WO2020088702A1

WO2020088702A1 - Methods of making nanocrystals with enhanced biological availability and formulation for such nanocrystals preparation for use in anticancer therapy

Info

Publication number: WO2020088702A1
Application number: PCT/CZ2019/050048
Authority: WO
Inventors: Haijun XIAO; Vladimir SEDLARIK
Original assignee: Tomas Bata University In Zlin
Priority date: 2018-10-30
Filing date: 2019-10-16
Publication date: 2020-05-07
Also published as: EP3968962A1; CN113423388A

Abstract

The method of nanocrystal preparation is based on the procedure of the crystalline nanoparticles preparation in powder form that comprises a) dissolving the protonated nitrogen containing derivatives of camptothecin and curcuminoids in an organic solvent leading to formation of self-assembled ionic complex followed by subsequent addition of nonionogenic injectable surfactant, b) transferring the obtained mixture into aqueous medium under continuous homogenization including mixing, shaking and/or using ultrasound, c) removing the solvent in order to obtain powder form, d) lyophilisation of the product with the previously added cryoprotectant. Composition for nanocrystal preparation through the method according to the invention consists of injectable nonionic surfactant and self-assembled ionic complex that contains curcuminoids and protonated nitrogen containing derivatives of camptothecin and its analogues in the molar ratios in the range between 10:1 and 1:10. Analogues of camptothecin include preferably camptothecin and homocamptothecin. Protonated nitrogen containing camptothecin derivatives preferably have the protonated nitrogen included in their chemical structure through at least one functional group including primary amine, secondary amine, tertiary amine, cyclic amine and/or their combinations. Utilization of nanocrystals prepared for the anticancer injectable drug application consists in the dissolution of 3 wt. % nanocrystals in powder form in the liquid medium based on distilled water with 5 wt. % of glucose.

Description

METHODS OF MAKING NANOCRYSTALS WITH ENHANCED BIOLOGICAL AVAILABILITY AND FORMULATION FOR SUCH NANOCRYSTALS PREPARATION FOR USE IN ANTICANCER THERAPY

Field of Invention

The invention describes method of preparation of nanocrystals based on ionic complexes forming self-assembled nanostructures and compositions prepared by this method. The composition and the obtained nanocrystals can be used for preparation of agents with enhanced biological availability that can be utilized especially in the field of preparation and application of cytostatics with reduced side effects.

State of the Art

The analogues of camptothecin and their derivatives are potent therapeutic agents for chemical treatment of various oncological diseases. These molecules and their active metabolites can specifically bind to topoisomerase I-DNA complex, thereby preventing reconnection of the single-stranded fracture and stopping DNA replication. Curcuminoids (curcumin (CAS 458- 37-7), desmethoxycurcumin (CAS 22608-11-3) and bis-demethoxycurcumin (CAS 24939-16- 0)) are natural polyphenols found in the extract of Curcuma longa, one of the widely used medicinal plants particularly in Asian countries. Curcuminoids show pharmacological effects, such as anti-inflammatory, antioxidant and antimicrobial properties. Furthermore, they are described through an anti-cancer effect, too.

However, the usage of camptothecin and its derivatives for chemotherapeutic treatment of cancer can cause side effects to oncological patients in form of stomach problems, nausea, diarrhoea. Another undesirable phenomenon reducing the effect of treatment is the gradual development of tumour cell tolerance to the drug.

Additionally, the hydrophobicity (very low water solubility), which can be also found in combination with other substances, significantly reduces the possibilities of biological effects of curcuminoids in the body. Another limitation of the wider biological utilization of curcuminoid therapeutic potential is their rapid metabolization causing a low rate of plasma protein binding described in WO 2010013224 (EP 2349237).

Patent CN102885800B (issued August 6, 2014) reveals that by combining irinotecan (a camptothecin derivative) with curcuminoids some of the above-mentioned side effects can be suppressed. The combination of camptothecin and curcuminoid in the form of nanocrystals is also discussed in patent application CN104546728, which describes the stabilization of nanocrystals by an amphoteric molecule, for example a poloxamer, and the possibility of its subsequent use in the preparation of injectable solutions. This takes into account the tendency of curcuminoids to metabolize rapidly. Nevertheless, a specific implementation of a combination suitable for injection is not described herein.

The aim of the invention is therefore to provide an easy-to-manufacture and stable composition intended for immediate use in injection applications which is based on curcuminoids and camptothecin derivatives, is suitable for use as an anticancer agent and gentle to the patient's gastrointestinal tract, i.e. does not show the above-mentioned side effects.

