WO2007063124A1

WO2007063124A1 - Use of a compound comprising a camptothecin derivative for preparing pharmaceutical formulations useful in the treatment of melanoma

Info

Publication number: WO2007063124A1
Application number: PCT/EP2006/069182
Authority: WO
Inventors: Monica Binaschi; Mario Bigioni
Original assignee: Menarini International Operations Luxembourg SA
Current assignee: Menarini International Operations Luxembourg SA
Priority date: 2005-12-02
Filing date: 2006-12-01
Publication date: 2007-06-07
Anticipated expiration: 2008-06-02
Also published as: ITFI20050246A1; AR057958A1; TW200731989A

Abstract

The use of a known compound of formula (I) is described for preparing pharmaceutical formulations useful in the treatment of melanoma.

Description

USE OF A COMPOUND COMPRISING A CAMPTOTHECIN DERIVATIVE FOR PREPARING PHARMACEUTICAL FORMULATIONS USEFUL IN THE TREATMENT OF MELANOMA

FIELD OF THE INVENTION

The invention relates to the field of pharmaceutical formulations, and in particular formulations for the treatment of human melanoma.

STATE OF THE ART

Melanoma is a malignant tumor which originates from melanocytes in the stratum basale of the epidermis and is a typical example of polyphasic tumor progression.

It develops in two growth phases, radial and vertical, sequential over time. The vertical growth phase is marked by the appearance of one or more cell clones that acquire the capacity for autonomous proliferation and cohesive growth with formation of aggregates or nodules which extend into the reticular dermis or subcutaneous layer. This phase, which arises in an unpredictable manner, is a significant moment in tumor progression in that the melanoma acquires invasive and metastasizing properties. The neoplasia spreads both via the lymphatic and blood systems.

As with other neoplasias, recent evidence suggests that melanoma arises from a transformed stem cell which is able to renew itself, to differentiate into progeny and to continue growing. It is a neoplasia that has been progressively increasing over the last 50 years in the white population all over the world; exposure to intense and intermittent ultraviolet irradiation is today considered to be an important etiologial factor in this disease. Melanoma, while still in the regional spreading stage, is a pathology of surgical interest in that there are currently no valid therapeutic options.

Instead chemotherapy, immunotherapy or radiotherapy are considered to be first choice treatments in the case of extra-regional spread of the disease.

In clinical practice, however, all testing carried out with traditional chemotherapeutic agents, with dacarbazine alone or combined with other factors, have not shown any benefit in the treatment of patients with melanoma at this stage of the disease. Melanoma therapy therefore represents a great challenge because of its high metastatic potential and pharmacoresistance.

Of the cytotoxic agents, camptothecins are the most specific and effective molecules in preclinical models. The molecular target of camptothecins is a nuclear enzyme, DNA topoisomerase I, which plays a key role in DNA replication, transcription and repair. DNA topoisomerase I acts by relaxing supercoils of the DNA double helix, incurring a breakage and subsequent religation in the single DNA strand. Camptothecins form a reversible ternary complex with DNA and the enzyme, which prevents DNA religation. A model based on the X-ray structure of the ternary complex was recently reported, in which camptothecin intercalates at the site of DNA breakage and the planar ring system mimics the pairing of DNA bases (Staker BL et al. PNAS₁ vol. 99, 2002; pp:15387-15392). The cytotoxic antitumor effect of this class of molecules takes place when the reversible ternary complex collides with the replication fork of DNA causing irreversible DNA breakages.

