WO1996025181A1

WO1996025181A1 - Process of preparing lymphography contrast agents

Info

Publication number: WO1996025181A1
Application number: PCT/US1996/001870
Authority: WO
Inventors: H. William Bosch; Donna M. Marcera; Ronald L. Mueller; Jon R. Swanson
Original assignee: Nanosystems L.L.C.
Priority date: 1995-02-14
Filing date: 1996-02-12
Publication date: 1996-08-22
Also published as: AU4867296A

Abstract

A process of preparing nanoparticulate contrast agents used in lymphography comprising the steps of: preparing a premix of the contrast agent and a surface modifier; and subjecting the premix to mechanical means to reduce the particle size of the contrast agent, the mechanical means producing shear, impact, cavitation and attrition.

Description

PROCESS OF PREPARING LYMPHOGRAPHY CONTRAST AGENTS

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to a process for preparing nanoparticulate contrast agents used in connection with lymphography.

Reported Developments

X-Ray contrast media containing an x-ray contrast agent are extensively used in radiographic diagnostic techniques to detect diseases of soft tissues of the body, such as cancer. ymphographic technique involves the x-ray examination of lymphatic vessels and nodes using contrast media which is injected directly into a lymphatic trunk or lymph node to provide visualization of the regional lymph structures or the media is injected into the tissues containing lymph capillaries from which it is transported to the lymphatic trunks and nodes.

The development of contrast agent for use in radiographic examination of the lymphatic system has been limited. It appears that in the U.S. the only commercially available contrast agent is ETHIODOL®, which consists of a mixture of ethyl esters of iodinated poppy seed oil. ETHIODOL® ^a thick oily consistency and its administration to the patient requires cannulation and massaging because it remains at the injection site in contact with local tissue for extended time periods which may cause toxicity problems. To minimize the problems the injection site must be massaged to promote lymphatic uptake. In addition, because of its oily consistency, administration of ETHIODOL®causes pain to the patient and it often causes inflammation. For these reasons lymphography with ETHIODOL®has been replaced to a great extent by computed tomography performed without contrast enhancing media.

Recently, the prior art has reported production and utilization of nanoparticulate crystalline substances found to be desirable in both pharmaceutical compositions for prevention and treatment of diseases and radiopaque compositions for detection of abnormalities in soft tissues used in conjunction with radiographic examinations. In both types of compositions bioavailability of the administered substance is one of the essential requirements for achieving the desired results.

Methods of making finely divided drugs have been studied and efforts have been made to control the size and size range of drug particles in pharmaceutical compositions. For example, dry milling techniques have been used to reduce particle size and hence influence drug absorption. However, in conventional dry milling, as discussed by Lachman, et al., The Theory and Practice of Industrial Pharmacy, Chapter 2, "Milling", p.45 (1986), the limit of fineness is reached in the region of 100 microns (100,000 nm) when material cakes on the milling chamber. Lachman, et al. note that wet grinding is beneficial in further reducing particle size, but that flocculation restricts the lower particle size limit to approximately 10 microns (10,000 nm) . However, there tends to be a bias in the pharmaceutical art against wet milling due to concerns associated with contamination. Commercial airjet milling techniques have provided particles ranging in average particle size from as low as about 1 to 50 mm (1,000 - 50,000 nm) .

Other techniques for preparing pharmaceutical compositions include loading drugs into liposomes or polymers, e.g., during emulsion polymerization.

However, such techniques have problems and limitations. For example, a lipid soluble drug is often required in preparing suitable liposomes. Further, unacceptably large amounts of the liposome or polymer are often required to prepare unit drug doses. Further still, techniques for preparing such pharmaceutical compositions tend to be complex. A principal technical difficulty encountered with emulsion polymerization is the removal of contaminants, such as unreacted monomer or initiator, which can be toxic, at the end of the manufacturing process.

U.S. Patent No. 4,540,602 (Motoyama et al.) discloses a solid drug pulverized in an aqueous solution of a water-soluble high molecular substance using a wet grinding machine. However, Motoyama et al. teach that as a result of such wet grinding, the drug is formed into finely divided particles ranging from 0.5 mm (500 nm) or less to 5 mm (5,000 nm) in diameter.

EPO 275,796 describes the production of colloidally dispersible systems comprising a substance in the form of spherical particles smaller than 500 nm. However, the method involves a precipitation effected by mixing a solution of the substance and a miscible non-solvent for the substance and results in the formation of non-crystalline nanoparticles.

