WO2000067802A1

WO2000067802A1 - Fatty acid-n-substituted indol-3-glyoxyl-amide compositions and uses thereof

Info

Publication number: WO2000067802A1
Application number: PCT/US2000/012752
Authority: WO
Inventors: Matthews O. Bradley; Charles S. Swindell; Forrest Anthony; Nigel L. Webb; Mark Fisher
Original assignee: Protarga Inc
Current assignee: Protarga Inc
Priority date: 1999-05-10
Filing date: 2000-05-10
Publication date: 2000-11-16
Anticipated expiration: 2001-11-10
Also published as: AU4834200A

Abstract

The present invention pertains to N-substituted indol-3- glyoxyl -amides that are conjugates of fatty acids and N-(pyridin-4-yl) -(1--(4-halobenzyl) -indol-3-yl) -glyoxyl- amides). The conjugates are useful in treating cancer.

Description

FATTY ACID - N-SUBSTITUTED INDOL-3-GLYOXYL-AMIDE COMPOSITIONS

AND USES THEREOF

Field of the invention The present invention pertains to N-substituted indol-3 -glyoxyl-amides that are conjugates of fatty acids and N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)-glyoxyl-amides). The conjugates are useful in treating cancer.

Background of the Invention Improving drug selectivity for target tissue is an established goal in the medical arts. In general, it is desirable to deliver a drug selectively to its target, so that dosage and, consequently, side effects can be reduced. This is particularly the case for toxic agents such as anti-cancer agents because achieving therapeutic doses effective for treating the cancer is often limited by the toxic side effects of the anti-cancer agent on normal, healthy tissue. The problems relating to lack of drug selectivity can be exemplified by Taxol®(paclitaxel). Recently, a new class of compounds known as N-substituted indol-3 -glyoxyl-amides were synthesized (see PCT WO98/09946, published March 12, 1998). Antiasthmatic, antiallergic and immunosuppressive/immunomodulating properties were associated with these compounds. More recently, investigators from ASTA Medica A.G., Germany (Applicants for the above-identified PCT), reported on the unique anti-cancer properties of one category of these N-substituted indol-3 -glyoxyl-amides, namely N-(pyridin-4-yl)-(l-(4- halobenzyl)-indol-3 -yl)-glyoxyl-amide) and N-(pyridin-4-yl)-( 1 -(4-chlorobenzyl)-indol-3 -yl)- glyoxyl-amide) in particular. N-(pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl- amide) appears to be a more potent anti-cancer agent in vivo than either taxol or vincristine. Its mechanism of action is believed to involve destabilization of microtubules. N-(pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide) has attracted strong scientific attention, not only because of its unique antiproliferative potency, but also because it is active against nearly all cancers against which it has been tested.

N-(pyridin-4-yl)-( 1 -(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide) ' s strength against cancers of diverse tissue origin also represents a significant drawback. An ideal anti cancer agent has tissue specificity, thereby reducing side-effects on normal (dividing) cells. N- (pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide) analogs with tissue specificity therefore are desired. Another drawback of N-(pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)- glyoxyl-amide) is its extreme insolubility. N-(pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)- glyoxyl-amide) has only been possible to be administered effectively by oral gavage. Fatty acids previously have been conjugated with drugs to help the drugs as conjugates cross the blood-brain barrier. For example, DHA (docosahexaenoic acid) is a 22 carbon naturally-occurring, unbranched fatty acid that previously has been shown to be unusually effective in crossing the blood-brain barrier. When DHA is conjugated to a drug, the entire drug-DHA conjugate is transported across the blood-brain barrier and into the brain.

DHA is attached via the acid group to hydrophilic drugs and renders these drugs more hydrophobic (lipophilic). DHA is an important constituent of the brain and recently has been approved as an additive to infant formula. It is present in the milk of lactating women. The mechanism of action by which DHA helps drugs conjugated to it cross the blood-brain barrier is unknown.

Another example of the conjugation of fatty acids to a drug is the attachment of pipotiazine to stearic acid, palmitic acid, enanthic acid, undecylenic acid or 2,2-dimethyl- palmitic acid. Pipotiazine is a drug that acts within the central nervous system. The purpose of conjugating pipotiazine to the fatty acids was to create an oily solution of the drug as a liquid implant for slow release of the drug when injected intramuscularly. The release of the drug appeared to depend on the particular fatty acid selected, and the drug was tested for its activity in the central nervous system.

Lipidic molecules, including the fatty acids, also have been conjugated with drugs to render the conjugates more lipophilic than the drug. In general, increased lipophilicity has been suggested as a mechanism for enhancing intestinal uptake of drugs into the lymphatic system, thereby enhancing the entry of the conjugate into the brain and also thereby avoiding first-pass metabolism of the conjugate in the liver. The type of lipidic molecules employed have included phospholipids, non-naturally occurring branched and unbranched fatty acids, and naturally occurring branched and unbranched fatty acids ranging from as few as 8 carbon atoms to more than 30 carbon atoms. In one instance, enhanced receptor binding activity was observed (for an adenosine receptor agonist), and it was postulated that the pendant lipid molecule interacted with the phospholipid membrane to act as a distal anchor for the receptor ligand in the membrane micro environment of the receptor. This increase in potency, however, was not observed when the same lipid derivatives of adenosine receptor antagonists were used, and generalizations thus were not made possible by those studies.

Summary of the Invention The present invention involves the unexpected finding that conjugates of a fatty acid and a N-substituted indol-3 -glyoxyl-amide, have a different selectivity relative to N- substituted indol-3 -glyoxyl-amide alone. The conjugates, in general, render the activity of N- substituted indol-3 -glyoxyl-amide selective for biliary tract cancer, brain cancer (including glioblastomas and medulloblastomas), breast cancer; cervical cancer; choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasms, including acute lymphocytic and myelogenous leukemia, multiple myeloma, AIDS associated leukemias and adult T-cell leukemia lymphoma, intraepithelial neoplasms, including Bowen's disease and Paget's disease, liver cancer, lung cancer, lymphomas, including Hodgkin's disease and lymphocytic lymphomas, neuroblastomas, oral cancer, including squamous cell carcinoma, ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma, skin cancer, including melanoma, Kaposi's sarcoma, basocellular cancer and squamous cell cancer, testicular cancer, including germinal tumors (seminoma, non- seminoma[teratomas, choriocarcinomas]), stromal tumors and germ cell tumors, thyroid cancer, including thyroid adenocarcinoma and medullar carcinoma, and renal cancer including adenocarcinoma and Wilms tumor ("targeted cancers"). The conjugates, also unexpectedly, restrict the activity of the N-substituted indol-3 -glyoxyl-amide even within these foregoing categories of cancer relative to that of N-substituted indol-3-glyoxyl-amide. The conjugates, further unexpectedly, reduce sharply the activity of a N-substituted indol-3 - glyoxyl-amide relative to that of N-substituted indol-3 -glyoxyl-amide in most cell lines of various tissue types, it is believed, other than bone, bone marrow, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, intraepithelium, kidney, liver, lung, ovaries, pancreas, prostate, rectum, skin, squamous cell epithelium, stomach, testicular tissue, and thyroid, thereby reducing potential side effects of the conjugates versus those of N-substituted indol-3-glyoxyl-amide. The therapeutic index of the conjugates is improved, versus that of N-substituted indol-3-glyoxyl-amide for targeted cancers. A preferred N-substituted indol-3 -glyoxyl-amide conjugated to a fatty acid according to the invention, is N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)-glyoxyl-amide). A preferred N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)-glyoxyl-amide) is N-(pyridin-4- yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide).

According to one aspect of the invention, novel compounds and pharmaceutical compositions are provided. Each pharmaceutical composition contains the novel compound, which is a covalent conjugate of a N-substituted indol-3 -glyoxyl-amide and a fatty acid having 8-26 carbons, in an amount effective to treat cancer, and a pharmaceutically acceptable carrier. Preferably, the fatty acid is an unbranched, naturally occurring fatty acid.

More preferably, the fatty acid has 14-22 carbons. Unbranched common naturally occurring fatty acids include C12:0 (lauric acid), C14.0 (myristic acid), C16:0 (palmitic acid), C16:l

(palmitoleic acid), C16:2, C18.0 (stearic acid), C18:l (oleic acid), C18.1-7 (vaccenic), C18:2- 6 (linoleic acid), C18:3-3 (α-linolenic acid), C18.3-5 (eleostearic), C18:3-6 (_-linolenic acid),

C18.4-3, C20:l (gondoic acid), C20.2-6, C20:3-6 (dihomo-y-linolenic acid), C20:4-3, C20:4-

6 (arachidonic acid), C20:5-3 (eicosapentaenoic acid), C22:l (docosenoic acid), C22.4-6

(docosatetraenoic acid), C22:5-6 (docosapentaenoic acid), C22.5-3 (docosapentaenoic acid),

C22:6-3 (docosahexaenoic acid) and C24:l-9 (nervonic). Highly preferred unbranched, naturally occurring fatty acids are those with between 14 and 22 carbon atoms. In some embodiments, the fatty acids are ω-3 fatty acids. The most preferred co-3 fatty acid is docosahexaenoic acid. In certain embodiments, the fatty acids are ω-6 fatty acids. The most preferred ω-6 fatty acid is linoleic acid. In one embodiment, the N-substituted indol-3 - glyoxyl-amides are N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)-glyoxyl-amide). In a preferred embodiment, the N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)-glyoxyl-amide) is N-(pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide).

