ITMI20071321A1 - CYCLODESTRINE-BASED NANOSPUGNE AS A VEHICLE FOR ANTITUMOR DRUGS - Google Patents

Description

Description of the patent for industrial invention entitled:

"CYCLODEXTRIN-BASED NANOSPONGES AS A VEHICLE FOR ANTI-CANCER DRUGS"

The present invention relates to pharmaceutical compositions comprising nanosponges based on cyclodextrins as a vehicle for water-insoluble anticancer drugs, in particular paclitaxel and other taxanes, camptothecin and tamoxifen.

BACKGROUND OF THE INVENTION

The solubility of a drug is often the limiting factor for its application in therapy. Therefore, new strategies are always being investigated to improve the solubility and release kinetics of active ingredients.

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

The lipophilic cavity gives the CD the ability to form stable inclusion complexes even in solution with organic molecules of suitable polarity and size.

For this reason, CDs have already been studied and have numerous applications in various fields of chemistry (pharmaceutical, analytical, catalysis, cosmetics, etc.) in which the characteristics of inclusion compounds are exploited. In pharmaceutical technology such complexes can be used to increase the dissolution rate, solubility and stability of drugs, or to mask unpleasant flavors or to convert liquid substances into solids, etc.

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

Particularly critical drugs under the formulation profile, especially because of their insolubility, are the tumor agents paclitaxel, docetaxel and in general the derivatives with a taxane structure, camptothecin and tamoxifen.

Paclitaxel is an important anticancer drug belonging to the family of taxanes, products of plant origin that perform their antineoplastic action by inhibiting the formation of the mitotic spindle.

Paclitaxel is practically 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 has allowed it to be administered intravenously. The components of the emulsion, however, are potent allergens that can cause severe hypersensitivity reactions. Also, the materials used for paclitaxel administration cannot be polyvinyl chloride.

Camptothecin is a plant-derived drug that inhibits topoisomerase I, an enzyme crucial in controlling cell growth. Camptoecin, topotecan and irinotecan have also been developed for the treatment of lung and colorectal cancers.

Tamoxifen is an estrogen antagonist used in the treatment of breast cancers and in the prevention of recurrence. In such a case, the drug is administered orally for long periods of time.

All these drugs pose bioavailability and / or formulation problems due to their low or no water solubility.

There is therefore a need to overcome these limitations by developing in particular simpler and less risky pharmaceutical formulations.

Description of the invention

It has now been found that hyper-crosslinked cyclodextrin polymers defined as "nanosponges" due to their particular "nanoporous" structure are capable of advantageously delivering hydroinsoluble drugs.

The cyclodextrin nanosponges allow to disperse otherwise insoluble drugs at the molecular level, stabilizing their structure and protecting them from the aggression of chemical agents. This can lead to a prolongation of the drug's efficacy, compared to the non-complexed form.

The invention therefore provides in its first aspect complexes of drugs selected from paclitaxel, docetaxel, tamoxifen, camptothecin and their derivatives with cyclodextrin nanosponges.

The invention also relates to pharmaceutical formulations that can be administered orally or parenterally comprising as an active ingredient said complexes in mixture with suitable vehicles or excipients.

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

Nanosponges can also be magnetized if prepared in the presence of compounds with magnetic properties.

The complexes of the invention are prepared by adding an excess of drug to an aqueous suspension of cyclodextrin nanosponges. After stirring the suspension for a time ranging from 1 to 8 hours, preferably at room temperature, the complex is recovered by filtering the excess of unsolubilized drug. The formation of the complex is demonstrated by DSC analysis.

The obtained complexes can be used directly for the preparation of oral or injectable formulations, using conventional techniques and excipients. For the injectable formulations, for example, the complex can be simply conveyed in sterile, physiological water or other aqueous solutions suitable for parenteral administration. For the oral route, the complexes can be dispersed in a matrix of excipients, diluents, lubricants, anti-adhesives suitable for the preparation of capsules and tablets. The dosages will depend on the type of complexed drug and will be at least equal to or more preferably lower than those currently proposed in clinical practice, by virtue of the improved bioavailability and pharmacokinetic characteristics.

The superiority of the formulations of the invention clearly emerges by comparing, for example, the antiproliferative action of paclitaxel in vitro with that of paclitaxel complexed with nanosponges ("Complex") through cell proliferation tests on neoplastic cell lines.

The following examples 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 finely homogenized diphenylcarbonate (DFC) are placed in a 250 ml flask. The temperature is gradually brought to 100 ° C under mechanical stirring and it is left to react for 4 h. At the end the reaction mixture solidifies and is washed numerous times with distilled water and subsequently with acetone to remove the unreacted DFC and the phenol present as reaction by-product. 20.2 g of nanosponge are obtained.

Example 2. Preparation of nanosponges

3 g of dextrin 20, 20.0 g of beta cyclodextrin and 17.12 g of diphenylcarbonate, previously finely mixed, are placed in a flask. The system is brought to 100 ° C in an ultrasonic bath and reacted for 4 h. At the end, the mass obtained is placed in excess of water, filtered and washed extensively with water and subsequently acetone to remove unreacted DFC and the reaction by-products. The filtrate is placed in a vacuum oven to dry at 80 ° C for 2 hours.

Example 2 bis. Preparation of nanosponges

As in example 2, but with the addition of 2.0 grams of Cobalt powder. In the end, nanosponge particles are obtained which maintain the complexing properties and which are able to bind to a magnet.

Example 3. Preparation of the inclusion complex

A fixed amount (5 mg) of nanosponges were weighed and dispersed in distilled water in a 25 ml flask with stirring. An excess of the drug, for example paclitaxel, was added and the suspension kept under constant stirring for 4 hours. Finally the suspension was filtered through a centrifuge filter. (MICROCON YM 30, Millipore Corporation Bedford MA 01730 U.S.A.) in order to separate the solubilized paclitaxel from the unsolubilized one. The filtrate was analyzed with HPLC to determine the paclitaxel content. The formation of the inclusion complex was verified by DSC analysis (Figure 1).

Example 4. Solubility studies

The determination of the solubility was carried out according to the Higuchi and Connors method. In particular 10 mg of paclitaxel were added to a flask containing an aqueous solution (10 ml) of different 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 flask shaken on a mechanical shaker at room temperature. After reaching equilibrium (48 hours) the suspension was filtered by centrifugation using a 3000 Dalton molecular cut filter (MICROCON YM 30, Millipore Corporation Bedford MA 01730 U.S.A.). The solution obtained was analyzed to determine the paclitaxel concentration by HPLC at 277 nm using acetonitrile: Water 62:38 v: v as mobile phase after lyophilization of the complex and extraction of paclitaxel in known quantities of methanol. The paclitaxel concentration in mg was plotted against the concentration of nanosponges in percent. The data were statistically processed using linear regression. The solubility analysis shows that the maximum solubilization is reached with 0.5% w / w of nanosponges suspension. Beyond this limit, no further increases in paclitaxel solubility are observed.

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

Example 5: activity test on cancerous cells

The test is based on cell incubation for periods of 24, 48 and 72 h, in the presence of scaled doses of the two formulations, taking care to administer, in any case, the same amount of active ingredient. The experiments were conducted on various cell lines, namely on the AT84 cells of murine spontaneous oral squamous carcinoma HSC and on the Cai cells of spontaneous human oral squamous carcinoma.

At the end of the incubation period, a solution of tetrazolium salts was added to the cells, metabolized only by living cells. After three hours of incubation, the excess salts were eliminated and DMSO was added to the cells, which causes a change in the color of the aforementioned reduced salts, thus allowing a colorimetric evaluation of the quantity of metabolized tetrazolium, directly proportional to the number of cells. lives. The data presented (relating to a part of the experiments carried out) are expressed as a percentage of live cells for each treatment compared to the controls, consisting of cells to which neither native nor complexed paclitaxel has been added. The data are the result of experiments conducted in octuplicate.

The range of concentrations used (always referring to the molarity of paclitaxel) was defined on the basis of the data in the literature for paclitaxel alone.

The data at 24 h did not show significant differences between the two treatment groups, while at 48 and 72 h there was a more important antiproliferative effect on the part of the Complex.

Figure 3 summarizes the data of the experiment on the AT84 line at 72 h. From them it is evident a much more pronounced antiproliferative action (almost three times) of the Complex compared to paclitaxel alone, which occurs mainly at lower doses. At higher doses the effect is similar.

Given the high toxicity of paclitaxel, the possibility of obtaining similar or higher therapeutic effects at lower doses allows the treatment to be prolonged, to increase its effectiveness and to limit side effects. Furthermore, the new formulation is simple, dispersed in distilled water and does not require the use of other organic substances, not even as solvents.

Similar results were obtained on the other cells, albeit at different concentration ranges, characteristic of each cell line.

