results of cell surface POP activity (FIG.1F). inhibition of both FAP and POP 27. The problem of mea (0208 Neither FAP nor POP activity was found in media Suring FAP and POP activities separately in biologic or patho that had been in contact with cells for three days, indicating logic scenarios where either may play a critical role, had that neither enzyme was shed or released (data not shown). received scant attention prior to the present work. When fresh buffer was again placed over the cells, both FAP 0206 Characterization of Tic-Pro-AFC as substrate: A and POP activities were found to be unchanged and immedi new substrate, Tic-Pro-AFC, was used for assessing APCE/ ately detectable, thereby Supporting the membrane location FAP, DPPIV and POP, each belonging to the same clade of of both enzymes. As can be seen in FIG.1B, WI-38 fibroblasts prolyl-specific serine proteinases and each with activity yielded higher FAP than POP activity. In FIG. 1C, immuno towards unique Substrates. The typical synthetic Substrate for blot analyses of lysates derived from a specified number of APCE/FAP or POP is benzyloxycarbonyl (Z)-GP-AMC for cells of each cell type, using the cellular content of C-tubulin which each enzyme exhibits a catalytic efficiency (k/K) of (50 kDa) and actin (43 kDa) as protein load controls, showed 6.7x10 and 9.5x10M's', respectively. Z-GP-AMC is not relative FAP levels in accord with those estimated by confocal cleaved by DPPIV; however, the latter does cleave GP-AMC microscopy of corresponding immunostained cells or by (k/K3.2x10 M's'). GP-AMC is a poor substrate for endopeptidase activity measurements. As shown in FIG. 2, APCE/FAP (k/K–1.1x10 MT's) and is not cleaved by amino acid sequence determinations for tryptic peptides of POP. Based on K. K. and k values in Tables 6 and 7. the isolated putative FAP protein band from each cell type Tic-Pro-AFC was readily cleaved by each enzyme, with k/ ensured that the 100 kDa band from WI-38 fibroblasts was K for APCE/FAP being slightly higher than POP and indeed FAP. slightly lower than DPPIV. Importantly, k/K values were (0209 FIG. 3 illustrates the effectiveness and rapidity of in keeping with those determined using Gly-Pro-AMC or FAP and POP inhibition by the pseudo-peptide inhibitor con Z-Gly-Pro-AMC as substrates; however, Tic-Pro-AFC struct, M83. Cultured W-138 cells were exposed to the inhibi proved more sensitive and gave more reproducible results. tor at either the beginning of the assay, or after incubation for Thus, Tic-Pro-AFC was chosen as the substrate for compar two hours with the substrate C95 that is cleaved by FAP or ing the potency and selectivity of the inhibitors towards POP. After an increasing fluorescent signal indicated the APCE/FAP, DPPIV, or POP accrual of significant proteolytic activity, the addition of even 0207. In FIG. 1A, confocal microscopy of immunostained nanomolar concentrations of inhibitor M83 instantly abol saponin-permeabilized WI-38 fibroblasts shows abundant ished endopeptidase activity as evidenced by lack of further FAP, most of which appears membrane-associated. As fluorescence increase. reported, FAP was absent in SV-40 transformed VA-13 fibro 0210. As shown in FIG. 4, three methods of assessment, blasts 5). FIG. 1B shows results for FAP and POP activity namely: confocal microscopy, Western blotting, and assays, using highly selective, specific Substrates and inhibi endopeptidase activity, indicated that normal human MCF tors designed and synthesized in the inventors lab 27. Over 12A breast cells or human breast carcinoma MDA-MB 436 lying media was first removed from each cell culture after cells contained far less FAP protein and FAP activity than which cells were washed with physiologic buffer and overlaid observed for WI-38 activated fibroblasts (FIG.1). While POP with fresh buffer. FAP or POP inhibitor was added, and each activity for MCF-12A cells and WI-38 fibroblasts was simi culture assayed with time for both FAP and POP activity. By lar, human MDA-MB436 breast cancer cells had about five using the specific substrate C95, which is cleaved only by times that amount. Human HCC1419 breast cancer cells pos US 2015/01 19330 A1 Apr. 30, 2015 26 sessed about the same amount of POP activity as MCF-12A human mesenchymal cells in FIG. 8A shows most of the normal breast cells; however, HCC1419 cancer cells con immunostained FAP in the cytosol, with relatively less in tained neither FAP protein nor FAP activity. Without wishing their membranes, which is opposite to what was observed for to be bound by theory, over-expression of POP in neoplasms WI-38 fibroblasts. Western immunoblots of mesenchymal has not been explained, but recent results of Myohanen et al. cell lysates (FIG. 8C) confirmed the large amount of FAP 36 suggest that POP is responsible for a second-step pro present and did identify POP protein as well (FIG. 8E). teolytic cleavage in the autoregulation of thymosin-B4 that LC/MS/MS analysis of tryptic peptides from digestion of the yields the derivative tetrapeptide, acetyl-SDKP, which is 100 kDa band in lysates of mesenchymal stem cells estab known to be a potent stimulator of angiogenesis. lished the protein band as a subunit of homodimeric FAP 0211. In FIG.5A, confocal microscopy of cultured normal (FIG. 2). When mesenchymal membrane and cytosolic frac human dermal microvascular endothelial cells (HMVEC-d) tions were separated and subjected to Western blotting, it was on plastic demonstrated that cell-associated FAP was only clear that FAP is abundant on membranes and is in the cytosol occasionally encountered (panel A shows a field containing a to a lesser extent (FIG. 8D). In contrast, the majority of POP rare positive cell). Western blotting of HMVEC-d lysates is in the cytosol, with a lesser amount found in the membrane lacked a band consistent with FAP protein (FIG.5C). Like (FIG. 8F). Mesenchymal cells and fibroblasts had about the wise, HMVEC-d were devoid of FAP activity, but HMVEC-d same amount of FAP and POP activity, which might be cultures did contain considerable POP activity, and as shown expected given that mesenchymal cells are believed precur in FIG. 6B, when grown on MatrigelTM and allowed to form sors of fibroblast. Assuming mesenchymal stem cell and tubules over an 