CA1112162A - Albumin microaggregates for radioactive scanning of reticuloendothelial systems - Google Patents

Abstract

ABSTRACT

Microaggregates of a complex of (1) albumin, particularly human serum albumin and (2) a metal reducing agent, particularly a stannous reducing agent, preferably formed in the presence of a stabilizing ligand, which complex, when labelled with technetium-99m provides an excellent agent for imaging reticuloendothelial systems (RES), particularly the liver, spleen, and bone marrow;
methods of making and using the same; a complex (physical or chemi-cal) of technetium-99m with such microaggregates and methods of using such latter complex.

Description

The present invention relates to an agent for radio-actively imaging the reticuloendothelial system (RES) of ver-tebrates, especially primates, particularly the liver, spleen and bone marrow. Such agents are someiimes referred to as radioactive RES imaging agents. More particularly the inven-tion relates to an RES agent comprising a ~c-labelled micro-aggregated complex of a reducing metal and albumin, particu-larly human serum albumin (HSA), to the unlabelled micro-aggregated complex as such and in the form of a kit, to a method of making the same and to a method of using the same for RES imaging.
At one time the most common commercial RES imaging agent was a radio-colloid (particle size of 0.001-0.05 micrometers (~m)) of gold, stabilized with gelatin, which, after injection into the blood stream,is removed by and collects in the RES to give a latent radioactive image there-of which can be converted into a visible image by the appropriate instrumentation.
This was displaced by 99mTc-labelled sulfur colloid stabilized with gelatin, most of which has a praticle size of <0.1-1.0 ~m, and which is presently still the most widely used radioactive RES imaging agent despite its disadvantages of (a) requiring a relatively large number of components, (b) requiring boiling and neutralization steps for labelling by the user at the use situs, and (c) not belng biodegradable.
Most of the sulfur colloid RES agents on the market do give sharp clear simultaneous images of the liver and spleen.
A 99mTc-labelled stannous hydroxide colloid has also been marketed as an RES agent but it has the disadvantage that it is difficult to prevent growth of the colloidal particles after labelling without the subsequent addition of stabilizers ' by the user at the use situs which make them unsatisfactory for RES imaging, i.e., they are not stable.
Another RES agent which has been marketed in small ;~
quantities is a 99mTc-labelled stannous phytate complex which, it is believed, is converted to an insoluble colloid by ~ -calcium in the blood stream, from which it is then removed by the RES. However, with this agent, difficulty has been encountered in obtaining a clinically acceptable clear, sharp image of the spleen simultaneously with the normal healthy liver. Accordingly, the use thereof has not become widespread.
The present invention provides a highly stable bio-degradable RES agent, which requires fewer components than the sulfur colloid RES agent, which does not require either a heating or neutralzing or any step by the user at the use situs other than addition of a 99mTc pertechnetate solution but yet which gives clinically acceptable clear, sharp images of both the liver and spleen simultaneously, and a novel method of making and using the same.
This is achieved by microaggregati~g albumin anaero-bically preferably human serum albumin (HSA), in the prescence of a reducing metal in ionic or physical or chemical complex form (hereinafter referred to simply as reducing metal), preferably a stannous chloride, stannous iodide, stannous fluoride or stannous bromide, to form microaggregated col-loidal particles of the albumin and reducing metal which are either labelled with 99mTc by admixture with a radioactive pertechnetate solution directly or which is freeze dried and sealed in a sterile pyrogen-free vial and stored until ready for use and is then labelled with 99mTc by admixture with a radioactive pertechnetate solution at the use situs.

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;2 Preferably the microaggregation is also carried out in the presence of an additional ligand, preferably in a water soluble form, for stabilizing the reducing metal against precipitation before aggregation. Preferred ligands are the diphosphonates, preferably methylenediphosphonate, hydroxy-ethylenediphosphonate and aminoethanediphosphonate, the phosphates, sùch as the polyphosphates, e.g., the pyrophos-phates, the aminocarboxylates, such as diethylenetriamine-pentaacetate salts; the polyhydroxycarboxylates, such as glucoheptonate, and the polycarboxylates, such as the salts of carboxymethylcellulose. To date the diphosphonates have been found most preferable. However, other known physio-logically and toxicologically compatible ligands for the particular reducing metal used are suitable. It has been found that the anion of certain useful reducing metal salts, such as stannous fluoride, itself, has a sufficiently high stability constant to preclude the necessity of an additional ligand. In such case, the fluoride anion itself functions as a stabilizing ligand for the stannous ion.
In accordance with the invention radioactive distri-bution by particle size of the microaggregated colloidal particles is as follows: at least 90-95, preferably at least 98, percent of the activity is associated with particles not more than 5 ~m. For good splenic imaging simultaneously with the liver not more than 40-60, more preferably not more than 40-50, and still more preferably not more than 10 to 40, percent is associated with particles less than 0.1, preferably 0.2 ~m. More preferably, for good simultaneous spleen and liver images, at least 40 to 60% and still more preferably more than 50% (the major portion) of the activity is associated with particles between 0.2 ~m and 5, more preferably 3 ~m. Good simultaneous spleen and liver images have been achieved where between 60 and 100% of the activity was associated with particles between 0.2 and 3 ~m provided more than 90-9~/O was associated with those not greater than 5 ~m. Good images of the liver with poorer simultaneous splenic images can be achieved where only 5 to 20% of the activity is associated with micro size particles between 0.2 and 5 ~m, provided more than 90-9~/O are not greater than 5 ~m, the remainder of the activity being associated predominantly with particles less than 0.2 ~m.
Radioactivity distribution by particle size is ob-tained by passing a known aliquot of a diluted suspension of the 99mTc-labelled microaggregated particles through a series of polycarbonate filter membranes (e.g., membranes sold under the name NUCLEPORE by Nuclepore Corporation assembled into NUCLEPORE filter housings according to the manufacturer's instructions and assembled in series into a stack of decreasing pore size (sometimes referred to as serial filtration tech-nique))and then measuring the radioactivity of the filtered particles within each filter housing and of the ultimate filtrat,e by conventional measurement technique9, dividing each amount by the total radioactivity and multiplying by 100 to obtain the percentage. The aliquot is diluted sufficiently to prevent or minimize occluding of the pores of thefilters which will reduce their effective size and thereby result in an incorrect measurement. A preferred technique for diluting the aliquot will be described herein-after. All particlesizes referred to herein are determined by this technique.
Although good liver imaging is achieved where vir-tually all of the particles are less than 0.2 ~m, simultaneous -` $~L12162 splenic imaging is most frequently poor.
Accordingly, it is preferred to achieve a particle size distribution in which as many particles as possible fall between 0.1 or 0.2 ~lm and 5 ~m, more preferably between 0.2 ~m and 3 ~m.
Broken down further, good simultaneous spleen and liver imaging results have been achieved with the following particle size distribution: not more than 4-10% greater than 5 ~m, not more than 15% larger than 3 ~Im, at least 20% over 1 ~m (mostly 1-3 ~m), at least 80 to 90% over 0.2 ~m, and preferably at least 40 to 50% between 0.4 and 3.0 ~Im, with not more than 5 to 10%, preferably no more than 5 to ~/0, mobile (relatively low molecular weight solubles) on saline ITLC.
From less than 15 minutes to greater than 60 minutes after injection, excellent visualization of both spleen and liver are achieved with no significant non-target distribu-tion at optimal acquisition for the target tissue. Marrow distribution, particularly in the vertebrae and pelvic area, can be imaged by ac~uisition for times beyond those which are optimal for liver and spleen, as is true of colloidal sulfur RES agents.
A 901ution of the albumin and reducing metal, prefer-ably with an additional ligand in the solution is heated at a controlled pH and temperature to form the microaggregates.
The aforesaid particle size distribution is achieved primarily by controlling the concentrations of the components, the pH and the heating conditions as described more fully hereinafter.
The reducing metal becomes bound to the albumin (it is believed that a physical or chemical complex is formed) which increases the selective binding efficiency of the Tc-99m to the denatured microaggregated albumin when the micro-aggregated albumin particles are subsequently labelled, to thereby provide increased RES uptake and clear RES imaging.
The function of the additional ligand is to increase the amount of the reducing metal which can be stabilized against hydrolysis (formation of insoluble hydroxides or hy-drated oxides of the reducing metal) before microaggregation.
In the course of denaturation of the albumin caused by heating, it is believed that conformational changes in the albumin ex-pose reactive groups which enhance the affinity of the albumin for the reducing metal, thus binding substantially greater amounts of the reducing metal to and within the microaggre-gates than would otherwise be achieved in the absence of the additional ligand or as compared to reacting the reducing metal with the albumin after it is microaggregated. In any event, the additional ligand contributes substantially to the excellent radioactive imagingof the RES. However, as afore-said, when the anion of the water soluble reducing metal salt has a sufficiently high stability constant to stabilize the reducing agent against hydrolysis, as in the case of the fluoride, the need for an additional ligand is obviated. In such cases the anion is in effect a stabilizing ligand for the reducing metal.
Where the microaggregates (~AA) are to be freeze dried for storage hefore use they are preferably admixed before freeze drying with a stabilizer solution ofsoluble undenatured (unaggregated) albumin (HSA) to aid in the dispersion (recon-stitution) of the solid freeze dried particles in the per-technetate solution when the latter is added thereto to labelthe microaggregates with Tc-99m for use thereof. In a ~ 6 --.. . . .