Ground of Invention

The aforesaid disadvantages and drawbacks of the above-mentioned biologically active substances used up to now for the treatment of oncological diseases are largely eliminated by the composition for the preparation of nanocrystals for the substances with enhanced biological availability according to the invention and the method of its preparation.

The ground of the method of preparation of nanocrystals according to the invention consists in that the preparation of nanocrystalline particles in powder form contains the following steps: a) dissolving the protonated nitrogen containing derivatives of camptothecin and curcuminoids in an organic solvent leading to formation of a self- assembled ionic complex followed by subsequent addition of nonionogenic injectable surfactant; b) transferring the obtained mixture into an aqueous medium under continuous homogenization, including mixing, shaking and / or using ultrasound; c) removing the solvent to obtain a powder form; d) lyophilisation of the product with the previously added cryoprotectant.

The organic solvent used in step a) is preferably dimethylsulfoxide (CAS 67-68-5), propylene carbonate (CAS 108-32-7), acetonitrile (CAS 75-05-8), acetone (CAS 67-64-1) , dimethylformamide (CAS 68-12-2), tetrahydrofuran (CAS 109-99-9), methylpyrrolidone (CAS 872-50-4), hexamethylphosphoramide (CAS 680-31-9), methanol (CAS 67-56-1), ethanol (CAS 64-17-5), acetic acid (CAS 64-19-7) or combinations thereof. The cryoprotectant used in step d) is preferably lactose (CAS 63-42-3), mannitol (CAS 69-65- 8), sucrose (CAS 57-50-1), trehalose (CAS 99-20-7), fructose (CAS 57-48-7), glucose (CAS 50-99-7), sodium alginate (CAS 9005-38-3), gelatin (CAS 9000-70-8) or combinations thereof.

The ground of the composition prepared by the method according to the invention consists in that the composition consists of an injectable nonionic surfactant and a self-assembled ionic complex containing curcuminoids and protonated nitrogen comprising camptothecin derivatives and analogues thereof in a molar ratio of components ranging from 10: 1 to 1: 10.

Camptothecin analogues preferably include camptothecin (CAS 7689-03-4) and homocamptothecin (CAS 186669-19-2). The protonated nitrogen is contained in camptothecin derivatives preferably in the form of at least one functional group including a primary amine, a secondary amine, a tertiary amine, a cyclic amine and / or a combination thereof. The primary amine-containing camptothecin derivative may be one or more substances from the following groups: 9-aminocamptothecin (CAS 91421-43-1), exatecan (CAS 171335-80-1), delimotecan (CAS 187852-63-7), namitecan (CAS 372105-27-6). Preferably, the secondary amine- containing camptothecin derivative is belotecan (CAS 256411-32-2). The tertiary amine- containing camptothecin derivative may be topotecan (CAS 123948-87-8) and / or lipotecan (CAS 1432176-87-8). Camptothecin derivatives containing a cyclic amine in their structure are represented by one or more substances from the group of: irinotecan (CAS 100286-90-6), lurtotecan (CAS 149882-10-0), afeletecan (CAS 215604-75-4), simmitecan (CAS 1247847-78- 4), elomotecan (CAS 220998-10-7).

The nitrogen-containing camptothecin analogue derivatives are preferably present in the composition in the form of salts with organic or inorganic acids selected from the group comprising hydrochloric acid (CAS 7647-01-0), sulfuric acid (CAS 7664-93-9), phosphoric acid (CAS 7664 -38-2), hydrobromic acid (CAS 10035-10-6), perchloric acid (CAS 7601-90- 3), methanesulfonic acid (CAS 75-75-2p, acetic acid (CAS 64-19-7), maleic acid (CAS 110- 16-7), tartaric acid (CAS 526-83-0), citric acid (CAS 77-92-9) or combinations thereof.

The ground of the use of the nanocrystals according to the invention for the preparation of an anticancer agent for injection applications consists in that the nanocrystals in the form of a powder are dissolved in a liquid medium which is distilled water containing 5% wt. glucose, wherein the weight ratio of nanocrystals to liquid medium is in the range of 0.01: 99.99 to 3:97.

Based on the ionic complexes according to the the present invention, injectable nanocrystalline self-assembled systems with therapeutic and anti-inflammatory effects and better tolerability in the body are produced. Combining them with the nonionic components according to the invention provides compositions for immediate or sequential use in injection applications, based on nanocrystals with cytostatic properties and exhibiting increased stability over a wide pH range.