18-hr period, significant amounts of both FAP fibroblast membranes are impermeable to C95 substrate and POP activities were expressed and easily detectable. POP because of the latter's positive charge and lack of hydropho expression began just before capillary-like tubules started bicity, cytosolic FAP activity in live cells would not likely forming and continued as the complexity of the tubule net contribute to measurement of membrane activity. work increased. Interestingly, detectable FAP expression as 0214 FIG. 9 shows the inhibition of lung cancer reflected by proteolytic activity began about 3-4 hours after xenografts in nude mice by compound M83. Mice were given tubule formation had clearly begun (i.e., 8 hours from plating a 50 ul injection into each hind leg consisting of human lung HMVEC-d) and then continued to increase during the subse adenocarcinoma epithelial cells (2x10 cells) suspended in quent 18-hr period of growth. These findings indicate the MatrigelTM. After ten days the tumors had grown to an esti involvement of POP in the initiation and propagation of ves mated 40 mm, so M83 treatment was initiated (day 0 in sel formation, with the timing of FAP expression synchro graph). For the M83 treatment group, mice were given a daily nized with ECM invasion by the forming capillary-type intraperitoneal injection of 26.5 micrograms of M83 dis tubules. While FAP mRNA is up-regulated coincident with solved in 20 microliters of sterile saline. The mice of the capillary formation 38, the observation that proteolytically control group were given an intraperitoneal injection of 20 active FAP protein becomes easily detectable and increases microliters of sterile saline. After ten days, tumors in the progressively with tubulogenesis has not been reported pre control group began to grow rapidly, while tumors in mice viously. treated with M83 failed to progress and by day 28 of treatment 0212. As shown in FIG. 7, when HMVEC-d and other the average tumor Volume was no greater than on day Zero. In selected cell types were stressed by removing hydrocortisone addition, one of the six tumors in the M83 treatment group (hyc) from growth media, FAP became substantially over had completely disappeared. The treatment groups consisted expressed as detected by Western blots of cell lysates and of three mice each with two tumors per mouse. FIGS. 10A corresponding increases in FAP proteolytic activity. Identifi and B are micrographs which demonstrate that application of cation of the over-expressed protein as FAP was validated by 50 um of compound J94 inhibited formation of capillary-like amino acid sequence determination as shown in FIG. 2. In tubes in MatrigelTM. FIG. 7A, the greatest over expression of FAP in response to 0215 FIG. 11 depicts results which show that J94 inhibi omission of hydrocortisone in growth media was seen with tor is effective in HCT 116 human colon cancer xenografts in MCF-12A normal breast cells. When hydrocortisone (hyc) immunodeficient mice treated with J94 or M83. Clearly dur was restored to concentrations used for growth of normal ing the 28-day treatment period, J94 diminished cancer cells in culture, over expression of FAP by MCF-12A cells growth by 75%, while M83 was slightly more effective than was totally reversed (FIG. 7B, compare MCF-12A (-) hyc this. Although not shown here, microscopy of control and and MCF-12A (-)hyc/(+)hyc). In contrast, removal of vari treated tumor specimens by Immunohistocytochemistry con ous growth factors (VEGF, bFGF, EGF, IGF) from growth firmed the presence of FAP and POP; demonstrated thicker media had no apparent effect, even after 42 days of culture. bundles of collagen Surrounding residual tumor; widespread 0213 Cancerous mesenchymal stem cells may be the best areas of tumor cell apoptosis within treated tumors; strong target in the search for a broadly applicable common denomi Suggestions of diminished capillary vascularity within treated nator or "pan-tumor approach for treating a large number of tumors; and lastly, raised the question of whether tumor cell cancers 40, 41. The inventors are aware of only one report autophagy might also have occurred. In every occasion of J94 showing FAP to be associated with mesenchymal stem cell or M83 treatment, xenografted human tumors, be they colon membranes as evidenced by immunoselection of mesenchy cancer or lung cancer, showed strikingly decreased rates of mal stem cells from human cryopreserved bone marrow with growth. a FAP monoclonal antibody 42. This prompted us to ques 0216 FAP is considered to be a potent diagnostic or thera tion whether mesenchymal stem cells, as the putative precur peutic target because it is (i) over-expressed by activated sor to activated stromal fibroblasts 43, also expressed pro stromal fibroblasts in epithelial-derived human malignancies teolytically active, membrane-bound FAP and if so, how 19-21 and (ii) absent in normal adult tissues and benign much relative to the activated fibroblast, and could it be tumors 19-21. Santos et al. 21 showed that targeted gene readily inhibited? Confocal microscopy of permeabilized disruption or pharmacologic inhibition of FAP proteinase US 2015/01 19330 A1 Apr. 30, 2015 27 activity slowed or halted tumor growth in mouse models of being cytosolic or inactive and hence are unlikely to cleave endogenous lung cancer and human colon cancer. In both extracellular substrates or to become inhibited by water tumor scenarios, cancer cell proliferation decreased, collagen soluble agents that must permeate the cell membrane. increased, and myofibroblast content and blood vessel den 0220. The results provided herein show that activated sity decreased, prompting the Suggestion that targeting fibro fibroblasts, i.e., those that are (i) rapidly dividing, (ii) highly blasts within the tumor microenvironment might be useful mobile, (iii) contain C-Smooth muscle actin (myofibroblasts), therapeutically. To date, however, studies of such relatively and (iv) manifest enhanced ECM deposition, also have non-specific putative inhibitors of FAP, e.g. Glu-boroPro or impressive amounts of FAP on their membranes with lesser Val-boroPro, show that both also inhibit physiologically amounts in the cytosol (FIGS. 1A and D). Cancer-associated important DPPIV. On a