Ha-paclitaxel conjugates for treating mesothelioma

Abstract

Described are HA-paclitaxel conjugates for local regional treatment of mesothelioma, i.e. malignant pleural mesothelioma, pericardial mesothelioma and peritoneal mesothelioma, preferably malignant pleural mesothelioma, and related pharmaceutical compositions.

Description

[0001] Inventive Purpose

[0002] Described are conjugates of hyaluronic acid with paclitaxel and compositions comprising or consisting of such conjugates and of a pharmacologically acceptable diluent / excipient for use in the treatment of mesothelioma, in particular malignant pleural mesothelioma. TECHNICAL FIELD

[0003] The term tunic or tunica refers to a specific tissue formed by a single layer of flat epithelium called mesothelium, which is sustained by underlying connective tissue, vascularized tissue and innervated tissue. The function of the tunic is to cover the organs of the thoracic and abdominal cavity, thus it is different in the splanchnic layer (the layer or lamella that covers the organs) and in the parietal layer (the outer layer or lamella that faces the wall and is lined on the wall); between the two layers a virtual cavity called tunic cavity is determined, which contains the appropriate amount of serous fluid produced by the mesothelium, while the underlying connective tissue provides the blood and nerve supply, the mesothelium being completely devoid of blood vessels and nerves.

[0004] Based on the organs or walls covered, the tunic is distinguished into:

[0005] • covering the heart and mediastinum, called pericardium;

[0006] • covering the lungs and chest wall, called pleura;

[0007] • covering the abdominal wall and the organs contained therein, called peritoneum;

[0008] • covering the testicles, called tunica vaginalis;

[0009] • finally covering the uterus, called perimetrium.

[0010] The pleura is therefore a double-walled tunic, with its parietal layer covering and adhering to the inner wall of the thoracic cavity and with its splanchnic layer covering and adhering to the distal wall of each lung.

[0011] The neoplasm produced by mesothelial cells is called mesothelioma and can therefore originate in the cavities around the heart and in the membranes covering the testicles, in addition to the thoracic and abdominal cavity, very rarely.

[0012] Mesothelioma is a rare tumor, even if its incidence is constantly increasing, with 2.2 cases per million people, but it represents less than 1% of all oncological diseases; it is a very aggressive neoplasm and the most frequent form is the one affecting the pleura: in this case it is defined as malignant pleural mesothelioma (MPM), which represents about 80% of all mesotheliomas, peritoneal, pericardial and testicular mesotheliomas being more rare; it is more common in men, the incidence of mesothelioma increases with age, with 75% of patients being over 65 years old.

[0013] The most important risk factor for pleural mesothelioma (but generally applicable to all types of mesothelioma) is represented by exposure to asbestos: in fact, the majority of these tumors affect people who have come into contact with this substance mainly at the workplace, or who have lived with people who worked with this substance. Asbestos is a natural mineral with fibrous structure, belonging to the chemical class of silicates, and due to its unique resistance to heat, it has been widely used in the past in roofing seals and insulations (in the material called Eternit), in ships and trains, in buildings (roof tiles, floor, paint...), in fireproof clothing, in cars (mechanical parts and paint) and in other fields.

[0014] When this mineral is broken, for example during the extraction and processing of the mineral or wear of materials containing it, a very fine powder is generated which remains suspended in the air even for long periods of time, and which is easily inhaled. This powder can damage mesothelial cells, causing (in some cases) cancer even decades after exposure. Other risk factors for mesothelioma are:

[0015] • Simian virus SV40, used in polio vaccines between 1955 and 1963;

[0016] • Thorium dioxide used between the 1920s and the 1950s;

[0017] • Radiation to the chest and abdomen.

[0018] (Sekido Y. et al., Carcinogenesis, 2013, 34(7): 1413-9; Remon J. et al., Cancer Treat Rev, 2013, 39(6): 584-91).

[0019] The treatment of mesothelioma depends overall on the site of the tumor, the stage of the disease, the age and general health of the patient. Standard treatment options include surgery, radiotherapy and chemotherapy.

[0020] In general, in stage I MPM, the tumor is confined to the parietal pleura and it is not present in the lymph nodes; in stage II, it also affects the visceral pleura, the lung or the diaphragm; in stage III, the tumor has invaded the first layer of the chest wall, a portion of the mediastinum or a point on the chest wall; it can also affect the outer surface of the pericardium and the lymph nodes on one of the two sides of the thoracic cavity; in stage IV, the tumor has reached other organs (metastases), such as the liver, the brain, the bone or the lymph nodes on the two sides of the thoracic cavity.

[0021] According to recent clinical studies, chemotherapy can be considered the only treatment able to (at least partially) prolong the life of the patient, although limited, improving its quality. The drugs approved for such use, usually used alone (or more often in combination) are pemetrexed Cisplatin and raltitrexed While mitomycin, vinorelbine and gemcitabine are still in the experimental phase, however, malignant pleural mesothelioma MPM (like all other forms of mesothelioma) is usually refractory to such treatments and the results obtained are modest (Stahel RA. et al., Ann Oncol., 2015, 26(8): 1649-60). Chemotherapeutic agents also have very debilitating side effects, so there are many uncertainties and even contraindications regarding the timing of the start of chemotherapy at the time of diagnosis of MPM or at the onset of early symptoms.

[0022] To date, there is no therapy capable of treating or significantly slowing down malignant pleural mesothelioma, only possible to reduce its tumor mass, thus prolonging the survival of the patient for a few months. The most successful surgery involves the early stages of the disease and combines preoperative chemotherapy with surgical intervention and subsequent postoperative radiotherapy. However, such a method can only be used for very young patients without concomitant disease. In any case, there are no adequate studies showing effective benefits in terms of prolonging life. Unfortunately, the average survival time after diagnosis is still 6-9 months without chemotherapy and 12 months with such therapy.

[0023] In view of the above reasons, new drug methods are being tested, such as immunotherapy or treatments below, which directly target the neoplasm by using drug delivery systems. In fact, such systems can transport the drug directly to the tumor site, allowing the treatment of the neoplasm with lower doses of chemotherapeutic agents than systemic therapy, thus having lower toxicity and producing a better quality of life of the patient, but especially, such systems, when truly effective, are able to significantly increase the life expectancy of cancer patients.

[0024] Hyaluronic acid is one of the polymers (and polysaccharides) used to form such drug delivery systems, both as an associative polymer and as a polymer chemically bound to many classes of antitumor drugs (Liao YH. et al., Drug Deliv., 2005, 12(6):327-42). HA is a heteropolysaccharide consisting of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine, which is a polymer with a molecular weight of 50,000 Da to 13 x 10 6Da is a linear polymer with a molecular weight that depends on its source and the method of preparation used. It is naturally found in gels surrounding cells, as an essential component of connective tissue in vertebrate organisms, in synovial fluid, vitreous humor, and in the umbilical cord. HA plays important roles in biological organisms, such as providing mechanical support for cells in many tissues, including skin, tendons, muscles, and cartilage; serving as a major component of the extracellular matrix; and also playing other roles such as tissue hydration, cell lubrication, migration, and differentiation (Weigel P. et al., J Theoretical Biol, 1986: 219-234).

[0025] In drug delivery systems using HA, conjugates of paclitaxel with hyaluronic acid (HA) derived from hydrazide molecules are known, which are linked to the carboxyl group of HA via an amide bond (Luo Y. et al., Biomacromolecules, 2000, 1(2):208-218); however, to date, there have been no preclinical or clinical trials that have demonstrated the effectiveness of such derivatives.

[0026] A system consisting of HA conjugated to paclitaxel via a spacer group (EP2045270) is also known: such conjugates have been tested in both in vitro (and particularly in bladder, breast, and ovarian cancer cells) trials demonstrating at least as much activity as the reference drug and in an in vivo clinical protocol for the treatment of noninvasive bladder cancer (currently underway) (IT102018000009731).

