WO2011078782A1

WO2011078782A1 - A composition comprising of melphalan derivatives and gemcitabine or etoposide useful in the treatment of cancer

Info

Publication number: WO2011078782A1
Application number: PCT/SE2010/051466
Authority: WO
Inventors: Joachim Gullbo; Rolf Larsson; Jack Spira; Emma Kristina Viktorsson
Original assignee: Oncopeptides Ab
Priority date: 2009-12-22
Filing date: 2010-12-22
Publication date: 2011-06-30

Abstract

The present invention is directed to a composition comprising melphalan, or a derivative thereof having essentially the biological activity of melphalan, and gemcitabine. The combination of melphalan or a derivative thereof and gemcitabine results in a synergistic effect when used for treating cancer. The invention therefore also relates to the use of the composition for the preparation of medicaments for the treatment and/or prevention of cancer, as well as methods for cancer treatment and/or prevention. The invention is also directed to a composition comprising a combination of melphalan, or a derivative thereof having essentially the biological activity of melphalan, and etoposide for the preparation of a medicament for the treatment and/or prevention of ovarian cancer, bladder cancer and/or lung cancer, as well as methods for treating and/or preventing such cancers.

Description

A composition comprising of melphalan derivatives and gemcitabine or etoposide useful in the treatment of cancer

Technical field

The present invention is within the field of cancer treatment and pharmaceutical compositions for the treatment and/or prevention of cancer. More particularly, the present invention is directed to cancer treatments utilizing a combination of a derivative of melphalan and gemcitabine or a combination of a derivative of melphalan and etoposide. Background art

Cancer is a major and often fatal disease. Accordingly, efforts to develop new therapies for cancer is a constantly ongoing in the research society. The vast majorities of cancers are present as solid tumors, e.g. lung cancer, breast cancer, prostate cancer, while the rest are hematological and lymphoid malignancies, e.g. leukemias and lymphomas.

Chemotherapy is often used in attempts to cure or palliate the disease. As cancer cells typically divide rapidly, chemotherapy usually acts by killing rapidly dividing cells. In the broad sense, most chemotherapeutic drugs work by impairing mitosis (cell division), effectively targeting fast-dividing cells. As these drugs cause damage to cells they are termed cytotoxic. Some drugs cause cells to undergo apoptosis (so-called "programmed cell death"). Often combination chemotherapy is used, when two or more drugs having different modes of action are used together in order to optimise the antitumoral effect, to minimise side effects and prevent resistance development. The results obtained with chemotherapy vary according to tumor type. Some tumors are very sensitive and the treatment has then a high probability of leading to cure.

Chemotherapeutic drugs can generally be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. The drugs affect cell division or DNA synthesis.

Gemcitabine is a nucleoside analogue used in chemotherapy. Gemcitabine is a

nucleoside analogue wherein the hydrogen atoms on the 2' carbon of deoxycytidine are replaced by fluorine atoms. Gemcitabine replaces one of the building blocks of nucleic acids, in this case cytidine, during DNA replication. Because of this growth of the tumor is arrested and apoptosis occurs. Gemcitabine also affects the activity of the enzyme ribonucleotide reductase (RNR) by binding to the RNR's active site and inactivating the enzyme. As RNR is directly involved in the cell's production of deoxyribonucleotides required for DNA replication and repair, the cell cannot replicate itself anymore due to the lack of deoxyribonucleotides, which results in apoptosis of the cell. Gemcitabine has been used to treat various forms of cancers, e.g. non-small cell lung cancer, pancreatic cancer, bladder cancer, ovarian cancer and breast cancer.

Etoposide inhibits the enzyme topoisomerase II. Etoposide has been used in the treatment of Ewing's sarcoma, lung cancer, testicular cancer, lymphoma, non-lymphocytic leukemia, and glioblastoma multiforme. Etoposide's chemical make-up derives from podophyllotoxin, a toxin found in the American Mayapple. The use of etoposide is associated with risk for damage to bladder and kidneys.

Alkylating agents, such as drugs derived from nitrogen mustard, that is bis(2- chloroethyl)amine derivatives, are used as chemotherapeutic drugs in the treatment of a wide variety of neoplastic diseases. Alkylating agents have the ability to covalently attach alkyl groups to electronegative sites in cells. Thus, these agents act by impairing cell function by forming covalent bonds with heteroatoms in biologically important molecules like RNA, DNA and proteins. Examples of alkylating agents are mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide, temozolomide and melphalan that chemically modify a cell's DNA.

The alkylating agents act by covalent interaction with nucleophilic heteroatoms in DNA or proteins. Difunctional agents are able to crosslink a DNA chain within a double helix in an intrastrand or interstrand fashion, or to crosslink between DNA and proteins. The crosslinking results in inhibitory effects on DNA replication and transcription with subsequent cell death. The drugs can be used as single agents or in combination with other antineoplastic agents. Alkylating agents seem to have some propensity for fast growing tissues. They exert effects in a broad spectrum of tumors. Side effects are mainly restricted to bone marrow and at very high doses also to the gastrointestinal tract.

Melphalan, or -[0/s-(2-chloroethyl)amino]phenylalanine, is a conjugate of nitrogen mustard and the amino acid phenylalanine, which was synthesised in the mid 1950s (US 3,032,584). This classic alkylating substance soon became a valuable drug in the chemotherapeutic field and is still of importance in the treatment of for example myeloma. Clinical use of melphalan in the treatment of metastatic melanomas has, however, had limited efficacy. In the search for a more selective action on malignant cells melphalan analogues have been synthesised.

Sarcolysine, m-[bis-(2-chloroethyl)amino]phenylalanine, was obtained by shifting the bis- (2-chloroethyl)amino group from the para- to the meta-position of phenylalanine. By covalent conjugation of different amino acids at the amino and carboxylate groups of sarcolysine, a peptide mixture known as Peptichemio (PTC) was prepared. PTC consisted of six different peptides (de Barbieri, "Proceedings of the symposium on Peptichemio", Milan, November 18, 1972). PTC was subsequently shown to be active on several tumor types as well as on tumors resistant to treatment with alkylating agents including melphalan and entered clinical trials with promising results. For the understanding of the effects and usage of PTC a serious disadvantage was the fact that it is a mixture of six peptides. The cytotoxic effects of each of the different peptides contained in PTC were therefore measured separately (Lewensohn et al. 1991) and a wide variation in the cytotoxicities of the peptides was found. One of the peptides, L-prolyl-m-L-sarcolysyl-L-p- fluorophenylalanine ethyl ester hydrochloride (P2) turned out to be more toxic to RPMI 8322 melanoma cells than any of the other peptides.

There is some confusion as to the nomenclature of the melphalan derivatives. When, in 1955, a British research group reported the synthesis and cytotoxic activity of a phenylalanine derivative substituted with a bis-(2'-chloroethyl)amino group in the para- position of the aromatic ring, the DL, L and D isomers were named merphalan, melphalan and medphalan, respectively. A Russian group at the same time independently named the racemic form (DL) of 4-[bis-(2-chloroethylamino)]phenylalanine sarcolysine. Later the term sarcolysine started to be used also for the meta phenylalanine mustard. This name confusion has continued, but today melphalan and sarcolysine are normally used for the para- and mera-derivatives, respectively.

Lopatin et al. disclosed that administration of the melphalan derivatives asaley and astyron, inhibited sarcoma 45 growth in rats. Astyron, or A/-acetyl-L melphalanyl-L-tyrosine ethyl ester, as well as asaley, or /V-acetyl-L-melphalanyl-L-valine ethyl ester, have an acetylated amino group, which generally means that the cytotoxicity of the compound to tumor cells has been considerably reduced compared to the non acetylated form. Romancva et al. refers to a compound called sarcolysine or salin, which according to Western nomenclature should rather be named L-melphalanyl-L-valine. This compound disrupted oxidative phosphorylation in mitochondria from rat liver homogenates and Jensen sarcoma cells.