molar basis both are 50% or less fibroblasts are activated fibroblasts that typically help form effective for inhibiting POP than FAP and, both undergo the stromal scaffolding of metastatic epithelial-derived tumor cyclization and have abbreviated survivals in vivo that detract microenvironments. Assays for FAP or POP activity in the from therapeutic potential. non-serum containing media in which cells grew, or in buffer 0217 Recently Kraman and associates 44 reported that washes of those cell cultures, were always devoid of pro absence of FAP-expressing cells in mice allowed effective teolytic activity, thereby supporting FAP and POP as mem vaccination strategies for immunological control of epithelial brane-associated proteins, with each in a conformation that cell-derived cancer growth. In that study, direct efforts were allows proteolytic activity to be easily and rapidly inhibited. not made to determine whether FAP proteolytic activity was Subtraction of the activity specifically inhibited by the POP necessary for immunosuppressive effects within the tumor inhibitor J94 provided an assessment of accessible FAP pro environment. In accord with both proteolytic and non-pro teolytic activity. Assuming equivalent recoveries of FAP from teolytic roles for FAP in malignant growth, Huang et al. 45 different cell types, the amount of FAP protein and FAP have recently suggested that FAP proteolytic function is proteinase activity in WI-38 fibroblasts exceeded that in any important in extracellular matrix degradation, but that other other cell, except for mesenchymal stem cells. Immunoreac undefined properties (possibly immunologic) of FAP may tive FAP protein samples recovered from WI-38 fibroblasts, promote tumor growth. While the function of FAP proteinase MDA-MB436 cancer cells, HMVEC-d deprived of hydrocor activity within malignancies has been poorly understood, tisone, or mesenchymal stem cells all had essentially identical most efforts to assess FAP as a therapeutic target have amino acid sequences to that established for FAP. Proteolytic involved inhibiting its proteinase activity. Characterizing the assays confirmed that viral-transformed VA-13 human fibro proteolytic activity of membrane-inserted FAP has been blasts lacked both membrane-associated FAP and POP activi unusually difficult, since a physiologic or pathologic Sub ties. strate has not been definitively identified. Having discovered 0221 Small amounts of FAP and POP activity and protein APCE and its only known physiologic Substrate, precursor in normal breast cells, about the same amount of FAP activity C-antiplasmin (CAP), the inventors took advantage of in MDA-MB436 human metastatic breast cancer cells, were APCE being essentially identical to FAP and used the amino demonstrated; however, the latter cells contained about 6 acid sequence Surrounding the Scissile bond of precursor times the amount of POP activity compared to normal breast CAP to design stable and highly effective water-soluble cells. It seems reasonable that within hormonally-induced inhibitors of APCE and FAP 27. In the process, it was cyclical tissue responses, an occasional normal breast paren discovered that a positively charged residue in the P6 or P7 chymal cell might be induced to express a small amount of position augmented the cleavage rate significantly 29, 46. FAP and POP, HCC1419 intraluminal primary breast cancer and this proved useful in developing a highly effective FAP cells contained barely detectable FAP by immunostaining, inhibitor, M83, and the fluorescent substrate C95 27, 29. and no assayable FAP proteinase activity; POP activity was Both manifested high affinity and good specificity for FAP; greatly reduced, but POP protein, likely intracellular, was however, these properties were also directed toward another easily demonstrable. Within a growing HCC1419 tumor, prolyl-specific serine protease family member, namely, POP, however, this does not preclude the possibility that requisite which the inventors, like others 33, 47, have found associ stroma within the breast cancer malignancy may express FAP ated with selected normal and cancer cell lines. and POP. The basis of abundant POP activity found on MDA 0218. To put the overall endopeptidase activity on cell MB436 breast cancer cells (FIG. 4) remains obscure, despite membrane surfaces in perspective, it was reasoned that FAP such increases having been noted before in several other and POP activities should each be quantitated. The C95 FAP malignancies. Larrinaga et al. 33 reported POP on cell and POP Substrate as well as the M83 inhibitor of both FAP membranes of various human cancers, but usually in amounts and POP were used in conjunction with a novel and highly not much different than those on corresponding normal cell specific J94 POP inhibitor, the latter having no effect on FAP, types; however, cytosolic POP within cancers cells was regu for estimating membrane-associated FAP or POP endopepti larly significantly increased beyond that in corresponding dase activity. POP cleaves selected peptides of < 30 residues. normal cells, which is in keeping with our finding for Most recently, as noted above, POP has been proposed to have HCC1419 breast cancer cells. a significant role in angiogenesis by cleaving a short deriva 0222 Endothelial cells (HMVEC-d) grown on plastic tive peptide from the ubiquitously tissue-distributed thy contained a rare FAP-positive cell by immunostaining, but no mosin B4, to yield the tetrapeptide compound, acetyl-SDKP. FAP protein by immunoblotting or proteinase assay. How that stimulates angiogenesis 35. ever, as confluence was achieved, significant POP activity 0219. Notably, the dipeptidase proteolytic activity of was expressed concordantly. As expected, tube formation did either membrane-inserted or soluble DPPIV is directed only not occur on plastic despite endothelial cell confluency; how toward amino-terminal dipeptides, and as expected, DPPIV ever, POP expression continued unabated. FAP was not neither cleaved C95 substrate nor was it inhibited by M83 or expressed during the 72-hr growth period (FIG. 5B). In con J94. Other prolyl dipeptidases have the added negative of trast, when grown on MatrigelTM, by about four hours, the US 2015/01 19330 A1 Apr. 30, 2015 28