As used in connection with the following conjugates described below, R is the organic substituent attached to the carboxyl-group in any one of the fatty acids described in the immediately preceding paragraph. The fatty acid, preferably, is an ω-3 fatty acid, and most preferably is DHA. Preferably, the covalent conjugate is selected from the group consisting of:

Conjugate 1

wherein X is H, F, Cl, Br or I; Y is -COR, -(CH₂)_nO₂CR, -(CH₂)_nNHCOR, -CO(CH₂)_nO₂CR, or -CO(CH₂)_nNHCOR, wherein n=l-22.

Conjugate 2

wherein X is H, F, Cl, Br or I;

Y is -O₂CR, -NHCOR, -(CH₂)_nO₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR, -O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR, -O(CH₂)_nNHCOR,

NHCO(CH₂)_nO₂CR, w -NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l-

22.

Conjugate 3

wherein X is H, F, Cl, Br or I;

NHCO(CH₂)_nO₂CR,

-NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l-

22.

Conjugate 4

wherein X is H, F, Cl, Br or I;

Y is -O₂CR, -NHCOR, -(CH₂)_nO₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)„O₂CR,

-O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR, -O(CH₂)_nNHCOR, w NHCO(CH₂)_nO₂CR,

-NHCO(CH₂)„NHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l-

22.

Conjugate 5

0 0

5 wherein X is H, F, Cl, Br or I;

Y is -O₂CR, -NHCOR, -(CH₂)_nO₂CR, -(CH₂)„NHCOR, -O₂C(CH₂)_nO₂CR, -O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR, -O(CH₂)_nNHCOR,

NHCO(CH₂)_nO₂CR,

-NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l- 22.

Conjugate 6

0 0

wherein X is H, F, Cl, Br or I;

NHCO(CH₂)_nO₂CR,

-NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l- 22.

Conjugate 7

wherein X is H, F, Cl, Br or I;

Y is -O₂CR, -NHCOR, -(CH₂)„O₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR, -O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR, -O(CH₂)_nNHCOR,

NHCO(CH₂)_nO₂CR,

-NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l-

22.

Conjugate 8

0 0

wherein X is H, F, Cl, Br or I;

NHCO(CH₂)_nO₂CR,

-NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l- 22.

wherein X is H, F, Cl, Br or I;

Y is -O₂CR, -NHCOR, -(CH₂)_nO₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR,

-O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR, -O(CH₂)„NHCOR,

NHCO(CH₂)_nO₂CR,

-NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l-

22.

wherein Y is -O₂CR, -NHCOR, -(CH₂)_nO₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR, -O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR, -O(CH₂)_nNHCOR,

NHCO(CH₂)_nO₂CR,

-NHCO(CH₂)_nNHCOR, NH(CH₂)„O₂CR, -NH(CH₂)„NHCOR, wherein n=l- 22.

Conjugate 11

0 0

wherein X is H, F, Cl, Br or I;

Y is -O₂CR, -NHCOR, -(CH₂)„O₂CR, -(CII₂)„NIICOR, -O₂C(CII₂)_nO₂CR, -O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR, -O(CH₂)_nNHCOR,

NHCO(CH₂)_nO₂CR, -NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l-

22.

In other embodiments, the covalent conjugate is conjugate 12:

Conjugate 12

wherein X is F, Cl, Br, I, or Y, wherein Yι-Yι (collectively Y) is selected from the group consisting of -H,-O₂CR,-NHCOR,-(CH₂)_nO₂CR,-(CH₂)_nNHCOR,-O₂C(CH₂)_nO₂CR,- O₂C(CH₂)_nNHCOR,-O(CH₂)_πO₂CR,-O(CH₂)_nNHCOR,-NHCO(CH₂)_nO₂CR,- NHCO(CH₂)_nNHCOR,-NH(CH₂)_nO₂CR,-NH(CH₂)_nNHCOR,-COR,-(CH₂)_nO₂CR,-

(CH₂)_nNHCOR, -CO(CH₂)_nO₂CR, and -CO(CH₂)_nNHCOR, wherein n=l-22, and wherein at least one Y is not Hydrogen (e.g., Yj) and is selected from the group consisting of -O₂CR,- NHCOR,-(CH₂)_nO₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR,-O2C(CH₂)_nNHCOR,-

O(CH₂)_nO₂CR, -O(CH₂)_nNHCOR,-NHCO(CH₂)_nO₂CR,-NHCO(CH₂)_nNHCOR,- NH(CH₂)_nO₂CR,-NH(CH₂)_nNHCOR,-COR,-(CH₂)_nθ₂CR,-(CH₂)_nNHCOR,-CO(CH₂)_nO₂CR, and -CO(CH₂)_nNHCOR, wherein n=l-22, the remainining Y groups (e.g.,Y₂-Y]₄) can be substituted, or preferably are unsubstituted and are Hydrogens. In further embodiments, in the covalent conjugate 12, at least two, at least three, or at least four Y groups are not Hydrogen and are selected from the group consisting of -O₂CR,-NHCOR,-(CH₂)_nO₂CR,- (CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR, -O₂C(CH₂)_nNHCOR,-O(CH₂)_πO₂CR,-O(CH₂)_nNHCOR,- NHCO(CH₂)_nO₂CR,-NHCO(CH₂)_nNHCOR,-NH(CH₂)_nO₂CR,-NH(CH₂)_nNHCOR,-COR,- (CH₂)_nO₂CR,-(CH₂)_nNHCOR,-CO(CH₂)_nO₂CR, and -CO(CH₂)_nNHCOR, wherein n=l-22, and the remainining Y groups can be substituted, or preferably are unsubstituted and are Hydrogens.

In other embodiments, the covalent conjugate is conjugate 13:

Conjugate 13

wherein X is F, Cl, Br, I, or Y, wherein Yι-Yι₃ (collectively Y) is selected from the group consisting of -H, -O₂CR, -NHCOR, -(CH₂)_nO₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR, - O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR, -O(CH₂)_nNHCOR, NHCO(CH₂)_nO₂CR, NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n-1-22, and wherein at least one Y is not Hydrogen (e.g., Yj) and is selected from the group consisting of -O₂CR, - NHCOR, -(CH₂)_nO₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR, -O₂C(CH₂)_nNHCOR, - O(CH₂)_nO₂CR, -O(CH₂)_nNHCOR, -NHCO(CH₂)_nO₂CR, -NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l-22, the remainining Y groups (e.g.,Y₂- Yι₃) can be substituted, or preferably are unsubstituted and are Hydrogens. In further embodiments, in the covalent conjugate 13, at least two, at least three, or at least four Y groups are not Hydrogen and are selected from the group consisting of -O₂CR,-NHCOR,- (CH₂)_nO₂CR,-(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR, -O₂C(CH₂)_nNHCOR,-O(CH₂)_nO₂CR,- O(CH₂)_nNHCOR,-NHCO(CH2)nO2CR,-NHCO(CH₂)_nNHCOR,-NH(CH2)_nO₂CR,- NH(CH₂)_nNHCOR, wherein n=l-22, and the remainining Y groups can be substituted, or preferably are unsubstituted and are Hydrogens. In any one of conjugates 12 or 13, when the at least one, the at least two, and/or the at least three Y group(s) is (are) not Hydrogen and is (are) selected from any of the foregoing sub-groups, the remaining Y groups may be also substituted, or preferably are unsubstituted and are Hydrogen substituents. One of ordinary skill in the art could identify other substituent molecules that can be utilized to substitute for the Y group(s), and maintain and/or enhance the conjugate's anti-cancer properties.

In other embodiments, the covalent conjugate is conjugate 14:

Conjugate 14

wherein X is selected from the group consisting of H, F, Cl, Br, I,-O₂CR,-NHCOR,- (CH₂)„O₂CR,-(CH₂)_nNHCOR,-O₂C(CH₂)_nO₂CR,-O₂C(CH₂)_nNHCOR,-O(CH₂)_nO₂CR,- O(CH₂)_nNHCOR,-NHCO(CH₂)_nO₂CR,-NHCO(CH2)nNHCOR,-NH(CH2)_nO₂CR, and - NH(CH₂)_nNHCOR, and Y is selected from the group consisting of -COR, -(CH₂)_nO₂CR, - (CH₂)_nNHCOR, -CO(CH₂)_nO₂CR, and -CO(CH₂)_nNHCOR, wherein n=l-22.

