CA2187749A1

CA2187749A1 - Microfluidization of calcium/oxyanion-containing particles

Info

Publication number: CA2187749A1
Application number: CA002187749A
Authority: CA
Inventors: Linda Meeh; William P. Cacheris
Original assignee: Individual
Current assignee: Mallinckrodt Inc
Priority date: 1994-04-11
Filing date: 1994-04-11
Publication date: 1995-10-19

Abstract

Methods of preparing solid apatite particles using a microfluidizer, for use in medical diagnostic imaging such as magnetic resonance imaging, X-ray, and ultrasound. The desired apatite particles are synthesized, passed through a microfluidizer, and purified to remove excess base, salts, and other materials used to synthesize the particles. The microfluidizer causes two high pressure streams to interact at ultra high velocities in a precisely defined microchannel. Microfluidization of preparations causes small ( 5µm) and uniform particles to be formed. Coating and purifying (especially by tangential flow filtration) the particles improves particle stability.

Description

wo 95l27437 2 1 8 7 7 ~ 9 PCT/US94103276 2lICROFr-UInI7'~TION OF cAI,cIT~ yy~NTr~-P~RTTrr.T.'!R
~R~ N~:~; TO R~T.ATT'n APP~IO'ATIONS
This invention is a cn~ttnl~tion-in-part of U.S.
Patent Application Serial No. 07/948,540, filed September 22, 1992, titled "Treated Apatite Particles for Medical Diagnostic Imaging, " which is ~nnt;nll~tion-in-part of ~.S.
Patent Application Serial No. 07/784,325, filed October 22, lO l99l, titled "Treated Apatite Particles for Medical Diag-nostic Imaging, n which applications are incorporated herein by ref erence .
BA~:~KUUNV OF T~E lNv~ N
This invention relates to the preparation of calci-um/oxyanion-cnnt~in;ng particles for use in medical diag-nostic imaging, such as magnetic resonance imaging ("MRI"), ultrasound, and X-ray. In particular, the present inven-tion is directed to the use of a micrnf1l~ ;7er for the 20 preparation of calcium/oxyanion-~nnt~ln;ng particles having a uniform small (~5 ~m) size distribution. The present invention also includes the use of tangential flow filtra-tion for particle purification.
The use of contrast agents in diagnostic ~;c;nP is 25 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 adminis-tering a contrast agent which is subst~nt;;i11y radiopaque.
30 In convf~nt;nnsll proton MRI diagnostics, increased contrast of internal organs and tissues may be obtained by adminis-tering compositions cnnt~;n;n~ paramagnetic metal species which increase the relaxivity of surrounding protons. In ultrasound diagnostics,;, r~ve:d contrast is obtained by 35 administering compositions having acoustic ; --l;in~
dif f erent than that of blood and other tissues .

