CA2120130A1

CA2120130A1 - Treated apatite particles for medical diagnostic imaging

Info

Publication number: CA2120130A1
Application number: CA002120130A
Authority: CA
Inventors: Edward A. Deutsch; Karen F. Deutsch; William P. Cacheris; William H. Ralston; David H. White; Steven R. Woulfe
Original assignee: Individual
Current assignee: Mallinckrodt Inc
Priority date: 1991-10-22
Filing date: 1992-10-21
Publication date: 1993-04-29
Also published as: US5468465A; DE69231627D1; AU2886492A; ATE198423T1; AU7034596A; US5344640A; ES2152932T3; WO1993007905A2; AU686523B2; AU674291B2; US5690908A; EP0610333B1; DE69231627T2; EP0610333A1; US5609850A; JPH07500823A; WO1993007905A3

Abstract

Treated apatite particles are disclosed for enhancing medical diagnostic imaging such as magnetic resonance imaging ("MRI"), magnetic resonance spectroscopy ("MRS"), magnetic resonance spectroscopy imaging ("MRSI"), X-ray diagnostic imaging, and ultrasound imaging. Novel coating and manufacturing techniques are disclosed to control particle size and particle aggregation resulting in compositions for organ specific imaging of the liver, spleen, gastrointestinal tract, or tissue disease states. Depending on the diagnostic imaging technique, apatite particles are treated to be paramagnetic, radiopaque, or echogenic. The apatite particles may also be fluorinated to form stable fluoroapatite compositions useful for 19F imaging. Also disclosed are diagnostic compositions and methods of performing medical diagnostic procedures which involve administering to a warm-blooded animal a diagnostically effective amount of the above-described apatite particles and then performing the medical diagnostic procedure.

Description

W O 93/07905 PC~r/US92/09032 TR~TED APATITF PP~RTIC~ES FOR
~DEDIC~iL DIAGNOSTIC I~L~GING :

: .
BACKGROUI~D OF THE I~rVENTION :

This invention relates to treated apatite particles and their use in medical diagnostic imaging techniques, such as magnetic resonance imaging ("MRI"), magnetic resonance spectroscopy ~"MRS"), magnetic resonance spectroscopy imaging (~MRSI"), X-ray, and ultrasound. The present invention also includes novel apatite particles, manufacturing methods, and coating compositions which prevent particle aggregation, improve particle stability, .and permit functionalization of the particle surface.
The use of contrast agents in diagnostic medicine is -rapidly growing. In X-ray diagnostics, for example, increased contrast of internal organs, such as the kidneys, the urinary tract, the digestive tract, the vascular system of the heart ~angiography), etc., is obtained by administering a contrast agent which is substantially radiopaque. In conventional proton NRI diagnostics, increased contrast of internal organs and tissues may be obtained by administering compositions containing paramagnetic metal species which increase the relaxivity of surrounding protons. In ultrasound diagnostics, improved contrast is obtained by administering compositions havin~
acoustic impedances different than that of blood and other tissues.
Often it is desirable to image a specific organ or tissue. Effective organ- or tissue-specific contrast agents accumulate in the organ or tissue of interest.
From the foregoing, it would be an important advancement in the art to provide organ specific medical diagnostic imaging agents. Specifically, it would be an improvement in the art to provide organ specific NRI, X-ray, and ultrasound contrast agents.
Such medical diagnostic imaging agents are disclosed and claimed herein.

.

WO 93/0790~ PCI/US9~/09032 a ~ ~

SU~lARY OF THE INVENTION
The present invention provides methods and compositions for improved medical diagnostic ima~ing. The imaging agents are derived from apatite particles S including, but not limited to, hydroxyapatite (sometimes referred to as Uhydroxylapatite''), fluoroapatite, iodoapatite, carbonate-apatite, and mixtures and derivatives thereof. As used herein, the term fluoroapatite includes pure fluoroapatite as well as mixtures of fluoroapatite, hydroxyapatite, iodoapatite, and carbonate-apatite. Likewise, hydroxyapatite, iodoapatite, and carbonate-apatite are intended to include the pure and mixed forms. Since hydroxyapatite is a natural bone constituent, it is well tolerated and generally safe.
By controlling the particle size and route of administration, organ specific imaging of the liver, spleen, gastrointestinal tract, or blood pool is obtained.
Typical particle sizes are in the range from about 10 nm to about 50 ~n depending upon the organ or disease state to be imaged, the mechanism of delivery of the particles to the organ or disease state, and the medical diagnostic ima~ing technique utilized. In addition, apatite particles within the range from 1 nm to about 50 nm may also be used to image the blood pool.
Depending on the diagnostic imaging technique, apatite particles are treated to be paramagnetic, radiopaque, or echogenic. For example, paramagnetic species may be incorporated into the apatite particles to improve magnetic resonance contrast, and radiopaque species may be incorporated into the apatite particles to provide X-ray contrast. Particle density, and corresponding echogenic characteristics, can be controlled to impart low or high acoustic impedance relative to blood. The apatite particles may also be fluorinated to form stable, fluoroapatite compositions useful for l9F imaging.

W093/07~5 PCT/US92/09032 2i2~a~Q :~' Incorporating a paramagnetic metal species in flouroapatite or hydroxyapatite particles may reduce l9F and proton relaxivity, thereby enhancing MRI, MRS, or MRSI.
Also disclosed are diagnostic compositions and methods of performing medical diagnostic procedures which involve administering to a warm-blooded animal a diagnostically effective amount of the above-described apatite particles and then performing the medical diagnostic procedure.

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods and compositions for improved medical diagnostic imaging. As used herein, medical diagnostic imaging includes magnetic resonance imaging (~MRI~), magnetic resonance spectroscopy (~MRS~), magnetic resonance spectroscopy imaging (~MRSI~
X-ray contrast imaging, and ultrasound imaging. The diagnostic imaging agents of the present invention are derived from apatite-like particles.
As used herein, apatite particles include apatite-like minerals of the general formula CanM~XrY~, where M is a paramagnetic, radiopaque, or radioactive metal ion or stoichiometric mixture of metal ions having a valence of 2+
or 3+, X is a simple anion, Y is a tetrahedral oxyanion, carbonate, tetrahedral anion, or mixtures thereof, m is from 1-10, n is from 1-10, and r and s are adjusted as needed to provide charge neutrality. Where M is a 2+ metal ion, then m + n = 10, and where M is a 3+ metal ion, then m + 1.5n = 10.
Possible metal ions which can be used in the apatite particles of the present invention include: chromium(III), manganese(II), iron(II), iron(III), praseodymium~III), neodymium(III), samarium(III), ytterbium~III), gadolinium~III), terbium~III), dysprosium(III), holmium(III), erbium(III), or mixtures of these with each other or with alkali or alkaline earth metals. Iypical W093/n7~5 PCT/US92/09032 1~ ~3~

simple anions which can be used in the apatite particles of the present invention include: OH-, F-, sr- I-, l~[CO32~], or mixtures thereof. The tetrahedral oxyanions used in the present invention may optionally include radiopaque metals or radioactive metals. Suitable tetrahedral oxyanions are non-oxidizing and stable to hydrolysis. Examples of suitable tetrahedral oxyanions for use in the present invention include: Aso43- ~ wo42- ~ MoO42- ~ vo43- ~ sio44- ~ and GeO44~.
By controlling the particle size, organ specific imaging of the liver or gastrointestinal tract is obtained.
When apatite particles having a size in the range from about 5 nm to about 2 ~m are injected into the vascular system, the particles collect in the liver or spleen (the RES system) because the normal function of these organs is to purify the blood of foreign particles. Once the particles have collected in the liver or spleen, these organs may be imaged by the desired medical diagnostic imaging techni~ue. Apatite particles having a larger size in the range from 200 nm to about 50 ~m may be used to image the gastrointestinal ("GI~) tract. Larger particles may be conveniently administered orally or rectally accordins to conventional administration techniques.
Depending on the diagnostic imaging technique, apatite particles are treated to be paramagnetic, radiopaque, or echogenic. For example, paramagnetic metal species may be incorporated into the apatite particles to improve magnetic resonance contrast, and radiopaque species may be incorporated into the apatite particles to provide X-ray contrast. Particle density, and corresponding echogenic characteristics, can be controlled to impart low or high acoustic impedance relative to blood. The apatite particles may also be fluorinated to form 9table, nontoxic fluoroapatite compositions useful for l9F imaging. The presence of a paramagnetic metal species in fluoroapatite 2120130 ~-or hydroxyapatite particles may reduce l9F and proton relaxivity, thereby enhancing MRI, MRS, or MRSI.

