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    • 专利摘要:
    Inclusion complex of natural and bioavailable origin for the treatment of diseases of parasitic origin. Inclusion complex comprising at least one compound active against parasitic diseases such as malaria, steviol glycoside and an aminoglycoside. In particular, the active compounds are artemisinin, a derivative thereof, curcumin, hydroxytyrosol or a combination of the foregoing. The present invention also relates to a method for obtaining said inclusion complex by ultrasound; as well as the inclusion complex for the treatment of parasitic diseases, in particular malaria. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2731554A1
    申请号:ES201830339
    申请日:2018-04-05
    公开日:2019-11-15
    发明作者:Gil Jesus Martin；Juan Celia Andres；Ben Eduardo Ruiz；Lebena Eduardo Perez
    申请人:Universidad de Valladolid；
    IPC主号:
    专利说明:

    [0001]
    [0002] COMPLEX INCLUSION OF NATURAL AND BIODISPONIBLE ORIGIN FOR
    [0003]
    [0004] TECHNICAL SECTOR
    [0005]
    [0006] The present invention falls within the scope of organic chemistry and finds its application in the pharmaceutical sector. More specifically, the present invention provides a new complex, in particular an inclusion complex or clathrate, which allows to increase the solubility and bioavailability of the compounds included therein. As a result, when the active compounds exhibit antiparasitic activity, the complex of the present invention allows the treatment of parasitic diseases, such as malaria, with an activity greater than that obtained from using the components separately.
    [0007]
    [0008] BACKGROUND OF THE INVENTION
    [0009]
    [0010] Parasites are living beings that live on other living beings, such as the human body, to feed and have a place to live. They can be contracted through food or contaminated water, the bite of an insect or through sexual contact. Some parasitic diseases are easier to treat than others.
    [0011]
    [0012] Parasites vary in size, from single-celled organisms called protozoa, to worms, which can be seen with the naked eye. The contaminated water supply can cause Giardias infections. Cats can transmit toxoplasmosis, dangerous for pregnant women. Other diseases, such as malaria, are common in certain parts of the world. Specifically, the latter, also called malaria, is produced by parasites of the genus Plasmodium and is the first in importance among debilitating diseases. It caused approximately 216 million cases of malaria in 2016 and 445,000 deaths, of which more than 85% are children in endemic areas of Africa. Most of the disease burden is recorded south of the Sahara.
    [0013]
    [0014] The symptoms of malaria are very varied, including fever, chills, sweating and headache. You may experience nausea, vomiting, cough, bloody stools, muscle aches, jaundice, blood clotting defects, shock, kidney failure or liver, central nervous system disorders and coma. Fever and chills are cyclic symptoms, repeating every two to three days.
    [0015]
    [0016] Plasmodium falciparum protozoan causes the most acute and severe malaria. Originates the kidnapping of red blood cells in venous microcirculation, prevents the passage of blood through the spleen and, therefore, causes their destruction; It presents alterations in the level of consciousness, coma, seizures, hypoglycemia, hyperinsulinemia in adults, metabolic acidosis, jaundice or hemorrhages that are signs of poor prognosis that require immediate medical action. The disease is serious in children and pregnant women.
    [0017]
    [0018] Antimalarial drugs vary from country to country, due to studies of antimalarial resistance that are carried out periodically and according to a protocol established by the local WHO agency. Resistance of parasites to several antimalarials has been observed. Resistance rates increase as the use of new antimalarials also increases. Among the commonly used medications, artemisinin derivatives stand out, since they have rapid blood schizonticidal action. Artemisinin is found in the Artemisia annua plant. It is a sesquiterpenic lactone that contains an unusual peroxide bridge. This peroxide seems to be responsible for the mechanism of action of the drug. Artemisinin derivatives form a family of drugs and have the fastest action of all common anti-malaria medications. Additionally, other terpenic compounds with antimalarial characteristics have been described, such as the so-called Mogrosides.
    [0019]
    [0020] Due to the short half-life of artemisinin, it should be used in combination with another antimalarial to prevent recrudescences of the infection. Thus, its use as monotherapy is discouraged by the WHO, since it has been found that malaria parasites have developed resistance to the drug. Therapies that combine artemisinin with some other drug (ACTs), usually organochlorine compounds, are the preferred treatments for their effectiveness, although they present tolerance problems for patients. The use of this medicine has also increased in treatments against vivax malaria and is the subject of research in studies of cancer treatments.