, preferred embodiment, the stabilizer solution also contains a non-ionic surfactant (preferably Pluronic F-68) to further aid in the dispersion of the solid freeze dried particles in the pertechnetate solution.
Also, buffers, such as sodium phosphate, are added (preferably with the unaggregated HSA and surfactant as part of the stabilizer solution) to achieve a pH sufficiently removed from the isoelectric point of the particles to stabilize the re-constituted preparation against particle growth when the per-technetate solution is subsequently added to the freeze dried particles to form the 99mTc-labelled albumin-Sn++ micro-aggregates dispersed in saline or other pharmaceutically and pharmacologically acceptable carrier for injection into the patient. Therefore, it is advantageous to add such a buffered stabilizer solution in conjunction with labelling, even if the product is to be utilized without freeze drying.
Among the most preferred ligands are the diphosphon-ates; of these methylene diphosphonate (MDP) and hydroxy-ethylene diphosphonate (HEDP) are preferred, but any of the di-phosphonates described in U.S. Patent No. 4,032,625 and German Offenlegungsschrift No. 2,424,296 can be used.
Of the phosphates, pyrophosphate (preferably sodium pyrophosphate) is preferred. However, orthophosphate, the linear polyphosphates and organic phosphates, such as the inosi-tolhexaphosphates may also be used.
Included among the aminocarboxylates which may be used are ethylenediaminetetraacetic acid (EDTA) salts and diethylene-triaminepentaacetic acid (DTPA) salts.
Although polyhydroxycarboxylates and polycarboxylates may function as weak ligands, they are not as suitable as those referred to above.

The stabilizing ligands which may be used are limited only by the ability to stabilize the reducing metal sufficiently against hydrolysis, and by toxicological considerations.
The most preferred albumin for human use is human serum albumin, although albumins from other species may be used for diagnostic applications for those respective species.
Although the stannous (Sn++) ion is preferred as a reducing metal, others, such as the ferrous (Fe +) ion and the monovalent copper ion (Cu ) can also be used, but without as good results. A11 these reducing metals can exist in at least two cationic redox states of which a lower valence charge is required for reduction of pertechnetate in subsequent labelling.
To microaggregate, a solution of a mixture of the al-bumin, ligand and reducing metal is heated rapidly to a tempera-ture between 70C and 100C, more preferably ~0-100C, and still more preferably between 85C and 99C. Optimum results have been achieved with temperatures between 90C and 99C. Higher temper-atures can also be used provided the pressure is increased suf-ficiently to prevent substantial boiling of the reaction mass.
Heating time may be between seconds and hours depending on the temperature and on the manner of heating. For example, heating by microwave energy or by radiofrequency heating or by induction heating requires only seconds whereas heating by immersion in boiling water or by passage through heating coils requires minu-tes. The maximum heating time is dictated by the fact that con-tinued heating after formation of the microaggregates (~AA) may cause an increase in the particle size of the HSA-reducing metal complex beyond 5 ~m and/or the production of soluble degradation products. Also boiling seems to increase particle size. The minimum heating time and temperature are dictated by the time and temperature required to achieve sufficient aggregation to obtain the micro particle size distribution desired as set forth above.

The maximum heating time and temperature are dictated by the time and temperature beyond which too many of the particles become larger than 5 ~m or degradation ensues. Using these guide lines, optimum heating temperature and time can easily be determined by routine testing of any given composition for any given manner of heating to provide the desired particle size distribution. Ex-cellent results have been achieved with heating times of 3 to 10 minutes at heating temperatures of 90-99C where heating was car-ried out in a hot water bath, with acceptable results beingh ach-ieved with heating times of 2-5 minutes using such a bath and temperatures.

It is believed that the albumin becomes denatured dur-ing this heating step, i.e., denaturation occurs simultaneously with microaggregation.
The microaggregation by heating is carried out at a pH
sufficiently removed from the isoelectric point of the albumin ~at which there is a 0 or near 0 charge on the molecules) to give the aforesaid particle size distribution. In this respect commercial HSA is comprised of a mixture of albumins with a distribution of isoelectric point5. Consequently, the iso-electric points of the mixture may vary from lot to lot. They have been reported to range from pH 4.8 to 5.5, however, the presence of charged compounds which tend to associate with the albumin may shift its apparent isoelectric point significantly.
They are believed to exert this effect, either by imparting their own charge to the albumin or by neutralizing some positively or negatively charged groups on that protein.
The closer the pH is to the isoelectric point the larger the aggregate particles, thus at isoelectric pH macro-aggregation or uncontrolled agglomeration occurs. As the pHis moved away from isoelectric pH toward the acid side and to-ward the alkaline side the particles become smaller and smaller.

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~1~2~i2 The optimum pH in the instant invention varies in accordance with the additional ligand which is used, It has been found that the desired particle size distribution of the micro-- 9a -- - . : - .
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~il;2162 aggregates can be achieved over a range of pHs, which again depends upon the particular additional ligand used, the most desirable ligand being that which provides the desired particle size over the widest pH range, thereby giving the least sen-sitive and most easily reproducible system. It has also been found that increasing ionic strength, e.g., by adding a neutral salt such as NaCl, during aggregation will shift the pH at which the desired particle size distribution is obtained further from the isoelectric point. Preferably, aside from the additional ligand and the reducing salt, the only other materials present which will affect the ionic strength of the preaggregated bulk solution (the mixture which is subjected to aggregation) are the acid, e.g., HCl, or base, e.g., NaOH, used for pH adjustment.
However, other materials which affect ionic strength may be pre-sent.
Although the desired particle size distribution can be achieved at a pH range on the acid side of the isoelectric pH
and on the alkaline side, the latter is preferred since micro-aggregation on the acid side presents other difficulties.
On the acid side of the isoelectric point the pH may range from about 3.5 to 4.5 and on the alkaline side of the iso-electric point it may range from about 5.4 to 9.5, more prefer-ably between 5.5 and 7.0 and still more preferably between 5.6 and 6.5, depending upon the additional ligand used, the iso-electric point of the particular albumin used and concentrations of components. However, where the apparent isoelectric point has been reduced by the presence of a charged bompound as aforesaid, to below 4.5, the optimum pH may be as low as 4.5 Under conditions explored with MDP, successful results have been achieved over a pH range of 5.4-6.6, more preferably 5.6-6.5 and optimum reproducible results have been achieved at ~1 .