Wo 95/27437 9 ~ PCr/uSg4/03276 2~87749 Of ten it is desirable to image or treat a specif ic organ or tissue. Effective organ- or tissue-specific diagnostic agents ~ 1 ~te in the organ or tissue of interest. Cnp~on~9;n~ patent application Serial No.
07/948,540, filed September 22, 1992, titled "Treated Apatite Particles for Medical Diagnostic Imaging, " which is incorporated herein by reference, discloses the preparation and use of apatite particles for medical diagnostic imag-ing. This patent application also describes methods for preparing apatite particles which provide organ- or tis8ue-specific contrast. By carefully controlling the particle size and route of administration, organ specific imaging of the liver, spleen, gastrointestinal tract, or blood pool is obtained .
In general, the apatite particles are prepared by modifying conventional methods for prepari~g lly-lLu~yd~c,tite (sometimes referred to as "hydroxylapatite") . For example, stoich; ~ric hydroxyapatite, CalO~OH),(PO~)6, i9 prepared by adding an ammonium phosphate= solution to a solution of calcium/ammonium hydroxide. Useful apatite particles may also be prepared by r-~pl~r~nr -calcium with par~ n~ic metal ions. Other apatite derivatives are ~lc:~aI~ by replacing the 0~~ with simple anions, including F-, Br~, I-, or ~ [Co3l~] .
VariQus techniques for controlling the particle size for certai~ calcium phosphate-rnnt~in-ng rn~rQlln (apatites) are disclosed in copending application Serial No. 07/948,540. For example, slower addition rates (intro-duction of the precipitating anion or cation), faster stirring, higher reaction temperatures, and lower concen-trations generally result in smaller particles. In addi-tion, sonication during precipitation, turbulent flow or imp;nf mixers, homogenization, and pH modification may be used to control particle size. Other means, such as 35 computer controlled autoburets, peristaltLc pumps, and 21 877~9 6yringes, may be used to control the release of precipita-ting ions to produce smaller particles.
Due to the small size and nature of apatite particles, they tend to aggregate. Particle aggregation may be inhibited by coating the particles with coating agents, while agglomerated particles may be disrupted by mechanical or rh~m; r~l means and then coated with a coating agent having an affinity for the apatite.
One preferred method of obtaining small, uniformly sized, --n~n~Re-doped apatite particles is to dropwise add a degassed solution of (NH~) 2HPO~ and NH~OH into a rapidly stirring degassed solution of Ca(NO3),-4H20 and Mn(NO3),-6H20.
The resulti~g apatite particles are then reacted with a solution of l-hydroxyethane-1,1-~11rhr~sFhr~nic acid (HEDP).
The smaller particles are separated f rom larger particles by repeated c~ntr;fll~r;ng and collection of the gllr~rrn;:t;~nt The particles are then washed to remove base and salts by centrifuging at a higher rpm, discarding the supernatant, r~ r~n~;nr, the solid pellet in water, and recentrifuging.
Although the foregoing procedure produces small-sized apatite particles having good size distribution and good medical diagnostic imaging properties, the repeated centri-fuging, decanting, and washing causes the process to be tedious and time-cAn~ nrJ It, therefore, would be a significant adv~n~- ~ in the art to provide an improved method for rapidly preparing calcium/oxyanion-rrnt~;n;ng particles for medical diagnostic applications having a controlled particle size distribution and good yield.
Such methods for preparing calcium/oxyaniOn-rrnt~;n;nrJ
particles are disclosed and claimed herein.
SUMMP RY OF TT~ Il~VENTION
The present invention provides methods of preparing calcium/oxyanion-c~nt~;nlnr, particles, including apatites 35 and apatite precursors, using a microfluidizer. The 21~77~9 particles thus prepared, are for uEe in medical diagno3tic imaging, such as magnetic r~Rnn~nr~ imaging, X-ray, and ultrasound applications. The desired calcium/oxyanion-cn"~in;"~ particles are synthesized, passed through a microfluidizer, and purified to remove excess base, salts, and other materials used to synthesize the particles. The mierofluidizer causes two high ~'e1~UL~: streams to interact at ultra high velocities in a precisely defined microehan-nel. Use of the mierofluidizer results in signifieant reduction in the average partiele size. Purifying the particles, preferably using ~n~n~;Al flow filtration, as well as coating the particles, improves particle stability.
R~T~ DES~RIPD:ON OF THE DRAWIl~G
Figure 1 i3 a graphical reE?res~"t~;r~" of the particle size distribution of r-n~nPR.Q-doped llydL~,~y~atite parti-cles prepared according to Example 8, before and after passing through a microfluidizer.
Figure 2 is a graphical representation of the osmolal-ity of a particulate suspension after sequential passes through a ~nrJ~n~ l flow filtration system as described in Example 8 .
D~3TAILED DES~RIPTION OF T~ NV~:N~
The present invention provides methods for preparing ealeium/oxyanion-c~nt~;nlng particles, including apatites and apatite precursors, especially hydroxyapatite, having uniform, small (~5 ~lm) particle size and uniform distribu-tion through use of a microfluidizer.
As used hereirL, ealeium/oxyanion-rr"~ particles inelude calcium phosphate ~inerals, apatites, and apatite precursors of the general formula CanMmX~Y" where M is a paramagnetic metal ion, radiopaque metal ion, radioactive metal ion, or stoichiometric mixture of metal ions, X is a 35 simple anion, Y is an oxyanion including tetrahedral Wo ssn7~37 P~ 776 ~1877~g oxyanions, ~rhnn~te~ or mixtures thereof, m is from O to 10, n is from 1 to 10, s is 2 1, and r is adjusted as needed to provide charge neutrality.
As used herein, apatite precursors include ~ ~ u~ds 5 within the scope of the above general formula having one or more amorphous phases which, when sintered, may become crystalline apatites.
Possible ~aL~~ t; c metal ions which can be used in the calcium/oxyanion-cnnt~;n;n~ particles of the present 10 invention include: chromium(III), r-n~nPçle(II), iron(II), iron(III), praseodymium(III), neodymium(III), samari-um(III), ytterbium(III), ~a~lnl;n;um(III), terbium(III), dysprosium(III), holmium(III), erbium(III), or mixtures of these with each other or with alkali or ~lk~ ;n~ earth 15 metals.
Certain radiopaque heavy metals, such as bismuth, tungsten, tantalum, hafnium, lanthanum and the 1Anth~n;des, barium, molybdenum, niobium, zirconium, and strontium may also be incorporated into particles to provide X-ray 2 0 contrast . The radiopaque metals are incorporated into the calcium/oxyanion-~-nnt~;n;ng particles in the same manner as paL~~ ylletic metal ions.
Typicai simple anions which can be used in the calci-um/oxyanion-cnnt;~;n;n~ particles oi the present invention 25 include: OH-, F-, Br~ I-, ~[CO3'-], or mixtures thereof. The tetrahedral oxyanions used in the present invention may optionally include radiopaque metals or radioactive metals.
Suitable tetrahedral oxyanions are nnnn~; ~; 71 nS and stable to hydrolysis. Examples of suitable tetrahedral oxyanions 30 for use in the present invention include: Pos3-, AsO~3~, WO~~, MoO~~, voS3~, sio4S-, and GeOs4~. Phosphate is a currently preferred tetrahedral oxyanion.
By controlling the particle size, organ specific imaging or therapy of the liver or gastrointestinal tract 35 is obtained. When apatite particles having a size in the wo 95n7137 PCTIUS94l03276 2~77g9 6 range from about 5 nm to about 5 ~m are in~ected into the vascular system, the particles collect in the liver or spleen (the RES system) because a normal function of these organs is to purify the blood of foreign particles. Once 5 the particles have collected in the liver or spleen, these organs may be imaged by the desired medical diagnostic imaging technique.
CPr~n~l;n~ on the diagnostic imaging techni~ue, calci-um/oxyanion cnnt~in;n~ particles are treated to be paramag-lO netic, radiopaque, or echogenic. For example, paramagneticmetal species may be incorporated into the particles to improve magnetic resonance contrast, and r~rl;np~ .o species may be incorporated to provide X-ray contrast. Particle density, and corr~pnn~l;n~ echogenic characteristics, can 15 be co~trolled to impart low or high acoustic i ~ nre relative to blood. The calcium/oxyanion-nnnt~;n1n~ parti-cles may also be fluorinated to form stable, nontoxic compositions useful for 19F imaging. The presence of a paramagnetic metal specieG in these particles may reduce 19F
20 and proton relaxivity, thereby onh~nr;n~ MRI, MRS, or MRSI.
Hydroxyapatite doped with a paramagnetic metal can be prepared by mixing a basic (pH 10-12) rhn~rh~te solution with a calcium/paramagnetic metal solution at native pH.
It has been found that the pdL ~n,otic ions incorporated 25 into the apatite particle tend to oxidize during particle synthesis. To prevent metal oxidation the amount of oxygen in the aqueous reactant solutions is minimized. Oxygen m;n;m;7at;on is obtained by synthesis at high temperature, such as 100C or by degassing the aSIueous reactant solu-30 tions with an inert gas such as argon, nitrogen, or helium.
AntinR;~l~nt~, such as gentisic acid and ascorbic acid,added during or after apatite particle synthesis may also be used to prevent metal ion oxidation . ~ ; n~ agents, such as NaBH~, have been found to reduce metal ions that are 35 lln~nt~nt;nn:q11y oxidized during apatite p~rticle synthesis.

