HK1186114A - Methods and compositions for cellular drug release - Google Patents

Description

Compositions and methods for intracellular drug delivery

[ technical field ] A method for producing a semiconductor device

The present invention relates to controllable drug delivery, in particular with respect to controlled release of drugs via cellular activity.

[ background of the invention ]

At present, the formulation of the sustained release medicine is more than the formulation of the sustained release medicine which is released within 2 to 3 days after injection, and the effect of long-acting sustained release is not achieved; or biphasic drug release with intermittent cessation of two weeks. For example, PCL, PLA, PLGA are used to form spheres, and drug molecules not entrapped (entrapped) in the spheres are released at the initial burst (initial burst) of administration, followed by a pause of about two weeks. The second phase of drug release occurs upon hydrolysis of the spheres.

Thus, to date, there remains a need in the art to address the deficiencies of the drug delivery formulations described above, particularly with respect to long-acting sustained release drug delivery.

[ summary of the invention ]

In one aspect, the invention relates to a composition comprising a therapeutically effective amount of an agent adsorbed onto mesoporous Hydroxyapatite (HAP) having a hydrophobic surface.

In another aspect, the present invention relates to a composition comprising: (a) medium pore size Hydroxyapatite (HAP); (b) acrylic acid grafted (graft) onto the surface of the medium-pore hydroxyapatite to form an acrylic acid-grafted medium-pore hydroxyapatite; (c) linoleic acid, which modifies the surface of the acrylic acid grafted medium-aperture hydroxyapatite to form a tail of hydrophobic hydrocarbon on the surface; and (d) a therapeutically effective amount of an agent adsorbed to the tail of the hydrophobic hydrocarbon.

In yet another aspect, the present invention relates to a method of producing intracellular drug release comprising: (a) providing a composition comprising a therapeutically effective amount of an agent adsorbed onto a mesoporous hydroxyapatite having a hydrophobic surface; (b) exposing the composition to a cell; (c) allowing the medium pore size hydroxyapatite to enter the cell and degrade in lysosomes of the cell, thereby desorbing the agent from the medium pore size hydroxyapatite; (d) releasing the desorbed agent from the lysosome into the cytoplasm of the cell; and (e) releasing the desorbed agent outside the cell.

The present invention will now be described in detail with reference to the following description of the preferred embodiments, which is provided for illustration purposes, and it is to be understood that various changes and modifications may be made without departing from the spirit and scope of the invention.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the invention and together with the description, serve to explain the principles of the invention. Reference characters used in the drawings correspond to like or similar elements in the embodiments.

[ description of the drawings ]

Fig. 1 is an X-ray diffraction (XRD) pattern of the intermediate pore size HAP nanoparticles.

FIG. 2 is a Scanning Electron Microscope (SEM) image (100000X) of medium pore size HAP.

FIG. 3 shows the results of differential thermal analysis-thermogravimetric analysis (TGA-DTA): (1) medium pore size HAP; (2) HAP-AA-LA; and (3) HAP-AA-LA-OLZ.

FIG. 4 shows the LDH analysis results of 3T3 cell line.

FIG. 5 shows the results of WST-1 analysis of 3T3 cell line.

Figure 6 is an in vitro drug release profile in cumulative amounts.

Figure 7 is an in vitro drug release profile in cumulative percentage.

Figure 8 is an in vivo drug release profile.

FIG. 9 is a schematic representation of an embodiment of the present invention, wherein the main reference numbers have the following meanings:

900 drug delivery system (composition)

902 medium pore size hydroxyapatite

904 acrylic acid grafted medium pore size hydroxyapatite

905 amphoteric Compounds

906 hydrophobic tail

907 hydrophilic head

908 pharmaceutical agent

Fig. 10 is a schematic diagram of another embodiment of the present invention.

[ detailed description ] embodiments

The terms used in the specification have the ordinary meanings in the technical field of the present invention. Some of the terms used in describing the invention and discussed below, or some of the terms discussed elsewhere in this specification, provide additional guidance to those who practice the invention in light of the description of the invention. For convenience, some terms will be emphasized in italics or parentheses, without detracting from the scope and definition of the terms. Also, the same may be referred to in more than one way, and thus, alias names or synonyms may be used for the terms discussed herein. Synonyms for some of the terms are provided herein, but the listed synonyms do not exclude the applicability of other synonyms. The embodiments used in this specification are only exemplary and are not intended to limit the scope of the present invention. The present invention is not limited to the embodiments provided in the present specification.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

As used herein, "about," "approximately," or the like, generally means that the recited value or range can encompass variations within 20%, preferably within 10%, and more preferably within 5%. Where numerical values are stated herein as approximate values, it is to be understood that variations in the numerical values are contemplated even if the term "about", "approximately", or the like is not expressly stated.

As used herein, when a value or a range is listed, one of ordinary skill in the art will understand that it is intended to cover an appropriate, reasonable range for the particular field relevant to the present invention.

E.g., having a size of from 500 to 3700 nanometers (nm), means that all integer unit values in this range have been disclosed as part of the present invention. Thus, unit values of 500, 501, 502, 1.... 1000, 1001, 1002, 3699, and 3700nm are all encompassed by the embodiments of the present invention.

As used herein, the term "mesoporous hydroxyapatite" means hydroxyapatite (Ca) having a mesoporous structure₁₀(PO₄)₆(OH)₂) And (3) granules.

The term "amphiphile" describes a compound having both hydrophilic (water-affinity, polar) and lipophilic (oil-affinity, hydrophobic) properties. Such compounds are referred to as amphoteric compounds (which may be ampphiclic or ampphithic in english).

Fatty acids are carboxylic acids with long aliphatic chains that are not branched, which may be saturated or unsaturated. Fatty acids have a hydrophobic tail, which is usually composed of long hydrocarbon chains of fatty acids, and a hydrophilic head. Fatty acids are distinguished by length and are generally divided into short, medium and long chains. Short Chain Fatty Acids (SCFA) are fatty acids with an aliphatic tail of less than 6 carbons, such as butyric acid. Medium Chain Fatty Acids (MCFA) are fatty acids with an aliphatic tail of 6-12 carbons, which can form medium chain triglycerides. Long Chain Fatty Acids (LCFAs) are fatty acids with an aliphatic tail of more than 12 carbons. Very Long Chain Fatty Acids (VLCFA) are fatty acids with an aliphatic tail of more than 22 carbons.

The terms "hydrophobic tail", "hydrophobic hydrocarbon tail", "hydrophobic tail region", and "lipophilic tail" are used interchangeably, e.g., a hydrophobic (lipophilic) fatty acid tail (chain).

Conventional pharmaceutically acceptable ionic or nonionic surfactants include Sodium Lauryl Sulfate (SLS), polyoxyethylene sorbitan monolaurate (Tween), cetyltrimethylammonium bromide (CTAB), polyoxyethylene castor oil (Cremophor), cetyltrimethylammonium bromide (HTAB), 4-octylphenol polyoxyethylene ether (Triton), nonylphenol acetate (Tergitol), cyclodextrin, and lecithin.

Adsorption, the attachment of atoms, ions, biological molecules or molecules to a surface in a gaseous, liquid or dissolved solid state. The term "adsorption" refers to the process of, or causing to proceed with, a material that accumulates on the surface of a solid to form a film.

Physiological saline solution (physiological saline solution) is a solution of one or more salts, and is essential for isotonicity with tissue fluid or blood. Among them, the sodium chloride solution of about 0.9% is particularly called normal saline (in english, physiological saline solution).

The term "treatment" as used herein includes both prophylactic and palliative treatment.

"pharmaceutically acceptable" means that the vehicle, carrier, diluent, excipient, and/or salt must be compatible with the other ingredients of the formulation and not deleterious to the recipient of the formulation.

The identity and amount of pharmaceutically suitable vehicles, carriers, diluents, excipients, and/or salts can be readily determined by one of ordinary skill in the art. May be selected depending on the type of drug desired and the method of administration.

The term "HAP-AA-LA-OLZ" denotes a complex (or nanocomplex) of medium pore size HAP-acrylic acid-linoleic acid-olanzapine.

Composite materials, often referred to simply as composites, or as composite materials; artificial or natural materials made of two or more constituent materials having significantly different physical or chemical properties.

In one aspect, the invention relates to a composition comprising a therapeutically effective amount of an agent adsorbed onto mesoporous Hydroxyapatite (HAP) having a hydrophobic surface.

In another aspect, the present invention relates to a composition comprising: (a) hydroxyapatite with medium aperture; (b) acrylic acid grafted on the surface of the medium-aperture hydroxyapatite to form an acrylic acid grafted medium-aperture hydroxyapatite; (c) linoleic acid, which modifies the surface of the acrylic acid grafted medium-aperture hydroxyapatite to form a tail of hydrophobic hydrocarbon on the surface; and (d) a therapeutically effective amount of an agent adsorbed to the tail of the hydrophobic hydrocarbon.

In yet another aspect, the present invention relates to a method of producing intracellular drug release comprising: (a) providing a composition comprising a therapeutically effective amount of an agent adsorbed onto a mesoporous hydroxyapatite having a hydrophobic surface; (b) exposing the composition to a cell; (c) allowing the medium pore size hydroxyapatite to enter the cell and degrade in lysosomes of the cell, thereby desorbing the agent from the medium pore size hydroxyapatite; (d) releasing the desorbed agent from the lysosome into the cytoplasm of the cell; and (e) releasing the desorbed agent outside the cell.

In an embodiment of the present invention, the composition has the following features: i) less than 10% of the agent is released from the medium pore size hydroxyapatite in a liquid having a pH of about 7.4; and ii) releasing the agent in a liquid having a pH of less than about 5 (≦ 5).

In one embodiment of the invention, the cell is present in an animal.

In another embodiment of the present invention, the desorbed pharmaceutical agent of step (e) is released into the bloodstream of the animal.

In another embodiment of the invention, the desorbed pharmaceutical agent is released into the bloodstream of the animal continuously without intermittent cessation for a period of 4 weeks or more.

In another embodiment of the invention, the desorbed pharmaceutical agent is released into the bloodstream of the animal for a sustained period of 5 weeks or more than 5 weeks.

In another embodiment of the invention, the cell is in the animal and the composition is exposed to the cell by intramuscular injection.

In another embodiment of the present invention, the cells include neutrophils, monocytes, macrophages, dendritic cells, and mast cells.

In another embodiment of the present invention, the method further comprises contacting Ca in the lysosome of the cell²⁺And PO₄ ^3-The ions increase.

In another embodiment of the present invention, the medium-pore hydroxyapatite includes hydroxyapatite particles, each particle having a size range of 500 to 3700 nanometers (nm).

In another embodiment of the present invention, the mesoporous hydroxyapatite having a hydrophobic surface comprises: a) hydroxyapatite with medium aperture; b) acrylic acid grafted to the surface of the medium-pore-size hydroxyapatite to form an acrylic acid-grafted medium-pore-size hydroxyapatite; and c) a pharmaceutically acceptable amphoteric compound which modifies the surface of the acrylic acid-grafted medium-diameter hydroxyapatite to form a hydrophobic hydrocarbon tail on the surface thereof.

In another embodiment of the present invention, the amphoteric compound comprises a fatty acid having 10 to 40 carbon atoms.

In another embodiment of the present invention, the fatty acid is selected from one or more of the group consisting of capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, elaidic acid, oleic acid, linoleic acid, polyunsaturated elaidolinoic acid (polyunsaturated elaidolinoic acid), polyunsaturated linolenic acid, elaidolinoic acid (elaidolinoic acid), polyunsaturated ricinoleic acid, arachidic acid, behenic acid, erucic acid, tetracosanoic acid, ceric acid (ceric acid), montanic acid (montanic acid), cerotic acid (melissic acid), and geminic acid (geddic acid).

In another embodiment of the present invention, the amphoteric compound comprises linoleic acid.

In one aspect, the present invention relates to a drug delivery composition comprising: (a) hydroxyapatite with medium aperture; (b) acrylic acid grafted to the surface of the medium-pore-size hydroxyapatite to form an acrylic acid-grafted medium-pore-size hydroxyapatite; (c) a pharmaceutically acceptable amphoteric compound, which modifies the surface of the acrylic acid grafted medium-aperture hydroxyapatite and forms a tail part of hydrophobic hydrocarbon on the surface; and (d) a therapeutically effective amount of an agent adsorbed onto the surface of the amphiphilic compound-modified and acrylic acid-grafted medium-diameter hydroxyapatite.

The medium pore size hydroxyapatite according to the present invention comprises a worm-like (worm) structure.

In another aspect, the present invention relates to a drug delivery composition comprising: (a) hydroxyapatite with medium aperture; (b) acrylic acid grafted to the surface of the medium-pore-size hydroxyapatite to form an acrylic acid-grafted medium-pore-size hydroxyapatite; and (c) a pharmaceutically acceptable amphoteric compound which modifies the surface of the acrylic acid-grafted medium-diameter hydroxyapatite and forms a tail of a hydrophobic hydrocarbon on the surface thereof.

In yet another aspect, the present invention relates to a drug delivery composition comprising: (a) a mesoporous hydroxyapatite having a hydrophobic surface; and (b) a therapeutically effective amount of an agent adsorbed to the hydrophobic surface of the medium pore size hydroxyapatite.

In one embodiment of the present invention, the mesoporous hydroxyapatite having a hydrophobic surface includes: a) hydroxyapatite with medium aperture; b) acrylic acid grafted to the surface of the medium-pore-size hydroxyapatite to form an acrylic acid-grafted medium-pore-size hydroxyapatite; and c) a pharmaceutically acceptable amphoteric compound which modifies the surface of the acrylic acid-grafted medium-diameter hydroxyapatite to form a hydrophobic hydrocarbon tail on the surface thereof.

In another embodiment of the invention, the composition further comprises a physiological saline, wherein the dosage form has a sustained release profile for at least 5 weeks.

In another embodiment of the invention, the medicament has a sustained release profile for at least 6 weeks.

In another embodiment of the invention, the agent has a sustained release profile in vivo for at least 5 weeks.

In another embodiment of the invention, the medicament has a sustained release profile for at least 7 weeks.

In another embodiment of the invention, the initial burst of the agent is less than 10% of the amount of the adsorbed agent.

In another embodiment of the present invention, the hydrophobic surface of the mesoporous hydroxyapatite comprises a fatty acid tail.

In another embodiment of the present invention, the fatty acid comprises between 16 and 26 carbon atoms.

In another embodiment of the present invention, the fatty acid comprises a long chain fatty acid.

In another embodiment of the present invention, the fatty acid comprises a very long chain fatty acid.

In another embodiment of the present invention, the medicament comprises an antidepressant.

In another embodiment of the invention, the antidepressant comprises olanzapine.

Olanzapine (abbreviated OLZ) is commonly used for the treatment of depression and schizophrenia. Olanzapine has this optimal property due to its natural hydrophobicity. OLZ, which is currently commercially available, is administered orally or intramuscularly once a day.

In another embodiment of the invention, the medium pore size hydroxyapatite does not contain water soluble polyvalent metal compounds and/or the calcium of the component of the hydroxyapatite is not substituted by other metals. According to the invention, the medium-pore hydroxyapatite of the composition is free of silica or polymer.

In another embodiment of the present invention, the amphoteric compound comprises a pharmaceutically acceptable surfactant.

In another embodiment of the invention, the surfactant comprises a polyoxyethylene glycolated natural or hydrogenated vegetable oil, or hydrogenated castor oil.

In another embodiment of the present invention, the pharmaceutically acceptable surfactant is selected from at least one of the following: sodium Lauryl Sulfate (SLS), polyoxyethylene sorbitan monolaurate (Tween), cetyltrimethylammonium bromide (CTAB), polyoxyethylene castor oil (Cremophor), cetyltrimethylammonium bromide (HTAB), 4-octylphenol polyoxyethylene ether (Triton), nonylphenol alcohol ester (Tergitol), cyclodextrin, and lecithin.

In another embodiment of the present invention, the agent is hydrophobic.

In another embodiment of the present invention, the composition further comprises a pharmaceutically acceptable vehicle, carrier, diluent, and/or excipient.

In yet another aspect, the present invention relates to a formulation comprising one of the aforementioned drug delivery compositions, wherein the medicament has a sustained release profile of not less than 2 weeks.

In one embodiment of the invention, the formulation has a sustained release profile in vivo for at least 4 weeks without intermittent cessation.

The above-mentioned mesoporous hydroxyapatite surface-modified and loaded with a drug (e.g., an antidepressant) is prepared in an injectable form and administered by intramuscular injection. The hydroxyapatite particles are held in the body by defense cells (macrophages, dendritic cells, mast cells, phagocytes) and enter lysosomes of the defense cells. Lysosomes have a pH of about 2-5 and rapidly dissolve the medium-pore hydroxyapatite particles and release the loaded agent into the cytoplasm.

The drug is then released from the defense cells and transported to the periphery of the cells, and then diffused to the local blood circulation system for subsequent therapeutic effect. The cell activity may be up to about 4 weeks or more until all the mesoporous hydroxyapatite particles at the injection site are captured. Thus, the drug release can be controlled by the cellular activity of the defence cells and consequently the goal of regular daily drug release is achieved.

Fig. 9 illustrates an embodiment of the present invention, a drug delivery system (composition) 900 having a hydrophobic tail 906 comprising: (a) medium pore size hydroxyapatite 902; (b) acrylic acid grafted to the surface of the medium pore size hydroxyapatite 902 to form an acrylic acid grafted medium pore size hydroxyapatite 904; (c) a pharmaceutically acceptable amphoteric compound 905 that modifies the surface of the acrylic acid-grafted medium-pore hydroxyapatite 904 to form a hydrophobic hydrocarbon tail 906 on the surface thereof; and (d) an agent 908. The amphiphilic compound 905 has a hydrophobic tail 906 and a hydrophilic head 907.

Fig. 10 illustrates intracellular drug delivery. A composition comprising the antidepressant olanzapine adsorbed on a particle size of about 0.5-4 μmOn medium-pore hydroxyapatite with hydrophobic surface. Following intramuscular injection, the particles enter the cells via phagocytosis. In the cytoplasm, the granules are engulfed by lysosomes. The lysozyme decomposes the medium-pore-size hydroxyapatite particles and causes Ca²⁺And PO₄ ^3-Ions are increased and the ions cause an increase in the osmotic pressure within the lysosome. Induction of H due to increased osmotic pressure₂O flows into lysosomes, causing the lysosomes to break. Olanzapine is then released from the lysosome and enters the cytoplasm. Olanzapine in the cytoplasm is expelled from the cell and once outside the cell, olanzapine can enter the blood stream and slowly circulate throughout the body until it reaches its destination. Thus, via the lysosome, the cell delivers olanzapine into the bloodstream of the patient.

Examples

The following exemplary apparatuses, devices, methods and related results according to the embodiments of the invention are not intended to limit the scope of the invention. The headings and sub-headings used in the examples are provided for the convenience of the reader and are not intended to limit the scope of the invention. Furthermore, there is no intention to be bound by any theory presented or suggested herein which should not be interpreted as limiting; as long as the present invention can be implemented in accordance with the teachings of the present invention, there is no need to consider any particular theory or particular scheme.

Materials and methods

Olanzapine was purchased from Santa Cruz Biotechnology, Inc (california, USA). Calcium hydroxide, 85% phosphoric acid, and ammonium hydroxide were all purchased from Riedel-de Haen (Seelze, Germany). Acrylic acid, linoleic acid, potassium persulfate and sodium metabisulfite were all purchased from Sigma-Aldrich (Wisconsin, USA). Methanol was purchased from Merck Chemicals (Darmstadt, Germany).

Synthesis and characteristics of hydroxyapatite nanoparticles with medium pore diameter

Mesoporous hydroxyapatite nanoparticles (Wu HC et al (2007) "A novel biological nanoparticle based on Hydroxyapatite. nanotechnology" 18(16), 165601, incorporated herein by reference in its entirety) were synthesized using a co-precipitation method. The chemical reaction of this synthesis is shown below:

10Ca(OH)₂+6H₃PO₄→Ca₁₀(PO₄)₆(OH)₂+18H₂O

adding 0.5M calcium hydroxide (Ca (OH)₂) Dispersed as a suspension and maintained at 80-85 ℃ during the reaction with a water bath. A stoichiometric amount (i.e. Ca/P molar ratio ═ 1.67) of 0.3M orthophosphoric acid (H)₃PO₄) The solution was added to the Ca (OH) at a rate of about 3 milliliters per minute (ml/min)₂And (3) suspension. When the H is₃PO₄Titration of the solution to Ca (OH)₂For suspension, 15g of albumin was rapidly stirred to form a foam, and then slowly added to the suspension. About 2ml of ammonium hydroxide (NH) was added₄OH) to adjust the pH of the mixture to 8.5-9. The mixture was stirred for about 2 hours, followed by aging for 20 hours while maintaining at 85 ℃. After 20 hours, the mixture was washed once with deionized water and three more times with methanol.

The mixture was then freeze dried in vacuo. The dried sample was calcined at 800 ℃ for decarburization. The crystallinity of the synthesized medium-pore-size HAP powder (nanoparticles, nanocomposite, or composite) was examined with an X-ray diffractometer (Rigaku, USA), with a diffraction angle set in the range of 10 to 60 °, and a scanning rate of 1 °/min. The surface morphology of the synthesized HAP nanoparticles with intermediate pore size was examined by Scanning Electron Microscopy (SEM) (Philips, USA).

Then a series of surface modification is carried out, and acrylic acid and linoleic acid are added. These HAP powders having a total weight of 2.5g and 100ml of deionized water were vigorously stirred, followed by N₂The bubbles were degassed for 30 minutes to obtain degassed HAP suspension. Adding a redox initiator (i.e., catalyst) potassium persulfate (K)₂S₂O₈，0.01g，0.38×10^-4mol) and sodium metabisulfite (Na)₂S₂O₅，0.01g，0.38×10^-4mol) and 1ml ofAfter rapid stirring with ionized water, the aforementioned degassed HAP suspension is added to obtain a mixture comprising the HAP and the catalyst. Mixing acrylic acid (C)₃H₄O₂，2.16g，3×10^-2mol) are added to the aforementioned mixture. The pH of the reaction mixture was adjusted to 9.5 with ammonium hydroxide (. about.9 ml). In N₂The reaction was carried out under continuous stirring at room temperature under an atmosphere for 3 hours. The mixture was centrifuged at 3000rpm for 10 minutes, washed three times with deionized water, and freeze-dried in vacuo. The following chemical reactions should occur when surface modification is carried out with acrylic acid.

Surface modification of acrylic free radical grafting to Medium pore size HAP on equimolar K₂S₂O₈/Na₂S₂O₅Occurs in the presence of a redox initiator. Possible reaction mechanisms are shown below. In S₂O₅ ^2-In the presence of S₂O₈ ^2-Will eventually degrade to 2SO₄ ^-. In addition, by removing hydrogen atoms, OH, on the surface of the mesoporous HAP nanoparticles^-And SO^4-Active groups on the HAP nanoparticles are induced. Because the amount of grafted acrylic acid is increased, the surface area of the medium pore size HAP is also increased.

(1) The initiation effect is as follows: decomposition of persulfate in liquid phase:

S₂O₈ ^2-+S₂O₅ ^2-→2SO₄ ^-

2SO₄ ^-+2H₂O→2^*OH+2SO₄ ^-+2H⁺

(2) group formation of HAP nano-surfaces:

after the above procedure was completed, the mixture weighed 125mgPlacing the medium-pore-size HAP powder subjected to olefine acid surface modification into a container with the size of 1 x 10^-4Torr (torr) vacuum for 5 hours. Then 1-2ml linoleic acid (C) was added to the system (i.e., the acrylic surface modified medium pore size HAP powder under vacuum) with a syringe₁₈H₃₂O₂) And stirred rapidly for 12 hours. OLZ (50mg) was dissolved in approximately 1-2ml of deionized water and added to the above system (i.e., acrylic surface modified medium pore size HAP powder to which linoleic acid had been added under vacuum) and stirred rapidly for 12 hours (Gardner I et al (1998) "A company of the Oxidation of Clozapine and olanzapine to Reactive microorganisms and the sensitivity of the enzyme metabolism to Human Leukocytes" Molecular Pharmacology, 53(6), 991. sup. 998). After linoleic acid reaction, the medium-pore-size HAP without the drug can be stored in a vacuum freeze drying way.

Drug loading

OLZ physically adsorb to the surface modified, mesoporous HAP. To examine the amount of the surface-modified medium-pore-size HAP pair OLZ captured, all organic materials were separately identified in N₂Calcination was performed at 600 ℃ under ambient conditions to obtain TGA-DTA results for (1) medium pore size HAP, (2) surface modified medium pore size HAP, and (3) surface modified medium pore size HAP with OLZ. The heating procedure in the TGA-DTA measurement is as follows:

(1) heating to 100 ℃ at a rate of 5 ℃/minute;

(2) this temperature was maintained for 20 minutes;

(3) heating to 600 ℃ at a rate of 5 ℃/minute; and

(4) this temperature was maintained for 20 minutes.

The mixture of OLZ and surface-modified medium pore size HAP nanoparticles (i.e., OLZ loaded surface-modified medium pore size HAP) was obtained by centrifuging the nanoparticle suspension at 1000rpm for 10 minutes, assuming no loss of HAP during centrifugation, the final product was approximately 155 mg. The ratio of LA to OLZ was 6.5: 3.5 as measured by weight loss during TGA-DTA analysis. The amount of drug captured was calculated by the following calculation (Vandervorort J and Ludwig A. (2004) "Preparation and evaluation of drug-loaded gelatin nanoparticles for topical ocular use" European Journal of pharmaceuticals and biopharmaceuticals, 57, 251-.

The amount of drug used for formulation was 50mg for drug loading.

In vitro cytotoxicity

The cytotoxicity of HAP-AA-LA-OLZ nanoparticles was evaluated with mouse embryonic fibroblast 3T3 cells. The cells were cultured in 10ml of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). The cells were incubated with different concentrations of HAP-AA-LA-OLZ nanoparticles (0, 0.05, 0.1, and 0.5mg/ml) at 37 deg.C and 5% CO₂Cultures were incubated under air for 24 and 72 hours at each concentration in a sextant (N ═ 6). Cell viability was calculated using an ELISA analyzer for LDH and WST-1 analysis and setting of absorbance at 420 nm. The relative cell viability in the presence or absence of HAP-AA-LA-OLZ nanoparticles was calculated.

In vitro drug delivery

Surface-modified medium pore size HAP loaded with olanzapine (HAP-AA-LA-OLZ) was placed in a separate container with 10ml of Phosphate Buffered Saline (PBS) pH 7.4 and placed in an incubator at 37 ℃. The solution was collected at a time point prior to the test, filtered through a 0.22 μm filter and then subjected to absorbance measurement at 226nm by a UV spectrometer (JASCOV-670). After each collection, 10ml of fresh PBS was added to the container. The absorbance of the sample is compared to a standard curve to determine the concentration of drug released in the solution and an in vitro drug release profile is plotted.

In vivo drug release

Male ICR mice (31-33g) were purchased from BioLasco Taiwan Co., Ltd. All animal experiments were performed according to the guidelines for laboratory animal care and use published by the institutional health. The drug was quantitated by intramuscular injection, and mice were sacrificed at intervals over 5 weeks and blood samples were collected. The blood sample was centrifuged at 800rpm for 10 minutes to obtain serum, and the absorbance at 252nm was measured. And comparing the light absorption value of the serum sample with a standard curve to determine the drug release concentration, and drawing an in-vivo drug release curve graph.

Results

The properties of the medium-pore HAP particles not surface-modified with acrylic acid and linoleic acid were examined with an X-ray diffractometer (Rigaku, USA). Figure 1 shows the three major peaks in the XRD pattern at 31.68 °, 32.12 °, and 32.82 °. The XRD pattern of HAP available from JCPDS (Catalog number 09-0432) is shown in the lower part of fig. 1, and comparison of the two shows that the medium-pore-size HAP of the present invention has an XRD pattern corresponding to the standard pattern. Therefore, it was confirmed that the intermediate pore size HAP particles synthesized by the present invention were actually HAP. The surface morphology of the medium-pore HAP particles was examined by SEM, and the SEM image (100000 ×) showed a worm-like medium-pore structure in the particles, as shown in fig. 2. The size of the medium pore size HAP (surface-modified) was measured with a particle size analyzer, the particle size ranged from 435 to 5174nm (data not shown), while the major size range of the particles fell within 400-500 nm.

FIG. 3 shows the TGA-DTA results: (1) medium pore HAP has a weight loss of 1.23%; (2) the surface modified medium pore size HAP had a weight loss of 70.77%; and (3) the surface modified medium pore size HAP loaded with OLZ had a weight loss of 38.48%. The average amount of drug in the nanoparticles was 10.395mg (N ═ 6), as follows:

(155-125.2)×0.3488＝10.395.

the amount of drug used for formulation was 50 mg. Therefore, the drug trapping rate was calculated to be 20.79%. In other words, 50mg of OLZ was added, but only 10.395mg was attached to or loaded into the medium pore size HAP.

Fig. 4 shows LDH assay results or cytotoxicity results of 3T3 cells on the first and third days. There was no significant difference in OD values between the control group (0mg/ml) and the experimental group (0.05mg/ml, 0.1mg/ml, and 0.2 mg/ml). Indicating that the HAP-AA-LA-OLZ particles have relatively low cytotoxicity to cells. FIG. 5 shows the results of WST-1 analysis of cell viability and cell proliferation of 3T3 cells, indicating that HAP-AA-LA-OLZ nanoparticles did not have a negative effect on cell viability and cell proliferation after the first and third days.

Fig. 6 and 7 show the 7-week release profile of OLZ released from the intermediate pore size HAP nanoparticles in vitro, showing the cumulative amount and cumulative ratio, respectively. The data show that the initial burst after 24 hours is low (5.12% of 10.395mg drug) and is an ideal, long-term, intramuscular dosage form for antidepressants. Cumulative drug release at weeks 4 and 7 was 1.467mg (14.11%) and 1.625mg (16.63%), respectively. The data show that the surface-modified intermediate pore size HAP nanoparticles according to the present invention extend OLZ release in vitro for up to 7 weeks.

Fig. 8 shows the 5-week release profile of OLZ released from the medium-pore size HAP nanoparticles in vivo. There was an initial burst within week 1 after intramuscular injection, consistent with OLZ reported in the literature as reaching a steady state one week after the first dose. It was noted that the drug concentration at week 5 tended to increase, probably due to the capture and disruption of HAP-AA-LA-OLZ by lysosomes within cells (e.g., macrophages).

In summary, the medium-pore size HAP nanoparticles are synthesized by coprecipitation, and the pore size thereof is examined by SEM image, and the particle size is in the range of 500-3700nm, which is suitable for pinocytosis. A series of surface modifications to the medium pore size HAP with acrylic and linoleic acids were performed, followed by loading OLZ. LDH and WST-1 cytotoxicity assays showed HAP-AA-LA-OLZ to have low cytotoxicity to cells. The in vitro drug release profile shows a low initial burst (5.12%) and a stable sustained release of the drug over 7 weeks. While the in vivo drug release profile shows an initial burst at week 1 and a stable sustained release of at least 5 weeks is observed.

The foregoing exemplary embodiments are provided to illustrate and describe the present invention and are not intended to limit or exclude the exact forms disclosed. Many modifications and variations are possible in light of the above teaching.

The embodiments and examples chosen and described are intended to explain the principles of the invention and its mode of operation, thereby enabling others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. It will be apparent to those of ordinary skill in the art that alternative embodiments may be chosen without departing from the spirit and scope of the invention. Accordingly, the scope of the invention is to be determined by the claims that follow.

Some references, which may include patents, patent applications, and various publications, are cited and discussed in this disclosure. The listing and/or discussion of such documents is for the purpose of clarity only and is not an admission that such documents are "prior art" to the present invention. All documents cited and discussed in this specification are incorporated herein by reference.

Claims (20)

1. A composition, comprising:

(a) hydroxyapatite with medium aperture;

(b) acrylic acid grafted to the surface of the medium-pore-size hydroxyapatite to form an acrylic acid-grafted medium-pore-size hydroxyapatite;

(c) linoleic acid, which modifies the surface of the acrylic acid grafted medium-aperture hydroxyapatite to form a tail of hydrophobic hydrocarbon on the surface; and

(d) a therapeutically effective amount of an agent adsorbed to the tail of the hydrophobic hydrocarbon.

2. The composition of claim 1, wherein the composition has the following characteristics:

i) less than 10% of the antidepressant is released from the medium pore size hydroxyapatite in a liquid having a pH of about 7.4; and

ii) releasing the antidepressant in a liquid having a pH below about 5.

3. A method of producing intracellular drug release comprising:

(a) providing a composition comprising a therapeutically effective amount of an agent adsorbed onto a mesoporous hydroxyapatite having a hydrophobic surface;

(b) exposing the composition to a cell;

(c) allowing the medium pore size hydroxyapatite to enter the cell and degrade in lysosomes of the cell, thereby desorbing the agent from the medium pore size hydroxyapatite;

(d) releasing the desorbed agent from the lysosome into the cytoplasm of the cell; and

(e) releasing the desorbed drug agent out of the cell.

4. The method of claim 3, wherein said agent comprises an antidepressant.

5. The method of claim 4 wherein in step (e) the desorbed pharmaceutical agent is released into the bloodstream of an animal.

6. The method of claim 5, wherein the desorbed pharmaceutical agent is released continuously without intermittent cessation for a duration of 4 weeks or more.

7. The method of claim 6, wherein the sustained release period of the desorbed pharmaceutical agent is up to 5 weeks or greater than 5 weeks.

8. The method of claim 3, wherein the cell is in an animal and the composition is exposed to the cell by intramuscular injection of the agent.

9. The method of claim 4, wherein the cells comprise neutrophils, monocytes, macrophages, dendritic cells, and mast cells.

10. The method of claim 3, further comprising contacting Ca in the lysosome of the cell²⁺And PO₄ ^3-The ions increase.

11. The method of claim 3, wherein the medium-pore size hydroxyapatite comprises hydroxyapatite particles, each particle having a size range of 500 to 3700 nanometers (nm).

12. The method of claim 3, wherein the mesoporous hydroxyapatite having a hydrophobic surface comprises:

a) hydroxyapatite with medium aperture;

b) acrylic acid grafted to the surface of the medium-pore-size hydroxyapatite to form an acrylic acid-grafted medium-pore-size hydroxyapatite; and

c) a pharmaceutically acceptable amphoteric compound is prepared by modifying the surface of the acrylic acid grafted medium-diameter hydroxyapatite to form a tail of hydrophobic hydrocarbon on the surface.

13. The method of claim 12, wherein the amphoteric compound comprises a fatty acid having 10 to 40 carbon atoms.

14. The method of claim 13, wherein the fatty acid is one or more selected from the group consisting of capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, elaidic acid, oleic acid, linoleic acid, polyunsaturated elaidic acid, polyunsaturated linolenic acid, elaidic linolenic acid, polyunsaturated ricinoleic acid, arachidic acid, behenic acid, erucic acid, tetracosanic acid, ceric acid, octacosanoic acid, cerotic acid, and gadoleic acid.

15. The method of claim 12, wherein the amphoteric compound comprises linoleic acid.

16. The method according to claim 3, wherein the medium pore size hydroxyapatite is free of water soluble polyvalent metal compounds and/or the calcium of a component of the hydroxyapatite is not substituted by other metals.

17. The method of claim 12, wherein the amphoteric compound comprises a pharmaceutically acceptable surfactant.

18. The method of claim 17, wherein the pharmaceutically acceptable surfactant comprises a polyoxyethylene-glycolated natural or hydrogenated vegetable oil, or hydrogenated castor oil.

19. The method of claim 17, wherein the pharmaceutically acceptable surfactant is at least one selected from the group consisting of sodium dodecyl sulfate (SLS), polyoxyethylene sorbitan monolaurate (Tween), cetyltrimethylammonium bromide (CTAB), polyoxyethylene castor oil (Cremophor), cetyltrimethylammonium bromide (HTAB), 4-octylphenol polyoxyethylene ether (Triton), nonylphenol ethyl alcohol ester (Tergitol), cyclodextrin, and lecithin.

20. A composition comprising a therapeutically effective amount of an agent adsorbed onto a medium pore size hydroxyapatite having a hydrophobic surface.