Preparation of APatite Particles Methods for preparing hydroxyapatite, having the formula Ca~0(OH)2~PO4)6, are well known in the art. Apatites in which the OH- is replaced with simple anions, including F-, Br~, I-, or %[Co32~], may be prepared by modifying the process for preparing hydroxyapatite. Apatite derivatives in which calcium is replaced by metal ions, such as paramagnetic, radiopaque, or radioactive metal ions, may also be prepared and used within the scope of the present invention. Useful apatites may also be prepared by replacing phosphate with oxyanions or tetrahedral anions containing radiopaque or radioactive metal species.
Stoichiometric pure hydroxyapatite has a Ca:P ratio of 1.67:1. The major impurity found in hydroxyapatite is tricalcium phosphate, Ca3(PO~) 2~ known as ~TCP~. This i.npurity can be detected by deviation from the 1.67:1 Ca:P
ratio ~for large amounts of impurity) or by X-ray diffraction for impurity le~els down to 1 percent.
Stoichiometric hydroxyapatite is prepared by adding an ammonium phosphate solution to a solution of calcium/
ammonium hydroxide. To minimize the amount of TCP formed, it i5 important to have excess calcium throughout the addition process.

Apatite Particles for MRI A~lications The techni~ue of MRI encompasses the detection of certain atomic nuclei (those possessing magnetic dipole moments) utilizing magnetic fields and radio-fre~uency radiation. It is similar in some respects to X-ray computed tomography t~CT~) in providing a cross-sectional display of the body organ anatomy with excellent resolution W093/07~5 PCT/US92/0~32 ~ ~9 ~3~

of soft tissue detail. The technique of MRI advantageously avoids the use of ionizing rad}ation.
The hydrogen atom, having a nucleus consisting of a single unpaired proton, has the strongest magnetic dipole moment of any nucleus. Since hydrogen occurs in both water and lipids, it is abundant in the human body. Therefore, MRI is most commonly used to produce images based upon the distribution density of protons and/or the relaxation times of protons in organs and tissues. Other nuclei having a net magnetic dipole moment also exhibit a nuclear magnetic resonance phenomenon which may be used in magnetic resonance applications. Such nuclei include carbon-13 (six protons and seven neutrons), fluorine-19 (9 protons and 10 neutrons), sodium-23 ~11 protons and 12 neutrons), and phosphorus-31 (15 protons and 16 neutrons).
In an MRI experiment, the nuclei under study in a sample (e.g. protons, l9F, etc.) are irradiated with the appropriate radio-frequency (~RF~) energy in a controlled gradient magnetic field. These nuclei, as they relax, subsequently emit RF energy at a sharp resonance frequency.
The resonance frequency of the nuclei depends on the applied magnetic field.
According to known principles, nuclei with appropriate spin when placed in an applied magnetic field (B, expressed generally in units of gauss or Tesla (10q gauss)) align in the direction of the field. In the case of protons, these nuclei precess at a frequency, F, of 42.6 MHz at a field strength of 1 Tesla. At this frequency, an RF pulse of radiation will excite the nuclei and can be considered to tip the net magnetization out of the field direction, the extend of this rotation being determined by the pulse, duration and energy. After the RF pulse, the nuclei ~relax~ or return to equilibrium with the magnetic field, emitting radiation at the resonant frequency. The decay of the emitted radiation is characterized by two relaxation W093/079~ PCT/US92/0~32 212~1~ 0 times, Tl and T2. Tl is the spin-lattice relaxation time or longitudinal relaxation time, that is, the time taken by the nuclei to return to equilibrium along the direction of the externally applied magnetic field. T2 is the spin-spin S relaxation time associated with the dephasin~ of the initially coherent precession of individual proton spins.
- These relaxation times have been established for various ~luids, organs, and tissues in different species of mammals.
For protons and other suitable nuclei, the relaxation times Tl and T2 are influenced by the environment of the nuclei (e.g., viscosity, temperature, and the like). These two relaxation phenomena are essentially mechanisms whereby the initially imparted radio-frequency energy is dissipated to the surrounding environment. The rate of this energy loss or relaxation can be influenced by certain other nuclei or molecules ~such as nitroxide radicals) which are paramagnetic. Chemical compounds incorporating paramagnetic nuclei or molecules may sub~tantially alter the T~ and T2 values for nearby nuclei having a magnetic dipole moment. The extent of the paramagnetic effect of the given chemical compound is a function of the environment within which it finds itself.
In general, paramagnetic ions of elements with an atomic number of 21 to 29, 42 to 44 and 58 to 70 have been found effective as MRI contrasting agents. Examples of suitable paramagnetic ions include chromium(III), manganese(II~, iron(II), iron(III), cobalt~II), nic~el(II), copper(II), praseodymium~III), neodymium(III), 3~ samarium(III), gadolinium~III), dysprosium III), and ytterbium~III). Certain molecules, such as nitroxide radicals, also exhibit paramaqnetic properties.
Paramagnetic metal ions may be incorporated into the apatite structure by replacement of calcium sites. Apatite doping in the range from about 1% to 100% is possible, ~; ' W093/07gO5 PCT/US92/~32 3`~

depending upon the particular metal species. In most cases, apatite doping with metal ions in the range from about 1% to 25~ is expected. Currently, the preferred metals from a toxicity and efficacy viewpoint are iron and manganese. With iron doped hydroxyapatite particles, any iron released from metabolized or solubilized particles would join the body~s pool of iron, with calcium and phosphate also going to their respective body pools.
Manganese is preferred because of its higher relaxivity properties and affinity for liver tissue. Moreover, the li.ver has a clearance mechanism for manganese, thereby reducing residual toxicity.
Metal doped hydroxyapatite is prepared by mixing a basic (pH 12) phosphate solution with a ~15 calcium/paramagnetic metal solution at native pH.
-~Alternatively, the calcium/paramagnetic metal solution could be basic (pH 12) if the solution also contains a ligand to prevent hydrolysis of the paramagnetic metal.
The ligand could either be left in the hydroxyapatite matrix or ~ashed out~ by sintering the hydroxyapatite between 200C and 1100C. Any strong chelating ligands may be used, such as polyamino polycarboxylic acid derivatives which are well known in the art.
It has been found that the paramagnetic ions incorporated into the apatite particle tend to oxidize during particle synthesis. To prevent metal oxidation, manufacturing techniques have been developed to minimize the amount of oxygen in the aqueous reactant solutions.
For example, two such manufacturing techniques are (1) synthesis at high temperature, such as 100C and (2) degassing the aqueous reactant solutions with an inert gas such as argon, nitrogen, or helium. An unexpected benefit of these techniques is the ability to prepar~ smaller particles, in the range from 50 nm to about 1 ~m.

W093/07~5 PCT/US92/09032 2l2al~

Antioxidants, such as gentisic acid and ascorbic acid, added durin~ apatite particle synthesis may also be used to prevent metal ion oxidation. Reducing agents, such as NaBH4, have been found to reduce metal ions that are unintentionally oxidized during apatite particle synthesis.
Paramagnetic apatite particles may also be prepared by adsorbing paramagnetic metal ions onto the particle surface. For example, manganese can be surface-adsorbed to hydroxyapatite particles by taking a slurry of hydroxy-apatite, adding Mn~NO3) 2 and applying energy, such asultrasonic power or heat, to the resulting mixture. The resulting mixture can be separated by either centrifugation and decantation or by filtration. The resulting solid is washed with large amounts of water to remove excess manganese. The same procedure may be used with other paramagnetic cations. The amount of manganese adsorbed onto the particle surface, as a percentage of the total calcium in the particle, is in the range from about 0.1% to about 10%. Such particles exhibit very high relaxivities and rapid liver enhancement in magnetic resonance imaging studies.
Paramagnetic metal species may also be adsorbed onto apatite particle surfaces through the use of bifunctional coating agents. Examples of possible bifunctional coating agents are chelating agents having one or more phosphonate groups capable of adsorption to the apatite particle surface. One currently preferred bifunctional coating agent is the functionalized polyphosphonate diethylenetri-aminepenta(methylenephosphonic acid), abbreviated DETAPMDP, having the following structure:

W093/07~5 PCT/US92/~9032 2~ 2-,N N ~
2-~p~ 2-~2 Once adsorbed to the apatite particle surface, the bifunctional coating agent may form complexes with paramagnetic metal ions. These particles also exhibit very high relaxivities and rapid liver enhancement in magnetic resonance imaging studies.
In some cases, the concentration of nuclei to be measured is not sufficiently high to produce a detectable MR signal. For instance, since l9F is present in the body in very low concentration, a fluorine source must be administered to a subject to obtain a measurable MR signal.
Signal sensitivity is improved by administering higher concentrations of fluorin~ or by coupling the fluorine to a suitable ~'probe" which will concentrate in the body tissues of interest. High fluorine concentration must be balanced against increased tissue toxicity. It is also currently believed that a fluorine agent should desirably contain magnetically equivalent fluorine atoms in order to obtain a clear, strong signal.
Fluoro~patites, useful as l9F imaging agents, are prepared by replacing the OH- with stoichiometric or non-stoichiometric ~uantities of F-. Fluoroapatites may also be synthesized with organic phosphate esters using the procedures described by M. Okazaki, ~Fluoridated Hydroxyapatites Synthesized With Or9anic Phosphate Ester,~
Biomaterials, Vol. 12, pp. 46-49, ~1991). It is currently believed that all of the fluorine atoms in fluoroapatite are chemically and magnetically e~uivalent. Since fluoroapatite has a high molar content of identical WO93/Q7905 PCT/US92/0~32 212Ul~(~

fluorine atoms, it may be advantageously used as a low concentration l9F MRI agent. Fluoroapatite may also be doped with paramagnetic metal species, as described above, to reduce l9F and proton relaxivity, thereby enhancing MRI, MRS, or MRSI.

