Acid-labile lipophilic prodrugs of cancer chemotherapeutic agents

Abstract

The present application discloses an acid labile lipophilic molecular conjugate of cancer chemotherapeutic agents and methods for reducing or substantially eliminating the side effects of chemotherapy associated with the administration of a cancer chemotherapeutic agent to a patient in need thereof.

Claims

1. A pharmaceutical composition comprising an acid labile lipophilic molecular conjugate (ALLMC) of the formula 1: ##STR00041## wherein: R is a 2 hydroxyl residue of a paclitaxel, a docetaxel or an abeo-taxane compound; R.sup.1 is hydrogen; R.sup.2 is a C.sub.5-C.sub.22 alkyl; Y is selected from O, NR or S wherein R is hydrogen or C.sub.1-C.sub.6 alkyl; Z is O or S; Q is O; and T is O; or an enantiomer, diastereoisomer or mixtures thereof; and a pharmaceutically acceptable salt thereof; wherein the pharmaceutical composition is formulated as a liquid formulation or solution for parenteral administration.

2. The pharmaceutical composition of claim 1, wherein the liquid formulation is a lipid emulsion comprising INTRALIPID for intravenous injection.

3. The pharmaceutical composition of claim 1, wherein the liquid formulation is a buffered, isotonic, aqueous solution.

4. The composition of claim 1, wherein the liquid formulation further comprises a suitable diluent selected from the group consisting of normal isotonic saline solution, 5% dextrose in water and buffered sodium or ammonium acetate solution.

5. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate is of the formula: ##STR00042##

6. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate of claim 1, where the conjugate is of the formula: ##STR00043##

7. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate of claim 1, where the conjugate is of the formula: ##STR00044##

8. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate of claim 1, where the conjugate is of the formula: ##STR00045##

9. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate of claim 1, where the conjugate is of the formula: ##STR00046##

10. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate of claim 1, where the conjugate is of the formula: ##STR00047##

11. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate of claim 1, where the conjugate is of the formula: ##STR00048##

12. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate of claim 1, where the abeo-taxane conjugate is of the formula: ##STR00049##

13. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate of claim 1, where the abeo-taxane conjugate is of the formula: ##STR00050##

14. The pharmaceutical composition of claim 1, wherein the acid labile lipophilic molecular conjugate of claim 1, where the abeo-taxane conjugate is of the formula: ##STR00051##

15. A method for the treatment of cancer in a patient comprising administering to the patient a therapeutically effective amount of a composition of claim 1, to a patient in need of such treatment.

16. The method of claim 15, wherein the cancer is selected from the group consisting of leukemia, neuroblastoma, glioblastoma, cervical, colorectal, pancreatic, renal and melanoma.

17. The method of claim 15, wherein the cancer is selected from the group consisting of lung, breast, prostate, ovarian and head and neck.

18. A method for reducing or eliminating the side effects of chemotherapy associated with the administration of a cancer chemotherapeutic agent to a patient when compared to the administration of a non-conjugated cancer chemotherapeutic agent, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an acid labile lipophilic molecular conjugate of the formula 1: ##STR00052## wherein: R is a 2 hydroxyl residue of a paclitaxel, a docetaxel or an abeo-taxane compound; R.sup.1 is hydrogen; R.sup.2 is a C.sub.5-C.sub.22 alkyl; Y is selected from O, NR or S wherein R is hydrogen or C.sub.1-C.sub.6 alkyl; Z is O or S; Q is O; and T is O; or an enantiomer, diastereoisomer or mixtures thereof; or a pharmaceutically acceptable salt thereof; wherein the pharmaceutical composition is formulated as a liquid formulation or solution for parenteral administration.

19. The method of claim 18, wherein the method provides a higher concentration of the cancer chemotherapeutic agent in a cancer cell of the patient, when compared to the administration of a non-conjugated cancer chemotherapeutic agent.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 depicts a graph of the stability of ART 273 when added to mouse plasma.

(2) FIG. 2 depicts a graph of the stability of ART 273 when added to rat plasma.

(3) FIG. 3 depicts a graph of the stability of ART 273 when added to human plasma.

(4) FIG. 4 depicts a graph of the stability of ART 488 when added to mouse plasma.

(5) FIG. 5 depicts a graph of the stability of ART 488 when added to rat plasma.

(6) FIG. 6 depicts a graph of the stability of ART 488 when added to human plasma.

(7) FIG. 7 depicts a graph of the stability of ART 488 in Liposyn when added to mouse plasma.

(8) FIG. 8 depicts a graph of the stability of ART 488 in Liposyn when added to human plasma.

(9) FIG. 9 depicts a graph of the stability of ART 198 when added to mouse plasma.

(10) FIG. 10 depicts a graph of the stability of ART 198 when added to rat plasma.

(11) FIG. 11 depicts a graph of the stability of ART 198 when added to human plasma.

(12) FIG. 12 depicts a graph of the stability of ART 489 when added to mouse plasma.

(13) FIG. 13 depicts a graph of the stability of ART 489 when added to rat plasma.

(14) FIG. 14 depicts a graph of the stability of ART 489 when added to human plasma.

(15) FIG. 15 depicts a graph of the stability of ART 489 in Liposyn when added to mouse plasma.

(16) FIG. 16 depicts a graph of the stability of ART 489 in Liposyn when added to human plasma.

(17) FIG. 17 depicts a graph of the stability of ART 467 when added to human plasma.

(18) FIGS. 18, 19 and 20 depict representative acid labile lipophilic molecular conjugates.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

(19) The following procedures may be employed for the preparation of the compounds of the present invention. The starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as the Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis, Mo.), or are prepared by methods well known to a person of ordinary skill in the art, following procedures described in such references as Fieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

(20) In some cases, protective groups may be introduced and finally removed. Suitable protective groups for amino, hydroxy, and carboxy groups are described in Greene et al., Protective Groups in Organic Synthesis, Second Edition, John Wiley and Sons, New York, 1991. Standard organic chemical reactions can be achieved by using a number of different reagents, for examples, as described in Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

(21) General Procedure for Synthesis of Acid Labile, Lipophilic Molecular Conjugates of Cancer Chemotherapeutic Agents.