Furthermore, precipitation techniques for preparing particles tend to provide particles contaminated with solvents. Such solvents are often toxic and can be very difficult, if not impossible, to adequately remove to pharmaceutically acceptable levels to be practical. U.S. Patent No. 4,107,288 describes particles in the size range from 10 to 1,000 nm containing a biologically or pharmacodynamically active material. However, the particles comprise a crosslinked matrix of macromolecules having the active material supported on or incorporated into the matrix. U.S. Patent No. 5,145,684 discloses a process for preparing particles consisting of a crystalline drug substance having a surface modifier or surface active agent adsorbed on the surface of the particles in an amount sufficient to maintain an average particle size of less than about 400 nanometers. The process of preparation comprises the steps of dispersing the drug substance in a liquid dispersion medium and applying mechanical means in the presence of grinding media to reduce the particle size of the drug substance to an average particle size of less than 400 nm. The particles can be reduced in the presence of a surface active agent or, alternatively, the particles can be contacted with a surface active agent after attrition. The presence of the surface active agent prevents flocculation/agglomeration of the nanoparticles. The mechanical means applied to reduce the particle size of the drug substance is a dispersion mill, the variety of which include a ball mill, an attrition mill, a vibratory mill and media mill, such as sand mill, and a bead mill.

The grinding media for the particle size reduction is spherical or particulate in form and includes: Zrθ₂ stabilized with magnesia, zirconium silicate, glass, stainless steel, titania, alumina and Zr0₂ stabilized with yttrium. Processing time of the sample can be several days long. This patent is incorporated herein in its entirety by reference.

To a more limited extent the prior art also utilized microfluidizers for preparing small particle- size materials in general. Microfluidizers are relatively new devices operating on the submerged jet principle. In operating a microfluidizer to obtain nanoparticulateε, a premix flow is forced by a high pressure pump through a so-called interaction chamber consisting of a system of channels in a ceramic block which split the premix into two streams. Precisely controlled sheer, turbulent and cavitational forces are generated within the interaction chamber during microfluidization. The two streams are recombined at high velocity to produce droplet shear. The so- obtained product can be recycled into the microfluidizer to obtain smaller and smaller particles. The prior art has reported two distinct advantages of microfluidization over conventional milling processes (such as reported in U.S. Patent No. 5,145,684, supra) : substantial reduction of contamination of the final product, and the ease of production scaleup.

Numerous publications and patents were devoted to emulsions, liposomes and/or microencapsulated suspensions of various substances including drug substances produced by the use of microfluidizers. See, for example:

1) U.S. Patent No. 5,342,609, directed to methods of preparing solid apatite particles used in magnetic resonance imaging, x-ray and ultrasound.

2) U.S. Patent No. 5,228,905, directed to producing an oil-in-water dispersion for coating a porous substrate, such as wood.

3) U.S. Patent No. 5,039,527 is drawn to a process of producing hexamethylmelamine containing parenteral emulsions.

4) G. Gregoriadis, H. Da Silva, and A.T. Florence, "A Procedure for the Efficient Entrapment of Drugs in Dehydration-

Rehydration Liposomes (DRVs), " Int . J. Pharm. 65, 235-242 (1990) . 5) E. Doegito, H. Fessi, M. Appel, F. Puisieux, J. Bolard, and J.P. Devissaguet, "New Techniques for Preparing Submicronic Emulsions — Application to Amphotericine-B, : STP Pharma Sciences 4, 155-162 (1994) .

6) D.M. Lidgate, R.C. Fu, N.E. Byars, L.C. Foster, and J.S. Fleitman, "Formulation of Vaccine Adjuvant Muramyldipeptides. Part 3. Processing Optimization, Characterization and Bioactivity of an Emulsion Vehicle, " Pharm

Res . 6, 748-752 (1989) .

7) H. Talsma, A.Y. Ozer, L. VanBloois, and D.J. Crommelin, "The Size Reduction of Liposomes with a High Pressure Homogenizer

(Microfluidizer) : Characterization of Prepared Dispersions and Comparison with Conventional Methods, " Drug Dev. Ind. Pharm. 15, 197-207 (1989) .

8) D.M. Lidgate, T. Trattner, R.M. Shultz, and R. Maskiewicz, "Sterile Filtration of a Parenteral Emulsion, " Pharm . Res . 9, 860-863 (1990) .

9) R. Bodmeier, and H. Chen, "Indomethacin Polymeric Nanosuspensions Prepared by Microfluidization, " J. Cont . Rel . 12, 223- 233 (1990) .

10) R. Bodmeier, H. Chen, P. Tyle, and P. Jarosz, "Spontaneous For atin of Drug-Containing Acrylic Nanoparticles, " J. Microencap, 8, 161-170 (1991) .

11) F. Koosha, and R.H. Muller, "Nanoparticle Production by Microfluidization, " Archiv Der Pharmazie 321, 680 (1988) . However, reports are few on reducing mean particle size (hereinafter sometimes abbreviated as MPS) of water insoluble materials for use in pharmaceutical/diagnostic imaging compositions.

The present invention is directed to a process incorporating the advantages of microfluidizer process over conventional milling processes along with utilizing formulation and/or process parameters necessary for successful particle size reduction of a pharmaceutical suspension prepared by microfluidization.

The primary forces attributed to microfluidization for producing either emulsions or dispersions, and for reducing the MPS of water insoluble materials include: shear, involving boundary layers, turbulent flow, acceleration and change in flow direction; impact, involving collision of solid elements and collision of particles in the chamber of microfluidizer; and cavitation, involving an increased change in velocity with a decreased change in pressure and turbulent flow. An additional force can be attributed to conventional milling processes of attrition, i.e., grinding by friction. In reference to conventional milling process it is understood that the process involves the use of gravity, attrition and /or media mills, all containing a grinding media.

SUMMARY OF THE INVENTION In accordance with the present invention, there is provided a process of preparing stable, dispersible drug nanoparticles consisting essentially of a crystalline drug substance having a surface modifier adsorbed on the surface thereon comprising the steps of: a) dispersing a drug substance in a liquid dispersion medium; and

b) subjecting the liquid dispersion medium to the comminuting action of a microfluidizer asserting shear, impact and cavitation forces onto the drug substance contained in the liquid dispersion medium for a time necessary to reduce the mean particle size of said drug substance to less than 400 nm.

In a preferred embodiment, there is provided a process for preparing a stable, nanoparticulate formulation for lymphographic imaging consisting essentially of ethyl ester of diatrizoic acid

(hereinafter sometimes referred to as EEDA) having a polymeric surfactant adsorbed on the surface thereon.

In accordance with the invention there is also provided a method for x-ray diagnostic imaging which comprises administering to a patient an effective contrast producing amount of a composition prepared by the process of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The Contrast Aσent

The x-ray contrast composition of the present invention comprises particles of ethyl ester of diatrizoic acid ( IN8883) , i.e., ethyl-3 , 5-diacetamido- 2, 4, 6-triodobenzoate having the structure

having a polymeric surface modifier adsorbed on the surface thereof in an amount sufficient to maintain an effective average particle size of less than 400 nm. Pharmaceutical compositions containing the particles exhibit excellent utility in lymphographic x-ray contrast imaging.

EEDA is poorly soluble in water, i.e., it has a solubility of less than 10 mg per ml of water. The preferred dispersion/suspension medium for imaging formulations containing EEDA is water. Other carriers include saline solutions and phosphate buffered saline solutions.

Surface Modifiers

The particles useful in the practice of the present invention include a surface modifier. Surface modifiers useful herein physically adhere to the surface of the x-ray contrast agent. Surface modifiers can be selected from known organic and inorganic agents, such as various polymers, natural products and surfactants.

Particularly preferred surface modifiers include polyvinylpyrrolidone, tyloxapol, poloxamers 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 sequential addition of propylene oxide and ethylene oxide to ethylenediamine, available from BASF, dextran, lecithin, dialkylesters of sodium sulfosuccinic acid, such as Aerosol OT, which is a dioctyl ester of sodium sulfosuccinic acid, available from American Cyanamid, Duponol P, which is a sodium lauryl sulfate, available from DuPont, Triton X-200, which is an alkyl aryl polyether sulfonate, availalbe from Rohm and Haas, Tween 80, which is a polyoxyethylene sorbitan fatty acid ester, available from ICI Speciality Chemicals, and Carbowax 3350 and 934, which are polyethylene glycols available from Union Carbide.

The Microfluidizer

Microfluidizers are available from Microfluidics International Corporation, Newton, MA. In the practice of the present invention Microfluidics International Corporation Model M-110Y was used, which is a laboratory scale microfluidizer equipped with a sanitary pressure transducer connected to a digital data acquisition system.

As indicated, the primary forces attributed to microfluidization by the microfluidizer for producing either emulsions or dispersions, and for reducing mean particle size of water insoluble materials are:

shear, involving boundary layers, turbulent flow, acceleration and change in flow direction; impact, involving collision of the particles processed with solid elements of the microfluidizer, and collision between the particles being processed; and cavitation, involving an increased change in velocity with a decreased change in pressure, and turbulent flow.

An additional force can be attributed to attrition, i.e., grinding by friction. The M-llOY laboratory scale microfluidizer consists of an air motor connected to a hydraulic pump which circulates the process fluid. The formulation stream is propelled at high pressures (up to 23,000 psi) through a specially designed interaction chamber which has fixed microchannels that focus the formulation stream and accelerate it to a high velocity. Within the chamber the formulation is subjected to intense shear, impact and cavitation, all of which contribute to particle size reduction. After processing, the formulation stream is passed through a heat exchanger coil and can be collected or recirculated through the machine. The microfluidizer was typically used in a continuous processing mode for one hour of total processing time. The heat exchanger and interaction chamber were externally cooled with a refrigerated circulating water bath.

The use of microfluidization in pharmaceutical dosage form development has largely been limited to processing of emulsions or liposomes as previously discussed.

The proςegs of MflHinq the Nanoparticu-lates

A general procedure for preparing the particles useful in the practice of this invention follows. The x-ray contrast agent selected is obtained commercially and/or prepared by techniques known in the art as described above, in a conventional coarse form. It is preferred, but not essential, that the particle size of the coarse x-ray contrast agent selected be less than about 100 mm, as determined by sieve analysis. If the coarse particle size of the contrast agent is greater than about 100 mm then it is preferred that the coarse particles of the contrast agent be reduced in size to less than 100 mm using a conventional milling method such as airjet or fragmentation milling. The coarse imaging agent selected 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 vary from about 0.1 - 60% w/w, and preferably is from 5 - 30% (w/w) . It is preferred, but not essential, that the surface modifier be present in the premix. The concentration of the surface modifier can vary from about 0.1 to 90%, and preferably is 1 - 75%, more preferably 20 - 60%, by weight based on the total combined weight of the drug substance and surface modifier. The apparent viscosity of the premix suspension is preferably less than about 1000 centipoise.

The premix then can be transferred to the microfluidizer and circulated continuously first at low pressures, then at maximum capacity having a fluid pressure of about 18,000 psi until the desired particle size reduction is achieved. The particles must be reduced in size at a temperature which does not significantly degrade the imaging agent. Processing temperatures of less than about 30 - 40_are preferred. As used herein, particle size refers to a weight average particle size of less than about 400 nm as measured by conventional particle size measuring techniques well known to those skilled in the art, such as sedimentation field flow fractionation, photon correlation spectroscopy, or disk centrifugation. By "a weight average particle size of less than about 400 nm" it is meant that at least 90% of the particles have a weight average particle size of less than about 400 nm when measured by the above-noted techniques. In preferred embodiments of the invention, the effective average particle size is less than about 250 nm. 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% and, more preferably, at least 99% of the particles have a particle size less than the effective average, e.g., 400 nm. In particularly preferred embodiments, essentially all of the particles have a size less than 400 nm.

Illustrative example and analysis follows:

Example 1

Premixes of 15% w/w EEDA and 1.0 - 3.5% w/w of a preferred surfactant (Pluronic F68; F108 and Poloxamine 908) were prepared by manually mixing EEDA and the surfactant. The premixes were processed through the microfluidizer. First, the samples were circulated continuously through the microfluidizer at low pressure for approximately 15 seconds, thus homogenizing the samples. After the initial mixing had taken place, a 12 ml aliquot was withdrawn for pre-processing analysis. The microfluidizer was then allowed to operate at maximum capacity at fluid pressures of about 18,000 psi. Every 15 minutes during processing a 1 ml aliquot of the formulation was removed for particle size analysis. After one hour had elapsed, additional samples were withdrawn for particle size and post- microfluidization analysis.

Particle Size Analysis

Particle size distributions were determined by photon correlation spectroscopy using a Coulter N4MD particle size analyzer. Correct operation of the analyzer was confirmed by measurement of latex calibration standards (198 nm) prior to each set of determinations. The samples were prepared by vortexing for 30 seconds before analysis; 5 ml of formulation was then placed in a plastic 1 cm cuvette and diluted with filtered distilled water. All samples were measured using a run time of 200 sec.

Formulation Analyses The surfactant degradation analyses were determined by liquid chromatography using a Waters 712 WISP Autosampler equipped with a refractive index detector. The column used was a Bio-Rad, Bio-Sil SEC 250 Gel Filtration HPLC column (catalog number 125- 0062) . The mobile phase consisted of 10% methanol in a 25 mM NaCl solution. The flow rate was 0.8 ml/min and the run time used was 22 min.

