WO2000078359A2 - Compositions for treating chemotherapy-resistant tumor cells - Google Patents

Abstract

The present invention provides methods and compositions for treating cancer and chemotherapy-resistant cancers comprising a chemotherapeutic agent conjugated to or coadministered with a peptide.

Description

COMPOSITIONS FOR TREATING CHEMOTHERAPY-RESISTANT TUMOR CELLS AND TARGETED CHEMOTHERAPY COMPOSITIONS

TECHNICAL FIELD

Compositions and methods for treating doxorubicin, paclitaxel, or

multidrug-resistant tumor cells are provided. The compositions include a

doxorubicin-peptide conjugate as well as peptide coadministered with

doxorubicin. The compositions of this invention also include a paclitaxel-

peptide conjugate as well as peptide coadministered with paclitaxel. The

conjugate compositions, as well as the coadministration therapy, will be

useful in treating both patients with doxorubicin, paclitaxel, or multidrug-

resistant cancer and those with normal cancer. The compositions of the

invention are also more effective, and require smaller doses, than the

chemotherapy agent alone because the peptide serves to target the

chemotherapy agent to the tumor cells.

BACKGROUND OF THE INVENTION

Doxorubicin is the most commonly used anticancer chemotherapeutic

agent, and it has the widest spectrum of antitumor effects. Nagy et al.,

Cytotoxic analogs of luteinizing hormone-releasing hormone containing

doxorubicin, PNAS 93:7269-7273 (1996). It is an anthracycline derived from

Streptomyces peucetius var. ccesius. Stan et al., Antineoplastic Efficacy of

Doxorubicin Enzymatically Assembled on Galactose Residues of a

Monoclonal Antibody Specific for the Carinoembryonic Antigen, Cancer Res.

59:115-121 (1998). It is has two regions, named adriamycinone and daunosamine. The structure of doxorubicin is shown in Figure 1.

Doxorubicin ("Dox") intercalates itself into double-stranded nucleic acids,

inhibiting DNA and RNA synthesis, and affects the stability of DNA-

topoisomerase II complexes. In order to be effective, Dox must accumulate in

the cell and reach certain threshold levels.

Paclitaxel is a common chemotherapeutic agent often used to treat

breast cancer. It is distributed by Bristol-Myers Squibb under the tradename

TAXOL™. It is a natural product with antitumor activity. It is obtained via a

semi-synthetic process from Taxus baccata (the Pacific Yew Tree). The

chemical name for paclitaxel is 5β,20-Epoxy-1 ,2α,4,7β,13α-hexahydroxytax-

11-en-9-one 4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzyol-3-

phenylisoserine.

The structure of paclitaxel is shown in Figure 2.

Paclitaxel acts as an antimicrotubule agent by promoting the assembly

of microtubules from tubulin dimers and stabilizing microtubules by preventing

depolymerization. This stability results in the inhibition of the normal dynamic

reorganization of the microtubule network that is essential for vital interphase

and mitotic cellular functions. In addition, paclitaxel induces abnormal arrays

or "bundles" of microtubules throughout the cell cycle and multiple arrays of

microtubules during mitosis.

The most significant problem oncologists face in the treatment of

cancer, is the existence of drug resistance in tumors resulting in decreased cytotoxicity of chemotherapy agents. Some cancers are drug resistant prior

to treatment, whereas others develop drug resistance during treatment.

In many instances, when a tumor develops drug resistance in

response to treatment with one agent, such as Dox or paclitaxel, cross-

resistance develops to structurally and functionally unrelated drugs such as

vinblastine and cisplatin. Choudhuri et al., Reversal of resistance against

doxorubicin by a newly developed compound, oxalyl bis(N-phenyl)hydroxamic

acid in vitro, Anti-Cancer Drugs 9:825-832 (1998). This pattern of resistance

is named multidrug resistance (MDR). Researchers have identified a gene

MDR1 , and its gene product, p-glycoprotein, in MDR tumors. P-glycoprotein

functions as an efflux pump, preventing accumulation of drugs and hence

reducing cytotoxicity. This mechanism is responsible for one type of

doxorubicin resistance in cancer cells. Rahman, Modulation of Multidrug

Resistance in Cancer Cells by Liposome Encapsulated Doxorubicin, J.

Liposome Res. 4:575-604 (1994).

Paclitaxel is a substrate for the multidrug resistant pump, which is also

termed gP170, and cells selected for high levels of resistance to this drug

have increased levels of gP170. Gonzalez-Garay, A β-Tubulin Leucine

Cluster Involved in Microtubule Assembly and Paclitaxel Resistance, J. Biol.

Chem. 274:23875-23882 (1999). Other paclitaxel resistance mechanisms

have also been proposed including changes in the expression of specific β-

tubulin genes and mutations in β-tubulin. Id. Other mechanisms for chemotherapy resistance include: glutathione

tranferances and detoxification mechanisms; topoisogenetic recombination,

DNA transcription, chromosome segregation; and DNA repair. Harris et al.,

Mechanisms of Multidrug Resistance in Cancer Treatment, Acta Oncological

31:205-213 (1992).

Researchers have experimented with numerous different strategies for

overcoming doxorubicin resistance. One such strategy involves the use of

nonionic amphipathic diesters of fatty acids or a reverse poloxmer to treat

MDR cancer. U.S. Pat. No. 5,681,812. Oligonucleotides specifically

hybridizable with nucleic acids encoding MDR associated proteins have also

been developed. U.S. Pat. No. 5,807,838. An hydroxamic acid derivative,

oxalyl bis(N-phenyl)hydroxamic acid, has also shown promising results in

reversing MDR. Choudhuh et al, Reversal of Resistance Against

Doxorubicin by a Newly Developed Compound, Oxalyl Bis(N-

phenyl) hydroxamic Acid in Vitro, Anti-Cancer Drugs, 9:825-832 (1998).