WO 9S1~7~37 PCT~US94/0327C
~77~9 Paramagnetic particles may also be prepared by adsorb-ing p~L _ -tiC metal ions onto the particle. For exam-ple, ~-n~n~Re can be adsorbed to hydroxyapatite particles by taking a slurry of hyd-.1~y-~atite and adding Mn(NO3) 2 5 with stirring. Applying energy, such as ultrasonic power or heat, to the resulting mixture may also facilitate the reaction. The resulting mixture can be separated by either centrifugation and rlr-r~nt~t;rn or by filtration. Any excess m=n~n~r- may be removed by washing with large 10 amounts of water. The r-n~nPSe adsorbed particles can then be stabilized against oxidation and particle agglomer-ation with a suitable coating agent. The same procedure may be used with other par~ n~t;c cations. The amount of --n~:'n'~qe adsorbed onto the particle surface, as a percent-15 age of the total calcium in the particle, is in the rangefrom about 0.1~ to about 5096. Such particles exhibit very high relaxivities and rapid liver ~nh;lnr ' in magnetic resonance imaging studies.
ao p~rticle Size Reduction and Production of ~articles of rTn;form Size ll~;n~r a Microfl~ ;7er It has been found that passing calcium/oxyanion-nt~;n;nS particles, including apatites and apatite~L~ ULSOL~, through a micrrf~ ;7~r results in dramatic 25 particle size reduction. A microfluidizer, such as those produced by Microfluidics Corporation, Newton, Massachu-setts, causes two high pressure fluid streams to interact at ultra high velocity. It is postulated that shear, impact and cavitation forces act on the fluid streams to 30 achieve submicron particle r~ rt;r,n with uniform distribu-tion. Fluid pressures typically range from 2000 psi to 30, 000 psi with some production size microfluidizers capable of h~n~l;n~ pre88ure8 up to 40,000 psi.
Experimental results suggest that particle size 35 reduction using a microfluidizer can be obtained from wo 95/27437 PCT/17Sg4/03276 apatite particles regardless of whether the particles are first stabilized with a coating agent or purified from the base, salts, and other c ,_ lq used to prepare the particles. The particles may be purified or unpurified, 5 coated or lln~ te~ when passed through the microfluidizer.
However, it appears that the ~icrofluidized apatite parti-cles show better stability with removal of the base, salts, and other ~ul~ds in the reaction mixture. The particles tend to become larger when stored in the basic reaction 10 solution, but growth of purified particles is either stopped or inhibited by purification of the particles from the mixture. Particle purification can be obtained by proce8ses such as repeated centrifuging and decanting, passing through a desalting colu~nn, and filtration, prefer-15 ably t~n~nt;Al flow filtration or ultrafiltration.
Pa~ti~le Coatinq Stabilized calcium/oxyanion-rr~ntA;n;n~ particles, including apatites and apatite LJrtuuL8uL~, are deslrable 20 for ~n vivo use as medical diagnostic imaging agents. Such particles tend to aggregate. Although the reasons calci-um/oxyanion-cnnt~;n;n~ particles aggregate is not fully understood, it has been foun-d that several different coating agents are able to inhibit particle aggregation.
25 For example, these particles may be stabilized by treatment with coating agents such as di- and polyphosphonate-con-taining Inrlq or their salts, such as ~ yd~u~yt:thane-1,l-diphosphonate (HEDP), pyrophosphate, Am;n~lph(~sphonatesi carboxylates and polycarboxylate-rtnt~;n;n~ rc _~u-lds such 30 as oxalates and citrates; alcohols and polyalcohol-contain-ing compoundsi . Ju~ld8 containing one or more phosphate, sulfate, or 8ulfonate moiety; and biomolecules such as peptides, proteins, antibodies, and lipids all have been shown to inhibit particle aggregation. Such coating agents wo gsl27437 Pcr/lrS94/03276 ~18~4g i~

