This is a continuation-in-part of co-pending U.S.

Patent Application Serial Number 07/934,131, filed August 21, 1992, the contents of which are hereby incorporated by reference.

The present invention relates to compositions having an anti-microtubule activity. In particular, the invention relates to modified taxol-based compositions, which, in certain cases, demonstrate prolonged anti- neoplastic activity.

Recently, taxol has been investigated as a possible anti-cancer agent. Taxol is a plant product derived in minute quantities from the needles and bark of the western pacific yew, Taxus brevifolia. In chemotherapy, taxol is known as an anti-microtubule agent and is thought to inhibit cell mitosis through the enhancement of the rate of microtubular assembly and prevention of microtubular depolymerization. Numerous studies performed to date indicate that taxol has a wide spectrum of activity against several malignancies. To date, the use of taxol, however, has been severely limited by, among other things, its short supply, poor water solubility and immunogenicity.

The pacific yew is a rare, slow-growing tree which is not typically cultivated. In addition, the anti- neoplastic portions of the tree are very minute. Extraction of these portions is complicated and costly. One solution to the problem of short supply has been suggested in U.S. Patent No. 5,019,504 which discloses an artificial media capable of producing the certain

desirable alkaloids found in the tree. Alternatively, synthetic derivatives such as taxotere and taxol intermediates have also been reported. See, for example, U.S. Patent No. 5,015,744 which discloses the preparation of taxol intermediates using oxazinones.

The poor water solubility of taxol has also hindered the development of pharmaceutical formulations. U. S . Patent Nos. 5,059,699, 4,960,790 and 4,942,184, contain suggestions for improving the water solubility of taxol. The '699 disclosure is directed to sulfonated 2' acryloytaxol and sulfonated 2' O-acyl acid taxol derivatives. The '790 reference discloses formation of taxol derivatives containing amino acid residues. The '184 patent discloses succinyltaxols. The increased solubility provided by these methods, however, tends to be short-lived .in vivo. The ester derivative solubilizing groups used to modify the molecules are quickly hydrolyzed, regenerating the poorly soluble, antigenic parent moieties.

Hypersensitivity reactions from taxol administration are known. See, for example, J. Clin Oncol 8:1263-1268 (1990) . Indeed, since taxol is extracted from a natural plant source, hypersensitivity reactions in a portion of the population are expected. Moreover, certain non- aqueous vehicles which have been suggested to deliver taxol in vivo have also been implicated in causing hypersensitivity reactions in mammals.

Finally, although taxol holds much promise as an anti-neoplastic agent, certain neoplasms have demonstrated resistance against it's activity. It would be highly desirable to provide novel anti-microtubule compositions with effectiveness against a wider range of

In view of the foregoing, it is an object of the present invention to address the shortcomings set forth above.

SUMMARY OF THE INVENTION

The present invention includes biologically active compositions having a taxol-like activity. The compositions have the structure:

(I)

wherein:

R _t = H or -CO-X, wherein X = alkyl, haloalkyl, substituted alkyl or aryl;

R ₂ = one of H, CH ₃ , alkyl, cycloalkyl, aryl, aralkyl, aryl(CH ₂ ) _n Y or (CH ₂ ) _n Y, wherein n = 1-12 and Y = OH, NH ₂ ,

CHO or COOR _t where R, = alkyl, substituted alkyl, phenyl or substituted phenyl, or an α-substituted polyalkylene oxide derivative; and

R ₃ = one of H, CH ₃ , alkyl, cycloalkyl, aryl, aralkyl or an en-substituted polyalkylene oxide derivative.

In those aspects of the invention where one or both of R ₂ and R ₃ is (are) α-substituted polyalkylene oxide derivatives, the derivatives are in a functionalized or

activated form. One preferred activated polymer is monomethoxy-polyethylene glycol or MPEG.

The present invention also includes methods of making the compositions described herein. The methods include reacting suitable taxol-based moieties with an activating reagent under conditions sufficient to form the biologically active composition. Some suitable reagents include: RjNCO where R ₂ is one of CH ₃ , alkyl, cycloalkyl, aryl, aralkyl, (CH ₂ ) _n Y, wherein n = 1-12 and Y = OH, NH ₂ , CHO or COOR, where R ₄ = alkyl, substituted alkyl, phenyl or substituted phenyl, or an α-substituted polyalkylene oxide derivative; and R ₂ R ₃ NC0C1 where R ₂ and R ₃ are independently one of

CH ₃ , alkyl, cycloalkyl, aryl, aralkyl or an α-substituted polyalkylene oxide derivative;

Methods of treatment using the compositions described herein are also disclosed.

DETAILED DESCRIPTION OF THE INVENTION

The compositions of the present invention are based on the premise that taxol and taxol-like molecules can be modified in the 7 position to provide improved variations of the naturally occurring alkaloids. The compositions are further described as having an anti-microtubule activity in vivo, especially as such action pertains to oncologic or anti-neoplastic activity as such activity is understood by those of ordinary skill in the art. For example, the compositions in some instances will demonstrate the ability to preferentially bind to and stabilize microtubules, thus interrupting cell mitosis.

While applicants are not bound by theory, it is believed that other anti-microtubule or oncolytic effects may also be observed in vivo with one or more of the compositions described herein.

For purposes of the present invention, the taxol component of the novel compositions can be selected from a wide variety of materials in addition to taxol per se harvested from naturally-occurring pacific yews and available from, for example, Calbiochem Corp. of San Diego, CA or ESCAgenetics Corp. of San Carlos, CA. For ease of description in the present invention, "taxol" will be understood to include all naturally occurring alkaloids as well as all synthetic and related moieties. A non-limiting list of suitable taxol-based moieties are described in Biochem. Biophys. Res. Comm. 124, 329 (1984); J. Med. Chem. 3_5, 145 (1992); J. of Nat'l Prod. 5_1, 298 (1988); J. Med. Chem. .32., 788 (1989) and S.B. Horowitz, et al Annals NY Acad. of Sci. 466, 733 (1986).

The artisan can also synthesize anti-micro tubule moieties such as taxotere based on need. Furthermore, 2' taxol esters can also be used. Since esters hydrolyze in the acidic environment of cancer cells, 2' taxol esters are useful as a prodrug. See J. Med. Chem. 3_2, 788 (1989) . While such pro-drug modifications are desirable in certain situations, it has been surprisingly found that the modifications described herein which are realized by conversion of the 7 OH to relatively stable carbamates provides novel compositions which are chemotherapeutically active. Moreover, 2 ' taxol esters can be modified in the 7 position if desired, to provide compositions which display both the prodrug and 1- carbamate features.

In addition to the preferred taxol- oieties described above, other moieties having anti-microtubule activity in mammals such as vinca alkaloids are modifiable with the polymers described herein. A review of anti-microtubule agents is set forth in Pharmac. Ther. 52, pp 35-84 (1991), the text of which is hereby incorporated by reference. The preferred taxol-based compositions of the present invention have the formula:

wherein:

Rj = H or -CO-X, wherein X = alkyl, haloalkyl, substituted alkyl or aryl; and R ₂ and R ₃ are independently one of H, CH ₃ , alkyl, cycloalkyl, aryl, aralkyl or an α- substituted polyalkylene oxide derivative.

In one aspect of the invention, one of R ₂ and R ₃ is an α-substituted polyalkylene oxide derivative. Alternatively, both R ₂ and R ₃ are α-substituted polyalkylene oxide derivatives. In each of these embodiments, the compositions have one or more of the following attributes:

1. improved solubility;

2. reduced antigenicity / immunogenicity;

3. prolonged circulating life; and

4. increased oncolytic activity against resistant cell lines when compared to the naturally occurring alkaloid or taxol-based derivatives not modified in accordance with this embodiment.

The attachment of the polymeric materials to taxol is preferably via a covalent linkage, and most preferably via a carbamate (urethane) linkage. Such linkages are preferred due to their stability, especially in aqueous based systems. It is to be understood, however, that linkage can be achieved via any suitable linking group containing at least one atom capable of joining the taxol moiety covalently to the polymeric material while substantially maintaining the activity of the taxol- containing substance. Alternative linking groups also include ethers. While it is preferred that at least one of R ₂ , and R ₃ is covalently attached to the taxol moiety, one or both may also be attached using reversible and/or ionic or non-covalent chemistries.