HMVEC-d cells began to align progressively in tubular struc mesenchymal stem cells, selected cancer cells, and endothe tures and POP expression continued to increase (FIG. 6B). lial cells as the latter participate in angiogenesis. (ii) In their Shortly after tube-like capillaries began forming, FAP activ membrane bound form, each enzyme is proteolytically active ity became demonstrable (FIG. 6B). By about 18 hours, well and easily accessible for efficient inhibition by a new soluble, defined capillary-like networks dominated. These results high affinity, selective pseudo-peptide inhibitor that meets prompted the speculation that expression of proteolytically structural requirements for the respective enzyme’s Sub active FAP might be synchronized with capillary growth to strate-binding region. (iii) Endothelial cells readily express foster invasiveness of developing microvasculature into the POP as they grow, and in addition, membrane-inserted pro ECM 37. Aimes et al. 38 noted endothelial expression of teolytically-active FAP is synthesized and expressed as tubu mRNA transcripts of several serine proteases, including FAP, logenesis occurs 38.50. (iv) FAP and POP are therapeutic in association with the nature of the culture Substratum and targets for a large number of cancers and the two new inhibi progression of angiogenesis. The finding of increasing FAP tors, M83 and J94, have use as therapeutics against com proteolytic activity demonstrates that the increased FAP monly encountered epithelial-derived cancers and their meta mRNA is actually translated during tubule formation. Several static foci. studies have suggested that endothelial cells within the devel 0226. Although the presently disclosed and claimed oping microvasculature of malignant tissues express FAP8, inventive concept(s) and the advantages thereof have been 39, 50. While it might be speculated that FAP's presence described in detail, it should be understood that various could result from growth of fibroblast-related pericytes that changes, Substitutions and alterations can be made herein accompany neovascularization, fibroblasts were never without departing from the spirit and scope of the presently observed in HMVEC-d cultures. These results indicate that disclosed and claimed inventive concept(s) as defined in the membrane-inserted proteolytically-active FAP is synthesized present disclosure. Moreover, the scope of the present appli and expressed by endothelial cells as they participate in cation is not intended to be limited to the particular embodi angiogenesis. ments of the processes, compositions of matter, means, meth 0223) As shown by immunoblotting and activity assays ods and steps described in the specification. As one of (FIG. 7), stress caused by removal of hydrocortisone from ordinary skill in the art will readily appreciate from the dis growth media stimulated both HMVEC-d and MCF 12A nor closure of the presently disclosed and claimed inventive con mal breast cells to over-express FAP, which was directly cept(s), processes, compositions of matter, means, methods, documented as the Source of proteolytic activity by its isola or steps, presently existing or later to be developed that per tion and amino acid sequence (FIG. 2). Notably, replenish form substantially the same function or achieve substantially ment of hydrocortisone returned MCF 12A expression of FAP the same result as the corresponding embodiments described to levels before its removal (FIG. 7). Similarly, WI-38 acti herein may be utilized according to the presently disclosed vated fibroblasts which are ordinarily grown in the absence of and claimed inventive concept(s). Accordingly, the presently hydrocortisone, showed a detectable decrease in FAP expres disclosed and claimed inventive concept(s) is intended to sion when hydrocortisone was added. Analogous effects with include within their scope all Such processes, compositions of hydrocortisone have been noted before with other cells, e.g., matter, means, methods, or steps. decreased hydrocortisone in cultures of selected normal human epithelial cells was associated with increased synthe TABLE 1 ses of both urokinase and tissue plasminogen activator 51. These data Suggest that cells are capable of altering expres Cyclic Amines and Proline Analogs sion of FAP if they should become stressed as might occur in 4-hydroxypyrrollidine-2-carboxylic acid (cis and trans) a rapidly expanding metastatic tumor microenvironment. 3-phenylpyrrollidine-2-carboxylic acid (cis and trans) 3-hydroxypyrrollidine-2-carboxylic acid (cis and trans) 0224 Mesenchymal stem cells give rise to progenitor adi 4-hydroxypyrrollidine-2-carboxylic acid (cis and trans) pocytes, bone cells and myocytes, and are also considered 2-ethylthiazolidine-4-carboxylic acid (cis and trans) precursive to fibroblasts 43.52. Conceivably, activated mes 2-methylthiazolidine-4-carboxylic acid (cis and trans) 2-phenylthiazolidine-4-carboxylic acid (cis and trans) enchymal stem cells could arise as a consequence of epithe 5,5-dimethylthiazolidine-4-carboxylic acid lial-mesenchymal cell transformation 53 and represent the thiazolidine-2-carboxylic acid (cis and trans) progenitor cell of the “tumor niche'. Some propose, however, thiazolidine-4-carboxylic acid (cis and trans) that mesenchymal stem cells originate from bone marrow, aZetidine-2-carboxylic acid (cis and trans) thiazolidine-2-carboxylic acid (cis and trans) move into a selected tissue and undergo malignant transfor thiazolidine-4-carboxylic acid (cis and trans) mation to produce cancer cells characteristic of that tissue amino-L-proline methyl ester 40.54. Others consider that a precursive cancer stem cell cyano-L-proline methyl ester might derive from a dormant multipotent cell unique to a 4-cyano-L-proline 3,4-dehydro-L-proline specific tissue. The amounts of FAP we observed for mesen Boronylproline chymal stem cells by confocal microscopy and Western blot 4-fluoro-L-proline ting (FIG. 8) were more than for WI-38 activated fibroblasts Nitrileproline (FIG. 1). The multiple roles of mesenchymal cells suggest lysyl piperidide N-(4-chlorobenzyl)4-0x0-4-(1-piperidinyl)-1,3-(s)-butane-diamine they may exist in an activated State more frequently than do bromocyclopentyl carboxylic acid fibroblasts. This may be particularly true with respect to mes chlorocyclopentyl carboxylic acid enchymal cell involvement in malignancies and account for fluorocyclopentyl carboxylic acid the large amount of FAP we observed in these cells. cis-3-methylproline cis-3-ethylproline 0225. The results provided herein allow the following con cis-3-isopropylproline clusions: (i) FAP and POP are both expressed on the mem cis-3-isopentanylproline branes of cells critical to tumor niche formation in primary homoproline tumors or metastases, namely: cancer-associated fibroblasts,

