CA2112905A1 - Ultrasmall non-aggregated porous particles entrapping gas-bubbles - Google Patents

Abstract

Ultra-small, substantially non-aggregated, non-crystalline particles of predetermined uniform size wich, when suspended in a liquid, contain entrapped gaseous bubbles are disclosed. These gaseous bubble particles are prepared by simultaneous co-precipitation of two compounds wherein one compound is substantially more soluble than the other in a given vehicle. When this vehicle is used for washing the co-precipitated particles, part of the soluble material is dissolved leaving a porous matrix. The porous particles then are dried and stored. The porous particles, which can be resuspended immediately prior to use, contain entrapped gas in the evacuated crevices or pores which is not displaced for a period of time because of surface tension of the suspending vehicle. The ultrasmall porous particles can be used as ultrasound contrast agents, such as ultrasound contrast agents in the blood vessels and soft tissue, including the liver, spleen, heart myocardium, kidney and brain of an animal, such as a human. The ultrasmall porous particles also can be used dry for applications where reduced weight is desirable.

Description

WO 93/00933 PCI /U~i92/05S92 21~29(~

ULTRASMALL NON-AGGREGATED POROIJS PARTICLES ENTRAPPING GAS-BUB~LES

Field uf the Invention The present invention relates to ultra-small, substantially non-aggregated porous particles of predet~rm med uniform size which, when reconsti-tuted contain entrapped gas bubbles. ~ The ultrasmall porous particles are suitable for use as fillers and the like~ and as ultrasound contrast ag nts when sus-pended in;a suitable ultrasound liquid. The present invention further rela~es to ~methods of making and~
using such ultrasmall, uniform~porous~parti~les~

, l~ckqround of_the_Inv ntion ~: Par~icles of compounds having low solu- ; :
bility in a disperslng medium are co~only used in ~a wide variety of applications, including pharmaceuti-15cals, c:eramics, paints, inks, dye~;, :lubricants~, pe~t-icides, insecticides, fungicides, fertilizers, chrom-:

~ :

W093/00933 PCT/~S92/05592 ~ s .
.'1,9~ 2-atography columns, cosmetics, lotions, ointments, and detergents. Aqueous dispersions o* particles are used in many cases to avoid hazards such as flammability and to.xicity associated with organic solvents. Such dispersions typically have a broad range of particle si~e.
In many cases, product performance is improved by controlling the particle size distri-bution. In general, smaller particles of a compound provide a more uniform dispersion and will dissolve faster than larger particles of the same compounds.
Control of particle size is, therefore, important in controlling the rate of solubilization.
Many drugs have been formulated as par-: 15 ticles for sustained-release following oral, aero-sol, subcutaneous, intramuscular, or other routes of administration. Particle size is one important factor a~facting the release rate of these' ~rug~.
Those skilled in the art can discern other examp1es for using particle size to contro1 product perfor-:
mance for the substances listed above.
~ rugs that are insoluble in water can have significant benefits when formulated as a stable suspension vf particles of less than three microns diameter. In this particulate form, the drug can be injected intravenously, cixculate in blood, and be preferentially accumulated in, for example, the , W093/00~33 PCT/US92/05592~
~ 129~

reticuloendothelial system, where it can facilitate normal reticuloendothelial functions such as detoxif-ication. Alternatively, the drug can reside in the reticuloendothelial cells where it is stored until solubilized or metabolized into an active form whlch ~irculates in blood to other tissues for efficacy.
This "slow" release of active drug can provide more constant drug concentrations in plasma over a period of hours, days, weeks, or months, resulting ln improve.d therapeutic efficacy. Biodegradable particles which are radiopa~ue or labelled with a radioisotope are useful for diagnoskic imaging of organs, such as liver and spleen, with high concentrations of fixed reticuloendothelial cells.
Solid biodegradabl~ particles ~also ~can be useful for diagnostic ultrasound imaging of the liver and spleen with;ultrasound~ These particles can be effective i~ they~ have a densi ~ ~or compressibility significantly di~ferent from :~ : 20 surr~unding tissue, thereby producing an impedance mismatch responsible for backscatter enhancement~
Since tumors and other leslons generally do not contain these fixed reticuloendothelial cells, :~ particles are not accumulated in these lesions so only tissue parenchyma backscatter is enhanced creating a larger difference in echogenicity between parenchyma and ~esion thereby facilitating lesion , W0~3/00933 PCT/~S92/05592 _ detection and diagnosis by ultrasound. See, for example, Parker, K.J. et al.: Ultrasound in Med. &
Biol., 13(9): 555-566 (1987).
By far the simplest form of ultrasound contrast agents is free gas bubbles~ Such bubbles may preexist in the liquid vehicle, or may be intro-duced via cavitation during the lnjection pha~e.
~hatever the mechanism may be, it appears that many liquids, when rapidly injected into ducts or ves-sels, are capable of generating a quantity of air bubbles which may produce sufficient echoes to cause partial or complete intraluminal sonographic opaci-fication.
The first report on the use of free gas bubbles appears to be that of Gramiak, R. and Shah, p.J.: ~ , 3:356-366 (1968).
They repor ed that they obtained ana~omic validation of the aortic origin of cardia~ ecboes by means of direct physiological saline injection during continu-~0 ous echocardiographic recording. The injection pro-duced a cloud of echoes which was dellneated by the parallel signal of the aortic root. See also, for ~xample, U.S. Patent No. 4,276,885. Kremkau, F.W.
et al.: Am. J. Roentqend.; 3:159: (1968) also re-ported :that they obtained intracardiac echoes Erom saline injection and from injection of autologous blood. They demon~trated that air bubbles may be W093/0093~ P~T/US92/05592 generated during the injecti~n process itself. Zis-kin, M.C. et al.: Investiaative Radioloqy, 7:500-505 (19~23 reported using a variety of liguids, such as renografin, carbonated water, and ether (which boils at body temperature) to demonstrate the pre-sence of echo,s in all cases, detected by enhanced Doppler signals from arteries. In recent years, numerous investigators such as Chiang, C.W. et al.:
Chest, 89(5):7z3-726 (1986), Rizayev, M.N. and Azatyan, T.S.: _eart J., 10(6):1308-1310 ~1985), Feinstein, S.B. et al.: J. Am, Coll. Cardiol., 3(1):14-20 (1984~, Konodo, 5. et al.: J. Am. Ço11.
Carldi~l~, 4:149-156 (1984j, Ten-Cate, F.J. et al~:
J. Am. Coll.: Cardiol., 3(1):21-27 (1984), Maurer, B.
e~ al.: Circu1ation,~ 69(2):418-429~ (19843, Arm-strong, W.F. et al.: J. ~m. ~Coll. _ ardiol., - 2(1):63-69 (1983):,~ Tei, C.: et al~ J. ~Am~ Coll.
~ardiol., ~ 3(1):39-46 (1984)~, ~Armstrong, W F. et ~ Circulation, 66(1~:l66-173 (19~2), Meltzer, ; 20 R.S. et al.: ~ Br. Heat J., 44(4):390-394~(1980a), Meltzer, R.S. et al.: ~ J. Clin. Ultras., 9(3? :127 131 (1981), Wise, N.X. et al.: ~ circulatiGfnl 63(5):1100-1103 (1983), Meltzer, R.S. et al.:
Ultraspund Med. _Biol., 6(3):263-269 ~1980b), have .
reported the use of indocyanine green for opaci-ficatlon of the common bile duct in cholangio-graphy. Presumably, microscopic air bubbles . .