stabilize the small particles by reducing further particle growth and promoting particle suspension.
When used in magnetic resonance imaging, particle relaxivity is ~nh~nrP~ by allowing more water accessible to 5 the particle surface. By limiting particle size and increasing the available surface area, relaxivity may be improved .
In addition to the coating agents ;A~ntif;~d above, conventional particle coating techniques may also be used 10 in the manuf acturing processes of the present invention .
Typical coating techniques are ;~ nt;f;ed in Int~rn~t;nn:ll Publication Numbers WO 85/02772, WO 91/02811, and European Publication Number EP 0343934, which are incorporated by ref erence .
For instance, agglomerated particles may be disrupted by mechanical 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 pre-ferred coating material. Albumin and surfactants, such as tween 80, have also been used to reduce particle aggrega-tion. One common characteristic of useful apatite coating agents is their ability to modify the particle surface charge, or zeta potential.
It will be appreciated that the calcium phosphate-containing particles within the scope of the present invention may be coated before, during, or after passage through the microf luidizer . When coated during passage through the microfluidizer, one fluid stream is the coating agent, while the other fluid stream is the particulate stream .
The currently preferred mechanical means for reducing particle size i8 microfl~ ;7~t1nn, but other means such as heating, sonication, other formg o~ particle energ;7~tir-n, such as irradiation, and chemical means, such as pH modifi-Wo 95/27437 PCT/US94/03276 ~187749 r cation or combinations of these types of treatment, such ab pE~ modification _ '-;nPd with 90nication may be used.
D; ~nn~tiC Pha~maceutical Por~--lAtions The calcium/oxyanion-cnnt~;n;n~ particles of this invention may be formulated into diagnostic compositions for parenteral administration. Por example, parenteral formulations advantageously contain a sterile aqueous solution or suspension of treated apatite or apatite precursor particles according to this invention. Variou8 techniques for preparing suitable pharmaceutical solutions and suspensions are known in the art. Such solutions also may contain pharmaceutically acceptable buffers and, optionally, electrolytes such as sodium chloride. Paren-teral compositions may be inj ected directly or mixed with a large volume parenteral composition for systemic adminis-tration .
The diagnostic compositions of this invention are used in a convl~nt; on~l manner in medical diagnostic imaging procedures such as magnetic resonance, X-ray, and ultra-sound imaging. The diagnostic compositions are adminis-tered in a sufficient amount to provide adequate v;Fnl~li7~-tion, 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, ~l-or-on~; n~ upon the diagnostic technique employed as well as the organ to be imaged.
The following examples are offered to further illus-trate the present inve~tio~. These examples are intended to be purely ~ ry and should not be viewed as a limitation on any claimed ~ ; 1 t .

W095/27437 r~uJ~ Ir7~76 ~1877~g r le 1 Preparation o~ v~.y~atite Particles Doped with M~, Treated with HEDP, Purified and Passed through Microf ~
~-n~slnP~e ~nnt~in;n~ llydru~ycl~atite particles were prepared by the following general procedure. A procedure is described for particles ~nnt;~;nin~ 10~ Mn (compared to the total metal content) but other percentages are also applicable .
Into a 1 L erlenmeyer flask were placed 10 . 5 g of (NHI,)2HPO~, 100 mL of concentrated NH~Orl and 350 mL of D.I.
water. The mixture was stirred for two hours with a continuous heavy argon flow (~ R;n~). In a separate 1 L erlenmeyer flask were placed 28 . 9 g of Ca (NO3) 2-4H20 and 2.4 g of Mn(NO3)i-6H20 in 400 mL of D.I. water. The metal nitrate solution was degassed with argon ~or 2 hours. The rhn~rhAte golution was then added dropwise to the rapidly stirred metal nitrate mixture over two hours with a peri-staltic pump. A cont;nllnus argon flow wa8 ~^-;nt~;n 20 throughout the course of the reaction. The reaction mixture was stirred for an additional two hours after the addition was complete . A solution of ~ . 3 mL of a 60 solution HEDP (acid form) in 25 mL of D.I. water was ed for 30 minutes then added in one ali~uot to the 25 lly-lr u~y~atite 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 30 water. The slurry was re-centrifuged at 2400 rpm and the milky s~lr~rn;lt~nt was collected. The solid was resuspended twice more and centrifuged at 2400 rpm. The three washes were combined and centrifuged at 7000 rpm for 30 minutes.
The resulting solid pellet was separated from the superna-35 tant by ~ nt~t;nn, and the pellet was washed (D.I. H20)and centrifuged three times, and the supernatants were _ _ _ _ _ . _ .. . ... _ . . _ .. _ . .. . ..

Wo 9~/27437 PCI/US94/03276 ... _f ~.P. . ..

discarded. After washing, the solid pellet was Qll~p~n~lpd in 3 0 ml~ of D . I. H,O .
The preparation was stored at room temperature for one month. The particle size was analyzed and found to be 280 nm (2.9 chi squared, 0.31 coefficient of variance). The particulate suspension was passed through a microfluidizer at apprn~;r~t~ly 5000 psi. After one pass through the microfluidizer, the particle size was reduced to 125 nm (0.43 chi s,quared, 0.35 coefficient of variance). After another pass through the microfluidizer at a pressure of approximately 10, 000 psi, the size did not change signifi-cantly, 144 nm (0 . 20 chi squared, 0 . 28 coefficient of variance). At three hours and 36 hours after passing through the microfluidizer, the particle gize ., ;n~
essentially constant at 159 nm and 148 nm, respectively.
r le 2 Preparation of H~ dL~.Y _ tite Particle~
Do~ed with Mn and Treated with HEDP and PasAed through Micrnf~ Ar U~purified ~nrJ~n~qe cnnt~;n;nrJ hydroxyapatite particles were prepared according to the ~L.,cedu~ of Example l, except that the particles were not purified by r.ontr; fl~ging~
t9~r:~ntlnJ, and waghing, but left in the base and salt solution. The particulate suspension (average size ~ 1 ~m, chi squared ~ 20) was pa3sed through a microfluidizer at apprn~;r~tf~ly 5000 psi. After one pass through the micro-fluidizer, the particle size was 87 nm (2.3 chi squared, 0.41 co~ff1r;~nt of=variance). After five passes through the microfluidizer at pressures from 5000 psi to 7000 psi, the particle size was 89 nm (0.88 chi squared, 0.37 coeffi-cient of variance).
The resulting particles were too small to pellet at 2400 rpm and were left in the base and salts. There was no indication that multiple passes through the microfluidizer made smaller particles, but it appears the unif ormity was _ _ .