Typically the covalent conjugates of the invention include only one fatty acid, although sometimes two, three, four, or more are possible.

According to another aspect of the invention, a kit is provided. The kit is a package which houses a container which contains a covalent conjugate of the invention and also houses instructions for administering the covalent conjugate to a cancer victim.

According to another aspect of the invention, a second kit is provided. This kit includes a package which houses a first container which contains a covalent conjugate of the invention and also houses a second container containing an anti-cancer agent other than the covalent conjugate. In the kits of the invention, the preferred fatty acids, bonds, covalent conjugate and anti-cancer agent other than the covalent conjugate are as described above.

According to another aspect of the invention, a method is provided for treating cancer.

The method involves administering to a subject in need of such treatment a covalent conjugate of an N-substituted Indol-3 -glyoxyl-amid and a fatty acid having 8-26 carbons in an amount effective to treat cancer. The preferred N-substituted indol-3 -glyoxyl-amid is N- (pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amid). The preferred fatty acids, bonds and covalent conjugates are as described above. The method also can involve co- administering to the subject an anti-cancer agent other than the covalent conjugate. Preferred anti-cancer agents are as described above.

According to one aspect of the invention, a conjugate composition for administration to a subject is provided. The composition includes at least one conjugate in a container for administration to a subject. The amount of the conjugate in the container is at least about 10% greater than the maximum tolerated dose (MTD) for the unconjugated at least one anti cancer compound. Preferably the amount of the conjugate in the container is at least about 20% greater than the MTD, 30% greater than the MTD, 40% greater than the MTD, 50% greater than the MTD, 75% greater than the MTD, 100% greater than the MTD, 200% greater than the MTD, 300% greater than the MTD, or 400% greater than the MTD for the unconjugated at least one anti cancer compound. In certain preferred embodiments, the container is a container for intravenous administration. In certain embodiments, the conjugate is not encapsulated in or in the form of a liposome.

According to still another aspect of the invention, methods for treating a subject having an abnormal mammalian cell proliferative disorder are provided. The methods include administering a composition including at least one fatty acid conjugate to the subject in an amount which is at least about 10% greater than the maximum tolerated dose (MTD) for the unconjugated at least one anti cancer compound. Preferably the amount of the at least one fatty acid-anti cancer compound administered is at least about 20% greater than the MTD, 30% greater than the MTD, 40% greater than the MTD, 50% greater than the MTD, 75% greater than the MTD, 100% greater than the MTD, 200% greater than the MTD, 300% greater than the MTD, or 400% greater than the MTD for the unconjugated at least one anti cancer compound. In certain embodiments the conjugate is not encapsulated in or in the form of a liposome.

In still another aspect of the invention, kits for administration of a fatty acid-anti cancer compound conjugate to a subject is provided. The kits include a container containing a composition which includes at least one fatty acid-anti cancer compound conjugate of the invention, and instructions for administering the at least one fatty acid-anti cancer compound conjugate to subject in need of such treatment in an amount which is at least about 10% greater than the maximum tolerated dose (MTD) for the unconjugated at least one anti cancer compound. Preferably the subject has an abnormal mammalian cell proliferative disorder.

Preferably the amount of the at least one fatty acid-anti cancer compound conjugate to be administered is at least about 20% greater than the MTD, 30% greater than the MTD, 40% greater than the MTD, 50% greater than the MTD, 75% greater than the MTD, 100% greater than the MTD, 200% greater than the MTD, 300%) greater than the MTD, or 400% greater than the MTD for the unconjugated at least one anti cancer compound. In certain preferred embodiments, the container is a container for intravenous administration. In certain embodiments the conjugate is not encapsulated in or in the form of a liposome.

A method for increasing the therapeutic index of anti cancer compounds in a subject is provided, according to another aspect of the invention. The method includes conjugating a fatty acid to an anti cancer compound as described herein to form a fatty acid-anti cancer compound conjugate of the invention; and administering the fatty acid-anti cancer compound conjugate to the subject. The therapeutic index of the anti cancer compound thus administered is improved relative to non-conjugated formulations of the anti cancer compound. Preferably the subject has an abnormal mammalian cell proliferative disorder, and the subject preferably is human. In certain embodiments the conjugate is not encapsulated in or in the form of a liposome.

In the foregoing methods, it is preferred that a dose of a fatty acid-conjugated anti- cancer compound is administered which exceeds the maximum tolerated dose of the unconjugated anti cancer compound. These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments.

Brief Description of the Drawings Figure 1 depicts a kit 11 comprising packaging 15, a first agent of the invention 17 (e.g., a container that contains a fatty acid conjugate of a N-substituted indol-3 -glyoxyl- amide, a second agent of the invention 19 (e.g., a container that contains a nonN-substituted indol-3 -glyoxyl-amide anticancer agent), and instructions 21, for utilizing such agents in therapeutic applications. Detailed Description of the Invention

N-substituted indol-3 -glyoxyl-amides are synthesized according to PCT

WO98/09946, published March 12, 1998, which is expressly incorporated herein by reference. A preferred N-substituted indol-3 -glyoxyl-amide according to the invention is N-

(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)-glyoxyl-amide) has the following structure:

0 0

wherein X is a halogen. A preferred N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)-glyoxyl- a ide) is N-(pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide). The preferred source of N-(pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide) is ASTA Medica A.G., Dresden, Germany.

The invention provides compositions of matter. Compositions according to one aspect of the invention comprise a conjugate of a fatty acid and a N-substituted indol-3- glyoxyl-amide. In this aspect of the invention, the fatty acids are polyunsaturated fatty acids. In some embodiments, the fatty acid is preferably a C16-C26 unbranched, naturally occurring fatty acid. The fatty acid can be selected from the group consisting of C8:0 (caprylic acid), C10:0 (capric acid), C12:0 (lauric acid), C14:0 (myristic acid), C16:0 (palmitic acid), C16:l (palmitoleic acid), C16:2, C18:0 (stearic acid), C18:l (oleic acid), C18:l-7 (vaccenic), C18:2- 6 (linoleic acid), C18:3-3 (α-linolenic acid), C18:3-5 (eleostearic), C18:3-6 (β-linolenic acid), Cl 8:4-3, C20:l (gondoic acid), C20:2-6, C20:3-6 (dihomo-y-linolenic acid), C20:4-3, C20:4- 6 (arachidonic acid), C20:5-3 (eicosapentaenoic acid), C22:l (docosenoic acid), C22:4-6 (docosatetraenoic acid), C22:5-6 (docosapentaenoic acid), C22:5-3 (docosapentaenoic ), C22:6-3 (docosahexaenoic acid) and C24:l-9 (nervonic). Particularly preferred is docosahexaenoic acid. In certain embodiments, the fatty acid can be linoleic acid, palmitic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, 2-octanoate, 2- hexanoate, CH₃-hexanoate, CH₃-butanoate, or oleic acid. In particularly preferred embodiments, the fatty acid is linoleic acid, palmitic acid, arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid. czs-docosahexaenoic acid (DHA) is a naturally occurring fatty acid. It is an unbranched chain fatty acid with six double bonds, all cis. Its structure is as follows:

DHA can be isolated, for example, from fish oil or can be chemically synthesized. These methods, however, can generate trans isomers, which are difficult and expensive to separate and which may present safety problems in humans. The preferred method of production is biological synthesis to produce the all cis isomer. The preferred source of DHA is from Martek Biosciences Corporation of Columbia, Maryland. Martek has a patented system for manufacturing DHA using microalgae which synthesize only a single isomer of DHA, the all cis isomer. Martek's patents include U.S. Pat. Nos. 5,374,657, 5,492,938, 5,407,957 and 5,397,591. DHA also is present in the milk of lactating women, and Martek's licensee has obtained approval in Europe of DHA as a nutritional supplement for infant formula.

It is known that DHA can be unstable in the presence of oxygen. To stabilize DHA and its conjugates it is important to add anti-oxidants to the material after it is synthesized. One method of stabilization is to make-up the newly synthesized material in the following solution:

100 g neat DHA-N-(pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide) plus 100 g of vehicle (100ml propylene glycol, 70 mg alpha-tocopherol, 5 mg dialaurylthiodipropionic acid, 50 mg ascorbic acid) prepared and held under argon in amber, sealed vials and stored at four degrees centigrade. The following anti-oxidants may also be employed: ascorbic acid, ascorbyl palmitate, dilauryl ascorbate, hydroquinone, butyated hydroxyanisole, sodium meta bisulfite, t-β carotene and α-tocopherol. A heavy metal chelator such as ethylenediamine tetra-acetic acid (EDTA) may also be used.

