WO2009003656A1

WO2009003656A1 - Cyclodextrin-based nanosponges as a vehicle for antitumoral drugs

Info

Publication number: WO2009003656A1
Application number: PCT/EP2008/005290
Authority: WO
Inventors: Francesco Trotta; Vander Tumiatti; Roberta Cavalli; Carlo Maria Roggero; Barbara Mognetti; Giovanni Nicolao Berta
Original assignee: Sea Marconi Technologies Di Vander Tumiatti S.A.S.
Priority date: 2007-07-04
Filing date: 2008-06-27
Publication date: 2009-01-08
Also published as: EP2175847A1; CA2692493A1; JP2010531827A; ITMI20071321A1

Abstract

Disclosed are complexes of hypercrosslinked nanoporous polymers of cyclodextrins with drugs selected from paclitaxel, docetaxel, tamoxifen, camptothecin and derivatives thereof.

Description

CYCLODEΞXTRIN-BASED NANOSPONGES AS A VEHICLE FOR ANTITUMORAL DRUGS

The present invention relates to pharmaceutical compositions comprising cyclodextrin-based nanosponges as a vehicle for antitumoral drugs which are insoluble in water, in particular paclitaxel and other taxanes, camptothecin and tamoxifen. BACKGROUND TO THE INVENTION

The solubility of a drug is often the factor that limits its clinical application. New strategies are therefore always under study with a view to improving the solubility and release kinetics of active constituents.

Cyclodextrins (CD) are non-reducing cyclic oligosaccharides consisting of 6-8 glucose molecules linked by a 1 ,4-α-glycoside bond, having a characteristic cone-frustum-shaped structure. The arrangement of the functional groups of the glucose molecules is such that the surface of the molecule is polar, while the inner cavity is relatively lipophilic.

The lipophilic cavity enables the CDs to form inclusion complexes which are stable even in solution with organic molecules of suitable polarity and dimensions.

CDs have therefore already been studied, and have numerous applications in various chemical fields (pharmaceuticals, analysis, catalysis, cosmetics, etc.) in which the characteristics of the inclusion compounds are exploited.

In pharmaceutical technology, these complexes can be used to increase the dissolution rate, solubility and stability of drugs, to mask unpleasant flavours or to convert liquid substances to solids, etc.

Various formulation approaches are used in the pharmaceutical field to improve solubility, such as the use of co-solvents, surfactants, complexes and particulate systems.

Drugs which are particularly critical in formulation terms, especially due to their insolubility, are the tumoral agents paclitaxel, docetaxel and derivatives with a taxane structure in general, camptothecin and tamoxifen. Paclitaxel is an important anti-tumoral drug belonging to the taxane family, products of plant origin which perform their antitumoral action by inhibiting the formation of the mitotic spindle.

Paclitaxel is substantially insoluble in water and biological systems, and therefore poses problems for the formulation of injectable formulations. Paclitaxel is currently formulated in the form of a lipid emulsion (Cremophor), which enables it to be administered intravenously. However, the constituents of the emulsion are potent allergens which can cause serious hypersensitivity reactions. Moreover, the materials used to administer paclitaxel cannot be made of polyvinyl chloride. Camptothecin is a drug of plant origin that inhibits topoisomerase I, a crucial enzyme in the control of cell growth. Topotecan and irinotecan have also been developed from camptothecin for the treatment of lung and colorectal tumours.

Tamoxifen is an antagonist of the oestrogens used to treat breast cancer and prevent flare-ups. In this case, the drug is administered orally for long periods.

All these drugs present problems of bioavailability and/or formulation, because their solubility in water is low or non-existent.

These limitations therefore need to be overcome, especially by developing simpler and less risky pharmaceutical formulations.

DESCRIPTION OF THE INVENTION

It has now been found that hypercrosslinked polymers of cyclodextrins, called "nanosponges" because of their particular "nanoporous" structure, can advantageously carry water-insoluble drugs.