Apatite Particles for X-raY Contrast A~lications The apatite particles described herein may also be adapted for delivery of radiopaque species into the body for X-ray contrast. Typical radiopaque species which may be incorporated into the apatite particles include heavy metals, iodine, or iodinated XRCM.
Iodoapatites are prepared by replacing the OH- of hydroxyapatite with stoichiometric or non-stoichiometric quantities of I-. Because iodine is substantially radiopaque, iodoapatites may be used as X-ray contrast media (~XR-~ ). By controlling the particle size, iodoapatite particles may be used to ima~e the liver or spleen of the RES or the gastrointestinal tract.
Commercially available XRCM, such as Ioversol, may be incorporated in apatite particles during particle precipitation. ~n the case of hydroxyapatite particles, the XRCM may be included in either the phosphate or calcium solution. The xRCM is preferably in sufficiently high concentration that upon precipitation of the apatite particles, the XRCM has a concentration in the particles in the range from about 1% to about 25%, by weight.
Certain radiopaque heavy metals, such as bismuth, tungsten, tantalum, hafnium, lanthanum and the lanthanides, barium, molybdenum, niobium, zirconium, and strontium may also be incorporated into apatite particles to provide X-ray contrast. The radiopaque metals are incorporated into apatite particles in the same manner as paramagnetic metal ions, described above.

W093/07~5 PCT/US92/0~032 Apatite Particles for Ultrasound A~lications Ultrasound is a medical diagnostic technique in which sound waves are reflected differently against different types of tissue, depending upon the acoustic impedance of these tissues. There is interest in being able to use some type of contrast agent to obtain an amplification of specific organs. Hydroxyapatite particles may be made echogenic by either of two mechanisms: (1) reflection off high density hydroxyapatite particles or (2) reflection off air trapped within low density hydroxyapatite particles.
Since hydroxyapatite is a porous material, small pockets of gas within the particles render them echogenic, with an impedance less than blood. An ultrasound contrast media would be provided in a two-vial kit form: one vial containing dry hydroxyapatite and the other vial containing a diluent.
For axample, appropriately sized particles would be synthesized using a volatile organic solvent and then dried by freeze-drying or lyophilization. The resulting dried particles would have pores filled with gas. Just prior to use, a second vial containing a specific volume of a sterile aqueous diluent, such as isotonic saline and/or buffer, can be aspirated and added to the vial of the dried hydroxyapatite. The slurry is then mixed and immediately injected. Enough gas remains in the pores to provide echogenicity in vivo for ultrasound contrast.
Alternatively, carbonate can be incorporated into the hydroxyapatite matrix. Appropriately sized particles would be dried as described above. The diluent vial would contain a weak biocompatible acid such as, but not limited to, acetic acid, citric acid, NaH2P0~, etc. The diluent and hydroxyapatite particles are mixed to allow the acid to react with the carbonate and form carbon dioxide in the particle pores according to equation A, below . The 2~f~0~

mixture would then be injected in vivo and ultrasound imaging of the desired vasculature would proceed.

co32- + 2H~ ~ CO2 + H2O (A) Echogenic hydroxyapatite particles with an impedance higher than blood may also be prepared. Prolonged heating or sintering at high temperatures can render hydroxyapatite, with or without additives, into a hardened, less porous material that is denser than blood. Dense hydroxyapatite particles can transmit sound faster than blood, thereby providing an echogenic material having an impedance higher than blood.
High impedance ultrasound contrast media may be provided as either a pre-mixed single vial formulation or a two-vial kit form. Appropriately sized particles formed and heated at optimum conditions would ultimately be formulated with a biocompatible aqueous diluent such as, but not limited to, isotonic saline and/or a buffer.
Although the foregoing discussion has focused on the use of treated hydroxyapatite particles for ultrasound contrast, it will be appreciated that other apatite particles may also be treated and used as ultrasound contrast agents.
Controllina the Particle Size and Aa~reqation Various techniques are available to control the apatite particle size. For example, slower mixing rates (introduction of the precipitating anion or cation), larger solution ~olumes, higher reaction temperatures, and lower concentrations generally result in smaller particles.
In addition, sonication during precipitation, turbulent flow or impingement mixers, homogenization, and pH
modification may be used to control particle size.

3~

Procedures for preparing monodispersed colloidal particles that are known in the art may be adapted for preparin~ submicron apatite particles. E. Matijevic, "Production of Monodispersed Colloidal Particles,~ Annual Review of Material Science, volume 15, pages 483-516, 1985, which is incorporated herein by reference, describes methods for controlling the release of precipitating anions and cations. For example, when urea, CO (NH2) 2~ is heated, hydroxide ions are slowly liberated which can cause precipitation of hydroxyapatite as submicron particles.
Likewise, precipitating cations can be released slowly by decomposition of metal complexes, such as organometallic compounds.
In addition to chemical means for controlling the release of precipitating ions, mechanical means, such as computer controlled autoburets, peristaltic pumps, and syringes, may also be used to control the release of ; precipitating ions. Commercially available autoburets are capable o~ releasing solutions at rates as low as 10 ~L/minute. In the future as computer controlled equipment improves, it is expected t`-~t even slower release rates may be obtained.
Due to the small size and nature of apatite particles, they tend to aggregate. Particle aggregation may be reduced by coating the particles. Although the reasons apatite particles aggregate is not fully understood, it has been found that several different coating agents are able to inhibit particle aggregation. For example, apatite particles may be stabilized by treatment with coating agents such as di- and polyphosphonate-containing compounds, such as hydroxyethyldiphosphonate (~EDP), pyrophosphate, aminophosphonates; carboxylates and polycarboxylate-containing compounds such as oxaltes and citrates; alcohols and polyalcohol-containing compounds;
phosphates and polyphosphate-containing compounds; sulfates W093/07~5 PCT/US92/09032 2 1 ~

and sulfate-containing compounds; sulfonates and sulfonate-containing compounds; and biomolecules such as peptides, proteins, antibodies, and lipids. Such coating agents stabilize the small apatite particles by reducing further particle growth and promoting particle suspension.
Stabilized apatite particles are desirable for in vivo use as medical diagnostic imaging agents. Apatite particle can also be stabilized by addition of small amounts of calcium sequestering anions, such as citrate and oxalate.
Such anions, which coordinate calcium, may effectively stabiliz~ small apatite particles.
When used in magnetic resonance imaging, particle relaxivity is enhanced by allowing more water accessible to the particle surface. By limiting particle size and increasing the available surface area, improved relaxivity is observed.
In addition to the coating agents identified above, conventional particle coating techniques may also be used in the manufacturing processes of the present invention.
Typical coating techni~ues are identified in International Publication Numbers WO 85~02772, WO 91~02811, and European Publication Number EP 0343934, which are incorporated by reference.
For instance, agglomerated particles may be disrupted by mechanic~l or chemical means and then coated with polymers such as carbohydrates, proteins, and synthetic polymers. Dextran having a molecular weight in the range from about 10,000 to about 40,000 is one currently preferred coating material. Albumin and surfactants, such as tween 80, have also been used to reduce particle aggregation. One co mon characteristic of useful apatite coating agents is their ability to modify the particle surface charge, or zeta potential.
The currently preferred mechanical means for disrupting or subdividing agglomerated particles is W093/07~5 PCT/US92/0~32 sonication, but other means such as heating, other forms of particle energization, such as irradiation, and chemical means, such as pH modification or combinations of these types of tre~tment, such as pH modification combined with sonication may be used.

Functionalized Apatite Particles Apatite particles may be prepared with coating a~ents containing reactive functional groups such as amine, active ester, alcohol, and carboxylate. Such functional groups may be used to couple apatite particles to paramagnetic metal chelates, to organ or tissue specific peptides or proteins, and to antibodies. An example of one possible coating agent having a reactive functional group is the following HEDP derivative:

~32-~32-Those skilled in the art will appreciate that other coating agents, modified to contain various reactive functional groups, may be used in the present invention.