    [0021]
    [0022] On the other hand, it is known that curcumin has antimalarial effects in vitro, but its zero bioavailability in vivo hinders the development of combinations with artemisinin. In fact, it also suffers from the same problem. In the Curcumin-Artemisinin study Combination Therapy for Malaria , Govindarajan Padmanaban et al., Antimicrob Agents Chemother. 2006 May, due to the fact that these substances are not soluble in water, they have to be dissolved in DMSO (dimethylsulfoxide), which is not practicable for in vivo studies with humans. Another drawback detected in the article is that they are not applied simultaneously by the same means, but rather that artemisinin is injected into blood and curcumin, previously dissolved in DMSO, has been introduced orally.
    [0023]
    [0024] In the Cytotoxic Effect of Curcumin on Malaria Parasite Plasmodium falciparum: Inhibition of Histone Acetylation and Generation of Reactive Oxygen Species , Long Cui, Jun Miao, and Liwang Cui, Antimicrob Agents Chemother. 2007 Feb, where curcumin is used as monotherapy, the effect generated is described so that the prooxidant activity of curcumin promotes the production of reactive oxygen species (ROS), which ultimately inhibit the activity of histone acetyltransferase (HAT) of the parasite, causing the necessary cytotoxicity in it.
    [0025]
    [0026] In the study Modulation of cerebral malaria by curcumin as an adjunctive therapy , Kunal Jain, Sumeet Sood, K. Gowthamarajan, The Brazilian Journal of Infectious Diseases, Jul 2013, up to 9 possible targets of the parasite on which curcumin acts are described. On the other hand, in the Ameliorative Effects of Curcumin on Artesunate-Induced Subchronic Toxicity in Testis of Swiss Albino Male Mice , Desai KR, Rajput DK, Patel PB, Highland HN, Dose Response, 2015 Jun, it is indicated that curcumin acts by attenuating the damage generated in the treatment of malaria with an artemisinin derivative, artesunate, which is on the List of Essential Medicines of the World Health Organization.
    [0027]
    [0028] On the other hand, patent application WO2012127287 A2 (Febris Bio Tech Ltd et al.) Describes the use of a composition comprising steviol glycosides for the treatment of malaria. Additionally, this composition may comprise other antimalarial agents such as artemisinin or one of its derivatives.
    [0029]
    [0030] To ensure that the ingested active compound is used by the body and is useful for the intended purpose, it is essential that it has adequate bioavailability. Unfortunately, oral administration of medications often suffers from problems of solubility and / or permeability, as well as stability problems that limit the shelf life of the active compound. therapy. The use of inclusion nanocomposites to improve the bioavailability of non-water soluble substances is a field of great breadth and in continuous technological progress, applicable to the pharmaceutical, food and cosmetic industries. These techniques need to evolve to meet the new demands of consumers and Science in general.
    [0031]
    [0032] Several attempts to encapsulate compounds with activity against malaria have been described in the state of the art in order to increase their solubility. In particular, the formation of complexes with cyclodextrins has been described (US2010179103 A1, WO03075904 A2 and Suvarna Vasanti et al. ( Complexation of phytochemicals with cyclodextrin derivatives - An insight. Biomedicine & Pharmacotherapy. April 2007. Vol. 88, pages 1122-1144 )); with liposomes (Isacchi Benedetta et al. ( Artemisinin and artemisinin plus curcumin liposomal formulations: Enhanced antimalarial efficacy against Plasmodium berghei-infected mice, European Journal of Pharmaceutics and Biopharmaceutics, April 2012, Vol. 80, No. 3, pages 528-534) ) and with lipids (Memvanga Patrick B et al. ( An oral malaria therapy: Curcumin-loaded lipid-based drug delivery systems combined with beta-arteether, Journal of Controlled Release, Dec 2013, Vol. 172, No. 3, pages 904 -913)).
    [0033]
    [0034] On the other hand, patent application WO2009126950 A2 (Univ Louisiana State & Agricultural and Mechanical College) describes the use of diterpene glycosides to increase the solubility of water insoluble substances such as curcumin or artemisinin, separately.
    [0035]
    [0036] DESCRIPTION OF THE INVENTION
    [0037]
    [0038] A first aspect of the present invention relates to a complex, in particular an inclusion complex or clathrate, comprising at least one active compound, a steviol glycoside (GL) and an aminoglycoside (AM).