wo 95(~7437 PcTnTS94AI3276 21877qg ` ~

increased . Twenty hours af ter passing through the micro -- fluidizer the particle size has increased to 713 nm (21.1 chi squared, 0.53 co~ff;~ nt of variance). Although the chi squared was large, ~nrl;~pt;n~ a poor fit to a gaussian 5 distribution, the co-~ff;f~i~nt of variance was small with 999~ of the particles less than 2 ~lm and 75~ less than 825 nm. The relaxivity (Rl) of these particles 2 hours after formation was apprnl~ir-tPly 22 mM ls~l.
r le 3 Preparation of ~IydL~Ay~atite Particles Doped wlth Mn and Passed through Micrsf~ 7~
UApurified with a simultaneous coaxial stream of IllEDP
Manganese cnnt~;n;~ hydLul~ycl~tite particles were 15 prepared according to the procedure of Example 1, except that the particles were not coated with ~EDP and were ~ot purified by centri~uging, ~ nt;n~, and washing, but left in the base and salt solution. The particulate suspension was passed as one stream into a microf luidizer . The other 20 microfluidizer stream consisted o~ a ~E~P solution prepared according to the procedure of Example 1. The two streams passed through the microfluidizer at a pressure of 10, 000 psi. The resulting particulate suspension had a particle size of 70 nm (2.4 chi s~uared, 0.42 coPff;r;~n~ of vari-25 ance). The particles were not purified ~rom base andsalts. Two hours after formation the particle size was 87 nm (1. 8 chi squared, 0 .41 cof~ff; ~ i~nt of variance) .
Thirty-six hours after _orr-t;nn the particle size was 903 nm (0.84 chi squared, 0.45 coPff;c;Pnt of variance) indi-30 cating the particles had grown uniformly to a large size.The relaxivity (Rl) of these particles was 24 mM~ls~l.