Drug substance degradation analyses were performed by high performance liquid chromatography using a Waters HPLC System equipped with an ultraviolet detector (1=239 nm) . The column used was a Beckman Ultrasphere ODS (part number 235329) . The mobile phase consisted of 50% methanol and 50% phosphate buffer (pH = 3); the flow rate was 1.0 ml/min and the run time was 7 min.

Trace metal contamination levels were measured by inductively coupled plasma mass spectrometry (ICP-MS) or atomic emission spectrometry (ICP-AES) for the following elements: Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, W, and Zr.

The results of the analysis were as follows:

Particle Size Reduction Microfluidization of 15% EEDA formulations for 60 min. yielded stable nanoparticulate dispersions with mean particle sizes in the range of 247-419 nm depending on the surfactant used. The most efficient size reduction occurred with 1% DOSS which yielded a mean particle size of 247 nm, with a standard deviation of 110 nm. The polyethylene oxide/polybutylene oxide surfactants B20-3800 and B20-5000 afforded slightly larger size distributions (293 and 266 nm receptively) . The Pluronic surfactants displayed a range of efficacy with respect to particle size reduction; mean diameters ranged from 279 nm (F127) to 419 nm (F108) .

Sy fac nt stability

Formulations which contained polymeric surfactants were analyzed for surfactant degradation by gel permeation chromatography. The Pluronic surfactants each displayed a small degree of decomposition which ranged from 1.4% (F68) to 6.3% (F108) . In comparison, no detectable quantities of degradation products were observed in the formulations which contained either B20-3800 or B20-5000.

Druσ Substance Analysis

All formulations were analyzed for drug substance degradation using high performance liquid chromatography. No detectable quantities of EEDA degradation products were observed for any of the formulations tested.

Trace Metal Concentrations The processed dispersions were analyzed for the presence of trace metal contamination which was found to be minimal. ICP-MS showed that for al formulations which contained polymeric surfactants, the concentrations of all trace metals assayed were very low. Levels of Al were less than 1 ppm, and the highest observed concentration of Fe was 1.1 ppm. The quantities of Cr and Ni did not exceed 0.32 and 0.13 ppm respectively. The formulation which contained DOSS had appreciably higher trace metal concentrations including 0.7 ppm Cr, 1.4 ppm Fe, and 0.36 ppm Ni. The process of the present invention represents a substantial improvement over media and ball milling in providing nanoparticulate drug formulations. The results of experiments show that microfluidization can be used to reduce particle size distribution of lymphography contrast agent in a very short period of time, without causing unacceptable decomposition of the drug substance or excipients. Further, the process introduces little or no contamination in the form of trace metals and therefore may be especially well- suited for use in the preparation of nanoparticulate parenteral products in general.

The invention has been described in detail with particular reference to the preparation of a lymphographic x-ray contrast agent, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

Claims

WBAT IS CIAIMEP jg;

1. A process for preparing particles consisting essentially of 99.9 - 10% by weight of a crystalline drug substance having a solubility in water of less than 10 mg/ml, said drug substance having a polymeric surface modifier adsorbed on the surface thereof in an amount of 0.1 - 90% by weight and sufficient to maintain an average particle size of less than about 400 nm, said method comprises the steps of:

preparing a premix of said crystalline drug substance and said surface modifier by mixing them; and

subjecting said premix to mechanical means to reduce the particle size thereof to less than 400 nm by the action of said mechanical means which produces shear, impact, cavitation and attrition.

2. The process of claim 1 wherein said crystalline drug substance is ethyl ester of diatrizoic acid.

3. The process of claim 1 wherein said surface modifier is a low molecular weight anionic surface active agent.

4. The process of claim 1 wherein said surface modifier is a polyethylene oxide/polypropylene oxide copolymer.

5. The process of claim 1 wherein said surface modifier is a polyethylene oxide/polybutylene oxide copolymer.

6. The process of claim 3 wherein said surface modifier is dioctyl sulfosuccinate. 96/25181 PCMJS96/01870

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7. A pharmaceutical composition for lymphographic examination comprising nanoparticles prepared by the process of claim 1 in combination with a pharmaceutically acceptable carrier.

8. A method for medical x-ray diagnostic imaging comprising administering to the body of a test subject an effective contrast producing amount of the drug substance particles of claim 1.

9. A method for medical x-ray diagnostic imaging comprising administering to the body of a test subject an effective contrast producing amount of the composition of claim 7.