In one aspect of the invention, the conjugate is prepared as a quaternary ammonium salt. The anion preferably is selected from the group consisting of I^", Cl^", OH^", F^" and Br^". Most preferably the anion is I^". Cancer patients could be evaluated to determine if conjugates 1 -18 are strongly active against the patient's cancer prior to selecting any of the conjugates 1-18 as the anti-cancer agent of choice for that patient.

The foregoing experiments establish that the conjugates of the invention will have altered specificity versus that of the N-substituted indol-3 -glyoxyl-amide alone, for cancer cell lines. Because of this altered specificity, it also is clear that the conjugates themselves are gaining access into the target cells (as opposed to simply releasing the N-substituted indol-3 -glyoxyl-amide into the environment outside of the cell). Thus, the fatty acid moiety appears to selectively target certain cell types as opposed to others. The ability of the conjugates to gain entry into the targeted cells was unknown prior to the invention, and the ability of the fatty acid moiety to selectively target certain cell types was unexpected.

Paclitaxel was first isolated from the bark of Taxus brevifolia (Wani et al., J. Am. Chem. Soc, 93, 2325, 1971). Its isolation and synthesis have been reported extensively in the literature. Applicants obtained paclitaxel from a commercial source, Hauser Laboratories, of Boulder, Colorado.

The compound of the invention described in Examples 3-9 below, "Taxoprexin™", is a covalent conjugate of DHA and paclitaxel. Its chemical structure, synthesis, purification and in vitro action are described in U.S Patne 5,795,909, the entire disclosure of which is incorporated by reference herein. The structure is shown as "conjugate 1 " in Example 1 of that patent.

The maximum tolerated dose (MTD) for any therapeutic compound is identified as part of its clinical evaluation. For example, phase I trials can include a determination of the maximum tolerated dose, dose limiting toxicities (DLT) and pharmacokinetics of a test compound. "Maximum tolerated dose," as used herein, refers to the largest dose of a pharmaceutical agent that an adult patient can take with safety to treat a particular disease or condition. Thus, the MTD for any Food and Drug Administration (FDA) approved therapeutic compound is known to those of ordinary skill in the art as a matter of the public record. The MTD for any particular therapeutic compound may vary according to its formulation (e.g., injectable formulation, implantable bioerodible polymer formulation, oral formulation), route of delivery (e.g., intravenous, oral, intratumoral), manner of delivery (e.g., infusion, bolus injection), dosing schedule (e.g., hourly, daily, weekly) and the like. The MTD frequently is defined as the highest dose level at which 50% of subjects administered with the drug develop a dose limiting toxicity. The doses for anti-neoplastic pharmaceutical agents found in the Physicians Desk Reference (PDR) are defined as the

MTD for those agents. The MTD is further defined to include only doses for drugs

(including anti-neoplasties) used as single agents and without additional cellular, genetic, pharmaceutical, or other agents added to alter the MTD. Other definitions which are clinically relevant and generally accepted will be known to one of ordinary skill in the art.

Measurement of maximum tolerated dose may be expressed as weight of drug per weight of subject, weight of drug per body surface area, etc. The MTD of anticancer compounds is frequently expressed as weight per square meters (mg/m²) of body surface area. For example, the MTD for paclitaxel infusion in humans is 225 mg/m². The most often used clinical tolerated dose is 175 mg/m . MTD also may be expressed as a dose relative to a time component, such as weight of drug per body surface area per day.

For therapeutics which have not yet been subjected to human clinical trails, or subjected to any determination of the MTD in humans (e.g., experimental or highly toxic compounds), one of skill in the art can estimate the MTD by using animal models. Calculation of MTD in animals may be based on a number of physiological parameters, such as death, particular toxicities, drug induced weight loss. Using death as an endpoint, the MTD may be the dose given test animals in which each member of the test group survived. Using toxicity as an endpoint, the MTD may be the dose at which moderate but not severe toxicity is observed. Using weight loss as an endpoint, the MTD may be the dose above which a certain percent change in body weight is induced. Other methods for determining MTDs using animal models and various endpoints are known to one of ordinary skill in the art. Correlation of animal MTDs to human MTDs for a therapeutic compound is an accepted practice in the pharmaceutical arts.

For example, it has been determined that a conjugate of DHA and paclitaxel (Taxoprexin™) has a maximum tolerated dose in animals (mice, rats and dogs) which is about 4-5 times greater (by weight) than paclitaxel alone or about 3-4 times greater (by molarity) than paclitaxel alone.

Thus the invention in another aspect provides compositions and formulations for administration to a subject, preferably a human subject, containing amounts of a fatty acid- anti cancer compound conjugate which exceeds the maximum tolerated dose for the unconjugated anti cancer compound. The fatty acid-anti cancer compound conjugate preferably is in a container for administration to a subject. Preferably the container is a container for intravenous administration, such as an IN bag. The amount of the fatty acid-anti cancer compound in the container is at least about

10%) greater than the MTD for the unconjugated compound. Preferably the amount of the fatty acid-anti cancer compound in the container is at least about 20%, 30%, 40%, 50%, 75%,

100%), 200%, 300%. or 400% greater than the MTD for the unconjugated at least one anti cancer compound.

Methods for administering these compositions to subjects having an abnormal mammalian cell proliferative disorder also are provided.

Kits containing fatty acid-anticancer compounds in amounts also are provided. The kits contain one or more containers with the conjugated anticancer compound along with instructions for mixing, diluting and/or administering the anticancer compound in amounts greater than the MTD for the unconjugated anticancer compound. The kits also can include other containers with one or more solvents, surfactants, preservatives and/or diluents (e.g. normal saline (0.9% NaCl), or 5% dextrose (D5W)), as well as containers for mixing, diluting, and/or administering the conjugates to a subject in need of such treatment. A kit embodying features of the present invention, generally designated by the numeral 11 , is illustrated in Figure 1. Kit 11 is comprised of the following major elements: packaging 15, a first agent of the invention 17 (e.g., a container that contains a fatty acid conjugate of a N- substituted indol-3 -glyoxyl-amide, a second agent of the invention 19 (e.g., a container that contains a nonN-substituted indol-3-glyoxyl-amide anticancer agent), and instructions 21 for utilizing such agents in therapeutic applications. Individuals skilled in the art can readily modify packaging 15 to suit individual needs.

The anti cancer compounds in the kit may be provided as liquid solutions, or as dried powders. When the compound provided is a dry powder, the powder may be reconstituted by the addition of a suitable solvent, which also may be provided. Liquid forms of the conjugates may be concentrated (for dilution prior to administration) or ready to administer to a subject.

As noted above, the therapeutic index is the ratio of the median toxic dose to the median effective dose. Conjugation of fatty acids to anticancer compounds to form a fatty acid-anticancer compound conjugate reduces toxicity of the anticancer compounds, and increases effectiveness as compared to the unconjugated anticancer compounds. Therefore the invention also provides methods for increasing the therapeutic index of anticancer compounds in a subject. The methods include conjugating a fatty acid to an anticancer compound to form a fatty acid-anticancer compound conjugate and administering the fatty acid-anticancer compound conjugate to the subject. The therapeutic index of the anticancer compound conjugate is improved relative to unconjugated formulations of the anticancer compound. Preferably the anticancer compound is a taxane, particularly paclitaxel or docetaxel.

Although the conjugate may be encapsulated in a liposome, it is preferred that the conjugate is not encapsulated by a liposome. The preferred subjects for the method are humans.

The conjugated anti cancer compounds described herein are less toxic and more effective than the corresponding unconjugated anti cancer compounds. Therefore the fatty acid-anti cancer compound conjugates can be administered in amounts which are equally toxic but more effective, or in doses which are equally effective and less toxic than the corresponding unconjugated anti cancer compounds. In general, conjugation of fatty acids to anti cancer compounds permits an increase in the maximum tolerated dose relative to unconjugated anti cancer compounds.

The compounds useful in the invention may be delivered in the form of anti-cancer cocktails. An anti-cancer cocktail is a mixture of any one of the compounds useful with this invention with another anti-cancer agent such as an anti-cancer drug, a cytokine, and/or supplementary potentiating agent(s). The use of cocktails in the treatment of cancer is routine. In this embodiment, a common administration vehicle (e.g., pill, tablet, implant, injectable solution, etc.) would contain both the conjugate useful in this invention and the anti-cancer drug and/or supplementary potentiating agent.

