X-ray contrast compositions useful in medical imaging.

IS 2 139 586 T3

DESCRIPTION

X-ray contrast compositions useful in imaging diagnostics.

This invention relates to X-ray contrast compositions for diagnostic X-ray imaging.

X-ray imaging is a well-known and extremely valuable tool for the early detection and diagnosis of different disease states in the human body. The use of contrast agents for image enhancement in diagnostic X-ray imaging procedures is widespread DP Swanson et al. in Pharmaceuticals in Medical Imaging, 1990, MacMillan Publishing Company, provides an excellent background on contrast agents and media in imaging diagnostics.

Briefly, in X-ray imaging, transmitted radiation is used to produce a radiograph based on the attenuation characteristics of all tissues. X-rays pass through different tissues and are attenuated by scattering, that is, reflection or refraction or absorption of energy. However, some organs of the body, vessels and anatomical sites have such low absorption of X-ray radiation that radiographs of these parts of the body are difficult to obtain. To overcome this problem, radiologists typically insert an X-ray absorbing medium containing a contrast agent into these body organs, vessels, and anatomic sites.

Currently available X-ray contrast agents generally do not have a direct release site or compartmentalization. Consequently, large amounts of contrast agent are generally required for imaging. It would be convenient to restrict the contrast agent to specific biological or anatomical compartments, such as blood, liver, kidney, or spleen. This will reduce the total amount of agent that needs to be administered to achieve the desired contrast enhancement.

Maximal enhancement of most blood vessels occurs during the so-called vascular phase of contrast medium kinetics, which occurs approximately within the first two minutes after intravascular infusion or bolus injection of contrast medium. This is because the plasma concentration of an intravascular contrast medium decreases rapidly as a result of vascular mixing, transcapillary diffusion of the medium from the circulation to the interstitial spaces, and renal excretion. Consequently, blood vessel imaging must be carried out in a narrow period of time, typically a few minutes after the infusion or injection of the X-ray contrast agent. Currently, there is no commercially available X-ray contrast agent for blood imaging that provides good contrast images over a long period of time. Therefore, multiple injections are often required to adequately visualize the vascular system. Furthermore, arteriography, as currently practiced, normally requires percutaneous or surgical catheterization, fluoroscopic localization, and multiple arterial bolus administrations to adequately visualize a particular vascular region.

The need for better visualization of the liver, kidney and spleen, particularly for the early detection of metastasis, has led to numerous attempts to develop a contrast medium that is accumulated by the mononuclear phagocyte system (MPS). In Handbook of Experimental Pharmacology, vol 73, Radiocontrast Agents, Chapter 13, "Particulate Suspensions as Contrast Media", Violante and Fischer describe and analyze the problems and complexities involved in the design and formulation of said medium. Since MPS from the liver and spleen are known to trap particles by phagocytosis, contrast agents in particulate form, such as emulsions of iodinated oils, for example iodinated ethyl esters from poppy seed oil, and liposomes have been proposed. containing water-soluble iodinated contrast agents for liver and spleen visualization. However, emulsions tend to be unacceptably thioxic when administered both intravenously and subcutaneously, and liposomes tend to require unacceptably high amounts of lipid to achieve adequate contrast enhancement. The MPS or Kuppfer cells of the liver, targeted by liposomes and emulsions, constitute approximately 5 percent of the total cell population, with the remainder being hepatocytic cells.

Radioactive thorium dioxide inorganic submicron particles have been used for liver visualization and have shown effective contrast enhancement in clinical trials. However, its use has not been continued due to extremely long retention of the particles in the liver. This, in combination with thorium's inherent radioactivity, has led to adverse side effects.

ES 2 139 586 T3 severe including neoplasm and fibrosis.

Violante et al., US Patent 4,826,689, describe a process for preparing non-crystalline amorphous particles of uniform size from water-insoluble organic compounds, in which the organic compound is dissolved in an organic solvent. In one embodiment, the ethyl ester of iodipamide is dissolved in dimethyl sulfoxide. However, solvent precipitation techniques, as described in US Pat. 4,826,689, to prepare particles, tend to provide solvent contaminated particles. Such solvents are often toxic and it can be very difficult, if not impossible, to adequately remove them to pharmaceutically acceptable levels for imaging. Additionally, amorphous materials and formulations tend to exhibit unacceptably poor stability and / or short shelf lives.

Motoyama et al., US Patent 4,540,602 discloses that a solid drug can be sprayed into an aqueous solution of a high molecular weight substance soluble in water, and that as a result of such grinding in moisture, the drug is Converts to finely divided particles with a diameter of 0.5 µm or less than 5 µm. However, it is not suggested that particles with a mean particle size of less than about 400 nm can be obtained. Furthermore, attempts to reproduce the wet grinding procedures described by Motoyama et al. resulted in particles having a mean particle size of more than 1 µm.

WO 90/07491 describes water insoluble iodinated carbonate esters which are described below, are useful as agents of and for the visualization of the liver and spleen. The particles of mean diameter of the order of 1.0 microns of the described esters are taken up by the reticuloendothelial system of the liver and spleen. However, such particles are prepared by conventional mechanical grinding or spray drying techniques or by solvent precipitation techniques as described in US Patent 4,826,689.

Currently, there is no completely satisfactory X-ray contrast agent on the market for imaging the liver and spleen. Each composition and / or contrast agent proposed for liver and spleen imaging has some disadvantage.

It would be desirable to provide better X-ray contrast compositions for imaging of vessels, anatomical sites, and organs of the body such as the liver and spleen. However, it would be highly desirable to provide intravenously administered X-ray contrast compositions that demonstrate effective blood imaging over extended periods of time.

We have discovered that surface-modified crystalline nanoparticles of water-insoluble X-ray contrast agents provide images of exceptional resolution and can be formulated for the best delivery to specific tissues or fluid sites, such as blood, liver , kidney, bone marrow, lymph nodes and spleen. However, the preferred X-ray contrast agents, when administered intravenously, provide effective imaging of blood within the vascular system for markedly prolonged periods of time.