Particular attention was paid to the lowest concentrations, at which paclitaxel did not seem to act if not 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, already at 1 nanomolar concentrations, where the non-complexed paclitaxel is not active or not very active, the Complex induces a reduction in proliferation of 30-50%.

The internal controls of each experiment are represented by the non-complexed nanosponges and by identical concentrations of DMSO, necessary for the solubilization of paclitaxel alone. None of these conditions showed any effect on cell proliferation (Figure 6 shows the data relating to the experiment performed on the Cai line at 48 hours).

The data obtained with the experiments conducted up to now on neoplastic cell lines would show a much more pronounced antiproliferative action of paclitaxel complexed with nanosponges than paclitaxel alone.

This data is particularly evident especially at very low concentrations (about 1 nM). Furthermore, the nanosponges alone have not been shown to have any antiproliferative or cytotoxic effects on the cell lines tested.

Example 6: determination of the bioavailability of the formulation

To evaluate the possible increase in the oral bioavailability of paclitaxel complexed with nanosponges, an experiment was conducted on 20 female Balb / c mice of six weeks.

The animals were randomly divided into two groups and, after anesthesia, the administration of 50 mg / kg of paclitaxel with Cremophor ("control" group) or the equimolar equivalent of the paclitaxel complex was carried out by means of an oral-gastric tube. nanosponges resuspended in physiological solution ("complex" group).

At predetermined intervals (30 ', 1, 2 and 4 h), the blood was taken from four animals for the complex group and one from the control group respectively, and the subsequent separation of the serum and its immediate freezing for HPLC analysis.

For the quantitative determination of paclitaxel in the plasma of mice after oral administration of the two formulations, the commercial one (Taxol) and the suspension of nanosponges, a reverse phase high pressure chromatography (HPLC) method was used. The instrument used for the analysis is Perkin Elmer (binary pump LC250) connected to a Perkin Elmer UV detector (LC-95). The measurements were carried out using a 10 mM ammonium acetate buffer mixture as mobile phase pH = 5.0: methanol: acetonitrile (50:10:40, v / v / v) while the stationary phase is represented by a Varian ODS column (250 mm x 4.6 mm). The analytical determination is carried out with a flow of 1 ml / min and the UV detector at a wavelength of 227 nm.

Preparation of biological samples: 200 μΐ of filtered water and 250 μΐ of methanol were added to a known volume of plasma (typically 25 μ). After vortexing the mixture is extracted three times with diethyl ether.

The ethereal phases are collected and evaporated to dryness under a nitrogen flow. The residue is taken up with a water solution: methanol: acetonitrile, 50:10:40, v / v / v) and after stirring injected directly into the HPLC.

Figure 7 reports the results obtained that prove the presence of elevated paclitaxel concentrations in the blood of mice 4 hours after oral administration.

Example 7

The cytotoxic activity of tamoxifen delivered in the nanosponges was determined on the MCL-7 (human breast cancer cells) cell line using the non-delivered drug as a control. The results are shown in Ligura 8.

Tamoxifen delivered in nanosponges showed greater cytotoxic activity than the active ingredient alone after a 48-hour incubation. In particular, by incubating the cells with tamoxifen at a concentration of 100 ng / ml, approximately 40% of surviving cells are obtained, while the percentage of surviving cells is greater than 70% with the non-conveyed drug.

Example 8

The cytotoxic activity of camptothecin complexed with nanosponges was determined on the cell line HCPC1 (hamster cheek pouch carcinoma) using the non-complexed drug as a control. The results are shown in Figure 9. Camptothecin delivered in nanosponges showed greater cytotoxicity than the active ingredient alone after 72 hours incubation. In particular, by incubating the cells with camptothecin at a concentration of 64 nM, there are less than 20% of surviving cells, while the percentage of surviving cells is greater than 90% with the drug not delivered.

Claims (8)

CLAIMS 1. Complexes of nanoporous cyclodextrin hyper-crosslinked polymers with drugs selected from paclitaxel, docetaxel, tamoxifen, camptothecin and their derivatives. 2. Complexes according to claim 1 wherein the hyper-crosslinked polymers are obtained from β-cyclodextrin. Complexes according to claim 1 or 2 wherein the drug is paclitaxel. 4. Complexes according to claim 1 or 2 wherein the drug is tamoxifen. 5. Complexes according to claim 1 or 2 wherein the drug is camptothecin. 6. Pharmaceutical formulations comprising the complexes of claims 1-5 in admixture with suitable carriers or excipients. 7. Formulations according to claim 6 suitable for parenteral administration. 8. Formulations according to claim 6 suitable for oral administration.