US 2015/01 19330 A1 Apr. 30, 2015 32

Data represent the best-fit value: the standard error, inhibition Fibroblast activation protein peptide substrates identified of APCE activity. from human collagen I derived gelatin cleavage sites. Bio chemistry 47:1076-1086. REFERENCES 0241 14. Niedermeyer J. Scanlan M. J. Garin-Chesa P. 0227. The following references, to the extent that they Daiber C, Fiebig HH, Old U, Rettig W.J. Schnapp A (1997) provide exemplary procedural or other details Supplementary Mouse fibroblast activation protein: molecular cloning, to those set forth herein, are specifically incorporated herein alternative splicing and expression in the reactive Stroma of by reference in their entireties. epithelial cancers. Int J Cancer 71:383-389. 0228 1. Garin-Chesa P. Old U, Rettig W J (1990) Cell 0242. 15. Christiansen VJ, Jackson KW. Lee KN, McKee Surface glycoprotein of reactive stromal fibroblasts as a P A (2007) Effect of fibroblast activation protein and potential antibody target in human epithelial cancers. Proc alpha2-antiplasmin cleaving enzyme on collagen types I. Natl Acad Sci USA 87: 7235-7239. Ill, and IV. Arch Biochem Biophys 457:177-186. 0229 2. Park J. E. Lenter MC, Zimmermann RN, Garin 0243 16. Huang Y. Wang S. Kelly T (2004) Seprase pro Chesa P. Old U, Rettig W J (1999) Fibroblast activation motes rapid tumor growth and increased microVessel den protein, a dual specificity serine protease expressed in reac sity in a mouse model of human breast cancer. Cancer Res tive human tumor stromal fibroblasts. J Biol Chem 274: 64:2712-2716. 36505-365.12. 0244 17. Cheng JD, Weiner LM (2003) Tumors and their 0230 3. Rettig W. J. Garin-Chesa P. Beresford HR, Oet microenvironments: tilling the soil. Commentary re: A. M. tgen H F, Melamed MR, Old U (1988) Cell-surface gly Scott et al., A Phase I dose-escalation study of sibrotu coproteins of human sarcomas: differential expression in Zumab in patients with advanced or metastatic fibroblast normal and malignant tissues and cultured cells. Proc Natl activation protein-positive cancer. Clin. Cancer Res., 9: Acad Sci USA 85:3110-3114. 1639-1647, 2003. Clin Cancer Res 9:1590-1595. 0231. 4. Niedermeyer J. Garin-Chesa P. Kriz M, Hilberg F, 0245 18. Kalluri R. Zeisberg M (2006) Fibroblasts in Mueller E. Bamberger U, Rettig W. J. Schnapp A (2001) cancer. Nat Rev Cancer 6:392–401. Expression of the fibroblast activation protein during 0246) 19. Pure E (2009) The road to integrative cancer mouse embryo development. Int J Dev Biol 45:445-447. therapies: emergence of a tumor-associated fibroblast pro 0232 5. Rettig W. J. Garin-Chesa P, Healey J. H. Su SL, tease as a potential therapeutic target in cancer. Expert Ozer HL, Schwab M. Albino A P. Old U (1993) Regulation Opin TherTargets 13:967-973. and heteromeric structure of the fibroblast activation pro 0247. 20. Hayward SW (2010) Preclinical assessment of tein in normal and transformed cells of mesenchymal and fibroblast activation protein as a target for antitumor neuroectodermal origin. Cancer Res 53:3327-3335. therapy. Future Oncol 6:347-349. 0233 6. Acharya PS, Zukas A, Chandan V. Katzenstein A 0248 21. Santos AM, Jung J, Aziz N, Kissil J. L. Puré E L. Pure E (2006) Fibroblast activation protein: a serine (2009) Targeting fibroblast activation protein inhibits protease expressed at the remodeling interface in idio tumor stromagenesis and growth in mice. J Clin Invest pathic pulmonary fibrosis. Hum Pathol 37:352-360. 119:3613-3625. 0234 7. Wang XM, Yao T W. Nadvi NA, Osborne B, McCaughan GW. Gorrell MD (2008) Fibroblast activation 0249 22. Kelly T. Adams J. Bachovchin W. Barton R, protein and chronic liver disease. Front Biosci 13:31 68 Campbell S. Courts S. Kennedy C, Snow R (1993) 318O. 0250 Immunosuppressive boronic acid dipeptides: corre 0235 8. Ge Y, Zhan F. Barlogie B, Epstein J. Shaughnessy lation between conformation and activity. JAm Chem Soc J, Jr., Yaccoby S (2006) Fibroblast activation protein (FAP) 11.5:12637-12638. is upregulated in myelomatous bone and Supports (0251 23. Rosenblum J S. Kozarich J W (2003) Prolyl myeloma cell survival. BrJ Haematol 133:83-92. peptidases: a serine protease subfamily with high potential 0236 9. Dohi O, Ohtani H, Hatori M. Sato E. Hosaka M, for drug discovery. Curr Opin Chem Biol 7:496-504. Nagura H. ltoi E. Kokubun S (2009) Histogenesis-specific 0252 24. Narra K. Mullins SR, Lee HO, Strzemkowski expression of fibroblast activation protein and dipepti Brun B, Magalong K. Christiansen V. J. McKee PA, dylpeptidase-IV in human bone and Soft tissue tumours. Egleston B. Cohen SJ, Weiner LM, Meropol NJ, Cheng J Histopathology 55:432-440. D (2007) Phase II trial of single agent Val-boroPro (Tala 0237 10. Bauer S. Jendro MC, Wadle A, Kleber S, Sten bostat) inhibiting Fibroblast Activation Protein in patients ner F, Dinser R, Reich A, Faccin E, Godde S, Dinges H. with metastatic colorectal cancer. Cancer Biol Ther Muller-Ladner U, Renner C (2006) Fibroblast activation 6:1691-1699. protein is expressed by rheumatoid myofibroblast-like syn (0253). 25. Goossens F. De M, I, Vanhoof G, Scharpe S oviocytes. Arthritis Res Ther 8:R171. (1996) Distribution of prolyl oligopeptidase in human 0238 11. Iwasa S. Okada K. Chen WT. Jin X, Yamane T. peripheral tissues and body fluids. Eur J. Clin Chem Clin Ooi A, Mitsumata M (2005) Increased expression of Biochem 34:17-22. seprase, a membrane-type serine protease, is associated (0254. 26. Liu JM, Kusinski M., Hie V. Bignon J. Hajem N, with lymph node metastasis in human colorectal cancer. Komorowski.J. Kuzdak K. Stepien H. Wazieczak-Bakala J Cancer Lett 227:229-236. (2008) Overexpression of the angiogenic tetrapeptide AcS 0239 12. Lee KN, Jackson KW, Christiansen VJ, Lee C DKP in human malignant tumors. Anticancer Res S. Chun J. G. McKee PA (2006) Antiplasmin-cleaving 28:2813-2817. enzyme is a soluble form of fibroblast activation protein. 0255 27. Lee K. N. Jackson K W. Christiansen V J, Blood 107:1397-1404. Dolence E. K. McKee PA (2011) Enhancement of fibrin 0240 13. Aggarwal S, Brennen WN, Kole TP Schneider olysis by inhibiting enzymatic cleavage of precursor E, Topaloglu O, Yates M. Cotter RJ, Denmeade S R (2008) alpha2-antiplasmin. JThromb Haemost 9:987-996. US 2015/01 19330 A1 Apr. 30, 2015