Wo 95127437 PCr/US94/03276 ,, ~ ,;: .
218774g le 4 Preparation of HYdL~Y~tite Particle~
Doped with Mn ~nd Pansed through Microf~ r Unpurified into Neutral HEDP Solut;nn Manganese rnnt~;n;n~ ~lydLu~Lyd~atite particles were ~=~aled according to the procedure of Example 1, except that the particle8 were not coated with HEDP and were not purified by centrifuging, ~r~nt;n~, and washing, but left in the base and salt solution. The particulate suspension was passed through a microfluidizer at 10,000 psi and into a beaker of neutral HEDP. The neutral HEDP solution was prepared from 8 . 3 mL of a 6096 solution HEDP (neutral form) in 25 mL of D. I . water.
The resulting particulate solution had an average particle size of 1333 nm (7.3 chi squared, 0.40 coefficient of variance). Two hour8 after fnr~-t;nn, the particle size was 884 nm (8.3 chi squared, 0.46 coefficient of variance).
The results suggest that the use of acidic HEDP is useful in the formation of small particles and the neutral form of HEDP may ~e used when larger particles are desired.
Examples 1-4 indicate that the particle size of manganese doped hydLu~;y~atite may be substAnt;~lly reduced by the shear, impact and cavitation forces present within the microfluidizer.
Example 5 Prepar~tion of Hyd ~y~ tite Particles Doped with l~n, Washed, Co~ted with Aminotri(methylene phoFrhnn;~
acid) (ATMP), and P~ssed through ~icrofl--;~;7~-~
M~ng~n~e rnnt~in1n~ llydLu~yd},dtite particles were L~L~:pdL~:d according to the ~Lucedult: of ~xample 1, except that the particles were not coated with HEDP and the particles were washed free of base and salts by centrifug-ing three times at 2400 rpm. Degassed water was used to wash the pelleted particles following centrifuging. An ATMP solution was prepared by mixing p.0027 moles or 1.6 mL
_ .. _ _ . . .. .... .. _ . . . . . . _ _ _ .. ... ... _ _ wo 9sr27437 PCT~U594~03Z76 of a 50~ aqueous 601ution with 25 mL D. I. E~20 and dega3sing for 30 minutes.under argon. The ~TMP solution was added dropwise to the washed particles resulting in a "white"
slurry. The slurry was passed through a microfluidizer at 5 10,000 psi. After passing through the microfluidizer, the particles had an estimated size of 84 nm (1. 3 chi squared, 0.52 coefficient of variance). There was some n~r;fli3t;nn of --n~n~e with time as evident from a brown appearance in the particles. After 5iX days there were two population8 10 of particles, 46 nm and ~2 ~Lm. The percentages of each component could not be ~ t~rm; n~d due to the limits of the particle analyzer and settling of the larger particles.
r le 6 PreparAtion of Hy~ y~tite P~rticl~s Doped with Nn, Coated with HEDP, Pas~ed through Microf~ Q~, and Purified Manganese rnnt~;n;nrJ hydroxyapatite particles were prepared according to the procedure of ~xample 1, except 20 that the particles were not coated with ~EDP and were not purified by centrifuging, r~.or~nt;n~, and washing, but left in the base and salt solution. An HEDP solution prepared according to the procedure of ~xampie 1 was added dropwise to the particles. The particle 8ize before passing through a microfluidizer was 1498 nm (13.4 chi squared, 0.93 co~ff;r;~nt of variance). After passing the particulate 8l~Frn~inn through the microfluidizer at 10, oOo psi the particle size was 62 nm (0.27 chi squared, 0.47 coefficient of variance) . About 2-3 hours after microf~ ; 7~tion~ one half of the particulate suspension was passed through a Sephadex 10 (S-10) desalting column to remove base, salts, and excess ligand. The " ; n; n~ particulate suspension was retained as a control. Following S-10 purification, the particle size was 78 nm (3.3 chi srluared, 0.44 coeffi-cient of variance). Six days later, the particle size of the S-10 purified sample was 100 nm (0.40 chi squared, 0.38 , _ _ _ . , , .. . ... ... ,, . . _ _ _ , _ _ _ Wo 9S/27~137 PCr/US94/03276 f`
21877 ~9 coefficient of Yariance). After 12 days, the size of the particles that.were pasaed through the microfluidizer but were not purified and stored in the base solution increased to 744 nm (4.22 chi scuared, 0.57 coefficient of variance).
5 In contrast, after 12 days the S-10 purified fraction had a particle aize of 77 nm (0.65 chi squared, 0.44 coeffi-cient of variance).
r le 7 Preparation of ~lL.,Ay~atite Pnrticle6 Doped with Mn, Coated with ATMP, P~s6ed through Microf~ r, and Purified Manganese rnnt~in;ng llydL~ y~atite particles were ld ' _d according to the procedure of ~xample 1, except that the particles were not coated with E~EDP and were not purified by centrifuging, decanting, and washing, but left in the base and salt solution. An ATMP solution was prepared by mixing O . 0027 moles or 1. 6 mL of a 50~ aqueous solution with 25 mL D.I. H~O and degassing for 30 minutes under argon. The ATMP solution was added dropwise to the particles. The particle size before passing through a microfluidizer was 1465 nm and difficult to analyze due to settling. After passing the particulate suspension through the microfluidizer at 10,000 psi the particle size was 85 nm (0.58 chi squared, 0.41 coefficient of variance). The particulate suspension was divided into two parts. One part was passed through a Sephadex 10 (S-10) desalting column to remove base, salts, and excess ligand. The re--;n;n~ part of the particulate suspension was retained as a control. Following S-10 purification, the particle size was 67 nm (0.25 chi squared, 0.44 coefficient of variance). Six days later, the particle size of the S-lO
purified sample was 131 nm (0.60 chi squared, 0.39 coeffi-cient of variance) . There were three pop~ ; nn~ in the S-10 fraction: 66 nm (459,i), 193 nm (38~6) a~d 665 nm (165f).
After 12 days, the fraction that was stored in base solu-_ _ _ _ _ _ _ _ ~ 2I877~g l~; 3 ;' tion had a particle size of 515 nm (0.50 chi squared, 0.47 coef f icient of .variance ) .
From the foregoing Examplea, it appears the apatite 5 particles are stabilized better with removal of the base, salts, and exce5s rhn~rh( n~te The particles tend to grow at a fast rate when 8tored in the reaction solution, but growth of purified particles is either stopped or inhibit-ed. There seems to be a preference for the formation of 10 smaller particles when the micro~ ; fl; 7~r experiments are carried out in the ba9e rather than the washed particles.
le 8 Preparation of H~d~ y~ ~tite PArticles Doped with ~Sn, Coatod with HEDP, Pas80d through Microf~ r, ~nd Puri~ied by TAn~On~ ;A1 Flow Filtration Manganese c~ nt~;n;n~ ilyd~u~ycl~atite particles were prepared by the following general ~L~cedu, ~ . A procedure is described for particles ~ nt~7n;n~ 109~ Mn but other percentages are also applicable.
Into a 1 L erlenmeyer flask were placed 10 . 55 g of (NHs)2HPOs, 100 mL of concentrated NH~OH and 300 ml, of D.I.
water. The mixture was stirred for one hour with a contin-uous heavy argon flow (degassing). In a separate 1 L
erlenmeyer flask were placed 28.9 g of Ca(NO3)2-4H,O and 2.42 g (0.01355 moles) of Mn(NO3)2-6H20 in 200 mL of D.I. water.
The metal nitrate solution was degassed with argon for one hour. The phosphate solution was then added dropwise to 3 0 the rapidly stirred metal nitrate mixture over 15 minutes with a peristaltic pump. A c~ntin~ argon flow was r~;nt~;n~1 throughout the course of the reaction. The reaction mixture was 8tirred f or an additional one hour after the addition was compiete. A solution of 5 g or 8.3 mL of a 609~ solution HEDP (acid form) in 20 mL of D. I .
water was degassed for 30 minutes then added dropwise to _ _ Wo 9512~437 PCrlUS94/03276 the llydLu~ydLlatite mixture. The resulting slurry wa6 stirred for 1.5 hours.
Two thirds of the reaction mixture wa6 passed through a microfluidizer at 10, 000 psi. The particle 6ize before pa66ing through a microfluidizer wa6 800 nm (27 chi 6quared, 0.92 co.off1rl~nt of variance). After pa66ing the particulate 6u6pen6ion through the micrQfluidizer, the particle 6ize wa6 53 nm (2.2 chi 6quared, 0.48 coefficient of variance) . The particulate 6u6pen6ion wa6 then purified to remove ba6e, 6alt6, and exce66 ligand by pa66ing it through a t~nJ~nt;~l flow filtration (9I t;--- referred to as "ultrafiltration") 6y6tem. The tangential flow filtra-tion 6y6tem was obtained from Koch Membrane Sy~tem6, Inc., Wilmington, Mi~R6;~rh--~ett6. After each filtration pa66, the o~ 1; ty wa6 mea6ured. A total of 10 filtration pa66e6 were made followed by a 3-fold rnnrPnt~ation 6tep.
Following filtration, the particle 6ize wa6 67 nm (0.43 chi 6quared, 0.44 coefficient of variance). After 12 day6, the 6ize of the particle6 that were pa66ed through the microfluidizer but were not purified and 6tQred in the ba6e 601ution increa6ed to 744 nm (4 . 22 chi 6~uared, 0 . 57 ro~ff;r;ent of variance). In contra6t, after 12 day6 the filtered fraction had a particle 6ize of 82 nm (2 . 7 chi 6r~uared, 0.41 ro~ff;rient of variance).
The re6ult6 of thi6 Example are illu6trated graphical-ly in Figure6 1 and 2.
r le ~3 Preparation ~t 100C of ll~ y _-tite Particle~ M~ f;~ by Surfac~
M,~I, Coating with ~EDP, P~;age through Micrsf~ Ar~ and Pnrification Calcium h-ydLu~y~l~atite particle6 are LJL~aL~d by the f ollowing procedure: ~
A solution rr,nt~;nln ~ 6.5 g o~ I.),HPO4 in 120 mL of D. I . water i6 treated with 60 mL of concentrated ~40 ~VO 95127437 2 1 8 7 7 ~ 9 PCT~US94/03276 , , .