The advantage of the composition according to the invention is the narrow particle size distribution and their stability, but also the increased water solubility of the hydrophobic curcuminoid molecules, which significantly enhances their use in injectable applications and the so-called biological availability - the possible actions inside the body. Thus, their therapeutic effect in the treatment of cancer is enhanced.

The nanocrystals based on the composition according to the invention can be prepared either in powder form without specific requirements for temperature, pressure or stabilizing additives, which is favorable from the viewpoint of commercial production of chemotherapeutic agents, or directly as a liquid substance for injectable applications.

Overview of Images in Drawings

The attached drawings show the chemical structures for the composition according to the invention and the obtained properties of the nanocrystals:

Fig. 1 - Chemical structures of amphiphilic camptothecin analogues and hydrophobic curcuminoids;

Fig. 2 - Fluorescence characteristics of self-assembled ionic complexes based on irinotecan hydrochloride and curcumin (Excitation (A), emission (B) and synchronous (C) fluorescence spectra of irinotecan with different curcumin concentrations in dimethylformamide at 25°C; (D) Stem-Volmer plot showing fluorescence quenching of irinotecan at 25 ° C; (E) Plot of (Io/ I- 1) versus logarithm of curcuminoid concentration (mol / L);

Note: Io and I represent the fluorescence intensity of irinotecan in the absence (I₀) and in the presence of (I) curcuminoids. (xo₎ and (x) are the fluorescence lifetimes of irinotecan in the absence and in the presence of curcumin, respectively; Ci is the concentration of irinotecan; Cc is the concentration of curcuminoids;

Fig. 3 - Chemical structures of protonated nitrogen containing camptothecin analogue derivatives; Fig. 4 - Representation of a typical hydrodynamic diameter (A) and zeta potential (B) of nanocrystals dispersed in distilled water and their scanning electron microscope images (C) with measuring scale showing the length of 200 nm; the yield of relevant camptothecin analogue derivatives from nanocrystals based on topotecan (TCN) / curcumin, belotecan (BCN) / curcumin and exatecan / curcumin (ECN);

Fig. 5 - Changes in nanocrystalline properties in suspension in water (A) at different pH values (after 8 hours at 37.5 ° C) and in phosphate buffer (B) at different times (pH = 7.4 , 37. 5 ° C);

Fig. 6 - XRD spectra on powder samples: A - irinotecan hydrochloride, B - curcumin, C - mixture of pure components of irinotecan hydrochloride and curcumin, D - nanocrystals based on self-assembled ionic complexes - final product, E - pure cryoprotectant mannitol after dissolution in water and subsequent evaporation, F - pure constituents of irinotecan hydrochloride and curcumin after dissolution in methanol and subsequent evaporation;

Figures 7 to 13 - procedure for verifying the anti-cancer effect of injectable irinotecan-curcumin hydrochloride (ICN) according to the invention in nano-form of HT-29 on a tumour produced by a subcutaneous graft applied in mouse (nude mouse) in vivo :

Fig. 7 is a schematic diagram of an experiment for testing anti-tumour efficacy using colorectal tumour graft applied in nude mice;

Fig. 8 - tumour volume applied in mice injected with PBS, I or ICN (PBS = Phosphate-buffered saline, Ph 7.4; I = irinotecan hydrochloride alone; ICN = combination I with curcumin according to the invention);

Figure 9 - Tumour volume change after injection of PBS, I or ICN;

Figure 10 - Tumour mass change after injection of PBS, I or ICN;

Fig. 11 - images of extracted tumours;

Fig. 12 - weight of tested animals after injection of PBS, I or ICN;

Fig. 13 - intensity of side effect in experimental animals (in the form of diarrhea); considerable difference between the ICN and I group, no difference between ICN and PBS group. The significance of the differences observed between the groups was analyzed using a two-way ANOVA analysis (parameters * p <0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

Detailed description of the detected dependences: Camptothecin analogue derivatives have fluorescent properties. It can be seen from Figures 2A, 2B and 2C that irinotecan (a nitrogen containing camptothecin derivative) exhibits an excitation peak at a wavelength of 385 nm and an emission peak at 428 nm. The addition of curcuminoid causes the quenching (decreasing intensity) of the excitatory, emission and synchronous spectra of irinotecan. The data obtained from the measured values within the quenching process analysed using the Stem-Volmer model (Fig. 2D) show changes in fluorescence intensities, but without changing its lifetime, indicating the formation of a complex with curcuminoid (static quenching) at a 1: 1 molar ratio (Fig. 2E). The above mentioned properties can also be observed for other protonated nitrogen-containing derivatives of camptothecin and its analogues, the structures of which are shown in Fig. 3. The above stated shows that curcuminoids and protonated nitrogen-containing derivatives of camptothecin and its analogues form at specific organic solvent environments ionic surfactants that are self-organized through intermolecular interactions (e.g. hydrophobic and electrostatic interactions).

The nanoparticles based on curcuminoid (7.4 mg) and another protonated nitrogen containing camptothecin derivative or salt thereof, e.g. topotecan hydrochloride (4.6 mg), belotecan (4.3 mg) or exatecan (5.3 mg), were - based on this finding - also prepared by the method according to the invention with similar results.

It has been found out that the parameters of nanocrystals prepared from the above mentioned ionic complexes are best controlled in the range of hydrodynamic diameter of 50-1000 nm with a polydispersity less than or equal to 0.2 in suspension. The zeta potential of nanocrystals ranges from -10 mV to +60 mV depending on the pH and ionic strength of the environment. The molar ratio of curcuminoid and protonated nitrogen-containing camptothecin derivatives and analogues thereof is in the range of 10: 1 to 1: 10.

However, the sizes of nanocrystals based solely on ionic complexes are strongly dependent on the pH and ionic strength of the surrounding environment, which may lead to their uncontrolled changes. Therefore, the addition of nonionic surfactants to the above mentioned compositions was further tested. The addition of non-ionic surfactants (e.g., cholesterol, poloxamer, polyoxylic castor oil and polysorbates) results in reaching the stability of nanocrystals over a wide pH range due to the impact of steric interparticle forces. Despite the fact that the surface tension of the particles decreases to almost zero value, there are shown only slight changes in their size. Nanocrystals based on a mixture of the above mentioned ionic and nonionic complexes exhibit an increase in solubilization resistance of at least 40% compared to nanocrystals based on only ionic complexes. The XRD spectra on powder samples (Fig. 6) show the creation of new crystalline structures formed during the preparation of the composition according to the invention.

The nanosuspension can be prepared by mixing nanocrystals in an injectable liquid containing, for example, 5 wt. % glucose and 0.96 % wt. sodium chloride.

The values and dependences shown in Figures 7 to 13 will be described and analysed directly in Example 11.

Examples

Example 1

Preparation of nanocrystals based on irinotecan and curcumin - structure see Fig. 1 and 3

Irinotecan hydrochloride (6.2 mg) and curcumin (7.4 mg) in a molar ratio of 1 : 2 were dissolved in 0.5 mL of dimethylsulfoxide at room temperature. Subsequently, 15 wt. % of poloxamer 105 as a nonionic surfactant was added. The resulting mixture was transferred to an aqueous medium - added to 30 mL of distilled water and stirred at 500 rpm for 5 min at room temperature. (In addition to water, an aqueous medium with a pH of max. 9 may be used for transfer to an aqueous medium.)

The obtained suspension was subjected to dialysis in order to remove the organic solvent; a membrane with a MWCO (molecular weight cutoff) of 500 D was used.

Dialysis was performed against distilled water for 3 hours. Before lyophilization, 20 % wt. mannitol was added, which served as a cryoprotectant.

The size of the resulting nanocrystals was around 100 nm (see Fig. 4A and 4B). The nanocrystals had a positive surface charge of +43 mV and showed a narrow size distribution represented by a polydispersity index of 0.107. According to the scanning electron microscope (SEM) analysis, the above mentioned nanocrystalline particles had an almost globular shape close to spheres and a smooth surface (Fig. 4C). Changes in the selected characteristics of nanoparticles (hydrodynamic diameter, polydispersity index and zeta potential) when exposed to different pH or ionic strength are shown in Fig. 5. The surface tension of nanoparticles decreased with the increasing acidity of the environment (pH less than 7). Negative zeta potential values of nanocrystals were monitored in an alkaline environment (pH greater than 7). On the other hand, there were slight changes in the size of nanocrystals and the breadth of their polydispersity as a result of further impact of repulsive steric forces. In addition, nanoparticles are stable even after 8 hours in phosphate buffer (pH 7.4) at 37.5 ° C, with a slight increase in particle diameter and their polydispersity. Their surface tension was below +10 mV.

The concentration of both bioactive substances was verified by high performance liquid chromatography. The detected proportion of bioactive substances in the particles was 70% by weight. The ratios between camptothecin derivatives and curcuminoids in nanoparticles were almost the same as the concentration ratio at the beginning of the reaction.

The combination of the ionic complex and the non-ionic surfactant resulted in nanocrystals having cytostatic properties that showed increased stability over a wide pH range.

Example 2

Preparation of nanocrystals of irinotecan and curcumin (ICN) - structure see Fig. 1 and 3

Irinotecan hydrochloride (6.2 mg) and curcumin (7.4 mg) at a molar ratio of 1 : 2 were dissolved in 0.4 mL of dimethylsulfoxide (DMSO) at room temperature to form a self-assembled ionic complex, to which 3 mg of cholesterol was subsequently added as nonionic surfactant. The organic solution thus obtained was then added to 30 mL of distilled water and stirred under the same conditions as in Example 1. The obtained suspension was dialyzed to remove the organic solvent; a MWCO 300 D membrane was used. Dialysis was performed against distilled water for 2 hours. Before lyophilization, 1% wt. mannitol serving as a cryoprotectant was added to the compound. In this way, nanocrystals were obtained in the form of a powder, from which a nanosuspension was prepared by dissolving in distilled water containing 5% wt. glucose.

Example 3

Preparation of nanocrystals based on topotecan and curcumin (TCN) - structure see Fig. 3

Topotecan hydrochloride (4.6 mg) and curcumin (4.0 mg) in a 1: 1 molar ratio were dissolved in 0.3 mL of dimethylformamide (DMF) at room temperature to form a self-assembled ionic complex, to which 1.3 mg of polysorbate 80 was subsequently added - serving as a nonionic surfactant. The organic solution thus obtained was then added to 15 mL of distilled water and stirred as in Example 1, after which the suspension obtained was subjected to dialysis to remove the organic solvent - MWCO 500 D membrane. Dialysis was performed against distilled water for 2 hours. Before lyophilization, 1% wt. mannitol serving as a cryoprotectant was added to the compound. A nanocrystalline powder was obtained from which the nanosuspension for application was prepared by dissolving in distilled water containing 5 % wt. glucose.

The parameters of the product obtained, as well as the products of Examples 4 to 10 below, will be listed and summarized in the final section of this chapter.

Example 4

Preparation of nanocrystals based on belotecan and curcumin (BCN) - structure see Fig. 3.

Belotecan hydrochloride (4.3 mg) and curcumin (3.5 mg) in a 1: 1 molar ratio were dissolved in 0.3 mL dimethylsulfoxide (DMSO) at room temperature to form a self-assembled ionic complex, to which 3.4 mg of nonionic surfactant- polyoxylated castor oil - was subsequently added. The organic solution thus obtained was then added to 15 mL of distilled water and stirred as in Example 1, and the suspension obtained was again dialyzed with a MWCO 500 D membrane. Dialysis was performed against distilled water for 1 hour. Before lyophilization, 1% wt. mannitol serving as a cryoprotectant was added to the compound. A powder was obtained from which the nanosuspension was subsequently prepared by dissolving in distilled water containing 5% wt. glucose.

Example 5

Preparation of nanocrystals based on exatecan and curcumin (ECN) - structure see Fig. 3

Exatecan mesylate (5.3 mg) and curcumin (7.0 mg) at a molar ratio of 1 :2 were dissolved in 0.4 mL DMF at room temperature to form a self-assembled ionic complex, to which 1.8 mg of a nonionic poloxamer 105 surfactant was subsequently added The organic solution thus obtained was then added to 20 mL of distilled water and stirred as in Example 1, the suspension obtained was dialyzed using a MWCO 300 D membrane. Dialysis was performed against distilled water for 3 hours. Before lyophilization, 1% wt. mannitol serving as a cryoprotectant was added to the compound. In this way, a nanocrystalline powder was obtained from which the nano suspension for use was prepared by dissolving the powder in distilled water containing 5% wt. glucose.

Example 6

Preparation of nanocrystals based on lipotecan and curcumin (LiCN) - structure see Fig. 3

Lipotecan hydrochloride (8.8 mg) and curcumin (3.5 mg) in a 1: 1 molar ratio were dissolved in 0.3 mL of DMF at room temperature to form a self-assembled ionic complex, to which 1.8 mg of nonionic surfactant poloxamer 105 was subsequently added. The organic solution thus obtained was then added to 30 mL of distilled water and stirred as in Example 1, the suspension obtained was dialyzed with a membrane of MWCO 500 D. Dialysis was performed against distilled water for 2 hours. Before lyophilization, 1% wt. mannitol serving as a cryoprotectant was added to the compound. In this way, a powder was obtained from which the nanosuspension was prepared before use by dissolving the powder in distilled water containing 5 wt. % glucose and 0.96 wt. % sodium chloride.