(22) Formation of Activated Intermediate Compounds:

(23) Compounds suitable for use for forming acid labile, lipophilic molecular conjugates of cancer chemotherapeutic agents may be prepared according to the general methods disclosed herein. In one aspect, solketal is reacted with an alkyl aldehyde or a dialkyl ketone in the presence of acid catalysis and an organic solvent to form the aldehyde solketal (acetal) derivative or the ketone solketal (ketal) derivative, respectively. According to the present method, 5-membered and 6-membered cyclic acetals may be prepared and may be isolated in substantially pure form by chromatography. In one aspect, the solvent is toluene and the reaction is performed at an elevated temperature, such as about 60 to 80 C. The acetal or ketal solketal derivative is subsequently activated by a reaction with an acid halide, such as 4-nitrophenyl chloroformate in the presence of base catalysis to form the corresponding activated derivative, such as the 4-nitrophenyl carbonate intermediate compound of the formula 3. In one aspect, the 4-nitrophenyl carbonate intermediate may be condensed with a HBCCA to form the acid labile, lipophilic molecular conjugate.

(24) ##STR00009##

(25) In another aspect solketal is first reacted with an acid halide such as 4-nitrophenyl chloroformate in the presence of base catalysis to form solketal nitrocarbonate which is subsequently reacted with an alkyl aldehyde or a dialkyl ketone in the presence of acid catalysis and an organic solvent to form the aldehyde solketal (acetal) derivative or the ketone solketal (ketal) derivative of formula 3, respectively. In one aspect, the solvent is toluene and the reaction is performed at RT. In one aspect, the 4-nitrophenyl carbonate intermediate may be condensed with a HBCCA to form the corresponding acid labile, lipophilic molecular conjugate.

(26) ##STR00010##

(27) In another aspect alcohol such as stearyl alcohol is reacted with vinyl acetate in the presence of a transition metal catalyst such as [Ir(cod)Cl].sub.2 and a base additive such as Na.sub.2CO.sub.3 to form the corresponding vinyl ether. In one aspect, the solvent is toluene and the reaction is performed at 100 C. In one aspect, the vinyl ether derivative may be condensed with a HBCCA to form the corresponding acid labile, lipophilic molecular conjugate.

(28) ##STR00011##
General Procedure for Synthesis of Alternative Acid Labile, Lipophilic Molecular Conjugates of Cancer Chemotherapeutic Agents:

(29) In one embodiment, the HBCCA may be reacted with the 4-nitrophenyl carbonate compound in the presence of a base, such as a catalytic amount of N,N-dimethyl-4-aminopyridine (DMAP) and pyridine, in an organic solvent, such as dichloromethane (DCM) at room temperature (RT), to form the desired acid labile, lipophilic molecular conjugate.

(30) ##STR00012##

(31) As shown in the scheme below, initial synthesis of activated acid labile, lipophilic molecular conjugate intermediates have been obtained by treating solketal with the aldehyde derived from the corresponding natural fatty acid followed by reaction with 4-nitrophenyl chloroformate.

(32) ##STR00013##

(33) However, this method result the formation of 5- and 6 membered conjugates along with their corresponding syn/anti isomers. Although both 5- and 6-membered acetals could act as lipophilic conjugate precursors, 3 sets of regio- and stereo isomers were isolated in the acetal formation step. In one embodiment, the desired acetal may be isolated in substantially pure form by chromatography. An alternate reaction sequence for the preparation of the 5-membered acetal is shown below. This route provides the 5-membered acetal and provides a method to access lipophilic conjugates of various candidate chemotherapeutic agents. The activated carbonate intermediate is further treated with the hydroxyl-bearing cancer chemotherapeutic agents to generate the corresponding acid labile, lipophilic molecular conjugate prodrugs of interest.

(34) ##STR00014##

(35) Alternative acid labile, lipophilic molecular conjugates of cancer chemotherapeutic agents may be formed by reacting a HBCCA with an alkyl vinyl ether in the presence of a halogenating agent, such as an NXS, such as N-bromosuccinimide (NBS) in DCM. In one aspect, the reactants are combined in solution at low temperatures, such as about 78 C., and the reaction is stirred and allowed to warm slowly to RT.

(36) ##STR00015##

(37) Other alternative acid labile, lipophilic molecular conjugates of cancer chemotherapeutic agents may be formed by reacting HBCCA with higher alkyl vinyl ethers (derived from natural fatty acids) in the presence of an acid catalyst such as pyridinium para-toluene sulfonate (PPTS). In one aspect, the reactants are combined in solution at RT to synthesize the corresponding acid labile lipophilic acetal prodrug.

(38) ##STR00016##
Formation of Acid-Labile Lipophilic Conjugates:

(39) Method A: A solution of the 4-nitrophenyl carbonate-solketal conjugate of formula 3 (0.21 mmol) in anhydrous (anh.) dichloromethane (1 ml) was added to a solution of HBCCA (0.2 mmol) and DMAP (0.3 mmol) in anh. dichloromethane (2 ml) and the reaction mixture was stirred at RT under nitrogen atmosphere (N.sub.2). The reaction progress was monitored by TLC/HPLC, upon completion, the reaction mixture was diluted with methylene chloride (DCM), washed with NH.sub.4Cl(s), water and brine. The organic layer was separated, dried over sodium sulfate and evaporated. The crude residue was purified by silica gel flash chromatography (SGFC) to obtain the conjugated prodrug.

(40) Method B: To a solution of alkyl vinyl ether (1.2 mmol, 6 eq.) and HBCCA (0.2 mmol, 1 eq.) in anh. DCM (8 mL, 0.025M), NBS (1 mmol, 5 eq.) was added at 15 C. under N.sub.2. The reaction mixture was stirred at 15 C. to 0 C. and the progress of the reaction was monitored by TLC/HPLC. Upon completion, the reaction mixture was diluted with DCM and the reaction mixture was washed with NaHCO.sub.3(sat.), water and brine solution. Organic layer was dried over sodium sulfate and evaporated. The crude residue was purified by SGFC to yield the conjugated prodrug.

(41) Method C: To a solution of alkyl vinyl ether (1.2 mmol, 6 eq.) and HBCCA (0.2 mmol, 1 eq.) in anh. DCM (8 mL, 0.025M), PPTS (0.02 mmol, 10 mol %) was added and the reaction mixture was stirred at RT under N.sub.2. The reaction progress was monitored by TLC/HPLC. Upon completion, the reaction mixture was diluted with DCM and the reaction mixture was washed with NaHCO.sub.3(sat.), water and brine solution. Organic layer was dried over sodium sulfate and evaporated. The crude residue was purified by SGFC to yield the conjugated prodrug.

(42) Characterization of Acid Labile Lipophilic Conjugates:

(43) Acid labile lipophilic conjugates were characterized by a combination of HPLC and High Resolution Mass Spectrometry. Specifics are provided with each compound.

