Title:  Method of making nanoparticles of substantially water insoluble materials

United States Patent:  6,623,761

Issued:  September 23, 2003

Inventors:  Hassan; EmadEldin M. (Coventry House, Apt. B2 8048 Oxford Ave., Philadelphia, PA 19111)

Appl. No.:  748803

Filed:  December 22, 2000

Abstract

This invention relates to a novel process of manufacture of nanoparticles of substantially water insoluble materials from emulsions. The emulsions have the ability to form a single liquid phase upon dilution of the external phase, instantly producing dispersible solid nanoparticles. The formed nanoparticles have average diameter of about 10 to 200 nm and are suitable for drug delivery and targeting of water insoluble therapeutic or diagnostic agents. Examples of such agents are methotrexate, progesterone, testosterone, prednisolone, and ibuprofen. Such agents can be used in a wide range of therapeutic and diagnostic treatments including treatment for cancer, hormonal therapy, and pain management.

SUMMARY OF THE INVENTION

According to one aspect, this invention provides a method of making nanoparticles of substantially insoluble water compounds and more specifically, nanoparticles of a water insoluble pharmaceutical compound (or "drug") from an emulsion in which a solution of said material forms the globules of the dispersed phase. These emulsions are readily transformed into a single uniform liquid phase, in which nanoparticles of the diagnostic or therapeutic agent are suspended, upon further dilution with the external or continuous phase. The resulting dispersed solid nanoparticles are generally less than 200 nm average diameter.

An advantageous feature of this invention is that therapeutic or diagnostic nanoparticles so produced can be utilized for intravascular injections to treat or diagnose local or systemic diseases. Another advantageous feature is that extravascular injections containing these particles can provide controlled release of the drug at the site of injection for prolonged drug effects, and minimize multiple dosing. Yet another advantage of this invention is improved drug transport across absorption barriers such as mucosal gastrointestinal barriers, nasal, pulmonary, ophthalmic, and vaginal membranes, and other distribution barriers, such as the blood--tissue and blood--tumor barriers of various organs and tissues. For example, anti-cancer nanoparticles of less than 50 nm diameter can migrate through the compromised, more permeable vascular bed to reach tumor tissues. Once the nanoparticles are inside the tumor tissue they will provide local cytotoxic action against the tumor cells. In the case of highly protected organs such as the brain, with its tight vascular bed surrounding the normal tissues, drug nanoparticles will preferentially concentrate in the tumor tissue, with minimal or no toxicity to the healthy brain tissue. A further advantage of this invention is the improved oral bioavailability of poorly absorbed drugs.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The formation of oil-in-water or water-in-oil emulsions is a well-known process. Emulsions suitable for generating nanoparticles of therapeutic or diagnostic agent in accordance with this invention comprise a dispersed or internal phase in which the agent is totally soluble, an appropriate emulsifier, e.g., a surfactant, and a continuous or external phase with limited solubilizing affinity to the dispersed phase.

While not intending to be confined by a particular theory, the nanoparticles described above appear to be formed by the following mechanism. After emulsification, the system comprises generally spherically shaped globules of agent surrounded by a protective sheath of emulsifier molecules. Those globules are dispersed throughout a bulk of the external phase. The emulsion will be intact as long as the protective sheath is intact and the external phase cannot dissolve the molecules of agent in the dispersed phase. Further dilution of the emulsion with the external phase will cause the sheath to become thinner, allowing the external phase to dissolve some or all of the internal phase globules. The dissolution of the internal phase globules in the external phase results in the production of nanometer-sized particles of the therapeutic or diagnostic agent. Aside from the novel procedure by which these nanoparticles are formed, a fundamental and unique feature of this invention is that precipitation of solid drug nanoparticles from the emulsion globules provides ultimate control over nanoparticle size because the resulting nanoparticles are less than or at least similar to the globule size of the initial emulsion.

Unlike the wet grinding technique, this invention can be practiced with a wide variety of therapeutic and diagnostic agents in either the crystalline or the amorphous state. Therapeutic and diagnostic agents with the following utilities can be employed in this invention:

Examples of agents that are useful include substances capable of treating or preventing an infection systemically or locally, as for example, antibacterial agents such as penicillin, cephalosporins, bacitracin, tetracycline, doxycycline, quinolines, clindamycin, and metronidazole; antiparasitic agents such as quinacrine, chloroquine and vidarabine; antifungal agents such as nystatin; antiviral agents such as acyclovir, ribarivin and interferons; anti-inflammatory agents such as hydrocortisone and prednisone; analgesic agents such as salicylic acid, acetaminophen, ibuprofen, naproxen, piroxicam, flurbiprofen and morphine; local anesthetics such as lidocaine, bupivacaine, benzocaine, and the like; immunogens (vaccines) for stimulating antibodies against hepatitis, influenza, measles, rubella, tetanus, polio and rabies; peptides such as leuprolide acetate (an LH-RH agonist), nafarelin and ganirelix.

Also useful is a substance or metabolic precursor thereof, which is capable of promoting growth and survival of cells and tissues or augmenting the functioning of cells, as for example, a nerve growth promoting substance such as a ganglioside, a nerve growth factor, and the like; a hard or soft tissue growth promoting agent such as fibronectin (FN), human growth hormone (HGH), a colony stimulating factor, bone morphogenetic protein, platelet-derived growth factor (PDGF), insulin-derived growth factor (IGF-I, IGF-II), transforming growth factor-alpha (TGF-a), transforming growth factor-.beta. (TGF-.beta.), epidermal growth factor (EGF), fibroblast growth factor (FGF) and interleukin-1 (IL-1); an osteoinductive agent or bone growth promoting substance such as bone chips and demineralized freeze-dried bone material; and antineoplastic agents such as methotrexate, 5-fluoroacil, adriamycin, vinblastine, cisplatin, tumor-specific antibodies conjugated to toxins and tumor necrosis factor.

Other useful substances include hormones such as progesterone, testosterone, and follicle stimulating hormone (FSH) (birth control, fertility-enhancement), insulin metal complexes and somatotropins; antihistamines such as diphenhydramine and chlorpheneramine; cardiovascular agents such as digitalis glycosides, papaverine and streptokinase; anti-ulcer agents such as cimetidine, famotidine and isopropamide iodide; vasodilators such as theophylline, B-adrenergic blocking agents and minoxidil; central nervous system agents such as dopamine; antipsychotic agents such as risperidone, olanzapine; narcotic antagonists such as naltrexone, maloxone and buprenorphine.

Preferred therapeutic and diagnostic agents are water insoluble anticancer drugs such as carmustine (BCNU), antiviral drugs such as azidothymidine (AZT) and other nucleosides, HIV Protease inhibitors such as saquinavir and retinovir immune-modulating agents such as cyclosporine, natural and synthetic hormones and hormone regulators such as contraceptives. Light imaging contrast materials for x-ray imaging such as iodinated materials (Iodepamide derivatives), Magnetic Resonance imaging contrast agents such as metal oxides (Iron Fe₃ O₄ and Fe₂ O₂) and markers for diagnostic nuclear medicine used in scintegraphy as radio-labeled Technetium sulphur or Technetium oxide. Other preferred therapeutic agents are steroidal and non-steroidal anti-inflammatory agents such as hydrocortisone, prednisolone, ketoprofen, celecoxib and ibuprofen. Further preferred therapeutic agents are centrally acting medicines such as antiseptics, antidepressants and sedatives and cardiovascular drugs such as anti-hypertensives and blood lipid lowering agents. Particularly preferred therapeutic agents are water insoluble anti-cancer drugs, hormones, analgesics, cardiovascular, antimicrobial or anti-viral agents. This technique is also suitable for immune modulators and drugs that are soluble in dilute acids or bases. Methotrexate is a preferred drug substance that is soluble in dilute alkaline solutions.

Once the principle of the invention is understood, people experienced in the field can select suitable emulsion systems for each agent. Preferred emulsion systems are triethyl citrate-water, dimethylsulphoxide-triglyceryl cabroate and ethyl citrate-water. Alkaline or acidic aqueous solutions comprising triethyl citrate are especially useful for emulsion systems for agents of pH-selective solubility. Acids in liquid form, such as hydrochloric, acetic, phosphoric, and lactic acids are preferred for acid soluble agents. Ammonium hydroxide, triethanolamine and ethylenediamine are preferred for alkali-soluble agents.

The choice of a suitable emulsifier or a combination of emulsifiers can readily be made by those in the field.

Surfactants

Surfactants which may be used for this purpose have preferably HLB value of 1 to about 20. Examples of them are as follows:

(a) Reaction products of natural or hydrogenated vegetable oils, and ethylene glycol; i.e., polyoxyethylene glycolated natural or hydrogenated vegetable oils: for example polyoxyethylene glycolated natural or hydrogenated castor oils. Surfactants commercialized under the trade names Cremophor RH-40, Cremophor RH60, Cremophor EL, Nikkol HCO-40 and Nikkol HCo-60 may be used in the composition according to the present invention. Cremophor RH40 and Cremophor El are preferred.