Example 7

Preparation of nanocrystals based on afeletecan and curcumin (ACN) - structure see Fig. 3

Afeletecan hydrochloride (9.0 mg) and curcumin (7.5 mg) at a molar ratio of 1 : 2 were dissolved in 0.5 mL DMSO at room temperature to form a self-assembled ionic complex, to which 2.5 mg of a nonionic surfactant poloxamer 105 was subsequently added. The organic solution thus obtained was then added to 40 mL of distilled water and stirred as in Example 1, and the suspension obtained was dialyzed with a MWCO 1000 D membrane to remove the organic solvent. Dialysis was performed against distilled water for 2 hours. Before lyophilization, 1% wt. mannitol serving as a cryoprotectant was added to the compound. Thus a powder was obtained from which a nanosuspension was subsequently prepared by dissolving in distilled water containing 5% wt. glucose in a weight ratio of 0.01:99.99 (lyophilisate: medium).

Example 8

Preparation of nanocrystals based on lurtotecan and curcumin (LuCN) - structure see Fig. 3

Lurtotecan dihydrochloride (6.0 mg) and curcumin (3.5 mg) in a 1 : 1 molar ratio were dissolved in 0.3 mL DMSO at room temperature to form a self-assembled ionic complex, to which 1.4 mg of a nonionic surfactant poloxamer 105 was subsequently added The organic solution thus obtained was then added to 15 mL of distilled water and stirred as in the previous examples, the obtained suspension was dialyzed using a MWCO 300 D membrane, the process was performed against distilled water for 2 hours. Before lyophilization, 1% wt. mannitol serving as a cryoprotectant was added to the compound. Within this procedure a powder was obtained from which the nanosuspension was prepared for purposes of application by dissolving in distilled water containing 5% wt. glucose in a weight ratio of 2:98 (lyophilisate: medium).

Example 9

Preparation of nanocrystals based on simmitecan and curcumin (SCN) - structure see Fig. 3 Simmitecan hydrochloride (6.2 mg) and curcumin (7.4 mg) at a molar ratio of 1:2 were dissolved in 0.4 mL of DMF at room temperature to form a self-assembled ionic complex, to which 2 mg of poloxamer 105 was subsequently added as a nonionic surfactant. The organic solution thus obtained was then added to 30 mL of distilled water and stirred as in the previous examples, and the suspension obtained was dialyzed using a MWCO 300 D membrane. Dialysis was performed against distilled water for 3 hours. Before lyophilization, 1% wt. mannitol serving as a cryoprotectant was added to the compound. In this way a powder was obtained from which the nanosuspension was prepared for application by dissolving in distilled water containing 5% wt. glucose in a weight ratio of 3:97 (lyophilisate: medium).

Example 10

Preparation of nanocrystals based on elomotecan and curcumin (EmCN) - structure see Fig. 3

Elomotecan hydrochloride (5.5 mg) and curcumin (7.4 mg) in a molar ratio of 1:2 were dissolved in 0.4 mL DMSO at room temperature to form a self-assembled complex, to which 2 mg of poloxamer 105 were subsequently added. The organic solution thus obtained was then added to 30 mL of distilled water and stirred as in the previous examples, the obtained suspension was dialyzed using a MWCO 300 D membrane. Dialysis was performed against distilled water for 3 hours. Before lyophilization, 1% wt. mannitol serving as a cryoprotectant was added to the compound. In this way a powder was obtained from which the application nanosuspension was prepared by dissolving in distilled water containing 5% wt. glucose in a weight ratio of 2:98 (lyophilisate: medium).

The compositions described in Examples 3 to 10 exhibited the following properties:

In the case of nanoparticles based on curcumininoid and other protonated nitrogen-containing derivatives of camptothecin and its analogues as shown in Examples 3 to 10, e.g. topotecan hydrochloride (TCN), belotecan hydrochloride (BCN), exatecan mesylate (ECN), lipotecan hydrochloride (LiCN) ), afeletecan hydrochloride (ACN), lurtotecan dihydrochloride (LuCN), simmitecan hydrochloride (SCN) or elomotecan hydrochloride (EmCN), they were discovered to have similar properties to irinotecan based particles in terms of size, size distribution, surface charge and morphology.