followed by 90 mI- of D.I. water. The resulting solution is stirred for 3 hPurs at room temperature.
Into a 3-neck 1 L round bottom flask er~uipped with a water cooled and low temperature condenser sequence (dry 5 ice/isu~v~dnol), mechanical stirrer and rubber septum are placed 19.4 g of Ca(N03)2-4H20 in 468 mL of D.I. water. The solution is heated to reflux. The phosphate mixture is added to the rapidly stirred calcium nitrate solution dropwise with a peristaltic pump over one hour. The heat 10 is removed when the addition is complete and the reaction mixture is cooled to room temperature. The hydroxylapatite slurry is stirred overnight at room temperature.
The pH of the reaction mixture is decreased from 9.53 to 8.50 with 169 ml of 1 N HCl. Manganese nitrate, Mn(N03)2-6H~0 (2.10 g) is added to the lly-lr~Ayd~dtite mixture and stirred for 1 hour and 15 minutes. The color of the slurry i8 pale tan. The mixture is pagsed through a tangential flow filter to remove excess n~n~n~Re nitrate from the apatite particles. The particulate slurry is then 20 treated with 0.5~ M HEDP (Ca/HEDP mole ratio=1.2) and stirred for 1.5 hours. The color of the mixture is pale pink/purple .
The HEDP treated llyd~v~yd~atite particulate suspension is passed through a micrr,f1~ er at a pressure of 5000 25 p8i. The particulate s-lRrf~nRinn i9 then purified to remove base, salts, and excess ligand by passing it through a tangential f low f iltration system .
r le 10 Prepar~tion of Mn-Doped H~IlvAyc.~tite Particle~
H~ving a Func~ no1 i 7r I Coating Agent, Pa~. age Through Microfl~ nd Purificatio~ }Iy Filtr tion This example describes the general preparation of lly.l"".y~dtite particles having a functi~n;ll;7~r~ coating agent where the fllnrtit~n~lized coating agent is defined as one with the ability to bind tightly to the particles and _ _ _ _ _ _ _ _ . . . . . . . _ . _, . , .. , , _ _ WO95/27437 . ~ ; ! PCT/US94/03276 218774~

colltains a pendant group to which other organic biomole-cules or organic may be attached. The particles are prepared by adding 0.1 to 100 mole 9~ of an appropriate coating agent to a slurry of- Mn(lI) substituted hydL~Lyd~a-til:e with 0.1 to 100 mole 96 Mn based on the Ca used in the reaction. The mixture iB stirred from 1 to 360 minutes at temperatures in the range from 4C to 100C. The particu-late suspension is passed through a microf luidizer at a pressure in the range from 2000 to 20, 000 psi, and the solid separated from the supernatant and purified from excess ions and coating agent by ta~gential flow filtra-tion . The solid may be treated with a metal salt ( 0 . 01 to 10 mole9~ based on the total metal in the preparation).
This is ~rP~ y appropriate if the coating agent con-tains a pendant ,-h,,l ~t i n~ group designed to capture and hold tightly the metal when subjected to in y~ and/or ln v'vo solutions. The resultant solid is purified to remove loosely attached coating agent or free metal/coating agent complex by tangential f low f iltration .
~cam~?le 11 Preparation of EI ~ ,Ay _ ~tite Particles by treatin with Diethylenetriamine-penta ~methyl~ . ,.h,~ .h. . .;
acid), Surf ace 1~ orh; ng Nn, PaElEling through 25 Nicrof~ ;7~r, and Pur;ft~t;,.n Calcium hydroxyapatite is ~L~ar~d by the ~ollowing procedure and treated with the polyphosphonate, diethylene-triaminepenta (methylPn-~rh~-srh~nl c acid) (abbreviated DETAPMDP) having the following formula:

z_Q p N~ CP3 35 A basic ammonium phosphate solution is ~ a:Led using 6.34 g of (NH,)2HPO~ in 120 mL of D.I. water. (~r7n~ ntrated wo ssr27437 PcTluss4/o3276 ammonium hydroxide (60 mL) is added followed by 90 ml of D. I . water. The mixture is stirred for 4 hours at room temperature .
A solution of 19. 0 g of Ca (N03) 2-4H20 in 468 mL of D. I .
5 water is placed in a 3 -neck 1 L round bottom f lask . The reaction setup includes a mechanical stirrer, water cooled and low temperature (dry ice/isopropanol) condenser ar-~ cul~ ~, and a rubber septum. The solution is heated to ref lux with rapid stirring . The basic phosphate solution 10 is added dropwise with a peristaltic pump over one hour The heat is removed af ter the addition is complete and the reaction mixture stirred overnight at room t~ .Lu~ e.
The hydroxyapatite slurry is treated with a solution of DETAPMDP (Ca/DETAPMDP mole ratio=l . l, pH of DETAPMDP
15 6.3) and stirred at room temperature for 2.5 hours. The phosphonate treated mixture iE then reacted with Mn(N03)2-6H20 (Ca/Mn mole ratio-2.3) and stirred for an additional 3 . 5 hours . The reaction mixture is passed through a microfluidizer at a pressure o~ 5000 psi and 2 0 purif ied by tangential f low ~iltration .
From the foregoing, it will be appreciated that the present invention provides an improved method for preparing solid calcium phosphate-c~nt~in;T-~ particles for medical diagnostic applications having a controlled particle size 25 distribution and good yield.
The invention may be: ' ~;ed in other specific forms without departing ~rom its spirit or essential characteris-tics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The 30 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. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging of body organs and tissues comprising the steps of:
(a) obtaining calcium/oxyanion-containing particles having the following general formula:
CanMmXrY?
wherein M is a paramagnetic metal ion, radiopaque metal ion, radioactive metal ion, or stoichiometric mixture of metal ions, X is a simple anion, Y is an oxyanion, tetrahedral oxyanion, carbonate, or mixtures thereof, n is from 1 to 10, m is from 0 to 10, s is 2 1, and r is adjusted as needed to provide charge neutrality; and (b) passing the calcium/oxyanion-containing particles through a microfluidizer.

2. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging 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+ and m is 1.

3. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 2, wherein M is manganese(II).

4. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 1, wherein the particles passed through the microfluidizer have a particle size in the range from about 5 nm to about 5 µm and are used for imaging the liver and spleen.

5. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 1, wherein the particles passed through the microfluidizer have a particle size in the range from about 1 nm to about 50 nm and are used for imaging the blood pool.

6. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 1, further comprising the step of coating the particles with a coating agent to stabilize the calcium/oxyanion-containing particles.

7. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 6, wherein the coating agent is selected from aminophosphonates, biomolecules, and compounds containing one or more phosphonate, carboxylate, phosphate, sulfate, or sulfonate moiety.

8. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 6, wherein the coating agent contains one or more phosphonate moieties.

9. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 6, wherein the coating agent is 1-hydroxyethane-1,1-diphosphonic acid and physiologically compatible salts thereof.

10. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 6, wherein the coating agent contains a reactive functional group.

11. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 10, wherein the reactive functional group is an amine, active ester, alcohol, or carboxylate functional group.

12. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 10, wherein the reactive functional group is capable of chelating a metal ion.

13. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 6, wherein the step of coating the particles with a coating agent is performed after the step of passing the particles through a microfluidizer.

14. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 6, wherein the step of coating the particles with a coating agent is performed during the step of passing the particles through a microfluidizer.

15. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 6, wherein the step of coating the particles with a coating agent is performed before the step of passing the particles through a microfluidizer.

16. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 1, further comprising the step of purifying the calcium/oxyanion-containing particles from base and salts used to synthesize the calcium/oxyanion-containing particles.

17. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 16, wherein the calcium/oxyanion-containing particles are purified by filtration.

18. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 16, wherein the calcium/oxyanion-containing particles are purified by tangential flow filtration.

19. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 16, wherein the calcium/oxyanion-containing particles are purified by passage through a desalting column.

20. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 16, wherein the step of purifying the calcium/oxyanion-containing particles is performed after the step of passing the particles through a microfluidizer.

21. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 16, wherein the step of purifying the calcium/oxyanion-containing particles is performed before the step of passing the particles through a microfluidizer.

22. A method of preparing a calcium/oxyanion-containing particle for use in medical diagnostic imaging as defined in claim 1, wherein the step of obtaining the calcium/oxyanion-containing particles is performed by passing reaction streams containing base and salts required to synthesize the calcium/oxyanion-containing particles through a microfluidizer.

23. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging of body organs and tissues comprising the steps of:
(a) obtaining particles having the following general formula:
CanMnm(OH)2(PO4)6 wherein m is from 1 to 10, n is from 1 to 10, and m+n=10;
(b) passing the particles through a microfluidizer; and (c) purifying the particles from base and salts used to synthesize the particles.

24. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 23, wherein the particles passed through the microfluidizer have a particle size in the range from about 5 nm to about 5 µm and are used for imaging the liver and spleen.

25. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 23, wherein the particles passed through the microfluidizer have a particle size in the range from about 1 nm to about 50 nm and are used for imaging the blood pool.

26. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 23, further comprising the step of coating the particles with a coating agent to stabilize the particles.

27. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 26, wherein the coating agent is selected from aminophosphonates, biomolecules, and compounds containing one or more phosphonate, carboxylate, phosphate, sulfate, or sulfonate moiety.

28. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 26, wherein the coating agent is 1-hydroxyethane-1,1-diphosphonic acid (HEDP) and physiologically acceptable salts thereof.

29. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 26, wherein the step of coating the particles with a coating agent is performed after the step of passing the particles through a microfluidizer.

30. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 26, wherein the step of coating the particles with a coating agent is performed during the step of passing the particles through a microfluidizer.

31. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 26, wherein the step of coating the particles with a coating agent is performed before the step of passing the particles through a microfluidizer.

32. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 23, wherein the apatite particles are purified by filtration.

33. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 32, wherein the apatite particles are filtered by tangential flow filtration.

34. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 23, wherein the apatite particles are purified by passage through a desalting column.

35. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 23, wherein the step of purifying the apatite particles is performed after the step of passing the particles through a microfluidizer.

36. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 23, wherein the step of purifying the apatite particles is performed before the step of passing the particles through a microfluidizer.

37. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging of body organs and tissues comprising the steps of:
(a) obtaining apatite particles comprising apatite or apatite precursor particles, a paramagnetic metal species adsorbed to the surface of the apatite particle, and a di- or polyphosphonate coating agent;
(b) passing the apatite particles through a microfluidizer; and (c) purifying the apatite particles from base and salts used to synthesize the apatite particles.

38. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 37, wherein the paramagnetic metal species is manganese(II), iron(II), iron(III), or mixtures thereof.

39. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging as defined in claim 37, wherein the phosphonate coating agent adsorbed onto the apatite particle surface is 1-hydroxyethane-1,1-diphosphonic acid (HEDP).

40. A method of preparing apatite or apatite precursor particles for use in magnetic resonance imaging comprising the steps of:
(a) preparing apatite or apatite precursor particles 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;
(c) forming a chelate complex between the bifunctional coating agent and the paramagnetic metal ion; and (d) passing the apatite particles through a microfluidizer; and (e) purifying the apatite particles from base and salts used to synthesize the apatite particles.

41. A method of preparing apatite or apatite precursor particles as defined in claim 40, wherein the bifunctional coating agent comprises polyphosphonate diethylenetriaminepenta(methylenephosphonic acid) having the following general structure: