KR20110009128A - Lipid-oil-water nanoemulsion delivery system for microtubule-interacting agents - Google Patents

Abstract

Pharmaceutical compositions that benefit from the treatment, diagnosis, and prevention of diseases, conditions, syndromes, or symptoms thereof, characterized by cell hyperplasia such as cancer in warm-blooded animals, including humans, and methods of using and preparing the same are preferred in diseased cells. Lipid nanoemulsions composed of lipid particles, each comprising at least one non-bilayer-forming lipid, which can optionally be actively endocytized, and prepared by a homogenized treatment;
An effective amount of at least one therapeutic or diagnostic microtubule-interacting agent associated with said nanoemulsion; And pharmaceutically acceptable carriers. In one preferred embodiment, the composition may be comprised of protein carrier molecules and / or emulsion enhancers such as surfactants, plant fats, solvents and combinations thereof.

Description

Lipid-Oil-Water Nanoemulsion Delivery System for Microtubule-Interacting Agents}

FIELD OF THE INVENTION The present invention relates to therapeutics and diagnostics, and more particularly to formulation of microtubule-interacting agents in lipid-oil-water nanoemulsions suitable for introduction into a patient, characterized by hyperproliferation comprising tumor cells. It relates to a formulation capable of increasing the active selector concentration into cells.

The role of drug distribution in the tumor and its microenvironment is poorly studied. Currently, high doses of the most potent drug are administered to observe the effects of the drug, resulting in diffusion and tissue distribution. However, increasing drug concentration in tumor mass does not always lead to an increase in cellular drug concentration or the ability to reach intracellular targets. Indeed, some drugs may have increased concentrations in tumor mass by conjugation with polymer, liposome encapsulation or solid lipid nanoparticle formulations, but this may actually inhibit cell uptake and distribution. In addition, the use of high doses to achieve tumor penetration by diffusion can result in side effects typically associated with high mortality tumor treatment. Therefore, it would be desirable to devise drug delivery techniques that would improve drug penetration through tumor cells and increase drug concentration in tumor cells while simultaneously presenting an improved safety and efficacy profile. However, while various approaches have been sought for the passive accumulation of drugs into tumors, few techniques are available for promoting active tumor cell uptake.

Lipid-soluble drugs can easily penetrate cell membranes and be transported through cells. Cells characterized by hyperproliferation, such as cancer cells, exhibit abnormal lipid metabolism, which is generally noted for preferentially high intake of lipids and fatty acids. Thus, for cancer, lipids have been shown to play essential roles in membrane structure, growth and metastasis, signal transduction and transport processes.

The phenomenon known as permeation and retention (EPR) effects on lipid particles, liposomes, synthetic nanoparticles and other macromolecular agents caused by the characteristic leakage properties of tumor vasculature is common in solid tumors and is, for example, polymer-conjugated. It has been sought for more selective targeting of anticancer drugs and tumor mass accumulation. The use of encapsulants such as polymeric vesicles, liposomes and solid polymers or lipid nanoparticles, each prepared in various compositions, formulations and structures and administered under various physiological conditions has been reported in the prior art. However, these agents only allow passive accumulation in the tumor. In addition, these agents are frequently rapidly removed from circulation by extensive co-accumulation in the reticuloendothelial system, and their use is often associated with cytotoxicity and enhanced immune responses.

Studies have shown that multiple receptor types and subtypes, transport proteins and lipid rafting phenomena have the same disease, but genetic characteristics are likely to be involved in lipid intake into cancer cells, even among altered patients. Lipid-based nanoparticles or HDL- or LDL-based drug carriers that mimic natural receptor ligands and increase targeting and drug uptake into cancer cells have been prepared with various therapeutic agents, but are optimized for the promotion of active tumor cell uptake for cancer therapy. Formulated formulations are a scarce reality.

U.S. Pat.Nos. 4684479 and 5215680 to D'Arrigo, incorporated herein by reference, form the gas or air-filled lipid coated microbubbles (LCMs), methods for their production and use as contrast agents for ultrasound and drug delivery. Describes the use. The production process for LCMs is based on simple mechanical shaking of water-soluble suspensions of specific chain lengths and fixed proportions of nonionic lipids such as saturated glycerides and cholesterol esters. In all cases, the majority of the added lipids (99%) aggregated or settled so that other lost material upon filtration had a yield of less than 1%. Still, this artificial LCM has been found to survive very long, lasting more than six months in vitro. In addition, LCM is small and flexible enough to pass across the fenestrated capillary wall of the tumor-tissue microcirculation, although its filtration yield is low. Of particular note is the highly-selective, temperature-dependent and apparent saturation uptake of LCM into rodent brain tumor cells as well as naturally occurring tumors of dogs, mimicking the natural uptake of certain lipids of cancer cells. However, to date no efficacy formulations of pharmaceutical mixtures utilizing LCM technology that provide minimal to no levels of side effects upon patient administration are present.

(폴리에톡실화된 피마자유) 및 에탄올의 1:1 혼합물에 제형되어 탁솔

(Taxol

)(브리스톨-마이어스 스퀴브 사)을 생성한다. 결정화할 수 있는 임의의 약물을 제거하기 위하여 직접 여과(in-line filtration)와 함께 약 12 시간 동안 안정적인 적절한 담체에 재구성이 요구된다. Taxol

은 신경독성을 보여서, 감각 기관에 영향을 주며 때로는 환자의 운동 신경병증을 동반한다. Cremphor EL

은 그 자체로는 현저한 부작용이 전혀 없기 때문에, 신경병증에 부분적인 원인이 된다.Paclitaxel is a type of drug known as taxanes and is mainly isolated from the bark of the Pacific yew and related species under the name Taxus brevifolia . Although allergic reactions are observed in the formulation of excipients, taxanes are known to be useful for the treatment of various cancers such as ovarian, breast, non-small cell lung and head and neck carcinoma. The difficulty in administering taxanes is that the drug is usually insoluble in water. Thus, paclitaxel is a Cremphor EL

(Polyethoxylated castor oil) and ethanol formulated in a 1: 1 mixture of ethanol

(Taxol

) (Bristol-Myers Squibb). Reconstitution is required in a suitable carrier that is stable for about 12 hours with in-line filtration to remove any drug that can crystallize. Taxol

Is neurotoxic, affecting the sensory organs and sometimes accompanied by motor neuropathy in the patient. Cremphor EL

Has no significant side effects on its own, which is partly responsible for neuropathy.

또는 두유 및 홍화씨유의 혼합물을 함유하는 Liposyn

과 같은 지질 에멀젼에서 불용성인 것으로 보고된다. 두유 또는 홍화씨유 어느 하나에서 파클리탁셀을 가열하거나 심지어 음파파쇄시에 가열하면 상기 약물의 상당량이 용해되지 않으며, 균질화 단계 중에 지질 에멀젼에 파클리탁셀을 첨가하면 똑같은 실망스러운 결과를 만나게 된다. 최대 15 ㎎/mL까지 파클리탁셀을 포함하는 에멀젼은 트리아세틴, L-알파-레시틴, 폴리소르베이트 80, 플루로닉 F-68, 에틸올레이트(ethyloleate) 및 글리세롤과 함께 제형된다. 그러나, 이러한 제형은 고도로 독성이 있으며 불안정하다. 또한, 파클리탁셀은 LCM 제형에서 Cremphor EL

및 에탄올과 혼합된다. 그러나, LCM의 종양 섭취 선택도가 약물 전달 관점에서 호기심을 자아내는 반면에, 선행 기술에 따른 제조 수율 및 유효 약물 용량(drug payload capacity)는 너무 낮아서 실제 이용되지 않는다. 또한, 파클리탁셀과 함께 Cremphor EL 및 에탄올을 사용하면 이 제형의 독성에 기여할 것 같다.Efforts to formulate paclitaxel in stable lipid emulsions have not been successful. The drug is mainly Intralipid containing soymilk

Or Liposyn containing a mixture of soy milk and safflower seed oil

It is reported to be insoluble in lipid emulsions such as Heating paclitaxel in either soy or safflower seed oil, or even during sonication, does not dissolve much of the drug, and adding the paclitaxel to the lipid emulsion during the homogenization step results in the same disappointing results. Emulsions comprising paclitaxel up to 15 mg / mL are formulated with triacetin, L-alpha-lecithin, polysorbate 80, Pluronic F-68, ethyloleate and glycerol. However, these formulations are highly toxic and unstable. In addition, paclitaxel can be used in CCM

And ethanol. However, while the tumor intake selectivity of LCM is curious in terms of drug delivery, the manufacturing yield and drug payload capacity according to the prior art are so low that they are not actually used. In addition, Cremphor EL with paclitaxel And ethanol are likely to contribute to the toxicity of this formulation.

In addition, production methods are complex and even slight changes in conditions can result in significant differences in particle size and potency characteristics. Common approaches include high-shear homogenization and ultrasound, high-pressure homogenization, hot homogenization, cold homogenization and solvent emulsification evaporation. In addition, aseptic filtration or sterilization by radiation can be complex. Thus, attempts to increase the effective drug, add the drug to ethanol and aqueous solutions, and high-shear homogenize to increase the solubility of lipids in the LCM result in less stable, non-gas-containing particles that contain little if any drug. Done.

It is highly desirable to administer the therapeutic agent with an appropriate delivery vehicle that simultaneously promotes accumulation and cellular internalization in the tumor mass but limits accumulation in healthy tissues. For more efficient delivery, systemic and healthy tissue concentrations of a therapeutic agent can be reduced while achieving better treatment results with the same or fewer or reduced side effects. Delivery of agents with inherent degrees of tumor cell selectivity offers other advantages. In addition, delivery carriers that allow for selective accumulation into tumor masses and endocytosis with various cancer cell types, although not limited to a single tumor type, will be particularly desirable and will allow safer and more effective treatment of cancer. In addition, delivery vehicles that allow improved effective drug dosages for therapeutic agents will be a significant advance in the industry.

Therefore, a clear, stable, readily prepared, biocompatible and potent formulation of microtubule-interacting formulations containing taxanes, such as taxel, which promotes uptake into tumor cells but exhibits minimal side effects. There is a demand.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for use in the treatment or diagnosis of diseases, conditions and syndromes characterized by hyperplasia of cells, such as cancer, which is rapid, highly selective and preferentially ingested into tumor cells.

It is another object of the present invention to provide a pharmaceutical composition for use in the treatment or diagnosis of diseases, conditions and syndromes characterized by cell hyperproliferation such as cancer causing minimal side effects upon administration.

It is still another object of the present invention to provide a pharmaceutical composition for use in the treatment or diagnosis of diseases, conditions and syndromes characterized by cell hyperproliferation, such as cancer, which can be readily prepared at the lowest possible cost and stored for the longest possible time. There is a purpose.

In order to achieve these objects, the present invention provides a pharmaceutical composition useful for the treatment, diagnosis or prevention of diseases, conditions or symptoms or symptoms thereof characterized by cell hyperplasia in warm-blooded animals, including humans, wherein the pharmaceutical composition is a cancer cell A lipid nanoemulsion consisting of lipid particles as defined below, uniformly dispersed in an aqueous phase that can be selectively and preferentially introduced into a diseased cell comprising: a lipid nanoemulsion; An effective amount of at least one therapeutic or diagnostic agent associated with the lipid nanoemulsion; And it provides a wide range of pharmaceutical compositions comprising a pharmaceutically acceptable carrier.

The lipid particles each comprise at least one non-bilayer-forming lipid. The appropriate lipid particles are the target of treatment or diagnostic agents for diseased cells characterized by hyperproliferation including cancer cells for improved therapeutic efficacy and bioavailability, while reducing or at least maintaining the dosage and frequency of administration necessary to achieve the desired therapeutic effect. It has been shown to significantly improve delivery and concentration. In addition, nanoemulsions exhibit exceptional physicochemical stability for extended periods of time, greatly facilitating the prepackaging of pharmaceutical compositions in stable, ready-to-administer form, thereby also providing similar active agents. This will eliminate the problems and inconveniences associated with bedside dilution and formulations currently made in prior art compositions.

The lipid particles are associated with pharmaceutically active therapeutic microtubule-interacting agents. Suitable microtubule-interacting agents include, for example, taxanes such as paclitaxel; Epothilones; Vinca alkaloids such as, for example, vincristine; Eleutherobins; Discodermolide; Dolastatins; Colchicine; Combrestatins; Phomopsin A; Halichondrin B; Spongestatin 1; Sarcodictyins; Laulimalides; And derivatives, analogs, congeners and combinations of each of the aforementioned agents. The microtubule-interacting agent is present in an amount sufficient to extinguish or at least arrest the growth of hyperproliferative cells.

In another aspect of the invention, a method of diagnosing, treating or preventing a disease, condition or syndrome or symptoms thereof characterized by cell hyperplasia including cancer in a warm blooded animal, including humans, the method comprising the pharmaceutical disclosed herein Provided is a method comprising administering an effective amount of a composition to a warm blooded animal.

In another aspect of the invention,

a) mixing the at least one non-bilayer-forming lipid with an effective amount of at least one diagnostic or therapeutic agent to produce a lipid fraction;

b) adding the lipid component to the aqueous phase to produce a dispersion; And

c) a method of preparing a pharmaceutical composition as disclosed herein, comprising stirring the dispersion under high-shear state sufficient to fully disperse the aforementioned lipids to form a lipid nanoemulsion composed of lipid particles. .

에서 제형된 파클리탁셀보다 본 발명에 따른 지질 입자들의 하나의 특정 혼합물에서 제형된 파클리탁셀을 더 많이 섭취한다는 것을 보여주는 그래프이다.
도 6은 LN 및 Cremophor EL

양쪽 모두에 대하여 2 시간의 약물 배양 이후에 파클리탁셀의 세포 섭취가 평탄역(plateau)에 도달하였음을 보여주는 그래프를 도시한다.
도 7은 파클리탁셀 양이 본 발명에 따른 지질 입자들에서의 세포 섭취에 대하여 포화되고 있는지의 여부를 결정하는 실험 결과를 도시한다.
도 8A 및 8B는 콜레스테롤이 본 발명에 따른 지질 입자들의 세포 섭취에 있어서 중요 성분임을 보여주는 그래프들을 도시한다.
도 9A 및 9B는 본 발명에 따른 지질 입자들에서 파클리탁셀이 Cremophor EL

에서의 파클리탁셀보다 더 많이 세포내이입 된다는 것을 보여주는 그래프들을 도시한다.
도 10A 내지 10D는 본 발명에 따른 지질 입자들에서의 파클리탁셀 대 파클리탁셀 단독의 세포독성을 보여주는 그래프들을 도시한다.
도 11은 파클리탁셀은 Cremophor EL

에서 제형될 경우보다 본 발명의 지질 입자들에서 제형될 경우 더 세포독성이 있다는 것을 도시한다.
도 12A 및 12B는 파클리탁셀은 Cremophor EL

에서 제형될 경우보다 본 발명의 지질 입자들에서 제형될 경우 현저히 더 높은 항-종양 활성을 갖는다는 것을 보여주는 그래프들을 도시한다.
도 13은 EmPAC, Abraxane

또는 Taxol

로 처리되고 파클리탁셀-특이적인 항체들로 염색된 A549 세포들의 대표적인 이미지들을 도시한다.The following drawings illustrate embodiments of the invention and are included by the full specification and claims and are not intended to limit the scope of the application.
1 shows particle size distribution and zeta potential for lipid particles loaded with 10% paclitaxel in 20% DMSO in accordance with the present invention.
FIG. 2 shows the mixing of paclitaxel into lipid particles according to the present invention by a graph plotting the percent solubility of paclitaxel-loaded lipids in sucrose density studies.
3A and 3B show graphs showing that cell lipid particle uptake according to the present invention can be quantified by fluorescence-activated cell sorting.
4A-4D show graphs showing cell lipid particle uptake according to the invention by tumor cell lines and SF-539 lung tumor cells for HT-29 colon.
5 shows that the tumor cells were Cremophor EL

It is a graph showing that intake of more paclitaxel formulated in one particular mixture of lipid particles according to the invention than paclitaxel formulated in.
6 shows LN and Cremophor EL

For both, a graph shows that the cell uptake of paclitaxel has reached a plateau after 2 hours of drug incubation.
7 shows experimental results for determining whether paclitaxel amount is saturated with respect to cell uptake in lipid particles according to the present invention.
8A and 8B show graphs showing that cholesterol is an important component in the cellular uptake of lipid particles according to the present invention.
9A and 9B show Cremophor EL containing paclitaxel in lipid particles according to the present invention.

Graphs show that endocytosis is greater than paclitaxel in.
10A-10D show graphs showing cytotoxicity of paclitaxel versus paclitaxel alone in lipid particles according to the present invention.
Figure 11 Paclitaxel Cremophor EL

It is shown that it is more cytotoxic when formulated in the lipid particles of the invention than when formulated in.
12A and 12B show Paclitaxel Cremophor EL

Shown are graphs showing that when formulated in the lipid particles of the present invention than when formulated in, it has significantly higher anti-tumor activity.
13 shows EmPAC, Abraxane

Or Taxol

Representative images of A549 cells treated with and stained with paclitaxel-specific antibodies are shown.

FIELD OF THE INVENTION The present invention relates generally to pharmaceutical compositions for the treatment, diagnosis or prevention of diseases, conditions or syndromes or their symptoms which are characterized by cell hyperplasia in warm-blooded animals. Such animals include mammalian animals such as humans, horses, cattle, and domestic animals, including dogs and cats, who suffer from diseases characterized by cell hyperproliferation including cancer and other pathological conditions and syndromes. The pharmaceutical compositions of the present invention comprise lipid particles and pharmaceutically acceptable carriers or excipients operably associated with at least one therapeutic or diagnostic microtubule-interacting agent because the lipid particles as defined below have improved charge capacity. Including nanoemulsions, which make the lipid particles particularly suited for selective delivery to diseased cells and tissues such as tumorous cells and tissues and to effective concentrations within the diseased cells and tissues.

The lipid particles of the nanoemulsion are structured to facilitate both passive accumulation and increase of active endocytosis into diseased cells and tissues including tumor cells and tissues. The lipid particles enter these cells through active metabolic uptake and passively accumulate in the vessel parts of diseased tissue. Thus, the lipid particles of the present invention provide a delivery vehicle that is selectively and preferentially targeted for uptake and endocytosis by cells characterized by hyperproliferation, including tumors and cancer cells. As used herein, "endocytosis" means that the lipid particles are actively ingested by the cell. Increased endocytosis levels combined with the high loading capacity of the particles for therapeutic or diagnostic agents deliver an effective amount of therapeutic or diagnostic agent to these targets to provide a powerful carrier for the treatment or diagnostics of the targets, thereby Induction of therapeutic benefit effects, induction of growth arrest, induction of differentiation or cell death. Thus, the lipid particles of the present invention not only enhance the delivery of therapeutic agents to diseased cells and tissues as compared to the delivery systems, especially in the prior art, but also reduce the amount of therapeutic agent required to achieve the desired efficacy.

The lipid particles of the present invention are exceptionally physicochemically stable over a prolonged period of time, thereby minimizing the loss of treatment or diagnostic agents due to undesirable precipitation, aggregation or insolubility commonly found in prior art delivery systems. Experience. In addition, these lipid particles can be controlled release; Improved drug stability; Active drug charge ability; Better compatibility with hydrophobic drugs; Relatively low biotoxicity; And other desirable features including low organic solvent content. In addition, the present lipid particles are relatively simple and convenient for preparation and administration.

The term "lipid particle" as used herein is meant to include any lipid-containing structures that are at least substantially-intact-intact particles that form part of the nanoemulsion, which are typically nanosized. . The term "substantially-unchanged" means that the particles retain their shape without a membrane as compared to liposomes. The lipid particles consist of at least one non-bilayer-forming lipid.

The lipid bilayer structure or arrangement is characterized by hydrophilic terminus (polar head region) and hydrophobic terminus (nonpolar tail) leading to amphiphilic molecules such as phospholipids, which tend to self-organize into two opposing layers of lipid molecules in aqueous solution and / or tend to. It is usually formed by certain kinds of lipids). The two opposite layers of the lipid molecules are arranged such that their hydrophobic ends meet with each other to form an oily core, while the hydrophilic ends meet with an aqueous solution on either side of the bilayer structure. In the present invention, the term "non-bilayer-forming lipid" includes lipids that lack such ability and / or tendency to form a lipid bilayer structure or arrangement in an aqueous environment. Examples of non-bilayer-forming lipids include lipids that are only weak polar, preferably lipids that are nonpolar or neutral. More preferred lipids in the present invention are neutral lipids.

The lipid particles of the present invention can be distinguished from the gas-containing microbubbles described in US Pat. Nos. 4684479 and 5215680 and also described, for example, in US Pat. Nos. 6565889 and 6596305, all of which are incorporated herein by reference. Structurally distinct from liposomes. In particular, the lipid particles are formed by a mixture of non-bilayer-forming lipids which are physiologically acceptable and, for example, at least substantially free of the presence of charged or polar lipids comprising phospholipids. Suitable examples of non-bilayer-forming lipids include glycerol monoesters of saturated and unsaturated carboxylic acids; Glycerol monoesters of saturated aliphatic alcohols; Sterol aromatic acid esters; Sterols; Terpenes; Bile acids; Alkali metal salts of bile acids; Sterol esters of aliphatic acids; Sterol esters of sugar acids; Esters of sugar acids; Esters of aliphatic alcohols; Esters of sugars; Esters of aliphatic acids; Sugar acids; Saponins; Sapogenin; Glycerol; Glycerol di-esters of aliphatic acids; Glycerol tri-esters of aliphatic acids; Glycerol diesters of aliphatic alcohols; Glycerol triesters of aliphatic alcohols; And lipids selected from combinations thereof.

In one embodiment of the invention, the lipid particles are selected from a group of non-bilayer-forming lipids that provide lipid particles with the sizes described below that promote high endocytosis levels when applied to diseased target tissues and cells. It is prepared by first forming the mixture. The lipid mixture is generally:

a) at least one first element selected from the group consisting of carboxylic acids containing about 9 to 18 carbon atoms and glycerol monoesters of aliphatic alcohols containing about 10 to 18 carbon atoms;

b) at least one second element selected from the group consisting of sterol aromatic acid esters;

c) at least one third element selected from the group consisting of sterols, terpenes, bile acids and alkali metal salts of bile acids;

d) sterol esters of aliphatic acids containing about 1 to 18 carbon atoms; Sterol esters of sugar acids; Esters of sugar acids and aliphatic alcohols containing about 10 to 18 carbon atoms, esters of sugar acids and aliphatic acids containing about 10 to 18 carbon atoms; Sugar acids and saponins; And at least one optional fourth element selected from the group consisting of sapongenins; And

e) at least one optional agent selected from the group consisting of glycerol, glycerol di- or tri-esters of aliphatic acids containing about 10 to 18 carbon atoms and aliphatic alcohols containing about 10 to 18 carbon atoms Contains 5 elements.

The above-described lipid mixture comprises only the presence of (a) to (c) elements, whereas it is more preferred to include (d) and / or (e) elements, which is a long term stability and uniformity of the size of the lipid particles. Sexuality is improved theoretically due to the presence of these two optional elements.

In one preferred embodiment of the invention, the five elements (including the two optional elements) which comprise the lipid mixture forming the lipid particles of the invention are (a) :( b) :( c) :( d) :( e) is combined in the weight ratio of (1-5) :( 0.25-3) :( 0.25-3) :( 0-3) :( 0-3), respectively.

The fifth element of the lipid mixture is described as comprising glycerol monoesters of saturated carboxylic acids containing about 10 to 18 carbon atoms, while the same as, but not limited to, 9-carbon oleic acid or elic acid Glycerol monoesters of mono- or polyunsaturated carboxylic acids containing about 9 to 18 carbon atoms which are not present are also contemplated as being useful in the construction of the lipid mixture.

The proportion of elements of the lipid mixture may vary depending on the type of cell and / or tissue targeted for delivery, the therapeutic or diagnostic agent being charged, the desired dosage of the therapeutic or diagnostic agent, the pharmaceutically acceptable carrier used, the mode of administration. It will be appreciated that some factors may be varied, including, but not limited to the presence of other excipients or additives, and the like. In addition, factors that allow the lipid particles to be selectively endocytized by targeted diseased tissues and cells may include the composition of the lipid mixture and the structure of the resulting lipid particles, as well as the size and molecular weight of the particles described below. Also includes.

The lipid particles of the present invention maintain a desirable particle size distribution, preferably most of the particles have a median average particle size of about 0.02 to 0.2 microns, preferably 0.02 to 0.1 microns, and a small amount of particles. It falls within this range and only some lipid particles reach about 200 nm. The particle size range that can be reached in the lipid particles of the present invention leads to improved physicochemical stability over an extended period of time, with substantially reduced undesirable aggregation and drug precipitation. This range is also particularly suitable for treating cancer; Large particles may be suitable for other uses (eg, targeting other types of cells or tissues). The range provided herein will be determined in part by the type and amount of lipid mixture employed and the added therapeutic or diagnostic agent.

The therapeutic or diagnostic agent employed in the present invention may be uncharged or charged, nonpolar or polar, natural or synthetic, and the like. As used herein, the term "therapeutic agent" affects microtubule production, structure, association, function, and destruction, thereby preventing diseases, conditions, symptoms, or symptoms thereof characterized by cell hyperproliferation, including cancer. And any substance including, but not limited to, drugs, hormones, vitamins, nutrients, substances, and the like useful for treatment. Thus, therapeutic agents useful in the present invention are all types of drugs, lipophilic polypeptides, cytotoxins, oligonucleotides, cytotoxic antitumor agents, anti-metabolic agents that affect microtubule production, structure, association, function and destruction , Hormones and radioactive molecules. The term "oligonucleotides" includes both antisense oligonucleotides and sense oligonucleotides (eg, nucleic acids known in the art as vector). Oligonucleotides may be "natural" or "modified" with respect to subunits or binding between subunits.

However, in certain embodiments of the present invention, the therapeutic agent may be, for example, paclitaxel, doxetaxel, cephalomannine baccatin-III, 10-deacetyl baccatin III, diacetyl Taxanes such as deacetylpaclitaxel and deacetyl-7-epipaclitaxel; Vinca alkaloids such as, for example, vincristine, vinblastine, vinorelbine, vindesine and the like; Epothilones; Eleuterobines; Discothemolide; Dolastatin; Colchicine; Combrestatin; Formomsin A; Haliconerin B; Spongestatin 1; Sarcodetic humans; Laulimides); Derivatives, analogs, homologs and combinations thereof of each of the aforementioned agents; And microtubule-interacting agents selected from the group consisting of similar drugs or substances known to exhibit microtubule-interacting activity.

Taxanes such as paclitaxel may also be used in small sustained release doses as anti-inflammatory agents. This use is particularly important in the field of biomedical instruments, such as stents, which are placed surgically in a patient. Some accumulation of cells around and inside the stent is desirable because this builds up a smooth cover and thereby includes the instrument in the artery itself, whereas the accumulation of cells can block internal channels and cause arterial recurrence. May cause stenosis. Thus, Boston Scientific Corporation produces a paclitaxel-eluting tubular stent system coated with a proprietary polymer that binds paclitaxel to the stent surface. The paclitaxel-polymer complex allows precise control of dosage and time-release characteristics for paclicktaxel, eluting a sufficient amount of drug to inhibit cell accumulation around the stent and restenosis around the stent and Significantly prevents blood vessel regeneration. In particular, where the therapeutic agent is a taxane or other microtubule-interacting agent, it is contemplated that the pharmaceutical compositions of the present invention will likewise be useful for regulating cell accumulation around surgically-implanted biomedical instruments.

The pharmaceutical compositions of the present invention show long-term physicochemical stability, such that the compositions are readily prepackaged in stable ready-to-administer dosage forms, thereby pre-dose bedside dilution and formulation typically associated with similar compositions in the prior art. Eliminates the need for The pharmaceutical compositions of the present invention exhibit desirable drug and emulsion stability over long periods of time (eg, at least 14 days at about 30 ° C. and at least 12 months at 4 ° C.). The pharmaceutical composition of the present invention comprises lipids in an amount of about 0.1 μg / mL to 1000 μg / mL, preferably about 10 μg / mL to 800 μg / mL and most preferably about 200 μg / mL to 600 μg / mL Contains particles. Typical concentrations of therapeutic or diagnostic agents based on the total volume of the pharmaceutical composition are at least 0.001% w / v, preferably 0.001% to 90% w / v and most preferably about 0.1% to 25% w /. may be v. The amount of therapeutic or diagnostic agent present in the pharmaceutical composition is about 0.001 μg / mL to 1000 μg / mL, preferably about 0.1 μg / mL to 800 μg / mL and most preferably about 60 μg / mL to 400 μg. / mL can be reached.

The pharmaceutical composition of the present invention may further comprise an emulsion-enhancer selected from plant-based fat sources, solvents, surfactants or combinations thereof. The emulsion-enhancing agents, when individually or in combination, theoretically reduce or minimize undesirable precipitation or aggregation of lipid particles, thereby actively affecting and promoting the active uptake of lipid particles into cancer cells, thereby improving stability. It has been found that the small particle size properties of lipid particles are maintained. In addition, the emulsion-enhancing agent should improve the physicochemical stability and drug-carrying capacity of the pharmaceutical composition of the present invention.

In a preferred embodiment of the present invention, the plant-based fat source is generally, for example, soybean oil, flaxseed oil, hemp seed oil, linseed oil, mustard oil, rapeseed oil, canola oil, safflower oil, sesame oil , Sunflower oil, grape seed oil, almond oil, apricot seed oil, castor oil, corn oil, cottonseed oil, coconut oil, hazelnut oil, neem oil, olive oil, palm oil, palm kernel oil, peanut oil, Plant-derived fatty acids in the form of vegetable oils such as pumpkin seed oil, rice bran oil, lake seed oil and mixtures thereof. More preferred vegetable oil is soybean oil.

The vegetable oil is generally present in an amount sufficient to allow high surface tension in the nanoemulsion which increases the probability of hydrophobic interaction with the plasma membrane of the target cell or receptor thereon. The plant fat source is about 0.001% v / v to 5.0% v / v, more preferably about 0.005% v / v to 4.0% v / v, and most preferably about 0.01% v / v to 2.5% v may be present in an amount of / v.

-80과 같은 세제 폴리소르베이트류이다.In another preferred embodiment of the invention, the surfactant is selected from nonionic surfactants. Examples of nonionic surfactants include sorbitan esters such as fatty-acylated sorbitan esters and their polyoxyethylene derivatives and mixtures thereof and poloxamer compounds 188, 182, 407 and 908), Tyloxapol, Polysorbate 20, 60, and 80, sodium glycolate, sodium dodecyl sulfate, and the like, and combinations thereof, including but not limited to Do not include mixtures thereof. More preferred nonionic surfactants are, for example, Tween

Detergent polysorbates such as -80.

Once formed, the surfactant is generally used in an amount sufficient to stabilize the interface between the hydrophobic and hydrophilic components of the nanoemulsion and prevent the hydrophobic components from coalescing so that the nanoemulsion does not significantly change in storage. Exists as. The surfactant may be from about 0.01% w / v to 4.0% w / v, more preferably from about 0.15 w / v to 3.0% w / v and most preferably from about 0.2% w / v to 2.5% w / v. May be present in amounts.

In another preferred embodiment of the invention, the solvent comprises a solvent which can be mixed with any pharmaceutically acceptable water, such as, for example, polar protic and polar aprotic solvents. The solvent is preferably 1,3-butanediol; Dimethyl sulfoxide; Alcohols such as methanol, butanol, benzyl alcohol, isopropanol and ethanol; And others. More preferred solvent is benzyl alcohol.

The solvent is generally present in an amount sufficient to control the degree of aggregation of the nonionic surfactant in the nanoemulsion. The solvent may be present in an amount of about 0.001% to 99.9% v / v, more preferably 0.005% v / v to 80% v / v and most preferably about 0.005% v / v to 70% v / v. have.

The compositions of the present invention do not alter or alter the basic pharmacological activity or chemical properties of the therapeutic or diagnostic agent, but the agent is delivered to diseased cells or tissues, including cancer cells or tissues, and the treatment or diagnosis is made by simply enhancing endocytosis. Give potency. Examples of teachings relating to the use of taxanes as therapeutic agents in the treatment of cancer are each described in US Pat. No. 6346543; 6384071; 6387946; 6395771; 6403634; And 6500858.

Fluid Materials Processors와 같은 적당한 전단-집중 균질화 혼합기 또는 균질화기를 사용하여 생성될 수 있다. 이렇게 생성된 나노에멀젼은 더 처리되어 인간을 포함하는 온혈 동물에 투여용으로 사용될 수 있는 더 정제된 형태를 생성할 수 있다.In general, the pharmaceutical compositions of the present invention are prepared by combining lipid particles with a therapeutic or diagnostic agent and thoroughly mixing them. The lipid mixture may be mixed with a surfactant combined with a plant fat source prior to mixing with a therapeutic or diagnostic agent that may be mixed with a solvent that may be mixed with water for dissolution. The lipid particle-therapeutic / diagnostic combination is then mixed with water, preferably purified water. The resulting mixture is then subjected to a high shear force typically produced in a conventional standard shear-intensive homogenizer mixer or homogenizer to produce a nanoemulsion comprising lipid particles dispersed in an aqueous phase. Sufficiently high shear forces are found in Microfluidizer, sold by Microfluidics of Newton, Massachusetts.

It may be produced using a suitable shear-intensive homogenizing mixer or homogenizer, such as Fluid Materials Processors. The nanoemulsions thus produced can be further processed to produce more purified forms that can be used for administration to warm blooded animals, including humans.

In some cases, it may be desirable to remove excessively large particles from the mixing process in order to maintain particle size distribution within the desired range. Suitable filtration systems, such as those purchased from Millipore, Waltham, Mass., Are available for this purpose. Therefore, the selection of a suitable filtration system can be a factor in controlling the particle size distribution of lipid particles within the desired range, which is within the skill of one of ordinary skill in the art. In addition, the nanoemulsion can be treated with dialysis to remove impurities, leaving only the dialysate retained for pharmaceutical use. Dialysis is a preferred method of removing any non-particulate lipid mixture components, drugs and / or solvents and achieving any desired buffer exchange or concentration. Slight molecular weight cutoffs of the dialysis membrane of 5000 to 500000, where molecular weights of 10000 to 300000 are preferred, can be used. Lipid particles produced as described when purified by dialysis to remove non-particulate drugs are such that the lipid particles can be introduced into cells targeted to, for example, C ₆ glioma cells. May be characterized in determining.

The composition of the present invention may further comprise a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers are known in the art and include, but are not limited to, antioxidants, buffers, chelating agents, flavoring agents, colorants, preservatives, absorption accelerators, antimicrobial agents, and combinations thereof that enhance bioavailability. Include those conventionally used in compositions. The amount of such additives depends on the desired properties, which can be readily determined by one skilled in the art.

The pharmaceutical compositions of the present invention may typically contain salts, buffers, preservatives and compatible carriers, optionally in combination with other therapeutic ingredients. When used in medicine, salts must be pharmaceutically acceptable, but non-pharmaceutically acceptable carriers can be readily used in the preparation of the pharmaceutically acceptable salts and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, palmilic acid, p-toluene sulfonic acid, tartaric acid, citric acid, methane sulfonic acid, formic acid, malonic acid , But are not limited to those prepared from succinic acid, naphthalene-2-succinic acid and benzene sulfonic acid. In addition, pharmaceutically acceptable salts may be prepared with alkali or alkaline earth metal salts, such as sodium, potassium or calcium salts of carboxylic acid groups.

The present invention also provides a method of treating or diagnosing a patient by delivering an effective amount of at least one therapeutic or diagnostic agent to a cell for carrying out the prevention, diagnosis or treatment of a disease, condition or syndrome or its symptoms characterized by cell hyperplasia. Or a method of diagnosis. Treatment of primary tumors by regulation of tumor cell proliferation, angiogenesis, metastatic growth, apoptosis and treatment of development of micrometastases after or with surgical removal; And improved cancer treatments are particularly contemplated, including radiological or other chemotherapy treatments of primary tumors. The pharmaceutical compositions of the present invention include primary or metastatic melanoma, lymphoma, sarcoma, lung cancer, liver cancer, Hodgkin's and non-Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer and breast cancer, prostate cancer, ovarian cancer and It is useful for cancer types such as adenocarcinomas such as pancreatic cancer.

For therapeutic and diagnostic applications, the pharmaceutical composition can be administered directly to the patient when combined with a pharmaceutically acceptable carrier. This method may be carried out by administering the therapeutic or diagnostic agent alone or in combination with an effective amount of another therapeutic or diagnostic agent that may or may not be a second microtubule-interacting agent. If not a microtubule-interacting agent, this second agent is a cytostatic agent, a folic acid inhibitor, an alkylating agent, a topoisomerase inhibitor, a tyrosine kinase inhibitor, grapephytotoxin, an anti-tumor antibiotic, a chemotherapeutic agent, apoptosis -Inducers and combinations thereof, but is not limited thereto. The therapeutic agents may further comprise metabolic inhibitory reagents. Many such therapeutic agents are known in the art. Combination therapies provide simultaneous, sequential or separate uses to treat such conditions as needed to amplify or secure patient response to the therapy.

The methods of the invention may be practiced using any mode of administration that produces an effective level of the active compound without causing medically acceptable and clinically unacceptable side effects. Although particularly suitable formulations for preferential administration are preferred, the compositions of the present invention may be inhaled, oral, topical, transdermal, nasal, ocular, lung, rectal in the form of aerosols, sprays, powders, gels, lotions, creams, suppositories, ointments, and the like. It may be formulated for transmucosal, intravenous, intramuscular, subcutaneous, intraperitoneal, intrathoracic, intrapleural, intrauterine, intratumoral or infusion methods or for administration. If such formulations are desired, other additives known in the art may be included to impart desired consistency and other properties to the formulation.

Those skilled in the art will appreciate that the particular mode of administering the therapeutic or diagnostic agent may be selected from a particular agent selected from; Whether the administration is for the treatment, diagnosis or prevention of a disease, condition, symptom muscle or symptoms thereof; The severity of the medical condition being treated or diagnosed; And the dosage required for therapeutic efficacy. For example, preferred modes of administering anticancer agents for the treatment of leukemia include intravenous administration, while preferred modes for treating skin cancer can include topical or intradermal administration.

As used herein, an "effective amount" refers to a dose or multiple doses of a therapeutic or diagnostic agent for which a desired therapeutic or diagnostic effect is reached. In general, an effective amount of a therapeutic or diagnostic agent will depend on the activity of the specific agent employed; Metabolic stability and length of action of the agent; The species, age, body weight, general health, nutritional status, sex and diet of the subject; Mode and time of administration; Discharge rate; If present, drug combination; And the degree of expression and / or severity of the particular condition being treated. The exact dosage can be determined by one of ordinary skill in the art without undue experimentation in single or multiple doses daily to produce the desired result, the dosage being determined to achieve the desired therapeutic effect or any complications. In this case it can be controlled by individual specialists. Importantly, the dosage of therapeutic agent used should be an amount sufficient to inhibit or kill tumor cells without substantially harming normal cells when used to treat cancer.

The therapeutic or diagnostic agent included in the pharmaceutical composition of the present invention may be prepared in any desired amount up to the maximum amount that can be dissolved by the given lipid particles and suspended in or operably associated with the particles. Can be. The amount of the diagnostic or therapeutic agent can range from 0.001 mg / mL to 1000 mg / mL, preferably from about 0.1 mg / mL to 800 mg / mL and most preferably from about 300 mg / mL.

In general, the lipid particles will be delivered in a manner sufficient to administer an effective amount to the patient. The dosage can range from about 0.1 mg / kg to 175 mg / kg, preferably from about 1 mg / kg to 80 mg / kg, and more preferably from 5 mg / kg to 60 mg / kg. Dosages may be administered in a single dose or in the form of separate divided doses, such as one to four or more times daily. If the response of the subject is insufficient at a particular dose, higher doses (or more potent doses by different but more localized delivery routes) may be employed to the extent of patient resistance. Multiple doses per day are contemplated to reach an appropriate systemic or target level of treatment or diagnostic agent.

In another embodiment of the present invention, microtubule-interacting agents can be used as in vitro diagnostic agents. As mentioned above, depending on the specific tumor cell or cell type being studied, other microtubule-interacting agents may be more or less effective at inhibiting different tumor types. Thus, for example, if the diagnosis or selection of the appropriate chemotherapeutic strategy can be different, testing of cultures of tumor cells in vitro with microtubule-interacting agents known to target specific tumor cell types Other approaches to identifying tumor types and effective treatments are provided.

In another aspect of the present invention, a method of preparing a pharmaceutical composition of the present invention is provided. The lipid mixture forms a lipid nanoemulsion in which the composition comprises a dispersion of lipid particles upon treatment with an aqueous phase, wherein the dispersed phase of the lipid particles is present in the form of macromolecules or clusters of small scale nanoscale molecules of a particular size. It is included in the said therapeutic or diagnostic agent in the quantity. In preparing the compositions of the present invention, the lipid mixture and the therapeutic or diagnostic agent are combined with an aqueous phase comprising water, preferably filtered water. The resulting mixture is treated so that it is typically in the range of up to 200 nm, a size that is particularly suitable for cancer treatment, but this is not always the case, and larger particles form lipid particles with a median average particle size range suitable for other uses. . The range obtained will be influenced in part by the lipid mixture employed, the type and amount of therapeutic or diagnostic agent added to the lipid mixture and the technique used to produce the lipid particles.

The pharmaceutical compositions of the present invention can be prepared using conventional dispersion-generating techniques or processes known in the art. Such techniques include, but are not limited to, high-shear homogenization, ultrasonic stirring or sonication, high-pressure homogenization, solvent emulsification / evaporation, and the like. In one embodiment of the present invention, lipid particles can be prepared through conventional high-pressure homogenization techniques using a suitable high-pressure homogenizer. Suitable size homogenizers are commercially available. High-pressure homogenizers are generally designed to push the fluid through a narrow gap, typically up to about several microns, at high pressures of about 100 to 2000 bar. Pressurized fluid is accelerated at very high speeds in excess of 1000 km / hr over very short distances. Pressurized fluids containing lipid mixtures encounter very high shear stresses and cavitation forces, effectively rupturing the lipid mixture and breaking it into particles in the submicron range. As described above, most of the lipid particles have a median average particle size of about 0.02 μ to 0.2 μ, preferably 0.02 μ to 0.1 μ, with a small amount of particles falling within the above range, in particular some lipid particles. Up to about 200 nm.

In another particular method of making lipid particles, the lipid mixture may be mixed with a vegetable fat source such as vegetable oil and a surfactant such as a nonionic surfactant to produce an aqueous phase. The therapeutic or diagnostic agent may be mixed with a solvent such as a solvent that may be mixed with water to produce a therapeutic or diagnostic agent. The lipid and therapeutic or diagnostic phase are then mixed and mixed together in the presence of an aqueous phase, preferably via sonication. The resulting mixture is then homogenized under high shear forces to produce the corresponding nanoemulsions of the invention. The nanoemulsions are then filtered through a 0.2 μm membrane to warm blooded animals, including humans, in need of treatment or diagnosis by sterilizing and / or removing impurities such as unused lipid substances such as extra therapeutic or diagnostic agents. To produce a purified form suitable for delivery as a pharmaceutical composition.

The following examples are provided to aid in understanding the pharmaceutical compositions of the present invention.

Example 1

EmPAC Formulation of

고압 균질화기(모델 M-110Y, Microfluidics 사, 매사추세츠 주 뉴턴 소재)를 사용하여 18000 psi에서 고압 균질화를 4 사이클 하여 제조되었다. 이전의 연구들은 파클리탁셀이 LN으로 최대 ~25% w/w 지질 농도로 혼합될 수 있다는 것을 보여주었지만, 상기 제형은 겨우 몇 시간 동안만 안정하여, 이러한 문제가 약물 혼합에 관한 것이라기보다는 안정성에 관한 것이라는 것을 알게 되었다. 1 mM 피로인산나트륨(pH 9.5)에서 제조된 LN은 적어도 2 개월 동안 안정했던 것을 알게 된 이후에, 1 mM 피로인산나트륨(pH 9.5)은 약물 혼합 연구에 대한 제 1 시험 매질로 선택되었다.Paclitaxel was chosen as the first drug candidate to be tested and formulated for commercial formulation growth onto lipid nanoparticles (LN). Paclitaxel / LN formulations were dissolved directly in water and 4% ethanol and then 110Y Microfluidics Microfluidizer

A high pressure homogenizer (Model M-110Y, Microfluidics, Newton, Mass.) Was used to produce four cycles of high pressure homogenization at 18000 psi. Previous studies have shown that paclitaxel can be mixed with LN up to ~ 25% w / w lipid concentration, but the formulation is stable for only a few hours, so this problem is not stable with drug mixing but rather with stability. I learned that it was about. After finding that LN prepared in 1 mM sodium pyrophosphate (pH 9.5) was stable for at least 2 months, 1 mM sodium pyrophosphate (pH 9.5) was selected as the first test medium for drug mixing studies.

If paclitaxel was mixed in 3 passes at 15000 psi with LN with 4% ethanol content in 1 mM sodium pyrophosphate, only the formulation with 5% drug loading was stable and even the last formulation was not stable for a significant period of time. I found out. On the second day after mixing, some turbidity was observed in the 5% loaded specimen, with complete precipitation on the third day. In contrast, immediate blurring was observed in samples of 33% and 16% drug loading. These findings are summarized in Table 1. Possible reasons for the reduced stability may be due to poor solubility of paclitaxel dissolved in water, low alcohol content (4%), high pH of the final solution (pH 11.5) and / or incompatible lipid concentrations.

Table 1 below relates to paclitaxel loading with LN prepared in 4% ethanol:

Lipid concentration (μg / mL ) % Drug Loading Particle Size (μm) zeta Potential (㎷) Explanation 200 0.0 0.149 -39.0 Sunny 200 5.0 0.101 -32.30 Sunny 200 16.2 - - Sedimentation 200 33.2 - - Sedimentation

고압 균질화기(모델 M-110EH, Microfluidics 사, 매사추세츠 주 뉴턴 소재) M-110 EH 상의 15000 psi 및 3 사이클로 10% DMSO에서 제조되었다.In such a case, it is possible to change the organic content of ethyl alcohol to improve the stability of the drug-mixed particles. Thus, LNs were first produced at different alcohol contents (eg 50% v / v, 25% v / v, 12.5% v / v). It was observed that even drug-free particles were not stable at ethanol content below 25% v / v. In addition, only LN at 12.5% v / v alcohol concentration was stable for at least one day. Thus, 10% paclitaxel was loaded with LN with 12.5% v / v alcohol content. After 3 hours, gel formation is observed, strongly suggesting that high concentrations of alcohol do not stabilize the paclitaxel formulation in LN. In addition, ethyl alcohol was generally used as a demulsifying agent. This may be the reason that even high percentages of ethanol do not help stable drug-load formulations. Among other solvents, sulfoxylated dimethyl (DMSO) is the solvent of choice because paclitaxel is known to have very good solubility in DMSO. Thus, paclitaxel-loaded specimens are a second machine, the Microfluidizer.

High pressure homogenizers (Model M-110EH, Microfluidics, Newton, Mass.) Were made in 10% DMSO in 15000 psi and 3 cycles on M-110 EH.

Two controls were used, one without paclitaxel and the other without lipid components. From the second control, it is clear that the lipid components play a role in the stability of the drug-mixed LN as a sample precipitated in the absence of lipid components. However, samples with 10% paclitaxel precipitated on day 2, while samples with 5% paclitaxel were observed to be clear. When performing high performance liquid chromatography (HPLC) tests on these samples, it was observed that paclitaxel was denatured in these samples.

Because paclitaxel is highly soluble in DMSO, the stability of LN prepared with paclitaxel has been tested at various DMSO concentrations. The concentration of 20% DMSO showed the highest stability for 200 μg / mL with 105 w / w paclitaxel. Representative particle size distributions and zeta potential values are shown in FIG. 1. Data on the stability of this modified formulation are shown in Table 2.

Stability of Paclitaxel-Loaded LN in Modified DMSO Formulations Lipid concentration (μg / mL ) % Drug Loading Particle Size (μm) zeta Potential (㎷) Explanation ^￥ 200 0 0.089 -46.36 Sunny 200 5 0.106 -38.71 Sunny 200 10 0.093 -46.46 Sunny 0 10 - - Sedimentation ^The samples were analyzed at room temperature after 24 hours.

The denaturation of paclitaxel mentioned above denatured almost 100% within 24 hours, which was assumed to be due to the high pH (11.0) in the formulation. The addition of 1 mM sodium pyrophosphate reduced the pH to 9.5. Therefore, the effect of pH control through 1 mM sodium pyrophosphate on the stability of lipid components as well as paclitaxel was studied.

At pH above 6.0, the formulation was found to be clear with no precipitation observed. However, above pH 6.0, paclitaxel denatured significantly. Thus, the optimal solution is to use 1 mM sodium pyrophosphate with pH adjusted to 6.0 using diluted phosphoric acid to maintain the physicochemical stability of the lipid components and paclitaxel in the formulation. All results are summarized in Table 3.

Effect of pH on the Stability of Lipid Nanoparticles ^¥ pH Geology (200 ppm ) Geology (200 ppm ) + 10% (w / w) Paclitaxel 3.0 Sedimentation - 4.0 Sedimentation - 5.0 Sedimentation - 5.5 Sedimentation - 6.0 Sunny Clear, Paclitaxel stable 6.7 - Sunny, paclitaxel denatured 7.0 - Sunny, paclitaxel denatured 9.5 - Sunny, paclitaxel denatured water - Clear, Paclitaxel stable ^The samples were analyzed at room temperature 24 hours after settling.

Therefore, since we found that the long-term stability of the formulation could be reached due to the adjustment of DMSO and pH, the next study was to go because filtration can sterilize the product during manufacture, so that the filtration can lead to lipid components and paclitaxel- Whether or not it affects drug titers of loaded LN samples.

After the initial screening for different types of membranes such as nylon, PVDF and PES, it was concluded that polyethersulfone (PES) hydrophilic membranes are the most suitable membranes for formulation filtration. When 10% paclitaxel-loaded LN prepared in 20% DMSO was filtered through a 0.22 μm PES membrane filter, at least 98% of the paclitaxel passed through the membrane. When lipid components of this same formulation were analyzed by ELSD, an average of 93% of all components passed through the membrane. Thus, it has been demonstrated that filtration improves not only the quality of the formulation but also the stability.

To determine whether paclitaxel was mixed with LN in the presence of 20% DMSO or if DMSO cleaves paclitaxel into the aqueous phase to prevent paclitaxel from mixing into LN, a ¹⁴ C- from LN using a sucrose density gradient was used. Separation of labeled paclitaxel was performed. Measuring the distribution of radioactivity coefficients gives data on which part contains paclitaxel. Thus, 200 μg / mL of LN was prepared in the presence of 10% w / w paclitaxel comprising some ¹⁴ C-labeled paclitaxel, which preparation was subdivided on a sucrose density gradient. Portions were collected for analysis of radioactivity counts to determine which parts contained paclitaxel. The precipitated portion was mainly observed at the bottom of the gradient, with the precipitated portion consisting mainly of insoluble paclitaxel. A band was observed in the gradient set to LN, and the radioactivity content was analyzed in both the precipitate as well as in the LN fraction. In the presence of lipids comprising LN, little radioactivity label was observed in the bottom precipitate. In the absence of lipids, greater amounts of precipitation are observed and much less radiolabel sections are observed in the sediments section.

As shown in FIG. 2, about 80% of paclitaxel was precipitated in the absence of lipids, whereas only 1.8% of paclitaxel was precipitated in the presence of lipids. This supports the view that most paclitaxel is mixed with lipids in LN.

In a clinical setting, intravenous infusion of about 2.5 L over 3 to 24 hours is generally considered safe and tolerable in most patients. Current clinical paclitaxel dosages range from 135 to 175 mg / m 2 over 3 to 24 hours. Assuming an average size of a male surface area of about 1.8 m 2, a paclitaxel concentration of about 97 μg / mL is required to achieve this dosage. To deliver a therapeutic dose of such paclitaxel within these time and volume parameters, paclitaxel must be at a sufficiently high concentration. Finally, formulations of paclitaxel-loaded LN specimens that achieve such dosages as benchmarks are preferred. However, since the present invention can selectively target tumor cells, the therapeutic dosage required for paclitaxel-mixed LN may in fact be much lower. Thus, more of the administered drug will disappear in tumor cells.

As is apparent from Table 4 below, it is possible to prepare up to 400 μg / mL by loading 10% paclitaxel with stable LN for at least 5 hours without any signs of aggregation. This preparation had a paclitaxel concentration of 40 μg / mL and was stable over the time required for intravenous administration.

After filtration through a 0.22 μm PES membrane, the samples dissolved in 10% paclitaxel in 20% DMSO in 1 mM sodium pyrophosphate (pH-6.0) loaded with LN were stable for at least 3 days. After changing the sodium pyrophosphate concentration to 0.5 mM, the sample was stable for 7 days. Initially, all samples were analyzed in filtered and unfiltered form for paclitaxel content and particle size distribution. Thus, these data suggest that paclitaxel formulations at concentrations of currently clinically utilized formulations can be prepared in LN.

-80으로 이루어진 제 2 파클리탁셀-LN 제형이 제조되었다. 이 제 2 제형에 대하여 수행된 안정성 연구에 따르면 400 ㎍/mL의 지질 및 일정 농도의 파클리탁셀을 사용한 제형은 적어도 24 시간 동안 안정하다는 것을 밝혔다. 결과들은 표 4에 정리되어 있다.Thus, 60 μg / mL paclitaxel; 300 μg / mL lipid mixture; 0.5% butanol; 0.5% soybean oil; And 0.25% Tween

A second paclitaxel-LN formulation consisting of -80 was prepared. Stability studies conducted on this second formulation revealed that formulations with 400 μg / mL of lipids and constant concentrations of paclitaxel were stable for at least 24 hours. The results are summarized in Table 4.

Effect of Lipid Concentration on Nanoparticle Stability Lipid concentration (μg / mL ) Paclitaxel  Charge (% w / w) Time after manufacture Explanation Average particle diameter (Nm) 200

20

0 hours ^§ Sunny 114 5 hours ^§ Sunny 113 24 hours ^￥ Sunny 126 400

10

0 hours ^§ Sunny 153 5 hours ^§ Sunny 145 24 hours ^￥ Sunny 180 600

5

0 hours ^§ Sunny 162 5 hours ^§ Sunny 170 24 hours ^￥ Muddy 286 ^§ Samples were stored at room temperature
^The samples were frozen at -80 ° C.

Specifically, as shown in Table 5, the second paclitaxel-LN formulation was stable at room temperature for at least 7 days and at 2-8 ° C. for at least 30 days.

Stability of Second Formulation Temperature Days after manufacture Particle diameter (μm) Room temperature

0 0.071 3 0.072 7 0.076 2 to 8 ° C



0 0.101 3 0.107 21 0.113 30 0.117 0 0.101

-80으로 이루어져 있다. 이 제 3 제형은 표 6에 정리되어 있는 바와 같이, 상온에서는 적어도 3 주 동안 안정하였고 2 내지 8℃에서는 8 일 넘게 안정하였다.However, the butanol was replaced with benzyl alcohol because butanol was induced to cause drowsiness in mice when injected (data not shown). Thereby, the third formulation comprises 60 μg / mL paclitaxel, 400 μg / mL lipid mixture; 0.06% Benzyl Alcohol, 0.5% Soybean Oil, and 0.25% Tween

It consists of -80. This third formulation was stable for at least 3 weeks at room temperature and over 8 days at 2-8 ° C., as summarized in Table 6.

Stability of the Third Formulation Temperature Days after manufacture particle diameter (Μm) Room temperature



0 0.073 8 0.071 12 0.071 16 0.070 21 0.072 2 to 8 ° C




0 0.068 15 0.069 30 0.070 60 0.071 70 0.071 82 0.070

Example 2

Emulsion ( EMULSIPHAN ) And EmPAC Cell intake

The above studies were conducted to determine whether human tumor cells ingested LN and whether human tumor cells exhibited differential ability to ingest LNs. Most tumor cell lines tested not only readily ingested fluorescent LNs, but also found that tumor cell lines derived from other tumor cell lines showed differential ability to ingest LNs.

고압 균질화기(모델 M-110Y, Microfluidics 사, 매사추세츠 주 뉴턴 소재)를 통해 처리되었다.The LN formulation used for this experiment was prepared as follows: The appropriate lipid was solubilized in 95% ethanol to 10 mg / mL by sonication for 10 minutes. Next, 100 μl of 0.5 mg / mL cholesteryl BODIPY-FL (Molecular Probes, Eugene, Oregon) was added to ethanol to 1 mL of the 10 mg / mL solubilized lipid. Finally, the lipid and cholesteryl BODIPY-FL mixture was added to 50 mL of a 1 mM sodium pyrophosphate solution dissolved in water to add 110 Y Microfluidics Microfluidizer.

The process was performed with a high pressure homogenizer (Model M-110Y, Microfluidics, Newton, Mass.).

To first demonstrate that the cellular uptake of fluorescence-labeled nanoparticles is directly proportional to the cellular fluorescence intensity through fluorescence activated cell sorting (FACS), the mean fluorescence intensity per cell is dependent on the concentration of LN as shown in FIG. 3A. It was determined that it is directly proportional and also proportional to the incubation time in the presence of LN as is apparent from FIG. 3B. Indeed, it was visually confirmed that the fluorescence intensity was proportional to the extent of fluorescence LN uptake with identically treated cells fixed on glass coverslips (data not shown). The LN was labeled with the fluorescent lipophilic dye DiO (Molecular Probes, Eugene, Oregon) that can be detected using filter sets for fluorescein. LN was prepared using 200 μg / mL lipids and 2.5 μg / mL DiO by microfluidization. Labeled LNs were added to C6 cells and incubated at 37 ° C. The medium was then removed and the cells washed with phosphate buffered saline (PBS), trypsinized and washed again before being fixed in 4% formaldehyde. LN was added at concentrations of 0, 12.5, 25, 50 and 100 μg / mL and incubated for 60 minutes. 50 μg / mL LN was added to the cells and incubated for 0, 5, 10, 15, 30 and 60 minutes before the medium was removed and processed for FACS analysis. It is clear that LN uptake can be assessed by FACS, as evident from the results shown in FIGS. 3A and 3B, respectively, showing that the fluorescence intensity per cell is directly proportional to the increased concentration and increased incubation time.

In order to determine whether cells from other cell lineages show a distinctive ability to assess the relative capacities of cells derived from other tumor cell types that ingest LN as well as the discriminative ability to ingest LN Were tested in tumor cell lines. Selected cell lines were selected from those used by the National Cancer Institute (NCI) Developmental Therapeutics Program In Vitro Screening. The NCI cell line panel consists of cell lines derived from numerous different human tumor lines, each with some different cell lines from the expressed human tumor lineage. Cell lines used were HS-578T, MDA-MB-231 and MX-1 breast cancer; H23, H460 and H522 lung cancer; SF-539 liver; And HT29, SW-620 and COLO205 crystalline tumor cell lines.

LN was prepared using 200 μg / mL lipid and 0.5% w / w cholesteryl-BODIPY-FL by microfluidization. Labeled LNs were added to the cells and incubated at 37 ° C. Thereafter, the medium was removed, cells were washed with PBS, trypsinized and washed again before being fixed in 4% formaldehyde. Samples were analyzed by FACS and average fluorescence intensity per cell was measured. Cells derived from the colon, breast, central nervous system (CNS) and lung were compared with cells of the HT-29 colon carcinoma cell line and SF-539 lung cancer cell line, which appear to consume low and high amounts of fluorescent LN, respectively. Fluorescent LN uptake by each LN-treated cell sample was obtained by subtracting fluorescence intensity from the same untreated cell line.

As shown in Figures 4A-4D, it is found that the comparison of LN uptake by cell lines derived from the NCI panel varies between cell lines of different lineages. Some variation was observed between cells from the same tumor lineage, but relative uptake was fairly consistent for each tumor lineage. For example, breast and lung tumor cell lines generally showed higher LN uptake than colon tumor cell lines showed. Maximum LN uptake was found in cell lines derived from lung cancer and the CNS. All results are summarized in Table 7.

Uptake of Lipid Particles by Tumor Cell Lines Tumor cell types Neon LN Relative cell intake of Tumor cell lineage Cell line FACS ^§ Fluorescence intensity per cell through Sample size breast

MDA-MB-231 120.02 ± 26.37 4 HS578T 100.65 ± 12.48 4 MX-1 86.51 ± 13.28 3 lungs


H460 93.60 ± 20.34 5 A549 48.82 ± 8.07 4 H23 189.03 ± 37.86 4 H522 184.67 ± 40.58 4 Central nervous system


SF-539 219.63 ± 23.33 10 SF-268 100.41 ± 7.17 3 SF-295 232.74 ± 45.79 2 U-87 234.07 ± 25.54 2 colon



HT-29 46.44 ± 5.68 12 COLO-205 35.17 ± 6.89 4 SW-620 32.11 ± 7.79 4 HCT-15 31.59 ± 16.66 3 Caco-2 52.87 ± 5.52 4 liver Hep-3B 537.69 ± 27.71 7 Uterus and drug resistant uterus

MES-SA 204.92 ± 39.38 4 MES-SA-DX5 180.57 ± 27.88 4 MES-SA-MX2 220.81 ± 32.85 6 ovary SKOV-3 65.81 ± 11.42 4 Pancreas
PAN 02 211.99 ± 35.76 4 PAN 03 73.07 ± 22.3 ^* 4

^§ Average fluorescence intensity per cell was obtained for each fluorescent LN-treated sample by comparing fluorescence and subtracting fluorescence from each same untreated cell line.

^£ ND- US measurements. Particle diameters cannot be measured due to high precipitation levels.

)에 제형된 Taxol

로 알려진 파클리탁셀과 비교되었다.Next, ingestion of paclitaxel formulated in LN (EmPAC) was performed in a series of experiments analyzing cultured tumor cell uptake of radiolabeled paclitaxel, which is a conventional carrier Cremophor / ethanol 1: 1 (Cremophor EL

Taxol formulated in

Compared to paclitaxel, known as.

에서 혼합되었다. 본 실험에 사용된 EmPAC 제형은 DMSO를 함유하는 제 1 제형이었다(실시예 1 참조). 동일한 농도의 파클리탁셀로 이루어진 이 제형들은 12-웰(well) 레플리케이트(replicate) 내의 SF539 신경교종 및 A549 폐암 세포들에 1 시간 동안 첨가되었다. 배지는 제거되었으며, 세포 단일층은 약물을 제거하고 톨루엔을 함유하는 동일한 부피의 섬광(scintillation) 칵테일을 사용하여 PBS에서 세척한 이후에 가용화되어 각각의 단일층 표본과 연관된 파클리탁셀을 측정하였다. 각각의 표본에 대한 방사능라벨 계수는 각각의 표본에 초기에 첨가된 총 방사능라벨에 대하여 차지하는 부분으로 표현되었다. 이 실험 결과들은 도 5에 입증된 바와 같이, SF539는 Taxol

보다 LN(즉, EmPAC)에서 제형된 파클리탁셀을 현저히 더 세포내이입하였다는 것을 시사하였다.In the first experiment, ¹⁴ C-labeled paclitaxel was mixed with LN (which forms EmPAC) and Taxol

Mixed in. The EmPAC formulation used in this experiment was the first formulation containing DMSO (see Example 1). These formulations, consisting of the same concentration of paclitaxel, were added to SF539 glioma and A549 lung cancer cells in 12-well replicates for 1 hour. The medium was removed and the cell monolayer was solubilized after washing in PBS using drug removal and the same volume scintillation cocktail containing toluene to determine paclitaxel associated with each monolayer sample. The radiolabel coefficients for each sample were expressed as fractions of the total radiolabel initially added to each sample. These experimental results are demonstrated in Figure 5, SF539 is Taxol

It was suggested that paclitaxel formulated in LN (ie EmPAC) significantly more endocytosis.

양쪽 모두에 대하여 관찰되었는데, 이는 이렇게 발생한 차이는 도 6에 도시된 바와 같이, Cremophor EL

제형에서의 파클리탁셀의 상대적인 비용해성으로 기인한 것이 아니였다는 것을 시사하는 것이다. 그러나, Taxol

보다는 이러한 세포들과 연관된 EmPAC에서 파클리탁셀이 현전히 더 많은 양이 있었다.This experiment was essentially repeated as previously described, except that cells were harvested at various times after being treated with a second formulation of EmPAC and incubated in the drug. An essentially equal amount of radiolabeled drug for each formulation was added to each cell sample. Raw coefficients of the radiolabelled drug associated with the cell monolayer are shown. In addition, paclitaxel uptake was analyzed as a function of time of drug incubation to assess the kinetics of cell uptake. Previous studies with SF539 glioma cells demonstrated that uptake increased over a period of 2 hours before reaching the plateau. Also, this is EmPAC and Taxol

Observations were made for both, and this difference occurred with Cremophor EL, as shown in FIG. 6.

It does not indicate that it was due to the relative insolubility of paclitaxel in the formulation. However, Taxol

Rather, there was a significant amount of paclitaxel in EmPAC associated with these cells.

Experiments were conducted in which the total unlabeled paclitaxel changed but the lipid and radiolabeled paclitaxel amount remained constant to determine whether paclitaxel or lipid mixtures in EmPAC could saturate cell uptake. As shown in FIG. 7, paclitaxel amounts are not entirely apparent from this particular experiment, but appear to saturate with respect to cell uptake. Since labeled paclitaxel is constant, while total paclitaxel changes, only small or large portions of total paclitaxel can be expressed. Therefore, even if the same number of drug-loaded particles are ingested, smaller amounts of labeled paclitaxel may be ingested.

As a result, a series of experiments were conducted to determine the contribution of each lipid component of LN to cell uptake. LN samples labeled with trace substitutions of ¹⁴ C-labeled cholesterol were prepared using 300 μg / mL lipid and 20% DMSO. All other ingredients increased proportionally to compensate for the missing ingredients. This sample was added to SF539 human glioma cells for 2 hours. In addition, the same experiment was conducted to prepare LN particles using single components mixed 1: 1 with cholesterol. 8A and 8B together confirm that cholesterol plays the most important role among all lipid components found in LN in promoting cell uptake.

에서 제형된 파클리탁셀로 2 시간 동안 처리되었다. 세포들은 이후 얼음-냉각 메탄올로 고정되고, 항-탁산 항체(Hawaii Biotech, 하와이 주 호놀룰루(Honolulu) 소재)로 염색한 다음 형광 Alexa Fluor 488 분자(Molecular Probes, 오레곤 주 유진 소재)에 접합된 제 2 항체로 처리하였다. 도 9A로부터 파클리탁셀이 세포내이입되었으며 미세소관으로 국소화된다는 것이 명백하다. 파클리탁셀은 생체내 미세소관과 결합하는 것으로 알려져 있기 때문에, 이는 Cremophor EL

에서 뿐만 아니라 EmPAC에서 제형된 파클리탁셀은 세포내이입된다는 것을 시사한다. 그러나, 각각의 표본에 대하여 수많은 세포 분야들의 세포내 형광 세기도 각 세포의 모서리들을 추적하고 추적된 경계 내에서 형광 세기를 정량화하여 정량화된다. 도 9B에 관찰된 바와 같이, EmPAC-처리된 세포는 Taxol

로 처리된 세포의 형광 세기의 대략 2 배의 형광 세기를 가져서, 이로 인하여 더 빨리 발견되었음을 알게 되었다.Although radiolabeled paclitaxel associated with cell monolayers was the result of endocytosis of paclitaxel, there was a formal possibility that the paclitaxel was located outside of the cell rather than endocytosis. To determine whether paclitaxel is endocytotic, A549 human lung tumor cells were treated with EmPAC or Cremophor EL.

Was treated for 2 hours with paclitaxel formulated in. Cells were then fixed with ice-cold methanol, stained with anti-taxane antibody (Hawaii Biotech, Honolulu, Hawaii) and then conjugated to fluorescent Alexa Fluor 488 molecules (Molecular Probes, Eugene, Oregon). Treated with antibody. It is clear from FIG. 9A that paclitaxel is endocytosis and localized to microtubules. Since paclitaxel is known to bind to microtubules in vivo, it is Cremophor EL

As well as in, it suggests that paclitaxel formulated in EmPAC is endocytosis. However, the intracellular fluorescence intensity of numerous cell fields for each sample is also quantified by tracking the edges of each cell and quantifying the fluorescence intensity within the traced boundary. As observed in FIG. 9B, EmPAC-treated cells were treated with Taxol

It was found that it had a fluorescence intensity of approximately twice the fluorescence intensity of the cells treated with, thereby discovering more quickly.

Example 3

EmPAC Cytotoxicity

중 어느 하나와 비교되었다.The purpose of this experimental set was to determine the relative tumor growth inhibition and cytotoxicity of EmPAC. EmPAC is paclitaxel or Taxol dissolved in DMSO as recommended by the manufacturer.

It was compared with either.

To determine whether paclitaxel mixed with LN retained the ability to kill cells, the cytotoxic effect of paclitaxel alone dissolved in DMSO stock solution was compared with equimolar amounts of EmPAC in numerous other cell lines. Relative cytotoxicity was assessed using the MTS cell proliferation assay. MTS is a tetrazolium compound bioreduced by lysate cells into soluble formazan product. The absorbance at 490 nm of the formazan product can be used to determine the relative number of synovial cells. Thus, the MTS can be used to assess the relative cytotoxic titers of EmPAC versus the relative cytotoxic titers of paclitaxel alone. The cell lines tested were subjected to uterine sarcoma cell line MES-SA and its drug-resistant sub-line MES-SA-DX5 to determine whether mixing of paclitaxel to LN affects the cytotoxicity of paclitaxel in drug resistant cells. It included. Thus, MES-SA, MES-SA-DX5, A549 and MX-2 cell lines were plated at subconfluent density. Equal molar concentrations of paclitaxel alone and EmPAC, diluted in tissue culture medium, were added to the cells the day after the cells were plated and incubated with the cells for 72 hours. The medium was replaced and the MTS test was performed according to the manufacturer's instructions (Madison, Wisconsin). Percent viability was calculated based on normalization from each cell line to untreated cells. Each data point in FIGS. 11A-11D represents the mean ± standard error of six samples.

As observed in FIG. 10A, EmPAC showed approximately weaker cytotoxicity compared to paclitaxel dissolved in DMSO in lower paclitaxel concentrations of MES-SA and A549 cells, but was worse than paclitaxel alone as observed in FIG. 10C. It was shown to kill more cell fractions at higher concentrations. This is because paclitaxel alone is extremely insoluble in aqueous media; It may be due to the fact that its hydrophobic nature causes paclitaxel to aggregate at higher concentrations, thereby excluding entry into the cells. Due to the mixing of paclitaxel to LN, paclitaxel is stable in aqueous medium, preventing aggregation and allowing higher concentrations of paclitaxel to enter the cells. In addition, EmPAC showed significantly higher cytotoxicity than paclitaxel alone in MX-1 breast cancer cells, as evident in FIG. 10D, and as shown in FIG. Cytotoxicity was shown. The mechanisms underlying this observation are unknown, but one hypothesis is that in the case of drug resistant cell line MES-SA DX-5, EmPAC is buried in lipids and at least initially treated with one component of LN, which is as efficient as paclitaxel alone. Is not pumped out of the cells. Lipid particles are taken into cells by specific mechanisms that may not be affected by the cellular mechanisms underlying drug resistance.

과 비교하였다. 본 실험에 사용된 EmPAC 제형은 Tween

-80 및 초음파분쇄에 의한 대두유의 존재하에 독점적인 지질 혼합물을 가용화시키고, 이후 이 혼합물을 부탄올에서 초음파분쇄로 가용화된 파클리탁셀로 첨가하여 제조되었다. 상기 혼합물은 110EH Microfluidics Microfluidizer

고압 균질화기(모델 M-110Y, Microfluidics 사, 매사추세츠주 뉴턴 소재) 상에서 미세유동화에 의하여 처리되었다. 이 파클리탁셀 제형은 약물 제거 전 한 시간 동안 SF539 신경교종 세포로 노출된 이후에 비교되었는데, 이는 파클리탁셀은 1 시간 미만의 생체이용가능한 생체내 반감기를 가지므로, 약물로의 단기 노출은 약물로의 연속적인 노출보다는 항-종양 약물로의 생체 종양 세포 노출을 모방하기 때문이다(Wiernik et al., 1987; Rowinsky et al., 1990).The above mentioned experiments were modified to convert EmPAC to Cremophor EL.

Compared with. EmPAC formulation used in this experiment was Tween

Proprietary lipid mixtures were solubilized in the presence of -80 and soybean oil by sonication and then added to paclitaxel solubilized by sonication in butanol. The mixture is 110EH Microfluidics Microfluidizer

Treatment was by microfluidization on a high pressure homogenizer (Model M-110Y, Microfluidics, Newton, Mass.). This paclitaxel formulation was compared after exposure to SF539 glioma cells for one hour before drug removal, since paclitaxel has a bioavailable half-life of less than one hour, so short-term exposure to the drug is continuous. Rather than exposure, mimicking live tumor cell exposure with anti-tumor drugs (Wiernik et al. al ., 1987; Rowinsky et al ., 1990).

사이의 통계학적 유의성은 학생 t-시험에 의하여 각각의 약물 농도에 대해 평가되었다. 도 11에 도시된 바와 같이, EmPAC 단독으로는 세포 사멸 및 세포 생존에 미치는 현저한 효과를 가지지 않는 반면에(자료 미도시), EmPAC는 Taxol

보다 신경교종 세포들에 현저히 더 세포독성이 있었다.After drug removal, cells were then allowed to proliferate for 72 hours further before survival and proliferation was examined by MTT. The MTT assay measures cell viability using mitochondrial dehydrogenase present in the active cells because the active cells are expected to have higher dehydrogenase activity than the killed or killed cells and thus have higher MTT activity levels. . Survival of each cell sample was calculated as the fraction for each control cell line not exposed to the drug. Percent survival is plotted as a function of drug concentration, with each curve fitted on a four-parameter logistic curve and EC ₅₀ values calculated. EmPAC vs Cremophor EL

Statistical significance between was assessed for each drug concentration by student t-test. As shown in FIG. 11, EmPAC alone does not have a significant effect on cell death and cell survival (data not shown), while EmPAC is a Taxol

It was significantly more cytotoxic to glioma cells.

Example  5

In the mouse tumor model EmPAC Tumor growth inhibition

로 처리되는 경우, Taxol

에서 균등한 용량의 파클리탁셀과 비교하여 EmPAC에 의한 종양 성장 억제가 현저히 더 많았다는 것이 관찰되었다.Nude mice implanted subcutaneously in H23 human lung tumors were treated with EmPAC or an equivalent dose of Taxol.

Taxol, if treated with

It was observed that the tumor growth inhibition by EmPAC was significantly higher compared to the equivalent dose of paclitaxel at.

Nude mice were implanted subcutaneously with H23 lung tumor cells allowed to grow for 25 days prior to drug administration. Animals with tumors received 2 mL of 60 μg / mL drug intraperitoneally at 25, 27, 29, 32, 34, 36, 39, 42 days respectively. Tumor volume was measured on each day of this injection. Final tumor volume measurements were 45 days after tumor transplantation. The compositions of the injected drug formulations are shown in Table 8.

Animal Experiments on H23 TGI group The number of animals Drug composition One 4 EmPAC
60 μg / ml paclitaxel
0.06% benzyl alcohol
0.5% soybean oil 2 4

60 ㎍/㎖ 파클리탁셀
50% Cremophor EL
50% 에탄올
5% 덱스트로스 Taxol

60 μg / ml paclitaxel
50% Cremophor EL
50% ethanol
5% dextrose 3 3 Carrier
0.06% benzyl alcohol
0.55 soybean oil 4 3 No medication ( Naive )

로 처리된 동물들은 나머지 실험을 수행하는 동안 정상(steady) 성장의 코스를 재개하기 전 최초 11 일 동안 종양 부피에서의 퇴보를 보였다. LN 대조로 처리된 동물 또는 미처리 동물의 종양은 연구 전체를 통해 정상 성장을 보였다. 따라서, EmPAC는 Taxol

이 한 것보다 더 오랜 시간 동안 종양 크기를 줄일 수 있었다. 도 12A의 자료는 약물 처리 코스에 걸쳐서 평균 종양 부피를 나타내도록 플롯팅되었다.As shown in FIG. 12A, over 20 days after the first injection of the drug, tumors in animals treated with EmPAC decreased in volume over the entire time period, while Taxol

Animals treated with showed a degeneration in tumor volume for the first 11 days before resuming the course of steady growth during the remaining experiments. Tumors from animals treated with LN controls or untreated animals showed normal growth throughout the study. Thus, EmPAC Taxol

Tumor size could be reduced for longer than this one. The data in FIG. 12A was plotted to show mean tumor volume over the course of drug treatment.

로 처리된 마우스들과 EmPAC로 처리된 마우스들 사이에 보여진다(p< 0.0005). 각 군은 평균 3 ± 표준오차를 나타낸다. 연구가 종결할 때에, EmPAC로 처리된 동물들의 종양은 약 71%(71.4 ± 2.4%) 만큼 퇴화된 반면에, Taxol

로 처리된 동물들의 종양은 약 19%(18.7 ± 0.9%) 만큼 퇴화되었다. 그러므로, 상기 자료는 EmPAC가 Taxol

보다는 현저히 더 항종양 활성을 갖는다는 것을 시사한다.In FIG. 12B, percent tumor degeneration was measured by comparing tumor volume differences between the first and last days of drug treatment. The statistically significant difference in tumor regression was Taxol

Between mice treated with and mice treated with EmPAC (p <0.0005). Each group represents an average of 3 ± standard error. At the conclusion of the study, tumors in animals treated with EmPAC were degraded by about 71% (71.4 ± 2.4%), while Taxol

Tumors of animals treated with were degraded by about 19% (18.7 ± 0.9%). Therefore, the data indicate that EmPAC is a Taxol

Rather than antitumor activity.

은 이전 TGI 실험들에서 주어진 용량보다 1 배 내지 4 배 더 투여되었다. 결과에 의하면 EmPAC는 대략 Taxol

의 4 배 정도의 효능이 있다.To compare tumor growth inhibition by EmPAC with different concentrations of paclitaxel, nude mice transplanted with H460 human lung tumor cells were injected with two times more EmPAC formulated with paclitaxel than in the third formulation of Example 1. In addition, Taxol

Was administered 1 to 4 times more than the dose given in previous TGI experiments. Results show that EmPAC is approximately Taxol

It is four times as effective.

이 H460 종양을 지닌 마우스들에 투여되었다. 마우스 종양은 이전 실험들에서보다 더 큰 크기로 자라도록 하였다. 동물들은 매주 3회 약물을 투여받았는데, 3 주 동안 2 일은 투여하지 않았으며, 종양이 컷오프(cutoff) 크기 한계 이상으로 도달한 동물은 자동으로 안락사시키도록 약물의 주사 이전에 종양이 측정되었다. 시간에 걸쳐 상대 종양 부피가 계산되었으며 시간의 함수로 플롯팅 되었다. T-테스트를 수행하여 다른 약물로 처리된 동물들 사이의 종양 성장 억제에 있어서 현저한 차이가 있는지의 여부를 결정하였다. 또한, 생존 곡선은 종말점이 컷오프 크기 한계에 도달한 종양으로부터 치료-관련 원인에 의하거나 안락사에 의한 동물 사망으로 정의되어 있는 곳에서 생성되었다.In this experiment, paclitaxel equivalent to EmPAC formulations of 60 and 137 μg / mL paclitaxel was used. In addition, Taxol is about four times the dose given in the first third EmPAC formulation (ie 60 μg / mL paclitaxel).

Mice with this H460 tumor were administered. Mouse tumors were allowed to grow to larger sizes than in previous experiments. Animals received the drug three times weekly, two days in three weeks, and tumors were measured prior to injection of the drug to automatically euthanize the animals whose tumors reached above the cutoff size limit. Relative tumor volume was calculated over time and plotted as a function of time. T-tests were performed to determine whether there was a significant difference in tumor growth inhibition between animals treated with different drugs. In addition, survival curves were generated where the endpoints were defined as animal death by treatment-related causes or euthanasia from tumors whose cutoff size limit was reached.

의 약 4 배 농도만큼 효능이 있다는 것을 시사한다.Unfortunately, the animals were not randomized, so control animals started with minimal tumors, while animals treated with higher doses of drug started with larger tumors before treatment. Nevertheless, the results indicate that EmPAC is a Taxol

It is as effective as about 4 times the concentration.

Example  6

EmPAC Pharmacokinetics  And biodistribution

내의 파클리탁셀을 중 어느 하나를 복강내 또는 정맥내로 주사하였는데, 각각은 방사능라벨링된 파클리탁셀을 함유한다.To determine the difference in EmPAC pharmacokinetics and in vivo distribution of EmPAC for intraperitoneal and intravenous, EmPAC or Taxol in nude mice transplanted with A549 lung tumors

Either of the paclitaxel injected either intraperitoneally or intravenously, each containing radiolabeled paclitaxel.

EmPAC formulations containing 14C-labeled paclitaxel were prepared as follows:

A) Buffer Preparation

1) A 1 mM sodium pyrophosphate solution was prepared by adding 466.1 mg of sodium pyrophosphate to 1 L of water.

2) Adjust the pH to 4.0 by adding diluted phosphoric acid.

3) Filter through a 0.22 μm PES filter.

-80을 10 mL의 이 완충액에 첨가한다.4) 10 μl 10% Tween

Add -80 to 10 mL of this buffer.

B) lipid stock solution.

1) Measure 10 mg lipid mixture powder and dissolve in 10 mL DMSO to prepare a 1 mg / mL lipid / DMSO solution.

2) Ultrasonic grinding until a clear solution is obtained.

C) high temperature paclitaxel:

1) Take 50 μl of hot paclitaxel.

2) Ethyl acetate is completely evaporated in an 80 ° C water bath.

D) Formulation: 800 μl of lipid / DMSO solution is added to the hot paclitaxel solution. Mix thoroughly and vortex Take this solution and add it to 3.2 mL of aqueous buffer.

에서의 파클리탁셀 사이의 유사한 약물동력학을 시사한다. 조직들이 주사 후 0.25, 0.75, 3, 6 또는 12 시간에 수거되는 유사한 실험들은 EmPAC 및 Taxol

은 본질적으로 동일한 약물동력학 프로파일을 가지며, 조직 파클리탁셀 레벨이 주사 후 약 3 시간에 최대에 도달한다. 또한, 상기 EmPAC 제형을 실시예 1에 기술된 제 3 제형으로 변경한 경우에, 결과는 다시 EmPAC 제형 #3은 Taxol

의 것과 동일한 약물동력학 프로파일을 갖는다는 것을 시사한다.Animals were euthanized at 0 (immediately after injection), 0.5, 1, 6 or 12 hours after injection. Blood was collected by cardiac puncture and from selected organs (e.g. whole brain, kidney, lung, liver, stomach, spleen, pancreas, tumor, muscle on the contralateral side of tumor, urine, feces, whole blood and tumor muscle under tumor). Tissue was collected. All tissues and blood were processed for counting radiolabeled drugs. All tissues were weighed and homogenized. Since the tissues were so small that they could not be accurately weighed by the scale used (the scale reads to one decimal place), the tissue homogeneous suspension containing the used buffer was weighed. Aliquots of the homogeneous suspension were weighed prior to scintillation measurement to determine the fraction of the total homogeneous suspension. Total ^14- C-labeled paclitaxel in total organ tissue was measured. The results suggest that there is no significant difference in pharmacokinetics between drugs injected intraperitoneally versus intravenous. These results also indicate paclitaxel vs Taxol in EmPAC.

Similar pharmacokinetics between paclitaxel in Similar experiments in which tissues were harvested at 0.25, 0.75, 3, 6 or 12 hours after injection were performed using EmPAC and Taxol.

Has essentially the same pharmacokinetic profile, and tissue paclitaxel levels reach a maximum about 3 hours after injection. In addition, if the EmPAC formulation was changed to the third formulation described in Example 1, the result was again that EmPAC formulation # 3 was Taxol

It has the same pharmacokinetic profile as that of.

Example 7

TAXOL®  And ABRAXANE ® to Formulated Paclitaxel  Compared

EmPAC in Formulated Paclitax Cell uptake of cells

Taxol® (paclitaxel dissolved in Cremophor EL®: ethanol 1: 1) has been used in clinical practice for many years to treat various cancer signs, including non-small cell lung cancer and breast cancer. Abraxane®, a formulation of paclitaxel formulated with HSA, is FDA approved for cancer treatment. The purpose of this study was to compare EmPAC to Taxol® and Abraxane® in the short-term exposure experiments with A549 human lung cancer and MDA MB 435 breast cancer cell lines exposed to each paclitaxel formulation for one hour. Was in determining whether or not. The cells were then stained with antibodies to paclitaxel and subsequently fluorescently-labeled secondary antibodies. Intracellular paclitaxel, which can be detected by intracellular fluorescence staining, was visualized by epifluorescence microscopy. Digital images of fluorescently labeled cells were captured with a cooled CCD camera. The fluorescence intensity of labeled cells was quantified using imaging software, and the average intracellular fluorescence intensity for cells in each experimental group was compared.

Substances and Methods

Cell line

A549 human non-small cell lung tumor cells purchased from the US Microbial Conservation Center (ATCC) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FCS) and 1: 3 to 1: Subcultured at 8 rates.

MDA MB 435 human breast cancer cells purchased from ATCC were cultured in Roswell Park Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FCS) and passaged at a ratio of 1: 3 to 1: 8.

Test item

(로트 #729537)은 오하이오 주 베드포드(Bedford) 소재 Ben Venue Laboratories 사로부터 구하였다.Taxol

(Lot # 729537) was obtained from Ben Venue Laboratories, Bedford, Ohio.

80 및 123.23 ㎎ 지질 혼합물 분말을 함유하는 초음파분쇄된 혼합물; 및 300 mL HPLC 급 물을 조합하고 1250 bar에서 고압 균질화에 의하여 에멀젼화하여 제조되었다. 이후 10 g의 덱스트로스가 이 용액 200 mL에 첨가되고, 초음파분쇄되어 용해되었으며 0.22 μM 필터를 통해 살균 여과되었다. 이로 인하여 생성된 생성물은 2.0% 대두유, 2.2% Tween

80, 400 ㎍/mL 지질 혼합물 분말, 0.1% 벤질 알코올 및 300 ㎍/㎖ 파클리탁셀(로트 # 28042/k1)을 함유하였다.EmPAC (batch # T343) was prepared using 90.42 mg paclitaxel dissolved by sonication in 0.3 mL benzyl alcohol; 5.6 g soybean oil, 6.61 g Tween

Sonicated mixture containing 80 and 123.23 mg lipid mixture powder; And 300 mL HPLC grade water were combined and emulsified by high pressure homogenization at 1250 bar. 10 g of dextrose was then added to 200 mL of this solution, sonicated to dissolve and sterile filtered through a 0.22 μM filter. The resulting product was 2.0% soybean oil, 2.2% Tween

80, 400 μg / mL lipid mixture powder, 0.1% benzyl alcohol and 300 μg / mL paclitaxel (lot # 28042 / k1).

(로트 #200495, 2007 년 4 월 만료; Abraxis BioScience 사)는 파클리탁셀 100 ㎎ 및 HSA 900 ㎎을 함유하는 분말로 공급되었다. Abraxane

분말은 이후 20 mL의 살균 PBS를 첨가하여 재구성되어 5 ㎎/mL 파클리탁셀 및 45 ㎎/mL HSA를 함유하는 현탁액을 생성하였다. 재구성된 Abraxane

은 안정성의 손실을 회피하기 위하여 패키지 지시사항에 따라 24 시간 이내에 실험용으로 활용되었다.Abraxane for injectable suspensions purchased from GlobalRx (Efland, North Carolina)

(Lot # 200495, expired April 2007; Abraxis BioScience) was supplied as a powder containing 100 mg of paclitaxel and 900 mg of HSA. Abraxane

The powder was then reconstituted by adding 20 mL of sterile PBS to yield a suspension containing 5 mg / mL paclitaxel and 45 mg / mL HSA. Reconstructed Abraxane

Was used for testing within 24 hours according to package instructions to avoid loss of stability.

reagent

1) Milli Q water

2) Benzyl Alcohol, Sigma, Lot # 02748PC

3) Soybean oil, Sigma, Lot # 074k0169

80, 시그마 울트라(Ultra), 로트#073K006414) Tween

80, Sigma Ultra, Lot # 073K00641

5) Paclitaxel (R & D), Indena, Lot # 28042 / k1

6) Dextrose (Anhydrous Powder), Fisher, Lot # 024291

7) Emulsion Tablet Lipid Mixture # 003/05

Antibodies

1) mouse anti-paclitaxel IgG. Catalog number. TA12; Hawaii Biotechnology, Aiea, Hawaii

2) Alexa Fluor 488-conjugated chicken anti-mouse IgG. Molecular Probes, Eugene, Oregon.

Other antibodies to stain substances

1) Nunc Lab Tek Cell Chamber Slide System. Nalge Nunc International Inc., Rochester, NY.

2) Tris buffered saline (TBS). Boston Bioproducts. Based in Worcester, Massachusetts.

Study design and procedure

The experiment was designed so that the investigator did not know about the identity of each test item. The test articles were placed in new test tubes and relabeled by a person not directly involved in the experiment; The identity of the test items was revealed after the results were calculated.

To cells grown to greater than 50% confluency, the medium was removed and the cell monolayer was briefly washed by adding 5 mL of PBS and then aspirating. 2 mL trypsin ethylenediaminetetraacetic acid (EDTA) was added to each flask, which was placed for 5 minutes in a tissue culture incubator. 10 mL of complete medium was added to stop the enzymatic reaction, and cells were re-suspended in a disposable pipette to release clumps. 10 μl of cells were added to 10 μl of 0.4% trypan blue solution and mixed, about 10-20 μl of this cell solution was placed in the chamber of the hemocytometer. Viable cell numbers were measured by observing and counting cell numbers excluding trypan blue in a square of the hemocytometer chamber at a total of 100 magnifications.

The volume of cells required was determined by the formula:

Volume of cells = (A x 10 ⁴  cell/ mL ) (Total volume of cells ml) ÷ (2 df (B cell / mL )

Where: df is the dilution factor; A is the number of cells measured on the hemocytometer; B is cell / mL, which is the concentration required for the experiment.

A549 lung tumors and MDA MB 435 breast cancer cells were spread on 8-well chamber slides at a concentration of 4 × 10 ⁴ cells / cm 2. Cells were incubated for 24 hours overnight before test items were added.

Test conditions containing 2.5 μM paclitaxel and control complete medium without test items were added to duplicate chamber slide wells and used to culture mammalian cells (5% CO 2 .95% O 2; 37 ° C.). Incubated for 1 hour. At the end of the incubation period, the cell samples were washed once with warm PBS plus 1 mL PBS, after which the warm PBS was aspirated. 3 ml of ice cooled methanol was added to each tissue culture well, and cells were fixed at about 20 ° C. for 15 minutes. Slides were placed in PBS containing 0.02% sodium azide until stained.

Cell samples were blocked for 1 hour with 10% normal chicken serum (NCS) in TBS, pH 8.0. After removal of the blocking solution, cells were incubated for 1 hour at room temperature in TBS containing 1: 100 diluted 2% NCS and mouse anti-paclitaxel. At the end of the primary antibody incubation, the cell samples were simply rinsed with TBS in the wash bottle and the entire chamber slide was immersed in Coplin staining containing TBS. Washing of the chamber slides was performed by stirring TBS for 10 minutes using a stir bar on a stir plate. Chamber slides were washed with three changes of TBS. Cells were stained for 30 minutes at room temperature with TBS containing 1: 100 diluted 2% NCS and chicken anti-mouse IgG conjugated to Alex Fluor 488. At the end of the secondary antibody culture, cells were simply rinsed with TBS in the wash bottle and washed as described above. Stained cells contain 14.3 μM 4 ', 6-diamidino-2-phenylindole, dihydrochloride (4', 6-diamidino-2-phenylindole, dihydrochloride: DAPI) and covered with Fluoromount G loading medium covered with coverslip (Southern Biotechnology Co., Ltd.) cells.

Slides and cells were observed with a fluorescence microscope excited at 480 nm and emitting at 520 nm to visualize Alexa Fluor secondary staining of anti-paclitaxel. DAPI chromosome staining was visualized using filters excited at 358 nm and emitting at 461 nm. Images were obtained using a Zeiss Axiocam HR digital camera and Axiovision image acquisition and analysis software. Images of paclitaxel staining and DAPI staining were captured simultaneously for each cell field using a Zeiss Axiocam HR digital camera. Intracellular fluorescence intensity per cell area of paclitaxel staining was measured using the Axiovision software. Images of 8 to 11 non-duplicate fields of cells, each containing 4 to 19 cells, were randomly taken from both duplicate wells in each sample. In all of these, a total of 123-147 cells were analyzed for each cell sample. Cell circumference was defined manually for each cell by digitally tracing the cell edges with the mouse cursor.

Calculation and statistical analysis

Data of fluorescence intensity and cell area were copied to an Excel spreadsheet, and the average fluorescence intensity per area for individual cells was calculated in Excel.

To calculate the mean cell fluorescence intensity, the following formula was used:

[( I _One / A _One ) + ( I ₂ / A ₂ ) + ( I ₃ / A ₃ ) + ... ( I _n Of A _n )] / n = average fluorescence intensity per cell area

Where I is the intensity of each cell area defined by manually tracing the cell circumference; A is the area of each defined cell area; And n is the number of cells sampled.

Average intracellular paclitaxel levels, defined as the mean fluorescence intensity per area for each treated sample, were compared between cells treated with different test articles.

Statistically significant differences in mean fluorescence intensity values between EmPAC-, Taxol®- and Abraxane®-treated cells were compared using the student t-test.

result

Average fluorescence intensity of 108-147 A549 cells treated with EmPAC, Taxol® or Abraxane®, respectively, was measured. The average fluorescence intensity per cell for each test article treated A549 cell sample is shown in Table 9. Cells not treated with the test article showed no specific staining (data not shown). Therefore, the fluorescence intensity of untreated cells was not measured. In addition, the fluorescence intensity of the MDA-MB-435 cells was rounded in shape when treated with paclitaxel, which was not measured because it made it difficult to visualize the plasma and the periphery of the cells.

Intracellular Fluorescent Paclitaxel in A549 Cells Exposed to Various Paclitaxel Formulations for 1 Hour                                       EmPAC Abraxane® Taxol® Relative intracellular fluorescence paclitaxel (mean density / area ± standard error) 116.73 ± 2.03 86.32 ± 1.54 111.97 ± 1.78 N = 106 N = 108 N = 147

§ Measured from 109-147 or more cells in 10-12 fields for each group.

Results of student t-tests comparing intracellular fluorescence paclitaxel intensity per cell area of paclitaxel formulations in A549 cells

EmPAC vs. Abraxane

Taxol

Vs Abraxane

EmPAC vs Taxol

P <0.00001 <0.00001 0.08

As shown in FIG. 13, all cells treated with EmPAC, Taxol® or Abraxane® showed fluorescence staining with a perinuclear fibrous pattern typical for microtubules. This suggests that anti-paclitaxel staining is specific because paclitaxel binds thermally with microtubules and is localized primarily on microtubules in cells.

Student t-test indicated that cells treated with either EmPAC or Taxol® had significantly higher mean intracellular paclitaxel levels compared to the levels of Abraxane® as indicated by mean fluorescence intensity per cell area value (Table 9 And 10; P <0.0001 for a comparison between EmPAC and Abraxane® and between Taxol® and Abraxane®). EmPAC also found that it had slightly higher intracellular paclitaxel levels than Taxol® (Table 9, 116.7 ± 2.0 and 112 ± 1.8, respectively) even though the difference was less than statistical significance (P = 0.08).

Discussion / Conclusion

The results of this study suggest that EmPAC and Taxol® formulations take up paclitaxel cells more significantly than Abraxane®. These results appear to be in agreement with the results of previous studies of growth inhibition by these paclitaxel formulations for cancer cell and tissue treatment, where EmPAC and Taxol® are larger than Abraxane® when exposed to cells for 1 hour shortly. Suggests cell growth inhibitory activity. Since these three formulations have the same active ingredient, one possible explanation for the differences between these three formulations can be explained by the carrier. In this case, the carrier of Abranxane® is capable of inhibiting cell uptake of paclitaxel as compared to the carriers of EmPAC and Taxol®.

This study is consistent with the results of Example 2, where in Example 2 we compared the cellular uptake of paclitaxel labeled with radioisotopes formulated in EmPAC as formulated in Taxol®. However, the results of Example 2 showed that uptake of paclitaxel of EmPAC-treated A549 cells was significantly higher than paclitaxel uptake of Taxol®-treated cells. Although this study observed more uptake of paclitaxel in EmPAC-treated A549 cells compared to paclitaxel uptake of Taxol®-treated cells, this difference was smaller than statistically significant. This difference between the results may be due to the fact that the EmPAC formulation was in both studies.

references

Ibrahim NK, Desai N, Legha S, Soon-Shiong P, Theriault RL, Rivera E, Esmaeli B, Ring SE, Bedikian A, Hortobagyi GN, and Ellerhorst JA. 2002. Phase I and pharmacokinetics study of ABI-007, a Cremophor-free, protein-stabilized, nanoparticle formulation of paclitaxel. Clin. Cancer Res . 8: 1038 1044.

Singla AK, Garg A, and Aggarwal D. 2002. Paclitaxel and its formulations. Int. J. Pharm. 235: 179-192.

It will be appreciated that the present invention relates to a delivery system in the form of a composition for delivering a therapeutic agent and a diagnostic agent comprising an anticancer agent for treating cancer cells or cancer tissue. Thus, all diseased tissues and cells that exhibit abnormal lipid metabolism and increased lipid uptake, including all anticancer agents, as well as cancer cells that can be treated with such therapeutics, are within the scope of the present invention.

The foregoing discussion discloses and describes only exemplary embodiments of the invention. Those skilled in the art will readily appreciate from the above discussion and the appended claims that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims. Also, while exemplary embodiments are represented herein, other skilled in the art may recognize other designs or uses of the present invention. Thus, while the invention has been described in connection with exemplary embodiments thereof, it will be apparent to those skilled in the art that many modifications may be made in both design and use, and the application may be modified or modified in any manner It is intended to include change. Therefore, it is manifestly intended that this invention be limited only by the foregoing claims and equivalents thereof.

Claims (69)

In pharmaceutical compositions useful for the treatment or prevention of diseases, conditions or syndromes or symptoms thereof characterized by hyperplasia in warm-blooded animals, including humans, the pharmaceutical composition comprises:
Lipid nanoemulsions composed of lipid particles each comprising at least one non-bilayer-forming lipid that can be preferentially and selectively introduced into a diseased cell;
An effective amount of at least one therapeutic agent associated with the nanoemulsion;
And a pharmaceutically acceptable carrier,
The pharmaceutical composition is a pharmaceutical composition, characterized in that produced by the homogenized treatment. The method of claim 1,
Wherein said therapeutic agent is a microtubule-interacting agent. The method of claim 2,
The microtubule-interacting agent is taxane, vinca alkaloid, epothilone, eleutherobin, dolastatin, combrestatin, sarcodictin ( sarcodictyin, laulimalide, discodermolide, colchicine, phomopsin A, halichondrin B; Pharmaceutical composition, characterized in that it is selected from the group consisting of spongeistatin 1 and its derivatives, analogs, congeners and combinations thereof. The method of claim 3, wherein
The taxanes are paclitaxel, cephalomannine, docetaxel, docatetaxel, baccatin-III, 10-diacetyl baccatin III, deacetylpaclitaxel, diacetyl -7-Epipaclitaxel and its derivatives, homologues, analogs and combinations thereof. The method of claim 3, wherein
The vinca alkaloid is selected from the group consisting of vincistine, vinblastine, vinorelbine, vindesine and derivatives, homologs, analogs and combinations thereof. Pharmaceutical compositions. The method of claim 1,
The amount of the therapeutic agent is a pharmaceutical composition, characterized in that at least 0.001% by weight based on the total volume of the pharmaceutical composition. The method according to claim 6,
The amount of the therapeutic agent is a pharmaceutical composition, characterized in that present in about 0.1% to 90% by weight based on the total volume of the pharmaceutical composition. The method of claim 1,
Pharmaceutical composition, characterized in that the non-bilayer-forming lipid is only weak polarity. The method of claim 8,
Wherein said non-bilayer-forming lipid is substantially nonpolar or neutral. The method of claim 8,
Pharmaceutical composition, characterized in that the non-bilayer-forming lipid is neutral. The method of claim 1,
Wherein said lipid particles consist of a mixture of non-bilayer forming lipids. The method of claim 1,
Wherein said lipid particles are stably dispersed in the aqueous phase for at least several days. The method of claim 12,
Wherein said lipid particles are stably dispersed in the aqueous phase for at least 14 days. The method of claim 13,
Wherein said lipid particles are stably dispersed in the aqueous phase for at least one month. The method of claim 14,
Wherein said lipid particles are stably dispersed in the aqueous phase for at least 6 months. The method of claim 15,
Wherein the lipid particles are stably dispersed in the aqueous phase for at least one year. The method of claim 1,
Pharmaceutical composition, characterized in that it further comprises an emulsion enhancer. The method of claim 17,
The emulsion enhancer is selected from the group consisting of surfactants, plant-based fat sources, solvents and combinations thereof. The method of claim 18,
The surfactant is a pharmaceutical composition, characterized in that the nonionic surfactant. The method of claim 19,
The nonionic surfactant is a pharmaceutical composition, characterized in that the detergent polysorbate. The method of claim 20,
The detergent polysorbate is Tween

Pharmaceutical composition, characterized in that -80. The method of claim 18,
The nonionic surfactant is a pharmaceutical composition, characterized in that the sorbitan ester. The method of claim 22,
The sorbitan esters are selected from the group consisting of fatty-acylated sorbitan esters and polyoxyethylene derivatives thereof and mixtures thereof. The method of claim 18,
Wherein said surfactant is present in an amount sufficient to prevent said nanoemulsion from being denatured during storage. The method of claim 24,
Wherein said surfactant is present in an amount of about 0.01% w / v to 4.0% w / v. The method of claim 25,
Wherein said surfactant is present in an amount from about 0.1% w / v to 3.0% w / v. The method of claim 18,
The solvent is a pharmaceutical composition, characterized in that the solvent can be mixed with water. The method of claim 27,
The solvent that can be mixed with water is a pharmaceutical composition, characterized in that selected from the group consisting of polar solvents and combinations thereof. 29. The method of claim 28,
Wherein said solvent is selected from the group consisting of polar aprotic solvents, polar protic solvents, and combinations thereof. The method of claim 29,
The polar aprotic solvent is a pharmaceutical composition, characterized in that dimethyl sulfoxide (dimethyl sulfoxide). The method of claim 29,
The polar protic solvent is a pharmaceutical composition, characterized in that the alcohol. The method of claim 31, wherein
Wherein said alcohol is selected from the group consisting of butanol, propanol, ethanol, methanol, benzyl alcohol and combinations thereof. The method of claim 18,
Wherein said solvent is present in an amount sufficient to control the degree of aggregation of said surfactant. The method of claim 33, wherein
Wherein said solvent is present in an amount from about 0.001% v / v to 99.9% v / v. 35. The method of claim 34,
Wherein said solvent is present in an amount from about 0.005% v / v to 80% v / v. The method of claim 18,
The vegetable fat source is a pharmaceutical composition, characterized in that the vegetable oil. The method of claim 36,
The vegetable oil is soybean oil, flaxseed oil, hemp seed oil, linseed oil, linseed oil, mustard oil, rapeseed oil, canola oil, safflower oil, sesame oil, sunflower oil, grape seed oil, almond oil, apricot seed oil, castor oil, Corn oil, cottonseed oil, coconut oil, hazelnut oil, neem oil, olive oil, palm oil, palm kernel oil, peanut oil, pumpkin seed oil, rice bran oil, lake seed oil and combinations thereof Pharmaceutical composition. 39. The method of claim 37,
The vegetable oil is a pharmaceutical composition, characterized in that the soybean oil. The method of claim 18,
Wherein said plant-based fat source is present in an amount that allows for higher surface tension in said nanoemulsion, thereby increasing the probability of hydrophobic interaction with the plasma membrane of the target cell. The method of claim 39,
Wherein said plant-based fat source is present in an amount of about 0.001% v / v to 5.0% v / v. The method of claim 40,
Wherein said plant-based fat source is present in an amount of about 0.005% v / v to 4.0% v / v. The method of claim 1,
The at least one non-bilayer-forming lipid is glycerol monoesters of carboxylic acids, aliphatic alcohols, sterol aromatic acid esters, sterols, terpenes, bile, alkali metal salts of bile acids, sterol esters of aliphatic acids. , Sterol esters of sugar acids, esters of sugar acids, esters of aliphatic alcohols, esters of sugars, esters of aliphatic acids, sugar acids, saponins, sapoge, glycerol, glycerol di-esters of aliphatic acids, aliphatic acids Pharmaceutical composition, characterized in that it is selected from the group consisting of glycerol tri-esters, glycerol diesters of aliphatic alcohols and glycerol triesters of aliphatic alcohols and combinations thereof. The method of claim 1,
The at least one non-bilayer-forming lipid is:
a) at least one first element selected from the group consisting of carboxylic acids containing about 9 to 18 carbon atoms and glycerol monoesters of aliphatic alcohols containing about 10 to 18 carbon atoms;
b) at least one second element selected from the group consisting of sterol aromatic acid esters; And
c) at least one third element selected from the group consisting of sterols, terpenes, bile acids and alkali metal salts of bile acids. The method of claim 43,
Pharmaceutical composition, characterized in that the glycerol monoesters of the carboxylic acids are saturated. The method of claim 43,
Pharmaceutical composition, characterized in that the glycerol monoesters of the carboxylic acids are unsaturated. The method of claim 43,
The lipid mixture has a weight ratio of (a) :( b) :( c) of (1 to 5) :( 0.25 to 3) :( 0.25 to 3) based on the total weight of the lipid particles Pharmaceutical compositions. The method of claim 43,
The lipid mixture is:
d) sterol esters of aliphatic acids containing about 1 to 18 carbon atoms; Sterol esters of sugar acids; Esters of sugar acids and aliphatic alcohols containing about 10 to 18 carbon atoms, esters of sugar acids and aliphatic acids containing about 10 to 18 carbon atoms; Sugar acids and saponins; And at least one fourth element selected from the group consisting of sapongenins; And
e) glycerol; Glycerol di- or tri-esters of aliphatic acids containing about 10 to 18 carbon atoms; And at least one fifth element selected from the group consisting of aliphatic alcohols containing about 10 to 18 carbon atoms. The method of claim 47,
The lipid mixture is based on the total weight of the lipid particles: (a) :( b) of (1-5) :( 0.25-3) :( 0.25-3) :( 0.25-3) :( 0.25-3) A pharmaceutical composition, characterized by having a weight ratio of (c) :( d) :( e). The method of claim 1,
Wherein said lipid particles are present in an amount of about 0.1 μg / mL to 1000 μg / mL. The method of claim 49,
Wherein the lipid particles are present in an amount of about 10 μg / mL to 800 μg / mL. 51. The method of claim 50 wherein
Wherein the lipid particles are present in an amount of about 200 μg / mL to 600 μg / mL. The method of claim 1,
Pharmaceutical composition, characterized in that the median average particle size of the lipid particles is at most 0.2 μ (micron). The method of claim 52, wherein
Pharmaceutical composition, characterized in that the median average particle size of the lipid particles is at most 0.02 μ to 0.2 μ. In a pharmaceutical composition useful for diagnosing a disease, condition, syndrome or symptom in warm-blooded animals, including humans, the pharmaceutical composition comprises:
Lipid nanoemulsions composed of lipid particles each comprising at least one non-bilayer-forming lipid that can be preferentially and selectively introduced into a diseased cell including cancer cells;
An effective amount of at least one therapeutic agent associated with the nanoemulsion;
And a pharmaceutically acceptable carrier,
The pharmaceutical composition is a pharmaceutical composition, characterized in that produced by the homogenized treatment. A method of treating or preventing diseases, conditions and symptoms thereof of a warm blooded animal, including a human,
A method comprising administering to the animal an effective amount of the pharmaceutical composition of claim 1. The method of claim 55,
Wherein said pharmaceutical composition is permeated onto the surface of a biomedical instrument surgically inserted into the body of said animal. The method of claim 56, wherein
And wherein said biomedical device is a stent. The method of preparing the pharmaceutical composition of claim 1, wherein the preparation method is:
a) mixing the at least one non-bilayer-forming lipid with an effective amount of at least one therapeutic agent to produce a lipid fraction;
b) adding the lipid component to the aqueous phase to produce a dispersion; And
c) stirring the dispersion under a high-shear state sufficient to completely disperse the at least one non-bilayer-forming lipid to form a lipid nanoemulsion consisting of lipid particles. The method of claim 58,
Adding an emulsion enhancer to the pharmaceutical composition. The method of claim 59,
And said emulsion enhancer is selected from the group consisting of surfactants, solvents, plant-based fat sources and combinations thereof. The method of claim 60,
Mixing the surfactant with the at least one non-bilayer-forming lipid prior to the step of mixing with the therapeutic agent. The method of claim 60,
Mixing the plant-based fat source with the at least one non-bilayer-forming lipid prior to the step of mixing with the therapeutic agent. The method of claim 60,
And mixing said solvent with said therapeutic agent prior to mixing with said at least one non-bilayer-forming lipid. The method of claim 58,
Said stirring step of treating said lipid mixture by a fluid homogenization process. The method of claim 64, wherein
Said fluid homogenization process is selected from the group consisting of high-shear homogenization, ultrasonic homogenization, high-pressure homogenization, and combinations thereof. The method of claim 58,
Sterilizing the nanoemulsion. The method of claim 58,
And treating the nanoemulsion by aseptic filtration. The method of claim 67 wherein
Wherein said treating step comprises passing said nanoemulsion through a 0.2 μ membrane. In a method of diagnosing a disease, condition, syndrome or symptoms thereof characterized by hyperplasia of cells in warm-blooded animals, including humans,
55. A method comprising administering to said warm blooded animal an effective amount of the pharmaceutical composition of claim 54.

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