Another strategy proposes the use of liposome encapulated doxorubicin to

overcome doxorubicin resistance. Rahman, Modulation of Multidrug

Resistance in Cancer Cells by Liposome Encapsulated Doxorubicin, J.

Liposome Res. 4:575-604 (1994).

Doxorubicin conjugates have also been developed. Doxorubicin

conjugated to a gallium-transferrin compound has been shown to reverse

drug resistance in breast cancer cell lines. Yang et al., Reversal of

doxorubicin resistance by doxorubicin-gallium-transferrin conjugate in human breast cancer cell lines, Proc. Am. Assn. Cancer Res. 40:4377 (1999).

Analogs to luteinizing hormone-releasing hormone ("LH-RH") have also been

conjugated to doxorubicin, resulting in a more potent, targeted anticancer

agent for tumors that possess receptors for LH-RH. Nagy et al., Cytotoxic

Analogs of Luteinizing Hormone-Releasing Hormone Containing Doxorubicin

or 2-Pyrrolinodoxorubicin, a Derivative 500-1000 Times More Potent, PNAS

93:7269-7273 (1996). Monoclonal antibodies have also been conjugated to

doxorubicin, resulting in more potent treatment compositions. Stan et al.,

Antmeoplastic Efficacy of Doxorubicin Enzymatically Assembled on Galactose

Residues of a Monoclonal Antibody Specific for the Carcinoembryonic

Antigen, Cancer Res. 59:115-121 (1999).

While doxorubicin conjugates have been considered previously, the art

has taught away from derivatization of the amino group of the doxorubicin on

the adriamycinone moiety. Neutralization of the dausonamine nitrogen of

doxorubicin has previously resulted in severe loss of cytotoxic activity. See

Nagy, Cytotoxic Analogs of Luteinizing Hormone-Releasing Hormone

containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times

more potent, PNAS 93:7269-7273 (1996); Zunino et al., Interaction of

Daunomycin and its Derivatives with DNA, Biochim. Biophys. Acta 227:489-

498 (1972). Such a modification was thought to inactivate the doxorubicin.

Instead, those in the art have followed a strategy more difficult to accomplish

involving modification of the CH₂OH group in the adriamycinone portion of

doxorubicin. A previous paclitaxel-peptide conjugate has been constructed with a

bombesin/gastrin-releasing peptide receptor-recognizing peptide: Gln-Trp-

Ala-Val-Gly-His-Leu (SEQ ID NO: 8). Safavy, Paclitaxel Derivatives for

Targeted Therapy of Cancer: Toward the Development of Smart Taxanes, J.

Med. Chem. 42:4919-4924 (1999).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for treating

a patient suffering from cancer, wherein a cancer chemotherapy agent is

selected, conjugated to a peptide, and administered to the patient.

It is a further object of the invention to provide a method of treating a

patient suffering from cancer, wherein a cancer chemotherapy agent is

selected, a peptide is selected, and the agent and peptide are coadministered

to the patient.

It is a further object of the invention to provide a composition for

treating a patient suffering from cancer, wherein the composition comprises a

chemotherapy agent and a peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (Doxorubicin) is a drawing of doxorubicin.

FIG. 2 (Paclitaxel) is a drawing of paclitaxel.

FIG. 3 (Doxorubicin - Peptide Conjugate) is a drawing of a doxorubicin-

peptide conjugate ("Dox-P") of the present invention, where the doxorubicin is

linked through its amino terminus to the carboxy terminus of the peptide

Cys(Acm)-Ser-Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6). The linkage is an amide linkage. The peptide's amino terminus is modified with an Acm group

for protection during the linkage with the doxorubicin, and the Acm group is

left on the conjugate.

FIG. 4 (Paclitaxel - Peptide Conjugate) is a drawing of a paclitaxel-

peptide conjugate (Paclitaxel-P) of the present invention, where the paclitaxel

is linked through a succinyl linker to the amino terminus of the peptide

Cys(Acm)-Ser-Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6). The linkage is an

ester-amide linkage, with the ester linkage being between paclitaxel and the

succinyl linker, and the amide linkage being between the succinyl linker and

the amino terminus of the peptide.

FIG. 5 (Effect of Dox and Dox-P on Drug-Resistant CHO Cell Viability)

is a dose response curve showing the activity of the Dox-P on doxorubicin

and multidrug-resistant CHO cells. As a control, doxorubicin alone ("Dox") is

applied to the cells.

FIG. 6 (Effect of Dox and Dox-P on CHO Cell Viability) demonstrates

that Dox-P appears to function more effectively against wild-type CHO cells

than Dox alone.

FIG. 7 (Effect of Doxorubicin Peptide Conjugate on Adhesion of B16-

F10 Melanoma Cells) show the effect of Dox-P on adhesion of B16-F10

melanoma cells (a non-resistant cell line). This shows that the conjugation of

the peptide with the doxorubicin does not destroy its adhesive activity. FIG. 8 (Effect of Doxorubicin (Dox), Doxorubicin-Peptide (Dox-P) on

Melanoma Tumor Development) shows that Dox, Dox-P, and peptide alone

all inhibit the development of lung metastasis of injected melanoma cells.

FIG. 9 (Effect of Ala-Ser-Val-Thr-Ala-Arg on Doxorubicin Toxicity of

Doxorubicin Resistant CHO Cells) shows the effect of coadministering the

peptide Ala-Ser-Val-Thr-Ala-Arg (SEQ ID NO: 2) with doxorubicin against

doxorubicin and multidrug-resistant cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to chemotherapeutic agents conjugated

to or coadministered with peptides. Chemotherapeutic agent-peptide

conjugates can be produced by linking the chemotherapeutic agent to

peptides using various methods of chemical synthesis. Chemotherapeutic

agents can also be coadministered with peptides of the present invention.

These two strategies can be used independently or they can be combined by

coadministering a chemotherapeutic agent-peptide conjugate with an

additional peptide of the present invention.

Various chemotherapeutic agents can be used in the present

invention. Those agents that tend to lose effectiveness due to resistant tumor

cells are preferable. Additionally, preferred compounds include those that

would benefit from targeted administration due to toxicity, for example.

Acceptable agents including the following:

• alkylating agents (bisulfan, carboplatin, cisplatin, thiotepa); • nitrogen mustards (melphalan, cyclophospamide, chlorambucil,

ifosfamide, mechlorethamine);

• nitrosoureas (carmustine, streptozocin);

• antibiotics (doxorubicin, bleomycin, danuorubicin, actinomycin D,

idarubicin, fludarabine, floxuradine, 5-flurouracil, antracylcine, plicamycin,

mitomycin C, mitoxantrone, cytarabine, cladribine);

• vinca alkyloids (vincristine, vinblastine);

• hormonal agonists and antagonists (androgens including: nilutamide

and testolactone; antiandrogens including: bicalutamide and flutamide;

antiestrogens including: anastrozole, toremitine, letrozole, and tamoxifen;

estrogens including: estradiol; gonadotropin releasing hormone analogs

including: leuprolide acetate and goserelin acetate; and progestins including:

medroxyprogesterone and megestrol); and • other agents (including

paclitaxel, camphothecan, topotecan, vincristine, vinblastine, colchicine,

methotrexate, mercaptopurine, irinotecan, B-methasone, dicarbazine,

aspartgenace, etoposide, germcitabine, altretamine, hydroxyurea, mitotane,

vinurelbine, L-asparginase, paclitaxel, docetaxal, tretinoin, temiposide, ricin,

cytoxin, saintopin, ellipticin, azatoxin, SQZ, dinalin, and VP16).

This invention is most useful when the chemotherapy agent is highly

toxic, as the peptide conjugation and/or coadministration strategy can both

reduce the toxicity of the agent and allow less composition to be administered

due to increased efficacy. Thus the invention is useful for treating resistant

cancers, but it is particularly beneficial to use them to treat nonresistant cancers. Moreover, by targeting the chemotherapy agents to the tumor cells

this invention allows for more effective treatment at lower doses. One

preferred composition of the present invention contains doxorubicin. Another

preferred composition contains paclitaxel.

Peptides of the present invention include peptide sequences from

thrombospondin that bind to the thrombospondin receptor. These peptides

can be selected from the amino acids at or near the receptor binding

sequence of thrombospondin, Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1), or

from other known peptides with thrombospondin activity. See, U.S. Patent

Appln. Serial Nos. : 08/476, 134 and 09/197, 770; and U. S. Patent Nos. :

5, 190,918; 5, 155,038; 5, 190,920; 5,200,397; 5,367,059; 5,506,208;

5,648,461; 5,426, 100; 5,654,277; 5,840,692; 5,357,041; and 5, 770,563.

Specifically, these include peptides binding to the TSP-1 receptor with an

affinity from about 10^"6 M to about 10^"10 M, preferably from about 10^"7 M to

about 10⁹ M, most preferably about 10^"8 M.

Peptides can also be identified by their capacity to bind to the

thrombospondin receptor. Such peptides can be developed and identified by

using a phage display peptide library kit, such as that available from New

England Biolabs (Beverly, MA). Phage display describes a selection

technique in which a peptide or protein is expressed as a fusion with a coat

protein of a bacteriophage, resulting in display of the fused protein on the

exterior surface of the phage virion, while the DNA encoding the fusion

resides within the virion. Phage display can be used to create a physical linkage between a vast library of random peptide sequences to the DNA

encoding each sequence, allowing rapid identification of peptide ligands for a

variety of target molecules (including receptors) by an in vitro selection

process called biopanning. This technique is carried out by incubating a

library of phage-displayed peptides with a plate (or bead) coated with the

target receptor, washing away the unbound phage, and eluting the

specifically-bound phage. The eluted phage is then amplified and taken

through additional cycles of biopanning and amplification to successively

enrich the pool of phage in favor of the tightest binding sequences. After 3-4

rounds, individual clones are characterized by DNA sequencing and ELISA.

Appropriate peptides can be made based on the oligonucleotide sequence

identified.

Additionally, random peptides can be screened for their ability to bind

to the thrombospondin receptor or for their ability to promote the activity of

doxorubicin paclitaxel, or other chemotherapeutic agents when conjugated

to or coadministered with them. They can be screened by the Affinity Sensor

System or the Adhesion Studies as discussed in the Examples below. Other

methods of screening for peptides that bind to the receptor would be readily

known to one of skill in the art.

The peptides can be from 3 to 100 amino acids in length, preferably 3

to 50, 3 to 20, or 4 to 11 amino acids in length, most preferably 4, 5, 6, 7, 9,

or 11 amino acids in length. The longer peptides may be useful as they could

include the heparin binding domains of the thrombospondin protein, which flank the Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1) region. Preferred peptides

include Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1), Cys(Acm)-Ser-Val-Thr-

Cys(Acm)-Gly (SEQ ID NO: 6), Ala-Ser-Val-Thr-Ala-Arg (SEQ ID NO: 2), Cys-

Ser-Val-Thr-Cys-Arg (SEQ ID NO: 4), and Ser-Val-Thr-Cys-Gly (SEQ ID NO:

5), and Val-Thr-Cys-Gly (SEQ ID NO: 7). Most preferable peptides include

Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1), Cys(Acm)-Ser-Val-Thr-Cys(Acm)-

Gly (SEQ ID NO: 6), and Ala-Ser-Val-Thr-Ala-Arg (SEQ ID NO: 2).

The Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1) and the Cys(Acm)-Ser-

Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6) are expected to behave similarly in the

present invention. The first peptide is subject to oxidation at the sulfur atoms

on the two cystine residues, and can become cyclized or linked to other

molecules of the peptide in a polymer fashion. The modified peptide remains

in the linear conformation. Other blocking strategies can be used to prevent

the peptide from oxidizing and forming disulfide bonds, as the oxidized

peptide may be less stable. These blocking strategies include alkylation with

a methyl or other alkyl group and acetylation.