W09~/00933 PCT/US92/0~592 _, ~
9 ~

contained in the liquid or generated during the injection phase is responsible for the observed e.ffects. Meyer-Schwickerath, M~ and Fritzsch, ~.:
Ultraschall Med., 7:34-36 (1986) have reported urologic applications of a new commercial agent which incorporates solid particles as microbubble c~rriers.
While free gas bubbles are extremely effi-cient scatterers of sound energy, ~heir utility is limited by the fact that they are effectively re~
moved by the lungs or by pressure changes in the heart~ Thus, it is impractical to use microbubb1es to elicit contrast in the soft ti~sue via venous injection.
In an ef~ort to overcome some of the limita-tions of free gas bubbles, encapsulated gas bubbles were manufactured:and in~ected directly into the car-otid artery in tumor bearing rabbits. See Carroll, B.A. et~al.: Investiqatlve Radiolo~y, 15(3):260-266 (1980~. These onsisted of nitrogen gas trapped in ~0 micron gelakin capsules. Carroll et al. report ultra~onic enhancement of tumor rim in rabbits with VX2 carcinoma. The large size of these particles ; did not allow their administration in the peripheral ,, : 25 circulation. Unfortunately, the :manufacturer of small (2-3 microns), ga5 filled capsules which could cl ar the lungs is difficult due to the extreme W093/00933 PCT/US92/0~92 2112~05 thinness of the capsule wall through which gas diffuses.
As another alternative, U.S. Patents, No.
4,442,~43, No. 4,657,756 and No. 4,681,119, disclose the use of asgr~gates as carriers of gas to produce microbubbles in blood to alter the transmission characteristics thereof to electromagnetic and sonic waves transmitted therethrough. Unfortunately, because the aggregates are of such ~ large size, i.e., on the order of between about 20-250 micr~ns,:
it is difficult if not impossible for such aggre-gates t~ travel pas~ the lungs and hear~, thereby limiting their use ulness as ultrasound contrast agents. In addition,~because~the solid material~s~
from which the aggregates are formed will generally~
:~ : solubilize in body fluids over a short period of time, their~ability to enhance ultrasound image~in~
the lungs and heart~ is~ short-lived:. ;Moreover, the :
individual particles from which the aggregate are~
formed have :little bubble generating capacity~in;~
their unaggregated form.
; Consequently, there is a demand in the :~
ultrasound industry for a contrast agent which:
offers enhanced gas bubble echogenicity with good long-term stability ~for arterial and organ ultra-sound image enhancement following intra~enous ~: injection.

3 PCr/l~S9~/0~592 g 8-SuD~ary of the I~vention In brief, the present invention alleviates and overcomes certain of the above~mentioned prob-lems and shortoomings of the present state of the art through the discovery of novel ultrasmall, substantially non-aggregatedt non-crystalline porous particles of substantially uniform size which, when reconstituted, contain entrapped gas bubbles, and methods of making and using same.
The novel ultrasmall, non-aggregated porous particles of the instant invention are uniquely suited for use as ultrasound image enhancers and for ultrasound measurements~ such as Doppler techniques, when reconstituted with suitable physiologically acceptable liquids. Quite amazingly, the novel ultra~mall non aggregated porous particles provide unique gas bubble echogenicity with good long-ter~
stability for arterial and organ ultrasound image enhancement ~ollowing intravenous in~ection, Even more amazingly, the ultrasmall porous particles:of the instant invention are able to accomplish this without having to form aggregates in order to develop microbubbles to enhance ultrasound imaging.
It has also been discovered, and quite surprisingly, that the novel ultrasmall, non-aggregated porous particles of the instant invention produce enhanced and long-term ultrasound back W0~3/00933 P~T/US92/05~92 21 ~29 ~5 _9_ scatter as a result of gas bubbles, such as air or helium bubbles, being trapped within their solid matrices or pores when they are suspended in a liquid. It is believed that the solid matrices or pores provide the stability for the unique sustained echogenicity which heretofore has not been achieved by the ultrasound contrast agents ~presently avail-able. And, because the novel ultrasmall porous particles of the instant invention have virtually no tendency to aggregate, they are uniquely~suited for use as contrast agents to enhance ultrasound images in the blood vessels and soft tissue or organs ;
throughout~ an animal. In other words,~ because the novel ultrasmall porous particles~have the capacity to circulate throughout the body of an animal, their suitability as ultrasound contrast agents greatly extends beycnd the lungs and heart into, for example, the liver,~ spleen, :~heart myocardium,~
kidney, brain and the like.:
In accordance with` the ~present invention,~ :
the ultrasmall, porous parti~les: of the instant invention are believed to be stable at ambient temperatures, have little to no tendency to .aggre- :
ga~e, and are non~toxic and physiologically accep-table when introduced into the bloodstream of living beings, such as humans. The:size of the ultrasmall, porous particles of the preSent invention typically --1 o--range up to about 10 microns, and preferably from about O.O1 microns to about 5.0 microns, and more preferably from about O.l microns to about 2.0 microns.
Preferred ultrasmall, substantially non-aggregated, non-crystalline porous particles of the inst~n~ nvention are iodipamide ethyl ester parti-cles ha~ring a substantially uniform mean diamster on the order of, for example, about 0.5 up to about 2 microns and the ~bility to entrap gas ~ubbles within their pores after resuspension in a lîquid vehicle.
In accordance with:a further feature~of the instant invention, the~ ultrasmall porous~ particles of substantially uniform size are~ made~ by,~ first, preparing a solution of two separate solid compounds~ :
in a ~uitable sol~ent for the two compounds, second, : infusing a precipitating liquid into the solution at~
a temperature between about -SOC and~ about~lOO-and at an in~usion rate of from~about O~.Ol ml/min.
to about 3000 ml/minO per unit volume of SO ml, the two solid compounds having essentially little solu-bility in th~ precipitating liguid and the solvent being miscible in the precipitating liquid, so as~to , produce~a suspension of precipitated solid~compounds in the form of substantially non-aggregated par-ticles with ~ substantially uniform mean parti:cle :~ diameter selec~ed from the range of up to about lO

. .

W093/00933 PCT/US92/~5~2 21~290~

microns, such that the particle size is directly related to the solution temperature and inversely related to infusion rate, third, separating the co-precipitated particles fr~m the solvent, and, fourth, washing the co-precipitated particles with a washing liquid which serves to selectively 501ubi-lize and remove the second compound as well as any remaining recidual solvent thereby producing par-ticles consisting of a porous matrix consisting of only, or mostly, the first compound. For example, when producing ultrasmall porous particles, such as ultrasmall porous iodipamide ethyl ester (~DE) particles, in accordance with the instant invention, the IDE porous particles formed a~ter the washing step are believed to be mostly IDE, but the matrix of the IDE particles ~ay include, ~or instance/ some : iodipamlc acid (IDA). The ultrasmall "porous"
particle suspension is then exposed to ~as, for example, at in~reas@d pressure, or dried to remove as much moisture as possible and to permit the porous particles to entrap gas within their porous matrices upon suspending the particles in liquid vehi ::les. The ultrasmall porous particles of the instant invention are much less dense than pure 2~ solid particles formed alone following the same procedure.