Diaqnostic Pharmaceutical Formulations The apatite particles of this invention are preferably formulated into diagnostic compositions for enteral or parenteral administxation. For example, parenteral formulations advantageously contain a sterile aqueous solution or suspen~ion of treated apatite particles according to this invention. Various techniques for preparing suitable pharmaceutical solutions and suspensions W093/0790s PCT/US92/09032 212~1 3~

are known in the art. Such solutions also may contain pharmaceutically acceptable buffers and, optionally, electrolytes such as sodium chloride. Parenteral compositions may be injected directly or mixed with a large volume parenteral composition for systemic administration.
Formulations for enteral administration may vary widely, as is well-known in the art. In general, such formulations include a diagnostically effective amount of the apatite particles in aqueous solution or suspension.
Such enteral compositions may optionally include buffers, .surfactants, thixotropic agents, and the like.
Compositions for oral administration may also contain flavoring agents and other ingredients for enhancing their organoleptic qualities.
The diagnostic compositions of this invention are used in a conventional manner in magnetic resonance, X-ray, and ultrasound procedures. The diagnostic compositions are administered in a sufficient amount to provide adequate visualization, to a warm-blooded animal either systemically or locally to an organ or tissues to be imaged, then the animal is subjected to the medical diagnostic procedure.
Such doses may vary widely, depending upon the diagnostic technique employed as well as the organ to be imaged.
The following examples are offered to further illustrate the present invention. These examples are intended to be purely exemplary and should not be viewed as a limitation on any claimed embodiment.

ExamPle 1 Pre~aratlon of Hydroxya~atite A calcium nitrate solution was prepared by adding 1,18 g Ca~NO3)2-4H20 to 20 mL deionized water such that the final lCa2~]=0.25 M. The calcium nitrate solution pH was adjusted to a pH of ll with ammonium hydroxide. An ammonium phosphate solution was prepared by adding 0.396 g W093/07~05 PCT/US92/09032 ~ ~r~a ~ 18 (NH4 )2HPO4 to 5 mL of deionized water. The pH of the ammonium phosphate solution was adjusted to a pH of 11 with ammonium hydroxide. The ammonium phosphate solution was injected into the calcium nitrate solution and vigorously stirred. The resulting precipitated particles were examined under a microscope and estimated to have particle sizes greater than 10 ~m.

Example 2 Pre~aration of Hydroxya~atite Hydroxyapatite particles were prepared according to the procedure of Example 1, except that the pH of the calcium nitrate solution was not adjusted to pH 11. The ammonium phosphate solution was injected into the calcium nitrate solution and vigorously stirred. The resulting precipitated particles were examined under a microscope and estimated to have particle sizes greater than 10 ~m. `-.
Exam~le 3 Pre~aration of Hydroxya~atite A calcium nitrate solution was prepared by adding O.68 g Ca(NO3)2-4H2O to 5 mL deionized water such that the [Ca2']=0.58 M. The calcium nitrate solution pH was adjusted to a pH of 11 with ammonium hydroxide. An ammonium phosphate solution was prepared by adding 0.22 g ~NH4)~HP04 to 10 mL of deionized water such that the [HPO42-]=0.17 M.
The pH of the ammonium phosphate solution was adjusted to 11 with ammonium hydroxide. The ammonium phosphate solution was dripped into a vigorously stirred calcium nitrate solution over 30 minutes. After mixing, the final [Ca2~=0.19 M. The resulting precipitated particles were examined under a microscope and estimated to have particle sizes of approximately 1 ~m.

W093/07905 PCT/~S92~0~32 2~ 2~13~

Example 4 ~re~aration of Hydroxya~atite Do~ed with a Paramagnetic Metal Ion A metal ion solution was prepared by adding 1.18 g Ca(NO3)2-4H2O and 0.202 g Fe~NO3)3-9H2O to 20 mL deionized water. An ammonium phosphate solution was prepared by adding 0.396 g (NH4) 2HPO4 to 5 mL of deionized water. The pH of the ammonium phosphate solution was adjusted to 11 with ammonium hydroxide. The ammonium phosphate solution was injected into the metal ion solution and vigorously stirred. The resulting precipitated particles were examined and found to have particle sizes greater than 10 ~- ..

Exam~le 5 Preparation of Fluoroapatite Fluoroapatite is prepared by mixing 5 mL of a 0.58 M
solution of calcium fluoride with 10 mL of a 0.17 M
ammonium phosphate solution at native pH. The calcium 2~ fluoride solution is dripped into a vigorously stirred ammonium phosphate solution over 30 minutes. The resulting precipitated particles are examined under a microscope and estimated to have particle sizes of approximately 1 ~m.

Exam~le 6 Preparation of Fluoroa~atite Fluoroapatite is prepared by mixing 5 mL of a 0.~8 M
solution of calcium nitrate with 10 mL of solution containing 0.17 M ammonium phosphate and 0.17 M ammonium fluoride. The calcium nitrate solution is dripped into a vigorously stirred ammonium phosphate and ammonium fluoride solution over 30 minutes. The resulting precipitated particles are examined under a microscope and estimated to have particle sizes of approximately 1 ~m.

W093/07~5 PCT/US92/~32 Example 7 Pre~aration of Fluoroa~atite Do~ed with a Parama~netic Metal Ion Fluoroapatite doped with a paramagnetic metal ion is prepared according to the procedure of Example 5, except that the calcium fluoride solution also contains 0.058 M
manganese nitrate. The calcium fluoride~manganese nitrate -solution is dripped into a vigorously stirred ammonium phosphate solution over 30 minutes. The resulting precipitated particles are examined under a microscope and estimated to have particle sizes of approximately 1 ~m.
,-:
ExamPle 8 PreDarat~on of Iodoapatite lS Iodoapatite is prepared by mixing 5 mL of a 0.58 M
~ solution of calcium iodide with 10 mL of a 0.17 M ammonium -~ phosphate solution at native pH. The calcium iodide solution is dripped into a vigorously stirred ammonium phosphate solution over 30 minutes. The resulting precipitated particles are examined under a microscope and estimated to have particle sizes of approximately 1 ~m~

Exam~le 9 Pre~aration of Iodoa~atite Iodoapatite is prepared by mixing 5 mL of a 0.58 M
solution of calcium nitrate with 10 mL of solution containing 0.17 M ammonium phosphate and 0.17 M ammonium iodide. The calcium nitrate solution is dripped into a vigorously stirred ammonium phosphate and ammonium iodide solution over 30 minute~. The resulting precipitated particles are examined under a microscope and estimated to have particle size9 of approximately 1 ~m.

~ .

W093~07905 2 t 2 D 1 3 ~ PCT/US92/0~032 Example 10 Pre~aration of Hydroxya~atite Do~ed with an XRCM
Hydroxyapatite particles doped with iothalamate me~lumine, an ionic XRCM, are prepared according to the procedure of Example 3, except that the calcium nitrate solution also contains O.058 M iothalamate meglumine salt.
Iothalamate has the following structure:
~ OH

J~N~ ~

~eglumlne Salt The ammonium phosphate solution is dripped using an autoburet into a vigorously stirring solution of calcium nitrate and iothalamate meglumine over 30 minutes. The resulting precipitated particles are examined under a microscope and estimated to have submicron particle sizes.

Example 11 Pre~aration of Hydroxya~atite Do~ed with a Radio~aque Heavy Metal Hydroxyapatite particles doped with tungsten are prepared according to the procedure of Example 3, except that the ammonium phosphate solution also contains 0.116 M
sodium tungstate, Na2WO4. The ammonium phosphate and sodium tungstate solution is dripped using an autoburet into a vigorously stirred solution of calcium nitrate over 30 minutes. The resulting precipitated particles are examined under a microscope and estimated to have submicron particle sizes.

~ 3~

Exam~le 12 Pre~aration of Hydroxya~atite Do~ed with Carbonate Carbonate-doped hydroxyapatite particles are prepared according to the procedure of Example 3, except that calcium carbonate was used instead of calcium nitrate. The ammonium phosphate solution is dripped using a computer controlled autoburet into a vigorously stirred calcium carbonate solution over 30 minutes. The resulting precipitated particles were examined under a microscope and estimated to have submicron particle sizes.

Exam~le 13 Pre~aration of ~igh Density Hydroxya~atite Hydroxyapatite particles are prepared according to the procedure of Example 3 and sintered at a temperature in the range from about 200C to about 1100C to harden and densify the particles. The dense particles can then be mixed with a suitable pharmaceutical carrier and administered as a high acoustic impedance ultrasound contrast media.

Example 14 Pre~aration at 100C of ~ydroxya~atite An ammonium phosphate solution was prepared by dissolving 10.56 grams ~NH4)2HPOq in 200 mL of D.I. water.
To this was added 100 mL of concentrated NH40H with stirring. A white precipitate formed which was dissolved by addition of 150 mL of H20. This solution was stirred for 3 hours at room temperature and then added dropwise (over 2 hours) via a peristaltic pump (Masterflex) to a 1000 mL
three-neck round bottom flask fitted with a dry ice/
isopropanol condenser on top of a standard water-jacketed condenser containing a solution of 31.5 grams Ca~NO3)2-4H2O
in 500 mL of H2O in boiling water stirred rapidly with a mechanical stirrer. Reflux was continued for two hours W~93/07905 PCT/US92/09~32 212 !~13 ~

after addition was complete and the mixture was allowed to cool to room temperature with stirring overnight. The reaction mixture was centrifuged at 2300 rpm and the nearly-clear supernatant discarded. The resulting white, pelleted solid was slurried with water and completely broken up by means of a vortex mixer. The mixture was again centrifuged and the cloudy supernatant collected.
The washing was repeated two separate times. All three washings were saved as was the remaining solid in the centrifuge tubes. The calcium/phosphorous ratio and particle size of the washed particles is summarized below:

Ca/P Ratio Particle size (std. dev.) wash 1: 1.65 663 (456) nm wash 2: 1.67 351 nm, 1853 nmt wash 3: 1.67 190 nm, 1069 nm' 'Bimodal distribution noted, no standard deviations given.

Exam~le 15 Pre~aration at 100C of HydroxyaDat~te Do~ed with Mn~II) This material was prepared according to the procedure of Example 14 except that a Mn~II) (as Mn~NO~)2-H20) was substituted mole-for-mole for Ca. For example, to synthesize 5% Mn incorporated into HA:
10.S6 grams ~NH~)2HPO~ was dissolved in 200 mL of D.I.
water. To this was added 100 mL of concentrated NH~OH with stirring. A white precipitate formed which was dissolved by addition of 150 mL of H20. This solution was stirred for 3 hours at room temperature and then added dropwise ~over 2 hours) via a peristaltic pump ~Masterflex) to a 1000 mL
three-neck round bottom flask fitted with a dry ice/
isopropanol condenser on top of a standard water-jacketed condenser containing a solution of 1.27 gram~ Mn(NO3)2.H~O
and 29.9 grams Ca~NO3)2-4H2O in 500 mL of H20 in boiling water stirred rapidly with a mechanical stirrer. Reflux .

W093/07~5 3~

was continued for two hours after addition was complete and the mixture was allowed to cool to room temperature with stirring overnight. The reaction mixture was centrifuged at 2300 rpm and the nearly-clear supernatant discarded.
The resulting off-white, pelleted solid was slurried with water and completely broken up by means of a vortex mixer.
The mixture was again centrifu~ed and the cloudy supernatant collected. The washing procedure was repeated two times. All three washings were saved as was the remaining solid in the centrifuge tubes. The particle size of the particles in the supernatant increased and the percentage of particles in the supernatant decreased (i.e., less cloudy supernatant). Solids from supernatants could be concentrated by further centrifugation at 7000 rpm. The average particle size was 449 nm with a standard deviation of 171 nm.

Exam~le 16 ~re~arat~o~ at 100C of ~ydroxya~atite ~articles 20Do~ed with Mn and treated with ~EDP
Manganese containing hydroxyapatite particles were prepared by the following general procedure (Mn/Ca mole ratios of < 0.33 can be used):
A solution containing 6.5 g of ~NHj)2HP0~ in 120 mL of deionized water was treated with 60 mL of concentrated ammonium hydroxide, NH~OH followed by 90 mL of D.I. water.
The resulting mixture was stirred at room temperature for 3 hours.
Into a lL 3-neck round bottom flask equipped with a water cooled/low temperature condenser sequence ~dry ice/
isopropanol bath), mechanical stirrer and rubber septum were placed 18.3 g of Ca(N03)2-4H20 and 0.7 g of MnlN03)2-XH2O
in 468 mL of D.I. water ~Ca/Mn mole ratio=l9~1, Ca + Mn =
0.081 moles). The resulting solution was heated to reflux.
The phosphate/hydroxide mixture was then added dropwise over approximatelY one hour with a peristaltic addition PCT/US92/0~32 2 1 2 ~ ~"

pump. The reaction mixture was cooled to room temperature and stirred overnight. The solution was then treated with 0.54 M HEDP (pH 6.6, 1-1.2 Ca/HEDP mole ratio) and stirred at room temperature for one hour.
The reaction mixture was then divided among six 50mL
plastic centrifuge tubes and centrifuged for 15 minutes at 2400 rpm. The procedure was repeated with the remainder of the reaction mixture. The almost clear supernatant was discarded and the solid in each tube resuspended to 50 mL
of volume with D.I. water and re-centrifu~ed. The milky wash was set aside and the solid washed twice more. The three washes were combined and then centrifuged at 7000 rpm for 30 minutes. The particles remained pelleted and the clear supernatant was decanted. The solid was resuspended in water and re-centrifuged three more times at 7000 rpm discarding the supernatant after each washing. After the centrifuge workup the solid particles were resuspended in 20-30 mL of D.I. water and then subjected to routine analysis.
Characterization of the particle suspension gave the following results:
size (average diamete~, nm): 258 relaxivity (mMolar~' sec~ 3.05 [Mn] (mole/liter): 0.11 lCa~ (mole/liter): 3.29 % Mn ~mole ~ relative to Ca): 3.35 In magnetic resonance imaging studies, a 95% enhancement of the liver was observed 4 hours post injection at a dose of 10 ~moles Mn/Kg animal body weight.
Exam~le 17 Preparat$on at room temperature of Hydroxya~at~te ~art$cles Do~ea w$th Mn and treatea with ~ D P
Manganese containing hydroxyapatite particles were prepared by the following general procedure. A procedure Wos3/07~ PCT/US92/0~32 ~ ~ ~ 26 is described for particles containing 10~ Mn but other percentages are also applicable.
Into a lL erlenmeyer flask were placed 10.5 g of (NH~)2HPO4, 100 mL of concentrated NH~OH and 350 mL of D.I.
water. The mixture was stirred for two hours with a continuous heavy argon flow ~degassing). In a separate lL
erlenmeyer flask were placed 28.9 g of Ca(NO3)2-4H20 and 2.4 g of Mn~NO~2-XH2O in 400 mL of D.I. water. The metal nitrate solution was degassed with argon for 2 hours. The phosphate solution was then added dropwise to the rapidly stirred metal nitrate mixture over two hours with a peristaltic pump. A continuous argon flow was maintained throughout the course of the reaction. The reaction mixture was stirred for an additional two hours after the lS addition was complete.
A solution of 8.3 g of a 60% solution HEDP (acid form) in 25 mL of D.I. water was degassed for 30 minutes then added in one aliquot to the hydroxyapatite mixture. The resulting slurry was stirred for 15 minutes~ The entire reaction mixture was centrifuged at one time at 2400 rpm for 15 minutes. The supernatant was discarded and the solid residue in each tube resuspended in water. The slurry was re-centrifuged at 2400 rpm and the milky supernatant was collected. The solid was resuspended twice 2S more and centrifuged at 2400 rpm. The three washes were combined and centrifuged at 7000 rpm for 30 minutes. The solid pellet was washed/centrifuged three times and the supernatants discarded. After washing, the solid pellet was suspended in 30 mL of D.I. H~O.
Characterization of the particulate suspension produced the following results:
size (average diameter, nm): 229 relaxivity (mMolar-l sec~ 29.4 tMn] ~mole/liter): 0.027 tca] (mole/liter): 0.377 . ~ ~
t PCT/US92/0~32 212~

~ Mn (mole % relative to Ca): 6.71 In magnetic resonance imaging studies, a 45% enhancement of the liver was observed immediately post injection at a dose of 10 ~moles Mn/Kg animal body weight.

Example 18 Pre~aration at room temperature of Hydroxya~atite Do~ed with 10% Mn(II~, Modified by Surface-Ad~orbed Mn(II) and H~D~ ~ddit~on An ammonium phosphate solution was prepared by . dissolving 5.3 grams (NH~)2HPO4 in 175 mL of D.I. water. To this was added 50 mL of concentrated NHqOH with stirring.
This solution was degassed for 2 hours (argon bubbling) with stirring and then added dropwise (over 2 hours) via a peristaltic pump (Masterflex) to a solution of 1.27 grams Mn(NO3)2-H20 and 14.5 grams Ca(NO~)2~4H20 in 200 mL of H20 that had also been deaerated for 2 hours with argon as it was stirred rapidly with a mechanical stirrer. Argon bubbling was continued during the addition. The reaction mixture was stirred for an additional 2 hours as the Ar bubbling continued. 1.27 g Mn(NO3)~-H20 in 25 mL of deaerated H2O was added in one portion to the reaction slurry, followed, after 15 minutes, by 4.3 grams of a 60%
HEDP solution in water dissolved in 10 mL of deaerated H2O.
The reaction mixture was centrifuged at 2300 rpm and the nearly-clear supernatant discarded. The resulting white, pelleted solid was slurried with water and completely broken up by means o~ a vortex mixer. The mixture was again centrifuged and the cloudy supernatant collected.
The washin~ was repeated two separate times. All three washings were saved as was the remaining solid in the cen~rifuge tubes.
In magnetic resonance imaging studies, a 30%
enhancement of the liver was observed immediately post injection at a dose of 10 ~moles Mn/Kg animal body weight.