    [0039]
    [0040] In this document "active compound" should be understood as any compound that has a therapeutic activity beneficial to humans or animals, in particular, this term refers to a compound with antiparasitic activity and, preferably, with antimalarial activity.
    [0041] The complex described herein may comprise one or more different steviol glycosides. The most important steviol glycosides are stevioside, rebaudioside A, B, C, D, E and F and dulcoside. While the stevioside molecule is a complex of three glucose molecules and an aglycone molecule called steviol, the rebaudioside A molecule contains a total of four glucose units, with the average glucose of the triplet connected to the central steviol structure.
    [0042]
    [0043]
    [0044]
    [0045] Stevioid Rebaudioside A
    [0046]
    [0047]
    [0048]
    [0049]
    [0050] Generally, the inclusion complex described herein comprises steviol glycosides, for example from stevia, with molecular weights between 800 to 1000 g / mol.
    [0051]
    [0052] Preferably, the aminoglycoside (AM) is selected from chitosan oligomer, glucosamine and a combination of the foregoing. In even more preferred embodiments, the molecular weight of the chitosan oligomer is 1000 to 10,000 g / mol and, even more preferably, between 1000 to 3000 g / mol.
    [0053]
    [0054] An important aspect of the present invention (found by DRX, DSC and SEM) is that the interactions between the components of the inclusion complex are weaker than shown with other complexes such as those formed by cyclodextrins, since the size of the complex is larger. In particular, the interactions between steviol glycosides, aminoglycosides and the active compounds in the complex of the invention, obtained by sonication by ultrasound as described hereinbelow, take place by hydrogen bonds. The hydrogen bond is a strong electrostatic force when many molecules are attached, as it provides great stability, but weaker than the covalent bond or ionic bond. Consequently, the compounds of the present invention have a greater ability to release the active compounds than could be obtained with cyclodextrins.
    [0055]
    [0056] In the appearance of the bonds through hydrogen bonds, the action of the ultrasound that is applied is essential. Otherwise, these links do not appear or do so very slowly, making the process described in this patent unfeasible.
    [0057]
    [0058] The inclusion complex described in this document has advantages over other vehiculization systems because it allows increasing the solubility of the compounds included therein, allowing their storage and administration, thus facilitating their application in the pharmaceutical sector. Another important advantage of this complex lies in the exclusive use of natural polymers and in the non-use of surfactants.
    [0059]
    [0060] Thus, the combination of steviol glycoside and aminoglycoside as forming agents of the complex of the invention provides high solubility (which can be 1000 times higher than that of the active compound separately) and solution stability. It has been observed that the inclusion complex formed solely with steviol glycoside as encapsulating agent precipitates over time. The addition of the aminoglycoside gives stability to the complex and, as a consequence, water solubility is maintained for a significantly longer time. In general, the contribution of the aminoglycoside improves the inclusion complex in two different aspects, on the one hand increasing the water solubility that had been obtained with steviol glycoside and on the other giving additional stability necessary for its useful life to be longer .
    [0061]
    [0062] In preferred embodiments of the present invention, the active compound present within the inclusion complex is a compound with activity against malaria. Preferably, this compound is selected from the group consisting of artemisinin, a derivative of artemisinin, curcumin, hydroxytyrosol and a combination of the foregoing. In In particular, the artemisinin derivative may be artemether, artesunate, or dihydroartemisinin (DHA).
    [0063]
    [0064] The inclusion of artemisinin in the inclusion complex described in this document allows to stabilize the peroxide group present in its structure, which is beneficial in order to use this active compound in the treatment of malaria, since it is intuited that this Functional group is particularly active against the causative agents of the infection, by inducing an oxidative process in their cell membranes, which causes phagocytosis and subsequent lysis.
    [0065]
    [0066] Preferably, two antimalarial agents are located inside the complex formed by steviol glycoside and aminoglycoside, one of them being artemisimine or one of its derivatives, in particular artemether, artesunate, or dihydroartemisinin; while the second active compound is selected from curcumin and hydroxytyrosol.