Anti-cancer agents include, but are not limited to, the following compounds and classes of compounds:

Antineoplastic agents such as: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA (N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin

Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate

Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine;

Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine;

Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin Hydrochloride;

Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon

Alfa-n3; Interferon Beta- I a; Interferon Gamma- I b; Iproplatin; Irinotecan Hydrochloride;

Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;

Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;

Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa;

Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper;

Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin

Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin;

Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride;

Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol;

Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;

Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium;

Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;

Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP-

53; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil

Mustard; Uredepa; Napreotide; Nerteporfm; Ninblastine; Ninblastine Sulfate; Nincristine;

Nincristine Sulfate; Nindesine; Nindesine Sulfate; Ninepidine Sulfate; Ninglycinate Sulfate;

Ninleurosine Sulfate; Ninorelbine Tartrate; Ninrosidine Sulfate; Ninzolidine Sulfate;

Norozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2'- Deoxyformycin; 9-aminocamptothecin; raltitrexed; Ν-propargyl-5,8-dideazafolic acid; 2- chloro-2'-arabino-fluoro-2'-deoxyadenosine; 2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlor ethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-N- nitrosourea (MNU); N, N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU); N-(2-chloroethyl)-N'- cyclohexyl-N-nitrosourea (CCNU); N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl-N- nitrosourea (MeCCNU); N-(2-chloroethyl)-N'-(diethyl)ethylphosphonate-N-nitrosourea

(fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin; Carboplatin; Ormaplatin; Oxaliplatin; C 1-973; DWA 2114R; JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6-Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide

9-amino camptothecin; Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4- Hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); 2-chlorodeoxyadenosine (2-Cda).

Other anti-neoplastic compounds include: 20-epi-l,25 dihydroxyvitamin D3 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1 antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1 axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bleomycin A₂; bleomycin B₂; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives (e.g., 10-hydroxy- camptothecin); canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4 combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8 cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B 2'deoxycoformycin (DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9- dioxamycin; diphenyl spiromustine; discodermolide; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epothilones (A, R = H; B, R = Me); epithilones; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide; etoposide 4'-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; mcrbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mithracin; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid

A + myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin

B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; podophyllotoxin; porfimer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1 ; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer. Antiproliferative agent: Piritrexim Isothionate. Antiprostatic hypertrophy: Sitogluside.

Benign prostatic hyperplasia therapy agent: Tamsulosin Hydrochloride. Prostate growth inhibitor: Pentomone. Radioactive agents: Fibrinogen 1 125; Fludeoxyglucose F 18; Fluorodopa F 18;

Insulin I 125; Insulin I 131; Iobenguane I 123; Iodipamide Sodium I 131 ; Iodoantipyrine I

131; Iodocholesterol I 131; Iodohippurate Sodium I 123; Iodohippurate Sodium I 125;

Iodohippurate Sodium 1 131; Iodopyracet I 125; Iodopyracet 1 131; Iofetamine Hydrochloride I 123; Iomethin I 125; Iomethin 1 131; Iothalamate Sodium I 125; Iothalamate Sodium 1 131 ;

Iotyrosine 1 131; Liothyronine I 125; Liothyronine I 131; Merisoprol Acetate Hg 197;

Merisoprol Acetate Hg 203; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Tc

99m Antimony Trisulfide Colloid; Technetium Tc 99m Bicisate; Technetium Tc 99m

Disofenin; Technetium Tc 99m Etidronate; Technetium Tc 99m Exametazime; Technetium Tc 99m Furifosmin; Technetium Tc 99m Gluceptate; Technetium Tc 99m Lidofenin;

Technetium Tc 99m Mebrofenin; Technetium Tc 99m Medronate; Technetium Tc 99m

Medronate Disodium; Technetium Tc 99m Mertiatide; Technetium Tc 99m Oxidronate;

Technetium Tc 99m Pentetate; Technetium Tc 99m Pentetate Calcium Trisodium;

Technetium Tc 99m Sestamibi; Technetium Tc 99m Siboroxime; Technetium Tc 99m Succimer; Technetium Tc 99m Sulfur Colloid; Technetium Tc 99m Teboroxime; Technetium

Tc 99m Tetrofosmin; Technetium Tc 99m Tiatide; Thyroxine 1 125; Thyroxine 1 131 ;

Tolpovidone 1 131; Triolein 1 125; Triolein 1 131.

Anti-cancer Supplementary Potentiating Agents: Tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone and citalopram); Ca⁺⁺ antagonists (e.g., verapamil, nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (e.g., prenylamine, trifluoroperazine and clomipramine); Amphotericin B; Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g., buthionine and sulfoximine) and Multiple Drug Resistance reducing agents such as

Cremaphor EL. The compounds of the invention also can be administered with cytokines such as granulocyte colony stimulating factor.

Preferred anticancer agents used in anti-cancer coctails (e.g., in combination with the agents of the invention) include (some with their MTDs shown in parentheses): gemcitabine (1000 mg/m²); methotrexate (15 gm/m² i.v.+ leuco. <500 mg/m² i.v. w/o leuco); 5-FU (500 mg/m²/day x 5days); FUDR (100 mg/kg x 5 in mice, 0.6 mg/kg/day in human i.a.); FdUMP;

Hydroxyurea (35 mg/kg/d in man); Docetaxel (60-100 mg/m ); discodermolide; epothilones; vincristine (1.4 mg/m²); vinblastine (escalating: 3.3 - 11.1 mg/m², or rarely to 18.5 mg/m²); vinorelbine (30 mg/m²/wk); meta-pac; irinotecan (50-150 mg/m², 1 x /wk depending on patient response); SN-38 (~100 times more potent than Irinotecan); 10-OH campto; topotecan

-\ 9 9

(1.5 mg/m /day in humans, 1 x iv LD10mice=75 mg/m ); etoposide (100 mg/m in man); adriamycin; flavopiridol; Cis-Pt (100mg/m² in man); carbo-Pt (360 mg/m² in man); 9 bleomycin (20 mg/m ); mitomycin C (20 mg/m ); mithramycin (30 μg/kg); capecitabine (2.5 g/m² orally); cytarabine (100 mg/m²/day); 2-Cl-2'deoxyadenosine; Fludarabine-PO₄ (25

9 9 9 mg/m /day, x 5days); mitoxantrone (12-14 mg/m ); mitozolomide (>400 mg/m ); Pentostatin; Tomudex.The compounds of the invention, when used alone or in cocktails, are administered in therapeutically effective amounts. A therapeutically effective amount will be determined by the parameters discussed below; but, in any event, is that amount which establishes a level of the drug(s) in the area of the tumor which is effective in inhibiting the tumor growth.

When administered, the formulations of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic ingredients. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); and phosphoric acid and a salt (0.8- 2% W/V).

Suitable preservatives include benzalkonium chloride (0.003-0.03%) W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W V) and thimerosal (0.004-0.02% W/V).

The active compounds of the present invention may be a pharmaceutical composition having a therapeutically effective amount of a conjugate of the invention optionally included in a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions are capable of being commingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

Compositions suitable for parenteral administration conveniently comprise a sterile preparation of the conjugates of the invention. This preparation may be formulated according to known methods. Formulations for Taxol and other taxanes can be found in Chapter 9 of

Taxol: Science and Applications, CRC Press, Inc., 2000 Corporate Boulevard, N.W., Boca Raton, FL 33431. In general, Taxol has been formulated as a 6 mg/ml cremophor EL (polyoxyethylated castor oil)/ethanol mixture, which is diluted to final volume with normal saline or 5% dextrose. A 15mg/ml solution of taxotere has been formulated in polysorbate 80 (polyoxyethylene sorbitanmonooleate)/ethanol mixture, diluted with 5% dextrose.

The sterile preparation thus may be a sterile solution or suspension in a non-toxic parenterally-acceptable diluent or solvent. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono ordi-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, PA.

The invention is used in connection with treating subject having, suspected of having, developing or suspected of developing cancer. A subject as used herein means humans, primates, horses, cows, pigs, sheep, goats, dogs, cats and rodents.

The conjugates of the invention are administered in effective amounts. An effective amount means that amount necessary to delay the onset of, inhibit the progression of or halt altogether the onset or progression of the particular condition being treated. In general, an effective amount will be that amount necessary to inhibit mammalian cancer cell proliferation in-situ. When administered to a subject, effective amounts will depend, of course, on the particular condition being treated; the severity of the condition; individual patient parameters including age, physical condition, size and weight; concurrent treatment; frequency of treatment; and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. Dosage may be adjusted appropriately to achieve desired drug levels, locally or systemically. Generally, daily oral doses of active compounds will be from about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected that IN doses in the range of about 1 to 1000 mg/m² per day will be effective. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Continuous IN dosing over, for example 24 hours or multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.

A variety of administration routes are available. The particular mode selected will depend of course, upon the particular drug selected, the severity of the disease state being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, sublingual, topical, nasal, transdermal or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous routes are preferred.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the conjugates of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquors or non- aqueous liquids such as a syrup, an elixir, or an emulsion.

Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the active compounds of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation. A long-term sustained release implant also may be used. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above. Such implants can be particularly useful in treating solid tumors by placing the implant near or directly within the tumor, thereby affecting localized, high-doses of the compounds of the invention.

The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate the embodiments of the invention and are not to be construed to limit the scope of the invention. Examples

Example 1: Synthesis of Fatty Acid - N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)- glvoxyl-amide) conjugates

Preparation of types 1-12 analogs, described below, refers specifically to the preparation of any of the foregoing conjugates of the invention except those where the Y group is attached to the Nitrogen next to the pyridin-4-yl group, as in conjugate 14 (or the Y₁₂ group in conjugate 12).

Type 1 analogs (i.e., those containing the -O₂CR subgroup), are prepared by reaction of the appropriate hydroxy-group-substituted parent drug with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 2 analogs (i.e., those containing the -NHCOR subgroup), are prepared by reaction of the appropriate amino-group-substituted parent drug with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4- dimethylaminopyridine or 4-pyrrolidinopyridine. Type 3 analogs (i.e., those containing the - (CH₂)„O₂CR subgroup) are prepared by reaction of the appropriate ω-hydroxy alkyl-group substituted parent drug with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 4 analogs (i.e., those containing the -(CH )_nNHCOR subgroup) are prepared by reaction of the appropriate ω-amino alkyl-group substituted parent drug with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 5 analogs (i.e., those containing the -O₂C(CH₂)_nO₂CR subgroup) are formed by reaction of the appropriate hydroxy-group-substituted parent drug with an ω- benzyloxy-(CH₂)_nCO₂H in the presence of dicyclohexylcarbodiimide or other carboxyl- activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine followed by hydrogenolytic removal of the benzyl protecting group and subsequent reaction of the deprotected intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 6 analogs (i.e., those containing the -O₂C(CH₂)_nNHCOR subgroup) are formed by reaction of the appropriate hydroxy-group-substituted parent drug with an ω-PhCH₂OCONH- (CH₂)_nCO₂H in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine followed by hydrogenolytic removal of the benzyloxycarbonyl protecting group and subsequent reaction of the deprotected intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl- activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 7 analogs (i.e., those containing the -O(CH₂)_nO₂CR subgroup) are prepared by reaction of the appropriate hydroxy-group-substituted parent drug with a 1-benzyloxy-ω-iodo-rø-alkane in the presence of base followed by hydrogenolytic removal of the benzyl protecting group and subsequent reaction of the deprotected intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 8 analogs (i.e., those containing the -O(CH₂)_nNHCOR subgroup) are prepared by reaction of the appropriate hydroxy-group-substituted parent drug with a 1 -azido-ω-iodo-«-alkane in the presence of base followed by hydrogenation and subsequent reaction of the primary amine intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 9 analogs (i.e., those containing the -NHCO(CH₂)_nO₂CR subgroup) are prepared by reaction of the appropriate amino-group-substituted parent drug with an ω-benzyloxy-(CH )_nCO₂H in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine followed by hydrogenolytic removal of the benzyl protecting group and subsequent reaction of the deprotected intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 10 analogs (i.e., those containing the -NHCO(CH₂)_nNHCOR subgroup) are prepared by reaction of the appropriate amino-group-substituted parent drug with an ω-PhCH₂OCONH- (CH₂)_nCO₂H in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine followed by hydrogenolytic removal of the benzyloxycarbonyl protecting group and subsequent reaction of the deprotected intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl- activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 11 analogs (i.e., those containing the -NH(CH₂)_nO₂CR subgroup) are prepared by reaction of the appropriate amino-group-substituted parent drug and a l-benzyloxy-ω-iodo-«-alkane followed by hydrogenolytic removal of the benzyl protecting group and subsequent reaction of the deprotected intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 12 analogs (i.e., those containing the -NH(CH₂)_nNHCOR subgroup) are prepared by reaction of the appropriate amino-groups-substituted parent drug with a l-azido-ω-iodo-«- alkane followed by hydrogenation and subsequent reaction of the primary amine intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Those skilled in the art will recognize other synthetic pathways to these fatty acid conjugates.

Preparation of types 13-17 analogs, described below, refers to the preparation of any of the foregoing conjugates of the invention where the Y group is attached specifically to the

Nitrogen next to the pyridin-4-yl group, as in conjugate 14 (or the Y₁₂ group in conjugate 12).

Type 13 analogs (i.e., those containing the -COR subgroup) are prepared by sequential reaction of the parent drug with sodium hydride or another base and a fatty acid- derived acid chloride. Type 14 analogs (i.e., those containing the JCH )_nO₂CR subgroup) are prepared by sequential reaction of the parent drug with sodium hydride or another base and a 1-benzyloxy-ω-iodo-w-alkane followed by hydrogenolytic removal of the benzyl protecting group and subsequent reaction of the deprotected intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4- dimethylaminopyridine or 4-pyrrolidinopyridine. Type 15 analogs (i.e., those containing the -(CH₂)_nNHCOR subgroup) are prepared by sequential reaction of the parent drug with sodium hydride or another base and an 1 -azido-ω-rø-alkane followed by hydrogenation and subsequent reaction of the primary amine intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type 16 analogs (i.e., those containing the -CO(CH₂)_nO₂CR subgroup) are prepared by sequential reaction of the parent drug with sodium hydride or another base and an ω-benzyloxy-(CH₂)_nCθ₂H-derived acid chloride followed by hydrogenolytic removal of the benzyl protecting group and subsequent reaction of the deprotected intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine. Type

17 analogs (i.e., those containing the -CO(CH₂)_nNHCOR subgroup) are prepared by sequential reaction of the parent drug with sodium hydride or another base and an ω-azido- (CH₂)_nCO₂H-derived acid chloride followed by hydrogenation and subsequent reaction of the primary amine intermediate with a fatty acid in the presence of dicyclohexylcarbodiimide or other carboxyl-activating agent and 4-dimethylaminopyridine or 4-pyrrolidinopyridine.

Those skilled in the art will recognize other synthetic pathways to these fatty acid conjugates.

Example 2: The Effects of Taxoprexin and Paclitaxel Against M 109 Lung Carcinoma in Mice

Syngeneic mice were injected with mouse lung tumor line M (Madison) 109 sub- cutaneously in the flank. Five days after tumor implantation, one day later than in the last example, when the tumors had grown ten-fold larger to 300 mg, taxoprexin (OD=120mg/kg/day x 5 days) or paclitaxel (OD=20mg/kg/day x 5 days) were injected as a bolus through the tail vein on each of five successive days. Both drugs were dissolved in 10%) cremophor EL/10% ethanol/80%) saline. Tumor volume was estimated from tumor width and length. Paclitaxel retarded tumor growth for about four days (LCK=1.0). In contrast, taxoprexin completely eliminated all measurable tumors in seven out of eight mice (C/T-l/8) at 120 mg/kg/day x 5 days, and in four out of seven mice at 80 mg/kg/day x 5 days. Histological examination of the tissue where the tumors had showed no tumor cells, only scar tissue. These data show that taxoprexin is curative in this model.

Example 3: Response of Human NCI-H522 Lung Tumor to Treatment with Taxoprexin and Paclitaxel in Mice

The Southern Research Institute studied the anti-tumor activity of taxoprexin against human NCI-H522 lung tumor growing in nude mice. The tumors were implanted sub- cutaneously. Tumor mass was determined by calculation from tumor length and width. The drugs were dissolved in 12.5% cremophor EL/12.5% ethanol/75% saline and delivered i.v. into the tail vein, once a day for 5 days, from day 15 to 19 after tumor implantation. The results show that taxoprexin at 50 mg/kg/day x 5 days and paclitaxel at 20 mg/kg/day x 5 days eliminated all measurable tumors in 10/10 mice. Example 4: The Pharmacokinetic Parameters of Taxoprexin and Paclitaxel

Rats were dosed for three minutes with 6.8mg/kg of taxoprexin through the tail vein. The drug was dissolved in 10% cremaphor EL/10% ethanol/80% saline. The serum concentrations of both taxoprexin and paclitaxel were measured in a reverse phase HPLC assay. Pharmacokinetic parameters were calculated from these data. Taxoprexin has - 100 fold lower clearance rate and volume of distribution (see Table 1).

Table 1.

Taxoprexin Pharmacokinetic Parameters in Rats Drug Clearance Plasma _\a Volume of Distribution (hr) (n=3)

Paclitaxel 28.2 ml/min/kg 4.8±2.6 4.3 L/kg

Taxoprexin^® 0.3 ml/min/kg 4.8±0.1 0.058 L/kg

Example 5: Plasma Concentration of Taxoprexin and Paclitaxel in Rats Following I.V. Administrations of Taxoprexin

Rats were given a 3 minute intravenous infusion of taxoprexin through the tail vein at 0 time. The drug was dissolved in 10% cremophor EL/10% ethanol/80% saline. The dose was 6.8 mg/kg. The concentrations in serum of both paclitaxel and taxoprexin as a function of time were measured in a reverse phase HPLC assay (see Table 2). Table 2.