Cyclodextrin nanosponges enable otherwise insoluble drugs to be dispersed at molecular level, stabilising their structure and protecting them against aggression by chemical agents. The result is that the drug can remain effective for longer than the non-complexed form.

A first aspect of the invention therefore relates to complexes of drugs selected from paclitaxel, docetaxel, tamoxifen, camptothecin and derivatives thereof with cyclodextrin-based nanosponges.

The invention also relates to pharmaceutical formulations which can be administered orally or parenterally, and which use said complexes, mixed with suitable vehicles or excipients, as active constituent.

Cyclodextrin-based nanosponges are prepared as described in EP 1786841 or WO 03085002, using a crosslinker/cyclodextrin ratio of 2-16, preferably 4. The cyclodextrins can be natural, preferably β-cyclodextrin, or partly chemically modified, such as methyl β-cyclodextrin, alkyloxycarbonyl cyclodextrins, etc. Nanosponges can also contain variable percentages (5-30% by weight) of linear dextrins.

Nanosponges can also be magnetized when they are prepared in the presence of compounds having magnetic properties. The complexes according to the invention are prepared by adding an excess of drug to an aqueous suspension of cyclodextrin-based nanosponges. The suspension is stirred for 1 to 8 hours, preferably at room temperature, and the complex is recovered filtering the excess of non- solubilised drug. Formation of the complex is demonstrated by DSC analysis. The complexes obtained can be used directly to prepare oral or injectable formulations, using conventional techniques and excipients. For injectable formulations, for example, the complex may simply be carried in sterile water, saline or other aqueous solutions suitable for the parenteral administration. For the oral administration, the complexes may be dispersed in a matrix of excipients, diluents, lubricants and anti-caking agents suitable for the preparation of capsules and tablets. The doses will depend on the type of drug complexed, and will be at least equal to, or more preferably lower than those currently recommended in clinical practice, due to their improved bioavailability and pharmacokinetics.

The superiority of the formulations according to the invention clearly emerges if, for example, the antiproliferative action of paclitaxel in vitro is compared with that of paclitaxel complexed with nanosponges ("Complex"), using a cell proliferation test on tumour cell lines.

The examples below illustrate the invention in greater detail.

Example 1. Preparation of nanosponges

17.00 g of anhydrous β-cyclodextrin, 0.93 g of anhydrous dextrin 20 and 80 g of diphenyl carbonate (DPC)₁ all finely homogenised, are placed in a 250 ml flask. The system is gradually heated to 100⁰C under mechanical stirring, and left to react for 4 h. At the end of this period the reaction mixture solidifies and is repeatedly washed with distilled water, and then with acetone, to remove the unreacted DPC and the phenol present as by-product of the reaction. 20.2 g of nanosponge is obtained. Example 2. Preparation of nanosponges

3 g of dextrin 20, 20.0 g of beta cyclodextrin and 17.12 g of diphenyl carbonate, all finely pre-mixed, are placed in a flask. The system is heated to 100⁰C under mechanical stirring, and left to react for 4 h. At the end of this period, the resulting mass is placed in an excess of water, filtered, and washed thoroughly with water followed by acetone to remove unreacted DPC and by-products of the reaction. The filtrate is placed in an oven under vacuum to dry at 80⁰C for 2 hours. Example 2 bis. Preparation of nanosponges

The procedure of Example 2 is followed, but adding 2.0 g of cobalt powder. The resulting nanosponge particles keep their complexing properties and are also able to bind to a magnet. Example 3. Preparation of inclusion complex

A fixed amount (5 mg) of nanosponges was weighed and dispersed in distilled water in a 25 ml flask under stirring. An excess of drug, such as paclitaxel, was added, and the suspension was maintained under constant stirring for 4 hours. At the end of this time the suspension was filtered through a centrifuge filter (MICROCON YM 30, Millipore Corporation, Bedford MA 01730 U.S.A.) to separate the solubilised from the unsolubilised paclitaxel. The filtrate was analysed by HPLC to determine the paclitaxel content. The formation of the inclusion complex was verified by DSC analysis (Figure 1). Example 4. Solubility studies

The solubility determination was conducted in accordance with the Higuchi and Connors method. In particular, 10 mg of paclitaxel was added to an Erlenmeyer flask containing an aqueous solution (10 ml) of various percentages of nanosponges (0.01 , 0.02, 0.03, 0.04, 0.05, 0.075, 0.09, 0.1 , 0.12, 0.15 and 0.2%). The Erlenmeyer flask was stirred on a mechanical shaker at room temperature. When a steady state was reached (48 hours), the suspension was filtered by centrifugation using a 3000 Dalton molecular filter (MICROCON YM 30, Millipore Corporation, Bedford MA 01730 U.S.A.). The solution obtained was analysed to determine the paclitaxel concentration by HPLC at 277 nm using acetonitrileiwater 62:38 v:v as mobile phase after freeze-drying of the complex and extraction of the paclitaxel in known amounts of methanol. The paclitaxel concentration in mg was plotted the percentage concentration of nanosponges. The data were statistically processed using linear regression. Solubility analysis shows that peak solubilisation is reached with an 0.5% w/w suspension of nanosponges. No further increases in the solubility of paclitaxel were observed above this limit.

In conclusion, 1 mg of drug is solubilised by 5 mg of nanosponge. (Figure 2).

Example 5. Test of activity against cancer cells

The test is based on incubation of the cells for periods of 24, 48 and 72 h in the presence of graduated doses of the two formulations, ensuring that the same amount of active ingredient is always administered. The experiments were carried out on various cell lines, and specifically on AT84 cells of spontaneous murine oral squamous-cell carcinoma HSC and CaI cells of spontaneous human oral squamous-cell carcinoma.

At the end of the incubation period a solution of tetrazole salts, which are only metabolised by live cells, was added to the cells. After three hour incubation the excess salts were removed and DMSO was added to the cells; this generates a colour change in said reduced salts, thus allowing a colorimetric evaluation of the amount of tetrazole metabolised, which is directly proportional to the number of live cells. The data presented (relating to some of the experiments effected) are expressed as a percentage of live cells for each treatment compared with the controls, consisting of cells to which neither native nor complexed paclitaxel was added. The data are the result of experiments conducted in octuplicate.

The concentration range used (always based on the molarity of paclitaxel) was defined on the basis of the data present in the literature for paclitaxel alone.

The data at 24 h do not indicate any significant differences between the two treatment groups, whereas at 48 and 72 h, the antiproliferative effect of the Complex was observed to be greater. Figure 3 shows the data for the experiment on line AT84 at 72 h. They clearly indicate that the antiproliferative action of the Complex is far more marked (nearly three times greater) than that of paclitaxel alone, and is mainly manifested at lower doses. The effect is similar at higher doses. In view of the high toxicity of paclitaxel, the possibility of obtaining similar or better therapeutic effects at lower doses would mean that the treatment may be prolonged, its efficacy increased and its side effects limited. Moreover, the new formulation is simple, dispersed in distilled water, and does not involve the use of other organic substances, even as solvents. Similar results were obtained with other cells, though at different concentration intervals characteristic of each cell line.

Attention focused in particular on the lower concentrations, at which paclitaxel did not seem to act, except in association with nanosponges; figures 4 and 5 show the results of experiments conducted on human cell lines at extremely low concentrations. In both cases, the Complex reduces proliferation by 30-50% at concentrations as low as 1 nanomole, where non- complexed paclitaxel is inactive or not very active.

The internal controls of each experiment were represented by non- complexed nanosponges and identical concentrations of DMSO, which is required to solubilise paclitaxel alone. None of these conditions had any effect on cell proliferation (figure 6 shows the data for the experiment conducted on the CaI line at 48 hours).

The data obtained with the experiments conducted to date on tumour cell lines suggest that the antiproliferative action of paclitaxel complexed with nanosponges is far more accentuated than that of paclitaxel alone.