Oftentimes, the polymers are activated in order to effect such linkages. By activation, it is understood by those of ordinary skill in the art that the polymer is functionalized to include the desired reactive group. See, for example, U.S. Patent Nos. 4,179,337 and 5,122,614, wherein the hydroxyl end-groups of polyalkylene glycols, are converted and activated into reactive functional groups to modify proteins and/or enzymes. The disclosure of each of the '337 and '614 patents is hereby incorporated by reference. These references, however, are directed to modifying enzymes and/or proteins via epsilon amino acid lysines. The differences in structure, function and effect between

these materials and taxol-based moieties are so substantial that the references are of little predictive value for the purposes of the present invention.

Alternative activated polymers include isocyanates as set forth in the parent U.S. patent application Serial Number 07/934,131, filed August 21, 1992 or the hydrazines set forth in commonly assigned PCT patent application bearing Publication No. W092/16555.

Polyethylene glycols are particularly preferred polymeric materials. Although polyethylene glycols come in a variety of molecular weights, molecular weight ranges of from about 200 to about 10,000, are usually selected for the purposes of the present invention.

Molecular weights of from about 2,000 to about 7,500 are preferred while molecular weights of about 5,000 are particularly preferred. Within this aspect of the invention, it is to be understood that alkyl-capped polyethylene oxides such as methoxypolyethylene glycols (MPEG) and bis-polyethylene oxides are also contemplated.

The polymeric substances included herein are also preferably water-soluble at room temperature. A non- limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers maintained.

As an alternative to PAO based polymers, effectively non-antigenic materials such as dextran, polyvinyl pyrrolidone, polyacrylamides polyvinyl alcohols,

carbohydrate-based polymers and the like. Those of ordinary skill and the art will realize that the foregoing list is merely illustrative and that all such polymeric materials having the qualities described herein are contemplated.

The polymeric materials are also effectively non- antigenic in mammals. For purposes of the present invention, "effectively non-antigenic" means all polymeric materials understood in the art as being nontoxic and not eliciting an appreciable immunogenic response in mammals.

In this aspect of the invention, the taxol-polymer conjugates retain at least a substantial portion of the anti-microtubule activity of the taxol moiety prior to conjugation. In most cases, however, the modified compositions demonstrate equipotent and even synergistic activity against certain neoplastic cells. See Examples below. For purposes of the present invention, the terms

"substantial portion of the activity" means that at least therapeutic effectiveness is maintained. While the conjugate may have less activity than the unmodified taxol-moiety in certain situations, the therapeutic effectiveness is nonetheless maintained. In any event, the advantageous properties of high aqueous solubility, substantially longer circulating life, and reduced antigenicity, either alone or in combination outweigh any decrease in activity in those situations where decreased but nonetheless therapeutically effective compositions result.

In those aspects of the invention where Rj and/or R ₂ are not α-substituted polyalkylene oxide derivatives,

that is, Rj and/or R ₂ is hydrogen, an alkyl, cycloalkyl, aryl, aralkyl, other 7 carbamate taxols are formed. While these compositions have utility in their own right, they can be further functionalized to include other moieties which would enhance the anti-microtubule composition. For example, the artisan can enhance aqueous solubility by adding moieties such as carboxylic, sulfonic, phosphonic acids or amines or other salt formers. Furthermore, when R ₂ = aryl(CH ₂ ) _n Y or (CH ₂ ) _n Y, wherein n = 1-12 and Y = OH, NH ₂ , CHO or COOR ₄ where R ₄ = alkyl, substituted alkyl, phenyl or substituted phenyl, the artisan is provided with an intermediary composition which can be further modified selectively in the 7 position. Those skilled in the art will recognize that when Y = OH, NH ₂ or CHO, these functional groups are added initially in a protected form such as a tetrahydropyranyl ether of the alcohol, carbobenzyloxy for the amine and dialkylacetal for the aldehydes. In this embodiment, an α-substituted polyalkylene oxide derivative can attached via an alkyl spacer to produce an active, highly water soluble anti-microtubule or cytotoxic composition.