PC~r/US92/09032 9 ~ 3 ~

Example 19 Pre~aratlon at 100C o Hydroxya~at~te ~artlcles Mod~f~ed by Surface-Adsorbed Mn(II~ and ~EDP Addition Into a 250 mL erlenmeyer flask were placed 6.3 g of (NH,)2HPO~ in 120 mL of D.I. water. Concentrated NH,O~ (60 mL) was added to the mixture followed by 90 mL of D.I.
water. The solution was stirred at room temperature for four hours.
,Into a lL 3-neck round bottom flask equipped with a water cooled and low temperature condenser sequence (dry ice/isopropanol), mechanical stirrer, and rubber septum were placed 19.0 g of Ca(NO~)2-4H2O in 468 mL of D.I. H2O.
The mixture was heated to reflux and the phosp~ate/ammonium hydroxide solution added dropwise with a peristaltic pump and rapid stirring over one hour. The hea~ing was removed when the addition was complete. The reaction mixture was cooled to room temperature then stirred overnight.
The pH of the hydroxyapatite slurry was adjusted from 9.50 to 8.70 with 8~ mL of 0.5 N HCl. 2.1 g o Mn(NO3)2-XH20 in 5 mL of H20 was added to the hydroxyapatite mixture and stirred for four hours. The reaction mixture became light brown in color. A solution of HEDP (0.54 M, Ca/HEDP mole ratio=1.1) was added and the resulting reaction mixture stirred at room temperature for 3 hours. The color of the slurry became purple/brown.
The reaction mixture was divided among six 50 mL
plastic centrifuge tubes and centrifuged for 15 minutes at 2400 rpm. The supernatant was deep purple and clear. The solid residue was washed~centrifuged three times with 50 mL
volumes of water per tube and the three washes combined.
The combined washes were centrifuged at 7000 rpm for 20 minutes. The solid pellets were washed/centrifuged three additional times discarding the supernatant after each centrifuge run. The white solid residue was suspended in 15 mL of D~I. H20 then subjected to routine analyses.

W O 93/07905 2 ~ 2 0 1 3 ~ PC~r/US92/09032 The analyses of the manganese adsorbed hydroxylapatite slurry gave the following results:
size (average diameter, nm): 259 relaxivity ~mMolar~' sec~'): 13.8 5[Mn) (mole/liter): 0.010 [Ca] (mole/liter): 1.60 Mn (mole ~ relative to Ca): 0.66 ExamPle 2Q
10Pre~aration at Room Tem~erature of ~ydroxya~atit;e Do~ed with 10~ Mn(II)~ Modif~ed by Sequential Addition of Mn(II) and H~DP with Wa~hing~ Between Ste~s The general procedure is the same as in Example 18.
Before addition of the additional Mn(NO3) 2~ however, the reaction mixture was pH adjusted from 9.8 to a lower pH
(7.5-9.5) and the mixture then centrifuged, the resulting solid washed with D.I. water, the Mn~NO3)~ added with stirring under argon bubbling, the resultant mixture centrifuged and the solid washed with water, In the final step the HEDP was added to the slurried solid and then the excess washed away with the supernatant during centrifugation.
In the preparation where the pH was adjusted to 9.5, 5.3 grams (NH4)2HPO~ was dissolved in 175 mL of D.I. water.
To this was added 50 mL of concentrated NH40H with stirring.
This solution was degassed for 2 hours (argon bubbling) with stirring and then added dropwise (over 2 hours) vi~ a peristaltic pump (Masterflex) to a solution of 1.~7 grams Mn(NO~)2-H.O and 14.5 grams Ca(NO~ qH~O in 200 mL of H20 that had also been deaerated for 2 hours with argon as it was stirred rapidly with a mechanical stirrer, Argon bubbling was continued during the addition. The reaction mixture was stirred for an additional 2 hours as the argon bubbling continued. The ~H of the reaction mixture was adjusted from 9.8 to 9.0 with 3 N HCl with rapid stirring - and argon bubbling.

W093/07~ PCT/US92/~32 3`~

1.27 g Mn(NO3)2-H2O in 25 mL of deaerated H2O was added in one portion to the reaction slurry, followed, after 60 minutes, by centrifugation and one washing of the resultant solid (via vortex mixing and recentrifugation). The solid was suspended in water and treated with 4.3 grams of a 60 HEDP solution in water dissolved in 10 mL of deaerated H20.
After 15 minutes the reaction mixture was centrifuged at 2300 rpm and the nearly-clear supernatant discarded. The resulting white, pelleted solid was slurried with water and completely broken up by means of a vortex mixer. The mixture was again centrifuged and the cloudy supernatant collected. The washing was repeated two separate times All three washings were combined and the solids from those washings pelleted by centrifugation at 7000 rpm. The resulting pellet was washed with water 3 times by suspension followed by centrifugation at 7000 rpm. The particles were analyzed and` found to have an average particle size of 251 nm and a relaxivity, R~ = 25 mM~lsec-~.

Exam~le 21 Pre~aration at 100C of Hydroxya~atite Particles Modified by Surface-Adsorbed ~n, Purified, then Treated with HEDP
Calcium hydroxyapatite particles were prepared by the following procedure:
A solution containing 6.5 g of ~NH~)2HPO~ in 120 mL of D.I. water was treated with 60 mL of concentrated NH,OH
followed b~ 90 mL of D.I. water. The resulting solution was stirred for 3 hours at room temperature.
Into a 3-neck lL round bottom flask eqllipped with a water cooled and low temperature condenser sequence ~dry ice/isoproPanol~ mechanical stirrer and rubber septum were placed 19.4 g of CatNO~)2-4H2O in 468 mL of D.I. water. The solution was heated to reflux. The phosphate mixture was added to the rapidly stirred calcium nitrate solution dropwise with a peristaltic pump over one hour~ The heat W093/07~ PCT~US92/~32 "? ~,i was removed when the addition was complete and the reaction mixture cooled to room temperature. The hydroxylapatite slurry was stirred overnight at room temperature.
The pH of the reaction mixture was decreased from 9.53 to 8.50 with 169 ml of lN HCl. Manganese nitrate, Mn~NOl)2-xH2O (2.10 g) was added to the hydroxyapatite mixture and stirred ~or 1 hour and 15 minutes. The color of the slurry became pale tan. The mixture was then centrifuged at 2400 rpm for 15 minutes. The clear ~olorless supernatant was discarded and the solid , washed/centrifuged with 3-50 mL aliquots of water at 2400 rpm for 15 minutes per run. Half of the solid residue was suspended in 200 mL of D.I. water and stirred vigorousl~
then placed in an ultrasonic bath for 10 minutes to break apart any large clumps. The solid slurry was then treated with 0.54 M HEDP (Ca/HEDP mole ratio=1.2) and stirred for 1.5 hours. The color of the mixture became pale pink/purple. The remaining half of the solid hydroxyapatite pellet was suspended in 200 mL of D.I. H20 and set aside for characterization and analyses.
The HEDP treated hydroxyapatite fraction was divided among six 50 mL plastic centrifuge tubes and centrifuged for 15 minutes at 2400 rpm. The supernatant was deep purple and slightly cloudy. The solid residue was suspended in H2O and centrifuged at 7000 rpm for 30 minutes.
The supernatant was discarded and the solid pellet washed/centrifuged three more times at 7000 rpm. The purified hydroxyapatite was suspended in approximately 30 mL of D.I. water then characterized. The results of the analyses are listed below.

W093/07~5 PCT/US92/09032 q ~
r~

HEDP treated untreated size (average diameter, nm): 216 34 ~ 100 relaxivity (mMolar-' secl): 38.3 0.78 [Mh] (mole/liter): 0.0025 0.016 [Ca] (mole/liter): 0.170 0.638 % Mn (mole % relative to Ca): 1.44 2.45 In magnetic resonance imaging studies, a 25 enhancement of the liver was observed immediately post injection at a dose of 10 ~moles Mn/Kg animal body weight.
' Exam~le 22 Pre~aration of Mn-DoDed Hydroxya~at~te Particle~
Having a Funct~onal~zed Coating Agent This example describes the general preparation of ~`
~15 hydroxyapatite particles having a functionalized coating ;~ agent. The particles are prepared by addin~ 0.1-100 mole % of an appropriate coating agent to a slurry of Mn~II) substituted hydroxyapatite with 0.1-100 mole % Mn based on the Ca used in the reaction. The mixture is stirred from 1 to 360 minutes at temperatuxes in the range from 4C to 100C and the solid separated from the supernatant by centrifu~ation. The resulting solid is collected or subjected to repeated washings with water to remove excess ions and coating agent. The solid, after resuspension in water, may be treated with a metal salt (0~01-10 mole~
based on Ca in the preparation). This is especially appropriate if the coating agent contains a pendant chelating group to capture and hold tightly the metal ~when subjected to in vitro and~or ln v vo solutions). The resultant solid is separated by centrifugation and washed 3 times ~ith water to remove loosely attached coating agent or free metal/coating agent complex.