(44) Preparation of ART 449

(45) A solution of the 4-nitrophenyl carbonate of docosahexaenoic alcohol (0.5 g) in anh. DCM was added to a solution of ART 273 (0.522 g) and DMAP (0.140 g) in anh. DCM (18 mL) at RT under N.sub.2 and stirred. Upon completion, the reaction was diluted with DCM, washed with saturated ammonium chloride solution (NH.sub.4Cl(s)), water and brine. The organic layer was separated, dried over sodium sulfate and evaporated. The crude residue was purified over silica gel to yield ART 449 as a white solid. TOF MS: m/z 1003.4859 (M+CF.sub.3C0.sub.2).sup.

(46) ##STR00017##
Preparation of ART 448

(47) A solution of the 4-nitrophenyl carbonate of 5-hexen-1-ol (0.1 g) in anh. DCM was added to a solution of ART 273 (0.207 g) and DMAP (0.051 g) in anh. DCM (5 mL) at RT under N.sub.2. Upon completion, the reaction was diluted with DCM, washed with NH.sub.4Cl(s), water and brine. The organic layer was separated, dried over sodium sulfate and evaporated. The crude residue was purified over silica gel to yield ART 448 as a white solid. TOF MS: m/z 789.2928 (M+CF.sub.3C0.sub.2).sup.

(48) ##STR00018##
Preparation of ART 473

(49) Cyclohexyl vinyl ether (0.24 mL) was added to a solution of ART 273 (0.230 g) and NBS (0.282 g) in anh. DCM (5 mL) at 78 C. under N.sub.2. Upon completion, the solution was evaporated and the crude residue purified over silica gel to yield ART 473 as a white solid.

(50) ##STR00019##
Preparation of ART 471

(51) Tert-Butyl vinyl ether (0.24 mL) was added to a solution of ART 273 (0.250 g) and NBS (0.307 g) in anh. DCM (5 mL) at 78 C. under N.sub.2. Upon completion, the solution was evaporated and the crude residue purified over silica gel to yield ART 471 as a white solid.

(52) ##STR00020##
Preparation of ART 472

(53) Octadecyl vinyl ether (0.448 g) was added to a solution of ART 273 (0.208 g) and NBS (0.255 g) in anh. DCM (5 mL) at 78 C. under N.sub.2. Upon completion, the solution was evaporated and the crude residue purified over silica gel to yield ART 472 as a white solid.

(54) ##STR00021##
Preparation of ART 470

(55) Ethyl vinyl ether (0.11 mL) was added to a solution of ART 273 (0.150 g) and N-bromosuccinimide (NBS, 0.170 g) in anh. DCM (5 mL) at 78 C. under N.sub.2. Upon completion, the solution was evaporated and the crude residue purified over silica gel to yield ART 470 as a white solid.

(56) ##STR00022##
Preparation of ART 489

(57) A solution of octadecyl solketal-4-nitrophenyl carbonate (0.750 g) in anh. DCM was added to a solution of ART 198 (0.754 g) and DMAP (0.238 g) in anh. DCM (30 mL) at RT under N.sub.2. Upon completion, the reaction was diluted with DCM, washed with NH.sub.4Cl(s), water and brine. The organic layer was separated, dried over sodium sulfate and evaporated. The crude residue was purified over silica gel to yield ART 489 as a solid. TOF MS: m/z 1031.4645 (M+CF.sub.3C0.sub.2).sup.

(58) ##STR00023##
Preparation of ART 488

(59) A solution of octadecyl solketal-4-nitrophenyl carbonate (0.53 g) in anh. DCM was added to a solution of ART 273 (0.507 g) and DMAP (0.168 g) in anh. DCM (30 mL) at RT under N.sub.2. Upon completion, the reaction was diluted with DCM, washed with NH.sub.4Cl(s), water and brine. The organic layer was separated, dried over sodium sulfate and evaporated. The crude residue was purified over silica gel to yield ART 488 as a solid. TOF MS: m/z 1003 4994 (M+CF.sub.3C0.sub.2).sup.

(60) ##STR00024##
Preparation of ART 332

(61) A solution of solketal-4-nitrophenyl carbonate (1.1 g) in anh. DCM was added to a solution of ART 273 (1.30 g) and DMAP (0.36 g) in anh. DCM (30 mL) at RT under N.sub.2. Upon completion, the reaction was diluted with DCM, washed with NH.sub.4Cl(s), water and brine. The organic layer was separated, dried over sodium sulfate and evaporated. The crude residue was purified over silica gel to yield ART 332 as a white solid. TOF MS: m/z 947.4601 (M+CF.sub.3C0.sub.2).sup.

(62) ##STR00025##
Preparation of ART 441

(63) DHA (0.2 g), DCC (0.157 g) and DMAP (0.006 g) were sequentially added to a solution of ART 273 (0.279 g) in anh. DCM (10 mL) at RT under N.sub.2. Upon completion, the reaction was diluted with DCM, washed with NH.sub.4Cl(s), water and brine. The organic layer was separated, dried over sodium sulfate and evaporated. The crude residue was purified over silica gel to yield ART 441 (0.2 g) as a white solid.

(64) ##STR00026##
Preparation of ART 467

(65) A solution of octadecyl solketal-4-nitrophenyl carbonate (1.75 g) in anh. DCM was added to a solution of paclitaxel (2.59 g) and DMAP (0.557 g) in anh. DCM (30 mL) at RT under N.sub.2. Upon completion, the reaction was diluted with DCM, washed with NH.sub.4Cl(s), water and brine. The organic layer was separated, dried over sodium sulfate and evaporated. The crude residue was purified over silica gel to yield ART 467 as a white solid. TOF MS: m/z 1306.5445 (M+CF.sub.3C0.sub.2).sup.

(66) ##STR00027##
Preparation of ART 151

(67) ART 151 was prepared by following the procedure as outlined in Method A. HPLC retention time 6.06, Method: Taxane conjugates_MKG4 (C18 column, MeOH/H.sub.2O/THF 95/3/2 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5 ml/min, 30 C., 14 min). +TOF MS: m/z 1239.6523 [M+18] (M+NH.sub.4.sup.+)

(68) ##STR00028##
Preparation of ART 152

(69) ART 152 was prepared by following the procedure as outlined in Method B. HPLC retention time 8.21, Method: Taxane conjugates MKG4 (C18 column, MeOH/H.sub.2O/THF 95/3/2 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5 ml/min, 30 C., 14 min). +TOF MS: m/z 1228.5654 [M+1] (M+H.sup.+)

(70) ##STR00029##
Preparation of ART 153

(71) ART 153 was prepared by following the procedure as outlined in Method C. HPLC retention time 7.05, Method: Taxane conjugates_MKG4 (C18 column, MeOH/H.sub.2O/THF 95/3/2 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5 ml/min, 30 C., 14 min). +TOF MS: m/z 1150.6485 [M+1] (M+H.sup.+)

(72) ##STR00030##

(73) ART 161 was prepared by following the procedure as outlined in Method A. HPLC retention time 4.88, Method: Taxane conjugates_MKG6 (C18 column, MeOH/H.sub.2O 95/5 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5 ml/min, 30 C., 16 min). +TOF MS: m/z 1235.6276 [M+18] (M+NH.sub.4.sup.+)

(74) ##STR00031##

(75) ART 207 was prepared by following the procedure as outlined in Method A. HPLC retention time 6.06, Method; Taxane conjugates_MKG17 (Synergy column, ACN/H.sub.2O 60/40 to 100% ACN 10 min, 2 min 100% ACN, 230 nm, 1.5 ml/min, 30 C., 15 min). +TOF MS: m/z 1220.6156 [M+1] and m/z 1237.6382 [M+18] (M+NH.sub.4.sup.+)

(76) ##STR00032##

(77) ART 156 was prepared by following the procedure as outlined in Method A. HPLC retention time 6.2, Method: Taxane conjugates_MKG4 (C18 column, MeOH/H.sub.2O/THF 95/3/2 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5 ml/min, 30 C., 14 min). +TOF MS: m/z 1176.6466 [M+1] and m/z 1193.6730 [M+18] (M+NH.sub.4.sup.+)

(78) ##STR00033##

(79) ART 162 was prepared by following the procedure as outlined in Method A. HPLC retention time 8.96, Method: Taxane conjugates_MKG16 (Synergy column, MeOH/H.sub.2O 75/25 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5 ml/min, 30 C., 15 min). +TOF MS: m/z 1189.6491 [M+18] and m/z 1172.6224 [M+1] (M+H.sup.+)

(80) ##STR00034##

(81) ART 208 was prepared by following the procedure as outlined in Method A. HPLC retention time 7.4, Method: Taxane conjugates_MKG19 (Synergy column, ACN/H.sub.2O 50/50 3 min, 80-100% ACN/H.sub.2O 10 min, 2 min 100% ACN, 230 nm, 1.5 ml/min, 30 C., 15 min). +TOF MS: m/z 1174.6306 [M+1] (M+H.sup.+)

(82) ##STR00035##

(83) ART 185 was prepared by following the procedure as outlined in Method C. HPLC retention time 6.42, Method: Taxane conjugates MKG15 (Synergy column, 70-100% ACN/H.sub.2O 10 min, 100% ACN 2 min, 230 nm, 1.5 ml/min, 30 C., 15 min). +TOF MS: m/z 1104.6648 [M+1](M+H.sup.+) and m/z 1126.6447 [M+18] (M+NH.sub.4.sup.+)

(84) ##STR00036##

(85) ART 137 was prepared by following the procedure as outlined in Method C. HPLC retention time 10.63, Method: Taxane (C18 column, ACN/H.sub.2O 50/50 to 100% ACN 10 min, 2 min 100% ACNH, 230 nm, 1.5 ml/min, 30 C., 16 min)

(86) ##STR00037##

(87) ART 164 was prepared by following the procedure as outlined in Method A. HPLC retention time 7.73, Method: Taxane conjugates_MKG6 (C18 column, MeOH/H.sub.2O 95/5 to 1000/% MeOH 10 min, 2 min 1000/MeOH, 230 nm, 1.5 ml/min, 30 C., 16 min). +TOF MS: m/z 1255.7506 [M+18] (M+NH.sub.4.sup.+)

(88) ##STR00038##

(89) ART 163 was prepared by following the procedure as outlined in Method A. HPLC retention time 7.56, Method: Taxane conjugates_MKG6 (C18 column, MeOH/H.sub.2O 95/5 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5 ml/min, 30 C., 16 min). +TOF MS: m/z 1251.7233 [M+18] (M+NH.sub.4.sup.+)

(90) ##STR00039##

(91) ART 209 was prepared by following the procedure as outlined in Method A. HPLC retention time 9.6, Method: Taxane conjugates_MKG18 (Synergy column, ACN/H.sub.2O 80/20 10 min, 100% ACN 2 min, 230 nm, 1.5 ml/min, 30 C., 15 min). +TOF MS: m/z 1253.7505 [M+18](M+NH.sub.4.sup.+)

(92) ##STR00040##
Cytotoxicity of Specific Compounds:
MTS Proliferation Assay Using SK-N-AS Cells

(93) Day 1:

(94) SK-N-AS cells were plated in appropriate growth medium at 510.sup.3 per well in 100 L in 96 well tissue culture plates, Falcon, one plate for each drug to be tested. Column 1 was blank; it contained medium, but no cells. The plates were incubated overnight at 37 C. in 5% CO.sub.2 to allow attachment.

(95) Day 2:

(96) Drug diluted in culture media was added to the cells at a concentration of 0.005 nM to 10 M, in quadruplicate. After 48-72 hours of drug exposure, the MTS agent was added to all wells and incubated 1-6 hrs (37 C., 5% CO.sub.2), depending on cell type, as per CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS), Promega. Plates were processed using a Bio-Tek Synergy HT Multi-detection microtiter plate reader at 490 nanometer wavelength and data were processed with KC4V.3 software. Data plots of drug concentration vs. absorbance were plotted and the concentration resulting in 50% inhibition (IC.sub.50) was extrapolated for each of the tested compounds.

(97) As summarized in Table 1, the IC.sub.50 value for each tested compound in the SK-N-AS cell line was determined. The clinical comparator drug, paclitaxel, was included in the experiment to allow comparison of the results of the candidate compounds to a clinically relevant standard in the taxane class.

(98) TABLE-US-00001 TABLE 1 IC.sub.50 (nM) Values in SK-N-AS IC.sub.50 (nM) Values in SK-N-AS (MDR Neuroblastoma) Compound IC.sub.50 ART 449 4.0 0.5 ART 448 5.0 0.7 ART 473 12.6 0.9 ART 471 261.6 12 ART 470 349.1 15 ART 488 0.33 0.1 ART 441 1.76 0.5 ART 472 1.19 0.5 ART 332 1.1 0.5 ART 273 2.0 0.5 ART 467 273.9 12 Paclitaxel 0.05 0.01 MTT Proliferation Assay Using Paired MDR+ and MDR Cell Lines

(99) A second evaluation of the cytotoxicity of the acid labile, lipophilic molecular conjugates was undertaken. The purpose of these experiments was to compare the toxicity of the conjugates in multidrug resistant cells and their parental susceptible lines to test the hypothesis that a subset of these compounds would exhibit a similar level of toxicity in the drug resistant lines as that observed in the parent susceptible cell line.

(100) MTT-based cytotoxicity assays were performed using human cancer cell lines and paired sublines exhibiting multidrug resistance. These lines included a uterine sarcoma line, MES-SA, and its doxorubicin-resistant subline, MES-SA/Dx5. See W. G. Harker, F. R MacKintosh, and B. I. Sikic. Development and characterization of a human sarcoma cell line, MES-SA, sensitive to multiple drugs. Cancer Research 43: 4943-4950 (1983); W. G. Harker and B. I. Sikic. Multidrug (pleiotropic) resistance in doxorubicin-selected variants of the human sarcoma cell line MES-SA. Cancer Research 45: 4091 4096 (1985).

(101) MES-SA/Dx5 exhibits a marked cross resistance to a number of chemotherapeutic agents including vinblastine, paclitaxel, colchicine, vincristine, etoposide, dactinomycin, mitoxantrone and daunorubicin and moderate cross resistance to mitomycin C and melphalan. However, resistance to bleomycin, cisplatin, carmustine, 5-fluorouracil or methotrexate is not observed. MES-SA/Dx5 cells express high levels of ABCB1 (MDR1) mRNA and its gene product, the P-glycoprotein. MES-SA and MES-SA/Dx5 were purchased from the American Type Culture Collection (ATCC, Manassas, Va.).

(102) The second set of cells tested, CCRF-CEM or simply CEM, were derived from the blood of a patient with acute lymphoblastic leukemia. G. E. Foley, H. Lazarus, S. Farber, B. G. Uzman, B. A. Boone, and R E. McCarthy. Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 18: 522-529 (1965). The subline CEM/VLB.sub.100 was developed to be resistant to up to vinblastine at 100 ng/ml. W. T. Beck, T. J. Mueller, and L. R. Tanzer. Altered surface membrane glycoproteins in Vinca alkaloid-resistant human leukemic lymphoblasts. Cancer Research 39: 2070-2076 (1979). Drug resistance is achieved by overexpression of the MDR1 gene. Resistance in the CEM subline designated CEM/VM-1-5, however, is atypical. M. K. Danks, J. C. Yalowich, and W. T. Beck. Atypical multiple drug resistance in a human leukemic cell line selected for resistance to teniposide (VM-26). Cancer Research 47: 1297-1301 (1987). The classes of drugs included in the classic multiple drug resistance phenotype are Vinca alkaloids, anthracyclines, epipodophyllotoxins and antibiotics. However, CEM/VM-1-5 cells retain sensitivity to the Vinca alkaloids despite resistance and cross-resistance to etoposide, anthracyclines and mitoxantrone. Danks, M. K.; Schmidt, C. A.; Cirtain, M. C.; Suttle, D. P.; Beck, W. T., Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26. Biochemistry 1988, 27, 8861-8869. Resistance in CEM/VM-1-5 cells is effected by over expression of the ABCC1 (MRP1) gene. CEM, CEM/VLB.sub.100 and CEM/VM-1-5 cells were obtained from Dr. WT Beck, University of Illinois at Chicago.

(103) TABLE-US-00002 TABLE 2 Summary of Testing Concentrations in Paired Cell Lines Summary of Testing Concentrations Compound Test Concentrations (ng/ml) ART 273 200, 40, 8, 1.6, 0.32, 0.064 ART 198 5,000, 1,000, 200, 40, 8, 1.6 ART 488 5,000, 1,000, 200, 40, 8, 1.6 ART 489 5,000, 1,000, 200, 40, 8, 1.6 Paclitaxel 25,000 5,000, 1,000, 200, 40, 8, 1.6 Vinblastine Doxorubicin

(104) TABLE-US-00003 TABLE 3 IC50 Results MESSA/ (nM) MESSA Dx5 Degree of CEM CEM/VLB.sub.100 Degree of CEM/VM-1-5 Degree of Compound (Hs) (MDR + Hs) resistance.sup.1 (HTL) (MDR + HTL) resistance.sup.2 (MDR + HTL) resistance.sup.2 ART 273 1.5 0.7 7 4 4.5 0.7 2 0 36 20 18 10 7 2 3 1 Q1 ART 488 1.5 0.7 7 5.7 4.3 1.8 4 0 34 12 9 4 8 4 2 1 Q1 prodrug ART 198 47.5 11 376 110 7.9 0.6 113 53 7186 1918 76 52 308 10 3 1 Q2 ART 489 5.5 3.5 17 5.7 4.3 3.8 21 2 670 71 33 0 24 4 1.2 0.07 Q2 prodrug Paclitaxel 9 7 19398 204 3105 2416 <11/2 3029/1295 >275/648 <11/8 4 Vinblastine 1.1 0.3 43 12 38.5 0.7 1 0.8 227 77 255 127 1.3 0.9 1.2 0.07 Doxorubicin 2 97 49 14 2100 150 3060 219 Data are expressed as IC.sub.50 values (nM). .sup.1Calculated by dividing the IC.sub.50 of the resistant lines by the IC.sub.50 of the sensitive MES-SA cells. .sup.2Calculated by dividing the IC.sub.50 of the resistant lines by the IC.sub.50 of the sensitive CEM cells. HTL means Human T-Lymphoblastoid; Hs means Human sarcoma.

(105) The observed cytotoxicity of the acid labile, lipophilic molecular conjugates demonstrates that they still possess the anti-cancer activity desired for them to retain utility as potential chemotherapeutic agents. It is especially noteworthy that the apparent degree of resistance expressed by the resistant cell lines is diminished by 20 to 50% for the acid labile, lipophilic molecular conjugates. This was an unexpected result.

(106) Stability of Acid Labile, Lipophilic Molecular Conjugates in Plasma:

(107) The stability of the acid labile, lipophilic molecular conjugates to hydrolysis in plasma was evaluated to determine their potential to release the active cancer chemotherapeutic agents into systemic circulation and thereby cause general off target toxicity (side effects). The conjugates were incubated with plasma of mouse, rat and human origin.

(108) HPLC grade Methanol from Fisher (Fair lawn, NJ, USA). Part No: A452-4 (074833). HPLC grade Water from Fisher (Fair lawn, NJ, USA). Part No: W5-4 (073352). Drug-free mouse, rat and human plasmas were purchased from Innovative Research Inc. (Southfield, Mich., USA). LIPOSYN I.V. (30% soybean oil and egg yolk phospholipids, Abbott Laboratories) Fat Emulsion from Hospira, Inc. (Lake Forest, Ill.).

(109) Preparation of Plasma Incubations:

(110) Each drug (ART 198, ART 273, ART 488 and ART 489) was prepared in triplicate in mouse, rat and human plasma individually at 10 pig/ml concentration and vortexed for 1 minute and placed in a water bath at 37 C. at a shake rate of 75 per minute. Samples were drawn at time points of 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 210, 240, 300, 360 and 480 minutes.

(111) Analytical Method for ART 198, ART 273, ART 488 and ART 489 Analysis in Plasma:

(112) Chromatographic separation of the compounds was performed on a Waters Acquity UPLC using a BEH C.sub.18 column (1.7 m, 2.150 mm). The mobile phase consisted of Methanol: 0.1% Formic acid (80:20). The flow rate was 0.3 ml/min; the sample injection volume was 5 L, resulting in a 3 minute run time.

(113) The MS instrumentation consisted of a Waters Micromass Quattro Micro triple-quadrupole system (Manchester, UK). The MS system was controlled by a 4.0 version of MassLynx software. Ionization was performed in the positive electrospray ionization mode. MS/MS conditions were the following: capillary voltage 3.02 kV; cone voltage 50 v; extractor voltage 5 v; and RF lens voltage 0.5 v. The source and desolvation temperatures were 100 C. and 400 C. respectively, and the desolvation and cone gas flow were 400 and 30 L/hr, respectively.

(114) The selected mass-to-charge (m/z) ratio transitions of the ART 198 used in the selected ion monitoring (SIM) were: for ART 198, 617 (M+K).sup.+, for ART 273, 589 (M+K).sup.+, for ART 488, 913 (M+Na).sup.+, and for ART 489, 957 (M+Na).sup.+. The dwell time was set at 200 msec. MS conditions were optimized using direct infusion of standard solutions prepared in methanol and delivered by a syringe pump at a flow rate of 20 L/min.

(115) Plasma Sample Preparation:

(116) Samples of 100 L were collected at time points of 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 210, 240, 300, 360 and 480 minutes respectively and the reaction was terminated with methanol. In a separate set of experiments the acid labile, lipophilic molecular conjugates were dissolved in a small amount of ethanol and diluted into a lipid emulsion (Liposyn) and added to mouse and human plasma before incubation and the hydrolysis of the conjugates was similarly measured. Collected plasma samples of 100 L containing drug were placed in separate Eppendorf micro centrifuge tubes for processing. Methanol (200 L) was added to extract the drug using the protein precipitation technique. The micro tubes were then vortex mixed for 10 minutes and centrifuged for 15 minutes at a speed of 10,000 rpm (Eppendorf 5415C centrifuge). The supernatant was collected and filtered using a 0.45 m filter (Waters 13 mm GHP 0.45 m) before analysis.

(117) UPLC/MS/MS analysis of blank mouse, rat and human plasma samples showed no endogenous peak interference with the quantification of ART 198, ART 273, ART 488 or ART 489.

(118) The weighted linear least-squares (1/x) regression was used as the mathematical model. The coefficient (r) for the compounds ranged from 0.9925 to 0.9999. The calibration range was selected according to the concentrations anticipated in the samples to be determined. The final calibration range was 10-12,500 ng/mL with a lower limit of quantification of 10 ng/mL.

(119) The repeatability and reproducibility bias (%) is within the acceptance limits of 20% at low concentration and 15% at other concentration levels with RSD's of less than 5% at all concentrations evaluated.

(120) The mean recoveries of the method were in the range of 86.22-99.83% at three different concentrations of the test drugs from plasma. These results suggested that there was no relevant difference in extraction recovery at different concentration levels.

(121) Incubations of ART 467 and Paclitaxel:

(122) A 0.2 ml aliquot from 210.6 g/ml stock solution of ART 467 was spiked into 3.8 ml of human plasma preincubated for 15 min (37 C.) and incubated in a reciprocating water bath at 37 C. Samples were drawn at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours.

(123) Analytical Method for ART 467 and Paclitaxel (Liquid Chromatography-Tandem Mass Spectrometry):

(124) Chromatographic separation was carried out using an ACQUITY UPLC liquid chromatograph (Waters Corporation, Milford, Mass., USA) consisting of a binary pump, autosampler, degasser and column oven. A mobile phase of methanol-acetonitrile (50:50, v/v) was pumped at a flow-rate of 0.4 ml/min through an ACQUITY UPLC BEH C.sub.18 column (1.7 m, 2.150 mm i.d., Waters Corporation) maintained at 25 C. 10 l of sample was injected and the run time was 3.0 min. The LC elute was connected directly to an ESCi triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source. The quadruples were operated in the positive ion mode. The multiple reaction monitoring (MRM) mode was used for quantification using MassLynx version 4.1 software. Mass transitions of m/z 876.2, 307.9; 882.2, 313.9; and 1216.5, 647.8 were optimized for paclitaxel Na.sup.+ adduct, .sup.13C6-paclitaxel adduct and ART 467 adduct respectively, with dwell time of 0.5 s. Nitrogen was used as nebulizing gas (30 l/h) and desolvation gas (300 l/h) with a desolvation temperature at 250 C., and argon was collision gas. The capillary voltage was set at 3.5 kV, and cone voltage at 90 V. The source temperature was set at 100 C.

(125) Plasma Sample Preparation:

(126) At the different time periods (0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h), 200 l aliquot of samples were taken and immediately added to 1.3 ml of cold TBME and subsequently 20 l of internal standard stock solution (80.7 g/ml in methanol) was added. Each tube was vortex mixed for approximately 2 min and then centrifuged at 13000 rpm for 10 min. 1.0 ml of resultant supernatant was transferred to another tube and dried under a stream of nitrogen gas at 35 C. Each dried residue was reconstituted with 200 l of methanol and vortex mixed for 0.5 min. After centrifugation at 13000 rpm for 10 min, the supernatants were transferred to HPLC autosampler vials, and 10 l aliquot of each sample was injected into LC-MS-MS.

(127) Samples were collected at various times and the percent remaining of the acid labile, lipophilic molecular conjugate of the cancer chemotherapeutic agent was determined along with the percent of the chemotherapeutic agent released from the hydrolysis of the conjugate. The results are presented in table format and graphically.

(128) Stability of Unconjugated ART 273 in Plasma:

(129) The intrinsic stability of unconjugated ART 273 in mouse, rat and human plasma was determined. Without reference to any particular kinetic model it is seen that approximately 30%, 54%, and 67% of the initial ART 273 remains after 480 minutes in mouse, rat and human plasma, respectively.

(130) TABLE-US-00004 TABLE 4 Stability of ART 273 in Plasma at 37 C. ART 273 in ART 273 in ART 273 in Mouse Plasma Rat Plasma Human Plasma Time, min ART 273 ART 273 ART 273 0 100 100 100 15 81.1 87.9 97.2 30 76.0 84.9 96.5 45 68.4 82.9 94.6 60 65.4 78.9 93.5 75 62.7 71.8 93.5 90 54.8 69.7 92.2 105 53.8 66.1 89.4 120 49.8 64.6 87.0 135 46.8 64.3 86.6 150 44.0 61.8 85.5 165 42.2 57.0 83.8 180 39.5 56.7 83.4 210 37.6 55.4 80.4 240 36.4 55.1 80.0 300 33.8 54.7 73.2 360 31.5 54.5 69.3 480 30.1 53.9 66.7
Stability of the ART 273 Conjugate, ART 488, in Plasma

(131) The intrinsic stability of the ART 273 Conjugate, ART 488, in mouse, rat and human plasma was determined. Without reference to any particular kinetic model it is seen that approximately 36%, 33%, and 44% of the initial ART 488 remains after 480 minutes in mouse, rat and human plasma, respectively. Also without reference to any particular kinetic model it is seen that the formation of ART 273 approximately equivalent to 36%, 32%, and 37% of the initial ART 488 is present after 480 minutes in mouse, rat and human plasma, respectively.

(132) TABLE-US-00005 TABLE 5 Stability of ART 488 in Plasma at 37 C. ART 488 in ART 488 in ART 488 in Mouse Plasma Mouse Plasma Human Plasma Time, ART ART ART ART ART ART min 488 273 488 273 488 273 0 100 0 100 0 100 0 15 91.2 3.3 90.7 2.2 90.8 1.2 30 85.7 7.9 80.6 8.7 89.1 7.3 45 81.3 10.7 79.8 10.1 87.8 9.3 60 75.0 11.3 78.3 11.7 87.9 10.3 75 73.2 12.2 78.0 12.4 87.9 11.2 90 65.2 13.2 77.5 13.2 87.1 12.5 105 58.8 14.4 73.7 14.0 86.2 13.4 120 56.4 16.3 69.5 16.3 85.3 15.1 135 56.2 18.2 69.1 19.5 84.0 19.7 150 55.0 19.2 68.7 20.0 82.7 19.9 165 53.7 22.5 64.0 22.1 81.1 23.3 180 53.7 26.1 63.8 74.7 78.6 26.5 210 52.4 27.9 63.6 25.5 78.1 28.1 240 50.3 28.7 60.4 26.7 76.5 29.3 300 48.2 29.3 53.7 28.0 59.3 30.8 360 45.6 30.1 48.7 29.0 59.8 32.2 480 35.7 35.6 33.3 32.2 43.6 36.6
Stability of the ART 273 Conjugate, ART 488, in Plasma when Added in a Lipid Emulsion:

(133) The intrinsic stability of the ART 273 Conjugate, ART 488, in mouse and human plasma was determined. Without reference to any particular kinetic model it is seen that approximately 89% and 88% of the initial ART 488 remains after 480 minutes in mouse and human plasma, respectively.

(134) TABLE-US-00006 TABLE 6 Stability of ART 488 in Plasma at 37 C. When Added in a Lipid Emulsion ART 488 ART 488 in Liposyn in Liposyn Time, in Mouse Plasma in Human Plasma min ART 488 ART 273 ART 488 ART 273 0 100 ND 100 ND.sup.a 15 98.7 ND 98.3 ND 30 98.2 ND 97.3 ND 45 97.4 ND 96.1 ND 60 96.9 ND 95.8 ND 75 97.0 ND 95.3 ND 90 98.3 ND 95.6 ND 105 96.0 ND 94.6 ND 120 95.2 ND 94.5 ND 135 93.8 ND 92.5 ND 150 93.1 ND 92.2 ND 165 92.9 ND 91.9 ND 180 91.8 ND 91.0 ND 210 91.7 ND 91.0 ND 240 91.4 ND 90.7 ND 300 91.3 ND 90.7 ND 360 90.0 ND 90.2 ND 480 88.5 ND 88.1 ND .sup.aND = None detected
Stability of Unconjugated ART 198 in Plasma:

(135) The intrinsic stability of unconjugated ART 198 in mouse, rat and human plasma was determined. Without reference to any particular kinetic model it is seen that approximately 26%, 30%, and 34% of the initial ART 198 remains after 480 minutes in mouse, rat and human plasma, respectively.

(136) TABLE-US-00007 TABLE 7 Stability of ART 198 in Plasma at 37 C. ART 198 in ART 198 in ART 198 in Time, Mouse Plasma Rat Plasma Human Plasma min ART 198 ART 198 ART 198 0 100 100 100 15 96.8 95.8 99.3 30 94.0 84.0 99.1 45 85.5 66.0 94.9 60 82.0 55.7 94.6 75 72.6 54.4 93.1 90 66.9 54.2 89.9 105 63.2 54.0 87.0 120 59.2 52.1 68.5 135 57.4 48.9 66.4 150 51.9 48.9 61.1 165 46.2 45.4 59.6 180 43.0 44.0 48.6 210 39.3 42.7 47.6 240 35.4 42.2 46.0 300 32.4 34.3 44.4 360 28.8 30.1 39.6 480 25.9 30.1 34.2
Stability of the ART 198 Conjugate, ART 489, in Plasma:

(137) The intrinsic stability of the ART 198 Conjugate, ART 489, in mouse, rat and human plasma was determined. Without reference to any particular kinetic model it is seen that approximately 34%, 34%, and 66% of the initial ART 489 remains after 480 minutes in mouse, rat and human plasma, respectively. Also without reference to any particular kinetic model it is seen that ART 198 equivalent to approximately 35%, 32%, and 20% of the initial ART 489 is present after 480 minutes in mouse, rat and human plasma, respectively.