    [0067]
    [0068] One of the advantages of the inclusion complex described here is that both steviol glycoside and aminoglycoside, encapsulating agents of the complex, are naturally occurring compounds that show activity against malaria. As a consequence, this complex is particularly useful for the treatment of said disease. Thus, in the case of using a combination of two active compounds as mentioned in the previous paragraph, the use of the complex of the present invention for the treatment of malaria takes place through the application of four compounds with antimalarial activity, i.e. , the present invention provides a tetratherapy for the treatment of malaria. It is significant that the presence of a polyphenol such as curcumin in the complex of the present invention prevents recrudescence, that is, the onset of the disease in the future.
    [0069]
    [0070] Additionally, curcumin and hydroxytyrosol are both phenolic compounds with a very similar behavior. In particular, both can act as hepatoprotectors, which is particularly advantageous in the treatment of malaria, since it is known that during such treatment the protozoa accumulates in the liver.
    [0071]
    [0072] In even more preferred embodiments, the inclusion complex described in this patent application comprises:
    [0073] i) between 10% and 90% artemisinin or one of its derivatives, and
    [0074] ii) between 90% and 10% curcumin or hydroxytyrosol;
    [0075] amounts expressed by weight with respect to the total weight of the active compounds i) and ii) located inside the inclusion complex.
    [0076]
    [0077] In particular, it is preferred that the inclusion complex comprises:
    [0078] i) between 35% and 65% artemisinin or one of its derivatives, and
    [0079] ii) between 65% and 35% curcumin or hydroxytyrosol;
    [0080] amounts expressed by weight with respect to the total weight of the active compounds i) and ii) located inside the inclusion complex.
    [0081]
    [0082] The solubility of the active compounds (CBA) increases with the concentration of steviol glycoside (GL), it being preferable that the ratio between CBA and GL is between 1: 6 and 1:10. Thus, it is considered preferable that the weight ratio between the different components of the complex is 1: 6: 1 to 1: 10: 1 between the CBA: GL: AM, the optimum ratio of 1 active compound (CBA) being: 6 steviol glycoside (GL): 1 aminoglycoside (AM), since this relationship allows the formation of supramolecular or toroid structures of clathrates in which the active compound is trapped by very weak or short-range molecular interactions with the glycoside molecule of steviol, for example, stevioside (see figure 1). If the concentration of steviol and / or aminoglycoside glycoside is increased, the solubilization of the active compound can be increased, but this also implies a greater economic expense by using a larger amount of the complexing agents of the complex.
    [0083]
    [0084] Preferably, the active compound comprised within the complex is a combination of artemisinin or one of its derivatives with curcurmine or hydroxytyrosol. In particular, it is preferred that the active compound be the combination of artemisinin and curcumin (ART / CUR), it being even more preferred that these compounds be in a 1: 1 weight ratio between them. As shown in Figure 1, when the active compound is the combination of artemisinin (ART) and curcumin (CUR) in a 1: 1 weight ratio, a co-amorphous aggregate can be formed, as proposed by Kuthuru Suresh, MK Chaitanya Mannava, and Ashwini Nangia *, A Novel Curcumin-Artemisinin Coamorphous Solid: Physical Properties and Pharmacokinetic Profile in RSC Advances (online publication).
    [0085] A second aspect of the present invention refers to a method for obtaining the complexes described above, where the method comprises the following steps:
    [0086] a) mixing by ultrasonic sonication at least one active compound to be encapsulated (CBA) with a steviol glycoside (GL), preferably in a hydroalcoholic medium;
    [0087] b) add an aminoglycoside (AM) to the solution obtained in the previous stage and mix by sonication by ultrasound; and
    [0088] c) isolate the CBA-GL-AM complex formed in step b) in the solid state, preferably by lyophilization.
    [0089]
    [0090] Preferably, the sonication mixing in steps a) and b) takes place between 10 to 20 kHz, the optimum value being 20 kHz. In particular embodiments of the present invention, these steps take place at a temperature between 15 and 50 ° C, more preferably between 20 and 40 ° C. To control that the temperature of the solution does not exceed 50 ° C, ultrasonic application can be performed intermittently, for example, in application periods of 2 minutes each. In the hydroalcoholic solution the alcohol can be methanol or ethanol, and the alcohol: water ratio can be 1: 1 (v / v).
    [0091]
    [0092] Additionally, the method may comprise a step c1), where the particular size of the inclusion complex CBA: GL: AM obtained in step c) is reduced to a particle size between 200 nm and 400 nm, preferably by a crushing or grinding process.
    [0093]
    [0094] The CBA-steviol glycoside complex formed in step a) can be isolated in solid form, for example, by lyophilization of the solution. This isolation can be done directly, without the need to adjust the pH. Generally, this isolated CBA-GL complex already has greater water solubility stability than the active compound without encapsulation. However, the incorporation of another encapsulation sphere formed by aminoglycosides, in particular chitosan oligomer, significantly improves its solubility in water, while improving the release control of CBA and, consequently, its bioavailability and absorption.
    [0095]
    [0096] In those embodiments in which the CBA-glycoside complex is isolated in solid state, the method may comprise an additional step, where the particle size of this Isolated complex is reduced to between 20 nm and 200 nm, preferably by a crushing or grinding process.
    [0097]
    [0098] Through a sonication process in step a) of the method, that is, the direct application of ultrasound on the solution comprising the mixture of CBA and steviol glycosides, preferably in hydroalcoholic medium, the glycoside molecules are oriented around the compound active to encapsulate, with weak interactions by formation of hydrogen bonds between the glycosides. Subsequently, the sonication process in the presence of aminoglycoside of step b) allows an additional encapsulation sphere to be formed, giving rise to the inclusion complex CBA-GL-AM by solid state isolation, preferably lyophilization. The size of the nanoparticles can be controlled by transmission electron microscopy (TEM) and / or scanning electron microscopy (SEM) and the strength of the bonds or interaction between the functional groups can be done by Fourier transform infrared spectroscopy (FTIR- ATR). The crystallinity characterization of the complex obtained can be followed by differential scanning calorimetry (DSC) or X-ray diffraction (DRX). The solubility and characterization studies of CBAs can be followed with a UV-VIS spectrophotometer and / or a mass chromatograph (HPLC-Masses).
    [0099]
    [0100] The solubility of CBAs in the complex of the invention increases with the concentration of glycosides. Thus, in the present invention it is preferred that the ratio CBA: steviol glycosides is between 1: 6 and 1:10 (weight: weight), it being even more preferable that this ratio CBA: glycosides is 1: 6 (weight: weight ), since it is with which adequate solubility is achieved at a lower cost.
    [0101]
    [0102] By way of theoretical explanation, but without binding character, a complex formed by artemisinin and curcumin in 1: 1 ratio as active compound (CBA) is represented in Figure 1, a first sphere formed by steviol glycoside in a 1: 6 ratio (CBA: GL), which is characterized by the presence of weak hydrogen bridge interactions between the molecules that favors the solubilization and subsequent vehiculization of artemisinin and curcumin. Although not shown in Figure 1, the inclusion complex of the present invention comprises successive spheres of coordination or self-assembly with aminoglycosides, in particular chitosan oligomers. These types of interactions contrast with the strong interactions that appear between cyclodextrins and CBAs (ibuprofen) that hinder their vehiculization (S. Pereva, T. Sarafska, S.
    [0103] Bogdanova, T. Spassov, Efficiency of “Cyclodextrin-lbuprofen” inclusion complex formation, Journal of Drug Delivery Science and Technology (2016)) (Figure 2).
    [0104]
    [0105] In preferred embodiments of the present invention, the method for obtaining the inclusion complex described herein comprises mixing by sonication between 10 kHZ and 20 kHZ, the CBA: GL complex with at least one aminoglycoside, preferably chitosan oligomers, in a solution hydroalcolic Then, by lyophilization of the hydroalcoholic solutions (directly and without adjusting the pH), the CBA-GL-AM complex that is subsequently subjected to milling can be isolated , so that the resulting complex has a nanoparticle size of less than 400 nm .
    [0106]
    [0107] Additionally, the present invention relates to the inclusion complex obtainable by the method described herein.
    [0108]
    [0109] The present invention also relates to the inclusion complex described in this patent application for use in medicine and, in particular, for the treatment of a parasitic disease such as malaria.
    [0110]
    [0111] In particular, when the complex comprises a combination of artemisinin or one of its derivatives, with curcumin or hydroxytyrosol, the present invention provides an inclusion complex that can be used as a tetratherapy against malaria, since the four ingredients comprising the complex have antimalarial activity, making it difficult to generate resistance. Additionally, all the ingredients of the complex do not present the tolerance problems associated with the majority of therapies currently available against malaria.
    [0112]
    [0113] BRIEF DESCRIPTION OF THE FIGURES
    [0114]
    [0115] Figure 1: Non-binding theoretical model of the formation of an inclusion complex or clathrate between stevioside and artemisinin-curcumin (ART-CUR), with stoichiometry 6 EST: 1 ART-CUR.
    [0116]
    [0117] Figure 2: Formation of toroid complexes of p-cyclodextrin with ibuprofen.
    [0118] Figure 3. FTIR-ATR spectra of absorbance of the starting products (ordered the spectra from top to bottom): 1st artemisinin (ART); 2nd curcumin (CUR); 3rd chitosan oligomers (OQ); 4th stevioside (ES).
    [0119]
    [0120] Figure 4. Absorbance FTIR-ATR spectra of the inclusion complexes obtained by lyophilization (ordered the spectra from top to bottom): 1st artemisinin curcumin (ART / CUR); 2nd artemisinin curcumin stevioside (ART / CUR-ES); 3rd artemisinin curcumin stevioside oligomers of chitosan (ART / CUR-ES-OQ); 4th centrifuged encapsulated complex and washing of chitosan oligomers with artemisinin curcumin stevioside (ART / CUR-ES-OQ).
    [0121]
    [0122] Figure 5. Photograph obtained by microscopy with 500x of the crushed compounds in ball mill: artemisinin (fig. 5a); artemisinin curcumin 1: 1 weight (fig. 5b); artemisinin curcumin stevioside 0.5: 0.5: 3 by weight (fig. 5c); Artemisinin curcumin stevioside oligomers of chitosan 0.5: 0.5: 3: 1 by weight (fig. 5d).
    [0123]
    [0124] Figure 6: Photograph obtained by microscopy with 500x of the precipitated and centrifuged compounds of amorphous co-products: artemisimine curcumin stevioside oligomers chitosan 0.5: 0.5: 6: 1 by weight.
    [0125]
    [0126] Figure 7: Artemisinin and curcumin DRX spectra (upper spectrum, M8) and further evolution of the DRX spectrum of artemisinin and curcumin mixtures with stevioside and chitosan oligomers (lower spectra, in descending order M7, M6 and M5) and finally (M3-1) the formation of the encapsulated and lyophilized and completely amorphous and water-soluble inclusion complex of artemisinin curcumin stevioside chitosan oligomers (0.5: 0.5: 6: 1).
    [0127]
    [0128] EXAMPLES
    [0129]
    [0130] A. PROCEDURE OF OBTAINING
    [0131]
    [0132] Example 1.- Preparation of the complex (ART / CUR-ES-OQ) comprising artemisinin and curcumin as a hydrophobic active compound (ART / CUR) with stevioside (ES) and chitosan oligomer (OQ) in a weight ratio: 1 ART / CUR: 10 ES: 1 OQ Section 1: Start from 1 gram of active compound (0.5 g artemisinin and 0.5 g curcumin) and disperse in 250 ml of absolute ethanol. On the other hand, 10 g of stevioside are weighed in 250 mL of distilled and deionized water. Both solutions are mixed and the resulting mixture is emulsified by directly introducing the sonicator head of the ultrasonic equipment into the solution (20 kHz frequency). The sonication is carried out for periods of 2 minutes each, and a time of 20 minutes, not allowing the solution to heat above 50 ° C. The resulting solution, protected from light, is filtered and / or centrifuged immediately to remove precipitates or impurities and stored cold 4-6 ° C overnight. It is subsequently lyophilized, obtaining the ART / CUR-ES complex. It is not recommended to alter the pH of the resulting solution so that undesirable precipitates do not form.
    [0133]
    [0134] Section 2: 1 g of the ART / CUR-ES complex obtained as described in section 1 is dissolved in 100 mL of water. This solution is added on a solution of a 2000-10000 low molecular weight chitosan oligomer polyelectrolyte (100 mg), dispersed in a solution of 100 mL of 0.4% tripolyphosphate (w / v) and sonicated to obtain the inclusion complex ART / CUR-ES-OQ. Subsequently, this complex is centrifuged and washed several times with distilled and deionized water, resulting in nanoparticles of ART / CUR-ES-OQ with an average diameter between 200 to 400 nm.
    [0135]
    [0136] Example 2.- Preparation of the complex (ART / HI-ES-OQ) comprising artemisinin and hydroxytyrosol as a hydrophobic active compound (ART / HI) with stevioside (ES) and chitosan oligomer (OQ) in a weight ratio: 1 ART / HI: 6 ES: 1 OQ
    [0137]
    [0138] Section 1: Start from 1 gram of active compound (0.5 g artemisinin 0.5 g hydroxytyrosol) and disperse in 250 ml of absolute ethanol. On the other hand, 6 g of stevioside are weighed in 250 mL of distilled and deionized water. Both solutions are mixed and the resulting mixture is emulsified by directly introducing the sonicator head of the ultrasonic equipment into the solution (20 kHz frequency). The sonication is carried out for periods of 2 minutes each, and a time of 20 minutes, not allowing the solution to heat above 50 ° C. The resulting solution, protected from light, is filtered and / or centrifuged immediately to remove precipitates or impurities and stored cold 4-6 ° C overnight. It is subsequently lyophilized to obtain the ART / HI-ES complex. It is not recommended to alter the pH of the resulting solution so that undesirable precipitates do not form.
    [0139] Section 2: 1 g of the ART / HI-ES complex obtained as described in section 1 is dissolved in 100 mL of water. This solution is added on a solution of a 2000-10000 low molecular weight chitosan oligomer polyelectrolyte (100 mg), dispersed in a solution of 100 mL of 0.4% tripolyphosphate (w / v) and sonicated to obtain the inclusion complex ART / HI-ES-OQ. Subsequently, this complex is centrifuged and several times with distilled and deionized water, resulting in nanoparticles of ART / HI-ES-OQ with an average diameter between 200 to 400 nm.
    [0140]
    [0141] B. CHARACTERIZATION OF THE COMPLEXES OF THE INVENTION
    [0142]
    [0143] The characterization of several complexes studied by different types of spectroscopy and microscopy is included below. In particular, the following inclusion complexes have been characterized:
    [0144] i. artemisinin: curcumin 1: 1 (by weight),
    [0145] ii. artemisinin: curcumin: stevioside 0.5: 0.5: 10 (by weight),
    [0146] iii. artemisinin: curcumin: stevioside: chitosan oligomers 0.5: 0.5: 10: 1 (by weight),
    [0147] iv. artemisinin: curcumin: stevioside: chitosan oligomers (0.5: 0.5: 10: 1) by weight, isolated centrifuged complex.
    [0148]
    [0149] 1.- Characterization by infrared spectroscopy with Fourier transform and Attenuated Total Reflectance ( ATR).
    [0150]
    [0151] The purity of the starting products has been analyzed by FTIR-ATR (Figure 3) and, in addition, the binary-ternary and quaternary mixtures of the inclusion complexes (i) to (iv) have been analyzed (Figure 4).
    [0152]
    [0153] The greatest differences are presented for the formation of binary complexes, and especially the loss of crystallinity of the starting products (artemisinin) and the formation of co-amorphous solids or inclusion complexes or molecular self-assembly.
    [0154] Figure 4 shows the FTIR-ATR spectra of absorbance of the inclusion complexes obtained by grinding and that the spectra are sorted from top to bottom):
    [0155]
    [0156] 1st Artemisinin Curcumin (ART: CUR) (upper part of figure 4). It is observed that the crystallinity of artemisinin and curcumin is maintained, despite having been subjected to crushing and / or grinding material below 50 microns. It can be seen how the peaks and their heights have hardly suffered alteration in the binary compound, in relation to the starting products.
    [0157]
    [0158] 2nd. Artemisinin Curcumin Stevioside (ART: CUR: ES) (upper central part of Figure 4). The FTIR spectrum shows that the stevioside added in the proportion 0.5: 0.5: 3 (ART: CUR: ES) by weight has experienced a considerable decrease in intensity and an increase in peak width or vibration of COC voltage, which implies a considerable increase in the degree of amorphous character and the formation of amorphous co-products.
    [0159]
    [0160] 3rd. Artemisinin Curcumin Stevioside Oligomers of Chitosan (ART: CUR: ES: OQ). The FTIR spectrum shows that products mixed in the 0.5: 0.5: 3: 1 ratio (ART: CUR: ES: OQ) experience a greater increase in amorphous character and at the same time improve the solubility of the new complex.
    [0161]
    [0162] 4th Encapsulated Complex of Chitosan Oligomers with Artemisinin Curcumin Stevioside (Art + Cur + Estev). The FTIR spectrum of the lyophilized complex shows a notable increase in amorphous character, and of a degree similar to the 3rd complex.
    [0163]
    [0164] 2.- Microscopy characterization of the new complexes
    [0165]
    [0166] In Figure 5, the evolution of the starting compounds, especially artemisinin (crystalline compound, figure 5a) to binary compounds such as artemisinin curcumin (1: 1) weight (figure 5b), ternary or crushed mixtures artemisinin curcumin are shown by microscopy techniques stevioside (0.5: 0.5: 6) weight (figure 5c); and quaternary or mixtures of artemisinin curcumin stevioside oligomers of chitosan (0.5: 0.5: 6: 1) weight (figure 5d) increasingly amorphous and more soluble in water.
    [0167] The formation of inclusion or self-assembly compounds greatly facilitates and increases the bioavailability of CBAs, due to inter and intramolecular interactions by hydrogen bonds between the stevioside molecule and the CBA (Artemisinin-Curcumin).
    [0168]
    [0169] In figures 6a and 6b the images with 500x of the centrifuged precipitates of amorphous co-products can be observed: 5th Artemisinin Curcumin Stevioside Oligomers Chitosan (0.5: 0.5: 6: 1) (upper) and 6th Artemisinin Curcumin Stevioside Oligomers Chitosan (0.5: 0.5: 6: 1) (lower).
    [0170]
    [0171] Figure 7 shows the highly crystalline and water insoluble artemisinin and curcumin DRX spectra (upper spectrum) and below the evolution of the DRX spectrum of mixtures with stevioside and chitosan oligomers (zone spectra) medium, increasingly amorphous) and finally the formation of the encapsulated and lyophilized and completely amorphous and water-soluble inclusion complex of Artemisinin Curcumin Stevioside Oligomers Chitosan (0.5: 0.5: 6: 1) (lower DRX spectrum).
    权利要求:
    Claims (12)
    [1]
    1. - Inclusion complex comprising at least one active compound, a steviol glycoside and an aminoglycoside.
    [2]
    2. - Inclusion complex according to claim 1, wherein the aminoglycoside is selected from the group consisting of chitosan oligomer, glucosamine and any combination of the foregoing.
    [3]
    3. - Inclusion complex according to any one of claims 1 to 2, wherein the active compound is selected from the group consisting of artemisinin, a derivative of artemisinin, curcumin, hydroxytyrosol and a combination of the foregoing.
    [4]
    4. - Inclusion complex according to claim 3, comprising a combination of active compounds i) and ii), wherein:
    i) between 10% and 90% artemisinin or one of its derivatives, and
    ii) between 90% and 10% curcumin or hydroxytyrosol;
    amounts expressed by weight with respect to the total weight of active compounds i) and ii).
    [5]
    5. - Inclusion complex according to any one of claims 1 to 4, wherein the ratio between the active compound (CBA), steviol glycoside (GL) and aminoglycoside (AM) in the complex is 1: 6: 1 at 1: 10: 1 (CBA: GL: AM).
    [6]
    6. - Method for obtaining the inclusion complex described in any one of claims 1 to 5, wherein the method comprises:
    a) mixing by ultrasonic sonication at least one active compound to be encapsulated (CBA) with a steviol glycoside (GL);
    b) add an aminoglycoside (AM) to the solution obtained in the previous stage and mix by sonication by ultrasound; and
    c) isolate the CBA-GL-AM complex formed in step b) in the solid state, preferably by lyophilization.
    [7]
    7. - Method for obtaining the inclusion complex according to claim 6, wherein steps a) and b) take place in a hydroalcoholic medium.
    [8]
    8. - Method for obtaining the inclusion complex according to any one of claims 6 to 7, wherein the sonication takes place between 10 kHZ and 20 kHZ.
    [9]
    9. - Method for obtaining the inclusion complex according to any one of claims 6 to 8, comprising a step c1) wherein the particle size of the complex isolated in step c) is reduced to between 200 nm and 400 nm.
    [10]
    10. - Inclusion complex obtained according to the method described in any one of claims 6 to 9.
    [11]
    11. - Inclusion complex according to one of claims 1 to 5 or 10 for use in medicine.
    [12]
    12. - Inclusion complex according to one of claims 1 to 5 or 10 for the treatment of malaria.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2009126950A2|2008-04-11|2009-10-15|Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College|Diterpene glycosides as natural solubilizers|
    WO2012127287A2|2011-03-18|2012-09-27|Febris Bio-Tech Limited|Compositions and methods for treating multi-drug resistant malaria|
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