In still another aspect of the invention, the compositions are further modified to include a targeting moiety that enhances accumulation of the taxol-based moiety in a desired location such as a tumor. In this regard, suitable targeting moieties include peptide sequences, mono- or polyclonal antibodies, single chain antigens (sFv's) , fusion proteins or the like. For example, a DNA binding peptide can be attached to the compositions via a properly functionalized R ₂ and/or R ₃ under conditions known to those of ordinary skill in the art.

The taxol-based compositions are prepared by reacting a taxol-based moiety depicted below as (II) or one of those described above with an activating reagent out under conditions which are sufficient to effect the desired 7 position modification yet maintain at least a portion of the therapeutic effect of the moiety.

Ri = alkyl, haloalkyl, substituted alkyl or aryl. Usually, in order to effect such 7 position modification, the 2' position must be blocked. The temporary formation of an acetate with the 2' OH has also been demonstrated to suffice. Blockage may be carried out in the manner described in Biochem. Biophys. Res. Comm 124 , 329 (1984) or J. Med. Chem. 35_, 145 (1992).

The activating reagents useful in the formation of the 7 carbamates are generally described as including:

1) R ₂ NCO where R ₂ is one of CH ₃ , alkyl, cycloalkyl, aryl, aralkyl, aryl(CH ₂ ) _n Y or (CH ₂ ) _n Y, wherein n = 1-12 and Y = OH, NH ₂ , CHO or COOR, where R, = alkyl, substituted alkyl, phenyl or substituted phenyl, or an α-substituted polyalkylene oxide derivative;

2) R ₂ NH ₂ where R ₂ is one of H, CH ₃ , alkyl, cycloalkyl, aryl, aralkyl or an α-substituted polyalkylene oxide derivative;

3) R ₂ R ₃ NH where R ₂ and R ₃ are independently one of CH ₃ , alkyl, cycloalkyl, aryl, aralkyl or an α-substituted polyalkylene oxide derivative; or

4) R ₂ R ₃ NCOCl where R ₂ and R ₃ are independently one of CH ₃ , alkyl, cycloalkyl, aryl, aralkyl or an α-substituted polyalkylene oxide derivative.

The conditions under which the anti-microtubule moiety and the activating reagent are reacted include: the use of nonhydroxylic solvents such as anhydrous toluene, tetrahydrofuran, 1,2-dichloroethane, CHC1 ₃ ,

CH ₂ C1 ₂ or mixtures thereof; carrying out the reaction at temperatures of from about 10-50 and preferably 25-35 degrees C; allowing the reaction to proceed for times of up to two or more days. Several reaction schemes are set forth below.

13

Another aspect of the present invention provides methods of treatment for various medical conditions in mammals. The methods include administering an effective amount of a taxol-based conjugate as described herein to the mammal. The compositions are useful for, among other things, treating neoplastic disease, reducing tumor burden, preventing metastasis of neoplasms and preventing recurrences of tumor / neoplastic growths.

The amount of taxol-based conjugate used in the methods described herein may generally be described as that amount which effectively achieves the desired therapeutic result in mammals. Naturally, the dosages of the various taxol conjugates will vary somewhat depending upon the taxol-based moiety and the non-antigenic polymer selected for conjugation. In general, however, the conjugate may be administered in amounts ranging from about 5 to about 500 mg/m ² per day, based the amount of the active taxol moiety in the conjugate. The range set forth above is illustrative and those skilled in the art will determine the optimal dosing of the conjugate selected based on clinical experience and the treatment indication.

The conjugates of the present invention can be included in one or more suitable pharmaceutical compositions for administration to mammals. The pharmaceutical compositions may be in the form of a solution, suspension, tablet, capsule or the like, prepared according to methods well known in the art. It is also contemplated that administration of such compositions may be by the oral and/or parenteral routes depending upon the needs of the artisan.

EXAMPLES

The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention.