Peptides of the present invention include those with unnatural or non-

amino acids. These peptides, which would be made by chemical synthesis,

include those with modified amino acids or other moieties in place of an

amino acid. Such other moieties could include fluorine, chlorine, organic

compounds such as alcohols, organic ring structures, and hydroxyacids.

Amino acids or peptides in the d-orientation can also be used, as can

peptides in the reverse orientation. One could also mimic the peptide with organic molecules having a similar structure and conformation. The inclusion

of unnatural or non-amino acids could be made to stabilize the peptide, block

metabolization (in the case of a fluorine), or to create a conformational

change in the peptide which would increase its binding affinity to the TSP-1

receptor.

One of the compositions of the present invention was produced by

linking the doxorubicin through its amino terminus to the carboxy terminus of

the peptide Cys(Acm)-Ser-Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6) through an

amide linkage. The peptide's amino terminus was modified with an acetyl

group for protection during the linkage with the doxorubicin, and the acetyl

group was left on the conjugate.

Another composition of the invention was produced by linking the

paclitaxel to the carboxy terminus of the peptide Cys(Acm)-Ser-Val-Thr-

Cys(Acm)-Gly (SEQ ID NO: 6) through a succinyl linker.

Other methods of conjugation would be readily apparent to one skilled

in the art. First, conjugation could occur though the primary hydroxyl of the

adriamycinone through an ester linkage with the carboxylic acid end of the

amino carboxylic acid. The amino group at the other terminus of the amino

acid linker can then be used to form an amide with the carboxylic acid of the

terminal glycine in the peptide.

Second, the amino group of the terminal cysteine of the

thrombospondin can be used to form an amide with one end of a dicarboxylic

acid while the other end of the dicarboxylic acid can be used with the amino group of the daunosamine fragment of the doxorubicin, thus forming a

bisamide. Alternatively, the same dicarboxylic acid might be used with the

amino group of the cysteine and the primary hydroxyl of the adriamycinone

aglycone of doxorubicin, thus forming an amidoester.

Another composition of the present invention was produced by linking

the paclitaxel through a succinyl linker to the amino terminus of the peptide

Cys(Acm)-Ser-Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6) through an ester-amide

linkage. The ester linkage is between the paclitaxel and the succinyl linker,

and the amide linkage is between the succinyl linker and the amino terminus

of the peptide.

The compositions of the present invention may be formulated in a

pharmaceutical composition, which may include carriers, thickeners, diluents,

buffers, preservatives, surface active agents, liposomes, or lipid formulations,

and the like. The pharmaceutical compositions may also include one or more

additional active ingredients such as other chemotherapy agents,

antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of

ways depending on whether local or systemic treatment is desired, and on the

area to be treated. Administration may be topically (including on the skin,

ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or

parenterally, for example by intravenous drip, subcutaneous, intratumor,

intraperitoneal, or intramuscular injection. With formulations for topical administration may include ointments,

lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

Conventional pharmaceutical carriers, aqueous, powder or oily bases,

thickeners, and the like may be necessary or desirable. Compositions for oral

administration include powders or granules, suspensions or solutions in water

or nonaqueous media, capsules, or tablets. Thickeners, flavorings, diluents,

emulsifiers, dispersing aids or binders may be desirable. Formulations for

parenteral administration may include sterile aqueous solutions optionally

containing buffers, liposomes, diluents and other suitable additives.

Dosing is dependent on the severity and responsiveness of the

condition to be treated, with course of treatment lasting from several days to

several months or until a cure is effected or a diminution of disease state is

achieved.

Optimal dosing schedules and dosing amounts can be calculated

based on the chemotherapy agent alone. The conjugated compound or the

coadministered compound can then be compared to the chemotherapy agent

alone, and the dosages can be adjusted accordingly. For instance, optimal

dosages are generally 10x below the lethal dose. The LD₅₀ (the dose that

kills 50% of the test animals) can be determined for the chemotherapy agent

alone as well as for the composition of the present invention. These

calculations should preferably be performed in two animal models. The

composition's half life can also be determined in one or more animal models,

and can be used to determine a dosing schedule. The differences between the LD₅₀ and the half life for the chemotherapy agent alone and the

composition of the present invention can be used to adjust the dosages.

After patients are treated, the dosages can be reduced in amount or

frequency if a patient exhibits signs of toxicity or increased if the patient

tolerates the dosage regime.

Optimal dosing schedules can also be calculated from measurements

of drug accumulation in the body. Persons of ordinary skill in the art can

easily determine optimum dosages, dosing methods, and repetition rates.

Optimum dosages may vary depending on the potency of the composition,

and can generally be estimated based on LD₅₀'s from in vitro studies. In

general, Dox-P conjugate dosage is from about 5 mg/kg to about 30 mg/kg,

more preferably about 10 mg/kg to about 15 mg/kg and the compositions may

be administered as a constant infusion using a pump or other device, over a

period of an hour or three hours to 24 hours, multiple times a day (e.g., 2 or 3

times a day), once daily, weekly, monthly, or yearly. The most commonly

used dosage of doxorubicin, when used in the absence of other

chemotherapy agents, is about 60 to about 75 mg/m² given every 21 days,

and when used in conjunction with other chemotherapy agents, is about 40 to

about 50 mg/m² given every 21 days.

In general, paclitaxel-p conjugate dosage is from about 50 mg/m² to

about 250 mg/m², preferably from about 100 mg/m² to about 200 mg/m², more

preferably from about 135 mg/m² to about 175 mg/m². This dosage can be

administered IV over about 1 hour to about 36 hours, preferably over about 3 hours to about 24 hours, and the compositions may be administered as a

constant infusion using a pump or other device, once weekly, once every two

weeks, once every three weeks, monthly, or yearly. Dosages of both dox-p

and paclitaxel-p can also be lower than the standard recommended dosages

because the peptide-conjugate allows for increased specificity, and thus a

lower dose.

EXAMPLES

The following examples are presented for illustrative purposes only and

are not intended to limit the scope of the invention in any way.