(b) Polyoxyethylene sorbitan fatty acid esters: e.g., mono- and tri-lauryl, palmityl, stearyl and oleyl esters; e.g. products of the trade name "Tween," which includes polyoxyethylene sorbitan mono-laurate (Tween), polyoxyethylene sorbitan mono-palmitate (Tween 40), polyoxyethylene sorbitan mono-oleate (Tween 80), etc. depending on the kind of fatty acid. Tween 20 and Tween 40 can be used preferably in the composition according to the present invention.

(c) Polyoxyethylene fatty acid esters: for example, polyoxyethylene stearic acid esters of the type known and commercially available under the trade name Myrj as well as polyoxyethylene fatty acid esters known and commercially available under the trade name "Cetiol HE."

(d) Polyoxyethylene-polyoxypropylene co-polymers: e.g. of the type known and commercially available under the trade names "Pluronic" and "Emkalyx."

(e) Polyoxyethylene-polyoxypropylene block co-polymers: e.g. of the type known and commercially available under the trade name "Poloxamer."

(f) Dioctylsuccinate, dioctylsodiumsulfosuccinate, di-[2-ethylhexyl]-succinate or sodium lauryl sulfate.

(g) Phospholipids, in particular lecithins: especially, soybean lecithin.

(h) Surfactants such as non-ionic polyoxyethylene fatty acid derivatives, in particular, polyoxyethylene sorbitan fatty acid esters (spans) such as sorbitan sesquiolate are preferred for use as emulsifiers.

Emulsification is usually performed by applying mechanical force to break down the internal phase liquid into small globules, in the range of 10 to 200 nm, more preferably less than 200 nm, and even more preferably less than 50 nm in diameter, and molecules of surfactant molecules forming a barrier between the globules and the bulk of the external liquid. Such mechanical force can be applied by mechanical stirring, ultrasonic probes, or by passing the emulsion components through narrow space, as in the case of colloidal mills, or through narrow tubes, valves or orifices. The preferred emulsification technique is passing the liquid through narrow tubes.

To obtain solid nanoparticles, the emulsion should be diluted to allow total miscibility of the liquid dispersed phase inside the continuous phase. The miscibility of the liquid dispersed phase is accompanied by the formation of the nanoparticles from the resulting one liquid phase system. The dilution step can be carried out with either an additional portion of the continuous phase solution or a liquid that is miscible with both the dispersed and continuous phases. Diluting with the same continuous phase solution is preferred.

Separation of nanoparticles can be performed using dialysis, filtration, centrifugation, or other known techniques. Nanoparticles can then be formulated into a suspension dosage form according to standard procedures in the art for injection or non-injection use. Nanoparticles can also be dried and reconstituted prior to use.

The composition of this invention enables sustained, continuous delivery of drugs, medicaments and other biologically active agents to tissues adjacent to or distant from an administration site. The biologically-active agent is capable of providing a local or systemic biological, physiological or therapeutic effect. For example, the agent may act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth or enhance bone growth, among other functions.

The nanoparticles of the invention can be administered using a pressure applicator such as injection into tissue through a syringe, needle, pump or similar article or by techniques known for delivering medication to parts of the human body, such as orally in a suspension, hard or soft capsules, or by topical application on the skin, to the eye, or into a mucous membrane or body cavity.

The nanoparticles of therapeutic or diagnostic agent are administered in an amount effective to provide the desired level of biological, physiological, pharmacological and/or therapeutic effect. The active agent may stimulate or inhibit a biological or physiological activity. There is generally no critical upper limit on the amount of the agent being administered. The concentration of the bioactive agent should not be so high that the composition has a consistency that inhibits its delivery to the administration site by the desired method. The lower limit of the amount of agent will depend on the activity of the agent and the period of time desired for treatment. The agent is gradually released by dissolution of the nanoparticles.

For further examples of agents that may be used in the present invention, see U.S. Pat. No. 5,324,519, the entire disclosure of which is incorporated by reference herein.

Claim 1 of 20 Claims

I claim as my invention:

1. A method of making nanoparticles of a substantially water insoluble therapeutic agent selected from the group consisting of progesterone, testosterone, methotrexate and ibuprofen, said method comprising the steps of:

(a) dissolving said therapeutic agent in a first liquid component of an emulsion system to form a solution;

(b) adding to the solution a second liquid component of an emulsion system and a surfactant functioning as an emulsifier to form a mixture;

(c) applying mechanical force to the mixture of (b) in order to transform the mixture into an emulsion comprising a continuous phase and a dispersed phase in which the continuous phase comprises the second liquid component of the emulsion system and the surfactant, and the dispersed phase comprises globules of the therapeutic agent dissolved in the first liquid component, said globules having a diameter of between 10 and 200 nm; and

(d) treating the emulsion formed in step (c) with an additional amount of the second component thereby transforming the emulsion into a liquid-solid suspension, whereby the solid phase comprises nanoparticles of the agent.

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