;2 a pH range of 5.7-6.35. Under conditions explored with pyro-phosphate, optimum results have been achieved over a pH range of 5.9-6.1.
The optimum pH for any particular ligand in any parti-cular concentration can be determined easily and routinely by aggreyating at different pHs away from isoelectric point until the desired particle size distribution is achieved.
The reducing metal, e.g., stannous chloride, may be added to the albumin and ligand solution as a solid or it may be added as a solution.
The maximum amount of reducing metal is that beyond which precipitation thereof occurs before aggregation of the albumin. The minimum amount is that necessary to reduce and bind sufficient Tc-99m to the aggregated albumin to achieve clinically acceptable RES uptake. These amounts can be readily determined for particular admixtures by routineexperiment. Very small amounts of reducing metal are effective for adequate re-ducing and binding of the Tc-99m to the aggregated albumin, e.g.
less than an 8:1 molar ratio of stannous to albumin, but be-cause it is easily oxidized, compositions using the minimum amount re~uired may lose their e~fectiveness over a period of time after storage, handling or use. Accordingly, an excess over the minimum amount for adequate binding of the Tc-99m is used. As the amount of reducing metal is increased there appears to be a point for any given combination of starting com-pounds at which binding effectiveness of the Tc-99m to the al-bumin no longer increases either initially or as a function of time up to 24 hours or more after labelling.
Keeping this in mind the molecular weight ratio of reducing metal, particularly Sn +, to albumin may vary over a wide range, i.e., from 8:1 to 80:1, preferably 30:1 to 50:1.

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It is preferred that the molecular weight ratio of Sn + to al-bumin not exceed 80:1. Excellent results have been achieved with molar ratios of Sn to albumin of from about 30:1 to 50:1.
The minimum amount of additional ligand is that required to avoid the formation of any substantial amounts of the insoluble hydroxide or hydrated oxide of the reducing metal for any given composition by binding the reducing metal. The maximum amount is that beyond which it commences to compete sub-stantially with the albumin for the Tc-99m when the micro-aggregates are admixed with the pertechnetate. Such ligands, when present in excess of that required to stabilize the re-ducing metal, particularly those with high affinity for 1'c, may react with the Tc-99m to form complexes which seek bone, kidney, or other non-target tissues, thereby increasing non-target up-take at the expense of RES uptake and reducing the effective-ness of the microaggregates as RES agents. Accordingly, the amount of ligand should not be in substantial excess of that amount which is required to stabilize the reducing metal. The minimum and maximum amounts of ligand can be easily determined by routine testing for insoluble hydroxides before aggregation and by observing the effect on bone uptake, kidney uptake, urinary excretion, and other non-target distribution by the aggregated product. Furthermore, the greater the concentration of ligand the narrower may be the pH range over which the desired particle size distribution is achieved during aggregation. Accordingly, for optimum results it is desirable to use little more than the minimum amount of ligand necessary to maintain the reducing metal in solution before aggregation.
The concentration of additional ligand and the maxi-mum and minimum amounts thereof also depends upon the ligand used, since some ligands, such as MDP, have a greater binding capacity (higher stability constant) and a lesser ionic -~ - 12 -.

;2 strength than others. A ligand which provides the widest range of pH's over which the desired particle size distribution --is achieved is the most desirable. The diphosphonates, parti-cularly MDP and HEDP, fall in that category. The maximum and minimum amounts of ligand also depends on the particular pH at which the aggregation is carried out.
The optimal amount of additional ligand is too small to have any appreciable buffering effect or to appreciably reduce the target to non-target activity ratio even though the ligand may be one which is known to seek non-target tissues.
It is interesting to note that the addition of ligand which is known to seek non-target tissues, enhances the target to non-target ratio.
Since it is the complexing moiety of the ligand which functions to bind the reducing metal, the concentration of lig-and is best expressed as a molecular weight ratio of such moiety to reducing metal ion, e.g., Sn . This ratio is strongly dependent upon the choice of ligand. For one such as methylene diphosphonate, which has a fairly high stability con-stant, such ratio is preferably between 0.6:1 and 1.2:1, for one such as glucoheptonate, which has a ~airly low stability constant, about 2-1/2 times that ratio is preferred.
Using MDP and stannous chloride and a pH range of 5.4 to 6.6 successful results have been achieved with a ligand:
SnC12.2H20 weight ratio of 0.5:1 to 1:1. If the pH is increased beyond this range the concentration of MDP may be increased to achieve the same biodistribution.
It is clear from the above that there is a functional interdependence between the choice and concentration of ligand and the optimal pH for obtaining particles of the proper size range during aggregation.

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~L~12~L~2 In a preferred embodiment of the invention the aggre-gation is carried out in the presence of a water soluble, pharmacologically and toxicologically acceptable surfactant of the same type which is preferably included in the stabilizer solution. Although good results have been achieved without the presence of a surfactant during aggregation it is preferred to use it as an additional safeguard since it is believed it may increase the reproducibility and stability of the aggregates with respect to particle size.
A wide variety of surfactants is suitable for use in the aggregation step and in the stabilizer solution. Prefer-ably the surfactants are of the non-ionic type and are solid at room temperature. The useful surfactants arethose which are non-toxic to blood components or tissues and preferably have a hydrophilic/lipophilic balance (HLB) of about 14 to about 40, more preferably about 27-30.5. When a surfactant is used in the aggregation step only a very small amount is usually used. Pre-ferably, the surfactant in the preaggregated bulk, when one is used, is present in an amount of about 0.1% to about 10% of the -20 albumin in such pr~3aggregation bulk, more preferably about 2-8%
by weight.
The surfactant dissolved in the stabilizer solution, as aforesaid, aids in the rapid dispersion (reconstitution) of the freeze dried albumin reducing metal aggregate in the per-technetate solution when the latter solution is added thereto for administering to the patient. The amount of surfactant used in the stabilizer solution is usually much greater than the amount used in the aggregation step.
Preferably the surfactant in the stabilizer solution is present in an amount of about 0.2% to 20%~ more preferably 1 to lOYo of the lyophilized composition (solid basis).

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Suitable surfactants for use during aggregation and in the stabilizer solution include Polysorbate 80, U.S.P., higher molecular weight polyethylene glycols such as Carbowaxes made by Union Carbide, and molecular combinations of polyoxy-ethylene and polyoxypropylene (ethylene oxide - propylene oxide block copolymers~, e.g., the Pluronics*, made by BASF Wyandotte.
See also, McCutcheon's Detergents and Emulsifiers, North American Edition (1973) at pages 213-217, where many commer-cially available surfactants having HLB numbers between about 14 and 40 are listed. Most preferrred are *trademark - 14a -, the Pluronics particularly Pluronic F-68, which has a mole-cular weight of about 8350, an HLB No. of 29.0 and i5 a solid at room temperature.
Volume ratio of unaggregated albumin stabilizer solution (containing surfactants and buffers when they are used) to the aggregated human serum albumin (Aggregated Bulk) may vary over a wide range. Excellent results have been achieved with ratios of from 1:1 to 1:3, a ratio of 1:2 being preferred.
Based on the final lyophilized solid composition the ratio of soluble albumin to aggregated albumin may range from about 3:1 to 20:1, more preferably 5:1 to 15:1.
The buffering compound is added to the aggregated bulk either as part of the stabilizer solution, or when such solution is not used, as such, to maintain the pH at a level sufficiently removed from the isoelectric point to stabilize the composition against particle growth from compounding through lyophilization, or after labelling. A suitable pH
range i.s between 7 and 9, preferably 8~0.5, for the reason set forth above. Any compatible pharmacologically and toxico-logically acceptable buffer compound can be used which does not compete significantly for the Tc-99m. Suitable buffers include mixtures of acids and salts of weak acids, such as the appropriate sodium salts of orthophosphoric acid.
In some instances, it may be desirable to add to the preaggregated bulk an electrolyte, such as alkali or alkaline earth metal soluble salts, e.g., NaCl, for adjustment of ionic strength.
The maximum amount of Tc-99m (pertechnetate) added to the albumin aggregates relative to the denatured and aggregated HSA is dictated by the fact that any substantial excess over that which becomes bound to the microaggregates has no B