W093/07~5 212 ~1 "l "

Example 23 Pre~arat~on of Hydroxya~atite Particle~ treated with Diethylenetriamine-~enta(methylene~ho~honlc acld) Followed by Surface Ad~orption of Mn Calcium hydroxyapatite was prepared by the following procedure then treated with the functionalized polyphosphonate, diethylenetriamine-penta(methylene-phosphonic acid), abbreviated DETAPMDP and having the following structure:

2~ 2-,N N ~
2-~p~ 2-~2-A basic ammonium phosphate solution was prepared using 6.34 g of (NH~)2HPO4 in 120 mL of D.I. water. Concentrated ammonium hydroxide (60 mL) was added followed by 90 ml of D.I. water. The mixture was stirred for 4 hours at room temperature.
A solution of 19.0 g of Ca(NO3)2-4H20 in 468 mL of D.I.
water was placed in a 3-neck lL round bottom 1ask. The reaction setup included a mechanical stirrer, water cooled and low temperature (dry ice/isopropanol) condenser arrangement, and a rubber septum. The solution was heated to reflux with rapid stirring. The basic phosphate solution was added dropwise with a peristaltic pump over one hour. The heat was removed after the addition was complete and the reaction mixture stirred overnight at room temperature.
The hydroxyapatite slurry was treated with a solution of DETAPMDP ~Ca/DETAPMDP mole ratio=l.1, pH of DETAPMDP
6.3) and stirred at room temperature for 2.5 hours. The phosphonate treated mixture was then reacted with W093~07905 PCT~US92/09032 J

Mn(NO3)2-XH2O (Ca/Mn mole ratio=2.3) and stirred for an additional 3.5 hours.
The reaction mixture was divided among six 50 mL
plastic centrifuge tubes and centrifuged at 2400 rpm for 15 minutes. The clear supernatant was discarded and the solid residue suspended in 50 mL of D.I. per tube and centrifuged at 2400 rpm. The milky suspension was decanted and set aside. The solid was washed/centrifuged twice more and the three washes combined. The milky suspension was re-centrifuged at 7000 rpm for 30 minutes. The clear supernatant was discarded and the solid pellet resuspended and centrifuged three additional times at 7000 rpm. The purified pellet was then suspended in 15 mL od D.I. water and analyzed. The follo~ing results were obtained.
size (average diameter, nm): 258 relaxivity (mMolar' sec~'): 20.3 [Mn] (mole/liter): 0.0013 lCa] (mole/liter): 1.921 ~ Mn (mole % relative to Ca): 0.07 In magnetic resonance imagin~ studies, a 30%
enhancement or the liver was observed immediately post injection at a dose of 10 ~moles Mn/Kg animal body weight~

Example_24 ~eplacement of Phos~hate with Arsenata in Pre~aration of Hydroxya~atite and Substituted Hydroxya~atites The procedure according to Example 17 is used except that 0.1-100 mole ~ arsenate is substituted for the phosphate. For example, 9.51 grams ~NH~)~H~O~ and 1.49 grams Na2AsO~ were dissolved in 400 mL of D.I. water. To this was added 100 mL of concentrated NH~OH with stirring. The rest of the procedure follows directly from Example 17.

W093/07905 21~ O 1 ' G PCT/US92/09032 Example 25 Replacement of Phos~hate with Vanadate in Pre~aration of Hydroxya~atite ana Substituted Hydroxya~atites The procedure according to Example 17 is used except that 0.1-100 mole percent vanadate is substituted for the phosphate. For example, 9.51 grams (NH4 )2HPO4 and 1.40 grams Na3VO4 were dissolved in 400 mL of D.I. water. To this was added 100 mL of concentrated NH40H with stirring. The rest of the procedure follows directly from Example 17.
ExamPle 26 Pre~aration at 100C of Mn-Do~ed Fluoroa~atite Particles Manganese fluoroapatite was prepared by the following general procedure. Into a 5-neck lL round bottom flask equipped with a mechanical stirrer, water cooled reflux condenser, adapter for pH electrode, and two rubber septa for addition of reagents were placed 10.3 g of Mn(OAC)2-4H20 in 200 mL of D.I. water. The solution was degassed with heavy argon bubbl_ns for 30 minutes. A solution of ammonium fluoride, NH4F (0.3 g) in 50 mL of D.I. water was prepared in a 125 mL erlenmeyer flask and degassed for 30 minutes with argon. Into a 250 mL erlenmeyer flask was placed 3.3 g of ~NH4)2HPO4 in 150 mL of D.I. water and degassed for 30 minutes before addition.
The manganese acetate solution was heated to reflux with rapid stirring (pH 6.6) and the NH4F and (NH4) 2HP04 solutions were added dropwise simultaneously with a peristaltic pump over 35 minutes. The solid precipitated among immediate addition of reagents and was pale pink in color. The pH of the reaction mixture dropped to 4.7 by the end of the reaction. The heati~g was stopped when the addition was complete. The reaction mixture was stirred at room temperature overnight.
The apatite slurry was divided among four 50 mL
plastic centrifuge tubes and centrifuged for 30 minutes at 2400 rpm. The clear supernatant was discarded, and the W093/07905 ~ PCT/US92/0~32 c a~ 3~

pale pink solid was resuspended and centrifuged for 30 minutes at 2400 rpm. The solid was washed and centrifuged twice more and the clear supernatants discarded. The purified solid pellet was suspended in 20 mL of D.I. water.

From the foregoing, it will be appreciated that the present invention provides organ specific medical diagnostic imaging agents for use in MRI, X-ray, and ultrasound.
The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

What is claimed is:

1. An apatite particle for use in magnetic resonance imaging of body organs and tissues having the following general formula:
CanMmXrYs wherein M is a paramagnetic ion or stoichiometric mixture of metal ions having a valence of 2+ or 3+, X is a simple anion, Y is a tetrahedral oxyanion, carbonate, tetrahedral anion, or mixtures thereof, m is from 1-10, n is from 1-10, where M is a 2+ metal ion, then m + n = 10, and where M is a 3+ metal ion, then m + 1.5n = 10, and r and s are adjusted as needed to provide charge neutrality.

2. An apatite particle as defined in claim 1, wherein M is selected from a group of elements having atomic numbers of 21-25, 27-29, 42-44, and 58-70 and a valence in the range from 2+ to 3+.

3. An apatite particle as defined in claim 1, wherein M is chromium(III), manganese(II), iron(II), iron(III), praseodymium(III), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), terbium(III), dysprosium(III), holmium(III), or erbium(III).

4. An apatite particle as defined in claim 1, wherein M is manganese(II), iron(II), iron(III), or mixtures thereof.

5. An apatite particle as defined in claim 1, wherein X is selected from the group consisting of OH-, F-, Br-, I-, ?[CO32-], or mixtures thereof.

6. An apatite particle as defined in claim 1, wherein X is a mixture of OH- and F-.

7. An apatite particle as defined in claim 1, wherein the particle size is in the range from about 5 nm to about 2 µm and are used for imaging the liver and spleen.

8. An apatite particle as defined in claim 1, wherein the apatite particles have a size in the range from about 200 nm to about 50 µm and are used for imaging the gastrointestinal tract.

9. An apatite particle as defined in claim 1, wherein the apatite particles have a size in the range from about 1 nm to about 50 nm and are used for imaging the blood pool.

10. An apatite particle as defined in claim 1, further comprising a coating agent to stabilize the apatite particles.

11. An apatite particle as defined in claim 10, wherein the coating agent stabilizes the apatite particles by modifying the surface charge and inhibiting particle aggregation.

12. An apatite particle as defined in claim 10, wherein the coating agent is selected from the group comprising di- and polyphosphonates, aminophosphonates, mono- and polycarboxylates, alcohols, mono- and polyphosphates, mono- and polysulfates, mono- and polysulfonates, and biomolecules.

13. An apatite particle as defined in claim 10, wherein the coating agent is a di- and polyphosphonate-containing compound.

14. An apatite particle as defined in claim 13, wherein the coating agent is hydroxyethyldiphosphonate (HEDP).

15. An apatite particle as defined in claim 10, wherein the coating agent is a peptide, protein, antibody, or lipid biomolecule.

16. An apatite particle as defined in claim 10, wherein the coating agent is an oxalate or citrate carboxylate-containing compound.

17. An apatite particle as defined in claim 10, wherein the coating agent comprises a reactive functional group.

18. An apatite particle as defined in claim 17, wherein the reactive functional group is an amine, active ester, alcohol, or carboxylate functional group.

19. An apatite particle as defined in claim 17, wherein the coating agent having a reactive functional group comprises:

20. An apatite particle as defined in claim 1, wherein the Y is selected from the group consisting of AsO43-, WO42-, MoO42-, VO43-, SiO44-, and GeO44-.