0256 28. Lee KN, Jackson KW, Christiansen VJ, Chung protein alpha identifies mesenchymal stromal cells from KH. McKee PA (2004) A novel plasma proteinase poten human bone marrow. BrJ Haematol 142:827-830. tiates alpha2-antiplasmin inhibition of fibrin digestion. 0272 43. Emura M, Ochiai A, Horino M, Arndt W. Blood 103:3783-3788. Kamino K, Hirohashi S (2000) Development of myofibro 0257 29. Lee KN, Jackson KW, Terzyan S, Christiansen blasts from human bone marrow mesenchymal stem cells V. J. McKee P A (2009) Using substrate specificity of cocultured with human colon carcinoma cells and TGF antiplasmin-cleaving enzyme for fibroblast activation pro beta 1. In Vitro Cell Dev Biol Anim 36: 77-80. tein inhibitor design. Biochemistry 48:5149-5158. (0273 44. Kraman M. Bambrough PJ, Arnold JN, Roberts 0258 30. Gorrao SS, Hemerly J P Lima A R, Melo RL, E. W. Magiera L. Jones J. O. Gopinathan A, Tuveson DA, Szeltner Z, Polgar L, Juliano MA, Juliano L. (2007) Fearon DT (2010) Suppression of antitumor immunity by 0259 Fluorescence resonance energy transfer (FRET) stromal cells expressing fibroblast activation protein-al peptides and cycloretro-inverso peptides derived from brady pha. Science 330:827-830. kinin as Substrates and inhibitors of prolyl oligopeptidase. 0274 45. HuangY. Simms A E, Mazur A. Wang S. Leon N Peptides 28:2146-2154. R, Jones B, Aziz N, Kelly T (2011) Fibroblast activation 0260 31. Szeltner Z, Polgar L. (2008) Structure, function protein-alpha promotes tumor growth and invasion of and biological relevance of prolyl oligopeptidase. Curr breast cancer cells through non-enzymatic functions. Clin Protein Pept Sci9:96-107. Exp Metastasis 6:567-579. 0261 32. Scanlan M.J, Raj BK, Calvo B, Garin-Chesa P. 0275 46. Christiansen VJ, Jackson KW. Lee KN, McKee Sanz-Moncasi MP Healey J. H. Old U, Rettig W J (1994) PA (2007) The effect of a single nucleotide polymorphism Molecular cloning of fibroblast activation protein alpha, a on human alpha 2-antiplasmin activity. Blood 109:5286 member of the serine protease family selectively expressed 5292. in stromal fibroblasts of epithelial cancers. Proc Natl Acad 0276 47. Duke-Cohan J S. Morimoto C, Rocker J A, Sci. USA 91:5657-5661. Schlossman SF (1995) A novel form of dipeptidylpepti 0262. 33. Larrinaga G. Perez I, Blanco L. Lopez J. I. dase IV found in human serum. Isolation, characterization, Andres L. Etxezarraga C, Santaolalla F. Zabala A. Varona and comparison with T lymphocyte membrane dipepti A, IrazustaJ (2010) Increased prolyl endopeptidase activ dylpeptidase IV (CD26). J Biol Chem 270: 14107-141 14. ity in human neoplasia. Regul Pept 163:102-106. (0277 48. Garcia-Horsman JA, Mannisto PT, Venalainen 0263. 34. Busek P. Stremenova J, Sedo A (2008) Dipepti JI (2007) On the role of prolyl oligopeptidase in health and dyl peptidase-IV enzymatic activity bearing molecules in disease. Neuropeptides 41:1-24. human brain tumors good or evil? Front Biosci 13:2319 2326. (0278 49. Cavallo-Medved D, Rudy D, Blum G, Bogyo M, 0264. 35. Liu JM, Garcia-Alvarez MC, Bignon J. Kusin Caglic D. Sloane B F (2009) Live-cell imaging demon ski M. Kuzdak K, Riches A. Wazieczak-Bakala J (2010) strates extracellular matrix degradation in association with Overexpression of the natural tetrapeptide acetyl-N-ser active cathepsin B in caveolae of endothelial cells during asp-lys-pro derived from thymosin beta4 in neoplastic dis tube formation. Exp Cell Res 315:1234-1246. eases. Ann NY Acad Sci 1194:53-59. (0279 50. Bhati R, Patterson C, Livasy C A, Fan C, 0265 36. Myohanen TT, Tenorio-Laranga J, Jokinen B, Ketelsen D. Hu Z, Reynolds E, Tanner C. Moore D T. Vazquez-Sanchez R. Moreno-Baylach MJ, Garcia-Hors Gabrielli F. Perou C M, Klauber-DeMore N (2008) man J. A. Mannisto P T (2011) Prolyl oligopeptidase Molecular characterization of human breast tumor vascu induces angiogenesis both in vitro and in vivo in a novel lar cells. Am J Pathol 172:1381-1390. regulatory manner. BrJ Pharmacol 163:1666-1678. (0280 51. Myohanen H, Virtanen I, VaheriA (2001) Elimi 0266) 37. Wang T. Shi W (2009) Expression of fibroblast nation of hydrocortisone from the medium enables tissue activation proteins in corneal stromal neovascularization. plasminogen activator gene expression by normal and Curr Eye Res 34:112-117. immortalized nonmalignant human epithelial cells. Biol 0267 38. Aimes RT, Zijlstra A, Hooper J D, Ogbourne S Chem 382:1563-1573. M, Sit ML, Fuchs S. Gotley DC, Quigley J. P. Antalis TM (0281 52. Mishra PJ, Mishra PJ. Humeniuk R. Medina D (2003) Endothelial cell serine proteases expressed during J, Alexe G. Mesirov J. P. Ganesan S, Glod J. W. Banerjee D vascular morphogenesis and angiogenesis. Thromb Hae (2008) Carcinoma-associated fibroblast-like differentia most 89:561-572. tion of human mesenchymal stem cells. Cancer Res 0268 39. Okada K. Chen WT, Iwasa S. Jin X, Yamane T. 68:4331-4339. Ooi A, Mitsumata M (2003) Seprase, a membrane-type (0282 53. Thiery J. P. Aclloque H, Huang RY, Nieto MA serine protease, has different expression patterns in intes (2009) Epithelial-mesenchymal transitions in develop tinal- and diffuse-type gastric cancer. Oncology 65:363 ment and disease. Cell 139:871-890. 370. (0283 54. Baguley BC (2006) Tumor stem cell niches: a 0269. 40. Karnoub A. E. Dash A B, Vo A P. Sullivan A, new functional framework for the action of anticancer Brooks MW, Bell G. W. RichardsonAL, Polyak K, Tubo R, drugs. Recent Pat Anticancer Drug Discov 1: 121-127. Weinberg R A (2007) Mesenchymal stem cells within 0284. 55. Momeni N, Nordstrom B M, Horstmann V. tumour stroma promote breast cancer metastasis. Nature Avarsei H, Sivberg B V (2005) Alterations of prolyl 449:557-563. endopeptidase activity in the plasma of children with autis (0270 41. Coghlin C, Murray G. I. (2010) Current and tic spectrum disorders. BioMed Central Psychiatry 5:27. emerging concepts in tumour metastasis. J Pathol 222:1- 0285 56. Myohanen TT Garcia-Horsman JA, Tenorio 15. Larang J, and Mannisto PT (2009) Issues about the Physi 0271 42. Bae S. Park CW, Son HK, Ju HK, Paik D, Jeon ological Functions of Prolyl Oligopeptidase Based on Its CJ, Koh GY, Kim J, Kim H (2008) Fibroblast activation Discordant Spatial Association with Substrates and Incon US 2015/01 19330 A1 Apr. 30, 2015 34

sistencies Among mRNA, Protein Levels, and Enzymatic Schizophrenia: effects of antidepressants, mood stabilizers, Activity. J. Histochem and Cytochem 57(9):831-848. and antipsychotic drugs. Psychiatry Res. 58:217-225. 0287 58. Mannisto P T Venalainen J. Jalkanen A, and 0286 57. Maes M. Goossnes F. Scharpe S., Calabrese J, Garcia-Horsman JA (2007) Prolyl oligopeptidaes: a poten Desnyder R, and Meltzer HY (1995) Alterations in plasma tial target for the treatment of cognitive disorders. Drug prolyl endopeptidase activity in depression, mania, and News Perspect (Abstract) 20(5).

SEQUENCE LISTING

NUMBER OF SEO ID NOS: 1.

SEO ID NO 1 LENGTH: 760 TYPE PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 1.

Met Lys Thir Trp Wall Lys Ile Wall Phe Gly Wall Ala Thir Ser Ala Wall 1. 5 15

Luell Ala Lell Luell Wall Met Ile Wall Lell Arg Pro Ser Arg Wall His 25 3 O

ASn Ser Glu Glu Asn Thir Met Arg Ala Lell Thir Luell Lys Asp Ile Lell 35 4 O 45

ASn Gly Thir Phe Ser Lys Thir Phe Phe Pro Asn Trp le Ser Gly SO 55 60

Glin Glu Luell His Glin Ser Ala Asp Asn Asn Ile Wall Tell Asn 65 70

Ile Glu Thir Gly Glin Ser Thir Ile Lell Ser Asn Arg Thir Met 85 90 95

Ser Wall Asn Ala Ser Asn Gly Lell Ser Pro Asp Arg Glin Phe Wall 105 110

Lell Glu Ser Asp Ser Lys Lell Trp Arg Ser Thir Ala 115 12O 125

Thir Tyr Ile Asp Luell Ser Asn Gly Glu Phe Wall Arg Gly Asn 13 O 135 14 O

Glu Lell Pro Arg Pro Ile Glin Lell Trp Ser Pro Wall Gly Ser 145 15 O 155 16 O

Lell Ala Wall Tyr Glin Asn Asn Ile Luell Glin Arg Pro 1.65 17 O 17s

Gly Asp Pro Pro Phe Glin Ile Thir Phe Asn Gly Arg Glu ASn Ile 18O 185 190

Phe Asn Gly Ile Pro Asp Trp Wall Glu Glu Glu Met Tell Ala Thir 195 2 OO

Tyr Ala Luell Trp Trp Ser Pro Asn Gly Phe Luell Ala Ala 210 215 22 O

Glu Phe Asn Asp Thir Asp Ile Pro Wall Ile Ala Ser Gly 225 23 O 235 24 O

Asp Glu Glin Pro Arg Thir Ile Asn Ile Pro Pro Ala Gly 245 25 O 255

Ala Asn Pro Wall Wall Arg Ile Phe Ile Ile Asp Thir Thir Pro 26 O 265 27 O

Ala Wall Gly Pro Glin Glu Wall Pro Wall Pro Ala Met le Ala Ser 27s 28O 285

Ser Asp Phe Ser Trp Lell Thir Trp Wall Thir Asp Glu Arg Wall 290 295 3OO

Lell Glin Trp Luell Lys Arg Wall Glin Asn Wall Ser Wall Tell Ser Ile US 2015/01 19330 A1 Apr. 30, 2015 35

- Continued

3. OS 310 315 32O Cys Asp Phe Arg Glu Asp Trp Glin Thir Trp Asp Cys Pro Llys Thr Glin 3.25 330 335 Glu. His Ile Glu Glu Ser Arg Thr Gly Trp Ala Gly Gly Phe Phe Val 34 O 345 35. O Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile Ser Tyr Tyr Lys Ile Phe 355 360 365 Ser Asp Lys Asp Gly Tyr Llys His Ile His Tyr Ile Lys Asp Thr Val 37 O 375 38O Glu Asn Ala Ile Glin Ile Thir Ser Gly Lys Trp Glu Ala Ile Asn. Ile 385 390 395 4 OO Phe Arg Val Thr Glin Asp Ser Leu Phe Tyr Ser Ser Asn Glu Phe Glu 4 OS 41O 415 Glu Tyr Pro Gly Arg Arg Asn Ile Tyr Arg Ile Ser Ile Gly Ser Tyr 42O 425 43 O Pro Pro Ser Lys Lys Cys Val Thr Cys His Lieu. Arg Lys Glu Arg Cys 435 44 O 445 Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr Ala Lys Tyr Tyr Ala Leu 450 45.5 460 Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser Thr Lieu. His Asp Gly Arg 465 470 47s 48O Thir Asp Glin Glu Ile Lys Ile Lieu. Glu Glu Asn Lys Glu Lieu. Glu Asn 485 490 495 Ala Lieu Lys Asn. Ile Glin Lieu Pro Lys Glu Glu Ile Llys Llys Lieu. Glu SOO 505 51O Val Asp Glu Ile Thr Lieu. Trp Tyr Lys Met Ile Leu Pro Pro Glin Phe 515 52O 525 Asp Arg Ser Lys Llys Tyr Pro Lieu. Lieu. Ile Glin Val Tyr Gly Gly Pro 53 O 535 54 O Cys Ser Glin Ser Val Arg Ser Val Phe Ala Val Asn Trp Ile Ser Tyr 5.45 550 555 560 Lieu Ala Ser Lys Glu Gly Met Val Ile Ala Lieu Val Asp Gly Arg Gly 565 st O sts Thir Ala Phe Glin Gly Asp Llys Lieu. Lieu. Tyr Ala Val Tyr Arg Llys Lieu. 58O 585 59 O Gly Val Tyr Glu Val Glu Asp Glin Ile Thir Ala Val Arg Llys Phe Ile 595 6OO 605 Glu Met Gly Phe Ile Asp Glu Lys Arg Ile Ala Ile Trp Gly Trp Ser 610 615 62O Tyr Gly Gly Tyr Val Ser Ser Lieu Ala Lieu Ala Ser Gly. Thr Gly Lieu. 625 630 635 64 O

Phe Lys Cys Gly Ile Ala Val Ala Pro Val Ser Ser Trp Glu Tyr Tyr 645 650 655

Ala Ser Val Tyr Thr Glu Arg Phe Met Gly Leu Pro Thr Lys Asp Asp 660 665 67 O

Asn Lieu. Glu. His Tyr Lys Asn. Ser Thr Val Met Ala Arg Ala Glu Tyr 675 68O 685 Phe Arg Asn. Wall Asp Tyr Lieu. Lieu. Ile His Gly Thr Ala Asp Asp Asn 69 O. 695 7 OO

Val His Phe Glin Asn. Ser Ala Glin Ile Ala Lys Ala Lieu Val Asn Ala 7 Os 71O 71s 72O US 2015/01 19330 A1 Apr. 30, 2015 36

- Continued

Glin Val Asp Phe Glin Ala Met Trp Tyr Ser Asp Glin Asn His Gly Lieu. 72 73 O 73 Ser Gly Lieu Ser Thr Asn His Leu Tyr Thr His Met Thr His Phe Leu 740 74. 7 O Lys Glin Cys Phe Ser Lieu. Ser Asp 7ss 760

1. A compound having the formula: 15. The compound of claim 1, further comprising a B-Xaa1-Sp-Xaa-Cyc (Formula II), 1-10mer peptide or oligopeptide extending from Cyc in the C-terminal direction. wherein: 16. The compound of claim 1, further defined as capable of B is at least one of aminobenzoyl (Abz), acetyl (Ac), ben binding to the active site of POP at a K-100 nM and capable Zoyl (BZ), benzyloxycarbonyl (Z), t-Butyloxycarbonyl of binding to DPPIV at a K-500 nM, and/or as having a K, (Boc), Fury lacryloyl (Fa), Methoxysuccinyl (MeOSuc), (DPPIV):K, (POP) ratio>500. Pyroglutamate (Pyr), Pyrazine, Phenylalanine, and Suc 17. (canceled) cinyl (Suc); 18. The compound of claim 1, wherein B is an acetyl, Xaa, is a positively-charged or negatively-charged amino pyroglutamate, or Succinyl: Xaa, is lysine; Sp is at least one acid; of Sp is leucine, isoleucine, Valine, or alanine, Xaa, is argi Sp is a spacer molecule having a length in the range of 0.3 nine; and Cyc is a boronyl proline or cyanopyrrolidine. nm to 2.5 nm, 19. A pharmaceutical composition comprising the com Xaa, is a positively-charged amino acid; and pound of claim 1 disposed within a pharmaceutically-accept Cyc is a boronyl proline, proline carbonitrile, nitrile pyr able carrier or vehicle. rolidone, or cyanopyrrolidine. 20. A method of inhibiting activity of prolyl oligopeptidase 2. The compound of claim 1, wherein Xaa, is C.B-diami (POP) in a cell or tissue which expresses POP comprising: nopropionic acid, O.Y-diaminobutyric acid, ornithine, B-ho administering to the POP-expressing cell or tissue a com moornithine, arginine, B-homoarginine, homoarginine, pound having the formula: lysine, homolysine, B-homolysine, histidine, aspartic acid or glutamic acid. B-Xaa1-Xaa-Cyc (Formula II), 3. The compound of claim 2, wherein Xaa, comprises a wherein: methylene group in Substitution for the carbonyl group adja B is at least one of aminobenzoyl (Abz), acetyl (Ac), cent Sp. benzoyl (BZ), benzyloxycarbonyl (Z), t-Butyloxycar 4. The compound of claim 1, wherein Xaa, is C. f-diami bonyl (Boc), Furylacryloyl (Fa), Methoxysuccinyl nopropionic acid, C.Y-diaminobutyric acid, ornithine, B-ho (MeOSuc), Pyroglutamate (Pyr), Pyrazine, Phenyla moornithine, arginine, B-homoarginine, homoarginine, lanine, and Succinyl (Suc); lysine, homolysine, B-homolysine, or histidine. Xaa, is a positively-charged or negatively-charged 5. The compound of claim 1, wherein Sp is selected from amino-acid; the group consisting of Y-aminobutyric acid; E-aminocaproic Sp is a spacer molecule having a length in the range of acid; 8-amino-3,6-dioxaoctanoic acid; 11-amino-3,6,9-triox 0.3 nm to 2.5 nm, aundecanoic acid; 14-amino-3,6,9,12-tetraoxatetradecanoic Xaa, is a positively-charged amino acid; and acid; C.-aminobutyric acid; 5-aminopentanoic acid; 6-amino Cyc is a boronyl proline, proline carbonitrile, nitrile hexanoic acid; 7-aminoheptanoic acid; 8-aminooctanoic pyrrolidone, or cyanopyrrolidine, acid; 3-(aminooxy)acetic acid; B-alanine; glycine; alanine; and whereinactivity of the POP in the POP-expressing cell threonine, tryptophan; tyrosine; methionine; leucine; isoleu or tissue is inhibited. cine; valine; serine; proline; ethylene glycol; PEG (wherein 21. The method of claim 20, wherein the POP-expressing n=1-6); propylene glycol; PPG, (wherein n=1-6); amino cells or tissues are cancer cells and/or activated fibroblast PEG-carboxy group (wherein n=1-6); amino-PPG-carboxy cells. (wherein n=1-6); and combinations thereof. 22. A method of inhibiting angiogenesis and/or treating 6. The compound of claim 5, wherein Sp is selected from cancer in a subject in need of Such therapy, comprising: the group consisting of ethylene glycol, PEG, (wherein n=1- administering a pharmaceutically-acceptable amount of a 6), propylene glycol, 8-amino-3,6-dioxaoctanoic acid, PPG, compound having the formula: (wherein n=1-6)), amino-PEG-carboxy group (wherein n=1-6), an amino-PPG-carboxy group (wherein n=1-6), and combinations thereof. wherein: 7. The compound of claim 5, wherein Sp is leucine, iso B is at least one of aminobenzoyl (Abz), acetyl (Ac), leucine, Valine, or alanine. benzoyl (BZ), benzyloxycarbonyl (Z), T-Butyloxy 8. The compound of claim 1, wherein Sp has a length in a carbonyl (Boc), Furylacryloyl (Fa), Methoxysuccinyl range of 0.6nm to 1.75 nm. (MeOSuc), Pyroglutamate (Pyr), Pyrazine, Phenyla 9-13. (canceled) lanine, and Succinyl (Suc); 14. The compound of claim 1, comprising an isostere bond Xaa is a positively-charged amino acid or negatively between Xaa, and Sp. charged amino-acid; US 2015/01 19330 A1 Apr. 30, 2015 37

Sp is a spacer molecule having a length in the range of 27. A method of inhibiting activity of prolyl oligopeptidase 0.3 nm to 2.5 nm, (POP) in a subject suffering from a disorder for which inhi Xaa- is a positively-charged amino acid, or Xaa is gly bition of POP provides a therapeutically-effective benefit, cine, D-alanine, D-serine, or D-threonine, with the comprising: provisio that when Xaa is glycine, D-alanine, administering to a subject in need of Such therapy a com D-serine, or D-threonine, Xaa is a positively-charged pound having the formula: amino acid; and Cyc is a boronyl proline, proline carbonitrile, nitrile B-Xaa1-Sp-Xaa-Cyc (Formula II) pyrrolidone, or cyanopyrrolidine. 23-25. (canceled) wherein: 26. A compound, wherein at least a portion of the com B is at least one of aminobenzoyl (Abz), acetyl (Ac), pound has the formula: benzoyl (BZ), benzyloxycarbonyl (Z), t-Butyloxycar bonyl (Boc), Furylacryloyl (Fa), Methoxysuccinyl B-Xaa1-Sp-Xaa, (Formula IIb), (MeOSuc), Pyroglutamate (Pyr), Pyrazine, Phenyla wherein: lanine, and Succinyl (Suc); B is at least one of aminobenzoyl (Abz), acetyl (Ac), ben Zoyl (BZ), benzyloxycarbonyl (Z), t-Butyloxycarbonyl Xaa, is a positively-charged or negatively-charged (Boc), Fury lacryloyl (Fa), Methoxysuccinyl (MeOSuc), amino-acid; Pyroglutamate (Pyr), Pyrazine, Phenylalanine, and Suc Sp is a spacer molecule having a length in the range of cinyl (Suc); 0.3 nm to 2.5 nm, Xaa, is a positively-charged or negatively-charged amino Xaa, is a positively-charged amino acid; and acid; Sp is a spacer molecule having a length in the range of 0.3 Cyc is a boronyl proline, proline carbonitrile, nitrile nm to 2.5 nmi; and pyrrolidone, or cyanopyrrolidine. Xaa, is a positively-charged amino acid. k k k k k