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EP3968962A1 - Procédés de fabrication de nanocristaux à disponibilité biologique améliorée et formulation pour une telle préparation de nanocristaux destinée à une utilisation en thérapie anticancéreuse - Google Patents

Procédés de fabrication de nanocristaux à disponibilité biologique améliorée et formulation pour une telle préparation de nanocristaux destinée à une utilisation en thérapie anticancéreuse

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Publication number
EP3968962A1
EP3968962A1 EP19816509.4A EP19816509A EP3968962A1 EP 3968962 A1 EP3968962 A1 EP 3968962A1 EP 19816509 A EP19816509 A EP 19816509A EP 3968962 A1 EP3968962 A1 EP 3968962A1
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EP
European Patent Office
Prior art keywords
nanocrystals
preparation
camptothecin
composition
derivatives
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19816509.4A
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German (de)
English (en)
Inventor
Haijun XIAO
Vladimir SEDLARIK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tomas Bata University In Zlin
Original Assignee
Tomas Bata University In Zlin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CZ2018591A external-priority patent/CZ308874B6/cs
Application filed by Tomas Bata University In Zlin filed Critical Tomas Bata University In Zlin
Publication of EP3968962A1 publication Critical patent/EP3968962A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • 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.
  • 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.
  • 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.
  • hydrophobicity very low water solubility
  • curcuminoid therapeutic potential 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.
  • 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).
  • 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.
  • 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 (
  • 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.
  • 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.
  • 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.
  • 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
  • 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);
  • Io and I represent the fluorescence intensity of irinotecan in the absence (I 0 ) 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);
  • TCN topotecan
  • BCN belotecan
  • ECN exatecan / curcumin
  • 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;
  • FIG. 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
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • SEM scanning electron microscope
  • 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.
  • 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.
  • DMSO dimethylsulfoxide
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • mannitol serving as a cryoprotectant was added to the compound.
  • 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).
  • 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).
  • compositions described in Examples 3 to 10 exhibited the following properties:
  • topotecan hydrochloride TCN
  • belotecan hydrochloride BCN
  • exatecan mesylate ECN
  • lipotecan hydrochloride LiCN
  • ACN lurtotecan dihydrochloride
  • SCN simmitecan hydrochloride
  • EmCN elomotecan hydrochloride
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • composition for the preparation of nanocrystals for medicaments with the enhanced biological availability 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.

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Abstract

L'invention concerne un procédé de préparation de nanocristaux basé sur le procédé de préparation de nanoparticules cristallines sous forme de poudre qui consiste à a) dissoudre de l'azote protoné contenant des dérivés de camptothécine et de curcuminoïdes dans un solvant organique conduisant à la formation d'un complexe ionique autoassemblé puis à ajouter un tensioactif injectable non ionogène, b) transférer le mélange obtenu dans un milieu aqueux sous homogénéisation continue comprenant le mélange, l'agitation et/ou l'utilisation d'ultrasons, c) éliminer le solvant pour obtenir une forme pulvérulente, d) lyophiliser le produit auquel le cryoprotecteur a été préalablement ajouté. La composition pour la préparation de nanocristaux par le procédé selon l'invention est constituée d'un tensioactif non ionique injectable et d'un complexe ionique autoassemblé qui contient des curcuminoïdes et de l'azote protoné contenant des dérivés de camptothécine et ses analogues dans les rapports molaires dans la plage entre 10:1 et 1:10. Les analogues de la camptothécine comprennent, de préférence, la camptothécine et l'homocamptothécine. Les dérivés de camptothécine contenant de l'azote protoné présentent de préférence l'azote protoné inclus dans leur structure chimique par l'intermédiaire d'au moins un groupe fonctionnel comprenant une amine primaire, une amine secondaire, une amine tertiaire, une amine cyclique et/ou leurs combinaisons. L'utilisation de nanocristaux préparés pour l'administration d'un médicament injectable anticancéreux consiste en la dissolution de 3 % en poids de nanocristaux sous forme de poudre dans le milieu liquide à base d'eau distillée comprenant 5 % en poids de glucose.
EP19816509.4A 2018-10-30 2019-10-16 Procédés de fabrication de nanocristaux à disponibilité biologique améliorée et formulation pour une telle préparation de nanocristaux destinée à une utilisation en thérapie anticancéreuse Withdrawn EP3968962A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CZ2018591A CZ308874B6 (cs) 2018-10-30 2018-10-30 Způsob přípravy nanokrystalů se zvýšenou biologickou dostupností a připravené nanokrystaly
CZ2019630 2019-10-09
PCT/CZ2019/050048 WO2020088702A1 (fr) 2018-10-30 2019-10-16 Procédés de fabrication de nanocristaux à disponibilité biologique améliorée et formulation pour une telle préparation de nanocristaux destinée à une utilisation en thérapie anticancéreuse

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EP3968962A1 true EP3968962A1 (fr) 2022-03-23

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