The concentration of bioactive components (ICN, TCN, BCN and ECN) - see Examples 1 to 5 in the prepared nanoparticles was verified by high performance liquid chromatography (HPLC). As can be seen from Fig. 4D, the detected proportion of bioactive substances in the particles was 70% wt. In the case of camptothecin and curcuminoid derivatives, their proportional presence in nanoparticles was almost identical with the initial ratio before the start of the reaction.

Changes in the selected characteristics of irinotecan-based nanoparticles were also observed under various environmental conditions. Fig. 5 shows the behaviour of nanoparticles at different pH or ionic strength. The surface tension of nanoparticles decreased with the increasing acidity of the environment (pH less than 7). Negative zeta potential values of nanoparticles were recorded in an alkaline environment (pH greater than 7). On the contrary, only minor changes occurred in the size of nanoparticles and the width of their polydispersity as a consequence of further impact of repulsive steric forces induced by the presence of nonionic complexes. In addition, nanoparticles are stable even after 8 hours in phosphate buffer (pH 7.4) at 37.5 ° C, with a slight increase in particle diameter and polydispersity.

Example 11

Preparation of the composition and experimental verification of the therapeutic effect - see Fig. 7 - 13.

Irinotecan hydrochloride 6.2 mg and curcumin 3.7 mg were dissolved in 0.4 mL dimethylsulfoxide (DMSO) at room temperature and 15% wt. d. poloxamer 105 was added. The obtained organic solution was added to 20 mL of distilled water with stirring (500 rpm) for 4 min at room temperature. The obtained suspension was transferred to a dialysis tube where MWCO (molecular-weight-cut-off) = 300 D, and dialyzed against distilled water to remove the organic solvent. Mannitol was added as a cryoprotectant. Irinotecan-curcumin powder was obtained by lyophilisation. By dissolving it in distilled water containing 5% wt. glucose, an anticancer emulsion for injection applications was obtained.

The anti-cancer effects of this injectable formulation were investigated in vivo using intestinal cells of the HT-29 line on a tumour created by subcutaneous graft of these cells applied in mouse (nude mouse). The experiment strategy is shown in Fig. 7.

Progress and monitoring:

Once the average tumour volume reached about 100 mm³, mice were randomly separated into three groups of 4-5 in each group and injected intravenously with 1) PBS, 2) I, 3) ICN (equivalent to the amount of irinotecan alone - 27.5 mg / kg) every other day for a total of 19 days. Course of treatment: Body weight and tumour volume were measured every 3 days. Tumour volume was calculated according to the formula Tv = length x width²/2. 5 days after interruption of medication, mice were sacrificed, by tumours were subsequently (day 25 from the start of the experiment) removed, weighed and photographed.

Monitoring of undesirable side effects: Diarrhea was reported according to the following scale: stool 0 - normal or none, 1 - slightly wet and soft, 2 - moderately soft and unshaped with moderate peri-anal contamination, 3 - intense, watery with critical contamination in the peri anal area.

Evaluation of therapeutic effects:

Compared to the PBS group in mice in both treatment groups, the average tumour volumes were effectively regulated and there were significant differences from the untreated group at day 9 (about 1 week after the first administration - Fig. 8). Tumour volume decline occurred in the treatment groups on day 15 (about 2 weeks after the first administration, Fig. 9). The treatment groups also show significant differences in tumour weight compared to PBS (blind) - Fig. 10. Photodocumentation of the removed tumours is shown in Fig. 11.

There was no statistical difference between the two treatment groups throughout the experiment. The results confirm that the efficacy of the combined nanoformulation of the ICN according to the invention is the same as the anti-cancer effect of irinotecan hydrochloride itself, and that the tumour regression effect is induced by irinotecan instead of curcumin in the ICN according to the invention. This can be attributed to a significant difference in half maximal inhibitory concentration of the both substances. Curcumin can generally be considered as biologically safe at the therapeutic level / concentration of irinotecan used.

In addition, no significant weight loss was observed in any of the mice throughout the whole experiment, which would indicate even minor side effects of the ICN nano-formulation according to the invention at the dosage used in the treatment of the tumour - Fig. 12.