21. A method for enhancing magnetic resonance images of body organs and tissues which comprises:
(a) administering to a patient, a diagnostically effective amount of apatite particles incorporating paramagnetic species therein, in a pharmaceutically acceptable carrier, said apatite particles having a general formula CanMmXrYs wherein M is a paramagnetic ion or stoichiometric mixture of metal ions having a valence of 2+ or 3+, X
is a simple anion, Y is an teterahedral oxyanion, carbonate, tetrahedral anion, or mixtures thereof, m is from 1-10, n is from 1-10, where M is a 2+ metal ion, then m + n = 10, and where M is a 3+ metal ion, then m + 1.5n = 10, and r and s are adjusted as needed to provide charge neutrality; and (b) imaging the organs and tissues using magnetic resonance techniques.

22. A method for enhancing magnetic resonance images as defined in claim 21, wherein the paramagnetic species is a metal ion is selected from a group of elements having atomic numbers of 21-25, 27-29, 42-44, and 58-70 and a valence in the range from 2+ to 3+.

23. A method for enhancing magnetic resonance images as defined in claim 21, wherein the paramagnetic species is chromium(III), manganese(II), iron(II), iron(III), praseodymium(III), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), terbium(III), dysprosium(III), holmium(III), or erbium(III).

24. A method for enhancing magnetic resonance images as defined in claim 21, wherein the paramagnetic species is manganese(II), iron(II), iron(III), or mixtures thereof.

25. A method for enhancing magnetic resonance images as defined in claim 21, wherein the paramagnetic species includes a nitroxide radical.

26. A method for enhancing magnetic resonance images as defined in claim 21, wherein the apatite particles have a size in the range from about 5 nm to about 2 µm and are used for imaging the liver or spleen.

27. A method for enhancing magnetic resonance images as defined in claim 21, wherein the apatite particles have a size in the range from about 200 nm to about 50 µm and are used for imaging the gastrointestinal tract.

28. A method for enhancing magnetic resonance images as defined in claim 21, wherein the apatite particles have a size in the range from about 1 nm to about 50 nm and are used for imaging the blood pool.

29. A method for enhancing magnetic resonance images as defined in claim 21, wherein the organs and tissues are imaged using proton magnetic resonance techniques.

30. A method for enhancing magnetic resonance images as defined in claim 21, wherein the organs and tissues are imaged using fluorine-19 magnetic resonance techniques.

31. A method for enhancing X-ray images of body organs and tissues which comprises:
(a) administering to a patient, a diagnostically effective amount of apatite particles incorporating a substantially radiopaque species therein, in a pharmaceutically acceptable carrier; and (b) imaging the organs and tissues using X-ray techniques.

32. A method for enhancing X-ray images of body organs and tissues as defined in claim 31, wherein the substantially radiopaque species is selected from the group consisting of iodine, iodinated X-ray contrast media, and radiopaque heavy metals.

33. A method for enhancing X-ray images of body organs and tissues as defined in claim 31, wherein the apatite particles have a size in the range from about 5 nm to about 2 µm and are used for imaging the liver or spleen.

34. A method for enhancing X-ray images of body organs and tissues as defined in claim 31, wherein the apatite particles have a size in the range from about 200 nm to about 50 µm and are used for imaging the gastrointestinal tract.

35. A method for enhancing X-ray images of body organs and tissues as defined in claim 31, wherein the apatite particles include iodoapatite.

36. A method for enhancing X-ray images of body organs and tissues as defined in claim 31, wherein the apatite particles include a substantially radiopaque heavy metal selected group consisting of bismuth, tungsten, tantalum, hafnium, lanthanum and the lanthanides, barium, molybdenum, niobium, zirconium, and strontium.

37. A method for enhancing ultrasound images of body organs and tissues comprising:
(a) administering to a patient, a diagnostically effective amount of echogenic apatite particles, in a pharmaceutically acceptable carrier;
and (b) imaging the organs and tissues using ultrasound techniques.

38. A method for enhancing ultrasound images of body organs and tissues as defined in claim 37, wherein the echogenic apatite particles include gas-filled pores.

39. A method for enhancing ultrasound images of body organs and tissues as defined in claim 37, wherein the echogenic apatite particles are prepared by mixing apatite particles, having a carbonate salt incorporated therein, with a weak biocompatible acid, such that carbon dioxide is formed within pores of the apatite particles.

40. A method for enhancing ultrasound images of body organs and tissues as defined in claim 37, wherein the echogenic apatite particles are dense particles substantially without pores.

41. A diagnostic composition for enhancing magnetic resonance imaging suitable for enteral or parenteral administration to a patient, which comprises:
a diagnostically effective amount of apatite particles having a size in the range from about 5 nm to about 50 µm, said apatite particles incorporating a paramagnetic species therein, said apatite particles having a general formula CanMmXrYs wherein M is a paramagnetic ion or stoichiometric mixture of metal ions having a valence of 2+ or 3+, X
is a simple anion, Y is a tetrahedral oxyanion, carbonate, tetrahedral anion, or mixtures thereof, m is from 1-10, n is from 1-10, where M is a 2+ metal ion, then m + n = 10, and where M is a 3+ metal ion, then m + 1.5n = 10, and r and s are adjusted as needed to provide charge neutrality; and a pharmaceutically acceptable carrier.

42. A diagnostic composition as defined in claim 41, wherein the paramagnetic species is a metal ion selected from a group of elements having atomic numbers of 21-25, 27-29, 42-44, and 58-70 and a valence in the range from 2+
to 3+.

43. A diagnostic composition as defined in claim 41, wherein the paramagnetic species is manganese(II), iron(II), iron(III), or mixtures thereof.

44. A diagnostic composition as defined in claim 41, wherein the paramagnetic species includes a nitroxide radical.

45. A diagnostic composition for enhancing X-ray contrast suitable for enteral or parenteral administration to a patient, which comprises:
a diagnostically effective amount of apatite particles having a particle size in the range from about 5 nm to about 50 µm, said apatite particles incorporating therein a substantially radiopaque species; and a pharmaceutically acceptable carrier.

46. A diagnostic composition as defined in claim 45, wherein the substantially radiopaque species is selected from the group consisting of iodine, iodinated X-ray contrast media, and radiopaque heavy metals.

47. A diagnostic composition as defined in claim 45, wherein the apatite particles have a size in the range from about 5 nm to about 2 µm useful for imaging the liver or spleen.

48. A diagnostic composition as defined in claim 45, wherein the apatite particles have a size in the range from about 200 nm to about 50 µm useful for imaging the gastrointestinal tract.

49. A diagnostic composition as defined in claim 45, wherein the apatite particles include iodoapatite.

50. A diagnostic composition for enhancing ultrasound contrast suitable for enteral or parenteral administration to a patient, which comprises:
a diagnostically effective amount of echogenic apatite particles having a particle size in the range from about 5 nm to about 50 µm, said apatite particles having a general formula Ca10X2(PO4)5, wherein X is OH, F, Br, I, ?CO3; and a pharmaceutically acceptable carrier.

51. A diagnostic composition as defined in claim 50, wherein the echogenic apatite particles include gas-filled pores.

52. A diagnostic composition as defined in claim 50, wherein the echogenic apatite particles are prepared by mixing apatite particles, having a carbonate salt incorporated therein, with a weak biocompatible acid, such that carbon dioxide is formed within pores of the apatite particles.

53. A diagnostic composition as defined in claim 50, wherein the echogenic apatite particles are dense particles substantially without pores.

54. Iodoapatite particles having a size in the range from about 5 nm to about 50 µm and incorporating a substantially radiopaque species therein, said apatite particles having a general formula Ca10X2(PO4)6, wherein X is I or mixtures of OH and I.

55. Carbonate-apatite particles having a size in the range from about 5 nm to about 50 µm and having a general formula Ca10X2(PO4)6, wherein X is ?CO3 or mixtures of ?CO3 and OH or F.

56. An apatite particle for use in magnetic resonance imaging of body organs and tissues comprising an apatite particle, a paramagnetic metal species adsorbed to the surface of the apatite particle, and a di- or polyphosphonate coating agent.

57. An apatite particle for use in magnetic resonance imaging as defined in claim 56, wherein the paramagnetic metal species is manganese(II), iron(II), iron(III), or mixtures thereof.

58. An apatite particle for use in magnetic resonance imaging as defined in claim 56, wherein the phosphonate coating agent is HEDP.

59. A method of preparing an apatite particle for use in magnetic resonance imaging comprising the steps of:
(a) preparing an apatite particle having a particle size in the range from about 5 nm to about 50 µm;
(b) adsorbing a bifunctional coating agent capable of forming a chelate complex with a paramagnetic metal ion onto the apatite particle surface; and (c) forming a chelate complex between the bifunctional coating agent and the paramagnetic metal ion.

60. A method of preparing an apatite particle as defined in claim 59, wherein the bifunctional coating agent comprises polyphosphonate diethylenetriaminepenta-(methylenephosphonic acid) having the following structure: