WO2020214106A1

WO2020214106A1 - Nano formulations comprising ceranib-2

Info

Publication number: WO2020214106A1
Application number: PCT/TR2019/050247
Authority: WO
Inventors: Devrim Gozuacik; Ozlem KUTLU; Nur Mehpare KOCATURK; Ayben KILISLIOGLU; Selcan KARAKUS; Mehtap KUTLU; Merve ILGAR; Ali Mohamed BEN TALEB; Ezgi TAN
Original assignee: Sabanci Universitesi; Sabanci Universitesi Nanoteknoloji Arastirma Ve Uygulama Merkezi; Istanbul Universitesi-Cerrahpasa Rektorlugu; Eskisehir Teknik Universitesi
Priority date: 2019-04-16
Filing date: 2019-04-16
Publication date: 2020-10-22

Abstract

The present invention provides novel nano formulations of ceranib-2 contained in a polymer matrix, method for producing the same, and pharmaceutical compositions comprising said nano formulations. In further aspects, the present invention provides said nano formulations for use in treatment of cancer.

Description

NANO FORMULATIONS COMPRISING CERANIB-2

Field of the Invention

The present invention relates to novel nano formulations comprising ceranib-2 contained in nanoparticle polymer matrix. In particular, the present invention pertains nano formulations comprising ceranib-2 contained in a polymer matrix, preferably of rosin or derivatives thereof, for increasing bioavalibility and reducing toxicity of the active ingredient. In another aspect, the present invention provides a novel method for producing the foregoing nanoparticles of ceranib- 2, and also the use of said particles for treatment of cancer.

Background of the Invention

Conventional therapies of cancer involve use of chemotherapy agents and radio therapy which in turn are highly toxic and non-tolerable for the healthy cells of a patient. Therefore, undesired and serious side effects are frequently encountered under the normal conditions of therapy. A further drawback of these therapies is that multiple drug resistance is developed against anti-cancer agents. In order to avoid the problems mentioned above, innovative approaches such as nanoforms of drug carriers have attracted particular attention of the scientists in order to reduce the side effects and improve bioavailability.

For instance, first generation nanomedicines have received widespread clinical approval over the past two decades, from Doxil® (liposomal doxorubicin) in 1995 to Onivyde® (liposomal irinoteean) in 2015 { Tran , S., DeGiovanni, P. ]., Piel, B., & Rai, P. (2017). Cancer nanomedicine: a review of recent success in drug delivery. Clinical and translational medicine , 6(1), 44.). There are also other methodologies suggesting the use of various types of nanoencapsulating agents. WO 16/210120 Al, for instance, mentions to lipid nanoparticles, dendrimers, D,L-lactide-co-glycolide nanoparticles, poly (D,L-lactide) nanoparticles, and polybutylcyanoacrylate nanoparticles. Likewise, US 2016/058886 A discloses nanoparticles being pH sensitive liposomes and incorporating cytotoxic ceramides. However, it is important to provide customized carriers for the specific therapeutic agent in order to obtain the desired drug delivery in particular diseases and reducing adverse effects.

One such agent that needs to be formulated with a customized carrier is ceranib-2 having the following formula:

Ceranib-2

Ceranib-2 is a well defined inhibitor of ceramidase enzyme. US 2018/0235951 Al, for instance, discloses effectiveness of ceranib-2 for treatment of breast cancer and lung cancer. Moreover, it is also reported in prior art that Ceranib-2 has stronger cytotoxicity as compared to that of C2 ceramide and cisplatin on glioma cells (Kus et al. , "Comparison of a ceramidase inhibitor (ceranib- 2) with C2 ceramide and cisplatin on cytotoxicity and apoptosis of glioma cells ", Turk J. Biol. (2018) 42: 259-265). Similarly, it is further reported that ceranib-2 is effective for treatment of tumors and showing particular synergism with carboplatin and paclitaxel (Ozer et al "The investigation of ceranib-2 on apoptosis and drug interaction with carboplatin in human non small cell lung cancer cells in vitro ", Cytotechnology, 2017).

For a successful therapy, it is important to understand the mechanism of action underlying a therapeutic agent In several studies, the interrelation between cancer and sphingolipids has been established. It is also thought that sphingomyelin existing in ample amounts in the cell membrane plays an important role in cellular injury or death. Various resources varify that ceramide, ceramide-1 phosphate, sphingosine and sphingosine-1 phosphate (SIP], all being composed of sphingolipids, have particular effect on cell proliferation, apoptosis, inflamation and cell cycle. Especially, ceramide and SIP inter alia are known to be stimulating genes for cell proliferation. There is a balance between ceramide and SIP levels in the cell. If the balance is in favour of ceramide, cell proliferation is prevented, and apoptosis and cell death are induced whereas if the balance is in favour of SIP, cell proliferation is induced and apoptosis is inhibited. Therefore, it is important to control the level of ceramide by stimulating the ceramidase enzyme. Notably, cancer cells express higher amounts of ceramidase enzyme which need to be supressed in a successful therapy. Accumulation of ceramide, on the other hand, is considered to be toxic for pancreatic beta cells and cardiomyocytes, and therefore plays an important role in the patogenesis of diseases such as diabetes, hypertension, cardiac failure and atheroslerosis. Therefore, it is important also to target cancer cells selectively for the inhibition of ceramidase enzyme. The inventors of the current invention have unexpectedly observed that ceranib-2 causes cardiovascular side efects in a combined therapy with other antineoplastic agents, especially with chemotherapy agents. Without wishing to be bound by a theory, this has been attributed to the fact that ceranib-2, without a suitable carrier and without targeting of the cancer cells, causes toxicity which is hardly tolerated in cardiac patients. Moreover, higher doses of ceranib-2 are needed in the state of the art applications which in turn increases toxicity.

Therefore, lack of a suitable nanocarrier for ceranib-2 in state of the art applications is delimiting the use of this valuable compound in therapy, and there is a long standing need for formulating ceranib-2 with suitable carriers for targeting cells, i.e. tumors in order to improve cytotoxicity, bioavailibility and tolerability. The prior references outlined above are mostly silent how to provide suitable carriers for ceranib-2 for targeting of cancer cells while maintaining effectivenes and safely of the treatment

These problems have been presently solved by nano-formulation of ceranib-2 in a polymer matrix of rosin or derivatives thereof. In particular embodiments, PEG is used along with the rosin in order to improve nanostructures obtained through the invention as will be explained in greater detail within the following description.

Brief Description of the Figures

Figure 1 shows a SEM micrograph of the nanoforms containing ceranib-2 in a carrier according to the present invention.

Figure 2 shows the effect of Ceranib-2 on cancer cells. SW480 and HeLa cells were treated with lipid solid form of Ceranib-2 and nanoform of Ceranib-2 in indicated concentrations, then the absorbance of formazan solution was measured by using enzyme-linked immunosorbent assay. Dimethyl sulfoxide (DMSO] was used as a carrier in negative CNT (control] Etoposide (50 mM] was used as a positive control. RE: Rosin ester, Cer-2: Ceranib-2. 1:500 (0,66 mM], 1:1000 (0,33 mM], 1:2000 (0,151 mM] Data were shown as mean ± SD of independent experiments, n=4, *p<0.05, ***p<0.001. Detailed Description of the Invention

In an aspect, the present invention provides novel nanoforms of ceranib-2 and a method for producing the same. In another aspect the invention provides said nanostructures for use in treatment of cancer.

As mentioned above, nanoforms of ceranib-2 are nano formulations comprising ceranib-2 contained nanoparticle polymer matrix. The matrix forming polymer is preferably rosin or a pharmaceutically acceptable derivative thereof capable of forming said polymer matrix.

Rosin is also called colophony, and is a solid resin generally obtained from pines. Rosin and its derivatives are highly biodegradable and biocompatible, and are perfectly suitable for microencapsulation and film formation with their amphiphilic properties. Rosin and derivatives thereof possess also antiviral, antitumour, antiulcer, antimicrobial, anxiolytic properties (Wasowski, C., & Marder, M. (2011 ). Central nervous system activities of two diterpenes isolated from Aloysia virgata. Phytomedicine , 18(5), 393-401 - Lee, C. L., Chiang, L. C., Cheng, L. H., Liaw, C. C., Abd El-Razek, M. H., Chang, F. R., & Wu, Y. C. (2009). Influenza A ( MINI ) antiviral and cytotoxic agents from Ferula assa-foetida. Journal of natural products, 72(9), 1568-1572). Being a natural product with low toxicity, rosin and derivatives thereof are approved by FDA and can be used as a food ingredient

The inventors have noted that rosin is not only advantageous because of low toxicity and matrix/film formation properties, but is also very suitable for comprising ceranib-2 and targeting cancer cells. This is important for eliminating lately discovered side effects in stand alone or combination therapies. Since the nanostructures disclosed herein allow use of ceranib-2 at lower dose levels, one skilled in the art shall be appreciating that containment of ceranib-2 is advantageous for improving safety of the treatment Besides, advantageous therapeutic benefits of rosin as mentioned above may improve effectiveness of the therapy.

It also noted that rosin is highly compatible with ceranib-2 and no side reactions occur between them. Stability of ceranib-2 is therefore preserved and bioavailibility is improved.

In the context of the present description, rosin derivatives refer to the polymers obtained from rosin which are suitable for carrying out objectives of the present invention. Rosin esters including glycerol, sorbitol and mannitol esters of rosin are particularly preferred. Most preferably glycerol ester of rosin is used. These esters provide excellent film formation and emulsification in the production procedure of the nanostructures.

In preferred embodiments, polyethylene glycol (PEG] is used along with the rosin or derivatives thereof for improving stability of the colloidal suspensions and preventing agglomeration. As can be appreciated by those skilled in the art, agglomerated particles of the nanostructures may cause lower bioavalibility. It is also highly biocompatible with ceranib-2 and rosin. PEG conjugates possess long half life, good tolerability, low toxicity and excellent properties as a carrier for targeting a particular group of cells. Hence, the inventors note that PEG is prominently advantageous for obtaining improved nano formulations of ceranib-2. In the context of the present invention, PEGs of low molecular weight are found to be advantageous. Thus, PEG may have an average molecular weight of 190-1050 g/mol. PEG 200 (MW: 190-210 g/mol], PEG 300 (MW: 285-315 g/mol] and PEG 400 (MW: 380-420 g/mol] are particularly preferred.

In the context of the present invention, nanoforms of ceranib-2 contained in the foregoing carrier system may have a particle size of 10 - 500 nm, more preferably 30 - 300 nm, and most preferably 40 - 180 nm as measured by SEM micrographs. The inventors have succeeded to obtain these nanostructures as shown in Fig. 1.

The present invention also provides a pharmaceutical composition comprising nanoforms of ceranib-2 as mentioned above along with at least one pharmaceutically acceptable excipient.

Still in a further aspect, the present invention provides a pharmaceutical combination of the foregoing nanoforms of ceranib-2 along with a antineoplastic agent which is preferably a chemotherapy agent The inventors have noted particular advantage of the nanoforms of the instant invention especially in combination therapies such that the required dosage of ceranib-2 is reduced and adverse effects are alleviated.

In an aspect, the present invention provides a novel method for producing nano formulations of ceranib-2 comprising the steps of:

dissolving ceranib-2 in a solvent,

admixing a matrix forming polymer with the ceranib-2 solution, and

obtaining nano formulations of ceranib-2 contained in a polymer matrix.

As mentioned above, the polymer is preferably rosin or a pharmaceutically acceptable derivative thereof capable of forming said polymer matrix. The inventors have noted that aforementioned nano formulations can be prepared in a better way by using a two phase system in order to ensure efficiency of the containment and prevent agglomeration. Accordingly;

a dispersing phase is provided whereby ceranib-2 is dissolved in a solvent and mixed with rosin or a derivative thereof,

a continuous phase is provided by mixing of PEG with water, and

finally dispersing phase is added to the continuous phase followed by homogenization.

The solvent used for solubilizing ceranib-2 can be a solvent or a system of solvents which can be DMSO, ethyl acetate or both. Therefore, the method of the present invention may further comprise the step of evaporating the solvent at the end for isolating nano formulations of ceranib-2.

In a further aspect, the present invention provides foregoing nanoforms of ceranib-2 for use in treatment of cancer. The cancer in the context of the present invention can be any of the group of lung cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer and cervix cancer as well as metastatic cancer.