Evaluation of undesirable side effects:

As can be seen from Fig. 13, diarrhea was significantly delayed in mice treated with irinotecan alone on day 10 after the first administration (p <0.01), with no distinct diarrhea in the PBS group (blind - no treatment) throughout the whole experiment, but also not in the ICN group (mice treated with the composition according to the invention). Reduction of diarrhea in mice in this group shows that the presence of curcumin together with irinotecan in the nanofonnulation according to the invention can reduce intestinal load and thus protect the intestines, thus increasing the patients’ comfort. This can be attributed either to the therapeutic effects of the curcumin molecule or to a change in the drug formulation from irinotecan alone to its combination with curcumin according to the invention.

Industrial applicability

The composition for the preparation of nanocrystals for medicaments with the enhanced biological availability, the method of the preparation of nanocrystals and the use of nanocrystals in injectable applications according to the invention will find use preferably in the field of production of medicaments with the increased requirement for biological availability and tolerability. It will be used especially in the field of preparation of cytostatics - anticancer agents.

Claims

1. A method of preparation of nanocrystals with enhanced biological availability, char acterized by that the procedure of preparation in the powder form contains the following steps: a) dissolving the protonated nitrogen containing derivatives 15of camptothecin and curcuminoids in an organic solvent leading to formation of self-assembled ionic complex followed by subsequent addition of nonionogenic injectable surfactant; b) transferring the obtained mixture into aqueous medium under continuous homogenization including mixing, shaking and/or using ultra-sound; c) removing the solvent in order to obtain powder form; d) lyophilisation of the product with the previously added cryoprotectant.

2. The method of preparation of nanocrystals according to claim 1, characterized b y that the organic solvent used in the step a) is dimethylsulphoxide, propylen carbonate, acetonitrile, acetone, dimethylformamide, tetrahydrofurane, methylpyrrolidone, hexamethylphosphoramide, methanol, ethanol, acetic acid or their combination.

3. The method of preparation of nanocrystals according to claim 1, characterized b y that the cryoprotectant used in step d) is lactose, mannitol, sucrose, trehalose, fructose, glucose, sodium alginate, gelatine or their combination.

4. A composition for preparation of nanocrystals with enhanced biological availability and nanocrystals prepared from this composition by the method according to claim 1, characterized by that the composition comprises of injectable nonionic surfactant and self-assembled ionic complex based on curcuminoids and protonated nitrogen containing camptothecin derivatives and its analogues in the molar ratio between 10:1 and 1:10.

5. A composition for preparation of nanocrystals according to claim 4, characteriz- e d b y that the camptothecin analogues contain protonated nitrogen and include camptothecin and homocamptothecin.

6. A composition for preparation of nanocrystals according to claim 4 and 5, charact erized by that the protonated nitrogen containing derivatives of camptothecin contain in their chemical structure at least one of functional groups including primary amine, secondary amine, tertiary amine, cyclic amine and/or their combinations.

7. A composition for preparation of nanocrystals according to claim 4 and 6, charact erized by that the derivatives of camptothecin, which contain primary amine included in their chemical structure, include one or more substances from the group of 9-aminokamptothecin, exatecan, delimotecan, namitecan.

8. A composition for preparation of nanocrystals according to claim 4 and 6, ch a r a c t - erized by that the derivatives of camptothecin, containing secondary amine in their chemical structure, include belotecan.

9. A composition for preparation of nanocrystals according to claim 4 and 6, ch a r a c t - erized by that the derivatives of camptothecin, containing tertiary amine in their chemical structure, include topotecan and/or lipotecan.

10. A composition for preparation of nanocrystals according to claim 4 and 6, charact erized by that the derivatives of camptothecin containing cyclic amine in their chemical structure, include one or more compounds from the group of irinotekan, lurtotecan, afeletecan, simmitecan, elomotecan.

11. A composition for preparation of nanocrystals according to claim 4, characteriz- e d by that the derivatives of nitrogen containing camptothecin analogues are present in form of salts of organic or inorganic acids selected from the group including hydrochloric acid, sulphuric acid, phosphoric acid, hydrobromic acid, perchloric acid, methanesulfonic acid, acetic acid, maleic acid, tartaric acid, citric acid or their combinations.

12. A composition for preparation of nanocrystals according to claim 4, characteriz- e d b y in that the injectable nonionogenic surfactant is based on cholesterol, poloxamers, polyoxyl castor oil, polysorbates or their combinations.

13. Utilization of nanocrystals prepared according to the claim 1 for the anticancer injectable drug application, characterized by that the nanocrystals in the powder form are dissolved in the liquid medium comprised of distilled water with 5 wt. % of glucose where the weight ratio of nanocrystals and liquid medium is in the range between 0.01:99.99 and 3:97.