Although this enzyme is not specific for the tumor cell, DNA topoisomerase I is recognised as a useful target and is exploitable for effective treatments. In this respect the ability of camptothecins to inhibit tumor growth in several tumors xenografted into nude mice is surprising. In line with this principle, a large number of camptothecins are in clinical trial. It is now evident that the clinical activity of this class of molecules is lower than that anticipated on the basis of pre-clinical activity and in order to improve the side-effects of classical camptothecins various strategies have been adopted. One strategy is to covalently bind a polymer to camptothecin. This system confers solubility, reduces binding to proteins, increases lactone stability and prolongs exposure to the drug by the slow release of the active principle. A compound is known which comprises a camptothecin derivative and can be defined as : Glycinamide, glycylglycyl-N-[3-[[(4S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy- 3,14-dioxo-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl]oxy]propyl]-, compd. with dextran carboxymethyl ether sodium salt (CAS registry number: 187852-63- 7) as is hereinafter defined as MEN 4901/T-0128, this compound having the general formula (I)

(I) where X represents a polymer carrier, carboxymethyl dextran (CM-dextran), bound through an amide bond to the glycine.

The active part of the molecule is represented by the camptothecin derivative, denominated T-2513 (CAS registry number: 187793-52-8) of formula (II)

(II) which is bound through a bridge of three glycine residues to the CM-dextran carrier. The compound of formula (I) thus defined has a weight average M.W. varying between 110000 and 160000 daltons, preferably between 123000 and 153000 daltons.

The percentage of T-2513 (compound of formula (II)) on the total weight of the compound is in the range from 4 - 6% wt/wt, preferably between 4.5 and 5.5% wt/wt.

The polymer carrier is a dextran which presents carboxymethylene groups bound through an ether bridge to the glucose units comprising the polymer backbone; the percentage of glucose units substituted by a carboxymethylene group to total number of units for each polymer varies from 35 to 55%, preferably from 40 to 45%. The number of glucose units which bear a carboxymethylene group to which the camptothecin of formula (II) is conjugated through a peptide linker varies on average from 1.5 to 3% of the total number of glucose units in the polymer, preferably from 1.9 to 2.1%. The intellectual property of the compound of formula (I) is claimed in patent EP757049 filed on 30/07/1996.

The polymer carrier CM-dextran is also known and can be prepared in accordance with the method described in WO9419376. The compound MEN 4901/T-0128 is a prodrug which undergoes metabolic activation in vivo by means of a proteolytic enzyme known as cathepsin B. Enzymatic activation performed by cathepsin B on the triglycine bridges releases the compound T-2513 with cytotoxic action which is thus able to carry out its toxic action on tumor cells. Experimental studies have shown that this compound is selectively captured by the tumor. The CM-dextran macromolecule carries the cytotoxic compound T-2513 to the tumor cell which is hence able to exert its pharmacological action. More recently, scientific data published in international journals have demonstrated the role of macrophage cells in the selective cytotoxicity of MEN 4901/T-0128. A large number of macrophages associated with the tumoral stroma, whose function is not fully understood, are known to be present at the tumor site. One of the main functions of this cell population is related to their phagocytic capacity, i.e. engulfing and digesting foreign bodies. Digestion is mainly achieved by lysosomal proteolytic enzymes such as the previously mentioned cathepsin B. It was thus shown that these macrophages internalize MEN 4901/T-0128 and, by means of cathepsin B, release the compound T-2513. It is released into the extra-cellular space and comes into contact with tumor cells which are subjected to all the cytotoxic potential of the compound T-2513 and incur cell death and/or apoptosis (Harada M. et al J. Of Controlled Release, 2000, 69: 389-397). Currently two camptothecins are approved for clinical use, topotecan and irinotecan.