W0~3/00933 PCT/U~92/05592 9~ -12-It should be appreciated by those versed in this art that when the dried ultrasmall, porous particles are reconstituted or suspended in a suit~
able liquid/ they are completely redispersed, but now a small amount of air or other gas~ such as helillm has been entrapped in the partic1e crevi~ces or pores where the second compound initia11y was present. The entrapped air, crevices or pores is belie~ed to remain for several hours, even after resuspension in a 1iquid vehic1eO Moreover, it is believed that because the crevices or pores are 50 small, the surface tension of the suspending li~uid does not permit rapid ~filling. Consequent~y, such reconstituted suspensions are uniquely~ suited~ for use as u1trasound contrast materials because the :
echogenic gas ~bubbles trapped within the solld~:
; ~ porous:partic1es are :sta~iliæed, e~en against: pres-sure changes in :the heart~ which t~ypically destroys other aompetitive ult:rasound contrasting agents~
currently avai1ab1e. ~ :
: In practicing the methods~ to produce~the ultrasmall porous particles of the instant inven-tion, a preferred weight ratio of the more solukle to the less so1uble compound in the washing solution ~ .
: 25 is from about Z:1 up to about 10:1. In addition, the amount of the more soluble compound dissolved and removed during the washing of the particles can :: :

W093/00933 2 1 1 2 9 o ~ PCT/~S92/05sg2 be from about 10% to about 100% of the amount of the more soluble compound present in the particles after precipitation, but before washing.
In one preferred embodiment, the less s~luble compound in the washing solution is iodi-pamide ethyl ester (IDE) and the more soluble compound in the washing solution is iod~pamic acid (IDA). The precipitating liquid is water at about pH 5 and the washing liquid is aqueous nO1% PVP at ~bout pH 11. When washing the co-precipitated particles in aocordance with the methods of the instant i nvention, washing can be continued until most or all IDA has been removed but most or al:l of the IDE remains which, for example, is preferably at a pH of about 3.3 to about 3.4 when producing ultrasmall porous IDE particlesa When washing is :
:: stopped at a pH of about 3.3 to 3.4, it is believed that the yields and~ echogeniaity of the~ultrasmall~
porous IDE:particl~s are enhanced.
: ~ 20 In accordance with a further feature of the~
present invention, the ultrasmall, porous particles~
may be coated with various substance , such as human serum albumin or selected antibodies, to alter the surface properties of the particles to improve, :for example, their biocompatibility or their ability to target a desired site.

PCTiUS92/05592 Accordingly, it can now be appreciated by those versed in this art that the present invention provides a solution to the ultrasound art that has long sought to overcome the shortcomings associated with the ultrasound contrast agents and methods available heretofore.
The above features and ad~antages of the present invention will be better understood with reference to the FIGS~, Detailed Description ahd Examples set out hereinbelow. It will also be understood that the ultrasmall, porous particles and methoda of this invention are exemplary only and are not to be regarded as limitations of~this invention.

: Brief DescriPtion of ~he:FIGS.
Examples of the present invention will now be more~fully described, :~with referen~e: ~o the ~: accompanying FIGS., wh~rein~
:
FIG. 1 depicts raw backscatter vaIues ~RMS
of in vitro: bubble/IDE particle suspensions a~ 5.2 mg/ml plotted versus the weight ratio :of iodipamic acid (IDA) to iodipamide ethyl es~er (IDEj in: a formulation mixture be~ore dissolving~away the IDA.
These data demonstrate increased echo-genicity (porosity~ with higher ratios of IDAjIDE;
FIG. 2:~depicts raw backscatter values (RMS) of in vitro bubblejIDE particle suspensions at 5.2!

W093/00933 21~ 2 9 0 r~ PCT/US92/0ss92 9.1 and 1301 mg/ml plotted versus time after mixing with bovine plasma. These data demonstrate in~
creased backscatter with increased particle concen-tration. These data also demonstrate the extremely S long persistence of this backscatter after mixing with bovine plasma compared with other bubble agents which survive only seconds to minutes;
FIG. 3 depicts raw backscatter values (RMS) of in vitro bubble/IDE particles at 5.2 mg/ml and standard solid IDE particles at 12.2 mg/ml plotted versus time. These data demonstrate the increased echogenicity obtained with bubble/IDE particles compared with that for solid IDE particles. This enhanced echogenicity is observed~even ~though the bubble/IDE particles are at less than half the concentration of the solid IDE partioles;
~:: FIG. 4 depicts B-scan image at 5 MHz of:
~: normal rabbit liver with gall bladder before lnfu-:
sion of the bubble/IDE contrast agent;
FIG. 5 depicts B-scan ~image at 5 :MHZ of normal rabbit liver with gall bladder,~ but :60 ~ : :
minutes following intra~enous infusion of the bubble/IDE contrast agent. The enhancem~nt of the parenchymal echogenicity was observed for ~at least 120 minutes after contrast administration;

W093/00933 ~9~ P~/U~92/05592 FIG. 6 is a graph of infusion rate (ml/
min.3 (of a~ueous pxecipitating liquid) as a func-tion of the product of stir rate (rpm) and total volume (liters) of the organic solution at a con-stant temperature; the relationship: aqueous infu-sion rate (ml/min.) = 23 + 0.14 [stir rate (rpm) X
volume organic solution(l)] defines the parameters for production of iodipamide ethyl ester particles of one micron diameter at a constant temperature (4C) and in dimethylsulfoxide/eth~nol;~
FIG. 7 is a graph showing iodipamide ethyl ester particle size as ~a fun~tion of temperature at~
a constant ratio of infusion rate of aqueous preci-pitating li~uid to [sl-ir ~rate (rpm)~ X volume~ o~
~ ~ 15 organlc solution~; ~
:~; FIG. 8 is a graph demonstrating the: effect on particle si2e of varying the infusion rate of agueous precipitating liquid at~constant temperature ~ ~ and ~tirring rate of an ~ iodipamids ~thyl: ester :~ 20. solution; and ~:
FIG. 9 depicts raw backscatter values:~(RMS~
o~ in vitro bubble-IDE particle~ suspensions at 5.2 mg/ml. These data demonstrate that ~ackscatter increases linearly with~concentration. ~ :~

. .

W093/00933 PCT/US92/~5592 21~2~

Detailed DescriPtion of the Invention By way of illustrating and providing a more complete appreciation of the present invention and many of the attendant ad~antages thereof, the S following detailed description is provid~d con-cerning the nov~l ultrasmall, substantially non-aggregated, non crystalline porous particles, and methods of making and using such particles.
This invention~ cvncerns the preparation of non-aggregated porous particles of a predetermined uniform size. One aspect of the invention concerns the preparation o~ uniform particles of a predeter-mined size in a vehicle in which the concentration of the compound in the vehicle is:greater than the 1~ solubility of the compound in that vehicle. The particles are formed by a carefully controlled precipitation of the compound into a guitable ; precipitating liquid from a solvent in which the compound i~ soluble.
: 20 A second a pect of this in~ention is the si~ultaneous co-precipitation of two compounds ; having significantly different solubilitie~s in designated washing solutions suc~ that the more - soluble compound can be dissolved and removed from the less soluble compound during the washing of the co-precipitated particles. The porous particles then can be coated to change the surface properties W093/~093~ 9~ PCT/US92/0~592 if so desired. The porous particles can then be dried to remove almost all moistur~ from the par-ticles leaving porous particles with lower density.
Such particles in dry form can be useful for high strength low weight materials.
Particles which are about one micron dia-meter or smaller have pores so small that they do not fill rapidly with liquid when the particles are xewetted. In this way it is possible to entrap air (or other gas) in the particles. Such particles can be useful as echogenic ultrasound contrast materials.
An important principle underlying this invention is the differential solubilities of the two compounds chosen for co-precipitation. For example, an ester and the acid from which it is synthesized may both be insoluble in aqueous solu-tions at, for example, pH 5-7. However, the acid may have signi~icantly higher solub:ility at pH 10~
such that washing at basic pH dissolves the acid leaving porous particles consisting solely,~ or mostly, of the ester compound. Other acid deri-vatives, such as amides can be substituted for ~the ester compound recognizlng that the wash solution may have to be at a lower pH to be effecti~e.
Similarly, two compounds :could be chosen for washing with an organic solvent as long as the W093/00933 PCTtUS92/05592 21~290~