Example 1 : Preparation of a Doxorubicin-Peptide Conjugate

A doxorubicin-peptide conjugate ("Dox-P") was produced by linking the

doxorubicin through its amino terminus to the carboxy terminus of the peptide

Cys(Acm)-Ser-Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6), through an amide

linkage. The peptide's amino terminus is modified with an Acm for protection

during the linkage with the doxorubicin, and the Acm group is left on the

conjugate. Depending on the conditions used for modification, the two OH

groups on the peptide may also be modified with an Acm. According to mass

determinations, however, the peptide used in these Examples had only one

additional Acm group. Applicants believe that the Acm group is on the amino

terminus of the peptide.

The method begins with a derivatized thrombospondin peptide, which

has its sulfur groups of the cysteine residues protected by acetamidomethyl

(Acm) groups. Under argon, the peptide (35 mg, 0.05 mM) was suspended in acetic anhydride (10 mL) and, with stirring, triethylamine (1 mL) was added.

Stirring was continued until dissolution was complete (about 1 hour). The

resulting solution was yellowish.

The flask was transferred to a room temperature water bath and the

solvents removed by pumping at about 10^"4 torr. Solvent removal took about

10 minutes. The residue was dissolved in water (about 5 mL) to hydrolyze

any anhydride that had been formed at the carboxylic acid end of the

thrombospondin peptide and the resulting aqueous solution evaporated to

dryness again. The procedure with water was repeated a second time. The

resulting residue was yellow-brownish.

The yellow-brown material was taken up in anhydrous ethanol and

then evaporated to dryness again at high vacuum to remove traces of water

which might have remained and the residue was then taken up in dry DMF (2

mL) and treated, successively, with N-hydroxysuccinimide (11.5 mg, 0.10

mM) and ethyl 3-(3-dimethylamino)-carbodiimide (19.1 mg, 0.10 mM). After

stirring the reaction mixture at room temperature for 2 hr, doxorubicin

hydrochloride C₂₇H₂₉NO₁₁, 10 mg, 0.017 mM) was added and the resulting

bright red solution allowed to stir at room temperature overnight.

The solvent was removed at reduced pressure (about 10^"4 torr) while

the flask stood in a warm water bath. The residue left in the flask was the

doxorubicin-peptide conjugate. Example 2: Dose Response Effect of Dox-P on Drug-Resistant CHO Cell Viability

Doxorubicin and multidrug-resistant CHO cells were cultured using

standard tissue culture conditions: 37°C, DMEM media, serum free

conditions, with 5% CO₂ for pH adjustment. The cells were treated for 24

hours with either doxorubicin or Dox-P, the peptide conjugate prepared in

Example 1 , at concentrations of 0.25, 0.5, 0.75, and 1.0 mM. Untreated cells

were used as a negative control.

Cell viability was measured using the ALAMAR BLUE™ assay

(available from Biosource International, Camarillo, CA). The assay

quantitatively measures the proliferation of cell lines and can establish the

relative cytotoxicity of chemical agents. The assay incorporates a

fluorometric/colorimetric growth indicator based on detection of metabolic

activity. The system incorporates an oxidation-reduction (redox) indicator that

both fluoresces and changes color in response to chemical reduction of

growth medium resulting from cell growth. This causes the redox indicator to

change from its oxidized, non-fluorescent, blue form to its reduced,

fluorescent, red form. Data can be collected using either fluorescence-based

instrumentation (530-560 nm excitation wavelength and 590 nm emission

wavelength) or absorbance-based instrumentation (570 nm and 600 nm).

Figure 4 shows the dramatic dose-response effect of the Dox-P

conjugate compared with the negligible effect of Dox alone on the resistant CHO cells. This figure demonstrates that the Dox-P conjugate provides an

effective treatment against doxorubicin and multidrug-resistant tumor cells.

Example 3: Effect of DOX and DOX-P on wild-type CHO cell viability

The study of Example 2 was performed, except wild-type CHO cells

were used instead of drug resistant CHO cells. This example, with results

illustrated in Figure 6, shows that the effectiveness of doxorubicin on wild-

type, nonresistant cells is not hindered by the conjugation with the peptide. In

fact, the data demonstrate that Dox-P appears to function more effectively

against wild-type CHO cells than Dox alone.

Example 4: In Vitro LD₅₀ (mM) of Dox and Dox-P Conjugate

The LD₅₀, the dose at which half of the cells die, was calculated from

Dox and Dox-P dose response curves for five different cell lines, as shown in

Table 1.

TABLE 1 : In Vitro LD₅₀ (mM) of Dox and Dox-P

15

20

The data for the first four cell lines demonstrates that doxorubicin's

effectiveness is not significantly altered by its conjugation to the peptide, and again the doxorubicin and multidrug-resistant CHO cell line shows that the

Dox-P can overcome resistance.

Example 5: Effect of Doxorubicin Peptide Conjugate on Adhesion of

B16-F10 Melanoma Cells

An adhesion study was performed to evaluate the adhesion of B16-

F10 melanoma cells (a nonresistant cell line) to doxorubicin and the

doxorubicin-peptide conjugate of Example 1. In a 96 well plate, duplicate

wells were covered with 40 μg/ml either TSP, Dox-P, the Cys(Acm)-Ser-Val-

Thr-Cys(Acm)-Gly (SEQ ID NO: 6) peptide, Dox, the scrambled peptide Val-

Cys-Thr-Gly-Ser-Cys (SEQ ID NO: 3), the Cys(Acm)-Ser-Val-Thr-Cys(Acm)-

Gly (SEQ ID NO: 6) peptide with a d orientation, or 1% bovine serum albumin

(BSA). The wells were dried out overnight and then blocked with BSA. 100

μ\ of a suspension containing 2 x 10⁵ B16-F-10 melanoma cells were plated in

the protein covered wells and incubated at 37°C for 20 minutes to 1 hour.