More specifically, in accordance with this invention, there is provided an X-ray contrast composition comprising:

a) particles consisting essentially of a non-radioactive, crystalline, organic X-ray contrast agent, having a surface modifier adsorbed on its surface, said non-radioactive, crystalline, organic, X-ray contrast agent having been wet ground , at an effective average particle size of less than 400 nm and having a coating of said surface modifier adsorbed to the surface of said particles, said surface modifier being present in an amount of 0.1 to 90% by weight, based on the total weight of the dry particles so as to maintain said effective average particle size and

b) a pharmaceutically acceptable vehicle.

According to this invention there is also provided the use of the above composition to prepare a medicament for X-ray imaging diagnosis.

This invention further provides a method for repairing the above-described X-ray contrast composition, which includes the steps of introducing a non-radioactive organic X-ray contrast agent, a liquid medium, a grinding medium, and optionally a surface modifier.

ES 2 139 586 T3 in a grinding vessel; wet grinding the contrast agent and then mixing a surface modifier with the liquid medium if the surface modifier was not present during grinding, to form particles having an average size of less than about 400 nm; and separating the particles from the container and the grinding medium.

An advantageous feature of this invention is that X-ray contrast compositions are provided that demonstrate effective imaging of blood within the vascular system for unexpectedly long periods of time, such as 2 hours or more.

Another advantageous feature of this invention is that X-ray contrast compositions are provided with enhanced delivery at specific anatomic sites, for example, the blood within the vascular system, the liver, the kidney, the bone marrow, the lymph nodes or the spleen. This allows better imaging of the site with lower amounts of the agent.

Another advantageous feature of this invention is that intravenous injectable X-ray contrast compositions are provided which do not require catheterization during angiography.

Yet another advantageous feature of this invention is that X-ray contrast compositions are provided which can be formulated with large loads of existing X-ray contrast agents and / or derivatives thereof.

Yet another advantageous feature is that desired levels of contrast, determined by a particular iodine content, can be achieved with lower amounts of X-ray contrast compositions of this invention compared to prior art compositions containing conventional carriers, for example. example polymers.

The X-ray contrast composition of this invention comprises particles of an organic X-ray contrast agent having a surface modifier adsorbed on its surface in sufficient quantity to maintain an effective average particle size of less than 400 nm.

The practical X-ray contrast agent of this invention is not radioactive and exists as a discrete, crystalline phase of an orgaonic substance. The crystalline phase differs from a non-crystalline or amorphous phase that results from solvent precipitation techniques as described in US Patent 4,826,689 noted above. The orgaonic substance may be present in one or more suitable crystalline phases. The invention can be carried out with a wide variety of non-radioactive, crystalline X-ray contrast agents. However, the X-ray contrast agent must be poorly soluble and poorly dispersible in at least one liquid medium. By "poorly soluble" is meant that the agent has a solubility in the liquid dispersion medium, eg water, of less than about 10 mg / ml, and preferably less than 1 mg / ml. The preferred liquid dispersion medium is water. Additionally, the invention can be carried out with other liquid media in which the contrast agent selected for X-rays is poorly soluble and dispersible, including, for example, aqueous saline solutions, such as phosphate buffered saline (PBS), plasma, mixture of aqueous and non-aqueous solutions, eg water and alcohol, and suitable non-aqueous solvents such as alcohol and glycerol.

The X-ray contrast agent can be an iodinated compound. The iodinated compound can be aromatic or non-aromatic. Aromatic compounds are preferred. The iodinated compound may comprise one, two, three or more aotomes of iodine per molecule. Preferred species contain at least two, and most preferably, at least three iodine atoms per molecule. Selected iodinated compounds may contain substituents that do not give the compound solubility, such as, for example, alkylureido, alkoxyacylamido, hydroxyacetamido, butyrolactamido, succinimido, trifluoroacetamido, carboxy, carboxamido, hydroxy, alkoxy, acylamino, and the like.

A preferred class of contrast agents includes various iodinated aromatic acid osters and amides. The osters are preferably alkyl or substituted alkyl osesters. The amides can be primary or secondary amides, preferably alkyl or substituted alkyl amides. The contrast agent may be, for example, an ester or amide of a substituted triiodobenzoic acid such as an acyl, carbamyl, and / or acylmethyl substituted triiodobenzoic acid. Representative illustrative examples of iodinated aromaotic acids include diatrizoic acid, metrizoic acid, iotalaomic acid, ioxaogly acid (hexabrix), ioxyitallamic acid, tetaiodoterephthaolic acid, and iodipamide. The use of poorly soluble derivatives of iodamide and ioprol is contemplated here.

IS 2 139 586 T3

The invention can also be carried out with poorly soluble derivatives, such as ester and ther derivatives, of nonionic hydroxylated X-ray contrast agents. Illustrative nonionic contrast agents include, but are not limited to, metrizamide; ioglunide; iopamidol; iopromida; iogulamide; iohexol, and other compounds described in US Patent 4,250,113; ioversol and other compounds described in US Pat. 4,396,598; nonionic triiodinated compounds, as described in Investigative Radiology vol 19, July-August 1984; and non-ionic dimers, as described in Radiology, 142: 115-118, January 1982. This invention can be carried out with poorly soluble derivatives of iodomethane sulfonamides, aromatic iodinated glucoanilides, 2-ketogulonamides, reverse amides, peptides, carbamates, osters, derivatives of glycoside and glucose, derivatives of benzamide, isophthalimides, bis compounds, and bis acylamides. -polyhydroxylated, as described in volume 73 of the Handbook of Experimental Pharmacology, entitled Radiocontrast Agents, edited by Sovak, 1984, Springer-Verlag, Berlin, pages 56-73.

Many of the iodinated molecules described above, in monomeric form, can also be prepared as dimers (sometimes referred to as bis compounds), tromers (sometimes referred to as tris compounds) etc., by techniques known in the art. It is contemplated that this invention may be carried out with poorly soluble iodinated compounds in monoomer, dimer, trimer, and polymer form. Sovak, cited above, pages 40-53, describes representative illustrative compounds.

The preferred classes of contrast agents have the following structural formulas:

<img file="ES2139586T3_D0001.tif" />

IS 2 139 586 T3

<img file="ES2139586T3_D0002.tif" />

(R = OH)

In the above structures, R can be OR¹ , _R2 ^/

N ^\_R3

OO alkylene -C-OR¹, oO- alkylene -C-OR¹;

in which R¹ is alkyl, and

R² yR³ they are independently H or alkyl.

Each alkyl group independently may contain 1-20, preferably 1-8, and more preferably 1-4 carbon atoms. The alkylene group preferably contains 1 to 4 carbon atoms, such as methylene, ethylene, propylene and the like, optionally substituted with for example an alkyl group, such as methyl and ethyl.

Particularly preferred contrast agents include diatrizoic acid ethyl ester, i.e. ethyl 3,5-diacetamido-2,4,6-triiodobenzoate, also known as 3,5bis (acetylamino) -2,4,6- ethyl triiodobenzoate or ethyl diatrizoate, having structural formula A above, wherein R = -OCH2CH3 (WIN 8883); the ethyl glycolate ester of diatrizoic acid, i.e., ethyl (3,5-bis (acetylamino) -2,4,6-triiodobenzyloxy) acetate, also known as ethyl diatrizoxyacetate, which has the above structural formula A, in which

R = OCH2-C-OCH2CH3

O (WIN 12901); and ethyl 2- (3,5-bis (acetylamino) -2,4,6-triiodobenzyloxy) butyrate also known as ethyl 2-diatrizoxybutyrate (WIN 16318), which has the structural formula A above in which

CH2CH3 ^|

R = OCH- (CO) -OCH2CH3

Furthermore, it is expected that the invention can be implemented together with the water-insoluble iodinated carbonate esters described in WO 90/07491.

IS 2 139 586 T3

The above-described X-ray contrast agents are known compounds and / or can be prepared by techniques known in the art. For example, the water-insoluble osesters and terminal amides of acids such as the above-described iodinated aromatic acids can be prepared by conventional alkylation or amidation techniques known in the art. The above-mentioned acids and other acids that can be used as raw materials are commercially available and / or can be prepared by techniques known in the art. The following examples contain illustrative examples of known syntactic techniques.

Particles useful in the practice of this invention include a surface modifier. The surface modifiers useful herein adhere phosphically to the surface of the X-ray contrast agent, but do not chemically react with the agent or with themselves. The individually adsorbed molecules of the surface modifier are essentially devoid of molecular crosslinking. Suitable surface modifiers can be selected from inorganic and orgaonic pharmaceutical excipients such as different polyomers, low molecular weight oligoomers, natural products and surfactants. Preferred surface modifiers include nonionic and anionic surfactants. Representative examples of surface modifiers include gelatin, caseone, lecithin (phosphatides), acacia gum, cholesterol, tragacanth, steaoric acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearolic alcohol, cetomacrogol emulsifying wax, osteres sorbitan, polyoxyethylene alkyl ethers, for example macrogol ethers such as ketomacrogol 1000, polyoxyethylene castor oil derivatives, Polyoxyethylene sorbiton fatty acid esters, for example commercially available Tweens®, polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecyl sulfate, calcium carboxymethylcellulose, carboxymethylcellulose, sodium hydrocellulosethyl cellulose, hydrocellulosethyl cellulosate, hydrocellulosethyl cellulose , non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinolic alcohol, and polyvinylpyrrolidone (PVP). Many of these surface modifiers are known pharmaceutical excipients and are described in detail in the Handbook of Pharmaceutical Excipients, jointly published by the American Pharmaceutical Association and The Pharmacuetical Society of Great Britain, the Pharmaceutical Press, 1986.

Particularly preferred surface modifiers include polyvinylpyrrolidone, tyloxapol, poloxomers such as Pluronic® F68 and F108, which are block copolymers of ethylene oxide and propylene oxide, and poloxamines such as Tetronic® 908 (also known as Poloxamine 908) , which is a tetrafunctional block copolymer derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine, available from BASF, dextran, lecithin, Sodium sulfosuccinic acid dialkylesters, such as Aerosol OT®, which is a dioctyl oster of sodium sulfosuccinic acid, available from American Cynamid, Duponol® P, which is a sodium lauryl sulfate, available from DuPont, Triton¹® X-200, which is an alkyl aryl polyether sulfonate, available from Rhom and Haas, Tween 80, which is a polyoxyethylene sorbitan fatty acid ester, available from ICI Specialty Chemicals, and Carbowax® 3350 and 934, which are polyethylene glycols available from Union Carbide. Surface modifiers that have been found to be particularly useful include Tetronic 908, the Tweens, Pluronic F-68, and polyvinylpyrrolidone. Other surface modifiers include: decanoyl-N-methylglucamide;

n-decyl β-D-glucopyranoside;

n-decyl β-D-maltopyranoside;

n-dodecyl β-D-glucopyranoside;

n-dodecyl β-D-maltoside;

heptanoyl-N-methylglucamide;

n-heptyl β-D-glucopyranoside;

n-heptyl β-D-thioglucoside;

n-hexyl β-D-glucopyranoside;

nonanoyl-N-methylglucamide;

n-nonyl β-D-glucopyranoside;

octanoyl-N-methylglucamide;

n-octyl β-D-glucopyranoside;

ES 2 139 586 T3 octyl β-thioglucopyranoside;

and the like.

A particularly preferred class of surface modifiers includes water-soluble or water-dispersible compounds having the formula _R5 ^|

CONCH2 (CHOH) xCH2OH ^/

L ^\

CONCH2 (CHOH) and CH2OH ^|_R6 in which ^/

LesR⁴-CH ^\

<img file="ES2139586T3_D0003.tif" />

<img file="ES2139586T3_D0004.tif" />

L 'is a chemical bond, -O-, -S-, -NH-, -CONH- or -SO₂NH-;

R⁴ is a substituted or unsubstituted hydrophobic alkyl, substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted aryl group;

each R⁵ yR⁶ independently they are hydrogen or an alkyl group having 1 to 4 carbon atoms;

each a and b independently is 0 or an integer from 1 to 3, provided that the sum of a and b is not greater than 3; and each x and y independently is an integer number from 3 to 7.

Preferred compounds within this class according to the above structure wherein R⁴ contains 6 to 36 carbon atoms, for example R⁴ is an n-alkyl group containing 6 to 18 carbon atoms, each R⁵ yR⁶ independently is a methyl, ethyl, propyl or butyl group and a is 0 and b is 0. This class of surface modifiers can be prepared by reacting a suitable dicarboxylic acid ester with a suitable monosaccharide amine, preferably in the absence of a solvent at a temperature from 140 to 200 ^ C.

Surface modifiers are commercially available and / or can be prepared by techniques known in the art. Two or more surface modifiers can be used in combination.

Particles useful in the practice of this invention can be prepared by dispersing a poorly soluble X-ray contrast agent in a liquid dispersion medium and wet grinding the agent in the presence of a grinding medium to reduce the particle size of the contrast agent at an effective average particle size of less than about 400 nm. The particles are

ES 2 139 586 T3 can be reduced in size in the presence of a surface modifier. Alternatively, the particles can be contacted with a surface modifier after abrasion.

A general procedure for preparing the particles, useful in the practice of this invention is as follows. The selected X-ray contrast agent is obtained commercially and / or prepared by techniques known in the art as described above, in a conventional coarse form. It is preferred, although not essential, that the particle size of the selected coarse X-ray contrast agent is less than 100 µm determined by screening analysis. If the size of the coarse contrast agent particles is greater than about 100 µm, then it is preferred that the coarse contrast agent particles be reduced in size to less than 100 µm using a conventional grinding method such as jet milling. air or fragmentation.

The selected coarse imaging agent can then be added to a liquid medium in which it is essentially insoluble to form a premix. The concentration of the agent in the liquid medium can range from about 0.1 to 60%, and preferably from 5 to 30% (w / w). It is preferable, but not essential, that the surface modifier is present in the premix. The concentration of the surface modifier can range from about 0.1 to 90%, and is preferably 1-75%, more preferably 10-60%, and more preferably 10-30% by weight based on the total combined weight of the substance. drug and the surface modifier. The apparent viscosity of the premix suspension is preferably less than 1 Pa.s.

The premix can be used directly by huomer milling to reduce the average particle size in the dispersion to less than 400 nm. It is preferred that the premix be used directly when using a ball mill for abrasion. Alternatively, the drug substance, and optionally, the surface modifier, can be dispersed in the liquid medium, using suitable agitation, for example a roll mill or a Cowles type mixer, until a homogeneous dispersion is observed in which there is no large agglomerates visible to the naked eye. It is preferred that the premix be subjected to such a dispersion step prior to grinding when using a mill with recirculation medium for abrasion.

The huomer milling can be carried out in any suitable dispersion mill, including, for example, a ball mill, an abrasion mill, a vibratory mill, and mills with milling media such as a sand mill and a pearl mill. A mill with grinding media is preferred because of the relatively short grinding time necessary to provide the desired result, ie, the desired reduction in particle size. For medium milling the apparent viscosity of the premix is ​​preferably about 0.1 Pa.s to about 1 Pa.s. For the ball mill, the apparent viscosity of the premix is ​​preferably about 0.001 to about 0.1 Pa.s. These intervals tend to give an optimal balance between effective particle fragmentation and erosion through the medium.

The grinding medium for the particle size reduction step can be selected from rigid medium preferably spherical or particulate in shape having a particle size of less than about 3 mm, and more preferably less than about 1 mm. Conveniently said medium is capable of providing the particles of the invention with shorter process times and imparts less stress to the grinding equipment. The selection of the material for the grinding medium is not believed to be crotic. However, the preferred medium has a density greater than about 3 g / cm³. Zirconium oxide, such as magnesia stabilized 95% ZrO, zirconium silicate, and glass grinding medium have been found to provide particles having levels of contamination that are believed to be acceptable for the preparation of contrast compositions for X-rays. However, other media, such as stainless steel, titania, alumina, and yttrium stabilized 95% ZrO are believed to be useful as well.

Abrasion time can vary greatly and depends mainly on the selected huomer grinding mill. For ball mills, process times of up to five days or more may be required. On the other hand, process times of less than 1 doa (dwell times of about one minute to several hours) have provided the desired results using a high shear medium mill.

The particles should be reduced in size at a temperature that does not significantly degrade the imaging agent. Process temperatures of less than about 30-40 ° C are generally preferred. If appropriate, the process equipment can be cooled with conventional cooling equipment. The procedure is conveniently carried out under temperature conditions

ES 2 139 586 T3 at ambient temperatures and at process pressures that are safe and effective for the grinding process. For example, ambient process pressures are typical of ball mills, abrasion mills, and vibratory mills. Process pressures greater than approximately 1.4 kg / cm² they are typical of the mill with medium.

The surface modifier, if not present in the premix, should be added to the dispersion after abrasion in an amount equivalent to that described for the premix. The dispersion can then be mixed, for example, by vigorous stirring. Optionally, the dispersion can be sonicated, for example using an ultrasound power supply. For example, the dispersion can be subjected to ultrasound energy with a frequency of 20-80 KHz for a period of time of approximately 1 to 120 seconds.

The relative amount of imaging agent and surface modifier can vary widely and the optimal amount of surface modifier may depend, for example, on the particular imaging agent and surface modifier selected, the critical micelle concentration of the surface modifier if it forms micelles, the hydrophilic lipophilic balance (HLB) of the stabilizer, the melting point of the stabilizer, its solubility in water, the surface tension of the stabilizer water solutions, etc. Preferably the surface modifier is present in an amount of about 0.1-10 mg per square meter of surface area of ​​the imaging agent. The surface modifier may be present in an amount of 0.1-90%, preferably 1-75%, more preferably 10-60%, and most preferably 10-30% by weight based on the total weight of the dry particle.

We have developed a simple selection procedure by which suitable surface modifiers and imaging agents can be selected that provide stable dispersions of the desired particles. First, coarse particles of an imaging agent of interest are dispersed in a liquid in which the agent is essentially insoluble, for example, 5% (w / v) water and ground for 60 minutes in a DYNO- MILL (model KDL, available from Willy A. Bachoffen AG Maschinenfabrik) under the following grinding conditions: Grinding vessel:

Stainless steel chamber with water jacket Premix flow rate:

250 ml / min

Available volume of the shredding container:

555 ml

Media volume:

472 ml

Media type:

0.5-0.75mm Lead Free Silica Glass Beads (Distributed by Glen Mills, Inc.)

Recirculation time:

240 min

Stay time:

min

Motor speed:

3000 RPM; tangential speed 595 m / min Shredding vessel cooling liquid:

Water

IS 2 139 586 T3

Cooling temperature:

10^or C

The ground material is then aliquoted and surface modifiers are added in concentrations of 2, 10, and 50% by weight based on the combined total weight of the imaging agent and surface modifier. The dispersions are then sonicated (1 minute, 20 KHz) to disperse the agglomerates and subjected to particle size analysis by examination with an ioptic microscope (1000 x magnification). If a stable dispersion is observed, then the process for preparing the particular surface modifying imaging agent combination can be optimized according to the above teachings. By stable it is meant that the dispersion does not present flocculation or agglomeration of particles visible to the naked eye at least 15 minutes, and preferably, at least two days or more after preparation. Furthermore, the preferred particles do not exhibit flocculation or agglomeration when dispersed in at least one or more of the following media: PBS, simulated gastrointestinal (GI) fluids, and plasma.

As used herein, particle size refers to a number for mean particle size by conventional particle size measurement techniques known to one of ordinary skill in the art, such as sedimentation field flow fractionation, spectroscopy. photin correlation, or disk centrifugation. By "an effective average particle size less than about 400 nm" is meant a particle size distribution in which at least 90% of the particles have a particle size of less than about 400 nm when measured by technical of weight-size indicated above. In preferred embodiments of the invention, the effective average particle size is less than about 300 nm, and more preferably less than about 250 µm. In some embodiments of the invention, an effective average particle size of less than about 200 nm has been achieved. With reference to the effective average particle size, it is preferred that at least 95% by weight, and preferably mine, at least 99% by weight of the particles have a particle size less than the effective average particle size, for example , 400 nm. In particularly preferred embodiments, essentially all of the particles are less than 400 nm in size. In some embodiments, essentially all of the particles are less than 250 nm in size.

As indicated in the following examples, the X-ray contrast composition of this invention comprising particles consisting of ethyl 3,5-diacetamido-2,4,6-triiodobenzoate having Tetronic® 908 adsorbed on their surface medium size particle size 166 and 188 nm) was particularly useful in imaging the blood and spleen. An X-ray contrast composition comprising particles consisting of diatrizoic acid ethyl glycolate ester crystals having Tetronic® 908 adsorbed on its surface (mean particle size 238 nm) was particularly useful in imaging the liver. . Efficient lymph node imaging has been achieved using X-ray contrast compositions comprising 170-315 nm size particles consisting of ethyl 3,5-diacetamido-2,4,6-triiodobenzoate with Tetronic¹® 908 adsorbed on its surface.

It is not fully understood why stable non-agglomerated dispersions of the above-described fine particles with narrow particle size distributions can be provided by the wet grinding technique according to this invention. Although the authors do not want to be bound by theoretical mechanisms, for intravenous administration it is postulated that the mean particle size together with the imaging agent, the selected surface modifier, and the degree to which the surface modifier remains adsorbed to the particle affects whether the agent remains in the blood or is directed to a specific site, for example it is taken up by the MPS. It is believed that certain very small particles, for example, some particles having an average particle size less than about 200 nm, tin preferentially predisposed to recirculation in the blood. Larger particles are believed to be preferentially taken up by MPS from the liver, spleen, and bone marrow.

The X-ray contrast compositions of this invention comprise the above-described particles and a carrier for it. . For example, the particles can be dispersed in an aqueous liquid that serves as a vehicle for the X-ray contrast agent. Other suitable carriers include liquid carriers such as mixed aqueous and nonaqueous solvents, eg, water and alcohols, and suitable nonaqueous solvents, such as alcohol; gels; gases, such as air, and dusts. The X-ray contrast composition may comprise about 1-99.9, preferably 2-45 and preferably 10-25% by weight of the particles described above, the remainder of the composition being carriers, additives and the like. Compositions of up to about 100% by weight of the particles are contemplated when the composition is in lyophilized form.

IS 2 139 586 T3

A process for preparing an X-ray contrast composition in accordance with this invention includes the steps of introducing a non-radioactive X-ray contrast agent, a liquid medium, a grinding medium, and optionally, a surface modifier into a container. crushing; wet milling to reduce the particle size of the contrast agent to less than about 400 nm; and separating the particles and optionally the liquid medium from the grinding vessel and the grinding medium, for example, by suction, filtration or evaporation. If the surface modifier was not present during the huomer grinding, it can be mixed with the particles later. The liquid medium, most often water, can serve as a pharmaceutically acceptable vehicle. Preferably the procedure is carried out under aseptic conditions. The X-ray contrast composition is then preferably subjected to a sterilization procedure. Sterilization can be carried out in the presence of polyethylene glycols, for example PEG 400, available from JT Baker Chemical Co., sodium dodecyl sulfate, and / or caprylic acid, which can minimize particle size growth during sterilization.

The dose of the contrast agent to be administered can be selected according to the techniques known to those skilled in the art, so that a sufficient contrast enhancing effect is obtained. Topical doses can range from 50 to 350 mg of iodine per kilogram of subject body weight, for many imaging applications. For some applications, eg lymphography, lower doses may be effective, eg 0.5-20 mg / kg.

The X-ray contrast composition may contain one or more conventional additives used to control and / or enhance the X-ray contrast properties. For example, thickening agents such as dextran or human serum albumin, buffers, viscosity regulation, suspending agents, peptizing agents, anticoagulant agents, mixing agents, and other drugs. A partial list of certain additives includes gums, sugars such as dextran, human serum albumin, sodium alginate, agar, dextrin, pectin, and sodium carboxymethylcellulose. Said additives, surfactants, preservatives and the like can be incorporated into the compositions of the invention.

A diagnostic imaging method for use in medical procedures in accordance with this invention comprises administering to the body of a test subject in need of X-rays an effective contrast-producing amount of the above-described X-ray contrast composition. In addition to human patients, the test subject can include mammalian species, such as rabbits, dogs, cats, monkeys, sheep, pigs, bovine animals, and the like. Then, at least a part of the body containing the administered contrast agent is exposed to X-rays to produce an X-ray image pattern corresponding to the presence of the contrast agent. Then the ray image diagram can be visualized. For example, any X-ray visualization technique, preferably a high contrast technique such as computerized tomography, can be applied in a conventional manner. Alternatively, the image pattern can be viewed directly on an X-ray sensitive silver halide photographic film-phosphor screen combination.

The compositions of this invention can be administered by a variety of routes depending on the type of procedure and the anatomical orientation of the tissue to be examined. Appropriate routes of administration include intravascular (arterial or venous) administration by catheter, intravenous injection, rectal administration, subcutaneous administration, intramuscular administration, intralesional administration, intraatecal administration, intracisternal administration, oral administration, administration by inhalation route. directly into a body cavity, eg, arthrography, and the like.

In addition to the preferred applications discussed above, i.e., for imaging the blood, liver, spleen, and lymph nodes, the X-ray contrast compositions of this invention are expected to also be useful as a contrast medium. angiographic, urographic contrast medium, myeloid contrast medium, gastrointestinal contrast medium, cholecystographic and cholangiographic contrast medium, arthographic contrast medium, hysterosalpingographic contrast medium, oral contrast medium and bronchographic contrast medium.

The present invention provides significant advantages compared to compositions prepared by solvent precipitation techniques, as described by Violante et al. in US Patent 4,826,689, which results in the formation of non-crystalline, solvent-contaminated particles. As noted above, such solvents are often toxic and can be very difficult, if not impossible, to remove to pharmacoeutically acceptable levels to be proactive. Solvent removal to pharmacoeutically acceptable levels often comes at such a significant cost.

ES 2 139 586 T3 bitive that is unacceptable from a commercial point of view. Violante et al. teach that the chemical precipitation process for providing particles was developed to avoid the problems of obtaining uniform particles of water-insoluble radiopaque contrast materials by conventional techniques. Indeed, the teachings of the patent depart from the present invention by the suggestion that physical methods for modifying and controlling particle size are problematic, that is, they result in preparations with unacceptably wide ranges of particle diameters and toxicity.

Furthermore, when compared to liposomes and emulsions, X-ray compositions according to the invention containing particulate contrast agent particles have a much higher iodine content. To achieve a desired level of contrast, provided by a particular amount of iodine, a small amount of material can be used. Furthermore, the X-ray compositions according to this invention are generally more storage stable than the lipid and amorphous compositions of the prior art.

The following examples illustrate the invention in more detail.

Example 1

Synthesis of WIN 8883 Ethyl 3,5-diacetamido-2,4,6-triiodobenzoate [R = -OCH₂CH₃]

In 8.11 liters of dry N, N-dimethylformamide, 1.01 kg (1.65 mol) of diatriazoic acid was added. To the vigorously stirred suspension 274 g (1.99 mol) of ground potassium carbonate were carefully added. During the addition there was significant gas evolution. Before all the suspended solids had dissolved, a second solid began to form towards the end of the carbonate addition. The mixture was stirred for 30 min at room temperature. Ethyl iodide (608 g, 3.90 mol) was added dropwise and the mixture was stirred overnight at room temperature, after which the reaction mixture was almost homogeneous. The reaction was poured into 25 liters of water, filtered and the solid was washed with water and dried under reduced pressure at 60 ° C to give 962 g (91% yield) of a white solid, mp 280-290 ° C (dec. ). Analysis for C₁₃H₁₃I₃N₂OR₄ calculated / found: C 24.32 / 24.27; H 2.05 / 1.93, N 4.36 / 4.28.

Preparation of nanocrystalline formulation of WIN 8883

A nanocrystalline formulation of WIN 8883 was prepared by putting 12.0 g of WIN 8883, 2.0 g of Tetronic 908 surfactant (BASF), and 100 ml of 1 mm ZrO beads (Zircoa, Inc.) in a glass bottle. 200 ml with a wide mouth (Qorpack). The ZrO beads were pre-treated by lamination in deionized water at 115 rpm for 24 hours followed by lamination in 1 M H2SO4 for 1 hour, rinsed with large amounts of distilled water, and air dried. 60 ml of deionized water was added and then the mixture was rolled in a US Stoneware shake mill (model # 784CVM) for 14 days at 115 rpm. At the end of this time, the suspension of particles was removed from the 1 mm ZrO medium by suction and / or filtration (see below) and placed in a 120 ml brown glass bottle. This suspension was then filtered through a series of filters (pore sizes 10 micrometers, 5 micrometers, 3 micrometers) by gravity (Nucleopore polycarbonate filters, Microstar, Inc.) in sterile plastic tubes (Kimble, flat top, graduated) .

Characterization of the properties of the nanocrystalline formulation of WIN 8883

In a suspension of WIN 8883 prepared as described above, particle size and zeta potential were characterized using a Malvern Zeta Sizer III instrument (Malvern Instruments, Ltd). A small aliquot of suspension was added to approximately 20 ml of 25 mM phosphate buffer at pH = 7.0 for analysis. This diluted sample was then introduced into the light scattering cell for photon correlation analysis. The size is recorded as a weight value of an intensity and was found to have a mean of 166 nm diameter with a zeta potential of -2.3 mV. The particle size distribution was surprisingly narrow.

These samples were then re-studied for physical stability by exposure to simulated gastric fluid (that is, prepared according to USP specifications, containing pepsin, pH adjusted to 2.5 with HCl); phosphate buffered saline (i.e. 25 mM sodium phosphate solution containing 0.9% NaCl purchased from Cellgro), and rat plasma (i.e. plasma obtained from white Sprague Dawley rats) Microscopic visualization of these added samples I confirmed for each fluid that no aggregation or flocculation occurred.

IS 2 139 586 T3

In Vivo Imaging Studies Using Nanocrystalline WIN 8883: 10% WIN 8883, 20% Tetronic 908, in Water

A suspension of WIN 8883 was prepared as described above, except 6.0 g of WIN 8883 and 1.2 g of T908 (ie, 10% w / vol WIN 8883) were used. The suspension was characterized as described above and exhibited a mean particle size of 166 nm and a zeta potential of -2.3 mV. In addition, the formulation passed all fluid stability tests and no adverse effects were observed after mouse injection. This formulation was injected by syringe into the ear vein of rabbits weighing approximately 3 kg to examine the effect on the formation of computed tomographic (CT) images of the rabbit. Imaging was carried out 5 min after injection, 30 min after injection, and 1 hour after injection in the kidney, liver and spleen areas. The dose was varied from 0.3 ml / kg to 3.0 ml / kg (from 30 mg / kg to 300 mg / kg) of compound or approximately from 16 mg 1 / kg to 160 mg 1 / kg which is a consideration. important in enhancing contrast for X-rays.

The images demonstrated an improvement in the X-ray density in the injection blood. This effect on the blood diminished at 30 min post injection and was essentially not discernible 1 h post injection. Contrast enhancement within the liver remained essentially constant over that time period. The spleen tended to improve X-ray density over time. The lower dose was minimally useful in this way, while the medium dose (i.e. 2 ml / kg or 200 mg / kg) was somewhat effective and the higher dose (i.e. 3 ml / kg or 300 mg / kg). kg) was the best for blood.

Example 2

In vivo imaging using nanocrystalline WIN 8883: 20% WIN 8883; 3.3% T908, in water

A suspension of WIN 8883 was prepared as described in Example 1 above, except that it was ground for 8 days. This suspension was characterized as in Example 1 and had a mean size of 180 nm. Stability in different fluids was determined as described in Example 1. Stability in PBS and rat plasma was observed with slight aggregation in GI fluid.

This formulation was injected by syringe into the ear vein of rabbits weighing approximately 3 kg to examine the effect on computed tomographic (CT) imaging of the rabbit. Imaging was carried out 5 min after injection, 30 min after injection, and 1 hour after injection in areas of the kidney, spleen, liver, and thoracic cavity. The dose was varied (0.5 ml / kg, 1.0 ml / kg, 1.5 ml / kg), (100 mg / kg, 200 mg / kg, and 300 mg / kg of compound) (59 mg l / kg, 118 mg l / kg, and 176 mg l / kg) respectively.

The images demonstrated a better X-ray density in the blood, liver, and spleen, especially at 5 min after injection. The improvement for blood was seen rapidly on images of the thoracic cavity within the chambers of the heart. This effect on the blood was greatly diminished at 30 min after injection and was essentially not discernible after 1 hour after injection. Contrast enhancement in the liver remained essentially constant over this time period. The spleen tended to improve X-ray density over time. These effects were seen at all dose levels with density decreasing with decreasing dose. Images taken 7 and 22 days after injection clearly demonstrated a decrease in X-ray density within the spleen. Example 3

In vivo studies using nanocrystalline WIN 8883: 20% WIN 8883; 3.3% T908, in PBS

A suspension of WIN 8883 was prepared as described except that PBS was used instead of water. Water was used to control the pH and achieve approximate equiosmolality with the blood. These particles were characterized as in Example 1 and had a mean size of 159 nm and a zeta potential of -3.5 V. The pH of this suspension was measured to be 9.5, while the osmolality was determined to be 249. mOsm / kg. While slight aggregation was observed in GI fluid, addition of this suspension in PBS or rat plasma did not give any aggregation or flocculation.

This formulation was injected by syringe into a vein of the ear of rabbits weighing approximately 3 kg, to examine the effect on the formation of computed tomographic (CT) images.

ES 2 139 586 T3 in the rabbit. Imaging was carried out 5 min after injection, 30 min after injection, and 1 hour after injection in areas of the kidneys, spleen, liver, and thoracic cavity. Dosages of 0.5 ml / kg, 1.0 ml / kg and 1.5 ml / kg (100 mg / kg, 200 mg / kg, and 300 mg / kg of compound) (59 mg l / kg, 118 mg l / kg and 176 (mg l / kg) respectively.

The images showed an increase in X-ray density in the blood, liver and spleen. The effect in the spleen was not very pronounced 5 min after the injection, while in the blood it improved a lot as evidenced by the chambers of the heart and the main blood vessels throughout the abdomen and the thoracic cavity. The spleen continued to improve over time (i.e. 30 min after injection) while blood remained almost constant over time indicating better performance (with respect to blood imaging) of this formulation versus to that observed in Examples 1 and 2. Liver intensity also remained or nearly constant over time. 1 an hour after the injection, the liver and blood began to decrease in density while the spleen continued to improve. Imaging at 7 days and 22 days after injection showed that the spleen had returned to pre-imaging X-ray densities over time.

Example 4

In Vivo Imaging Studies Using Nanocrystalline WIN 8883: 20% WIN 8883, 3.3% T908, Phosphate Buffer

A suspension of WIN 8883 was prepared as described in Example 1 except that a moderately concentrated phosphate buffer (0.1M, pH = 7.5) was used as the solution phase instead of water alone. This resulted in the suspension being at physiologically acceptable pH and osmolality (ie pH = 7.5; 344 mOsm / kg). In addition, the grinding time decreased to 22 hours. The suspension was characterized as in Example 1 and resulted in a mean particle size of 258 nm.

Images of this formulation were formed as described in Examples 2 and 3. The images demonstrated very little improvement in blood with great improvement in liver and some improvement in spleen and kidney at 5 min after injection. injection. This represents a significant effect that is believed to be the result in part of the particle size of these formulations. No mortality was observed in the rabbits with the injection or during the course of the experiment. Any improvement that might have occurred in the blood at 5 min after injection was absent at 30 min after injection. The improvement of the liver and kidney remained evident both at 30 min and 1 hour after the injection, although it diminished after 1 hour.

Example 5

In vivo studies using nanocrystalline WIN 8883: 10% WIN 8883; 1% surfactant; 1% polysorbate 20 (i.e., Tween 20), and 5% mannitol

A suspension of WIN 8883 was prepared as described in Example 1, except that only 6 g of WIN 8883 was used (i.e. 10% WIN 8883) and the surfactants used included C18H37CH2 (CON (CH3) CH2 (CHOH) 4CH2H) 2 with a structure including an alkyl chain (tail) and 2 acyclic sugar moieties as the hydrophilic part (i.e. head groups) with 0.6 g / 60 ml and a polysorbate 20 (Tween 20) with 0 , 6 g / 60 ml. In addition, the solution was made equi-osmolar with the blood by adding 5% mannitol to the suspension after completing the initial grinding procedure. This suspension was characterized as described in Example 1 and the suspension was found to be stable in GI, PBS and rat plasma.

Images of this formulation were formed as in Example 1, except that only the medium dose was administered (ie 1.5 ml / kg animal). The images showed improvement in liver X-ray contrast and that there was no improvement in blood at 5 min after injection, 30 min after injection or 1 hour after injection. The rabbits did not show adverse effects with the administration of this formulation.

Example 6

Synthesis of WIN 12901 (3,5-bis (acetylamino) -2,4,6-triiodo-benzyloxy) ethyl acetate [R = OCH₂- (CO) CH2CH3]

IS 2 139 586 T3

To 175 ml of dry N, N-dimethylformamide (DMF) 63.6 g (0.100 mol) of sodium diatrizoate and 14.7 g (0.120 mol) of ethyl chloroacetate were added and the mixture was heated on a steam bath for 6 h. The reaction was filtered while warm to room temperature and diluted to 500 ml with water. The mixture was cooled and filtered, and the collected solid was washed with water. The solid was then dissolved in 350 ml of hot DMF, filtered and added to an equal volume of water. The mixture was cooled, filtered, washed with water, and the solid was dried at 100 ° C overnight to give 53.0 g (76% yield) of a white powder, mp 269.5-270.5 ^ C. Analysis for C₁₅H₁₅I₃N₂OR₆ calculated / found: C 25.73 / 25.80; H 2.15 / 2.77; I 54.4 / 53.8.

In Vivo Imaging Studies Using Nanocrystalline WIN 12901: 10% WIN 12901, 2% T908, in Water

A suspension of WIN 12901 was prepared as described in Example 1, except that 6 g of WIN 12901 (ie 10% WIN 12901) and 1.2 g of T908 (ie 2% T908) were used. The suspension was ground for 4 days. These particles were characterized as described in Example 1. The mean particle size that was measured was 238 pm. Evaluation of fluid stability as described in Example 1 suggested stability in GI, PBS, and rat plasma.

Images were formed with this formulation as described in Example 1. The images demonstrated an enhancement of blood X-ray contrast, liver contrast and kidney contrast 5 min after injection, liver contrast enhancement and kidney at 30 min after injection and slightly less improvement in liver and kidney 1 hour after injection. The density in the liver was particularly surprising.

Example 7

In Vivo Imaging Studies Using Nanocrystalline WIN12901: 20% WIN12901, 3.3% T908, 100 mM Phosphate Buffer

A suspension of WIN 12901 was prepared as described in Example 1, except that phosphate buffer at pH 6.5 was used instead of water. This was used to control the pH and osmolality of this formulation. These particles were characterized as described in Example 1 and had a mean size of 289 nm. The pH of the samples was 6.5 and the osmolality 344 mOsm / kg. Fluid stability was evaluated as described in Example 1. This formulation was found to be stable in PBS, GI, and rat plasma.

Images were formed with this formulation as described in Example 1, except that a dose of 1.5 ml / kg animal weight was administered. Imaging studies demonstrated improvement in liver imaging at 5 and 30 min after injection with no real improvement in blood. Retention in the spleen was minimal and dissipated 2 hours after injection. Improvement in the kidney was observed at 30 minutes and 60 minutes after the injection. The liver appeared to return to original X-ray density 1 hour after injection.

Example 8

Synthesis of WIN 16318 Ethyl 2- (3,5-bis (acetylamino) -2,4,6-triiodobenzyloxy) butyrate

CH2CH3 ^|

R = OCH- (CO) -OCH2CH3

In 500 ml of dry N, N-dimethylformamide, 159 g (0.250 mol) of sodium diatrizoate and 54.5 to (0.280 mol) of ethyl 2-bromobutyrate were added. The mixture was heated on a steam bath for 20 h, cooled to room temperature, and poured into 3 liters of dilute ammonium hydroxide. The solid was filtered, washed with water, and air dried. The solid was then purified by crystallization in 50% aqueous ethanol (after treatment with decolorizing charcoal) giving two crops that were dried at 100 ° C overnight to give 121 g (66%) of a white powder, mp 288-290 ^ C (desc). Analysis for C₁₇H₁₉I₃N₂OR₆calculated / found C 28.05 / 25.36; H 2.63 / 2.55; I 52.3 / 52.3.

IS 2 139 586 T3

In Vivo In Vivo Imaging Studies Using Nanocrystalline WIN 16318:

10% WIN 16318, 2% Tween 80, 100 mM phosphate

A nanoparticle suspension of WIN 16318 was prepared as described in Example 1, except that only 6 g of WIN 16381 (i.e. 10% WIN 16318) and 1.2 g of a different surface modifier were added, that is say Tween 80 (ie 2% Tween 80) and the liquid phase was made up to 100 mM in phosphate buffer (pH 7.5). This was done to control the pH and osmolality of the formulation. These particles were characterized as described in Example 1. A mean size of 219 nm was measured after 14 days of milling. The pH of the formulation was 7.8 and the osmolality was 348 mOsm / kg. These formulations were monitored for stability in the fluids described in Example 1 and demonstrated stability in PBS and rat plasma.

Example 9

Lymphographic Imaging Using Nanocrystalline WIN 8883

A suspension prepared as described in Example 1 was used to image the lymphatic system (about 3 kg rabbits) by computed tomography (CT). The suspension was dosed by percutaneous administration through the pad of the paws of the rabbits, of 0.03 ml / kg of body weight of the animal and the images were formed 9 hours after the administration. The CT images demonstrated an enhancement of the X-ray contrast of the lymph nodes responsible for the cleansing of the anatomical areas of the rabbit into which the staformulation was injected. Better density was observed for times as long as 1 week after which the X-ray density of the synthetic ganglia returned to normal levels.

Example 10

The images were formed with a formulation prepared as described in Example 3 with a particle size of 169 pm, in rabbits with a dose of 3 ml / kg (100 mg / kg of compound; 352 mg l / kg). Bloodstream imaging at this dose was excellent until at least 2 hours after administration.