two compounds have significantly different solu-bilities in that solvent.
This invention can be practiced utilizing compounds which differ in solubility in a given wash solution by orders Qf magnitude. The difference in solubility, however, can be small - a factor of two or less - but washing conditions must be a~justed to~
create the porous particles.
Varying the ratio of more~-soluble to less soluble components can alter the properties of the resultant particles~ For example, varying the ratio of iodipamic acid (IDA) to iodipamide ethyl ester (IDE~ ~rom 2:1 to 8:1 significantly ~increases: the echogenicity of the resultant particles,~:~as shown in FIG. 1. These data~ ustrated in FIG. 1 were ac-: ~uired at 4.7 mg/ml. ~ ~
: Varying the concentration of particles also affects the echogenic properties of ~a~ suspension.
As shown :in FIG. 2, the RMS backscatter~of particles in plasma increased from~0.4 to 1.2 when the concen-t~ation o~ parti-les i~creased from 5~.2 to 13.1 mg/ml.
The ultrasmall porous particles of the : , instant invention can be coated with substances to ;; 25 alter their surface properties. For examplej coat-ing the:particles with serum albumin can improve the biocompatibility of such ~articles. Coating with W093/00933 ~ ~ P~T/US92/0~59 antibodies may improve the targeting of particles to a desired site. Coatings altering the wetability~
charge, or permeability of the particle surface of such particles may bP applied prior to drying to achieve the desired surface characteristics.
Drying can be accomplished by any of a number of techni~ues known to one skilled ln the art. Vacuum, spray, or lyophilization may be utilized depending on the compound(s) and appli-cation for these particles.
After drying, the particles can be recon-stituted with ~ny liquid in which he particles are not soluble. For ultrasonic contrast agents, the liguid generally will be water or some other suit-able aqueous solution. Qther liquids may be appro-priate for other applications.
Methods other than drying can be utilized~
to introduce gas into the porous ~articles For ; example,~ supersaturation ~and~ rectified diffusion under acoustic irradiation can utilize ~the porous particles as nuc]ei for gas~cavity~formation~in~situ.
; The preferred IDE/ID~ particles are be~
lieved to be useful ~or diagnostic imaging with both computer tomography (CT~ and ultrasound. The ~iodine~
in the IDE solid matrix is the effec~ive att~enuator for CT while the antrapped air is the effective scat~erer for ultrasound. Similar particles could W~93/00933 PCT/USg2/0559~
2112~l~U~

be useful for magnetic resonance imaging as well.
This would be possible by reconstituting the dried partic~es with a liquid containing a paramagnetic material, such as gadolinium chelates. ~fter allow-ing sufficient time for equilibration of the para-magnetic material into the pores of the particles, the particles could be useful for enhanced magnetic resonance imaging of tissues, e.g. liver, in which the biodistribution of the entrapped~ paramagnetic "_. 10 material is contro~led by the particle :rather than the bulk liquid biodistribution. It is believed tha~ this can be accomplished by suspending the dried porous particles in a paramagnetic material, such as gadolinium EDTA (Gd EDTA). For example, ~: 15 once he porous partices have keen suspended in gadolinium EDTA,~ they can be introduced into an animaI in an effecti~e amount such that the porous particles and Gd EDTA will accumulate~ ln~ the ~`~ phagocytiG cells in the liver,~ while the remaining Gd EDTA will distribute normally and~clear~ quickly from~ the animal. The Gd EDTA accumulated in the liver will remain in the phagocytic cells: for a period of time to enhance the liver parenchyma :
:~ - during~ imaging. Tum9r and metastatic cells will, however, no~ be enhanced upon imaging thereby improving their detection. Other combinations, W093/00933 ~ PCT/US92/05592 e.g., diagnostic and therapeutic agents, could be utilized in the practice of this invention.
The physical chemical princip1es thought to be involved in this invention suggest that the free energy of the system is higher when the compound is d~ssolved in the organic solvent than when the compound exists in the particulate or crystalline state. During precipitation the compound will naturally convert to tha crystalline ~orm - the ~- 10 lowest ~ree energy state - unless it is trapped in the metastable particulate form, a condition where its free ener~y is intermediate between the solution and the crystalline phases. When properly prac-ticed, this invention enabl~s the trapping of the compound in the metastable particle state, pre-cluding transformation to the crystalline state.
The size distribution of particles formed during co-precipitation can b~ correlated with ~the time interval between onset and completion of co-precipitation. It is belie~ed~ that a very short time interval results in the production of uniformly sized particles, while a very long time interval results in a broad particle size distribution.
Intermediate conditions produce intermediate par-ticle size d1stributions.
An important parameter for utilization of this invention is the solubility of the compounds in ~ . ., -232 1 12 ~ U ~
the precipitating liquid. Thus, compounds having essentially little aqueous solu~ility, i.e~, com pounds which ~ave an a~ueous solubility of less than one part in ten thousand, may be precipitated in an a~ueous solution in order ~o ~btain an excellent yield~ Compounds which are more w~ter-soluble can also use an aqueous precipitating liquid. However,~
the higher the solubllity of the compounds,~ the greater the probability that som~ of the compounds . 10 will dissolve in the a~u~ous phase and transfoxm to the more stable crystalline state. Also, redis-solution ln the a~ueous phase can~lead to a broaden-ing o~ the particle size distribution. For these reasons, it is preferred that :an a~ueous pre~lpi-tating liquid be used for compounds having a water-solubili of less than one part in ten thousand. : :
It has been found that it is possible to prepare:
: suspensions of compounds:which::are poorly soluble in :
aqueous solutions, i.e.,~ have a solubility from about one part per ten thousand to about one part~
per one hundred which provide excellent yields by using an acceptable precipikating liquid in which the compounds have even less solubility:than water.
The difference in the solubiIity of the compounds:in water as compared to the precipitating liquid need not be large in or.der to be significant in terms of W093/0~'~33 ~5~ PCTJUS92/~559%~ `

yield. In order to make particles of a uniform and predeterminPd size, a solution of the solid com-pounds in a suitable solvent is prepared. The solution may be diluted with a non-solvent that does not cause the compounds to precipitate. A preci-pitating liquid is also prepared, preferably with a surfactant, in su~ficient quantity to both co-precipitate the compounds and stabilize the re~.ul ting suspension of particles~ of the compounds . 10 against aggregation. The precipitating li~uid may be used alone when compounds which do no~ aggregate are used. The precipitating liquid is in~used into the solution in which the compound~ are ~dissolved under carefully controlled~ conditions,~including:
~ 15 the rate of stirring of the organic solution;, the : : rate~o~ infusion of the aqueous solution, the volume vf the organic solution and the:temperature of the - solutions and the suspensionL:~ The precipitating liquid~may be infused, for:~example, through: a needle : 20 of standard gauge.
In investigation~ of varying ~parameters~to adjust for particle size, three usable relationships were discovered~ diluting the solution~with:more :
~: ~ of the non-solvent produces larger particles, and ~ diluting~ with less of the non-solvent produces smaller pa:rticles; (2) higher temperatures of the :
:: :

"

:

W0~3/00933 P~T/VS92/055~2 211~3(~5 solution during precipitation produce larger par-ticles, and lower temperatures of the solution during precipitation produce smaller particles; and (3) at a given stirring rate of the organic solu-tion, faster infusion rates of precipitating li~uid produce smaller par icles while slower infusion rates produce larger particles.
When the co-precipitation:is complete, the - uniformly sized particles are:~washed :to:remove the ~- 10 solvent, i.e. by centrifugation,~ filtration, etc,;
and the soluble compound to produce the porous particles of the instan*~invention. In most ca~es, the particles ~shou3.d be separated from~the solvent quickly to prevent transformation to a~ crystalline form.
AqueouB p:recipitating liquids ~are useful ~or many compounds, including but not: limited to organic compounds~such~as~iodipamide ~ethyl :ester, iothalamate ethyl~ ester, iosefamate~ ethyl ester,~
: 20 2,~2'~ 4 4'-te~ra-hydro~ybenzophenone~, RS~ nitro~
cellulose, progesterone, ~: beta-2,4,6-triiodo-3-dimethyl formamidinophenyl propionic acid ethyl ester~, N-(trifluoroacetyl) Adrimycin 14 valerate, : ~ :
1,2 diaminocyclohexane malinate platinum (II)~
norethi~terone, acetyl 5alicylic acid, wafarin, :
heparin-tridodecyl methyl ammonium chloride complex,~
sul~ametho~azole, cephalexin, prednisolone acetate t W0 93/OOg33 ~` PCr/US92/055g2 ~26-diazepam, clonazepam, methidone, naloxone, disul-firam, . mercaptopurine, digitoxin, primaguine, mefloquine, atropine, scopolamine, thi~zide, furosemide, propanalol, methyl methacrylate, poly methyl methacrylate, 5-~luorodeo ~ ridine, c~tosine arabinoside, acyclovir, and levonorgestrel; and inorganic compounds such as aluminum chloride hexahydrate, the oxides of iron, copper, manganese and tin.
~-~ 10 Compounds which are better suited for precipitation using a non-a~ueous precipitating liquid include organic compounds such as mitin domide, hydrolytically unstable compounds such as 1 isopropylpyrrolizine (IPP, or carbamic acid, lS (l-methylethyol)-,(5-(3, 4-dichlorophenol7-2, 3-dlhydro-l,H-pyrrolizine-6-,7-diyl) bis(-methylene ester); and inorg~nic compounds such as iron citrate, iron iodate~, calcium pyrophosphate, calcium sa~icylate, platinum dichloride and ~sodium pyrophosphate.
The first step is to prepare a solution o:~
:~ two compounds, one compound being of interest, in a suitable solvent for the compounds. This can occur by simply dissolving the compounds in the solvent of choice.
The solvent is chosen to suit the com-pounds. For example, dimethylformamide (DMF) is a , W093/0~33 PCT~VS92/05592 2~2~30~

solv~nt for iothalamate ethyl ester (IEE) and iosefamate ethyl ester (IFE), and dimethylsulfoxide (DMSO) is a solvent for iodipamide ethyl ester (IDE) and IEE. ~MS0 is also a suitable solvent for com-pounds such as mitindomide. Another suitable solvent for many compounds, ~nd especially IPP, is tetrahydrofuran (THF).
The solution i5 then optionally diluted with a non-~olYent that doe~ not cause the compounds . 10 to precipitate. :The non-solvent causes greater dispersion of khe dissolved molecules of the com-pounds in the liquid~phase. Greater dilution of the solution with non-solvent produce~ larger particles, and less dilution of~ the solution with non-solvsnt produces smaller part~icles.
The non-solvent should not precipi~ate the compounds whan it is added to the solution. Low~r aliphatic alcohols, such as ethanol, are e~fec~tive non-sol~ents for solutions of IDE and IEE in DMS0.
For the ethyl esters of triiodoben~oic acid, propor-tions of non-solvent to solven~ at a ratio o~ 2: or more can produce 1 to 3 micron sized particles (d~pending on other parameters); and ratios of ~less than 2 can produce submicron particles, at least as applied to DMS0 solutions diluted with ethanol~
To co-precipitate the compounds ~rom the solution in a desired particle size, preferably a W093~0933 PCT/US92/05592 28- :
solution of a surfactant is prepared in sufficient quantity to effect complate precipitation of the compounds and to stabilize the resulting suspension of particles of the compound against aggregation.
The surfactant provides the stabili~ation against aggregation, while a suitable precipitatiny agent causes the co-precipitation of the compounds.
Presence of extra surfactant solution is advisa~ls to en~ure stabilization so that the co-precipitated . 10 particles suspended in liquid do no aggregate, forming agglomerates of an improperly large size.
While surfactants are used in most cases, some compounds appear to form stabl~, substantially non-aggresated particles without the use of sur-factants. Examples of such non-aggregating compounds:are certain heparin complexes.
It is tho~ght that particles :wi~h relatively high surface charge are les~ likely to require ~urfactant in the precipitating solution.
The sur~ace charge o~ a particle is sometim~s referred to as its zeta potential, a measurement of charge which falls off w~th distance. There may be a threshold zeta potential above which no surfactant is needed, but below which, surfactant is needed to keep the precipitating particles from aggregating.
The zeta potential is directly correlated with the polarity or net charge of a compound. Thus, the W093~00933 PCT/VS~i/05592 21~.9~305 need for surfactant in the precipitating solution may be predicted from the extent of the charge or polarity of the compound employed in the method of the invention. F~r example, heparin complexes are highly charged, and form stable non-aggregated particles when precipitated with water.
Generally, such a théory notwithstanding, empirical methods will suffice; that is, a co-precipitation may first be performed with~water, and ~, 10 if aggregation occurs, then a co-precipitation in the presence of surfactant is indica~ed. Surfac-tants are chosen for their compatibility with the compounds and their ability to stabilize a suspen-sion of compound particles. For work with IEE and IDE drug~, a solution of 5% polyvinylpyrrolidone (C-30), 0.1% poly-vinylpyrralidone (C-1~), or ~0~1%
: human serum albumin is preferred. Also 0.1%
Pluronic F-68, [Poloxamer 188, a ::
poly(oxyethylene-co-oxypropylene) polymer~, a 0,33%
gelatin, 0.33% gelatin plus 0~6% Hetastarch, 0.33%
g~latin plus 0.002% propylene glycol, : and 0.3%
gelatin plus 2% sucrose, or other surfactants known to one skilled in the art can be used.
To co-precipltate par~icles of the com-pounds in the desired sizes, the precipitating liquid and the ~olution are combined under controlled conditions of temperature, ratio of WOg3/00933 ~ 3 PCT/VS92/05592 infusion rate to stirring rate, and the proportion of non-solvent to solvent in the dispersed solution~
Preferably, the solution being infused with - precipitating liquid is agitated. This can be accom-plished by stirxing, ~haking, by the infusion itself and by other techniques ~nown to those skilled: in the art. ~his effect can also~ be achieved by com~
bining a stream of precipitating li~uid with a stream of the solution.
f.10 The co-precipitation of the compounds occurs exothermically, heating the solution and the resulting suspension~ The temperature of the solution and resulting suspension is controll d ~o achieve the particle size of precipitate tha~ is:
: 15 desired. Higher solution temperatures during precipitation produce larger particles,~ and lower solution temperatures dur~ing precipitation produce smaller particles. Since many compounds are le s soluble at lower temperatures,~ it is generally preferred to conduct the infusion of precipitating liquid at a low temperature in order to maximize yield. The lower limit of the temperature at which co-precipitation can be conducted is, of course dependen~ upon the freezing point of the solvent, : 25 preclpikating liquid, as well as economic concerns.
Alsoj faster infusion rates ~t constant stirring rate of organic sol~tion produce smal~er W093/00933 2 1 1 2 9 ~ ~ PCT/us92/0~592 particles, and slower infusion rates produce larger particles.
FIGS. 6-8 show the ef~ects on particle size of varying parameters during precipitation of IDE
from a ~MS0 ~olution diluted with l part solution to 2 parts ethanol using an aqueous solution of 5%
polyvinylpyrrolidone at different infusion rates and temperatures.
FIG. 6 shows that as the vol~me and stlr-. 10 ring rate of the organic~compound~iodi:pamide ethyl ester and dimethyl sulfoxide/ethanol solution are increased, t~e infusion rat~ of aqueous surfactant solution must be increased proportionally~as defined by: infusion~ rate (ml/min.l = 23 ~+ 0.14~ [volume (liters)~ X stir ra~e~ (r.p.m.)~ to~ produce particles of 1 micron diameter~at 4C.
; FIG. 7 shows ~hat at a constant ra~io of infuslon rate to~ t;stir~ rate X~ volume~, increased ; precipitation temperature produces~larger particles.
FIG~ 8 plots 3 points~for;rate~of infusion of the precipitating ~1iquid~ into the~ ~organic~
solution to approximate the curve by~ which larger~
particles are formed from slower injection rates, showing that at a constant ratio of temperature ~o [stir rate X volume], particle side ~is ~inversely :
related to the rate of infusion of the precipitating ~ ~ liquid.