Paclitaxel and Taxoprexin^® plasma concentration (ng/ml) following administration of Taxoprexin^® in Rats

Time (hr) Paclitaxel Taxoprexin^

0 200 100,000

1 100 95,000

2 70 90,000 5 40 70,000

24 10 40,000

Example 6: Plasma and Tumor Concentrations of Paclitaxel Derived from an I.V. Dose of 50 mg/kg of Taxoprexin to Mice Bearing M 109 or M 5076 Tumors

Mice with tumors derived from Ml 09 or M5076 were given a bolus does of taxoprexin through the tail vein at 0 time. The drug was dissolved in 10% cremophor EL/10%) ethanol/80% saline. Mice were sacrificed and tumors immediately excised as a function of time after injecting the drug. Tumor tissue was homogenized and paclitaxel extracted. The concentration of paclitaxel was measured in a reverse phase HPLC assay. Blood was collected at the same time intervals and the amount of paclitaxel determined. The results show that after 24 hours the concentration of paclitaxel derived from taxoprexin is about 3 μM, 40 times higher than the plasma concentration, 70 nM. Each data point is the mean of three measurements (n=3). NOTE: Paclitaxel has a tι_/ of <8 hours in the same tumor system. Example 7: Dose Comparisons (MTD and Est LDgn. of Taxoprexin and Paclitaxel in Various Species Except Humans

Dose comparisons for paclitaxel and taxoprexin were made in mice, rats and dogs. The maximum tolerated dose (MTD) for mice, rats and dogs were about 4-5 times higher for taxoprexin than for paclitaxel on a mg/kg basis, or 3-3.5 times higher on a molar paclitaxel equivalent basis. Dose limiting toxicity for rats and dogs is due to decreases in platelets, neutrophils and lymphocytes. Taxoprexin is less toxic to mice, rats and dogs than is paclitaxel(see Table 3). Table 3.

Dose Comparisons: Paclitaxel vs. Taxoprexin in various species

Species Dose (mg/kg)* Dose ratio. -Taxoprexin' /Paclitaxel

Taxoprexin Paclitaxel Based on Based on Taxane

Weight Molarity**

Mouse MTD = 100 x 5 = MTD = 20 x 5 5 3.6 500 =100

Rat Est LD₄₀ = 420 LD₄₀ = 85 5 3.6 Dog MTD = 80 Est MTD = 20 4 2.9

Average 4.7 3.4

* MTD is Maximum Tolerated Dose

**MW of Taxoprexin^® = 1164; MW of Paclitaxel = 854; MW ratio = 0.73

The foregoing data establish, surprisingly, safety implications of dose and pharmacokinetic advantages of taxoprexin. The higher MTD of taxoprexin compared to paclitaxel is believed to lead to greater safety of taxoprexin with much greater efficacy. The smaller volume of distribution for taxoprexin is believed to lead to less damage by taxoprexin in peripheral tissues including, but not limited to, nerves, hair follicles, GI cells, etc. The longer residence time of taxoprexin in tumors is believed to lead to fewer required dosing cycles for optimum therapeutic efficacy, which is believed to lead to decreased systemic toxicity. Taxoprexin thus appears to have a 100 fold lower clearance rate and volume of distribution than paclitaxel. In addition, levels of paclitaxel in tumors treated with taxoprexin remain stable for 24 hours, whereas such levels in tumors treated with paclitaxel have stable levels for less than 8 hours. Finally, taxoprexin was shown to cure 3/8 mice of the human HCT colon tumor, while paclitaxel cured 0/8. HCT is a paclitaxel resistant tumor.

Other aspects of the invention will be clear to the skilled artisan and need not be repeated here. All patents, published patent applications and literature cited herein are incorporated by reference in their entirety. We claim:

Claims

1. A compound, comprising: a covalent conjugate of N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)- glyoxyl-amide) and a fatty acid having 12-26 carbons.

2. The compound of claim 1, wherein the N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3- yl)-glyoxyl-amide) is N-(pyridin-4-yl)-(l -(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide).

3. The compound of claim 1, wherein the covalent conjugate is

70

wherein X is F, Cl, Br, I, or Y, wherein Y is selected from the group consisting of -H, -O₂CR, -NHCOR, (CH₂)_nO₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR, -O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR,

75 O(CH₂)_nNHCOR, -NHCO(CH₂)_nO₂CR, -NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, NH(CH₂)_nNHCOR, wherein n=l-22, and wherein at least one Y is not H and is selected from the group consisting of -O₂CR, NHCOR₅-(CH₂)_nO₂CR,-(CH₂)_nNHCOR,-O₂C(CH₂)_nO₂CR,-O2C(CH2)„NHCOR,- O(CH₂)_nO₂CR,

-O(CH₂)_nNHCOR,-NHCO(CH₂)_nO₂CR,-NHCO(CH₂)_nNHCOR,-NH(CH₂)_nO₂CR, and NH(CH₂)_nNHCOR, wherein n=l-22, the remainining Y groups are unsubstituted and are H. The compound of claim 1, wherein the covalent conjugate is

wherein X is F, Cl, Br, I, or Y, wherein Y is selected from the group consisting of -H, -O₂CR, -NHCOR, - (CH₂)_nO₂CR, -(CH₂)_nNHCOR, -O₂C(CH₂)_nO₂CR, -O₂C(CH₂)_nNHCOR, -O(CH₂)_nO₂CR, - O(CH₂)_nNHCOR, -NHCO(CH₂)_nO₂CR, -NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, - NH(CH₂)_nNHCOR, wherein n=l-22, and wherein at least two Y groups are not H and are selected from the group consisting of -θ₂CR,-NHCOR,-(CH₂)_nO₂CR,-(CH₂)_nNHCOR,-O₂C(CH₂)_nO₂CR,-O₂C(CH₂)_nNHCOR,- O(CH₂)_nO₂CR,-O(CH₂)_nNHCOR,-NHCO(CH₂)_nθ2CR,-NHCO(CH₂)_nNHCOR, NH(CH₂)_nO₂CR, -NH(CH₂)_nNHCOR, wherein n=l-22, the remainining Y groups are unsubstituted and are H.

The compound of claim 1, wherein the covalent conjugate is

0 0

wherein X is selected from the group consisting of H, F, Cl, Br, I, -O₂CR, -NHCOR, -

(CH₂)_nθ₂CR,-(CH₂)_nNHCOR,-O₂C(CH₂)_nO₂CR,-θ2C(CH2)nNHCOR,-O(CH2)_nO₂CR,-

O(CH₂)_nNHCOR,-NHCO(CH₂)_nO₂CR,-NHCO(CH₂)_nNHCOR,-NH(CH₂)_nO₂CR, and

-NH(CH₂)_nNHCOR, wherein n= 1-22, and wherein Y is selected from the group consisting of -COR, -(CH₂)_nO CR, -

(CH₂)_nNHCOR, -CO(CH₂)_nO₂CR, and -CO(CH₂)_nNHCOR, wherein n=l-22.

6. The compound of claims 1, 2, 3, 4, or 5 wherein the fatty acid is an unbranched, naturally occurring fatty acid.

70

7. The compound of claim 6, wherein the fatty acid has 16-22 carbons.

8. The compound of claim 7, wherein the fatty acid is docosahexaenoic acid.

75 9. A pharmaceutical composition comprising the compound of claim 1, 2, 3, 4, or 5 in an amount effective to treat cancer, and a pharmaceutically acceptable carrier.

10. A pharmaceutical composition comprising the compound of claim 6 in an amount effective to treat cancer, and a pharmaceutically acceptable carrier.

20

11. A pharmaceutical composition comprising the compound of claim 7 in an amount effective to treat cancer, and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition comprising the compound of claim 8 in an amount 25 effective to treat cancer, and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 9, further comprising an anti-cancer agent other than the covalent conjugate.