(138) TABLE-US-00008 TABLE 8 Stability of ART 489 in Plasma at 37 C. ART 489 in ART 489 in ART 489 in Mouse Plasma Rat Plasma Human Plasma Time, ART ART ART ART ART ART min 489 198 489 198 489 198 0 100 0 100 0 100 0 15 95.7 1.7 93.2 3.1 99.3 0.1 30 88.6 6.1 75.8 14.1 98.8 0.6 45 84.8 10.0 74.3 16.3 98.4 0.9 60 79.2 14.6 75.0 18.1 97.4 1.1 75 78.1 16.7 74.4 20.5 94.6 1.2 90 70.1 18.2 74.4 20.8 93.7 2.4 105 68.0 20.3 73.7 21.4 93.0 3.2 120 64.1 21.3 69.9 21.9 91.9 5.1 135 63.2 22.1 68.5 22.3 91.7 6.5 150 59.4 25.1 67.3 22.9 90.9 7.2 165 54.7 26.4 63.0 23.6 90.4 8.5 180 51.6 27.6 63.0 24.5 90.1 9.6 210 50.3 29.7 62.7 25.0 89.0 12.3 240 47.5 32.0 61.7 25.3 86.7 14.2 300 41.1 34.1 55.4 26.1 84.1 16.3 360 38.1 34.3 48.2 28.0 78.7 19.5 480 34.0 34.7 34.3 32.3 65.9 20.4
Stability of the ART 198 Conjugate, ART 489, in Plasma when Added in a Lipid Emulsion:

(139) The intrinsic stability of the ART 198 Conjugate, ART 489, in mouse and human plasma was determined. Without reference to any particular kinetic model it is seen that approximately 73% and 77% of the initial ART 489 remains after 480 minutes in mouse and human plasma, respectively.

(140) TABLE-US-00009 TABLE 9 Stability of ART 489 in Plasma at 37 C. When Added in a Lipid Emulsion ART 489 ART 489 in Liposyn in Liposyn Time, in Mouse Plasma in Human Plasma min ART 489 ART 198 ART 489 ART 198 0 100 ND 100 ND 15 98.0 ND 98.4 ND 30 97.9 ND 93.9 ND 45 97.4 ND 92.7 ND 60 91.4 ND 88.2 ND 75 90.3 ND 87.9 ND 90 87.9 ND 87.5 ND 105 80.7 ND 86.4 ND 120 80.4 ND 86.4 ND 135 79.9 ND 84.7 ND 150 79.2 ND 84.6 ND 165 78.7 ND 83.7 ND 180 78.2 ND 82.9 ND 210 75.6 ND 82.0 ND 240 74.7 ND 81.4 ND 300 73.7 ND 80.2 ND 360 73.0 ND 78.2 ND 480 72.9 ND 76.6 ND
Stability of the Paclitaxel Conjugate, ART 467, in Plasma:

(141) The intrinsic stability of the paclitaxel conjugate, ART 467, in human plasma was determined. Without reference to any particular kinetic model it is seen that approximately 41% of the initial ART 467 remains after 1440 minutes in human plasma. Also without reference to any particular kinetic model it is seen that paclitaxel equivalent to approximately 16% of the initial ART 467 is present after 1440 minutes in human plasma.

(142) TABLE-US-00010 TABLE 10 Stability of ART 467 in Human Plasma at 37 C. ART 467 in Human Plasma Time, min ART 467 Paclitaxel 0 100.0 0.0 30 86.3 0.7 60 78.0 1.7 120 76.0 2.7 180 75.0 3.8 240 73.7 5.5 360 72.8 8.2 480 70.5 10.3 600 68.2 12.4 720 64.6 14.1 1440 41.3 15.5

(143) Dissolution of the acid labile, lipophilic molecular conjugates ART 488 and ART 489 in a lipid emulsion before addition to plasma enhanced the stability of the conjugate to hydrolysis by the plasma medium dramatically (summarized in Table 11). That the acid labile, lipophilic molecular conjugates remained within the lipid emulsion and did not leak into the plasma phase of the incubation is evident from the lack of release of the free drug from the conjugates. No detectable concentrations of free drug could be observed in the incubations wherein the conjugates were first dissolved in the lipid emulsion before addition to the incubation medium (see Table 6 and Table 9).

(144) TABLE-US-00011 TABLE 11 Drug Stabilization by Incorporation in a Lipid Emulsion % of Initial Drug Remaining After 480 Minutes Mouse Rat Human Plasma Plasma Plasma ART 273 30.1 53.9 66.7 ART 488 35.7 33.3 43.6 ART 488 in 88.5 NP 88.1 Liposyn ART 198 25.9 30.1 34.2 ART 489 34.0 34.3 65.9 ART 489 in 72.9 NP.sup.a 76.6 Liposyn .sup.aNP = Experiment not performed
Estimation of Maximum Tolerated Dose (MTD) of Acid Labile, Lipophilic Molecular Conjugates in the Mouse:

(145) Stock solutions of ART 198 and 273 and their respective acid labile, lipophilic molecular conjugates (ART 489 and ART 488, respectively) were prepared in ethanol and then diluted into a lipid emulsion (INTRALIPID, 20% Soybean Oil, 1.2% egg yolk phospholipids, 2.25% glycerin and water) and injected intravenously into mice at various doses in milligrams per kilogram. The animals were observed daily for signs of toxicity and/or death for a period of 30 days. The MTD was defined as survival of the dosed mice for the full 30 day observation period.

(146) The MTD of ART 198 was determined to be 4.0+/1.0 mg/kg; the MTD of ART 273 was determined to be 1.0+/0.5 mg/kg; the MTD of ART 489 was determined to be 3.1+/1.0 mg/kg; and the MTD of ART 488 was determined to be 4.0+/0.5 mg/kg.

(147) The observed similarity of MTD for ART 198 and its acid labile, lipophilic molecular conjugate ART 489, or in the case of ART 273, the increase from an MTD of roughly 1 mg/kg for ART 273 to roughly 4 mg/kg for its acid labile, lipophilic molecular conjugate ART 488 is surprising in light of their observed in vitro cytotoxicities. In in vitro cytotoxicity evaluations, the acid labile, lipophilic molecular conjugates of ART 273 are routinely observed to be nearly an order of magnitude (10) more potent than ART 273. The MTD determination results suggest that the acid labile, lipophilic molecular conjugates of cancer chemotherapeutic agent may be more useful for treating patients due to reduced toxicity.