: .

W~93~00g33 PCT/U~9~05592 ~9Q~

When FIGS~ 6-8 are considered together, they show clearly that higher temperatures and slower mixing rates produce larger piarticles, and lower temperatures and faster mixing rates produce smaller particles. Another paraneter than can be varied to affect particle size is the amount of diluti~n of the solution before co-precipitation occurs.
When the co-precipitation is complete, ex~ra surfactant solution can be added to further stabi~ize the suspended particles against agglomera-tion. The extra solution can be added at a rapid rate, since essentially all the compounds are now co-precipitated in unif:ormly sized particles. The lS precipitated particles are promptly separated from the solvent to prevent redissolving and repreci-pitation of particl~s at undesirable~sizes. Cen-trifuging is a preferred way to ~perform the separ-, ation.~ Other methodsl including membrane filtra-tion, ~ reverse osmosis, and;~others known to persons~
skilled in ~he art may also be used to remove undesired substances.
Promptly after separating the particles, the particles are washed or rinsed or titrated with a solution in which one of the two compounds initi-ally di~ssolved is soluble to remove solvent and excess surfa~tant and to remove or extr ct such W093/00933 2 1 1 2 ~ ~ ~ P~/usg2/o5592 solubilized compound from the precipitated particles and dried as discussed hereinabove.
The dried porous particles prepared accor-ding to the method outlined above may be resuspended in an appropriate suspension vehicle which may be aqueous or non-aqueous solution, as the situation requires. FOr example, where the porous particles formed compri6e a pharmaceutical compound for parenteral administration, t~e porous parti~les are u~timately resuspen~ed in an aqueous solution, such as a sterile saline solution. In so doing, however, gas bubbles, such as air bubbles, will be entrapped in the crevices or pores o~ the particles. In other instances, the particles may be suspended in a carrying agent such as an ointmen~, gel, or the : like. Preferably, the ~ompound: has the same range of solubility in the suspension vehicle as in the preclpitating 1i~3[uid.
It should be understood to those versed in this art that the methods and~ ultrasmall, non-aggrega ed solid particles disclosed in U.S.
Patents, No. 4,826,689 and No~ 4~997,454, provide teachings upon which the instant invention has improved, and therefore are incorporated herein by reference in their entireties.
Examples of ultrasmall, su~stantially non-aggregated porous particles of the present wOg3/00933 ~ P~T/US92/0~92 invention will now be further illustrated with reference to the following examples.

~xample I
Ultrasmall, non-aggregated porous iodipamide ethyl ester (IDE) particles havi~g a substantially uniform mean diameter o~ about 0.5 microns were prepared as re~ited hereunder :and suspended in bovine plasma-distilled water (1 solution and placed in a smalI plastic pipette.
,~ --A pulse echo technique was used to determine relative back catterO A wide band 10 MHz center fre~uency, Panametrics transducer (1.3 cm diameter 5 focus), driven by a ~SR Pulser, was used ~o obtain rf scan lines. For in y~ measurements~
~: 15 the mean backscatter (root mean square, RMS) was computed for eight uncorrelated: scan linesj: each corresponding to about;4 mm in length. ~
:
~: In vi*ro:analysis of the bubble/IDE par-: ticle suspènsion reveals that backscatter increases monotonically with concentration~, as shown in FIG. 9.
In vitro analysis of the bu~ble/IDE par ticIe suspension and a standard solid IDE particle suspension after mixing with b~vine: p~asma, re~eals a much higher backscatter from the bubble/IDE
particles than from the solid IDE particles, as shown in FIG. 3. Moreover, these data demonstrate W093/00933 2 1 ~ 2 ~ ~J S PCT~US92/05592 that the high echogenicity of the bubble/IDE
particles is sustained for hours after mixing with bovine-plasma.
To prepare the bubble/ID~ suspension, about 250 mg of solid iodipamide ethyl est~er (IDE) and 500 mg of iodipamic acid ~ID~) were added to a 50 ml beak2r with 1" x 5/16" teflon coated stir bar.
Approximately 5 mls of dimethylsul~oxide (DMSO) were added and stirred f or about 10-15~ minO until dis solvedO To the soluti.on, approximately 6.25 mls of absolute ethanol were added. The beaker was then cooled in an acetone/dry ic~ bath. While oooling, the mixture was stirred quickly,~ but without splashing. Approximately 4.5 mls of water at about pH 5 in about 0.5 ml increments were slowly~injected into the mixture while maintaining the ~emperature thereof at about ooc. Approximately 8 mls of water :
a~ abou~ pH 5 was th~n in us~d into the mixture::at a rate of about 5 mls~min. starting at about 0C.
Thus, a total of 12~5 mls of water was in~used~into : the mlxture. A:Harvard Infusion pump and a ~60::cc plastîc syringe and 19 gauge infusion set ~(butter~
fly~ were used to infuse the water. Co-precipita-tion occurred after about 6.5 mls of water were in~used into the mixture and at a temperature of about 1C. Following co-precipitation, the suspensi~n was s~abilized by adding about 150 mls of WOg3/00933 ~ PCT/US92/05~92 1~ polyvinylpyrrolidone (PVP) at about pH 7~4 and kept at a temperature of about 10C. The suspension was then poured into a 250 ml centrifuge bottle (approximately 31.25 mls) and centrifuged at approximately 2500 rpm for about 30 min. The supernate was discarded. The precipitate in the centrifuge bottle was then repeatedly washed with approximately 31.25 mls of about 0.1% PVP at about pH 11 until the wash reached a pH of about 10 to , 10 extract the IDA from the IDE particles to generate the ultrasmall, non-aggregated, porous, non-crystalline IDE particles~ The particles where then ~reated with human serum alb~min according to Exam~le III and then lyophyllized according to the procedure set forth in Example I~.

Exam~l~ II
New Zealand white rabbits (Hazelton Labora-tories) weighin~ 2-4 Kg were anesthekized, and injected intravenously at a rate of about 1 ml/mln.
with a 8-10 ml ~depending on rabbit weight) suspen-sion o~ iodipamide ethyl ester (IDE) porous par-~icles having a substantially uniform mean diameter of about 0.5 microns. The final concentration of the bubble/IDE particle suspension was approximately 100 mgjml. The injected dose of the bubbl /IDE
particle suspension was approximately 250 mg IDE/Kg W093/OOg~3 2 1 1 2 9 o ~j P~T/~92/055~2 rabbit body weight. The IDE particles contained air bubbles trapped within their rev~ces or pores.
The rabbits were scanned periodically.
B-scan images of rabbit liver with and without the bubble/IDE particle suspension are shown in FIGS. 4 and S. These images were obtained from a 5.0 MHz Acuson Scanner with all settings held~constant over a 120 min. examination.
Liver echogen}city following intravenous ~, 10 administration of the bubble/IDE particIe suspen ion is markedly enhanced as compared with no contrast agent, as evidenced by FIGS. 4 and 5. Results in these rabbits demomstrate ~hat the stabilized/
echogeni~:gas can be deliv~red to the li~er, raising :~ 15 the backscatter well above the levels obtained with no contrast ag n~ or with stan~ard solid ;IDE
particles.: Impro~ing liver lesi~n ~detection by enhanced ultrasound may now be possi~le~ with~ the~
bubble/IDE particles.
To prepare the bubble/IDE particle suspen- ::
. :
sion, about 5000 mg of ~olid iodipamide ethyl ester ~IDE) and about 10,000 mg of iodipamic acid (IDA~
were added to a 800 ml beaker with 2" X 5/16" teflon coated stir ~ar. Approximately 100 mls of dimethyl-sulfo~ide IDMS0) wera added and stirr~d ~or about 10-15 min. until dis591ved. To the solution, approximately 125 mls of absolute ethanol were W093/00~33 ~ PCT/US92/0~592 -3~-added. The beaker was then cooled in an acetone/dry ice bath. While cooling, the mixture was stirred ~uickly, but without splashing. Approximately 90 mls of water ai`t about pH 5 at about 10 ml increments were slowly injected into the mixture while main-taining the ~emperature thereo~ at about 0C.
Approximately 160 mls of water at about pH 5 was then infused into the mixture at a ra$e o~ 100 mljmin. starting at about O~C. ~Thus~ a total of 250 . 10 mls of water was infused into the mixture. A
Harvard Infusion pump and a 60 cc plastic syringe and 19 gauge infusion set (butterfly) were used to infuse the water. C'o-precipitation occurred after about }25 mls of water were in~used into the mixture ; 15 and at a tempera ure ;of a~out 20C. ~ Following :
co-precipitation, the suspension was stabili~ed by adding lS0 mls of abou~ 1~ polyvlnylpyrrolidone (PVP) at about pH 7.4 a~d kept at a temperature at about 10C. The suspension~ was then poured into five~250 ml centrifuge bottles (approx~imately 125~ml per~bottle) and centrifuged at approximately 2500 rpm for about 30 min. The supernate was di5-charged. The precipltates remaining in the~ five centrifuge bottles were then repeatedly washed with approximately 125 mls of about 0.1% PVP at about p~
: 11 until the wash reached a pH of abou~ 10 to extract th~ IDA from the particles and to form the W093/00933 2 I 12 ~ j PCT/US92/ossg2 . -3g-ultrasmall, non-aggregated, p~rous, non-crystalline IDE particles. The particles were then treated with human serum albumin according to Example III and then lyophyllized according to the procedure set forth in Example IV.

~xample_III
The IDE particles of Examples I and II were coated with human serum a1bumin to improve the bio-. compatibility o~ such particles.
To coat the IDE particles, an appropriate amount of the IDE suspenæion (e.g. 250 mg or 5 g) is pipetted into a ~0 ml centrifuge tube. The tube is centrifuged at 2500 rpm for 30 min. and the supernatent discardecl. To 5 g or 250 mg of the IDE
porous particl~s, add lO0 mls or 5 mls, rQspec-tively, of about 25~ human serum albumin (HSA) and mix thoroughly with vortex mixer. A preferred HSA/IDE ratio in accordance with the instant invention is about 2:l to about 5:l. Let the HSA
mixture stand at room temperature for a~out two hours. After standing, mix thoroughly with a vortex mixer approximate1y every 30 min., and then store at 40C overnigh~. Centrifuge the stored mixture at about 3000 rpm for 120 min. Discard supernate. Add by pipette, approxima~ely 2,0 mls for 2S0 mg of IDE

W0~3/00~33 ~ PCT/US92/05592 or 15 mls for 5 g of IDE of about 0.1% P~P at about pH 7.4 and mix thoroughly with ~ortex mixture.

ExamPle IV
To lyophilize the IDE particles of Examples I or II, place centr7fuge tubes in dry ice/acet~ne bath to freeze suspension as quicXly as possible~
To facilitate a thin ~rozen layer, the suspension may be rolled onto the lower sides of the centrifuge . tu~
Once frozen, place frozen tubes in lyophi lizer as quickly as possible and begin lyophili-zation. Typical lyophilizer se~tings include:
freeze-dryer temperat.ure e~ual about -55-C; shelf temperature is equal to about -40C; and vacuum is at about 10-50 micrometer mercury. The frozen suspension should be lyophilized until ~he moisture remainlng is about 2~ or less. When removing from the lyophilizer, immediately cap the centrifuge : tuhes and store in a refrigerator at about 5~C until needed.
Alternatively, to lyophilize the IDE
particles of Examples I or II, a compound such as mannitol, or other suitable agent known to one skilled in the art to facilitate lyophilization cake formation, should be added to the suspending vehicle. Up to about 4 ml of suspension then is W0~3/00933 2 1 1 2 ~ ~ ~ P~/us92/05~92 added to about lO ml lyophilization ~ials (or equivalent ratio for larger volumes~ and flash freeze the vial contents. The ~ials then are placed in a lyophilizer for vacuum drying. A profile that works includes a condenser of about -5soc and a vacuum of about 10-50 micrometers of mercury~ ~he shelf temperature is adjusted to about -40C for about 48 hours the~ increas2d to about 15C for abou 24 hours followed~by about~8 ~hours at about . 10 20C. Other pro~iles could be acceptable as long as the moisture remaining is about 2% or less. The vîals should be seal~d,~removed from the lyophilizer and stored at about 5C until needed.:

Exa~ple y:
: To prepare the 1:6 bubbicles (porous particles) ~suspension (Example 16, Table I), about ~: lo grams~of iodipamide~ethyl ester (IDE)~and about 60 grams~of iodipamic ~acid~(IDA)~ were~added~to~a two liter beaker. Approxima~ely tw~: hund~ed milliliters 20 ~ of dimethylsulfoxide~(DMSO~ ~ere;~added:~to the beaker~
and stirréd for approximately one~hour until all ~f :~:
the` solids dissolved. About two hundred and fifty . ~ : milIiliters of ethanol~ was~ then~ added~ to~the solution a~d the solution was :mixed. The: beaker : ^ 25 containing the~solution was then placed in a dry ice/acetone bath and stirred quickly while it cooled;

~ ' ~:

W0~3/~0!)33 ~ PCI/U592/0~92 to about 10 ~ C . Two hundred andL twenty milliliters of water ( about pH 5 ) was then infused at a rate of about 150 ml~min by means of a Harvard Infusion pump, about 60 cc plastic syringe, and 19 gauge butterfly set~ The water (about pN 5) was infused in about twenty milliliter increments into the stirring beaker, allowing the temperature to return to about 10C after each increment. The solution in the beaker was then cooled to about 8~C and about 60 milliliters of water (abou~ pH 5) was infused at a rate of about lO0 ml/min, so that the precipitation would occur at a total infused volume of about 250 ml and a temperature of about 9C. An add~tional 220 ml of water (about pH 5) was then in~used at a rate :of about 150 mljmin into the beaker. The co-precipitated bubbicles ~por~us particles~ were then stabilized by the addition~of about 300 ml of 1% PVP (about pH 7~4).
:~ Ths material was :then poured into six 250 ~0 ml centrifuge bottles (about 208 nl per bottle) and centrifuged at about 2500 rpm f or about 45 minutes.
The supernatant was discarded and each bottle was resuspended in about 125 ml of about 0.1% PVP (about pH ll). The bottles were then centrif~ged (about 2500 rpm for about 30 minutes~ twice more and resuspended in both about 125 ml of about 0.1% PVP
(about pH ll) per bottle and finally about 50 ml of .