Further aspects and advantages of the present invention shall be apparent from the following Examples and appended claims.

Example 1 - Preparation of the nanoforms of Ceranib-2 in rosin

A dispersing phase and a continuous phase have been prepared and combined as follows:

Dispersing Phase: Ceranib-2 is dissolved in DMSO. Rosin ester powders were added to the ceranib- 2 solution under room temperature and continuous mixing with a magnetic stirrer for five minutes. Then the mixture was added ethyl acetate under continuous mixing.

Continuous Phase: PEG 400 was dissolved in distilled water under room temperature and the mixture was then saturated with ethyl acetate.

The dispersing phase in its entirety was added to the same volume of continuous phase dropwise and this procedure was carried out under sonication in a homogenizer for two minutes. The ethyl acetate in the mixture was evaporated under 40 °C for two hours. The remaining solution was isolated and put into glass vials. Example 2 - Effect of Ceranib-2 nanoform on viability of cancer cells

The effect of Ceranib-2 nanoform was analyzed on Dukes' type B, colorectal adenocarcinoma cell line (SW480] and cervix adenocarcinoma cell line (HeLa] Cell viability was analyzed by a mitochondrial function-based MTT [3-(4, 5-dimethylthiazol-2yl]-2, 5-diphenyltetrazolium bromide] assay. Different concentrations of lipid solid form of ceranib-2 and nanoform of Ceranib- 2 ranging from 0,66 to 0,151mM were added in each well and cells were incubated for 24 h at 37 °C in a humidified incubator with 5 % of CO2 in air. After incubation period 20 pL of MTT solution (5 mg/mL] was added into each well followed by further incubation for 2 h in humidified incubator. The medium was then aspirated, and cells were lysed with DMSO to dissolve formazan crystals. MTT is a yellow tetrazolium salt reducing to purple formazan. The absorbance of formazan solution was measured on an enzyme-linked immunosorbent assay (ELISA] plate reader (iMark Microplate Reader, Bio-Rad] at wavelength of 570nm and 655 nm. The statistical significance of differences between groups was assessed by two-tailed Student’s t-test Data were represented as means of ±S.D. of 4 independent experiments. Values with p< 0.05 were considered as significant (Fig. 2]

Claims

1. Nano formulations comprising ceranib-2 contained in a polymer matrix.

2. The nano formulations according to claim 1 wherein the polymer matrix comprises rosin or a pharmaceutically acceptable derivative thereof capable of forming said polymer matrix.

3. The nano formulations according to claim 2 wherein the rosin derivative in the matrix is a rosin ester.

4. The nano formulations according to claim 3 wherein the rosin ester is selected from the group consisting of glycerol, sorbitol and mannitol esters of rosin.

5. The nano formulations according to claim 1 further comprising PEG.

6. The nano formulations according to claim 5 wherein the PEG has an average molecular weight in between 190-1050 g/mol.

7. The nano formulations according to claim 6 wherein the PEG is PEG 400.

8. The nano formulations according to claim 1 wherein said nano formulations have a particle size up to 500 nm.

9. A method for producing nano formulations according to any of the preceeding claims comprising the steps of: dissolving ceranib-2 in a solvent,

admixing a matrix forming polymer with the ceranib-2 solution, and

obtaining nano formulations of ceranib-2 contained in a nanoparticle polymer matrix.

10. The method according to claim 10 comprising the steps of: providing a dispersing phase whereby ceranib-2 is dissolved in a solvent and mixed with a matrix forming polymer,

providing a continuous phase whereby PEG is mixed with water, and

adding the dispersing phase to the continuous phase.

11. The method according to claim 10 or 11 wherein the matrix forming polymer comprises rosin or a pharmaceutically acceptable derivative thereof capable of forming said polymer matrix.

12. The method according to claim 11 wherein the derivative is a rosin ester selected from the group consisting of glycerol, sorbitol and mannitol esters of rosin.

13. Nano formulations according to any of the claims 1 to 8 for use in treatment of cancer.

14. Nano formulations for use according to claim 13 wherein the cancer is selected from the group of lung cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer and cervix cancer as well as their metastatic forms.

15. A pharmaceutical composition comprising the nano formulations according to any of the claims 1 to 8 and further comprising at least one pharmaceutically acceptable excipient.