The non-adherent cells were removed and the wells were washed with a

Hepes buffer. The adherent cells were fixed with 2.5% glutaraldehyde for 10

minutes and stained with 0.2% Giemsa. The stain was washed off and the

cells were counted in a field of 1 mm square. Cells adhering to BSA were

considered background.

The data are displayed in Figure 7. This experiment shows that the

peptide conjugation does not destroy, and in fact may potentiate, the

adhesive activity of doxorubicin. While not wishing to be bound by any theory, it may be that the peptide's affinity to the thrombospondin receptor

increases the adhesive effect of doxorubicin to the cell.

Example 6: Effect of Dox and Dox-P on Melanoma Tumor Development

This experiment was designed to evaluate whether the conjugation of

the peptide to doxorubicin altered its ability to prevent wild-type, nonresistant

tumor development in mice. Melanoma tumor cells were injected into mice.

The animals (5 in each group) were treated intraperitoneally 24 and 96 hours

after tumor implantation with either buffer alone, Dox-P, the Cys(Acm)-Ser-

Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6) peptide, or Dox, at a concentration of

18 μM/kg. The mice were sacrificed and the melanoma tumor colonies on the

lung were counted. The resulting data are displayed in Figure 8. Additionally,

the tumor colonies in the Dox-P and the Dox groups were 2-5 fold smaller

than in the buffer group. This demonstrates that both Dox and Dox-P were

effective at preventing tumor development of this cancer that metastasizes to

the lungs. Administration of the peptide alone, interestingly, also resulting in

a lower number of tumors than buffer alone; however, the tumors in the

peptide group had a similar size to the buffer group. These results indicate

that the peptides binding to the receptor also effect tumor development,

perhaps by inhibiting metalloprotease activity.

Example 7: Toxicity of Dox versus Dox-P

A toxicity study was performed using mice to evaluate the comparative

toxicity levels of Dox and the Dox-P conjugate. The two compounds were

administered to mice at a concentration of 30 and 68 mg/kg, respectively. Both compound have the same number of doxorubicin molecules per weight

of the animal, due to the extra mass of the peptide. The toxicity of the

compounds were evaluated and the data are presented in Table 2. These

surprising data shows that the conjugation of the peptide to doxorubicin

appears to lessen its toxicity and increase its lethal dose.

TABLE 2: Toxicity Comparison

¹ One mouse died from the treatment and the remaining two were so significantly ill that standard animal treatment protocols required their euthanization.

Example 8: Effect of Ala-Ser-Val-Thr-Ala-Arg (SEQ ID NO: 2) on

Doxorubicin Toxicity

Doxorubicin and multidrug-resistant CHO cells were treated with a

7coadministration of peptides and doxorubicin. In this example the peptides

were not linked to the doxorubicin, but were only administered at the same

time. The data are shown in Figure 9. The Cys(Acm)-Ser-Val-Thr-

Cys(Acm)-Gly (SEQ ID NO: 6) peptide did not show any effect when

coadministered with the doxorubicin. Surprisingly, however, the Ala-Ser-Val-

Thr-Ala-Arg (SEQ ID NO: 2) peptide shows a dose-response effect when

administered with doxorubicin, overcoming the resistance mechanisms. While not wishing to be bound by theory, it may be that the Ala-Ser-

Val-Thr-Ala-Arg (SEQ ID NO: 2) peptide may cause this effect by binding to

the MDR pump, inactivating it or decreasing its effectiveness. It may also

bind to an additional receptor which mediates drug interaction with the cell.

The Ala-Ser-Val-Thr-Ala-Arg (SEQ ID NO: 2) peptide may also interact with

the membrane of the cell such that it modifies the membrane or MDR pump

so as to increase permeability of the doxorubicin into the cell or decrease the

cells ability to pump it out.

Example 9: Evaluation of Random Peptides by the Affinity Sensor

System

A peptide's ability to bind to the thrombospondin receptor can be

evaluated by the following cuvette study. One can evaluate binding of

peptides to the thrombospondin receptor using the Affinity Sensor System,

Cambridge, UK. This is an optical binding method that uses a cuvette to

which either peptide or receptor is covalently coupled. A laser beam is used

to detect bound proteins to the protein-derivatized cuvette surface. This

method is highly sensitive and measures both the association and

dissociation rate constants for ligand receptor interactions. The instrument

assumes that one molecule of receptor binds one molecule of peptide and

calculates the dissociation constant (K_D) according to the following

relationships: ¹) ^k _ass [R-[peptide]=k_dlss[R-peptide] at equilibrium, where k_ass is the

second order rate constant for association and k_dιss is the first

order rate constant for dissociation

2) K_D= [R][peptide]/[R-peptide] = k_dιss/k_ass

3) [R-peptide]_t= [R-peptide]_eq[1-exp(-k_ont)], where the instrument

response measure in arc seconds is proportional to receptor-

peptide complex R-peptide].

4) k_on = k_ass[L] + k_dιss, where k_on is the pseudo-first order rate

constant for receptor-peptide interaction.

Thus, one can couple about 2 μg of receptor through its amino groups

to COOH groups on the cuvette surface. Unreacted groups on the cuvette

surface can be then blocked with ethanolamine and albumin. Peptides to be

evaluated can then be applied to the cuvette at concentrations above 189 nM

in HEPES buffered saline, pH 7.00, which should show saturable binding after

7 min. The dissociation constant of can then be calculated from a plot of the

pseudo first order rate constant for association versus the concentration of

the receptor. Thrombospondin can be used as a positive control.

Example 10: Transient Transfection and Cell Adhesion Assay

Bovine Aorta Endothelial Cells (BAEC) and MDA-MB-231 cells, breast

carcinoma cells, are transfected with purified DNA encoding for the receptor

by the Wizard Plus Kit (Promega, WI). The DNA is incorporated into the cells

using the Superfect transfection reagent (Qiagen, CA). Cells are plated in 6

well plates and upon 80% confluency transfection is performed. 12 μ\ of the reagent is used as well as 2.5 μg of the DNA, with minimal concentration of

0.1 μg/μl. Superfect-DNA complex formation is performed in a serum free

and antibiotic free medium. Cells are incubated at 37°C for 3-4 hours. Then

media is changed and 48 hours post transfection they are harvested for the

adhesion assays.

For the adhesion assay, in a 96 well plate, duplicate wells are covered

with either a peptide to be evaluated (40 μg/ml), thrombospondin "TSP-1" (40

μg/ml), fibronectin (40 μg/ml), or and 1% bovine serum albumin (BSA). The

wells are dried out overnight and then blocked with BSA. 100 μl of a

suspension containing 2 x 10⁵ cells are plated in the protein covered wells

and incubated at 37°C for 20 minutes to 1 hour. The non-adherent cells are

removed and the wells are washed with a Hepes buffer. The adherent cells

are fixed with 2.5% glutaraldehyde for 10 minutes and stained with 0.2%

Giemsa. The stain is washed off and the cells are counted in a field of 1 mm

square. Cells adhering to BSA are considered background while cells

adhering to TSP-1 and fibronectin are the positive control. The data can be

used to evaluate which random peptides bind to the thrombospondin

receptor.

Example 11 : Preparation of a Paclitaxel-Peptide Conjugate

A paclitaxel-peptide conjugate ("Paclitaxel-P") was produced by the

following method. TAXOL™ (paclitaxel) from the Pacific Yew Tree was

purchased from ICN Biomedical Research Products, a division of ICN

Pharmaceticals, Inc. and Aldrich Chemical Company (Costa Mesa, CA). The hexapeptide (Acm)Cys-Ser-Val-Thr-(Acm)Cys-Gly (SEQ ID NO: 6) was

custom synthesized by Peptidogenics Research & Co., Inc. Acm is an

acetamidomethyl protecting group for the SH of cysteine.

2'-Succinyltaxol was prepared from paclitaxel by a method similar to

that described in the literature. Magri, N.F., J. Nat. Products 51:298 (1988);

Safavy, A., J. Med. Chem. 42:4919 (1999). A mixture of paclitaxel (0.0168 g,

0.0196 mmol) and succinic anhydride (0.0324 g, 0.032 mmol) in pyridine (2

mL) was stirred at ambient temperature for 3.5 hours. The solvent was

removed at reduced pressure and the residue stirred with water (2 mL). The

resulting white solid was collected by filtration, washed with water and

recrystallized from 1 :1 water/acetone to give 13.9 mg of product. This was

used to prepare the activated ester.

The activated ester (N-hydroxysuccinimide ester of 2' succinyltaxol)

was prepared by a method similar to that used in Anderson, G.W., J. Am.

Chem. Soc. 86:1839 (1964). A mixture of 2' succinyltaxol (0.0105 g, 0.011

mmol), N-hydroxysuccinimide (0.0029 g, 0.025 mmol) and 1-ethyl-3(3-

dimethylaminopropyl)carbodiimide (0.0031 g, 0.0161 mmol) in N,N'-

dimethylformamide (0.4 ml) was stirred at ambient temperature overnight.

The solvent was removed at reduced pressure. Examination of the residue

by thin layer chromatography (SiO₂, 10% methanol/90% chloroform) revealed

the absence of the more polar 2'-succinyltaxol and the presence of a less

polar N-hydroxysuccinimide ester. The white solid residue containing the

activated ester was used without further purification in the conjugation step. To the activated ester in N.N'-dimethylformamide (0.4 mL), was added

a solution of the hexapeptide (Acm)Cys-Ser-Val-Thr-(Acm)Cys-Gly (0.0076 g,

0.0106 mmol) and NaHCO₃ (0.0020 g, 0.0238 mmol) in N,N'-

dimethylformamide (0.4 mL) and water (0.4 mL). The mixture was stirred at

ambient temperature overnight and the solvent removed at reduced pressure.

The resulting residue was subjected to preparative thin layer chromatography

(1000 micron SiO₂, 1 :1 EtOAc/CH₃OH). Several bands were observed

including starting material. A polar band was extracted from the silica gel

using the elution solvent to develop the plate. Removal of the solvent gave a

very viscous, clear, thick gel material. Comparative thin layer

chromatography revealed that it was not starting material.

Example 12: Evaluation of the Paclitaxel-P Conjugate

The effectiveness of the paclitaxel-p conjugate can be tested through a

variety of means. A cytotoxicity assay can be used to evaluate the

effectiveness of the conjugate versus paclitaxel alone. For example, normal

cancer cells (such as human breast cancer cells) and paclitaxel-resistant cells

can be cultured using standard tissue culture conditions: 37°C, DMEM

medium, serum free conditions, with 5% CO₂ for pH adjustment. The cells

may then be treated for 24 hours with either paclitaxel alone or the paclitaxel-

p conjugate prepared in Example 11 , at concentrations of 0.25, 0.5, 0.75, and

1.0 mM. Untreated cells can be used as a negative control.

Cell viability can then be measured using the ALAMAR BLUE™ assay

(available from Biosource International, Camarillo, CA). The assay quantitatively measures the proliferation of cell lines and can establish the

relative cytotoxicity of chemical agents. The assay incorporates a

fluorometric/colorimetric growth indicator based on detection of metabolic

activity. The system incorporates an oxidation-reduction (redox) indicator that

both fluoresces and changes color in response to chemical reduction of

growth medium from cell growth. This causes the redox indicator to change

from its oxidized, non-fluorescent, blue form to its reduced, fluorescent, red

form. Data can be collected using either fluorescence-based instrumentation

(530-560 nm excitation wavelength and 590 nm emission wavelength) or

absorbance-based instrumentation (570 nm and 600 nm).

Example 13: In Vitro LD₅₀ (mM) of Paclitaxel and Paclitaxel-P Conjugate

The LD₅₀, the dose at which half of the cells die, can be calculated from

paclitaxel and paclitaxel-p dose response curves for various cell line,

including B16-F10 melanoma, lewis lung carcinoma, human breast

carcinoma, wild type CHO cells, and paclitaxel resistant cells, and multidrug

resistant cells. An example of a paclitaxel resistant cell line is the SKOV-3TR

ovarian cancer cell line. Feller et al., Discovery of differentially expressed

genes associated with paclitaxel resistance using cDNA array technology,

Clin. Caner Res. 5(11):3445-53 (1999).

Example 14: Effect of Paclitaxel-Peptide Conjugate on Adhesion of B16-

F10 Melanoma Cells

An adhesion study can be performed to evaluate the adhesion of B16-

F10 melanoma cells (a nonresistant cell line) to paclitaxel and the paclitaxel- peptide conjugate of Example 11. This study could also be performed with a

breast cancer cell line. In a 96 well plate, duplicate wells are covered with 40

μg/ml either TSP, paclitaxel-p, the Cys(Acm)-Ser-Val-Thr-Cys(Acm)-Gly (SEQ

ID NO: 6) peptide, paclitaxel, the scrambled peptide Val-Cys-Thr-Gly-Ser-Cys

(SEQ ID NO: 3), the Cys(Acm)-Ser-Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6)

peptide with a d orientation, or 1 % bovine serum albumin (BSA). The wells

are dried out overnight and then blocked with BSA. 100 μl of a suspension

containing 2 x 10⁵ B16-F-10 melanoma cells is plated in the protein covered

wells and incubated at 37°C for 20 minutes to 1 hour. The non-adherent cells

are removed and the wells are washed with a Hepes buffer. The adherent

cells are fixed with 2.5% glutaraldehyde for 10 minutes and stained with 0.2%

Giemsa. The stain is washed off and the cells are counted in a field of 1 mm

square. Cells adhering to BSA were considered background.

Example 15: Effect of Paclitaxel and Paclitaxel-P on Melanoma Tumor

Development

This experiment can evaluate whether the conjugation of the peptide to

doxorubicin alters its ability to prevent wild-type, nonresistant tumor

development in mice. Melanoma tumor cells are injected into mice. The

animals (5 in each group) are treated intraperitoneally 24 and 96 hours after

tumor implantation with either buffer alone, paclitaxel-p, the Cys(Acm)-Ser-

Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6) peptide, or paclitaxel, at a

concentration of which is lower than the LD₅₀, preferably 10 fold lower than

the LD₅₀. The mice are sacrificed and the melanoma tumor colonies on the lung are counted. The LD₅₀ can be determined experimentally by treating

groups of mice with increasing doses of paclitaxel and identifying the

concentration at which half of the animals die after an acute treatment

regimen.

Example 16: Toxicity of Paclitaxel Versus Paclitaxel-P

A toxicity study can be performed using mice to evaluate the

comparative toxicity levels of paclitaxel and the paclitaxel-P conjugate. The

paclitaxel is administered to mice at a concentration which is lower than the

LD₅₀, which can be determined as in Example 15, preferably 10 fold lower

than the LD₅₀. The paclitaxel-p conjugage is administered at a dose that

yields the same number of paclitaxel molecules per weight of the animal, due

to the extra mass of the peptide. The toxicity of the compounds are then

evaluated.

Example 17: Effect of Ala-Ser-Val-Thr-Ala-Arg (SEQ ID NO: 2) on

Paclitaxel Toxicity

Paclitaxel and multidrug-resistant CHO cells are treated with a

coadministration of peptides and paclitaxel. In this example the peptides are

not linked to the paclitaxel, but are only administered at the same time. It is

expected that the Cys(Acm)-Ser-Val-Thr-Cys(Acm)-Gly (SEQ ID NO: 6)

peptide will not show any effect when coadministered with the paclitaxel.

Other embodiments of the invention will be apparent to those skilled in

the art from consideration of the specification and practice of the invention

disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention

being indicated by the following claims. All documents cited herein are

incorporated by reference.

Claims

CLAIMSWe claim:

1. A method of treating a patient suffering from cancer, wherein a cancer

chemotherapy agent is selected, conjugated to a peptide, and administered to

the patient.

2. The method of claim 1 , wherein the method is used to treat a patient

with drug resistant cancer.

3. The method of claim 1 , wherein the method is used to treat a patient

with cancer to prevent drug resistance from occurring.

4. The method of claim 1 , wherein the chemotherapy agent is selected

from the group consisting of doxorubicin and paclitaxel.

5. The method of claim 1 , wherein the peptide targets the composition to

the cancer to be treated.

6. The method of claim 1 , wherein the peptide binds to a receptor for

thrombospondin.

7. The method of claim 1 , wherein the peptide is Cys(Acm)-Ser-Val-Thr-

Cys(Acm)-Gly (SEQ ID NO: 6).

8. A method of treating a patient suffering from cancer, wherein a cancer

chemotherapy agent is selected, a peptide is selected, and the agent and

peptide are coadministered to the patient.

9. The method of claim 8, wherein the chemotherapy agent is selected

from the group consisting of doxorubicin and paclitaxel.

10. The method of claim 8, wherein the peptide targets the composition to

the cancer to be treated.

11. The composition of claim 8, wherein the peptide is Ala-Ser-Val-Thr-Ala-

Arg (SEQ ID NO: 2).

12. A composition for treating a patient suffering from cancer, wherein the

composition comprises a chemotherapy agent and a peptide.

13. The composition of claim 12, wherein the composition is used to treat a

patient with drug resistant cancer.

14. The composition of claim 12, wherein the chemotherapy agent is

conjugated to the peptide.

15. The composition of claim 12, wherein the peptide binds to a receptor

for thrombospondin.

16. The composition of claim 15, wherein the peptide is Cys(Acm)-Ser-Val-

Thr-Cys(Acm)-Gly (SEQ ID NO: 6).

17. The composition of claim 15, wherein the peptide is Ala-Ser-Val-Thr-

Ala-Arg (SEQ ID NO: 2).