W093/00933 2 1 ~ 2 9 o ~ PCT/US92/OS592 about o.~ PVP (about pH 11) per bottle. About one hundred and twenty five milliliters of about 0.1 N
sodium hydroxide was then added to each bottle. The bottles were then washed eleven times by centrifuging at about 2500 rpm for about 30 minutes and resuspending with about 0.1~ PVP (about pH
7.4). Once all of the material was washed, the material was again centri~uged and resuspended in about 0.1% PYP, about 10% mannitol ~about pH 7.4), _. 10 diluted with the mannitol solution to about 50 mg/ml, and lyophilized in doses of 4 ml per lO ml vial as described in Example IV.
Table I hereinafter illustrates the produc tion of ultrasmall porous IDE particles ~in accor dance with Example: I :and V. More parti~cularly, Examples 6-lO in Table I generally follow the :: :
: procedure outlined in Example I, whereas Examples 14~in Table I also;follow Example I procedures : generally, but i~lustrate~a vàriation in the IDE/IDA
~ 20 ratio. Example lS, as compared to Example 13, in :~ Table I ~shows how the diameter of the ~porous~
: particles can be increased by : increasing~
temperature. Examples ~5 and 16 demonstrate how . porous particles production can be scaled~up while :
maintainin~ constant particle diameter.

: ~ :

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~'93/00933 2 1 ~ 2 ~ O S

The presant invention may, of cours~, be carried out in other specific ways than those her~in set for~h without departing from t~e spirit and essential characteristics of the invention. The present embodiments~are, there~ore, to be considered in all respects as illustrative and not rè~trictive and all changes coming w~ thin the mean and equiva-lency range of the appended claims are intended to be embraced therein.
~, 10 Having descri~ed our invention, we claim:

::

: :

: ~

Claims

(1) Ultrasmall, substantially non-aggregated, non-crystalline porous particles having substantial uniformity in size selected from a particle diameter range of up to about 10 microns, said particles having the ability to trap gas bubbles within its porous matrix when suspended in a liquid and to enhance medical images of blood vessels and soft tissue when introduced into the blood stream of an animal.
(2) Ultrasmall porous particles according to claim 1 wherein the mean particle diameter is selected from the range of from about 0.01 microns to about 5.0 microns.
(3) Ultrasmall porous particles according to claim 1 wherein the mean particle diameter is selected from the range of from about 0.1 microns to about 2.0 microns.
(4) Ultrasmall porous particles according to claim 1, said particles being porous iodipamide ethyl ester particles.

(5) Ultrasmall porous particles according to claim 1, said particles being suspended in a liquid and having gas bubbles entrapped within their porous matrices.
(6) Ultrasmall porous particles according to claim 1, said particles having a coating on their surfaces.

(7) Ultrasmall, substantially non-aggregated, non-crystalline porous particles having substantial uniformity in size selected from a particle diameter range of up to about 10 microns, said particles being produced by the process comprising:
(a) preparing a solution which includes first and second solid compounds in a suitable solvent for the two compounds wherein;
(b) infusing a precipitating liquid into the solution at a temperature between about -50°C
and about 100°C at an infusion rate of from about 0.01 ml/min. to about 3000 ml/min. per 50 ml unit volume of solution, the two solid compounds having essentially little solubility in the precipitating liquid and the solvent being miscible in the precipitating liquid, so as to produce a suspension of precipitated amorphous, non-crystalline solid compounds in the form of substantially non-aggregated particles of a uniform size selected from a particle diameter range of up to about 10 microns, the particle size being directly related to the solution temperature during precipitation and inversely related to the infusion rate; and (c) washing-the particles in a suitable washing liquid, the first compound having essen-tially little solubility in the washing liquid and the second compound being soluble in said washing liquid, so that the second compound is extracted from the first compound to produce the ultrasmall, substantially non-aggregated, non-crystalline porous particles.
(8) Ultrasmall porous particles according to claim 7, said process further including the step of:
separating the co-precipitated particles from the solvent before said washing or during said washing.
(9) Ultrasmall porous particles according to claim 7, wherein additional precipitating liquid is added to the suspension before the particles are washed.
(10) Ultrasmall porous particles according to claim 8, wherein the particles are separated by centrifugation, membrane filtration, or reverse osmosis.

(11) Ultrasmall porous particles according to claim 7, wherein the precipitating liquid is a surfactant solution.

(12) Ultrasmall porous particles according to claim 7, wherein a solution is prepared such that the concentration of the two solid compounds are near their solubility limits in the solvent.

(13) Ultrasmall porous particles according to claim 7, wherein the first solid compound is iodi-pamide ethyl ester and the second solid compound is iodipamic acid, the solvent is DMSO, the precipi-tating liquid is water at a pH of about 5.0, and the washing liquid is an aqueous solution of polyvinyl-pyrrolidone at a pH of about 11.

(14) Ultrasmall porous particles according to claim 7, wherein the mean particle diameter is selected from the range of from about 0.01 microns to about 5.0 microns.

(15) Ultrasmall porous particles according to claim 7, wherein the mean particle diameter is selected from the range of from about 0.1 microns to about 2.0 microns.

(16) Ultrasmall porous particles according to claim 7, wherein the particle size distribution has a maximum relative standard deviation of about 40%.
(17) Ultrasmall porous particles according to claim 7 wherein the weight ratio of the second compound soluble in the washing fluid to the first compound insoluble in the washing fluid is in the range of fro- about 2:1 to about 10:1.
(18) Ultrasmall porous particles according to claim 7, said process including the further step of drying said ultrasmall porous particles.
(19) Ultrasmall porous particles according to claim 7, said ultrasmall porous particles being suspended in a liquid and having gas or a medically useful compound entrapped within their porous matrices.
(20) Ultrasmall porous particles according to claim 7, said process including the further step of coating said ultrasmall porous particles.

(21) A sterile injectible fluid composition c matter in unit dosage form and adapted for injectio into the blood stream of an animal for providing ga bubble echogenicity for blood vessels and sof tissue ultrasound imaging, said composition com prising a suspension of said ultrasmall porou particles as recited in claim 1 suspended in carrier liquid which is non-toxic and physio logically acceptable and in which the ultrasmal porous particles are at least temporarily stable.
(22) A composition of matter according to clai 21 wherein the ultrasmall porous particles ar iodipamide ethyl ester particles.
(23) A composition of matter according to clai 22 wherein the iodipamide ethyl ester particles ar substantially uniform in size having a mean particl diameter size of about 0.5 microns.
(24) A composition of matter according to clai 21 wherein said ultrasmall porous particles ar coated.

(25) A method for enhancing ultrasound imaging in the blood vessels and soft tissue of an animal, said method comprising introducing the composition of matter as recited in claim 21 into an animal in an effective amount; and scanning the animal with an ultrasound scanner to generate an ultrasound image.

(26) A method of forming ultrasmall, substan-tially non-aggregated, non-crystalline porous particles having substantial uniformity in size selected from a particle diameter range of up to about 10 microns, said method comprising:
(a) preparing a solution which includes first and second solid compounds in a suitable solvent for the two compounds;
(b) infusing a precipitating liquid into the solution at a temperature between about -50°C
and about 100°C at an infusion rate of from about 0.01 ml/min. to about 3000 ml/min. per 50 ml unit volume of solution, the two solid compounds have an essentially little solubility in the precipitating liquid and the solvent being miscible in the precipitating liquid, so as to produce a suspension of precipitated amorphous, non-crystalline solid compounds in the form of substantially non-aggregated particles of a uniform size selected from a particle diameter range of up to about 10 microns, the particle size being directly related to the solution temperature during precipitation and inversely related to the infusion rate; and (c) washing the particles in a suitable washing liquid, the first compound having essen-tially little solubility in the washing liquid and the second compound having greater solubility in said washing liquid, so that the second compound is extracted from the first compound to produce the ultrasmall, substantially non-aggregated, non-crystalline porous particles.
(27) A method according to claim 26 including the further step of separating the co-precipitated particles from the solvent before said washing.
(28) A method according to claim 26 including the further step of drying said ultrasmall porous particles.
(29) A method according to claim 26 including the further step of coating said ultrasmall porous particles.
(30) A method according to claim 26 including the further step of suspending the ultrasmall porous particles in a liquid.

(31) A method according to claim 26 including the further step of introducing a gas or a medically useful compound into said ultrasmall porous particles.