HA-PACLITAXEL CONJUGATE FOR TREATMENT OF MESOTHELIOMA

Abstract

HA-paclitaxel conjugate for use in the loco-regional treatment of the mesothelioma, i.e., the malignant pleural mesothelioma, pericardial mesothelioma and peritoneal mesothelioma, preferably malignant pleural mesothelioma, and relative pharmaceutical compositions are described.

Claims

1. A method of loco-regional treatment of mesothelioma which comprises administering to a patient a HA-paclitaxel conjugate, wherein said hyaluronic acid-paclitaxel conjugate has an ester bond between the carboxyl of hyaluronic acid (HA) and a spacer, in turn bound by an ester bond through its carboxyl to the hydroxyl group to the carbon C2′ of paclitaxel, wherein the spacer introduced is 4-bromobutyric acid and wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate is within the range that varies from 15% to 21% weight/weight ( w/w).

2. The method according to claim 1, wherein the mesothelioma is malignant pleural mesothelioma, pericardial mesothelioma and peritoneal mesothelioma.

3. The method use according to claim 1, wherein the mesothelioma is malignant pleural mesothelioma.

4. The method according to claim 1 , wherein the administration route is intrapleural, or intraperitoneal or intrapericardial.

5. The method according to claim 1, wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate ranges from 16% to 20% w/w.

6. The method according to claim 1, wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate is equal to 20% w/w.

7. The method according to claim 1, wherein HA used for the synthesis of the HA-paclitaxel conjugate is a fermentative HA with an average weight MW ranging from 140,000 to 250,000 Da.

8. A pharmaceutical composition, essentially consisting of an HA-paclitaxel conjugate, wherein said hyaluronic acid-paclitaxel conjugate has an ester bond between the carboxyl of hyaluronic acid (HA) and a spacer, in turn bound by an ester bond through its carboxyl to the hydroxyl group to the carbon C2′ of paclitaxel, wherein the spacer introduced is 4-bromobutyric acid and wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate is within the range that varies from 15% to 21% weight/weight ( w/w) , and one or more pharmacologically acceptable diluents/excipients.

9. The pharmaceutical composition according to claim 8, wherein said pharmaceutical composition is formulated in sterile, isotonic water, containing 5% w/v of glucose.

10. The method according to claim 2, wherein the administration route is intrapleural, or intraperitoneal or intrapericardial.

11. The method according to claim 3, wherein the administration route is intrapleural.

12. The method according to claim 2, wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate ranges from 16% to 20% w/w.

13. The method according to claim 3, wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate ranges from 16% to 20% w/w.

14. The method according to claim 4, wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate ranges from 16% to 20% w/w.

15. The method according to claim 2, wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate is equal to 20% w/w.

16. The method according to claim 3, wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate is equal to 20% w/w.

17. The method according to claim 4, wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate is equal to 20% w/w.

18. The method according to claim 5, wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate is equal to 20% w/w.

19. The method according to claim 2, wherein HA used for the synthesis of the HA-paclitaxel conjugate is a fermentative HA with an average weight MW ranging from 140,000 to 250,000 Da.

20. The method according to claim 3, wherein HA used for the synthesis of the HA-paclitaxel conjugate is a fermentative HA with an average weight MW ranging from 140,000 to 250,000 Da.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0026] Object of the present invention is the HA-paclitaxel conjugate for use in the loco-regional treatment of mesothelioma, therefore malignant pleural mesothelioma, pericardial mesothelioma and peritoneal mesothelioma, in particular for use in the treatment of MPM, wherein such HA-paclitaxel conjugate has an ester bond between the carboxyl of hyaluronic acid (HA) and a spacer, in turn bound by an ester bond through its carboxyl to the hydroxyl group to the carbon C2′ of paclitaxel, wherein the spacer introduced is the 4-bromobutyric acid, and wherein the derivatization degree of paclitaxel in the HA-paclitaxel conjugate is within the range that varies from 15% to 21% weight/weight (w/w), and preferably from 16% to 20% w/w.

[0027] Hereinafter in the present description, derivatization (or esterification) degree of the above-mentioned conjugate means the weight percentage of paclitaxel with respect to the weight of the HA-paclitaxel conjugate.

[0028] Therefore, 100 mg of conjugate with derivatization degree comprised between 15% and 21% w/w, will contain 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg, or 21 mg of chemotherapeutic paclitaxel depending on the derivatization degree indicated (to further exemplify this, the derivatization degree at 20% w/w contains 20 mg of paclitaxel per 100 mg of conjugate); it is however obvious to the skilled person that, at the end of such industrial synthesis processes, a small variation in the weight ratios between the molecules can occur, therefore hereinafter the Applicant, describing and claiming the derivatization range of the above-mentioned conjugate comprised between 15% and 21% w/w, intends to claim all the reported percentage values comprising ±1%: by way of example, the degree of 20% w/w is accordingly to be intended as 20% ± 1%.

[0029] The HA-paclitaxel conjugate for the above-claimed use was prepared according to a synthesis process known in the state of the art as described in EP2045270 and, for the derivatization degree of 20%, improved in IT 102018000009731.

[0030] In broad terms, in such synthesis process of the above-mentioned conjugate, the spacer/linker 4-bromobutyric acid is introduced between hyaluronic acid and paclitaxel through the formation of the ester bond between the carboxyl of hyaluronic acid and the spacer, said spacer being in turn bound (always by ester bond) through its carboxyl to the hydroxyl group to the carbon C2′ of paclitaxel, more precisely the carboxyl group of the 4-bromobutyric acid, through activation with an activating agent and in the presence of a catalyst, forming an ester bond with the hydroxyl function to C2′ carbon of paclitaxel; subsequently the intermediate thus obtained reacts with a salt of HA, under suitable conditions, giving rise to the nucleophilic substitution of COO— of HA to the carbon bound to the bromine of the spacer-linker. In this way, the ester bond is formed between the HA and the spacer-linker previously bound to paclitaxel.

[0031] The HA used for the synthesis of such HA-paclitaxel conjugate can derive from any source, for example by extraction from rooster combs (EP0138572, WO2018020458), by fermentative route (EP0716688), or by biotechnological route (EP2614088, EP2614087) and having a weight average molecular weight (MW) ranging from 400 to 3x10.sup.6 Da, particularly from 400 to 1x 10.sup.6 Da, even more particularly from 140.000 to 250.000 Da (weight average molecular weight means the one calculated with the “intrinsic viscosity” method (Terbojevich et al., Carbohydr Res, 1986, 363-377).

[0032] Preferably, the HA used for the synthesis of the HA-paclitaxel conjugate for use according to the present invention is a fermentative HA with an average weight MW comprised between 140.000 and 250.000 Da.

[0033] Preferably, the HA-paclitaxel conjugate for use according to the present invention has an average derivatization degree of 20% w/w.

[0034] A further object of the present invention is also a pharmaceutical composition consisting essentially of the HA-paclitaxel conjugate described above, associated with pharmacologically acceptable diluents/excipients, preferably a pharmaceutical composition formulated in sterile, isotonic water, containing 5% w/v of glucose for use in the loco-regional treatment of mesothelioma, therefore of the malignant pleural mesothelioma, pericardial mesothelioma and peritoneal mesothelioma, preferably for use in the treatment of malignant pleural mesothelioma.

[0035] Such pharmaceutical composition is thus formulated as, sterile and isotonic, aqueous solution, consisting essentially of the conjugate itself.

[0036] The Applicant describes and claims the conjugate for the use above in the loco-regional treatment of all the known and above-cited mesothelioma forms and preferably the use in the loco-regional treatment of MPM, since the synergistic effect of HA and paclitaxel chemically bound in the conjugate object of the invention, both in tests carried out in vitro and in vivo vs non-conjugated paclitaxel, is demonstrated below.

[0037] The results obtained, both in terms of IC.sub.50 for in vitro studies, and expressed as bioluminescence values as index of the tumor mass size in the treated animals, surprisingly demonstrate the pharmacological potency of the HA-paclitaxel conjugate and the synergistic effect of the hyaluronic acid conjugate to chemotherapeutic agent vs non-conjugated paclitaxel in the treatment of mesothelioma, in particular of the pleural mesothelioma.

[0038] Such result is even more important considering the loco-regional treatment intended to be claimed for such conjugate: this administration route, i.e., intrapleural, or intraperitoneal and intrapericardial, in fact allows the administration of the conjugate directly to the tumor mass at lower/analogous doses or even higher than the reference chemotherapeutic agent, in order to obtain a therapeutic effectiveness higher than the drug reference maintaining a high safety profile.

[0039] The HA-paclitaxel conjugate remains indeed as depot in the administration cavity directly in contact with the tumor mass for prolonged times, thus allowing its concentration mainly at the action site (and not at systemic level), maximizing in this way the interaction with the tumor without damaging the surrounding tissue, reducing the side effects. The drug selective transport mechanism to the tumor cell for the high expression of CD44 on the surface of such cells is also used, through the “targeting” action of hyaluronic acid.

[0040] Animal testing described below especially demonstrates the effectiveness in vivo of such HA-paclitaxel conjugate, wherein such effectiveness will result in humans not only in the increasing of sick-person survival, but also in a better quality of life for a category of patients which, as previously reported, have an extremely limited life expectancy even after the chemotherapy treatment.

Example 1

Cytotoxicity of the HA-Paclitaxel Conjugate in Mesothelioma Cells vs Paclitaxel

[0041] Experimental procedure: the cytotoxic activity of the HA-paclitaxel conjugate object of the invention has been evaluated in vitro by performing the MTT assay on cell lines of human mesothelioma commercially available H2052, H28 and H2452, wherein all such cell lines express CD44 receptor for hyaluronic acid. The conjugate used for such trial has been prepared according to IT 102018000009731, therefore with an average derivatization degree of 20% w/w starting from a fermentative HA with an average weight MW comprised between 140.000 and 250.000 Da.

[0042] Cells were plated (Day 0) in multiwell plate of 96 flat-bottomed wells (3000 cells per well) in EMEM culture medium (SIGMA, Saint Louis, Missouri, USA) and subsequently they were incubated for 24 hours at 37° C. in the presence of 5% CO.sub.2. The next day (Day 1) the medium was changed providing the cells with fresh EMEM medium containing alternatively paclitaxel, as a control, or the HA-paclitaxel conjugate in suitable dilutions. After two days of incubation (Day 3), each well containing the cells was treated with 100 .Math.l of reagent 3-2,5-diphenyltetrazolium bromide (5 mg/ml in DMEM) for 90 minutes. Subsequently, at the removal of the reagent from each well and the corresponding addition of 100 .Math.l of DMSO, was measured the absorbance of the formazan salt (blue, produced by the reaction of breaking the MTT tetrazolium ring, performed by the mitochondrial enzyme “succinate dehydrogenase” present only in the viable cells) at the wavelength of 570 nm by a multiplate reader. Such MTT assay, widely known to the skilled person, allows to evaluate the viability of the cells treated with the conjugate vs those treated with paclitaxel alone and therefore to determine the susceptibility of the above-mentioned cells to the tested samples. Data were then normalized to control and the IC.sub.50 value was calculated in .Math.g/ml of conjugate (expressed in terms of paclitaxel-equivalents) compared to non-conjugated paclitaxel.

[0043] Results are summarized in the table below where it is clearly evident that the HA-paclitaxel conjugate shows an activity (therefore an antitumor effectiveness) up to 65-70 times greater than the one of non-conjugated paclitaxel.

TABLE-US-00001 Normalized IC.sub.50, .Math.g/ml HA-paclitaxel paclitaxel H2452 0.0122 0.8 H28 0.033 0.787 H2052 0.0342 0.225

Example 2

In Vivo Trial in Immunodeficient NSG Mice

[0044] The cell lines of human mesothelioma H2052 were transduced with a lentiviral vector (method by which the genes can be inserted into cells using the lentivirus) containing a bidirectional promoter allowing the high and coordinated expression of two reporter genes for the synthesis of luciferase and 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 luciferin which oxidizing emits energy (electrons) in the form of light radiation and such reaction has an ultra-high sensitivity; the eGFP derives from the jellyfish Aequorea victoria, and it is capable of emitting bright green light when exposed to certain wavelengths; both such proteins are used below as a “marker system” for human mesothelioma cells H2052 transduced as above, in order to identify the tumor mass that is formed in animals injected with such cells and to determine its dimensions and the growth over time through suitable bioluminescence sensor.

[0045] Cells were incubated for 6 hours in a mixture of complete medium (RPMI + 10% FBS, 1% HEPES, L-glutamine and penicillin/streptomycin) containing the above-mentioned vector and protamine sulphate. At the end of 6 hours of incubation, supernatants were re-integrated with complete medium. After 5 days, the effect of the transduction was evaluated through flow cytometry.

[0046] Cells were then expanded and sub-divided in order to isolate the population with the higher expression of reporter genes. Such cells transduced and selected were then injected into the peritoneum of NSG mice, (i.e., immunodeficient that therefore allow engraftment of a wide range of human cancer cells), 10 mice/sample, which were then subjected to the following treatment: [0047] 1° sample treated with HA-paclitaxel 40 mg/Kg, by intraperitoneal (ip) loco-regional administration; [0048] 2° sample treated with paclitaxel 10 mg/Kg, by intravenous administration (iv) for 3 treatments, switching then to ip administration due to the very poor tolerability of iv treatment with paclitaxel of NSG mice; [0049] 3° sample treated with paclitaxel 10 mg/Kg, by intraperitoneal (ip) loco-regional administration; [0050] 4° not-treated sample; [0051] by weekly administration for 10 weeks.

[0052] All treated animals were monitored through detection of bioluminescence emitted by their tumor zone treated with HA-paclitaxel conjugate, in order to assess over time, the dimension (and therefore the growth) of the tumor with reference to animals untreated with any chemotherapeutic agent and to those treated with non-conjugated paclitaxel.

[0053] Results have been plotted in FIG. 1 where it is clearly evident that the luminescence (expressed in photons/sec, Ph/sec) of the untreated animals reaches average values in the order of magnitude of 1x10.sup.12 on the 100° day of survival, indicating therefore the dimension/growth reached by the untreated tumor; 2° and 3° sample, both treated with non-conjugated paclitaxel with different ways of administration, are compared vs such controlled sample: this comparison indicates an important result for both, since the average luminescence values reached prove to be in the order of magnitude of 1x10.sup.11, therefore lower than untreated, indicating a reduced tumor mass compared to the control.

[0054] However, the result of the HA-paclitaxel conjugate is entirely surprising since from the 14° to the 125° day of survival, the luminescence drops dramatically and remains negligible over time. These completely unexpected data show the significant reduction in the tumor both vs the untreated control and vs animals treated with non-conjugate chemotherapeutic agent administered iv and ip, demonstrating the total effectiveness of the conjugate itself. In FIG. 1 luminescence datum related to the HA-paclitaxel conjugate is represented in the third out of the four columns corresponding to the days of survival reported in abscissa.

[0055] In FIG. 2 data obtained after 139 days of observation are reported instead, where it is clearly evident that bioluminescence detection in Ph/sec emitted by their treated tumor zone and therefore representative of the tumor mass, remains around 12x10.sup.11 in mice treated with non-conjugated paclitaxel, while in mice treated with the conjugate such value is extremely lower and close to 0, indicating therefore an almost complete reduction in the tumor mass.

[0056] Through the results discussed above, the Applicant demonstrated the pharmacological potency and therefore the effectiveness of the HA-paclitaxel conjugate in the loco-regional treatment of mesothelioma, particularly of MPM, peritoneal and pericardial mesothelioma, since it is capable of determining the significant reduction of tumor mass, accordingly increasing patient survival and his quality of life.