30 14. The pharmaceutical composition of claim 13, wherein the anti-cancer agent is selected from the group consisting of Aminoglutethimide; Asparaginase; Bleomycin; L-Buthiamine Sulfoxide; Busulfan; Camptothecin; Carboplatin; Carmustine (BCNU); Chlorambucil; Cisplatin (cis-DDP); Cyclophosphamide; Cytarabine HCI; Dacarbazine; Dactinomycin; Daunorubicin HCI; Doxorubicin HCI; Edatrexate; Estramustine phosphate sodium; Etoposide (V16-213); Floxuridine; Fluorouracil (5-FU); Flutamide; Gallium Nitrite; Hydroxyurea

(hydroxycarbamide); Idarubicin; Ifosfamide; Interferon-α-2a, -α2b; Leuprolide acetate

(LHRH-releasing factor analogue); Lomustine (CCNU); Mechlorethamine HCI (nitrogen mustard); Megestrol; melphalan; Mercaptopurine; Methotrexate (MTX); Mitomycin; Mitotane (o.p' -DDD); Mitoxantrone HCI; Octreotide; Plicamycin; Prednisone; Procarbazine

HCI; Streptozocin; Tamoxifen citrate; Taxanes; Taxoids; Thioguanine; Thiotepa; Tiasofuran;

Topotecan; Vinblastine sulfate; Vincristine sulfate; Amsacrine (m-AMSA); Azacitidine;

Hexamethylmelamine (HMM); Interleukin 2; Mitoguazone (methyl-GAG); Methyl glyoxal bis-guanylhydrazone (MGBG); Paclitaxel; Pentostatin; Semustine (methyl-CCNU); Taxol; Taxotere; Tamoxifen; and Teniposide (VM-26).

15. A kit comprising a package housing a container containing the compound of claims 1, 2, 3, 4, or 5, and also housing instructions for administering to a cancer victim the covalent conjugate.

16. A kit comprising a package housing: a first container containing the covalent conjugate of claim 1, 2, 3, 4, or 5, and a second container containing an anti-cancer agent other that the covalent conjugate.

17. A method for treating a subject having or suspected of having cancer, comprising: administering to a subject in need of such treatment an amount of a covalent conjugate of N- (pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)-glyoxyl-amid) and a fatty acid having 12-26 carbons in an amount effective to treat cancer.

18. The method of claim 17, wherein the N-(pyridin-4-yl)-(l-(4-halobenzyl)-indol-3-yl)- glyoxyl-amide) is N-(pyridin-4-yl)-(l-(4-chlorobenzyl)-indol-3-yl)-glyoxyl-amide).

19. The method of claim 17, wherein the covalent conjugate comprises the pharmaceutical preparation of claim 9.

20. The method of claim 17, wherein the covalent conjugate comprises the pharmaceutical preparation of claim 10.

21. The method of claim 17, wherein the covalent conjugate comprises the pharmaceutical preparation of claim 11.

22. The method of claim 17, wherein the covalent conjugate comprises the pharmaceutical preparation of claim 12.

23. The method of claim 19, further comprising administering to the subject an anticancer agent other than the covalent conjugate.

24. The method of claim 23, wherein the anti-cancer agent is selected from the group consisting of Aminoglutethimide; Asparaginase; Bleomycin; L-Buthiamine Sulfoxide; Busulfan; Camptothecin; Carboplatin; Carmustine (BCNU); Chlorambucil; Cisplatin (cis-DDP); Cyclophosphamide; Cytarabine HCI; Dacarbazine; Dactinomycin; Daunorubicin HCI; Doxorubicin HCI; Edatrexate; Estramustine phosphate sodium; Etoposide (VI 6-213); Floxuridine; Fluorouracil (5-FU); Flutamide; Gallium Nitrite; Hydroxyurea (hydroxycarbamide); Idarubicin; Ifosfamide; Interferon Alfa-2a, Alfa 2b; Leuprolide acetate (LHRH-releasing factor analogue); Lomustine (CCNU); Mechlorethamine HCI (nitrogen mustard); Megestrol; melphalan; Mercaptopurine; Methotrexate (MTX); Mitomycin; Mitotane (o.p'-DDD); Mitoxantrone HCI; Octreotide; Plicamycin; Prednisone; Procarbazine HCI; Streptozocin; Tamoxifen citrate; Taxanes; Taxoids; Thioguanine; Thiotepa; Tiasofuran; Topotecan; Vinblastine sulfate; Vincristine sulfate; Amsacrine (m-AMSA); Azacitidine; Hexamethylmelamine (HMM); Interleukin 2; Mitoguazone (methyl-GAG); Methyl glyoxal bis-guanylhydrazone (MGBG); Paclitaxel; Pentostatin; Semustine (methyl-CCNU); Taxol; Taxotere; and Teniposide (VM-26).

25. A fatty acid-anti cancer compound conjugate pharmaceutical composition for administration to a subject, comprising at least one fatty acid-anti cancer compound conjugate in a container for administration to a subject, wherein the amount of the fatty acid- anti cancer compound in the container is at least about 10% greater than the maximum tolerated dose (MTD) for the unconjugated at least one anti cancer compound wherein the conjugate is a compound of claim 1.

26. The fatty acid-anti cancer compound conjugate composition of claim 25, wherein the amount in the container is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% greater than the MTD for the unconjugated at least one anti cancer compound.

27. The fatty acid-anti cancer compound conjugate composition of claim 25, wherein the amount in the container is at least about 200% greater than the MTD for the unconjugated at least one anti cancer compound.

28. The fatty acid-anti cancer compound conjugate composition of claim 25, wherein the amount in the container is at least about 300% greater than the MTD for the unconjugated at least one anti cancer compound.

29. The fatty acid-anti cancer compound conjugate composition of claim 25, wherein the amount in the container is at least about 400% greater than the MTD for the unconjugated at least one anti cancer compound.

30. The fatty acid-anti cancer compound conjugate composition of claim 25, wherein the container is a container for intravenous administration.

31. The fatty acid-anti cancer compound conjugate composition of claim 25, wherein the fatty acid is a C 16-22 unbranched, naturally occurring fatty acid.

32. The fatty acid-anti cancer compound conjugate composition of claim 25, wherein the fatty acid is docosahexaenoic acid

33. A method for treating a subject having an abnormal mammalian cell proliferative disorder, comprising administering to the subject a fatty acid-anti cancer compound conjugate composition in an amount which is at least about 10% greater than the maximum tolerated dose (MTD) for the unconjugated at least one anti cancer compound, wherein the conjugate is a compound of claim 1.

34. The method of claim 33, wherein the amount of the fatty acid-anti cancer compound conjugate composition administered is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% greater than the MTD for the unconjugated at least one anti cancer compound.

35. The method of claim 33, wherein the amount of the fatty acid-anti cancer compound conjugate composition administered is at least about 200% greater than the MTD for the unconjugated at least one anti cancer compound.

36. The method of claim 33, wherein the amount of the fatty acid-anti cancer compound conjugate composition administered is at least about 300%) greater than the MTD for the unconjugated at least one anti cancer compound.

37. The method of claim 33, wherein the amount of the fatty acid-anti cancer compound conjugate composition administered is at least about 400%) greater than the MTD for the unconjugated at least one anti cancer compound.

38. The method of claim 33, wherein the fatty acid is a C16-C22 unbranched, naturally occurring fatty acid.

39. The method of claim 33, wherein the fatty acid is docosahexaenoic acid.

40. A kit for administration of a fatty acid-anti cancer compound conjugate composition to a subject, comprising a container containing at least one fatty acid-anti cancer compound conjugate, and instructions for administering the at least one fatty acid-anti cancer compound conjugate to subject in need of such treatment in an amount which is at least about 10% greater than the maximum tolerated dose (MTD) for the unconjugated at least one anti cancer compound, wherein the conjugate is a compound of claim 1.

41. The kit of claim 40, wherein the amount of the at least one fatty acid-anti cancer compound conjugate is at least about 20%, at least about 30%>, at least about 40%, at least about 50%), at least about 75%, or at least about 100%) greater than the MTD for the unconjugated at least one anti cancer compound.

42. The kit of claim 40, wherein the amount of the at least one fatty acid-anti cancer compound conjugate is at least about 200% greater than the MTD for the unconjugated at least one anti cancer compound.

43. The kit of claim 40, wherein the amount of the at least one fatty acid-anti cancer compound conjugate is at least about 300%> greater than the MTD for the unconjugated at least one anti cancer compound.

44. The kit of claim 40, wherein the amount of the at least one fatty acid-anti cancer compound conjugate is at least about 400% greater than the MTD for the unconjugated at least one anti cancer compound.

45. The kit of claim 40, wherein the fatty acid is a C16-C22 unbranched, naturally occurring fatty acid.

46. The kit of claim 40, wherein the fatty acid is docosohexaenoic acid.

47. A method for increasing the therapeutic index of anti cancer compounds in a subject, comprising conjugating a fatty acid to a compound from claim 1 to form a fatty acid-anti cancer compound conjugate; and administering the fatty acid-anti cancer compound conjugate to the subject, whereby the therapeutic index of the compound of claim 1 is improved relative to non-conjugated formulations of the compound of claim 1.

48. The method of claim 47, wherein the fatty acid is a C16-C22 unbranched, naturally occurring fatty acid.

49. The method of claim 47, wherein the fatty acid is docosohexaenoic acid.

50. The method of claim 47, wherein the subject is human.