Topotecan is approved for the treatment of ovarian and lung cancers, while irinotecan is used in colon carcinoma therapy. Hence, no camptothecins are currently registered for treating melanoma and a phase Il clinical trial (Cancer Investig. 1997; 15:318-320) with topotecan and melanoma did not show any activity. Pharmaceutical compositions containing compounds of general formula (I) or their analogues for inhibiting or preventing malignant tumor metastases were described in WO03015826, but melanoma has never been cited as being a tumor potentially vulnerable to attack and neither has the surprising activity that MEN 4901/T-0128 shows against this tumor ever been surmised. DESCRIPTION OF THE INVENTION

MEN 4901/T-0128 is surprisingly found to be very active in inducing substantial and prolonged growth regression of human metastatic melanoma characterized by an extremely aggressive phenotype and by a natural resistance to treatment with common currently used chemotherapeutic agents. The gold standard for this tumor pathology is considered to be dacarbazine, a drug initially synthesized as a selective inhibitor of purine synthesis and subsequently found to be an alkylating agent. The percentage clinical response to dacarbazine is between 15% and 20% with less than 5% complete responses, with a median response duration of 4 months. Dacarbazine, when administered to mice at the optimal dosage of 100 mg/kg for three consecutive administrations 7 days apart, is unable to inhibit tumor growth, which is actually found to match that observed in the control group. In mice xenografted with human metastatic melanoma, the compound MEN 4901/T-0128, characterized as a topoisomerase I inhibitor, after a single intravenous administration at a dose of 80 mg/kg of the corresponding active principle, shows tumor volume regression equivalent to a 95% inhibition of tumor volume when compared to the volume measured in the control group. The compound MEN 4901/T-0128 shows an extremely varied and interesting spectrum of antitumor activity and, in xenografted mice, also proves to be active on other tumor histotypes. However, the action demonstrated on metastatic melanoma appeared to be particularly effective when compared with other tumors. In particular, good antitumor activities have been observed in other gastrointestinal tumors using the same administration means, but with less tumor volume inhibition than observed with metastatic melanoma. Furthermore, in this latter case antitumor action was found to be extremely long lasting and displayed significant tumor growth inhibition even months after tumor implantation. Translated into clinical terms, this signifies that MEN 4901/T-0128 has a direct action on minimum residual disease and hence on disease-free survival. The activity of MEN 4901/T-0128 on metastatic melanoma is found to be greater than other camptothecins currently used for chemotherapy of solid tumors when studied under the same conditions. Despite lrinotecan (Aventis Pharma) being the most effective camptothecin for solid tumor treatment in current clinical use and also proving effective for treating metastatic melanoma xenografted into nude mice, it is found to have a lower activity than MEN 4901/T-0128. lrinotecan inhibits tumor volume growth by 80% whereas MEN 4901/T-0128 achieves 95%. In addition, MEN 4901/T-0128 has shown a more prolonged and enduring action than irinotecan. Consequently, even in the form of pharmaceutically acceptable salts, MEN 4901/T-0128 is an effective compound for treating human primary and metastatic melanoma, particularly metastatic melanoma.

The pharmaceutical formulations used are prepared in accordance with methods well known to the expert of the art and are commonly described in the Pharmacopoeia.

Administration of MEN 4901/T-0128 to a patient needing treatment can be undertaken by the routes normally used for treating this pathology, preferably by injection, particularly intravenous. One pharmaceutical form suitable for administration by injection is an aqueous solution, generally contained in vials. Alternatively the formulation can be in solid form to be dissolved in an aqueous solution at the time of treatment. A particularly advantageous form can be a lyophilized solid obtained from an aqueous solution comprising the active principle which is lyophilized for improved stability during storage and can be reconstituted as an aqueous solution at the time of therapeutic use.

The pharmaceutical forms used, in addition to the active principle, can possibly contain certain excipients commonly used in the art to include by way of example pairs of salts suitable as buffers, such as salts containing the pair (HPO₄)^2" /(H₂PO₄)^" for the phosphate butter or citric acid/citrate for the citrate buffer, and stabilizers, antioxidants etc.

In terms of active principle concentration, the pharmaceutical forms used to treat metastatic melanoma contain a MEN 4901/T-0128 quantity from 200 to 2000 mg per dosage unit.

The preferred dosage for the treatment of metastatic melanoma can range from 500 mg/m² to 3600 mg/m² of active principle per single administration; said administration can be repeated after 4-8 weeks for 4-6 times. MEN 4901 /T-0128 can be effectively used as a chemotherapeutic treatment of metastatic melanoma, before or after surgical removal of the tumor mass. Combined therapies can also be used, in a simultaneous or sequential manner, in which formulations containing MEN 4901/T-0128 are administered with other therapies currently in use for melanoma, such as radiotherapy, conventional chemotherapy, biochemotherapy and immunotherapy such as antitumor vaccinations. In particular, chemotherapeutic agents usable in combination with MEN 4901/T-0128 can be chosen from: Dacarbazine, Temozolomide, Carmustine, Fotemustine, Hydroxyurea, Tamoxifen, Cisplatin, Vinca alkaloids and Taxanes; biochemotherapeutic agents such as: BCG (Bacillus Calmette-Guerin or BCG Pasteur), lnterleukin-2, α-lnterferon; antitumor vaccines such as Melacine and Canvaxin. Example 1

5g of MEN 4901/T-0128 are dissolved in 50 ml of deionised water. 0.3 g of sodium hydrogen phosphate, 0.3 g of citric acid are added and the solution is brought to pH 6-6.5 by small additions of a 0.5M sodium dihydrogen phosphate solution or citric acid solution. Water is added to achieve a total of 100 ml. The solution is filtered and sub-divided into 5 ampoules which are then lyophilized. Example 2: Cytotoxic activity of MEN 4901/T-0128 in human cell lines of various histotype. The in vitro cytotoxic effect of MEN 4901/T-0128 in various human tumor cell lines was evaluated after 48 hours' incubation. Table 1 shows the percent survival of cells treated with 0.5 mg/ml of MEN 4901/T-0128 compared with the control cells. The results in table 1 indicate that the cytotoxic activity of MEN 4901/T-0128 is greater in the melanoma cell line (1205Lu) than in human pancreatic (ASPC-1), monocytic leukemia (THP-1) and colon (HT-29) tumor cell lines. Table 1

Cytotoxic effect of MEN 4901 /T-0128

Example 3: Activity of cathepsin B in human cell lines of various histotype. Cathepsins are proteolytic enzymes which are highly expressed in tumor cells. Previous results (Anti-Cancer Drugs, 17(10): 1119-1126, November 2006) have shown that human cathepsin B is able to release T-2513 from MEN 4901/T-0128 at pH 3-5, hence suggesting an intracellular release of the active principle. In table 2 cathepsin B activity is given, expressed as the fluorescent intensity obtained by a fixed number of cells. The cells, lysed in 0.1% triton X-100, were incubated for 1 hour with 200 μm of a substrate specific for cathepsin B (Z-arg- arg-AMC). The tabulated results show that the level of enzymatic activity of the metastatic melanoma cells is greater than the tumor histotypes portrayed (respectively, monocytic leukemia, pancreatic, oesophagal, colon, ovarian). Table 2

Cathepsin B activity in human cell lines

The activity given is normal compared with the enzymatic activity measured in

THP-1 cells

Example 4: Antitumor activity of MEN 4901/T-0128 on a human metastatic melanoma line xenografted subcutaneously in athymic mice.

MEN 4901/T-0128 was assayed for its in vivo biological activity on a human melanoma model (1205Lu) xenografted into athymic mice in accordance with previously described methods.

Tumor growth inhibition values are given in table 3, obtained after a single intravenous administration of MEN 4901/T-0128 when the tumor in the animal had achieved an evident and measurable volume. The compound was injected at the doses of 20, 40 and 80 mg/kg (expressed as equivalents of T-2513).

The table shows a significant dose-effect relationship with increasing dose, with a maximum antitumor effectiveness obtained at a dosage of 80 mg/kg. Table 3

Dose-effect relationship of MEN 4901/T-0128 in metastatic melanoma Groups Dose Treatment TVI% LCK (Ig)

(mg/kg) schedule

MEN 4901/T-0128 80 single 95 3.3

" " 30 single 85 0.7

" " 20 single 71 0.6

Example 5: Antitumor activity of MEN 4901/T-0128 towards various human tumor histotypes. The MEN 4901/T-0128 of the present invention was tested for its biological activity in vivo on models of human tumor xenografted into athymic mice in accordance with a method widely described in the literature (Anticancer Drug Development Guide, Beverly A. Teicher, Humana Press, 1997). The activities in terms of percent tumor volume inhibition measured in the group treated with MEN 4901/T-0128 compared with the tumor volume measured in the control group are given in table 4. When the tumor in the animal had achieved an evident and measurable volume, the compound MEN 4901/T-0128 was administered by single intravenous injection, at a dose corresponding to 80 mg/kg of active principle. The data given show that the more promising antitumor activity is obtained in the melanoma model (1205Lu) rather than in the pancreatic (ASPC- 1) or oesophagal (OE-21) models.

Table 4 Antitumor activity of MEN 4901/T-0128 on human tumors xenografted into immunosuppressed animals

Tumor histotype TVI %

1205Lu 95

ASPC-1 67

OE-21 70

TVI = Tumor Volume Inhibition calculated in accordance with the following formula:

100 - [(median tumor volume in treated group: median tumor volume in control group) x 100]

Example 6: Activity on metastatic melanoma compared with other camptothecins and dacarbazine.

The biological activity of MEN 4901/T-0128 on the previously described human melanoma model was compared with the activity of a known camptothecin such as irinotecan and with dacarbazine (the only chemotherapeutic agent currently used for metastatic melanoma therapy).

Table 5 shows the activities in terms of percent inhibition of tumor volume measured in the two groups treated with MEN 4901/T-0128 and irinotecan and decarbazine compared with tumor volume measured in the control group. When the tumor in the animal attains an evident and measurable volume, the animals are divided randomly into different groups and treated as follows: the compound MEN 4901/T-0128 is administered with a single intravenous injection at a dose corresponding to 80 mg/kg of active principle, lrinotecan is administered by intravenous injection following a schedule given in the literature (F. Lavelle et al. Seminars in Oncology, Vol.23, No 1 , Suppl. 3, 1996; pp: 11-20) at the maximum tolerated dose (MTD), i.e. 60 mg/kg, with an i.v. administration every four days for a total of 4 administrations giving a total dose of 240 mg/kg. Dacarbazine is administered at an optimal dosage for a mouse, that is 100 mg/kg for three consecutive administrations 7 days apart, as given in the literature (DJ Dykes et al., Contributions to Oncology. Basel, Karger, 1992, Vol. 42: 1-22). The results show that with the described schedules, MEN 4901/T-0128 has a greater antitumor activity than irinotecan and dacarbazine.

Table 5

Antitumor activity of MEN 4901/T-0128, irinotecan and dacarbazine on human melanoma xenografted into immunosuppressed animals

TVI %

MEN 4901/T-0128 Irinotecan Dacarbazine

1205Lu 95 80 6 TVI = Tumor Volume Inhibition calculated in accordance with the following formula:

Example 7: Activity of MEN 4901/T-0128 on a model of pulmonary metastasis in athymic mice xenografted subcutaneously with cells of human metastatic melanoma.

The human metastatic melanoma model when inoculated subcutaneously in athymic mice presents the peculiar characteristic of metastatizing in the lungs of the athymic mice a long time (40-50 days) after the subcutaneous implantation. According to the literature, metastatization of the human line in athymic mice is a relatively rare occurrence, however some human tumor lines such as those utilized in our studies can present this phenotype (Anticancer Drug Development