[0027] The conjugation with HA makes paclitaxel soluble, and therefore paclitaxel can be administered without various hypersensitivity reactions that typically occur during drug use due to the presence of Cremophor EL (currently used as a solubilizing solvent in standard clinical regimens) because the active ingredient is lipid-soluble. Its existence is essential. Paclitaxel is an anticancer agent (Huizing MT et al., Cancer Inv., 1995, 13:381-404), which exerts its antiproliferative effect by acting on the organization of microtubules in the cytoskeleton system, preventing their normal reorganization during mitosis (Manfredi JJ et al., J Cell Biol, 1982, 94:688-696); its main therapeutic indications are the treatment of breast cancer, lung cancer, ovarian cancer, bladder cancer, prostate cancer, and endometrial cancer, and Not including Treatment of mesothelioma.

[0028] In the HA-paclitaxel conjugate, the bond between paclitaxel and HA allows the drug to reach directly the membrane surface of tumor cells characterized by the overexpression of the HA receptor CD44 (Isacke CM. et al., Int J Biochem Cell Biol, 2002, 34:718-21). Therefore, paclitaxel conjugated to HA is able to bind specifically to CD44 of tumor cells, thus being able to enter the cytoplasm, where it is activated by the hydrolysis of the bond drug / HA. This drug selective transport mechanism is defined as "targeted" to the target cell. Inventive Details

[0030] The aim of the present application is an HA-paclitaxel conjugate for the local area treatment of mesothelioma, therefore of malignant pleural mesothelioma, pericardial mesothelioma and peritoneal mesothelioma, in particular for the treatment of MPM, wherein the HA-paclitaxel conjugate has an ester bond between the carboxyl of hyaluronic acid (HA) and a spacer, in turn bound to the hydroxyl on carbon C2' of paclitaxel by means of the ester bond through its carboxyl, wherein the spacer introduced is 4-bromobutyric acid, and wherein the degree of derivatization of paclitaxel in the HA-paclitaxel conjugate ranges from 15% to 21% weight / weight (w / w), and preferably from 16% to 20% w / w.

[0031] In the present description hereinafter, the degree of derivatization (or esterification) of the above conjugate means the percentage by weight of paclitaxel with respect to the weight of the HA-paclitaxel conjugate.

[0032] Therefore, 100 mg of conjugate with a degree of derivatization of 15% to 21% w / w will contain 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg, or 21 mg of the chemotherapeutic agent paclitaxel, depending on the degree of derivatization indicated (to further illustrate this, at a degree of derivatization of 20% w / w, 100 mg of conjugate contain 20 mg of paclitaxel); however, it is obvious to the skilled person that small variations in the weight ratio between molecules can occur at the end of such an industrial synthesis process, therefore in the following, the Applicant intends to claim all the reported percentage values comprising ± 1% when describing and claiming the above conjugate with a degree of derivatization ranging from 15% to 21% w / w: as an example, a degree of 20% w / w therefore intends 20% ± 1%.

[0033] The HA-paclitaxel conjugate for the above claimed use is prepared according to the synthesis method known in the state of the art described in EP2045270, and improved in IT 102018000009731 for a degree of derivatization of 20%.

[0034] Broadly speaking, in such a synthesis process of the above conjugate, the spacer / linker 4-bromobutyric acid is introduced between hyaluronic acid and paclitaxel by forming an ester bond between the carboxyl group of hyaluronic acid and the spacer, which in turn binds (always by means of an ester bond) to the hydroxyl group on the carbon C2' of paclitaxel through its carboxyl group, more precisely the carboxyl group of 4-bromobutyric acid is activated with an activating agent and forms an ester bond with the hydroxyl function on the C2' carbon of paclitaxel in the presence of a catalyst; subsequently, the intermediate thus obtained is reacted with a salt of HA under suitable conditions, a nucleophilic substitution of the COO- of HA on the carbon to which the spacer-linker bromide is bound occurs. In this way, an ester bond is formed between HA and the spacer-linker previously bound to paclitaxel.

[0035] The HA used for the synthesis of such HA-paclitaxel conjugates can be derived from any source, for example by extraction from combs (EP0138572, WO2018020458), by fermentation routes (EP0716688), or by biotechnological routes (EP2614088, EP2614087), and has a weight average molecular weight (Mw) of 400 Da to 3 x 10 6 Da, in particular 400 Da to 1 x 10 6 Da, even more particularly 140.000 Da to 250.000 Da (weight average molecular weight means weight average molecular weight calculated with the "intrinsic viscosity" method (Terbojevich et al., Carbohydr Res, 1986, 363-377).

[0036] Preferably, the HA used for the synthesis of the HA-paclitaxel conjugates for use according to the present application is a fermentation HA, which has an average molecular weight Mw of 140.000 Da to 250.000 Da.

[0037] Preferably, the HA-paclitaxel conjugates for use according to the present application have an average degree of derivatization of 20% w / w.

[0038] A further object of the present application is also a pharmaceutical composition consisting essentially of the above described HA-paclitaxel conjugate with a pharmacologically acceptable diluent / excipient, preferably a pharmaceutical composition formulated in sterile isotonic water containing 5% w / v of glucose, for the treatment of mesothelioma, thus for the local area treatment of malignant pleural mesothelioma, pericardial mesothelioma and peritoneal mesothelioma, preferably for the treatment of malignant pleural mesothelioma.

[0039] Therefore, such a pharmaceutical composition is formulated as a sterile and isotonic aqueous solution consisting essentially of the conjugate itself.

[0040] The Applicant describes and claims for use in the above local area treatment of all known forms of mesothelioma and above cited and preferably for use in the local area treatment of MPM the conjugate, since in both tests carried out in vitro and in vivo the pharmacological efficacy of the HA and paclitaxel chemically bound in the conjugate of the present application with respect to the non-conjugated paclitaxel is Synergistic effect This is demonstrated below.

[0041] IC of the in vitro study 50 The results obtained both in terms of IC50 and as a representation of the bioluminescence values as an index of the tumor mass size in the treated animals, surprisingly demonstrate the pharmacological efficacy of the HA-paclitaxel conjugate and the synergistic effect of the hyaluronic acid conjugate on the chemotherapeutic agent with respect to the non-conjugated paclitaxel in the treatment of mesothelioma, in particular pleural mesothelioma.

[0042] Such results are even more important considering that the local area treatment for which such conjugate is intended to be claimed, i.e. intrapleural, or intraperitoneal and intrapericardial, in fact allows to administer the conjugate directly to the tumor mass at lower / similar or even higher doses than the reference chemotherapeutic agent, in order to obtain a higher therapeutic effectiveness than the drug reference, maintaining high safety.

[0043] The HA-paclitaxel conjugate in fact remains in direct contact with the tumor mass in the administration cavity for a prolonged time as a depot, thus allowing its concentration mainly at the site of action (and not at systemic level), in this way maximizing the interaction with the tumor without damaging the surrounding tissues, reducing side effects. By the "targeting" action of the hyaluronic acid, a mechanism of selective drug transport to the tumor cells is also used due to the high expression of CD44 on the surface of the tumor cells.

[0044] The animal tests described below demonstrate in particular the in vivo effectiveness of such HA-paclitaxel conjugate, which will lead to an increase in the survival of patients and to a better quality of life of a class of patients who, as previously reported, have an extremely limited life expectancy even after chemotherapy treatment.

[0045] Example 1

[0046] Cytotoxicity of HA-paclitaxel conjugates in mesothelioma cells relative to paclitaxel

[0047] Experimental procedure: the cytotoxic activity of the HA-paclitaxel conjugates of the present application has been evaluated in vitro by means of MTT assay on commercially available H2052, H28 and H2452 human mesothelioma cell lines, all of which express the CD44 receptor of hyaluronic acid. The conjugates used for such tests have been prepared starting from a fermentation HA having an average weight MW of 140.000 Da to 250.000 Da, according to IT 102018000009731, thus having an average degree of derivatization of 20% w / w.

[0048] The cells were plated (day 0) in EMEM medium (SIGMA, Saint Louis, Missouri, USA) in 96 flat-bottomed wells of a multiwell plate (3000 cells per well) and then they were incubated at 37°C in the presence of 5% C02for 24 hours. The next day (day 1) the medium was changed, providing the cells with fresh EMEM medium, which optionally contained paclitaxel as control or the HA-paclitaxel conjugate in the appropriate dilution. After two days of incubation (day 3), each well containing the cells was treated with 100 μl of reagent 3-2,5-diphenyl tetrazolium bromide (5 mg / ml in DMEM) for 90 minutes. Subsequently, upon removal of the reagent from each well and corresponding addition of 100 μl of DMSO, the absorbance of formazan (blue, produced by the reaction of the breaking of the MTT tetrazolium ring, carried out by the mitochondrial enzyme "succinate dehydrogenase" present only in living cells) was measured by means of a multiplate reader at a wavelength of 570 nm. Such MTT assay, widely known to the skilled person, allows to evaluate the viability of the cells treated with the conjugate with respect to the cells treated with paclitaxel alone, and thus to determine the sensitivity of the above-mentioned cells to the sample under test. The data were then normalized to the control and the IC 50 values of the conjugate were calculated in μg / ml (expressed as paclitaxel equivalent) compared to non-conjugated paclitaxel.

[0049] The results are summarized in the table below, where it is clearly indicated that the HA-paclitaxel conjugate shows an activity (and thus antitumor effectiveness) 65-70 times higher than non-conjugated paclitaxel.

[0050] Normalized IC 50 , pg / ml

[0051] HA-paclitaxel Paclitaxel H2452 0.0122 0.8 H28 0.033 0.787 H2052 0.0342 0.225

[0052] Example 2

[0053] In vivo testing in immunodeficient NSG mice

[0054] The human mesothelioma cell line H2052 was transduced with a lentiviral vector containing a bidirectional promoter that allows the high and synergic expression of two reporter genes (by this method it is possible to use Lentivirus The gene was inserted into the cell for the synthesis of Fluorescence Luciferase and eGFP (eGFP, enhanced green fluorescent protein), as described in Amendola et al., Nat Biotechnol 2005, 23: 108-16: Luciferase is an enzyme that catalyzes the oxidation of fluorescein, which emits energy (electrons) in the form of light radiation, and such a reaction has a super-high sensitivity; eGFP is derived from the jellyfish Aequorea victoria and is able to emit a bright green light when exposed to certain wavelengths; the two such proteins were used as "marker system" of the human mesothelioma cells H2052 transduced as described above, in order to identify the tumor masses formed in the animals injected with such cells and to determine their size and growth over time by means of suitable bioluminescence sensors.

[0055] The cells were incubated in a mixture of complete medium (RPMI + 10% FBS, 1% HEPES, L-glutamine and penicillin / streptomycin) containing the above-mentioned vectors and protamine sulfate for 6 hours. At the end of the 6-hour incubation, the supernatant was recombined with complete medium. After 5 days, the effect of transduction was evaluated by flow cytometry.

[0056] The cells were then expanded and subdivided in order to isolate populations with higher reporter gene expression. The cells Transduction and Selected Such cells Injection into the peritoneum of NSG mice (i.e. immunodeficient, thus allowing the transplantation of various human cancer cells) 10 mice / sample Figure 1 , Abrupt drop were then subjected to the following treatments:

[0057] 1 ° sample treated with HA-paclitaxel 40 mg / Kg, by local administration intraperitoneally (ip);

[0058] 2 ° sample treated with paclitaxel 10 mg / Kg, 3 treatments by intravenous administration (iv), then switched to ip administration due to the poor tolerance of NSG mice to iv treatments of paclitaxel;

[0059] 3 ° sample treated with paclitaxel 10 mg / Kg, by local administration intraperitoneally (ip);

[0060] 4 ° untreated sample;

[0061] administration every week for 10 weeks.