EXAMPLE 1

PREPARATION of 2' ACETYL 7-PEG 5000 CARBAMATE TAXOL USING mPEG-NCO SCHEME 1

In this Example, mPEG-NCO was prepared in situ by placing 515 mg (0.010 m ol) of mPEG-NH ₂ in a 100 ml round bottom flask and undergoing drying by azeotropic toluene distillation followed by conversion to mPEG-NCO as described in the parent application. The FTIR showed the isocyanate peak at 2263 cm ^"1 . The reaction mixture was cooled to room temperature before adding 60 mg of 2 ' - acetyltaxol prepared as described in Biochem. Biophys. R.S. Commun. 124, 329-336 (1984) and 10 mg Sn(II) octoate. The reaction was followed by HPLC on a C ₈ reverse phase column with 3:1 methanol-H ₂ 0 as eluent. The reaction appeared to be complete when about 75% of 2'-acetyltaxol was converted to the corresponding PEG- derivative. The reaction product was evaporated to near dryness and precipitated with ether. Most of the unreacted 2'-acetyltaxol and any 2', 7-diacetyltaxol present remained in the ether phase. The ether was decanted, and the precipitate was recrystallized from 20 ml of 2-propanol. The 7-carbamate-PEG derivative was isolated by centrifugation, washing with two 20 milliliter portions of 2-propanol, and finally drying under high vacuum to obtain 508 mg of product with less than 1% 2'-acetyltaxol and some non-functionalized PEG.

The FTIR spectrum of the purified compound had all the characteristic peaks of PEG in addition to peaks at 1748.6, 1741.2, 1726.5, 1663 cm ^"1 which are characteristic of the 2'-acetyltaxol molecule.

EXAMPLE 2 PREPARATION OF 7-GLYCINE CARBAMATE ALTERNATE SCHEME 1 In this Example, 58 mg of 2'-acetyltaxol (0.067 mmol) was placed in 25 ml round-bottomed flask. Chloroform (ca 5.4 ml) was distilled directly over phosphorus pentoxide into this flask. Next, 138 mg of ethyl isocyanatoacetate (1.07 mmol) and dibutyltin dilaurate (300 mg) were added to the flask and the reaction mixture was heated to 40°C. The reaction was followed by HPLC on a C ₈ reverse phase column with 3:1 methanol-water as the eluent. After 16 hours, the 2'- acetyltaxol peak disappeared completely and a new peak corresponding to the product appeared.

The mixture was concentrated to near dryness, precipitated with 25 ml of hexane, and then centrifuged. The supernatant contained only a very small amount of the desired product. The precipitate was dried under high vacuum and further purified by HPLC on a preparative C ₈ column to yield 50 mg of product which was characterized by NMR. The product was subjected to hydrolysis by NaHC0 ₃ (20 mg) in 3:1 methanol-water (8 ml). After 30 minutes at room temperature, a new peak of lower retention time began to appear in the HPLC trace which corresponded to the hydrolysis of 2'-acetyl group (II) . After 2 hours of further hydrolysis, a third peak appeared corresponding to the removal of the ethyl group of the side chain at 7 position (III) • The reaction was

stopped when the ratio of II to III was about 40:60 because longer reaction times led to undesired hydrolysis of the taxane skeleton.

The product was isolated by extraction into methylene chloride. The mixture of II and III was separated from any impurities by HPLC.

EXAMPLE 3 PEGYLATION OF 2'-ACETYL TAXOL USING MPEGNH,

SCHEME 2 2'-acetyltaxol (78mg, 89 μmol) was dissolved in 2 ml of dry 2-dichloroethane, and to this solution were added 13.2 mg of triphosgene (44.5 mmol) and 7.6 mg of pyridine (96.8 xmol) 7.6 mg. The reaction was followed by HPLC using the disappearance of 2'-acetyltaxol and appearance of a new peak with higher retention time. More triphosgene and pyridine were added until this conversion to chloroformate was greater than 80%. At this time one equivalent each of mPEGNH ₂ and pyridine were added to complete the reaction. The resulting composition was found to have an HPLC peak which corresponded to the peak obtained for 2' -acetyl-7-PEG urethane taxol prepared using mPEG-NCO described in Example 4 of the parent application herein. Confirmation of the compound was achieved by "spiking" with pure 2'-acetyl-7-PEG-(5000)- Carbamate taxol. The relative intensity of the HPLC peak increased, confirming that the product obtained by the two methods were the same.

EXAMPLE 4

SCHEME 3 In this Example, the procedure of Example 3 was repeated except that N-methyl-PEG-amine was employed instead of the mPEGNH ₂ to yield the N-methyl-carbamate derivative of taxol.

EXAMPLE 5

Functionalization of 2'-Acetyltaxol with N-Methyl PEGamine. SCHEME 4

In this example, 100 ml of chloroform was added to 1 g (0.20 mmol) of mPEGNCH ₃ .HCl. The solution was dried by azeotropic distillation using molecular sieves placed in the side-arm of the Dean-Stark trap. After removal of 100 ml of chloroform, the reaction was cooled to room temperature and triphosgene (22 mg, 0.074 mmol), and triethylamine (42 mg, 0.42 mmol) were added, and the mixture was refluxed for 16 hours. An aliquot was withdrawn and checked by IR after removal of the solvent. The IR spectrum showed a strong peak at 1702 cm ^-1 indicating the formation of the carbamyl chloride. Next, 25 mg of 2'-acetyltaxol(0.029 mmol) followed by 0.02 ml of triethylamine was added to the flask and refluxed. After 16 hours, 6% conversion occurred. Additional chlorocarbamate (1 g) prepared separately as described above was added and reflux continued for 40 hours. A 10% yield of the desired product was obtained. The retention time of this peak on the C ₈ column was similar to that of 2'-acetyltaxol PEGylated using MPEGNCO indicating that the desired conjugation had occurred.

EXAMPLE 6 In this Example, the activity of several taxol compositions against various malignant human cell lines was compared. Compositions A, B and C were dissolved in DMSO prior to dilution for cell testing. The inhibitory concentrations (IC ₅₀ ) were determined using standard procedures and after 72 hours, the results were reported. All cell lines are obtained from the ATCC.

A549 = HUMAN LUNG CARCINOMA

BT20 = HUMAN BREAST CARCINOMA C4i = HUMAN CERVICAL CARCINOMA A375 = HUMAN MALIGNANT MELANOMA

IC _;ft MICROMOLAR QUANTITIES OF TAXOL DERIVATIVES

BT20 C4l A375

CARBAMATE ^* TAXOL

E. 2'-ACETYL-7-PEG(500 CARBAMATE TAXOL

F. 2'-OH-7-PEG(5000) CARBAMATE TAXOL

* as a 60:40 mixture of ester to acid

It can be seen from the above table, the 7- carbamates showed significant activity against these cell lines. Even though samples D, E and F were less active than the non-carbamate taxols, these compositions nonetheless have significant utility as therapeutic compounds for a variety of neoplastic diseases.

EXAMPLE 7

In this Example, the same taxol derivatives A-F described above in Example 6 were evaluated in two more malignant cell lines. Cell line P388/0 is a doxorubicin- sensitive mouse lymphoid neoplasm; P388/ADR is a doxorubicin-resistant mouse lymphoid neoplasm. Both were obtained from the Southern Research Institute, Birmingham, AL. The results are set forth below in Table II.

TABLE II

COMPARATIVE ACTIVITY OF PEG-TAXOL

IC- MICROMOLAR QUANTITIES OF TAXOL DERIVATIVES

* as a 60:40 mixture of ester to acid

Referring now to Table II, it can be seen that the compositions maintained activity against these cell lines in spite of the substantial 7 position modifications. What is especially noteworthy is that Sample E was essentially as active against the resistant strain as it was against the non-resistant line. The 2'-OH derivative also showed substantial retained activity against this resistant cell line. While applicants are not bound by

theory, it is believed that the PEG modification may overcome certain tumor cell resistance in a mode of action separate from the composition's prolonged in vivo disappearance time.

EXAMPLE 8 In this Example, the water solubility of the taxol carbamates prepared in accordance with the present invention is demonstrated. Each of the derivatives tested demonstrated superior water solubility when compared to unmodified molecule. Indeed, the 7- carbamate-PEG taxol was over 600 times more soluble in water than the unmodified material.

TABLE III

WATER SOLUBILITY COMPARISON

COMPOSITION AQUEOUS SOLUBILITY Taxol 0.5 mg/ml

7-PEG(5000)Carbamate Taxol > 300.0 mg/ml

7-Glycine-Carbamate Taxol ~1.0mg/ml

While there have been described what are presently believed to be the preferred embodiments of the invention, those skilled in the art will realize that changes in modification may be made there to without departing from the spirit of the invention. It is intended to claim all such changes and modifications as all within the true scope of the invention.