Method for producing nano-particles with modified surface having anti-cancer effect

The present invention relates to particulate anti-cancer agents, to pharmaceutical compositions containing such particles, and to the preparation thereof. The present invention makes it possible to use these particles or compositions in the treatment of cancers,

The therapeutic ratio (therapeutic index, "safety index") is the degree of selectivity of an active agent to produce the desired effects, which is defined as the ratio of the average lethal dose to the average effective dose [LD50 / ED5Q; see Goodman and Gilman; The Pharmacological Basis of Therapeutics, 8th Edition, pp. 68-69. pp.]. Essentially, the therapeutic ratio of all anti-cancer agents is low; for example, even less than about 1.0. Increasing the therapeutic ratio, for example by reducing toxicity or increasing efficacy, would provide a wider opportunity for physicians to administer anticancer agents to their patients in need of such treatment. It follows that the development of methods to reduce the toxicity and / or efficacy of anticancer agents, and thus to increase the therapeutic ratio of these agents, can be very important in the treatment of cancer.

In addition, administration of intravenous (iv) bolus injections of water-insoluble drugs, such as water-insoluble anticancer agents, is problematic. Implementation of injectable pharmaceutical forms of sparingly soluble active ingredients

- 3 very serious problems_Λ Thus, it is highly desirable to formulate intravenous bolus injection (bolus injectable) formulations of sparingly soluble drugs, e.g.

It has been found that formulations containing anticancer agents in the form of surface-modified nanometer-sized particles (hereinafter, nanoparticles) have lower toxicity and / or higher potency.

In particular, the present invention enables the formation of substantially crystalline anticancer particles comprising a surface adsorbent in an amount sufficient to maintain an average particle size of less than about 1000 nanometers.

The invention also makes it possible to formulate anti-cancer formulations containing the particles described above.

The invention further provides the use of the particles described above or compositions containing such particles for the preparation of a medicament for the treatment of cancer.

A particularly valuable feature of the present invention is that its use can provide anticancer compositions having reduced toxicity and / or improved efficacy.

Another advantage of the present invention is that it enables the formulation of low soluble anticancer agents which can be administered by intravenous bolus injection and circulate in the blood for a prolonged period after injection. The present invention is based in part on the recognition that

- 4 that surface modified anticancer nanoparticles show reduced toxicity and / or enhanced efficacy. Although the present invention is primarily directed to the most preferred classes of active ingredients, i.e., the most preferred anticancer agents, including immunosuppressive agents, the invention may also be applicable to other substances that are poorly water soluble, particularly to other classes of active pharmaceutical ingredients.

The particles of the present invention consist of an anticancer drug. The particles contain one or more distinct crystalline phases of the anticancer agent. The crystalline phase is different from the amorphous, i.e., non-crystalline phase, which results from solvent precipitation processes used to produce particles of submicron size. (See, for example, U.S. Patent No. 4,826,689.)

The present invention is practicable in a wide variety of anticancer agents; however, the anticancer agent used must be poorly soluble and dispersible in at least one liquid medium. By low solubility is meant that the solubility of the active ingredient in a liquid dispersant such as water is less than about 10 mg / ml, preferably less than 1 mg / ml at processing temperature such as room temperature. A preferred liquid dispersion medium is water; however, the invention may also be practiced with other liquid media in which the anticancer agent is dispersible, such as aqueous saline solutions, safflower oil, and solvents such as ethanol, tert-butanol, hexane and glycol. The pH of the aqueous dispersion media can be adjusted by known methods,

The anticancer agents are preferably selected from the group consisting of alkylating agents, antimetabolites, natural substances, hormones and antagonists, and agents of various classes, such as radiation sensitizing agents.

Exemplary alkylating agents include those of the bis (2-chloroethyl) amine group, such as chloromethine, chlorampbutyl, melphalan, uramastine, nanomustine, mechlorethamine oxide, cyclophosphamide, ifosfamide and triphosphamide; alkylating agents belonging to the group of substituted aziridines are, for example, tretamine, thiotepa, triazziquon and mitomycin; alkylsulfonate alkylating agents include, for example, busulfan, piposulfan and piposulfam; examples of N-alkyl-N-nitrosourea derivatives include carmustine, lomustine, semustine and streptozotocin; other alkylating agents of the mitobronitol, decarbazine and procarbazine type are also contemplated.

Suitable antimetabolite-type agents include: folic acid analogues such as methotrexate; pyrimidine analogs such as fluorouracil, floxuridine, tegafur, cytarabine, idoxuridine and flucytosine; and purine derivatives such as mercaptopurine, thioguanine, azathioprine, thiamiprine, vidarabine, pentostatin and puromycin.

Natural materials include, for example, Vinca alkaloids such as vinblastine and vincristine;

epipodophyllotoxins such as etoposide and teniposide; antibiotics such as adriamycin, daunomycin, doctinomycin, daunorubicin, doxorubicin, mitramycin, bleomycin and mitomycin.

Suitable enzymes are, for example, L-asparaginase.

Examples of biological response modifiers include α-interferon, camptothecin, taxol; and retinoids such as stearic acid.

Hormones and their antagonists include, for example, adrenocytosic steroids such as prednisone; progestins such as hydroxy-progesterone caproate, medroxyprogesterone acetate and megoestrole acetate; estrogens such as diethylstilbestrol and ethinylestradiol; antiestrogens such as tamoxifen; androgens such as testosterone propionate and fluoxymesterone; anti-androgens such as flutamide; and gonadotropin releasing hormone analogues such as leuprolide.

Examples of mixed-group agents that can be used in the invention include radiation-sensitizing agents such as 1,2,4-benzotriazine-3-amine-1,4-dioxide and 1,2,4-benzotriazine-7-amine. l, 4-dioxide; and platinum coordination complexes such as cisplatin and carboplatin; anthracenediones such as mitoxantrone; substituted ureas such as hydroxyurea; and andrenocorticoid inhibitors such as mitotane and aminoglutethimide.

The anticancer agent may also be an immunosuppressive agent such as cyclosporine, azathioprine, sulfasalazine, methoxsalen and thalidomide.

The anticancer agents useful in practicing the present invention are known compounds and / or can be prepared by methods known per se.

The anticancer agent may be used alone or in combination with one or more other anticancer agents.

The particles of the present invention contain, as described above, an anticancer agent which carries on its surface an adsorbed surface modifier. Surfactants which are physically adherent to the surface of the anticancer agent but are not chemically bound to the anticancer agent are believed to be useful.

Suitable surface modifiers are preferably selected from known organic or inorganic pharmaceutically acceptable carriers (excipients). Examples of such carriers are various polymers, low molecular weight oligomers, substances of natural origin, and wetting agents (surfactants). Preferred surfactants are nonionic and anionic wetting agents (surfactants). Important examples of excipients are: gelatin, casein, lecithin (phosphatides), acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl ester, cetomacrogol, cetomacrogol, oxyethylene) alkyl ethers such as macrogol ethers such as cetomacrogol 1000, polyoxyethylene castor oil derivatives.

polyoxyethylene sorbitan fatty acid esters such as commercially available Tween® formulations, polyethylene glycols, polyoxyethylene stearates, colloidal silica, phosphates, sodium dodecyl sulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (in short: PVA) and polyvinylpyrrolidone (PVP for short). For a detailed description of these excipients, see Handbook of Pharmaceutical Excipients, published by the American Pharmaceutical Association, The Pharmaceutical Society of Great Britain and the Pharmaceutical Press (1986). Surface modifiers are commercially available and / or can be prepared by methods known in the art. A combination of two or more surface modifiers may also be used.

Particularly preferred surface modifiers are PVP, tyloxapol, poloxamers such as Pluronic ™ F68, F108 and F127 which are copolymers of ethylene oxide and propylene oxide (available from BASF); furthermore, poloxamines such as Tetronic ™ 908 (T908), which is a tetrafunctional block copolymer formed by sequential addition of ethylene oxide and propylene oxide to ethylenediamine (available from BASF); dextran, lecithin, Aeresol 0T ™ (AOT), a dioctyl sodium sulfosuccinate (available from American Cyanamid); Duponol ™ P, chemically sodium lauryl sulfate (available from DuPont), Triton ™ χ-200, chemically alkylaryl polyether sulfonate (Rohm and Haas); Tween 20, 40, 60 and 80, chemically polyoxyethylene sorbitan fatty acid esters (available from ICI Specialty Chemicals); Span 20, 40, 60 and 80, which are sorbitan esters of fatty acids; Arlacel 20, 40, 60 and 80, which are sorbitan esters of fatty acids (available from Hercules Inc.); Carbowax ™ 3550 and 934 Chemically Polyethylene Glycols (Unión Carbide); Crodasta ™ F-110, a mixture of sucrose stearate and sucrose distearate (Croda Inc.); Crodesta SL-40; hexadecyltrimethylammonium chloride (CTAC); bovine serum albumin; and SA90HCO, chemically^C18^C37^CH2[^CON(^CH3)^CH2(^CHOH)4^CH2^OH12 · We have found that PVP, Pluronic F-108, PVA and acacia are particularly suitable surface modifiers.

The surface modifying agent is adsorbed on the surface of the anticancer agent in an amount sufficient to maintain an effective average particle size of less than about 1000 nm. The surface modifying agent does not chemically react with the anticancer agent; furthermore, the individually (individually) adsorbed molecules of the surface modifying agent are substantially free of intermolecular (intermolecular) crosslinking.

In this specification, particle size is the mean particle size, as measured by conventional particle size determination methods, well known to those of ordinary skill in the art. Suitable methods for determining this size include, for example, sedimentation, free-flow fractionation, photon correlation spectroscopy, or disk-plate centrifugation. An effective average particle size of less than about 1000 nm means that at least 90% of the particles have a mathematically average particle size of less than about 1000 nm, as measured by the above methods. In particularly preferred embodiments of the invention, the effective average particle size is less than about 400 nm; in some embodiments of the invention, the effective average particle size is less than about 300 nm. It is advantageous for the effective average particle size to have at least 95%, more preferably at least 99%, of the particles having a size smaller than the effective average, e.g. 1000 nm. The invention is particularly advantageous when substantially all of the particles have a size of less than 1000 nm.

U.S. Patent No. 4,540,602 to Motoyama et al. Discloses that a solid active ingredient may be pulverized in an aqueous solution of a water-soluble, high molecular weight material, and as a result of such wet comminution, the active ingredient is dispersed into fine particles having a diameter of 0.5 µm. but less than 5 μιη, however, it is not reported that particles having an average size of less than about 1 µm were produced. Reproduction of the grinding procedures described by Motoyama et al. Resulted in particles having an average particle size well in excess of 1 μιπ.

The particles of the present invention may be prepared by a process comprising the following steps. An anticancer agent is dispersed in a liquid dispersant and, by mechanical means, in the presence of a grinding medium, the particle size is reduced to reduce the effective average particle size of the anticancer agent to less than about 1000 nm. The particle size may be reduced in the presence of a surface modifier. Alternatively, the particles may be contacted with the surface modifier after the grinding process.

The following is a detailed description of a general method for preparing the particles of the present invention. The anticancer agent is a compound which is commercially available and / or can be prepared in the usual coarse particle form by methods known in the art. It is preferred, but not essential, that the particle size of the anticancer agent containing the coarse particles, as measured by screening, be less than about 100 µm. If this coarse particle size is greater than about 100 µm, it is preferable to reduce the anti-cancer drug particles to a size less than 100 µιπ by a conventional milling process, such as by spraying or grinding.

The selected coarse-grained anticancer agent may be added to the liquid medium, which is substantially insoluble, to form a premix. The concentration of the anticancer agent in the liquid medium may be in the range of about 0.1 to 60%, preferably 5 to 30% by weight. The presence of a surface modifier in the premix is ​​advantageous but not essential. The concentration of the surfactant may be from about 0.1 to about 90% by weight, preferably from 1 to 75% by weight, more preferably from 2 to 60% by weight, based on the total weight of the active agent and the surface modifier. The premix suspension preferably has an apparent viscosity of less than about 1000 centipoise.

Subsequently, the premix can be subjected directly to mechanical grinding, whereby the average particle size in the dispersion is reduced to less than 1000 nm. If a ball mill is used for grinding, the premix is ​​preferably used directly. However, it is also possible to mix the anticancer agent and optionally the surface modifying agent in a liquid medium, for example in a roll mill or a Coxles mixer, until a homogeneous dispersion is obtained in which larger sets are not visible to the naked eye. Preferably, the premix is ​​subjected to such a pre-dispersing step by using a recirculating medium for abrasive grinding.

A mechanical means for reducing the particle size of the anticancer agent is preferably a dispersion mill. Dispersion mills suitable for this purpose include, for example, ball mill, abrasive mill, vibrator mill, planetary mill, and medium mills such as sand mill and medicinal mill. Among these, it is advantageous to use media mills, since this results in the required grinding in a shorter time, i.e. the desired reduction in particle size. For medium milling, the apparent viscosity of the premix is ​​preferably in the range of about 100 to about 1000 centipoise. These ranges are selected so that the best balance between effective particle milling and media-induced erosion is achieved.

In the step of reducing the particle size, a rigid medium may be selected as the grinding medium, preferably consisting of spherical particles having an average size of less than about 3 mm, more preferably less than 1 mm in diameter. These media desirably allow the particles of the present invention to be reduced with a shorter machining time and less wear on the grinding equipment. In our view, the selection of the quality of the grinding media is not critical; however, zirconium oxide (such as 95% ZrO) in magnesium oxide stabilized form, and zirconium silicate and glass particles such as grinding media, become particles that are considered to be acceptable in the formulation of pharmaceutical compositions. It is expected that other media, such as stainless steel, titanium, alumina, and 95% ZrO stabilized with yttrium will also be used. Preferably, a medium having a density greater than about 2.5 g / ml is used.

Grinding time can vary over a wide range and depends primarily on the particular mechanical equipment and machining conditions. When using ball mills, processing time may be up to 5 days or longer. On the other hand, the application of a high shear grinding medium required less than 1 day processing time (residence time ranging from 1 minute to several hours).

The particle size reduction must be carried out at temperatures that do not cause significant degradation of the anticancer agent. Generally, it is preferred that the machining temperature be lower than about 30-40 ° C. If desired, the processing equipment may be cooled by conventional means. The process is preferably carried out at ambient temperatures at processing pressures that are safe and effective in the milling process. For example, when using a ball mill, a rubbing mill and a shaker mill, we generally work at ambient pressure; medium milling generally involves pressures of up to about 140 kPa.

Surface modifier - if not present in the premix, must be added to the dispersion prior to grinding in the amount described above for the premix. The dispersion can then be mixed, for example, by vigorous shaking. Optionally, the dispersion may also be subjected to an ultrasound treatment step, for example using an ultrasound energy source. For example, the dispersion may be subjected to ultrasound treatment at a frequency of 20 to 80 kHz for about 1 to 120 seconds.

The relative amount of anticancer agent and surfactant may vary over a wide range; the optimal amount of surface modifying agent may depend, for example, on the particular anticancer agent and the surface modifying agent selected; furthermore, from the critical micellar concentration of the surface modifier (if it forms micelles), the anticancer activity

- from a surface area of ​​15 material and similar factors, the surface modifier is preferably 1 m of the surface area of ​​the anticancer agent.²in an amount of about 0.1 mg to about 10 mg. The surface modifier may be present in an amount of from 0.1 to 90% by weight based on the total weight of the dry particles, preferably from 0.5 to 80% by weight, more preferably from 1 to 60% by weight.

A simple screening procedure has been developed to select compatible surfactants and anticancer agents which form stable dispersions containing the desired particles. For this purpose, the coarse particles of an anticancer agent are first dispersed in a liquid, such as water, in which the anticancer agent is substantially insoluble; 2% w / v of the anticancer agent. The resulting mixture is ground in a rolling mill for 120 hours, keeping the following grinding conditions:

Grinding vessel:

Useful volume of grinding vessel:

Volume of medium:

Type of medium:

Grinding time:

Volume of dispersion:

Speed ​​(rpm):

250 ml of glass

250 ml

120 ml

1.0mm Pre-Purified Zirconia Beads (available from Zircoa Inc.)

120 hours ml

92 / min • · * «

- We work at 16 room temperature,

The dispersion (pulp) is separated from the grinding medium by conventional means, for example by pouring the dispersion from the vessel or using a pipette. The separated dispersion is aliquoted and 2-50% by weight of the surfactant is added based on the total weight of the anticancer agent and surfactant. The dispersions are then sonicated for 20 minutes at 20 kHz or vortexed in a multi-tube apparatus for 1 minute to disperse the assemblies. The particle size is then determined, for example, by photon correlation spectroscopy (FCS) and / or optical microscopy (at 1000x magnification). If a stable dispersion is observed, the combination of the particular anticancer agent and surfactant may be optimized as described above. The dispersion is stable if it does not flake and its particles do not visibly accumulate, and when observed under optical microscope at 1000x magnification for at least 15 minutes, preferably at least 2 days or longer after its production, and particles are not visibly visible. accumulate. Further, the preferred particles do not flocculate or accumulate when dispersed in 0.1 N hydrochloric acid and / or phosphate buffered saline at pH 7.4 or in rat plasma.

The dispersion thus obtained is stable and consists of the liquid dispersant and the particles described above.

A dispersion of nanoparticles modified on their 17 surfaces can be sprayed onto sugar globules or a pharmaceutically acceptable carrier in a fluidized bed spray device in a manner known to one of ordinary skill in the art.

The anticancer pharmaceutical compositions of the present invention comprise the above-described particles and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art; for example, non-toxic physiologically acceptable carriers, adjuvants or diluents for parenteral injection, oral administration (solid or liquid), rectal administration, nasal administration, intramuscular and subcutaneous administration.

Treatment of a mammal with a composition as described above comprises administering to a mammal in need of such treatment an effective amount of the anticancer composition described above. The dosage of the anticancer agent selected for treatment should be chosen to produce the desired therapeutic response in a particular formulation and according to the particular mode of administration. A person skilled in the art can readily determine the required daily dose: this dose will depend upon the particular anticancer agent, the desired therapeutic effect, the route of administration, the duration of treatment required, and other factors.

It is particularly preferred that the "" * 1 according to the present invention

- the anticancer formulations 18 exhibit reduced toxicity and / or greater efficacy as shown in the examples below, moreover, the particles of the present invention circulate permanently in the blood as a storage site,

The present invention also provides anticancer agents which have not previously been injectable can be effectively administered in the form of nanoparticles converted to nanoparticulate compositions of the present invention by injection, such as by intravenous bolus injection.

The invention is further illustrated by the following non-limiting examples.

Examples 1-4

Preparation of nanoparticles from piposulfan

First example

Piposulfan (available from Eastmen Kodak) in a mixture of 0.33% polyoxyethylene sorbitan monooleate, Tween 80 (ICI Americas Inc., Wilmington DE) and 0.67% sorbitan monooleate (Span 80 ICI) 1 mm of zirconia beads were milled for about 96 hours to give particles of approximately 240 nm in diameter. The final concentration of piposulfan in the suspension is 10 mg / ml. The resulting particles are stable in rat plasma: they do not flake and do not accumulate.

Grinding is carried out under the following conditions. A coarse-grained suspension of piposulfan is prepared by placing 300 mg of the active ingredient in a 120 ml yellow container.

.... _t... ,...

• « * · ·«« ··· • ·»···· « · ·

- 19 containing 60 ml of pre-purified zirconia beads 1 mm in diameter and 30 ml of 1% Tween 80 / Span 80 (1: 2). The wetting agent solution is prepared by first measuring exactly 333 mg of Tween 80 in a volumetric flask, then adding sterile water for injection to dissolve or disperse the wetting agent and then making up to 100 ml. The zirconium oxide beads are purified by first rinsing with 1 N sulfuric acid and then rinsing several times with deionized water. The medium was dried in a vacuum oven at about 100 ° C for the next day.

The sealed primary container is placed in a secondary lined aluminum casing that provides a tight seal and is ground in a rolling mill at 144 rpm for approximately 96 hours. After the milling time, the dispersion is separated, the particles are separated from the medium and the particle size is determined by photon correlation spectroscopy (PCS). The stability of the resulting particles against rat plasma was examined by optical microscopy at 1000x magnification. The final pH of the preparation is 6.

Assay for Control A (unground product): A coarse distribution suspension of 40 mg bulk piposulfan was dispersed in water in the presence of 3% ethanol and 1% Tween 80. The resulting suspension should not be used for intravenous injection.

The product of Example 1 was evaluated for efficacy in female female mice with a mean body weight of 22 g.,. »· '· *» • 9 «·· ♦» »·

- 20 inoculated with Day 16 early-stage breast cancer adenocarcinoma. The preparation was injected over several days starting on day 1. The antitumor effect was evaluated by measuring tumor mass and compared with control animals in the fall. The results are summarized in the table below.

<td>Treatment Treatment</td><td>Total doses</td><td>Loss</td><td>T / C</td><td>Pig Skin</td>

<td>way *</td><td>mg / kg</td><td>crowd%</td><td> %</td><td>cell Death</td>

<td>Controls ---</td><td> —</td><td> +5,5</td><td> —</td><td> —</td>

<td>Example 1 iv</td><td> 356</td><td> -5,5</td><td> 0</td><td> 2,75</td>

<td>material iv</td><td> 220</td><td> -5,5</td><td> 2</td><td> 2,75</td>

<td>(243 nm) iv</td><td> 137</td><td> -1,8</td><td> 2</td><td> 2,25</td>

<td>arc</td><td> 85</td><td> 0</td><td> 18</td><td> 1,0</td>

<td>Control A sc</td><td> 800</td><td> -10,8</td><td> 0</td><td> 2,1</td>

Symbols and abbreviations used in the table:

* Treatment route: iv: intravenous; sc: subcutaneous

The product of Example 1 of the invention prepared in Example 1 was directly injected as a suspension of 10 mg / ml. No signs of acute toxicity were observed following a single injection at a dose of 78 mg / kg.

···· ···· j ··· J ··· • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 9 •••

- 21 Tumor weight in treated animals

T / C = ____________________________________%

Mass of tumor in control animals

A lower value means better efficiency. 0% means healing; <10% is considered very active; 10-42% shows moderate efficiency; > 42% is considered ineffective>

log 10 cell death = (TC) / 3.32 (Td), where

T is the number of days completed for the central tumor to reach 1000 mg in treated animals,

C is the time taken in days for the central tumor to reach 1000 mg in control animals,

Td is the duration, expressed in days, necessary to double the volume of the tumor.

Healing (tumor-free animals) is excluded from the (TC) calculations.

Example 1 demonstrates that a composition of the present invention has reduced toxicity and increased efficacy compared to the prior art formulation and can be administered by intravenous bolus injection.

2-4. examples

The grinding procedures described in Example 1 were repeated except that the ratio of Tween 80 to Span 80 was 2: 1. Particles of average size 297 nm were obtained.

·· «· V • · *« · · ··· Μ · «

V · ···· »4 · · ·» · ·· ··· ·· «

- 22 “

The grinding procedures described in Example 1 were repeated, except that the ratio of Tween 80 to Span 80 was 1: 1. This yields particles having an average size of 380 nm.

The grinding procedure described in Example 1 was repeated except that Tween 60 and Span 60 were used as surface modifying agents in a 1: 1 ratio. This yields particles of average size 302 nm.

Stable piposulfan nanoparticles were also prepared using bovine serum albumin as a surface modifier.

5-7. examples

Preparation of nanoparticles from camptothecin

5th example

About 60 ml of pre-purified, 1 nm zirconia beads are placed in a 120 ml wide mouth, round-bottomed yellow dish, first with 0.35 g of Tetronic 908 (BASF) followed by 35 ml of camptothecin (95% pure). from Sigma Chemicals). To the resulting mixture was added 35 ml of water for injection, and the vessel was sealed and mounted on a roller mill. Grinding is performed by rotating the vessel at 100 rpm for 7 days.

At the end of the milling process, a 100 μΐ aliquot was partially determined using a Malvern Zetasizer instrument. According to this definition, the average particle size was 240 nm.

6th example

The procedure described in Example 5 is repeated, with the exception that PVA (having a molecular weight of 30-70 kD) is used in place of Tetronic 908. This results in particles of 256 nm.

&lt;, example

The procedure described in Example 5 was repeated except that Tetronic 908 was replaced with acacia gum. This results in particles of 298 nm.

The effects of formulations containing camptothecin nanoparticles were evaluated in two rodent tumor models. Mammalian adenocarcinoma 16 / C and pancreatic adenocarcinoma 03 were used. The antitumor effect was evaluated by determining the tumor mass of experimental and control animals.

First Effect studies on 03 adenocarcinoma of the pancreas

<td>The example</td><td>The dosage</td><td>Dose</td><td>Loss</td><td>agent</td><td>T / C</td>

<td>number</td><td>path</td><td>mq / kq</td><td>% w: w</td><td>(Degradation)</td><td> %</td>

<td>B control</td><td>sc</td><td> 60</td><td> -24,1</td><td> 6/6</td><td> -</td>

<td></td><td>sc</td><td> 40,2</td><td> -21,8</td><td> 5/5</td><td> -</td>

<td></td><td>sc</td><td> 26,7</td><td> -18,2</td><td> 5/5</td><td> -</td>

<td></td><td>sc</td><td> 18</td><td> -10,9</td><td> 1/5</td><td> 62</td>

<td> 6.</td><td>i. V</td><td> 83,1</td><td> -16,7</td><td> 1/4</td><td> 14</td>

<td></td><td>i. V</td><td> 78,2</td><td> -14,6</td><td> 1/4</td><td> 55</td>

<td></td><td>i. V</td><td> 48,6</td><td> 8,3</td><td> 0/4</td><td> 0</td>

<td></td><td>i. V</td><td> 24,3</td><td> - 4,2</td><td> 0/4</td><td> 18</td>

<td>The example</td><td>The dosage</td><td>Dose</td><td>Loss</td><td>agent</td><td>T / C</td>

<td>number</td><td>path</td><td>ma / kq</td><td>% w: w</td><td>(Degradation)</td><td> %</td>

<td>PVA control</td><td>arc</td><td></td><td> + 6,3</td><td> 0/4</td><td> 100</td>

<td> 7,</td><td>i. V</td><td> 93,5</td><td> -16,7</td><td> 1/4</td><td> 7</td>

<td></td><td>i. vo</td><td> 46,8</td><td> -14,6</td><td> 0/4</td><td> 17</td>

<td></td><td>arc</td><td> 23,4</td><td> - 8,3</td><td> 0/4</td><td> 11</td>

<td colspan="6">acacia</td>

<td>control</td><td>i. V</td><td> —</td><td> 0,0</td><td> 0/4</td><td> 60</td>

Control preparation B contained 1% coarse-grained camptothecin, 3% ethanol and 1% Tween 20. The control preparation B could only be administered subcutaneously and was ineffective at the lowest subcutaneous dose (18 mg / kg). Control B was toxic in 1 out of 5 animals. In contrast, the formulations of the invention containing camptothecin in the form of nanoparticles, at doses ranging from 24 to 93 mg / kg, were administered intravenously, were safe and effective.

Second Effects on a 16 / C murine adenocarcinoma rodent model

<td>The example is</td><td>dosage</td><td>Dose</td><td>Loss</td><td>agent</td><td>T / C</td>

<td>number</td><td>path</td><td>mer / ka</td><td>Tome%</td><td>(Degradation)</td><td> %</td>

<td>B control</td><td>sc</td><td> 60</td><td> -23,5</td><td> 5/5</td><td></td>

<td></td><td>sc</td><td> 30</td><td> -20,9</td><td> 5/5</td><td> -</td>

<td></td><td>sc</td><td> 15</td><td> -18,3</td><td> 3/5</td><td> 14**</td>

<td> 5.</td><td>arc</td><td> 65</td><td> -17,4</td><td> 0/5</td><td> 23</td>

<td></td><td>i. V</td><td> 33</td><td> -18,7</td><td> 1/5</td><td> 33</td>

<td></td><td>arc</td><td> 16</td><td> - 2,2</td><td> 0/5</td><td> 63</td>

<td>T908 control</td><td>arc</td><td> —</td><td> + 4,3</td><td> 0/2</td><td> 100</td>

<td> 6.</td><td>arc</td><td> 65</td><td> -21,7</td><td> 5/5</td><td> -</td>

<td></td><td>arc</td><td> 33</td><td> -15,7</td><td> 0/5</td><td> 100</td>

<td>PVA control</td><td>arc</td><td></td><td> + 4.3</td><td> 0/2</td><td> 100</td>

T908 and PVA controls contained 1% aqueous solutions of the respective surface modifying agents. Control B was administered subcutaneously and was toxic at all dose levels. Effective doses of camptothecin nanoparticulate compositions of the present invention were administered intravenously.

**% T&amp;C for control animals was determined by number of surviving animals.

In this case N = 2.

Testing blood and tumor clearance '

Blood clearance and tumor distribution in the murine adenocarcinoma 16 / C rodent tumor model were investigated to determine whether greater efficacy is related to its pharmacokinetic properties.

Tumor bearing mice were injected with 10 mg / ml camptothecin (formulated as in Examples 5 and 6) via the tail vein and a control formulation containing 5 mg / ml camptothecin in 0.1 N sodium hydroxide solution was added as a solubilizing agent. At various times after injection, 5, 30, 60 minutes at 2, 4, 8, 16, 24 and 48 hours after injection, the animals were gently killed and blood samples were taken and the tumor excised. The drug concentration of the samples was quantitatively analyzed by HPLC. The results show that the compositions of the present invention affect the clearance of the active ingredient from the circulating blood storage and the tumor.

Tl / 2 (half-life) in blood and tumor

The preparation

In the blood

In the tumor

6th The product of Example 1c has a clock> 48 hours

5th The product of Example 1c has a clock> 48 hours

control

1.6 hours

13.5 hours

In the embodiment of the present invention, the elimination half-life and the half-life of camptothecin in the tumor were significantly prolonged. From this it was concluded that the pharmacokinetic parameters of the camptothecin nanoparticulate formulation are directly related to the potentiation of the active ingredient.

8-10. examples

Preparation of nanoparticles from etoposide

8th example

The procedure of Example 5 was repeated except that 1.7 g of etoposide was combined with 1.7 g of PVA and milled for 14 days. This results in a particle size of 310 nm. These particles are stable in acid and plasma.

9th example

The procedure described in Example 8 was repeated except that Pluronic F-198 (BASF) was used in place of PVA. This results in a final particle size of 312 nm. These particles are stable in acid and plasma.

10th example

Ethoposide (2%) was ground in sterile water for 7 days. The milled slurry was mixed with 2% Pluronic F127 in a 1: 1 mixture and vortexed before determining the particle size. This results in a final particle size of 277 nm. The suspension appeared to be stable in simulated gastric juice, phosphate buffered saline (PBS, pH 7.4 in short) and in rat plasma.

Effectiveness studies

Formulations of etoposide nanoparticles were evaluated in two separate efficacy studies evaluated in Pancreatic Adenocarcinoma 03 (PANC = 03). A 2% non-aqueous etoposide solution as control C was prepared in the Physicians'Desk Reference 46th Edition, 741-743. pages using the formula. As described above, the anti-tumor activity was evaluated by measuring the tumor mass of experimental and control animals. These studies suggested that etoposide formulations of the present invention can be used to administer high doses of etoposide without signs of serious toxic reactions.

First Effect of nanoparticulate etoposide on PANC 03.

Rodent daganatmodellen

<td>The example</td><td>The dosage</td><td>Dose</td><td>Loss</td><td>agent</td><td>T / C</td>

<td>number</td><td>travel a</td><td>MCI / kq</td><td>% w: w</td><td>(Degradation)</td><td> %</td>

<td>C control</td><td>i. V</td><td> 120</td><td> -24,0</td><td> 0/5</td><td> 4,0</td>

<td></td><td>i. V</td><td> 75</td><td> - 4,0</td><td> 0/5</td><td> 20,0</td>

<td> 7.</td><td>i. V</td><td> 160</td><td> -12,0</td><td> 0/5</td><td> 18,0</td>

<td></td><td>i. V</td><td> 100</td><td> 0,0</td><td> 0/5</td><td> 32,0</td>

<td></td><td>arc</td><td> 62</td><td> 2,0</td><td> 0/5</td><td> 42,0</td>

<td> 8.</td><td>i. V</td><td> 160</td><td> -12,0</td><td> 0/5</td><td> 26,0</td>

<td></td><td>arc</td><td> 100</td><td> + 2,0</td><td> 0/5</td><td> 35,0</td>

<td></td><td>arc</td><td> 62</td><td> + 4,0</td><td> 0/5</td><td> 35,0</td>

<td>The example</td><td>The dosage</td><td>Dose</td><td>Loss</td><td>agent</td><td>T / C</td>

<td>number</td><td>travel a</td><td>MCJ / KCl</td><td>% w: w</td><td>(Degradation)</td><td> %</td>

<td> 9,</td><td>arc</td><td> 170</td><td> -18,5</td><td> 1/5</td><td> 16</td>

<td></td><td>i. V</td><td> 85</td><td> - 2,0</td><td> 0/5</td><td> 35</td>

<td></td><td>i. V</td><td> 43</td><td> + 2,5</td><td> 0/5</td><td> 41</td>

11th-16th examples

Production of nanoparticles from taxol

11th example

Approximately 18 ml of pre-purified zirconium oxide medium (particle size 1 mm) was placed in a 3 ml yellow glass cup, to which 240 mg of taxol (Sigma Chemicals) and 180 mg of Tween 20 were added, followed by 12 ml of water for injection. The cup was sealed and kept on a roller mill for 11 days. This results in a particle size of 327 nm. The composition thus obtained is stable against PBS pH 7.4 and rat plasma.

12th example

Example 11 is repeated except that PVA (molecular weight 30-70 kD) is used instead of Tween 20. The final result is 365 nm particles.

The above samples were evaluated in efficacy studies in mice bearing early-stage 16 / C breast carcinoma. The antitumor effect was evaluated by comparing the tumor weight of taxol treated animals with that of untreated animals. Toxicity was determined by dose escalation assays while observing animal mortality and weight loss. All samples were administered intravenously.

<td>The example</td><td>Dose</td><td>Loss</td><td>decay</td><td>Middle tumor</td><td>T / C</td>

<td>number</td><td>ma / kcr</td><td>Tome%</td><td></td><td>on the 11th</td><td> %</td>

<td>D ”control</td><td> -</td><td> + 6,1</td><td> —</td><td>1539 mg</td><td> —</td>

<td>Example 11</td><td></td><td></td><td></td><td></td><td></td>

<td>products</td><td> 108,5</td><td> - 1,5</td><td> 0/5</td><td> 1528</td><td> 13</td>

<td>Example 12</td><td></td><td></td><td></td><td></td><td></td>

<td>products</td><td> 108,5</td><td> - 3,0</td><td> 0/5</td><td> 201</td><td> 99</td>

A control sample of taxol could not be obtained. In contrast, the taxol formulation in the form of Cremophore EL produced a single dose of 25 mg / kg of direct death. In contrast, the nanoparticulate taxol formed in the compositions of the present invention could be injected at a dose of 88 mg / kg without any appreciable adverse effects.

A taxol suspension prepared in a manner similar to Example 11 was treated separately with a plurality of surface modifiers. After the addition of the new surface modifier, the mixture was maintained in a vortex mix and then evaluated for particle size and fluidity stability. Each suspension contained 1% taxol and 0.75% Tween 80. The results are summarized in the table below.

<td colspan="2" rowspan="2">Example // surface modifier</td><td rowspan="2">Concentration %</td><td rowspan="2">Size nm</td><td colspan="2">Fluidity stability</td>

<td>In PBS</td><td>Rat in zma</td>

<td>13o</td><td>CTAC</td><td> 0,25</td><td> 364</td><td>OK</td><td>flocculation</td>

<td> 14.</td><td>AOT</td><td> 0,25</td><td> 322</td><td>SA *</td><td>SA</td>

<td> 15.</td><td>F68</td><td> 0,5</td><td> 297</td><td>SA / S</td><td>SA / S</td>

<td> 16.</td><td>T908</td><td> 0,5</td><td> 313</td><td>SA / S</td><td>SA / S</td>

* SA = slight aggregation

17th-18th examples

Preparation of Nanoparticles from 1,2,4-Benzotriazine-7-amine-1,4-dioxide

Transfer approximately 60 ml of the pre-purified zirconium oxide medium containing particles of 1 mm size into a 120 ml yellow vessel, add 1.5 g of 1,2,4-benzotriazine-7-amine-1,4-dioxide and 28.5 ml. water for injection. The vessels were sealed, mounted on a roller mill and run at 95 rpm for 48 hours. Based on PCS analysis, the particle size is 322 nm, but larger particles are expected. Grinding was continued for another 5 days.

Studies were carried out with 0.5 ml of benzotriazine amine dioxide pulp prepared with 0.5 ml of a 6% surfactant solution above. The final concentration of the active ingredient is 2.5% and that of the surface modifiers is 3%. Nanoparticulate slurries of benzotriazine amine dioxide stabilized surface modifier were subsequently treated with either PBS (pH 7.0) or 0.1 N hydrochloric acid (pH 1). Optical microscopy observations were made to determine fluidity stability. Here are the results,

<td rowspan="2">The example number</td><td rowspan="2">Surface modifying agent</td><td colspan="2">Stability</td><td rowspan="2">In human plasma</td>

<td>at pH 1</td><td>pH 7</td>

<td>Example 17</td><td>PVP (12kD)</td><td>fine</td><td>fine</td><td>fine</td>

<td>products</td><td>(BASF)</td><td>distributed</td><td>distributed</td><td>distributed</td>

<td>Example 18</td><td>acacia</td><td> —</td><td>SA / S</td><td>SA / S</td>

<td>products</td><td></td><td></td><td></td><td></td>

SA / OK means slight aggregation or good

The results indicate that stable nanoparticles can be formed from 1,2,4-benzotriazine-7-amine-1,4-dioxide.

19-22. examples

Preparation of Nanoparticles from 1,2,4-Benzotriazine-3-amine-1,4-dioxide

7.5 ml of pre-purified zirconia containing 1 mm particles, 18.75 mg of 1,2,4-benzotriazine-3-amine-1,4-dioxide and 3.75 ml of water in a 15 ml yellow vessel placed. After milling for 11 days, the nanoparticulate slurry is separated from the medium. To each 100 μΐ aliquot of the suspension is added 100 μΐ of a 2% wetting solution to give a concentration of 0.25% and a 1% wetting agent. The mixture is kept in a vortex mix and the particle size is determined. Fluid stability was evaluated by microscopy by mixing 10 μΐ suspension with 90 μΐ rat plasma. Our results are summarized in the table below,

<td>The number of the example</td><td>surface Modifier agent</td><td>particle Size nm</td><td>Fluidity stability in rat plasma</td>

<td>Example 19</td><td>PVP (12kD)</td><td> 134</td><td>finely divided</td>

<td>products</td><td></td><td></td><td></td>

<td>Example 20</td><td>acacia</td><td> 344</td><td>SA / S</td>

<td>products</td><td></td><td></td><td></td>

<td>Example 21</td><td>Tween 80</td><td> 128</td><td>finely divided</td>

<td>products</td><td></td><td></td><td></td>

<td>Example 22</td><td>T908</td><td> 130</td><td>aggregates</td>

<td>products</td><td></td><td></td><td></td>

23rd example

Preparation of Nanoparticles An ml of pre-purified zirconium oxide medium was placed in a 60 ml yellow flask, 1 g of trans-retinoic acid (from Sigma), 470 mg of tyloxapol and 15 ml of water were added. The mixture was milled on a roller mill for 15 days to achieve a final particle size of 140 nm. This nanoparticulate suspension is stable in both rat plasma and simulated gastric juice.