This finding is particularly evident at very low concentrations (approx. 1 nM). Nanosponges alone did not exhibit any antiproliferative or cytotoxic effect on the cell lines tested. Example 6. Determination of bioavailability of the formulation

To evaluate whether there is any increase in the oral bioavailability of paclitaxel complexed with nanosponges, an experiment was conducted on 20 six-week-old female Balb/c mice. The animals were divided at random into two groups, and 50 mg/kg of paclitaxel with Cremophor ("control" group) or the equimolar equivalent of the paclitaxel-nanosponge complex resuspended in saline solution ("complex" group) was administered by orogastric probe, after anaesthesia.

At pre-set intervals (30 min., 1 , 2 and 4 h), blood samples were taken from four animals in the complex group and one in the control group; the serum was then separated and immediately frozen for HPLC analysis.

A reverse-phase high-pressure chromatography (HPLC) method was used to quantitate the paclitaxel in the plasma of the mice after oral administration of the two formulations, ie. the commercial formulation (Taxol) and the nanosponge suspension. The instrument used for the analysis was a

Perkin Elmer binary pump (LC250) connected to a Perkin Elmer UV detector

(LC-95). The measurements were taken using a buffer mixture of 10 mM ammonium acetate pH = 5.0:methanol:acetonitrile (50:10:40, v/v/v) as mobile phase, while the stationary phase was represented by a Varian ODS column (250 mm x 4.6 mm). The analytical determination was performed at a flow rate of 1 ml/10 min, with the UV detector at the wavelength of 227 nm.

Preparation of biological samples: 200 μl of filtered water and 250 μl of methanol were added to a known volume of plasma (generally 25 μl). After stirring on a vortex, the mixture is extracted three times with diethyl ether. The ether phases are collected and evaporated to dryness under nitrogen. The residue is taken up with a water:methanol:acetonitri!e, 50:10:40, v/v/v solution, and injected directly into the HPLC after stirring.

Figure 7 shows the results obtained, which demonstrate the presence of high concentrations of paclitaxel in the blood of the mice 4 hours after oral administration. Example 7

The cytotoxic activity of tamoxifen carried in nanosponges was determined on the MCF-7 cell line (human breast cancer cells), using the drug without the vehicle as control. The results are shown in Figure 8.

Tamoxifen carried in nanosponges exhibited greater cytotoxic activity than the active constituent alone after 48 hours' incubation. In particular, if the cells are incubated with tamoxifen at the concentration of 100 ng/ml, approx. 40% of the cells survive, whereas the percentage of surviving cells exceeds 70% when the drug is used without the vehicle. Example 8

The cytotoxic activity of camptothecin complexed with nanosponges was determined on the HCPC1 (hamster cheek pouch carcinoma) cell line, using the non-complexed drug as control. The results are shown in Figure 9.

Camptothecin carried in nanosponges exhibited greater cytotoxic activity than the active constituent alone after 72 hours' incubation. In particular, if the cells are incubated with camptothecin at the concentration of 64 nM, under 20% of the cells survive, whereas the percentage of surviving cells exceeds 90% when the drug is used without the vehicle.

Claims

1. Complexes of hyper-crosslinked nanoporous polymers of cyclodextrins with drugs selected from paclitaxel, docetaxel, tamoxifen, camptothecin and derivatives thereof.

2. Complexes as claimed in claim 1 , wherein the hyper-crosslinked polymers are obtained from β-cyclodextrin.

3. Complexes as claimed in claim 1 or 2, wherein the drug is paclitaxel.

4. Complexes as claimed in claim 1 or 2, wherein the drug is tamoxifen.

5. Complexes as claimed in claim 1 or 2, wherein the drug is camptothecin.

6. Pharmaceutical formulations comprising the complexes claimed in claims 1-5, mixed with suitable vehicles or excipients.

7. Formulations as claimed in claim 6, suitable for the parenteral administration.

8. Formulations as claimed in claim 6, suitable for the oral administration.