Guide, Beverly A. Teicher, Humana Press, 1997). For the purposes of the study, human tumor cells were inoculated subcutaneously into athymic mice by the same methods seen in the dose-effect correlation study. When the tumor has attained a measurable volume, the animals were randomised and treated intravenously with MEN 4901/T-0128, using the most effective dose of 80 mg/kg. Two additional groups were then introduced for a comparison of activities with known drugs. One was dacarbazine, the drug of choice in melanoma treatment, and the other was irinotecan (CPT-11) one of the most active camptothecins, currently used in antiblastic chemotherapy. The study was considered to be at its conclusion when tumor volume reached its limit of 10% of total animal weight. The animals were then killed and the lungs excised, fixed in formaline and microscopically analysed to observe the presence of metastases. As shown in table 6, dacarbazine was found to be ineffective for controlling the appearance of lung metastases whereas MEN 4901/T-0128 was found to be particularly active in inhibiting their growth. Furthermore the anti- metastatic activity of MEN 4901/T-0128 was attained at a much lower dosage than that used for CPT-11 , indicating that the product has greater potency for equal effectiveness.

Table 6 Antitumor and anti-metastatic activity of MEN 4901/T-0128.

Groups Treatment Median tumor Observation by

(mg/kg) volume (mm³)* microscopy

Carrier - 2186 ± 134 Multifocal metastasis

Dacarbazine 300 2244 ± 322 Multifocal metastasis

CPT-11 240 1129 ± 241 -

MEN 4901 80 638 ± 313 -

* volume of tumor nodule obtained by subcutaneous inoculation of metastatic melanoma cells.

Claims

1. Use of a compound of general formula (I)

(I) wherein X represents a polymer carrier of carboxymethyl dextran (CM-dextran) bound through an amide bond to glycine, for preparing pharmaceutical compositions for the treatment of human primary and metastatic melanomas.

2. Use as claimed in claim 1 , wherein the weight average M.W. of said compound of general formula (I) can vary from 110000 to 160000 daltons and the percentage of the compound of formula (II)

(H) on the total weight of the compound is within the range 4-6% wt/wt.

3. Use as claimed in claim 2, wherein the weight average M.W. of the compound of general formula (I) can vary from 123000 to 153000 daltons and the percentage of the compound of formula (II) on the total weight of the compound is within the range 4.5 to 5.5% wt/wt.

4. Use as claimed in claim 1 , wherein said pharmaceutical compositions are of injectable type and are chosen from: aqueous solution, solid dissolvable in an aqueous solution at the time of use or a lyophilized solution to be reconstituted in an aqueous solution at the time of use.

5. Use as claimed in claim 1 , wherein the active principle is present in a quantity from 200 to 2000 mg per single dosage unit.

6. Use as claimed in claim 1 , wherein said pharmaceutical formulations are provided for the treatment of human melanoma before or after surgical removal of the tumor mass.

7. Use as claimed in claim 1 , wherein said pharmaceutical formulations are useful in combined administration with other therapies, given simultaneously or sequentially, chosen from radiotherapy, chemotherapy, biochemotherapy and immunotherapy.

8. Use as claimed in claim 7, wherein said formulations are useful in combined administration with other chemotherapeutic agents, given simultaneously or sequentially, chosen from: Dacarbazine, Temozolomide, Carmustine, Fotemustine, Hydroxyurea, Tamoxifen, Cisplatin, Vinca alkaloids and Taxanes.

9. Use as claimed in claim 7, wherein said formulations are useful for simultaneous or sequential administration with biochemotherapeutic agents chosen from: BCG, lnterleukin-2, α-lnterferon.

10. Use as claimed in claim 7, wherein said formulations are useful for simultaneous or sequential administration with antitumor vaccines chosen from: Melacine and Canvaxin.

11. Pharmaceutical composition as claimed in claim 1 , comprising a compound of general formula (I) as active principle for treating human primary and metastatic melanomas.

12. Method for the treatment of human melanoma wherein from 500 mg/m² to 3600 mg/mg² of the compound of formula (I) claimed in claim 1 are administered per single administration.