[0062] All the treated animals were monitored by detecting the bioluminescence emitted by the tumor area of the animals treated with the HA-paclitaxel conjugate, in order to evaluate the size of the tumor (and therefore the growth) over time with respect to the animals not treated with any chemotherapeutic agent and to the animals treated with non-conjugated paclitaxel.

[0063] The results have been plotted in Phase where it is clearly shown that the luminescence (expressed in photons / second, Ph / sec) of the untreated animals reaches an average value of the order of 1 x 1010 12 on the 100th day of life, thus indicating the size / growth reached by the untreated tumor; the comparison of the 2° and 3° samples (both treated with non-conjugated paclitaxel in different ways of administration) with respect to such a control sample indicates an important result for both, since the average luminescence value reached proves to be of the order of 1 x 109 11 , thus lower than the untreated, indicating a reduction of the tumor mass with respect to the control.

[0064] However, the results of the HA-paclitaxel conjugate are completely unexpected, since from the 14th day of life to the 125th day the luminescence Relative to untreated controls and relative to animals treated with non-conjugated chemotherapeutic agents administered iv and ip, tumors were significantly reduced and remains negligible over time. These completely unexpected data indicate, Figure 1 Figure 2 ​ proving the overall effectiveness of the conjugate itself. In ​ the luminescence data related to the HA-paclitaxel conjugate are indicated in the third column of the four columns corresponding to the days of life reported in the abscissa.

[0065] The data obtained after 139 days of observation are reported in ​ where it is clearly shown that in the mice treated with non-conjugated paclitaxel the bioluminescence detection in Ph / sec emitted by their treated tumor area and therefore representative of the tumor mass remains at about 1-2 x 1010 11 , while in the mice treated with the conjugate this value is extremely low and close to 0, thus indicating a practically complete reduction of the tumor mass.

[0066] With the above discussed results, the Applicant has proved the pharmacological efficacy and effectiveness of the HA-paclitaxel conjugate in the local area treatment of mesothelioma, in particular MPM, peritoneal and pericardial mesothelioma, since it is able to determine a significant reduction of the tumor mass, thus increasing the patient survival and its quality of life.

Claims

1. Use of a hyaluronic acid (HA)-paclitaxel conjugate in the manufacture of a medicament for local regional treatment of mesothelioma, wherein said hyaluronic acid-paclitaxel conjugate has an ester bond between the carboxyl group of hyaluronic acid (HA) and a spacer, in turn bound to the hydroxyl group on carbon C2' of paclitaxel by means of the ester bond through its carboxyl group, wherein said spacer introduced is 4-bromobutyric acid, and wherein the degree of derivatization of paclitaxel in said HA-paclitaxel conjugate ranges from 15% to 21% weight / weight (w / w), wherein said HA used for the synthesis of said HA-paclitaxel conjugate is a fermentation HA having a weight average molecular weight of 140,000 Da to 250,000 Da.

2. Use according to claim 1, wherein said mesothelioma is malignant pleural mesothelioma, pericardial mesothelioma and peritoneal mesothelioma.

3. Use according to claim 2, wherein said mesothelioma is malignant pleural mesothelioma.

4. Use according to any one of claims 1-3, wherein the route of administration of said medicament is intrapleural, or intraperitoneal and intrapericardial.

5. Use according to any one of claims 1-3, wherein said degree of derivatization of paclitaxel in said HA-paclitaxel conjugate ranges from 16% to 20% w / w.

6. Use according to any one of claims 1-3, wherein said degree of derivatization of paclitaxel in said HA-paclitaxel conjugate is equal to 20% w / w.

7. Use according to any one of claims 1-3, wherein said medicament consists of said HA-paclitaxel conjugate and of a pharmacologically acceptable diluent / excipient.

8. Use according to any one of claims 1-3, wherein said medicament is formulated in sterile isotonic water containing 5% w / v of glucose.

Images