Kupczyk-Subotkowska et al. disclosed derivatives of melphalan designed to enhance the accumulation of melphalan into cancer cells. A number of dipeptides comprising melphalan and valine or glutamic acid were tested and it was concluded that the cellular uptake of said dipeptides as well as of their esters probably took place via passive diffusion rather than by an amino acid or oligopeptide transporter. WO01/96367 discloses new alkylating di- and tripeptides based on the melphalan unit and one or two additional amino acids or amino acid derivatives. These melphalan derivatives were demonstrated to have an improved efficacy on a variety of tumor types.

One problem with the usage of bifunctional alkylating agents (as well as with other chemotherapeutic agents) is primary, i.e. intrinsic, and secondary, i.e. acquired, tumor resistance to the treatment. Attempts have been made to circumvent resistance by increasing the dose of alkylating agent administered to the patient. It is, however, unclear how much this increases the effective dose at the tumor cell level. However, there is still an urgent need for new antitumor drugs in a wide variety of tumor diseases, especially in tumors showing primary resistance to conventional therapy, and/or in tumor diseases that have developed resistance, secondary resistance, after having responded to treatment with conventional cancer chemotherapeutics. Summary of invention

The object of the present invention is to overcome or at least mitigate some of the disadvantages of the prior art. This object is in a first aspect obtained by a composition comprising a derivative of melphalan of formula I

wherein is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino,

cycloalkylamino or arylamino;

R₂ is

wherein R₃ is independently NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl, N(CH₂CH₂CI)₂

N0₂, F, CF₃ or H, and n is 1 or 2;

X is

(II)

wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid, or H,

or a pharmaceutically acceptable salt thereof; and

gemcitabine, or a pharmaceutically acceptable salt thereof,

optionally in admixture with a pharmaceutically acceptable carrier, excipient and/or adjuvant.

The invention is also directed to a kit comprising

a first container comprising the melphalan derivative as defined herein or a

pharmaceutically acceptable salt thereof and a second container comprising gemcitabine; or a pharmaceutically acceptable salt thereof.

The combination of the derivative of melphalan and gemcitabine was demonstrated to result in a synergistic effect when used for treating cancer compared to when the respective compounds were used alone. The invention therefore is also directed to the composition or kit as described herein for use as a medicament and the use of the composition or the kit for the preparation of a medicament for the treatment and/or prevention of cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer and a method of treating such cancer comprising administering the composition in a therapeutically effective dose to a subject in need thereof. Preferably, in all aspects of the invention when a combination therapy using a melphalan derivative or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof is used, the melphalan derivative and the gemcitabine are administered simultaneously or immediately after each other.

In another aspect, the invention is directed to the use of a pharmaceutical composition comprising

a derivative of melphalan of formula I

wherein is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino,

cycloalkylamino or arylamino;

R₂ is

^"(R₃)n

(IV) wherein R₃ is independently NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl, N(CH₂CH₂CI)₂, N0₂, F, CF₃ or H, and n is 1 or 2;

X is

(II)

wherein R₅ is H; R₄ is a natural or modified cyclic or aromatic amino acid, or H,

or a pharmaceutically acceptable salt thereof; and

etoposide or a pharmaceutically acceptable salt thereof;

for the manufacture of a medicament for the treatment and/or prevention of ovarian cancer, bladder cancer and/or lung cancer.

Also, the invention is directed to a kit comprising a first container comprising a derivative of melphalan as defined in the herein, or a pharmaceutically acceptable salt thereof, and a second container comprising etoposide, or a pharmaceutically acceptable salt thereof.

The invention is further directed to a method for treating and/or preventing ovarian cancer, bladder cancer and/or lung cancer comprising administering a composition or a kit comprising the derivative of melphalan as defined above and etoposide in a

therapeutically effective dose to a subject in need thereof.

Brief description of drawings

Figure 1 : structural formulas for the melphalan derivatives L-melphalanyl-L-p- fluorophenylalanine ethyl ester (J1 ), L-melphalanyl-L-p-fluorophenylalanine isopropyi ester (JV28) and L-prolinyl- L-melphalanyl- L-p-fluorophenylalanine ethyl ester (J3).

Figure 2: The combination effects of J1 and gemcitabine in the parental A2780 cell line (Fig. 2a), cisplatinum resistant cisA2780 (Fig. 2b) and primary tumor cells from a patient with mesothelioma (Fig 2c). Figure 3: Combination of J1 and gemcitabine in A2780 ovarian cancer xenografts in vivo. A2780 ovarian cancer cells were injected into SCID BALB c mice and allowed to form tumors. When the tumors reached the mean weight of 225±54 mg treatment with vehicle (black diamond), 5 mg/kg Gemcitabine (yellow squares) or a combination of J1 (2 mg/kg (blue squares), 4 mg/kg (red circles) and 8 mg/kg (black triangles) and gemcitabine 5 mg/kg were applied twice a week for two weeks. Tumor growth was monitored every third day during the entire experiment and is depicted as tumor weight calculated by the formula Tumor weight= length x width². The number of mice in each treatment group is given in Table 5. Statistical significance was reached for a combination of J1 8 mg/kg + gemcitabine 5mg/kg using Mann-Whitney test. Fig. 3a: Median values of tumor weight are shown. Fig. 3b: Mean values of tumor weight are shown and the bars represent SEM. Fig. 4. Combination of J1 and Gemcitabine in A2780CIS (cisplatinum resistant cell line) ovarian cancer xenografts in vivo. Triangles: 5 mg/kg gemcitabine, squares: 8 mg/kg J1 , and crosses: combination of J1 (8 (mg/kg) and gemcitabine (5 mg/kg). Fig. 5. The effect on PARP cleavage of J1 or melphalan alone or in combination with gemcitabine in A2780 ovarian carcinoma cells. A2780 ovarian carcinoma cells were treated with indicated doses of J1 or melphalan either alone or in combination as outlined in (A). The cleavage of PARP by caspase activation was examined with western blot at 48h post treatment start (B; C). Upon caspase activation the full length PARP disappear and a cleavage fragment with lower molecular weight is observed. GAPDH was used as loading control (B, C, Left). The ratio between cleaved and full length PARP was densitometrically quantified after correction for loading differences using GAPDH (B and C, right). B; J1 ; C: Melphalan. Fig. 6. The effect of scheduling on J1 and Gemcitabine combination treatment. A2780 ovarian carcinoma cells were treated with indicated doses of J1 either alone or in combination with gemcitabine as outlined in Fig. 5A. The cleavage of PARP by caspase activation was examined with western blot at 48h post treatment start (left). Upon caspase activation the full length PARP disappear and a cleavage fragment with lower molecular weight is observed. GAPDH was used as loading control. The ratio between cleaved and full length PARP was densitometrically quantified after correction for loading differences using GAPDH (bottom).

Fig. 7. A2780 cells were treated with either 10 μΜ or J1 or Melphalan for indicated time periods. The intracellular concentration of Melphalan was measured. The ratio of Mel ng/ml between J1 10 μΜ or Melphalan 10 μΜ was calculated at indicated time points. As can be seen the ratio is at 60 min 12-fold which is in the same range as was used for combo (0.1 vs 1 ). Fig. 8 shows the effect of scheduling. S1A shows the strong synergistic effect on apoptosis and celldeath when the drugs are given concomitantly. In contrast when scheduling is used, where the drugs are given 24 hours apart (experiment S3), shows no synergy but only an additive effect. Detailed description of invention

The present inventors have surprisingly found that combination cancer chemotherapy using the combination of

a. a derivative of melphalan having the formula I

wherein Ri is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino, cycloalkylamino or arylamino;

R₂ is

wherein R₃ is independently NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl, N(CH₂CH₂CI)₂, N0₂, F, CF₃ or H, and n is 1 or 2;

X is

(II)

wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid, or H,

or a pharmaceutically acceptable salt thereof; and

b. gemcitabine or a pharmaceutically acceptable salt thereof,

results in an unexpected improvement of antitumoral activity.

In the present context "derivative of melphalan" or "melphalan derivative" may be used interchangeably when referring to a derivative of melphalan as defined in the above paragraph or other derivatives of melphalan defined herein. Alternatively, a pharmaceutically acceptable salt of such a melphalan derivative may be used. Preferably, for all aspects of the invention, the melphalan derivative is the derivative denoted "J1 " or a pharmaceutically acceptable salt thereof. J1 is described in more detail elsewhere herein. Gemcitabine's lUPAC name is 4-amino-1 -[(2 R,4 R,5f?)-3,3-difluoro-4-hydroxy-5-

(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one. In the context of the present invention also pharmaceutically acceptable salts thereof may be used.

In all aspects of the invention when gemcitabine is used together with the derivative of melphalan, the composition of the invention may also comprise a mixture of two or more derivatives of melphalan of formula I.

In a first aspect the present invention consequently relates to a pharmaceutical composition comprising

a. a derivative of melphalan having the formula I

wherein Ri is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino,

cycloalkylamino or arylamino;

R₂ is

X is

(II) wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid, or H,

or a pharmaceutically acceptable salt thereof; and

b. gemcitabine or a pharmaceutically acceptable salt thereof,

optionally in admixture with one or more pharmaceutically acceptable carrier(s), excipient(s) and/or adjuvant(s).

Preferred for the present invention is a composition comprising one or more of the melphalan derivatives L-melphalanyl-L-p-fluorophenylalanine ethyl ester (J1 ), L- melphalanyl-L-p-fluorophenylalanine isopropyl ester (JV28), and /or L-prolinyl- L- melphalanyl- L-p-fluorophenylalanine ethyl ester (J3); or pharmaceutically acceptable salts thereof, and gemcitabine or a pharmaceutically acceptable salt thereof, optionally in admixture with a pharmaceutically acceptable carrier, excipient and/or adjuvant. The melphalan derivatives used in the present invention have previously been disclosed in WO01/96367. The N-terminus of the melphalan molecule should preferably not be protected as amide or carbamate, this means that R₄ in formula I above should preferably not be a protecting group, such as formyl, acetyl or propionyl, or benzoyl, as the protected form of the compound in general has a lower cytotoxic activity than the corresponding free form. Natural amino acids refer to amino acids that are normally existing and exerting their functions in living organisms. Modified amino acids refer to amino acids that in some way have been modified into a different chemical structure and chemical composition than a natural amino acid. An example of a natural cyclic amino acid is proline. Examples of aromatic amino acids are phenylalanine, tyrosine, tryptophan, and histidine.

Particularly preferred for the present invention is a composition comprising L-melphalanyl- L-p-fluorophenylalanine ethyl ester (J1), or a pharmaceutically acceptable salt thereof, and gemcitabine or a pharmaceutically acceptable salt thereof, optionally in admixture with a pharmaceutically acceptable carrier, excipient and/or adjuvant. J1 is also preferably used in other aspects of the invention.

Consequently such composition comprises the derivative of melphalan as a first antitumoral component and gemcitabine as a second antitumoral component. The present inventors have found that by using a combination cancer therapy utilizing these two compounds, a synergistic increase in antitumoral activity can be seen. This has the advantage of increasing the antitumoral effect when using this combination of compounds to treat and/or prevent cancer. Also, in this way it may be possible to decrease the dose of the respective compound used, thereby decreasing the negative side effects that otherwise may occur when using the compounds alone. In addition, the risk for development of resistance to the drug is reduced as two drugs are used simultaneously, as the tumor therefore has to develop resistance to both compounds in order to escape treatment. Also, due to the more effective treatment and/or prevention of the tumor, drug resistance is less likely to develop as the tumor is more rapidly and effectively treated.

The use of the combination therapy with a derivative of melphalan and gemcitabine disclosed in the present application results in cell death and/or reduced growth rate of tumor cells, and consequently a reduction and/or complete disappearance of tumor cells in a subject.

As demonstrated in Examples 1 and 3, the combination of the melphalan derivative J1 and gemcitabine, even allowed the treatment of cisplatinum resistant cells both in vitro (Example 1 ) and in vivo (Example 3). Normally, the development of cisplatinum resistance causes a cross-resistance to also other alkylating agents to develop. It is therefore highly surprising that such a cross-resistance can be circumvented by using the combination of a derivative of melphalan and gemcitabine. In all aspects of the invention the melphalan derivative and/or the gemcitabine may be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts for all aspects of the invention are, for instance, an acid-addition salt of a compound of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, nitric,

methansulphonic, sulphuric, phosphoric, trifluoroacetic, para-toluene sulphonic, 2- mesitylen sulphonic, citric, acetic, tartaric, fumaric, lactic, succinic, malic, malonic, maleic, 1 ,2-ethanedisulphonic, adipic, aspartic, benzenesulphonic, benzoic, ethanesulphonic or nicotinic acid. The compounds may exist as solvates or hydrates. It is to be understood that the compounds disclosed herein and/or the compositions of the present invention

encompasses all such solvates or hydrates.

The compounds may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵l) or carbon-14 (¹⁴C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

A preferred melphalan derivative for all aspects of the invention is also disclosed in WO01/96367 and has the formula V

cycloalkylamino or arylamino;

R₃ is NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl, N(CH₂CH₂CI)₂, N0₂, F, CF₃ or H; and R₄ is a natural or modified cyclic or aromatic amino acid, or H; as well as pharmaceutically acceptable salts thereof. An especially interesting group of melphalane derivatives for all aspects of the present invention are peptides of the formula I or V, wherein R₃ is F.

Dipeptides are peptides of the formula I or V, wherein is alkyloxy; R₃ is F, CF₃, H, OH, O-alkyl, N0₂, N(CH₂CH₂CI)₂, NH-acyl or NH₂; and R₄ is H.

Tripeptides are peptides of the formula I or V, wherein is alkyloxy; R₃ is F, CF₃, H, OH, O-alkyl, NH-acyl, N0₂, N(CH₂CH₂CI)₂ or NH₂; and R₄ is a natural or modified cyclic or aromatic amino acid. The dipeptide derivatives disclosed in WO01/96367 can be synthesised from tert- butoxycarbonyl(Boc)-protected melphalan as disclosed therein. Also, WO01/96367 discloses the preparation of tripeptide derivatives, in which Boc-protected amino acids were coupled to the melphalan containing dipeptide derivative using EDC/NMM/HOBt as coupling reagents (EDC is triethylamine or 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride, NMM is N-methylmorpholine and HOBt is 1 -hydroxybenzotriazole). Examples of preferred derivatives of melphalan for all aspects of the present invention are L-melphalanyl-L-p-fluorophenylalanine ethyl ester (J1 ), L-melphalanyl-L-p- fluorophenylalanine isopropyl ester (JV28), L-prolinyl- L-melpha!anyl- L-p- fluorophenylalanine ethyl ester (J3) (Fig. 1) and pharmaceutically acceptable salts thereof. These compounds have all been disclosed previously in WO01/96367, which also provides methods for their preparation. J1 , JV28 and J3 are all derivatives of melphalan which in the body are transformed into melphalan. In WO 01/96367 these melphalan derivatives were demonstrated to have an increased cell killing activity against tumors, even when used at lower concentrations than melphalan. In addition, melphalan resistance could be circumvented. In the present context, J1 , JV28 and J3 are used interchangeably with their chemical names L-melphalanyl-L-p-fluorophenylalanine ethyl ester (J1 ), L-melphalanyl-L-p-fluorophenylalanine isopropyl ester (JV28), and L-prolinyl- L- melphalanyl- L-p-fluorophenylalanine ethyl ester (J3), respectively.

Particularly preferred for all aspects of the invention as the derivative of melphalan is L- melphalanyl-L-p-fluorophenylalanine ethyl ester (J1 ).

The melphalan derivative is preferably administered in an amount in the range of about 10-135 mg, such as 20-100 mg, for example 50-75 mg total amount of melphalan derivative per administration. Gemcitabine is preferably administered in an amount in the range of about 250-2000 mg/m² body surface area. The compositions or kits of the invention comprising a derivative of melphalan and gemcitabine should therefore have a concentration such that these amounts of the respective compounds can be administered. Such a composition or kit therefore has about 10-100 mg/ml, preferably about 20-50 mg/ml, of melphalan derivative in a suitable solvent. The amount of gemicitabine in the composition or the kit of the invention is about 33-42 mg/ml, such as about 36-40 mg/ml, preferably about 38 mg/ml. The composition of the invention comprising a melphalan derivative and gemcitabine may be administered daily, every second or third day, weekly, every second or third week or even as a high single dose (e.g. before transplantation) depending on the subject and cancer form to be treated. More preferably, gemcitabine is administered in an amount in the range of about 500- 1250 mg/m² body surface area. Gemcitabine may also be administered in an amount in the range of about 700-1000 mg/m² body surface area. Therefore, more preferably a composition or a kit of the invention comprising a melphalan derivative and gemcitabin comprises a melphalan derivative at a concentration of about 20-50 mg/ml and gemcitabine at a concentration of 33-42 mg/ml.

Preferably, the pharmaceutical composition only has the melphalan derivative and gemcitabine as antitumoral agents. More preferably, J1 , JV28 and/or J3 and gemcitabine are the only antitumoral substances in such a composition. Most preferably, J1 and gemcitabine are the only antitumoral substances in such a composition. Such

compositions may of course still have one or more pharmaceutically acceptable carrier(s), excipient(s) and/or adjuvant(s).

For all aspects of the invention the melphalan derivative and gemcitabine, or a

pharmaceutically acceptable salt of any of these may be combined with carrier(s), excipient(s) and/or adjuvant(s).

The carrier or the excipient could be a solid, semi-solid or liquid material that could serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are known in the art. The composition comprising a derivative of melphalan and gemcitabine is preferably administered intravenously. It is also possible to administer the composition in body cavities, such as instillation in the bladder, or in peritoneal or pleural cavities.

The compositions of the invention may also be denoted as a pharmaceutical composition. Such compositions are prepared in a manner known to a person skilled in the

pharmaceutical art.

As mentioned above, when in the form of solutions or suspensions, the compositions of the invention may also comprise at least one of the following (pharmaceutically

acceptable) adjuvants: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents, antioxidants such as ascorbic acid or sodium bisulfite, organic solvents, buffers such as acetates, citrates or phosphates, and agents for adjustment of the tonicity such as sodium chloride or dextrose.

The present invention also is directed to a kit comprising a first container comprising a melphalan derivative of formula I

wherein is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino,

cycloalkylamino or arylamino;

R₂ is

X is

wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid, or H,

or a pharmaceutically acceptable salt thereof; and

a second container comprising gemcitabine; or a pharmaceutically acceptable salt thereof.

Preferably, each container in such a kit only has the melphalan derivative, and

gemcitabine, as antitumoral substances. A kit according to the present invention may also comprise one or more device(s), such as a syringe, for administering the respective drugs of the two containers. Also, the kit may contain instructions for the sequential, separate or simultaneous administration of the melphalan derivative and gemcitabine to a patient in need thereof. Preferred derivatives of melphalan for the kit are described above.

Preferably, in a kit the first container comprises the melphalan derivative L-melphalanyl-L- p-fluorophenylalanine ethyl ester (J1 ), L-melphalanyl-L-p-fluorophenylalanine isopropyl ester (JV28) and/or L-prolinyl- L-melphalanyl-L-p-fluorophenylalanine ethyl ester (J3) and the second container comprises gemcitabine. The melphalan derivative is preferably J1 . Preferably, the kit consists of a first container comprising J1 and second container comprising gemcitabine. The melphalan derivative and the gemcitabine may be in admixture with a pharmaceutically acceptable carrier, excipient and/or adjuvant in their respective containers. The containers may comprise the melphalan derivative and gemcitabine, respectively, in dry or liquid form. Also, the carrier(s), excipient(s) and/or adjuvant(s) may be present in one or more further separate containers for mixing with the melphalan derivative and the gemcitabine, respectively, before administration to a subject. The concentrations of the melphalan derivative and gemcitabine in their respective containers in the kit are e.g. as specified above for the composition comprising a melphalan derivative and gemcitabine. The containers can be made of any suitable material for harbouring its constituents, such as glass or plastics. The skilled person knows how to select the proper material for the container. When the melphalan derivative and the gemcitabine are provided in a kit of parts, the same dosages, administration routes, administration schemes etc. are used as disclosed elsewhere herein when a composition comprising a melphalan derivative and gemcitabine is used. Another aspect of the invention is directed to a composition as described above or a kit as described above comprising a derivative of melphalan and gemcitabine for use as a medicament. Consequently the invention is also directed to a pharmaceutical composition comprising

a. a derivative of melphalan of formula I

(I ) wherein Ri is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino,

cycloalkylamino or arylamino;

R₂ is (R₃) ',η

X is

wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid, or H or a pharmaceutically acceptable salt thereof, and

b. gemcitabine or a pharmaceutically acceptable salt thereof,

optionally in admixture with one or more pharmaceutically acceptable carrier(s), excipient(s) and/or adjuvant(s), for use as a medicament.

The invention is therefore also directed to kit comprising a first container comprising a. a derivative of melphalan of formula I

wherein is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino, cycloalkylamino or arylamino;

R₂ is

X is

wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid, or H, or a pharmaceutically acceptable salt thereof; and

b. a second container comprising gemcitabine; or a pharmaceutically acceptable salt thereof,

for use as a medicament.

Preferred melphalan derivatives for compositions and kits for the aspect of their use as a medicament and in therapy are the same as the ones described above. Preferably, such a composition for use as a medicament comprises J1 , J3 and/or JV28 and gemcitabine, or pharmaceutically acceptable salts thereof, optionally in combination with one or more pharmaceutically acceptable carrier(s), excipient(s) and/or adjuvants. Most preferably, such a composition for use as a medicament comprises J1 and gemcitabine. Thus, preferably in all aspects of the invention wherein a melphalan derivative is used in combination with gemcitabine, the melphalan derivative is J1.

The present application is also directed to the use of a composition or a kit as described above comprising a derivative of melphalan and gemcitabine for the manufacture of a medicament for the treatment and/or prevention of cancer. The present invention is also directed to a composition or kit as described above comprising a derivative of melphalan and gemcitabine for use for treating and/or preventing cancer. Consequently the invention is also directed to the use of a composition comprising

a. a derivative of melphalan of formula I

cycloalkylamino or arylamino;

2 is -T(R₃)n

X is

wherein R₅ is H;

b. gemcitabine or a pharmaceutically acceptable salt thereof,

for the preparation of a medicament for the treatment and/or prevention of cancer.

The invention is therefore consequently also directed to the use of a kit comprising a first container comprising

a first container comprising a melphalan derivative of formula I

wherein Ri is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino

cycloalkylamino or arylamino;

R₂ is

wherein R₃ is independently NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl, N(CH₂CH₂CI)₂ N0₂, F, CF₃ or H, and n is 1 or 2; X is

wherein R₅ is H;

a second container comprising gemcitabine; or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment and/or prevention of cancer.

Particularly preferred for such use is a composition or kit comprising J1 , J3 and/or JV28, in particular J 1 , and gemcitabine, or pharmaceutically acceptable salts of these.

Otherwise, preferred melphalan derivatives, compositions and kits for these aspects of the invention are described above. The cancer forms particularly preferred to be treated and/or prevented are ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer. As mentioned above, a synergistic antitumoral effect is achieved by a combination therapy with the melphalan derivative and gemcitabine. In order to achieve this synergistic effect, the melphalan derivative and gemcitabine may be administered to a subject either sequentially, preferably immediately after each other, with either of the compounds first, or simultaneously. Consequently, the present application is also directed to the use of a composition comprising a melphalan derivative, or a pharmaceutically acceptable salt thereof, and gemcitabine, or a pharmaceutically acceptable salt thereof, for the

preparation of a medicament for the treatment and/or prevention of cancer wherein said melphalan derivative or pharmaceutically acceptable salt thereof, and said gemcitabine, are administered sequentially, preferably immediately after each other. The present application is also directed to the use of a melphalan derivative, or a pharmaceutically acceptable salt thereof, and gemcitabine, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment and/or prevention of cancer wherein said melphalan derivative or pharmaceutically acceptable salt thereof, and said gemcitabine, are administered sequentially, preferably immediately after each other.

Alternatively, another aspect of the invention is directed to the use of a kit as described herein comprising a melphalan derivative and gemcitabine for the preparation of a medicament for the treatment and/or prevention of cancer wherein the melphalan derivative, or pharmaceutically acceptable salt thereof, and the gemcitabine, or pharmaceutically acceptable salt thereof are administered sequentially, preferably immediately after each other. The invention is also directed to a derivative of melphalan as defined herein or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof or kit comprising a melphalan derivative and gemcitabine for use for the treatment and/or prevention of cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer, wherein said derivative of melphalan and gemcitabine are to be administered sequentially, preferably immediately after each other. Preferred melphalan derivatives for these aspects of the invention are as described above.

The inventors have surprisingly found that when the melphalan derivative or a

pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof are administered simultaneously, or immediately after each other, a particularly high synergistic effect of the combination of the melphalan derivative and the gemcitabine in the cancer treatment and/or prevention is achieved. Thus, preferably, the melphalan derivative and the gemcitabine are administered simultaneously or immediately after each other.

Consequently, the invention is also directed to the use of a derivative of melphalan as defined herein or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prevention of cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer, wherein said derivative of melphalan and gemcitabine are to be administered simultaneously.

The invention is further directed to a derivative of melphalan as defined herein or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof for use for the treatment and/or prevention of cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer, wherein said derivative of melphalan and gemcitabine are to be

administered simultaneously.

Cancer forms for which the combination therapy using a melphalan derivative, or a pharmaceutically acceptable salt thereof, and gemcitabine, or a pharmaceutically acceptable salt thereof, are particularly useful for are ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer. However, the present invention is not limited to the treatment and/or prevention of these cancers but may also used in the treatment and/or prevention of other cancer forms. The subject to be treated using the combination therapies as disclosed herein is in any vertebrate subject, such as a mammal. In particular the combination therapy is may be used to treat and/or prevent cancer in humans or domestic animals.

The present application is also directed to a method for treating and/or preventing cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer, comprising administering a derivative of melphalan, or a

pharmaceutically acceptable salt thereof, and gemcitabine, or a pharmaceutically acceptable salt thereof, in a therapeutically effective dose to a subject in need of such a treatment. Alternatively, such a method for treating and/or preventing cancer comprises administering a kit comprising a first container comprising

a melphalan derivative, or a pharmaceutically acceptable salt thereof; and

a second container comprising gemcitabine; or a pharmaceutically acceptable salt thereof in a therapeutically effective dose to a subject in need of such a treatment. The melphalan derivative and the gemcitabine may be administered in the form of a composition comprising these compounds as defined herein. Preferred melphalan derivatives for this aspect of the invention are as described above.

The invention is also directed to a method for treating and/or preventing cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer, comprising administering a derivative of melphalan as defined herein or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in a therapeutically effective dose to a subject in need thereof, wherein said administration of said derivative of melphalan and gemcitabine is performed simultaneously. Alternatively, the melphalan derivative and the gemcitabine may be administered sequentially, preferably immediately after each other.

In a method for treating and/or preventing cancer according to the above the melphalan derivative is preferably given in doses of 10-135 mg, such as 20-100 mg, for example 50- 75 mg per administration. Gemcitabine is preferably administered in an amount in the range of about 250-2000 mg/m² body surface area, such as 500-1250 mg/m² body surface area, for example about 700-1000 mg/m² body surface area.

Another aspect of the invention is directed to the use of a composition comprising a. a derivative of melphalan of formula I

(I ) wherein is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino,

cycloalkylamino or arylamino;

R₂ is

N0₂, F, CF₃ or H, and n is 1 or 2;

X is

wherein R₅ is H;

b. etoposide, or a pharmaceutically acceptable salt thereof;

for the manufacture of a medicament for the treatment and/or prevention of ovarian cancer, bladder cancer and/or lung cancer. Another aspect of the invention is directed to a composition comprising a derivative of melphalan as defined above, or a pharmaceutically acceptable salt thereof and etoposide, or a pharmaceutically acceptable salt thereof, for use for treating and/or preventing ovarian cancer, bladder cancer and/or lung cancer. Preferred melphalan derivatives for these aspects of the invention are the same as those described above. As in other aspects of the invention, particularly preferred are the melphalan derivative J1 , J3 and JV28, in particular J1 , or pharmaceutically acceptable salts thereof. Preferably, the compositions of this aspect of the invention only have the derivative of melphalan and etoposide as antitumoral agents. Such compositions may of course still have one or more pharmaceutically acceptable carrier(s), excipient(s) and/or adjuvant(s). Pharmaceutically acceptable salt suitable for this aspect of the invention are the same as the ones described above.

Etoposide may be administered as etoposide or as its prodrug etoposide phosphate. Etoposide is an inhibitor of the enzyme topoisomerase I I. It is used for the treatment of cancers such as Ewing's sarcoma, lung cancer, testicular cancer, lymphoma, non- lymphocytic leukemia, and glioblastoma multiforme. Etoposide (CAS registry number 33419-42-0) chemically derives from podophyllotoxin, a toxin found in the American Mayapple.

Suitable pharmaceutically acceptable carrier(s), excipient(s) and/or adjuvant(s) for all aspects of the invention wherein etoposide is combined with the melphalan derivative are the same as those described above for the combination of the melphalan derivative and gemcitabine.

In all aspects of the invention when etoposide is used together with the melphalan derivative, the composition of the invention may also comprise a mixture of one or more derivatives of melphalan. The invention is also directed to a kit comprising a first container comprising

a melphalan derivative of formula I

wherein R is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino

cycloalkylamino or arylamino;

R₂ is

X is

wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid, or H, or a pharmaceutically acceptable salt thereof; and a second container comprising etoposide, or a

pharmaceutically acceptable salt thereof. The invention therefore is also directed to the use of such a kit for the preparation of a medicament for the prevention and/or treatment of ovarian cancer, bladder cancer and/or lung cancer, and such a kit for use for treating and/or preventing ovarian cancer, bladder cancer and/or lung cancer. Preferably, each container in such a kit only contains the melphalan derivative and etoposide as antitumoral substances. Preferred melphalan derivatives for this aspect of the invention are the same as those described above. Most preferably, the derivative of melphalan for this aspect of the invention is J1 , J3 or JV28, in particular J1. Such a kit may also comprise one or more device(s), such as a syringe, for administering the respective drugs of the two containers to a subject. Also, the kit may contain instructions for the sequential, separate or simultaneous administration of the melphalan derivative and the etoposide to a patient in need thereof. Preferred derivatives of melphalan for the kit are the same as those described above for other aspects of the invention. Preferably, in a kit the first container comprises the melphalan derivative J1 , J3 or JV28, in particular J1 , and the second container comprises etoposide. Preferably, the kit consists of one container comprising J1 , J3 or JV28, in particular J1 , and second container comprising etoposide. The melphalan derivative and the etoposide may be in admixture with a pharmaceutically acceptable carrier, excipient and/or adjuvant in their respective containers. The containers may comprise the melphalan derivative and the etoposide, respectively, in dry or liquid form. Also, the carrier(s), excipient(s) and/or adjuvant(s) may be present in one or more further separate containers for mixing with the melphalan derivative and the etoposide, respectively, before administration to a subject.

Another aspect of the invention is directed to a method for treating and/or preventing ovarian cancer, bladder cancer and/or lung cancer comprising administering a

pharmaceutical composition as described above comprising a. a melphalan derivative of formula I

R₂ is

X is

wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid,

or H, or a pharmaceutically acceptable salt thereof; and

b. etoposide, or a pharmaceutically acceptable salt thereof,

or a kit as described above comprising

a first container comprising a melphalan derivative of formula I as described above, or a pharmaceutically acceptable salt thereof; and

a second container comprising etoposide, or a pharmaceutically acceptable salt thereof, in a therapeutically effective dose to a subject in need thereof. Preferred melphalan derivatives for this aspect are the same as those described above. Most preferably the melphalan derivative is J1 , J3 or JV28, in particular J1. Preferably, the melphalan derivative and the etoposide are administered simultaneously or immediately after each other. A composition, or kit, comprising a melphalan derivative, and etoposide is preferably administered intravenously. The melphalan derivative and the etoposide may be administered to a subject

sequentially, with either of the compounds first, or simultaneously. If sequential administration is used, the compounds may be administered immediately after each other.

Consequently, the invention is also directed to the use of a derivative of melphalan as defined herein or a pharmaceutically acceptable salt thereof and etoposide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prevention of ovarian cancer, bladder cancer and/or lung cancer, wherein said derivative of melphalan and etoposide are to be administered

simultaneously.

The invention is further directed to a derivative of melphalan as defined herein or a pharmaceutically acceptable salt thereof and etoposide or a pharmaceutically acceptable salt thereof for use for the treatment and/or prevention of ovarian cancer, bladder cancer and/or lung cancer, wherein said derivative of melphalan and etoposide are to be administered simultaneously.

Preferably the amount of the melphalan derivative in such a composition or kit comprising etoposide and a melphalan derivative is about 3-7 mg/ml, such as about 5 mg/ml. The dose of the melphalan derivative to be administered when used together with etoposide is about 0.2-1 mg/kg, preferably about 0.3-0.4, or about 10-30 mg/m² body surface area, preferably about 12-16 mg/m² body surface area. Preferably etoposide in such a composition or kit is about 15-25 mg/ml, preferably about 20 mg/ml, and the dose administered is about 100-500 mg/m² body surface area, such as about 100-500 mg/m² body surface area or about 50-200 mg/m² body surface area per day five days in a row or about 100 mg/m² body surface area per day three days in a row. Often the treatment is repeated every three to four weeks, i.e. with a new five days treatment period. Experimental section

Example 1 : Evaluation of the antitumor activity of the melphalan derivative J1 in combination with gemcitabine Aim

To study the antitumor activity of J1 in combination with gemcitabine on a human tumor cell line from ovarian carcinoma and a cisplatinum resistant subline.

Cell lines

The A2780 ovarian carcinoma cell line and its cisplatinum resistant subline A2780/cis was obtained from ECACC and were cultivated in RPMI 1640 supplemented with 10% heat- inactivated fetal calf serum, 2 mM glutamine, 100 Mg/ml streptomycin, and 100 U/ml penicillin (Sigma). Compounds

J1 (L-melphalanyl-L-p-fluorophenylalanine ethyl ester hydrochloride) was supplied by Oncopeptides AB (Stockholm, Sweden) as a 100 mM solution in DMSO. Gemcitabine (Eli Lilly, Sweden AB, Box 721 , 169 27 Solna, Sweden) and the reference compound doxorubicin (Meda AB, Box 906, Solna, Sweden) was obtained from the local hospital pharmacy.

Experimental setup

Cytotoxicity assay procedure (FMCA)

The fluorometric microculture cytotoxicity assay (FMCA) is a total cell kill assay that was used to investigate the cytotoxic activity of J1-gemcitabine combinations (Lindhagen E. et al. 2008). FMCA is based on measurement of fluorescence generated from hydrolysis of fluorescein diacetate (FDA) to fluorescein by cells with intact plasma membranes. Briefly, 384-well microtiter plates (NUNC, Roskilde, Denmark) were prepared with drug solutions in triplicates at 10 times the desired drug concentrations. Cell suspensions were seeded into the drug-prepared plates with 4 000 cells per well and plates were then incubated for 72 h. After incubation, the plates were washed, FDA is added, and after 50 min of incubation the fluorescence generated (excitation 480 nm) was measured at 538 nm in a fluorometer (Fluorostar Optima, BMG Technologies). The fluorescence is proportional to the number of cells with intact plasma membrane present in the well. Plates were automatically handled by a SAIGAN Core System for high throughput screening. Calculation of combination effects

An additive model for estimating combination effects was used. The additive model predicts the effect of a combination to be equal to the product of its constituents. Thus if a drug combination is composed of single drugs producing SI values of 40% and 60%, respectively the combination would be expected to result in a SI value of 24% (0.4 x 0.6). An observed effect of a combination higher than predicted by the model indicate synergism whereas a lower value represents a subadditive effect. A ratio between the observed SI value and the SI predicted by the additive model was calculated for all combinations. If the ratio (index) exceeded 1 .1 the interaction was classified as subadditive, below 0.9 synergistic and between 0.9 and 1.1 were additive. The interval for the additive interaction was set to account for the intra-assay variability.

Results

The combination effects of J1 and gemcitabine in the parental A2780 cell line (n=2) are shown in Fig. 2a and Table . All three combinations demonstrated clear synergy with a mean interaction indices ranging from 0.2 to 0.68 (mean value 0.42). In contrast the reference compound doxorubicin produced only additive interactions (interaction indices ranging from 0.91 to 1.04) in the same cell line (Table 3). Also in the cisplatinum resistant subline J1 and gemcitabine produced synergistic interactions with indices ranging from 0.09 to 0.41 (mean value 0.26, Fig. 2 b and Table 2). Again the reference combination of J1 and doxorubicin demonstrated only subadditive to additive interactions (interaction indicies ranging from .04 to 1.1 1 , Table 4). Finally, strong synergy was observed also when J1 and gemcitabine combinations were tested on primary tumor cells from a patient with mesothelioma (Fig. 2 c).

Conclusions

Evaluation of the antitumor activity of the alkylating prodrug J1 in combination with gemcitabine demonstrated unexpected synergistic interactions, not only in cisplatinum sensitive but also in cisplatinum resistant tumor cells, which might be utilised in the clinical setting. Table 1. Effect of J1 and gemcitabine (G) combinations in parental human ovarian carcinoma A2780 cells

Table 2. Effect of J1 and gemcitabine (G) combinations in cisplatin resistant human ovarian carcinoma A2780 cells

Table 3. Effect of reference compound doxorubicin (D) and J1 combinations in parental human ovarian carcinoma A2780 cells

Table 4. Effect of reference compound doxorubicin (D) and J1 combinations in parental human ovarian carcinoma A2780 cells

Example 2: Addition of gemcitabine to J1 treatment enhances antitumour activity of J1 in a mouse xenograft model of ovarian carcinoma

Background and aim

In vitro studies on A2780 ovarian carcinoma cells showed that combining J1 with gemcitabine induced a synergistic effect with respect to tumor cell kill. The aim of these animal studies were to demonstrate that adding a subtoxic dose of gemcitabine to J1 improve J1 -antitumour activity on ovarian cancer cells also in vivo.

Experimental design

Drugs

As vehicle for gemcitabine physiological saline solution was used whereas for J1 Glucose solution (50 mg/ml) was applied. Animals whose tumors were used as controls received equal volume vehicle alone. Gemcitabine (Gemzar™, Eli Lilly, Sweden AB, Box 721 , 169 27 Solna, Sweden) was obtained as powder and a stock solution of 36 mg/ml was prepared in physiologic saline. Just prior to administration the stock solution was further diluted in physiologic saline to concentrations indicated in Table 5. Gemcitabine was given in an intraperitoneal route with an administration volume of 200 μΙ_.

A stock solution of 50 mg/ml was prepared in dimethyl acetamid (DMAc) and just prior to administration the stock solution was diluted to the indicated concentrations using Glucose solution (50mg/ml). J1 was given in an intravenous route with an administration volume of 100 μΙ_. Mice and tumor xenografts

For the animal experiments human epithelial ovarian cancer cell line A2780 (obtained from ATCC, US) were used for grafting in a tumor xenograft model. 2x 10⁶ A2780 cells were inoculated in 0.2 ml saline subcutaneously in the right flank of 7 week old SCID mice with a BALB c background. The tumor weight was followed and estimated using the formula: Tumor weight= length x width² as described in (Gallo et al., 2006). Treatment started when the tumor reached about 200 mg and given twice a week for two weeks given a total of 4 administrations as indicated in Table 5. For the experiment subtoxic doses of J1 and gemcitabine were used determined in preparatory experiments (data not shown). A concomitant administration of J1 and gemcitabine was used.

Results

Co- administration of J1 and gemcitabine increases J1 antitumor activity in vivo

Previous analysis in vitro have indicated that addition of gemcitabine potentiate J1- induced antitumour cell kill in ovarian carcinoma A2780 cells. With the aim to examine if addition of gemcitabine could potentiate the antitumor activity of J1 also in vivo the same cell line, A2780, was used for in vivo tumor xenograft experiments. For this purpose A2780 cells were inoculated subcutaneously into the flank of SCID mice and allowed to form tumors of the size of about 200 mg. In preparatory experiments sub toxic doses of J1 and gemcitabine has been assessed (data not shown) and were used for the combination experiments as indicated in Table 5. In total four administrations were done and the total doses for each drug is given in Table 5.

Table 5. Treatments for the A2780 ovarian xenograft experiment

Test Compound Dose No of Total dose No of group (mg/kg/day) doses (mg/kg) animals

1 - 0 (vehicle) 4 4

2 Gemcitabine 5 4 20 5

3 J1 2 4 8

4 J1 4 4 16

5 J1 8 4 32

6 Gemcitabine+J1 5+2 4 20+8 4 Gemcitabine + J1 5+4 4 20+16

Gemcitabine + J1 5 + 8 4 20 + 32

Tumor weight was followed three times a week and calculated as described in Material and Methods section. As can be seen in Figure 3, addition of a subtoxic dose of gemcitabine i.e. 5 mg/kg to a subtoxic dose of J1 i.e. 2, 4 or 8 mg/kg all caused an increased inhibition of growth of A2780 ovarian xenografts (Fig.2 A-B). This was evident both when median as well as mean value of ovarian cancer tumor weight was used (Fig. 2A and B respectively).

Conclusion

In this study it was examined if J1 and gemcitabine also in vivo could act in synergy with respect to growth inhibition of ovarian cancer cells. Indeed the results demonstrated that addition of gemcitabine to a subtoxic dose of J1 increases the potential of J1 to inhibit ovarian cancer cell growth. Example 3: Treatment of cisplatinum resistant tumors with the combination of the melphalan derivative J1 and gemcitabine in vivo

Combination of J1 and gemcitabine cause inhibition of A2780CIS (cisplatinum resistant cell line) ovarian cancer xenografts in vivo (see Fig. 4). A2780CIS (cisplatinum resistant) ovarian cancer cells were injected into SCID BALB c mice and allowed to form tumors. When the tumors reached the mean weight of 225±54 mg treatment with 5 mg/kg gemcitabine (triangles), 8 mg/kg J1 (squares) or a combination of J1 (8 (mg/kg) and gemcitabine (5 mg/kg, crosses) were applied twice a week for three weeks. Tumor growth was monitored every third day during the entire experiment and is depicted as tumor weight calculated by the formula Tumor weight= length x width². The number of mice in each treatment group was 5. Mean values of tumor weight are shown in Fig. 4 and the bars represent SEM. At day 20 all of the animals in the monotherapy groups with J1 or gemcitabine alone were dead, while 4 out 5 of the animals in the combination treatment group still were alive.

The data show that combination treatment of J1 with gemcitabine can overcome the cisplatinum resistancy and control tumor growth. Also, this experiment demonstrates a much higher tolerance to the combination treatment as the majority of the mice survived the treatment, as compared to when J1 and gemcitabine were used separately for treatment.

Example 4. Demonstration of synergistic effect of the combination of J1 and gemcitabine using PARP cleavage

Material and Methods:

Cell culture and drug treatment

The human ovarian cancer cell line A2780 (obtained from European Collection of Cell Cultures (ECACC), Health Protection Agency Culture Collection, and United Kingdom). The cells were maintained in RMPI-medium supplemented with 10% fetal calf serum (FCS) (both from Invitrogen) at 37 °C in a humidified atmosphere containing 5% carbon dioxide. The prodrug of melphalan, J1 , was from Oncopeptides AB, Stockholm, Sweden. The parental drug melphalan was purchased as Alkeran^® from the Swedish Pharmacy (Apoteket AB, Sweden). Either drug was dissolved in 99.5% ethanol with 2% HCI and then diluted in cell culture media to the indicated final concentrations. For both drugs the working solutions were freshly made prior to experiments. Gemcitabine was obtained from Lilly AB, Sweden and diluted according to the manufacturer's instruction and further diluted in cell culture media to the final concentration indicated.

Analysis of apoptotic signaling by PARP cleavage on western blot

To analyze the effect of the single and combination treatment in A2780, cells apoptosis was analyzed using poly (ADP-ribose) polymerase (PARP) cleavage on western blot as an endpoint. During apoptosis the 1 16 kDa full length PARP is cleaved by caspases most notably the executor caspase-3 into an 89 kDa fragment (Patel et a/., 1996). The disappearance of full length PARP and the appearance of the c-terminal 89 kDa cleavage fragments on western blot are widely used as a biochemical marker of ongoing apoptotic signaling and were used in the current analysis using an antibody recognizing both full length and cleaved PARP (Santa Cruz Biotechnology). Western blot extracts were made from untreated or treated A2780 cells by lysing the cells in RIPA buffer (50 mM Tris-HCI, pH 7.4, 150 mM NaCI, 1 mM EDTA, 0.1 % Na-deoxycholate, 1 % NP-40) containing protease and phosphatase inhibitor cocktail tablets (Roche). After a 10 min-incubation on ice, the lysates were cleared from insoluble cell debris by centrifugation at 13000 rpm 4°C for 15 minutes. For analysis with western blotting 50 pg of total cell lysates were resolved on a 4-12%Bis Tris Gel using MOPS SDS running buffer (NuPAGE, Invitrogen). The proteins were after separation on gel transferred onto nitrocellulose membranes (Thermo Scientific) during 90 min at 30V. After blocking for 60 min at RT in Odyssey blocking buffer (LI-COR Biosciences) immunoblotting was performed with the above described PARP antibody over night. After washing in TBS-T buffer the membranes were incubated for 30 min with a goat anti rabbit IR Dyes 800 CW (Odyssey LI-COR). Bands were visualized in the 800 channel on the ODYSSEY (LI-COR) and quantified using the Odyssey software. To correct for loading differences between the samples an antibody against GAPDH (Trevigen) followed by goat anti rabbit IR Dyes 800 CW (Odyssey LI-COR) and quantification as above. Results

A combination of J1 and Gemcitabine causes increased cleavage of PARP than either agent alone

Apoptotic signaling in response to J1 alone, gemcitabine alone and in combination at 48h post treatment using different schedules was analyzed (Fig. 5A). The effect of treatment on apoptotic signaling was measured as PARP cleavage, a substrate for caspase-3 activity. As can be seen, J1 in combination with gemcitabine caused an increased cleavage of PARP (as can be seen by the disappearance of full length PARP and appearance of the 89 kDa cleavage product) than either treatment alone in these A2780 ovarian cancer cells (Fig. 5B, left, S1 a). The ratio between cleaved and full length PARP was analyzed in the different treatments and a higher ratio indicate more apoptosis and hence cell death. Thus it was found that a combination of 0.1 μΜ J1 and 0.025 μΜ gemcitabine had a significant higher PARP ratio indicative of a higher amount of cell death (Fig. 5B, right). A combination of melphalan and gemcitabine causes less PARP cleavage than a combination of J1 and gemcitabine

Next it was examined if the parental substance melphalan showed similar synergistic effect with gemcitabine on PARP-cleavage (Fig. 5C). Two different concentrations of melphalan were used 0.1 and 1 μΜ respectively to a fixed concentration of gemcitabine. At 48h post treatment, a cleavage fragment of PARP was also observed when melphalan was used (Fig. 5C). However, it was not observed the same increase in PARP cleavage fragment appearance when melphalan of either 0.1 and 1 μΜ was used with gemcitabine as was observed with 0.1 μΜ J1 (compare Fig. 5B, S1 a with Fig. 5C S1 b and S1 c respectively). This was also confirmed when the ratio between cleaved and full length PARP was quantified (Fig. 5C, right). Thus at equimolar doses, a combination of J1 and Gemcitabine was clearly more effective than a combination of melphalan and Gemcitabine (compare Fig. 5B, S1 a with Fig. 5C). Moreover, a 10-fold increase in melphalan concentration still did not reach the effect observed between J1 and gemcitabine (Fig. 5B S1 a with Fig. 5C).

Concomitant addition of J1 and gemcitabine is better with respect to PARP cleavage then using either J1 or gemcitabine for 24h prior to addition of the other agent

Next it was examined if scheduling of the different substances influence this effect. The scheduling scheme is outlined in Fig. 5A. Interestingly, as described above, when J1 was added concomitantly with gemcitabine a increased cleavage of PARP, indicative of increased apoptotic signaling was observed (compare the lanes of J1 , gemcitabine with lane S1 a in Fig. 5B). Next it was analyzed if addition of J1 for 24h prior to gemcitabine addition would influence this synergistic effect. As can be seen, a reduced PARP cleavage was observed in S2 compared to S1 a (Fig. 6A). Next the opposite was tried, thus gemcitabine was added for 24h and then J1 was added for another 24h (Fig. 6B). Adding gemcitabine prior to J1 further reduced the combination effect (Fig. 6B). Thus the data suggest that concomitant scheduling of J1 and gemcitabine exerts a more pronounced effect on PARP cleavage and hence cell death.

Comments to the results

J1 vs melphalan in combination with gemcitabine

In these experiment a higher cytotoxic effect on A2780 cells (measured as increased PARP cleavage) is seen when gemcitabine is combined with J1 than if it is combined with melphalan. This effect is likely an effect of increased loading of melphalan intracellular^ when J1 is used.

Treatment with J1 results in 12 fold higher amount of intracellular melphalan than treatment equimlar amounts of melphalan. Increasing melphalan amount can not compensate for the synergy effect.

In these experiment cells were treated with 10 μΜ of either J1 or Melphalan. Fig. 7 shows the amount of Melphalan accumulated intracellular^ following up to 60 minutes. J1 gives an intracellular accumulation of melphalan that is considerable higher than treatment with Melphalan. This shows the increased rate of cellular uptake achieved with J1. The ratio is plotted in the bar diagram and shows that the increase is up to 12 times higher when treating with J1 . It should be noted that in the combination experiments described previously the amount of melphalan used was 10X that of J1 (i.e. 1 uM vs 0.1 uM resp.). The 10X increase in Melphalan concentration did not not result in an increase in PARP cleavage and showed only additive effect. This is in sharp contrast to the combination experiments using 0.1 uM J1 and combining with Gemcitabine. Here a strong synergy was shown. Synergistic effect of J1 and Gemcitabine is only seen with concomitant treatment and not with scheduling (treatment on separate occasions)

Figure 8 shows the effect of scheduling. S1A shows the strong synergistic effect on apoptosis and celldeath when the drugs are given concomitantly. In contrast when scheduling is used, where the drugs are given 24 hours apart (experiment S3), shows no synergy but only an additive effect.

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6. WO01/96367

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FASEB J 10: 587-97.

Claims

1. A composition comprising a derivative of melphalan having the formula I

wherein R-i is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino, cycloalkylamino or arylamino;

R₂ is

wherein R₃ is independently NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl,

N(CH₂CH₂CI)₂, N0₂, F, CF₃ or H, and n is 1 or 2;

X i

(ll)

wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid, or H,

or a pharmaceutically acceptable salt thereof; and

gemcitabine, or a pharmaceutically acceptable salt thereof,

2. A composition according to claim 1 , wherein the derivative of melphalan is L- melphalanyl-L-p-fluorophenylalanine ethyl ester (J1 ), L-melphalanyl-L-p- fluorophenylalanine isopropyl ester (JV28) and/or L-prolinyl-L-melphalanyl-L-p- fluorophenylalanine ethyl ester (J3); or pharmaceutically acceptable salts thereof.

3. A composition according to claim 2, wherein the derivative of melphalan is L- melphalanyl-L-p-fluorophenylalanine ethyl ester (J1 ).

4. A kit comprising

a first container comprising a derivative of melphalan as defined in any one of claim 1 -3, or a pharmaceutically acceptable salt thereof; and

5. A composition as defined in any one of claims 1 -3 or a kit according to claim 4 for use as a medicament.

6. Use of composition as defined in any one of claims 1 -3 or a kit according to claim 4 for the manufacture of a medicament for the treatment and/or prevention of cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer.

7. Use of a derivative of melphalan as defined in any one of claims 1-3 or a

pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prevention of cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer, wherein said derivative of melphalan and gemcitabine are to be administered simultaneously.

8. A composition as defined in any one of claims 1 -3 or a kit according to claim 4 for use for treating and/or preventing cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer.

9. A derivative of melphalan as defined in any one of claims 1-3 or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof for use for the treatment and/or prevention of cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer, wherein said derivative of melphalan and gemcitabine are to be administered simultaneously.

10. A method for treating and/or preventing cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, and/or breast cancer, comprising administering a composition as defined in any one of claims 1-3 or a kit according to claim 4 in a therapeutically effective dose to a subject in need thereof.

1 1 . A method according to claim 10, wherein said administration of said derivative of melphalan and said gemcitabine is performed simultaneously.

12. Use of a combination of a derivative of melphalan of formula I

R₂ is

wherein R₃ is independently NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl,

N(CH₂CH₂CI)₂, N0₂, F, CF₃ or H, and n is 1 or 2;

X i

wherein R₅ is H;

R₄ is a natural or modified cyclic or aromatic amino acid,

or H, or a pharmaceutically acceptable salt thereof; and

etoposide or a pharmaceutically acceptable salt thereof,

13. Use according to claim 12, wherein the derivative of melphalan is L-melphalanyl-L- p-fluorophenylalanine ethyl ester (J1 ), L-melphalanyl-L-p-fluorophenylalanine isopropyl ester (JV28) and/or L-prolinyl- L-melphalanyl- L-p-fluorophenylalanine ethyl ester (J3) or pharmaceutically acceptable salts thereof.

14. Use according to claim 13, wherein the derivative of melphalan is L-melphalanyl-L- p-fluorophenylalanine ethyl ester (J1 ).

15. A composition comprising a derivative of melphalan as defined in any one of claims 12-14, or a pharmaceutically acceptable salt thereof; and

etoposide, or a pharmaceutically acceptable salt thereof, for use for treating and/or preventing ovarian cancer, bladder cancer and/or lung cancer.

16. A kit comprising

a first container comprising a derivative of melphalan as defined in any one of claims

12-14, or a pharmaceutically acceptable salt thereof, and

a second container comprising etoposide, or a pharmaceutically acceptable salt thereof.

17. A method for treating and/or preventing ovarian cancer, bladder cancer and/or lung cancer comprising administering a composition as defined in any one of claims 12- 14 or a kit according to claim 16 in a therapeutically effective dose to a subject in need thereof.