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beneficial effect and should be avoided for reasons having nothing to do with the invention, namely because the amount of radioactive material injected into the body should be kept to the minimum required. However, a slight excess over that which becomes bound to the microaggregates may be used. Based upon primate studies, up to l~/o free pertechnetate would pro-bably not impair clinical utility. The minimum amount is dictated by that amount required to give clinically accept-able images. The molecular weight ratio of Tc-99m to aggregated HSA (based on molecular weight of the HSA before aggregation) may be as great as 0.25 or greater.
The radioactive dosage of the Tc-99m labelled micro-aggregates of the invention may vary from 0.01 to 50 mCi (millicuries) per patient, but preferably is from 1 to 8.
Preferably, the volume of the pertechnetate solution added to the final bulk as such, or after freeze drying, may vary from 1-10, preferably 1-8, ml containing 1-300 or more, preferably 5-50, mCi per milligram of denatured albumin.
The pertechnetate solution is usually the eluate from a conventional Tc-99m generator but it need not be.
Better binding of the reducing metal to the HSA and more homogeneous microaggregates are achieved with Le~
oxidation of the reducing metal by admixing it and the additional ligand with the HSA before aggregation, with resulting improved RES imaging. It is believed that more intimate contact between the HSA and reducing metal is achieved because, as the HSA opens up during heating, the freshly exposed binding sites thereof react with the reducing metal ~, -16-before and during aggregation and the reducing metal becomes an intimate part of the microaggregate particles.
In any event, as aforesaid, it has been found that better results are achieved when the HSA is aggregated in the presence of the reducing metal. However, the presence of the reducing metal during aggregation presents problems which are not present when HSA is aggregated without the presence of the reducing metal and which are overcome by the presence of the ligand during aggregation, by control of pH and heating condi-tions during aggregation, and by maintaining anaerobic condi-tions throughout.
One such problem is to achieve the particle size dis-tribution desired, another is the binding of the required amount of reducing metal to the denatured albumin; another is to avoid the formation of insoluble reducing metal hydroxides or hydrated oxides.
The water or other pharmaceutically acceptable carrier used to form the various solutions and in which microaggregation occurs is preferably apyrogenic distilled water which has been treated to reduce the oxygen contained therein.
Also, preferably all the .~teps of the process are car-ried out under anaerobic conditions, i.e., in the absence of oxygen, as for example under a nitrogen atmosphere.
In the preferred embodiment the mixture of stabilizer solution and denatured HSA-reducing metal microaggregates, con-taining the ligand, when one is used, are freeze-dried in con-ventional manner in sterile non-pyrogenic containers or vials which are sealed and marketed in the form of a kit which can be used at the use situs by adding the prescribed amount of radio-active pertechnetate to the vial.
With respect to the technique for determining the radioactive distribution of the microaggregates by particle size,an appropriate technique which has been used for diluting the aliquots of Tc-99m labelled microaggregates for passage through the filters arranged in series is by adding to the labelled microaggregates, i.e., the product resulting from the addition of the pertechnetate solution to the lyophilized aggregates, a dilution of the stabilizer solution in a volume ratio of 9 parts diluted stabilizer solution to 1 part of labelled aggre-gates. This ratio may vary over a wide range so long as the finally diluted aliquot does not unduly occlude the pores of the filter membranes. The stabilizer solution is diluted by adding a sufficient amount of saline solution thereto to dilute it to about the same solids concentration as that of the label-led product. The particle size distributions referred to herein were obtained by this technique. However, other techniques can be used.
Although the stabilizer solution is not required when the aggregated bulk is to be used without freeze drying (in such case the buffer can be added as such), it is preferred to add the stabilizer soluiton with buffer in any event to stabllize the particle size upon addition of the pertechnetate solution.
The process of the invention can be carried out as a batch process, a semi-continuous process or a continuous process using a relatively large heating surface to volume ratio.
Preferably the preaggregated bulk is filtered through a sterilizing membrane, e.g., an 0.22 ~m sterilizing membrane, before aggregation and the stabilizer solution is filtered through a sterilizing filter before being added to the aggre-gated bulk.

Also preferably the final bulk (the mixture of the aggregated bulk and the stabilizer solution) is passed through ~1' ~,,.

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a 3 ~m filter to remove particles greater than 3 ~m and is then dispensed into vials and lyophilized.
The aggregated bulk of the invention has a milky to hazy appearance depending on the conditions used in the aggrega-tion step. At the lower pHs within the acceptable pH range it may be opaque. When the pH is increased toward the middle of the range it becomes more lightly milky with slight translucence, and as the pH continues to be increased within the range it become hazy.

To 90 ml of mixing low oxygen water is added anaero-bically the following: 0.6 ml Human Serum Albumin, 25% (25g/-100 ml) Salt Poor, U.S.P., the HSA weighing 0.15 grams, 3 ml sodium methylene diphosphonate (MDP) solution (0.5 grams methyl-ene diphosphonic acid dissolved in 100 ml .OS N sodium hydroxide, 11.15 ml .05 N sodium hydroxide for pH adjustment, 0.5 ml stan-nous chloride solution (4.2 grams stannous chloride dihydrate plus 1.5 ml 12N hydrochloric acid diluted to 100 ml with low oxygen water), and 0.6 ml of a 1% a~ueous solution of Pluronic*
F-68, an ethylene oxide-propylene oxide block copolymer nonionic surfactant. The pH of the solution is 6.1. Aliquots of this solution filtered anaerobically through an 0.22 ~m sterilizing membrane and heated for 3.5 minutes in a water bath at about 99C yield a milky suspension of microaggregates of the HSA
and Sn++.
To approximately 50 ml of low oxygen water is added anaerobically 5.7 grams disodium orthophosphate heptahydrate (buffer), 12 ml 25% HSA and 0.33 grams Pluronic*F-68. After dissolving the solids, the stabilizer solution is diluted to 100 ml with low oxygen water and is passed through a sterilizing filter anaerobically.
*trademark 3.3 ml of the above sterile stabilizing solution is anaerobically mixed with 6.7 ml of the sterile milky suspension of microaggregates. 1 ml aliquots of this formulation, which has a pH of 8 are dispensed aseptically into sterile non-pyrogenic 10 ml serum vials. The vials are freeze dried (lyo-philized) in a conventional manner and under aseptic conditions to remove water. This provides solid microaggregates of the complex (chemical or physical) of denatured HSA and Sn++. Each vial contains 1 milligram of microaggregated particles of com-- plexed denatured and aggregated HSA, 10 milligrams of non-ag-gregated HSA, 0.1 milligrams of SnC12, 0.1 milligrams of MDP, 10 milligrams of phosphate buffer (expressed as disodium ortho-phosphate) and 1.14 milligrams of Pluronic*F-68 surfactant.
The vials are sealed and stored until ready for use at which time the stannous microaggregated alburnin contained therein is labelled with Tc-99m.
To prepare the Tc-99m labelled aggregates, five mils of fresh radioactive sodium pertechnate (about 100 mCi, al-though effective labelling iq obtained from less than 1 mCi to greater than 300 mCi), removed as a sterile non-pyrogenic eluate from a sterile NEN Tc-99m generator in an 0.9/0 saline solution, is added aseptically to each vial, the vial is shaken to dissolve the soluble components and disperse the microaggregate particles in the saline solution thereby recon-stituting the freeze-dried product and labelling the microag-gregates.
Aseptic techniques and sterile, non-pyrogenic ingre-dients and containers are used at all steps.

*trademark , Activity distribution by particle size of the microaggregates revealed the following_in one such preparation: _ _ Particle Size% of Total -~

>5llm 0.5 3 - 5~lm 1.5 0.2 - 3~1m 71.1 <0.2~m 26.9 1-5 mCi of this dispersion of Tc-99m labelled stannous micro-aggregates were injected into adult mice intravenously.
Fifteen minutes after intravenous injection the mice were sacrificed and the various organs, (liver, spleen, etc.) were counted by conventional gamma ray counting techniques to determine uptake of Tc-99m by each organ.

Biodistribution in the mice 15 minutes after intravenous n ~ s as follows:- _ Organ% of Injected Dose per Or~an Liver 87.8 Spleen 2.6 Lungs 0.5 Carcass 5.8 - includes bone marrow Kidneys 1.0 G~ I. Tract 0.6 Remainder 1.6 1-5 mCi of similar preparations were injected intra-venously into primates (baboons). Liver-qpleen imaging was per-formed in conventional manner during the first 30 minutes after injection, using a gamma scintillation camera (Picker Dyna Camera II)o Simultaneous sharp, clear images of the liver and spleen were obtained with low background. Lung and soft tis-sue uptake was minimal. Excellent images of the bone marrow can also be obtained with appropriately longer exposure than that used for liver and spleen. These images were as good as those which can be obtained with sulfur colloid RES agent and would be clinically useful for accurate diagnostic purposes.

'.

Although biodistribution of radioactivity in mice can be relied on to predict in primates the selective uptake by the RES as a whole (liver, spleen and bone marrow) and contrast between up-take of the RES as a whole and uptake by the other organs, it does not correlate with the liver-spleen uptake ratio in pri-mates, and hence with the quality of the splenic image indivi-dually and simultaneously with the liver image in primates. See Int. J. of Applied Radiation and Isotopes 1977, Vol. 28, pp 123-130. Accordingly, to predict this ratio and quality in humans it is necessary to image the RES of a primate such as the monkey or baboon.
The aforesaid bioditribution in mice and the RES radio-active images in primates shows that the radio-labelled stannous microaggregated albumin preparationsof this example are excel-lent RES agents with excellent simultaneous spleen-liver definition.
Substantially the same results were achieved where the freeze-drying step was omitted, also good results may be achieved where the stannous microaggregated albumin i9 used directly without being freeze-dried and without addition of the stabili-zing buffered HSA-Pluronic*F-68 solution but with addition of sufficient buffer to achieve the same pH. However, when the microaggregates are freeze-dried without addition of such sta-bilizer solution it is difficult to redisperse them upon ad-dition of the pertechnetate solution. Whether or not the micro-aggregates are freeze-dried, in the absence of the stabilization provided by the buffer, the particle size may increase in an uncontrolled fashion in pertechnetate-physiological saline.
In each of the following examples all the mixing and filtering and heating steps are carried out anaerobically, as in this example, and in each the 1 ml aliquots dispersed in the *trademark ,~

~L~1216Z
10 ml sterile, non-pyrogenic vials, are subjected to the same steps as in Example 1, i.e., the aliquot may be freeze-dried and labelled , or it may be labelled without freeze-drying,and the labelled aliquot is analyzed for activity distribution by particle size as above. Biodistribution in mice and radioactive images of monkeys, when tested by these procedures, as indicated in each example, are performed in the same'way as in Example 1.

To 45 ml of mixing low oxygen water is added the fol-lowing 0.3 ml 250/o HSA (75 milligrams HSA), 0.25 ml stannous chloride solution (as in Example 1), 0.3 ml 1% Pluronic*F-68 Solution, 11.2 mg diethylenetriaminepentaacetic acid, and 1.65 ml O.lN sodium hydroxide. The pH of the solution is 6.54. Ali-quots of this solution, filtered through an 0.22 ~Im sterilizing membrane and heated for 3.5 minutes at about 99C, yield a light milky suspension of microaggregates of the denatured HSA and Sn++.
3.3 ml of a stabilizing solution compounded as in Example 1, is mixed anaerobically with 6.7 ml of the milky 9us-pension. 1 ml aliquots of this formulation, which ha8 a pH of8, are dispensed into sterile, non-pyrogenic 10 ml serum vials as in Example 1. Test data on one such preparation, processed and labelled as described in Example 1 are as follows:
Activity distribution bY particle size:

Particle Size % of Total . _ >5um 1.1 . 3 - 5~m 1.1 ' 0.2 - 31~m 68.4 <0.2LIm 29.5 To 45 ml of mixing low oxygen water is added the fol-lowing 0.3 ml 1% Pluronic ~-68 solution, 0.3 ml 25% HSA ( 75 *trademark ,, -milligrams HSA), 0.25 ml stannous chloride solution (4.2 grams stannous chloride plus 3 ml 12N hydrochloric acid, diluted to 100 ml with low oxygen water). 16.7 ml of this solution is mixed with 1 ml of a 1% aqueous solution of sodium pyrophosphate (10-2 milligrams Na4P207.10H20) and 0.55 ml .025N sodium hydro-xide solution. The pH of the solution is 5.85. Aliquots of this solution, filtered through an 0.22 ~lm sterilizing membrane and heated for.3.5 minutes in boiling water give a milky sus-pension of microaggregates of denatured HSA.
~o approximately 50 ml of low oxygen water is added 5.7 grams Na2HP04.7H20, 12 ml 25% HSA, and 0.33 grams Pluronic*
F-68. After dissolving the solids this stabilizing solution is diluted to 100 ml with low oxygen water, and is passed through a sterilizing filter.
3.3 ml of the above stabilizing solution is mixed anaerobically with 6.7 ml of the milky suspension. 1 ml ali-quots of this formulation, which has a pH of 8, are dispensed into 10 ml serum vials. Test data on one such preparation, pro-cessed and labelled as described after Example 1, are as follows:
ActivitY distribution by_particle 9ize:

Particle Size% of Total . . .
>5~m 1.0 3 - 5~m 1.0 0.2 - 3~ 69.0 <0.2~m 29.0 To 290 ml of mixing low oxygen water is added the fol-lowing: 1 ml of an aqueous solution containing 0.2 grams Plu-ronic* F-68 per 5 ml, 4 ml 25% HSA (1 gram HSA), 3 ml of a stannous chloride solution (4.64 grams stannous chloride di-hydrate plus 2 ml 12N hydrochloric acid, diluted to 100 ml with low oxygen water), 0.66 grams Na2HP04.7H20 in 15 ml low oxygen *trademark ,j,~, - ~12~6~2 water, and 4.04 ml 5 Molar sodium chloride solution. The pH of the solution is 6.5. After anaerobic filtration through an 0.22 m sterilizing membrane, it is passed sequentially and continu-ously through two heat exchangers. This provides a semi-con-tinuous method for aggregation which can be made fully continuous by use of metered and continuous flow of the compoundingmaterials into a mixing chamber with continuous flow therefrom through the filter and heat exchangers with the exit from the heat exchangers being continuously metered into a mixing chamber into which the stabilizer solution is continously metered and mixed with the resulting mixture being continuously passed through the 3 ~m filter anddispensed into vials which may be continously loaded into the freeze dryer. The first heat exchanger is heated by fluid maintained at about ~9C, and the second is cooled by fluid at ambient temperature, from which the aggregated products are collected anaerobically. The bulk average residence time in each heat exchanger ranges from 3 to 8 minutes, and yields a milky suspension of microaggregates of stannous denatured HSA.
~ To 10 ml sodium phosphate solution (9.5 grams Na2-HPo4.7H2o per 100 ml) is added 2 ml 25% HSA ~0.5 gra~q HSA), 6.25 ml Pluronic*F-68 solution (4.0 grams per 100 ml), and water to a total volume of 35 ml of stabilizer solution. After mixing well, deoxygenation, and sterilizing filtration, 15 ml of the milky suspension is added, followed by thorough mixing once again.
1 ml ali~uots of this formulation, which has a pH of 8, are dispensed into 10 ml serum vials. Test data of such preparation labelled as described in Example 1, are as follows:

*trademark - ~iL2~62 Activity distribution by particle size Paticle Size % of Total _ >3~m 7 1 - 3~m 16 0.2 - l~m 57 <0.2~m 18 Biodistribution in mice, 15 minutes after intravenous administration:
.
Organ% of Injected Dose per Organ Liver 89.3 Spleen 1.1 Lungs 1.0 Carcass 7 7 Kidneys 0 5 G; I. Tract0 2 ~ Remainderb . 2 To 45 ml of mixing low oxygen water is added the fol-lowing 0.3 ml 25% HSA (75 milligrams HSA), 0.25 ml stannous chloride solution (4.2 grams stannous chloride dihydrate plus 3 ml 12N hydrochloric acid diluted to 100 ml with low oxygen water), 0.3 ml 1% Pluronic*F-68 solution, and 3.7 ml 1% sodium pyrophosphate solution (prepared as in Example 3). The pH is 5.71. Aliquots of this preaggregation bulk solution, filtered through an 0.22 ~m sterilizing membrane and subjected to sufficient microwave heating, e.g., 20 seconds, give a milky suspension of microaggregates of stannous denatured HSA.
This milky suspension is formulated with an HSA-sodium phosphate-Pluronic*F-68 stabilizing solution to give a pH8 formulation, after which it is dispensed into vials and labelled with 7 ml 99mTc-sodium pertechnetate all as in Example 3. The following results were obtained on one such preparation:
Activity distribution bY particle size:

Particle Size% of Total >5um 2.1 3 - 5~m 3.1 0.2 - 3~m 84.0 <0.2~m 10.8 *Trademark 4~
,p, ' `

$~

Biodistribution in mice 15 minutes after intravenous administration~
Organ% Injected Dose per Organ Liver 85.9 Spleen 4.3 Lungs 0.5 - Carcass 5.3 Kidneys 1.0 G. I. Tract 0.7 Remainder 2.3 EXAMPLES 6 and 7 To 417 ml of mixing low oxygen water are added the following 16.3 ml (0,75 grams) of purified HSA (delipidized by acidification and activated ch`arcoal, as described in copending Canadian Patent appLication Serial No. 29g,753, filed March 23, 1978, Eugene L. Saklad, followed by ultrafiltration, to give an albumin concentration of 4.6%), 2.5 ml stannous chloride solu- ,~
tion (4.2 grams stannous chloride dihydrate plus 3 ml 12N
hydrochloric acid diluted to 100 ml with low oxygen water) and 3 ml 1% Pluronic*F~68 solution. The resulting solution is mixed well with 25.6 ml of a 1% aqueous solution of sodium pyrophos-phate (prepared as in Example 3) and 34.2 ml of .025N sodium hydroxide. 150 ml of the resulting solution is filtered through an 0.22 ~Im sterilizing membrane. This filtered pre-aggregation bulk, having a pH of 5.62, is reserved for Example 6.
An additional 3.4 ml 0.025N sodium hydroxide is added to the ~emaining solution while maintaining agitation. The pH
is 6.11. Filtration is performed as above. This pre-aggregation bulk is reserved for Example 7.

. .

Aliquots of both pre-aggregation bulks are heated for 3.5 minutes in a hot water bath at 99C to form microaggregates.

ApPearance Example 6 ExamPle 7 milky X
hazy X

*trademark -. . . , . . ' : -Formulated with stabilizing solution (pH of stabilized formulation ~) dispensed into vials and labelled with 5 ml 9 Tc-sodium pertechnetate, all as in Example 3, the following results were obtained:
Activity distribution by particle size:

. . . ... _ Particle Size % of Total - ~ mple 7 >5~m 1.9 0.7 - -' 3 - 5~m 2.8 1.3 1 - 3~m 32.7 1.3 0.2 - l~m 50.5 4.6 <0.2~m 12.2 92.1 Biodistribution in mice 15 minutes after intravenous administration:
Orqan % of Injected Dose Per Organ Examp e 6Example 7 Liver 86.0 88.3 Spleen 3.3 2.2 Lungs 0.8 0.6 Carcass 6.2 7.3 Kidneys 1.0 0.7 G. I. Tract 1.0 0.3 Remainder 1.7 0.6 Imaginq in Monkeys:
Organ Qualit of Ima e Liver Good Good Spleen Good Poor Bone Marrow Good Good Deterioration in the quality of splenic imaging resul-ting from a sharp reduction in particle size caused by increas-ing the pH in Example 7 is evident from the correlation in Examples 6 and 7 between activity distribution by particle size (also reflected in appearance of aggregated bulks) and image quality in healthy primates.

-Tb45 ml of mixing low oxygen water is added the fol-lowing: 0.3 ml 25% HSA (75 milligrams HSA), 1.5 ml hydroxy-.:
,: .

ethylenediphosphonate (HEDP) solution (an aqueous solution con-taining 0.21 grams HEDP in 30 ml), 0.25 ml stannous chloride so-lution (4.2 grams stannous chloride dihydrate plus 3 ml 6.15N
hydrochloric acid diluted to 100 ml with low oxygen water), 0.3 ml 1% Pluronic*F-68 solution and 5.42 ml 0.05N sodium hydroxide solution. The pH of the solution is 5.90. Aliquots of this preaggregation bulk solution, filtered through an 0.22 ~m steri-lizing membrane and heated for 3.5 minutes in a hot water bath at 99C, form a milky suspension of microaggregates.
Formulated with stabilizing solution (pH of formula-tion is 8), dispensed into vials and labelled with 5 ml 99mTc-sodium pertechnetate, all as in E~ample 3, the following results were obtained:
ActivitY distribution by particle size:

Particle Size% of Total _ _ _ _ ~3~m 8.9 1.3~m 2.1 0.2 - l~m 61.2 -<0.2~m 27.8 Biodistribution in mice 15 minutes after intravenous administration-. _ Orqan k Iniected DoRe Per Organ Liver 92.8 Spleen 2.6 Lungs 0 4 Carcass 5.1 Kidneys 0.5 G. I. Tract 0.5 Remainder 2.3 The lyophilized stannous microaggregated albumin of the instant invention has a very long shelf life of at least one year at ambient temperatures (when protected from light). It is also stable for at least 24 hours after. labelling although it iQ preferred that it be used within 8 hours after labelling when stored under refrigeration, for patient safety, because it *trademark .~j;r~, ~,,,~"

lZ~2 contains no preservative.
9mTc-labelled biodegradable stannous-macroaggregates (greater than 5 ~m) of HSA macroaggregated at or near the iso-lectric point of the HSA at which the particles have a 0 or near 0 charge (achieved by aggregating at the isolectric pH of about 4.8-5.5 and at a relatively high ion concentration) have been used commerically for radioactively imaging the lungs. They are also described in the patent literature. See U.S. Patent Nos. 3,987,157; 3,863,004 and 3,872,226. However such agents are not suitable as RES agents since these larger size particles are removed from the blood stream by the lungs before they reach the liver, spleen and bone marrow. See also U.S. Patent No.
4,024,233 and United States Pharmacopia XIX page 488.
An RES scanning kit of tin-containing Tc-99m-labelled minimicroshperes (mean diameter of 1 um and 98% by weight less than 3 ~Im) of HSA maintained at a pH of 2.4 with a glycine-HCl buffer and said to be offered by 3M, is reported in Journal of Nuclear Medicine Vol. 16, No. 6, 1975, pp. 543. It is believed that these minimicrospheres are precipitated from a liquid HSA
emulsion in an oi~ bath. It is not ~nown at what point the tin is added. See als0 U.S. Patent No. 3,937,668 and Int. J. of App. Rad. and Isotopes, 1970, Vol. 21, pp 155-167.
Also, it has been suggested in the literature to ultrasonically disintegrate radioiodinated and 99mTc labelled macroaggregates of HSA to micro-size particles. (J.N.M. Vol.
13, 1972 pp 260-65; J.N.M. Abstract, Vol. 12, No. 6, 1971, pp 373, J.N.M. Abstract Vol. 10, No. 6, pp 453-4, Int. J. of Applied Radiation and Isotopes, 1975 Vol. 26, pp 31-32). How-ever, there is no mention in these reports of the presence of a reducing metal during aggregation or a ligand therefor, and such products require a disintegrating step.

.

. ~ . . : .
:

1~L1;216Z
There is also reported in J.N.M. Vol. 12, No. 6, arti-cle commencing on pp 372 and ending on pp 373, Tc-99m and 131I
labelled microaggregates of albumin but there is no mention of the presence of any reducing metal and ligand therefor and no teaching of how the microaggregates are formed.
In J.N.M. Vol. 10, No. 6, 1969 pp 454, there is descri-bed methods of labelling macro and microaggregates of serum albu-min using ferric ions and ascorbic acid with no teaching of how the microaggregates are formed and with no mention of the pre--10 sence of a reducing metal and ligand therefor.
In J.N.M. Vol. 9, No. 9, 1968 pp 482-5, a method of making microaggregates of HSA is described followed by radio-iodinating them. There is no mention of a reducing metal and ligand therefor. In J.N.M., Vol. 11, No. 6, 1970 pp 387 there is described a method of preparing Tc-99m labelled microaagre-gates of albumin, particle size about 0.5 ~m, in which the micro-aggregates are apparently formed from Tc-99m labelled unaggre-gated albumin (the unaggregated albumin was labelled with Tc-99m utilizing ferric chloride, ascorbic acid and acetate buffer), there is no mention of the presence of a reducing metal and ligand therefor.
In J.N.M~ Vol. 12, No. 6, 1971 pp. 467-8 there is des-cribed a Tc-99m-labelled stannous hydrcxide colloidal suspension used in liver imaging, but no mention of HSA or a stabilizing ligand in the preparation so-described.
J.N.M. Vol. 11, No. 10 pp 580-85, 1970 describes micro-aggregation of HSA followed by labelling with technetium pre-reduced with ferric chloride and ascorbic acid. There is no mention of the presence of a reducing metal or ligand therefor during aggregation.

' ~ ' ' . .

The following literature references describe iodi-nated HSA aggregates: Br. J. Exp. Path. Vol. 38, 1957, pp 35-48, Report of Scientific Exhibit entitled "Colloidal~
Radioalbumin Aggregates For - 32 ~

` ~ lllZ16Z

IlOrgan Scanning" 10th Annual ~eeting, Nuclear Medicine Society, i~Montreal, Canada, June 26-29, 1963; J.N.M., Vol. 5, p~. 259-275, ,, 1964; J. Lab. & Clin. Med., Vol. 51, No. 2, pp. 230-39, 1958;
"Dynamic Clinical Studies With Radioisotopes" USAEC Division of Tech. Info., pp 285-317, June 1964; Invest. Radiol. Vol. 1, 1966, ¦pp. 295-300; Report of G. V. Taplan et al, entitled "Preparation of ¦Colloidal Suspensions of ~uman Serum Albumin I131 For Estimating Liver Blood Flow and Reticulo-endothelial System Functions in Man", UCLA Report No. 481 Biology and rlediciné~ June 23, 1961, Univer-'sity of California, Los Angeles School of Medicine.
U. S. Patent No. 4,054,645 describes the use of a stannous fluoride reducing agent with a number of different tarqet seeking ligands, one of which is albumin, but there is no teaching of micro-aggregating albumin in the presence of a stannous salt and an "additional non-target-seeking ligand. .
U. S. Patent No. 4,057,615 describes the use of a reducing agent complex with various target-seeking excipients ~an improperly chosen term for target seeking ligands)wherein the association 'constant of the reducing agent anion for technetium is le9~ than that of the excipient. There is no teaching of microaggregating in the presence of a reducing metal and non-target seeking ligand.
Although some of the aforesaid literature references refer to Tc-99m-labelled microaggregates of albumin as radioactive imagin agents, there is no such agent presently on the market for ~ES

2~ imaging,and colloidal sulfur continues to be the maJor ~ES agent onl the market despite its disadvantages and even though Tc-99m labelleld macroaggregates have become a major factor in the lung imaging market.

i' ,~

Claims (61)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition for labelling with Tc-99m for radio-active imaging comprising microaggregates of albumin and a reducing metal.

2. A composition according to claim 1, said composition also comprising an additional stabilizing ligand for said reducing metal.

3. A composition for labelling with Tc-99m for radio-active imaging comprising microaggregates of albumin and a reducing metal, said composition also comprising an additional stabilizing ligand for said reducing metal selected from the group consisting of a phosphonate, a phosphate, an amino-carboxylate, a polyhydroxycarboxylate and a polycarboxylate.

4. A composition according to claim 3, said reducing metal being Sn++ and said ligand being a diphosphonate.

5. A composition according to claim 2, at least 90%
of said microaggregates being not greater than 5 µm in particle size.

6. A composition according to claim 5, at least 40%
of said microaggregates being between 0.2 and 5 µm in particle size.

7. A composition according to claim 5, at least 40%
of said microaggregates being between 0.2 and 3 µm.

8. A composition according to claim 7, said reducing metal being stannous and at least the major portion of said microaggregates being between 0.2 and 3 µm in particle size.

9. A composition according to claim 5, no more than 50% of said microaggregates being less than 0.2 µm in particle size.

10. A composition according to claim 2, said albumin being human serum albumin and said reducing metal being stannous.

11. A composition according to claim 10, wherein said composition is stabilized with undenatured albumin and a buffer.

12. A composition according to claim 11, also contain-ing a non-ionic surfactant.

13. A composition according to claim 10, micro-aggregated at a pH between 4.5 and 9.5 but on the alkaline side of the apparent isoelectric point of said albumin.

14. A composition according to claim 13, said pH being between 5.4 and 7Ø

15. A composition according to claim 10, said com-position being in the form of a freeze-dried solid.

16. A composition according to claim 1, at least 90% of said microaggregates being not greater than 5 µm in particle size.

17. A composition for labelling with Tc-99m for radio-active imaging comprising microaggregates of albumin and a reducing metal, at least 90% of said microaggregates being not greater than 5 µm in particle size, and at least 40% of said microaggregates being between 0.2 and 5 µm in particle size.

18. A composition according to claim 17, no more than 50% of said microaggregates being less than 0.2 µm in particle size.

19. A composition according to claim 1, said albumin being human serum albumin, said reducing metal being stannous, said microaggregates being microaggregated at a pH of 3.5-9.5, said composition being stabilized with undenatured human serum albumin and a buffer and said stabilized composition being freeze-dried.

20. A radioactive imaging agent comprising Tc-99m labelled microaggregates of albumin and a reducing metal, wherein at least 90% of said microaggregates have a particle size not greater than 5 µm and at least 40% of said microaggregates are between 0.2 and 5 µm.

21. An agent according to claim 20, also comprising a stabilizing ligand for said reducing metal.

22. A radioactive imaging agent comprising Tc-99m labelled microaggregates of human serum albumin and a stannous reducing metal, said agent also comprising a stabilizing ligand for said reducing metal selected from the group consisting of phosphonates, phosphates, aminocarboxy-lates, polyhydroxycarboxylates and polycarboxylates.

23. A radioactive imaging agent comprising Tc-99m labelled microaggregates of albumin and a reducing metal, said agent also comprising a diphosphonate stabilizing ligand for said reducing metal.

24. An agent according to claim 23, said ligand being hydroxyethylene diphosphonate.

25. An agent according to claim 23, said ligand being methylene diphosphonate.

26. An agent according to claim 22, not substantially more than 50% of said microaggregates being less than 0.2 µm.

27. An agent according to claim 21, at least 40% of said microaggregates being between 0.2 and 3 µm.

28. An agent according to claim 21, the major portion of said microaggregates being between 0.2 and 3.0 µm.

29. An agent according to claim 23, admixed with non-aggregated albumin, buffer and surfactant.

30. An agent according to claim 20, said albumin being human serum albumin, said reducing metal being stannous, at least 90% of said microaggregates having a particle size no greater than 5 µm and not substantially more than 50% of said microaggregates being less than 0.2 µm.

31. A radioactive imaging agent comprising Tc-99m labelled microaggregates of human serum albumin and a stannous reducing metal, at least 90% of said micro-aggregates having a particle size no greater than 5 µm and not substantially more than 50% of said microaggregates being less than 0.2 µm, said agent stabilized with non-aggregated human serum albumin and buffer, said micro-aggregates being microaggregated at a pH of 4.5-9.5 but on the alkaline side of the apparent isoelectric point of said albumin.

32. An agent according to claim 31, said pH being between 5.4 and 7Ø

33. An agent according to claim 31, said agent com-prising methylene diphosphonate and said pH being between 5.6 and 6.5.

34. A method for making an agent for labelling with Tc-99m for radioactive imaging, said method comprising micro-aggregating albumin in the presence of a reducing metal.

35. A method according to claim 34, said micro-aggregation being carried out in the presence of a stabilizing ligand for said reducing metal.

36. A method for making an agent for labelling with Tc-99m for radioactive imaging, said method comprising microaggregating human serum albumin in the presence of a stannous reducing metal, said microaggregation being carried out in the presence of a stabilizing ligand for said reducing metal selected from the group consisting of a phosphonate, phosphate, an aminocarboxylate, a poly-hydroxycarboxylate and a polycarboxylate.

37. A method according to claim 35, said micro-aggregation being carried out at a pH at which at least 90% of the microaggregates have a particle size not greater than 5 µm.

38. A method according to claim 37, said micro-aggregation being carried out at a pH at which not sub-stantially more than 50% of the microaggregates have a particle size below 0.2 µm.

39. A method according to claim 37, said aggregation being carried out at a pH at which at least 40% have a particle size between 0.2 and 5 µm.

40. A method according to claim 37, said aggregation being carried out at a pH at which at least 40% of the microaggregates have a particle size between 0.2 and 3 µm.

41. A method according to claim 37, said aggregation being carried out at a pH at which at least the major portion of the microaggregates have a particle size between 0.2 and 3 µm.

42. A method according to claim 35, said aggregation being carried out at a pH between 4.5 and 9.5 but on the alkaline side of the apparent isoelectric point of said albumin.

43. A method according to claim 42, said pH being between 5.4 and 7Ø

44. A method according to claim 36, said ligand being a diphosphonate.

45. A method according to claim 36, said ligand being hydroxyethylene diphosphonate.

46. A method according to claim 36, said ligand being methylene diphosphonate.

47. A method according to claim 46, said microaggregates being carried out by heating at a pH of 5.4 to 6.6, at which at least 90% of the microaggregates have a particle size not greater than 5 µm and at least 40% have a particle size between 0.2 and 3 µm, said reducing metal being stannous and said albumin being human serum albumin.

48. A method according to claim 34, said micro-aggregation being carried out by passing a solution of undenatured human serum albumin and said reducing metal through a plurality of sequentially arranged heat exchangers in a semi-continuous manner.

49. A method according to claim 34, said reducing metal being stannous and said albumin being human serum albumin, said aggregation being carried out at a pH at which at least 90% of the microaggregates have a particle size not greater than 5 µm and not substantially more than 50% have a particle size below 0.2 µm.

50. A method according to claim 49, said micro-aggregation being carried out by heating at a pH between 4.5 and 9.5 but on the alkaline side of the apparent iso-electric point of said albumin at which at least the major portion of said microaggregates have a particle size between 0.2 µm and 3.0 µm.

51. A method according to claim 50, said pH being between 5.4 and 7Ø

52. A method according to claim 50, said pH being between 5.4 and 6.6.

53. A method according to claim 34, said aggregation being carried out by heating albumin and said reducing metal by radiofrequency heating.

54. A method according to claim 34, said aggregation being carried out by heating albumin and said reducing metal by microwave heating.

55. A method according to claim 34, said aggregation being carried out by heating albumin and said reducing metal by induction heating.

56. A preaggregated bulk, containing human serum albumin to be aggregated to microaggregates for radioactive labelling for use as an RES imaging agent, comprising a solution of unaggregated and undenatured human serum albumin, reducing metal and stabilizing ligand for said reducing metal, said bulk having a pH of between 4.5 and 9.5 but on the alkaline side of the isoelectric point of said albumin, the amount of ligand being sufficient to stabilize said stannous ions and prevent precipitation thereof before micro-aggregation but insufficient to provide any substantial non-RES target seeking properties when the microaggregates sub-sequently formed from said bulk are radioactively labelled and utilized for RES imaging.

57. A bulk according to claim 56, said pH being between 5.4 and 7Ø

58. A bulk according to claim 57, said reducing metal being stannous.

59. A preaggregated bulk containing human serum albumin to be aggregated to microaggregates for radioactive labelling for use as an RES imaging agent, comprising a solution of unaggregated and undenatured human serum albumin, stannous reducing metal and stabilizing ligand for said reducing metal, said bulk having a pH of between 5.6 and 6.5 but on the alkaline side of the isoelectric point of said albumin, the amount of ligand being sufficient to stablilize said stannous ions and prevent precipitation thereof before microaggregation but insufficient to provide any substantial non-RES target seeking properties when the microaggregates subsequently formed from said bulk are radioactively labelled and utilized for RES imaging, said ligand being a diphos-phonate selected from the group consisting of methylene di-phosphonate and hydroxyethylene diphosphonate.

60. A method for making an agent for labelling with Tc-99m for radioactive imaging, said method comprising micro-aggregating albumin in the presence of a reducing metal, at a pH between 3.5 and 9.5 but at a pH away from the apparent isoelectric point of said albumin.

61. A method according to claim 60, wherein said reducing metal is stannous and said albumin is human serum albumin, said step of microaggregating comprising heating said albumin and said reducing agent at said pH, thus forming microaggregates at least a major portion of which have a particle size between 0.2µm and 5.0µm, and at least 90% of which have a particle size not greater than 5.0µm.