US 3663687 A
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3,663,687 BIODEGRADABLE PARENTERAL MICROSPHERULES Roger L. Evans, Sunfish Lake, Minn., assignor to Minnesota Mining and Manufacturing Company, St. Paul, Minn. No Drawing. Filed June 26, 1968, Ser. No. 740,038
Int. Cl. A61k 27/04 US. Cl. 424-1 16 Claims ABSTRACT OF THE DISCLOSURE Tiny substantially spherular particles having a smooth outer surface and essentially void-free interior, are produced consisting essentially of solid, cold-water insoluble material comprising a physiologically acceptable, parenterally metabolizable protein, said spherules being capable of being made radioactive after their preparation, and said radioactivity being substantially non-leachable upon short term exposure to cold water. They can be used as carriers for substances, e.g. radioisotopes, which are administered parenterally for diagnostic, prophylactic or therapeutic purposes. On administration in this way, they provide a predeterminable dwell time and thereafter are broken down or solubilized by the body fluids over a predeterminable period ranging from minutes to several days. Thereafter the radioisotopic or other material is excreted from the body thus terminating exposure to the carried substance.
BACKGROUND OF THE INVENTION Field of the invention It has heretofore been known to encapsulate natural products for food or pharmaceutical use in proteinaceous materials such as gelatin and albumin, and small sperical particles of such encapsulated materials have been made, e.g. by processes such as those disclosed in US. Pats. 3,137,631; 3,016,308; 3,202,731; 2,800,457 and the like. These prior art processes, however, either produce capsular materials wherein a central core is surrounded by a thin shell, e.g. albumin or gelatin; or (for purposes of obtaining materials that can be handled and/ or stored under adverse conditions) result in severe denaturization of the protein so that its solubility and other properties are impaired. Such materials are not suitable for parenteral administration in the animal organism. Similarly, while the use of radioisotope-labeled particulates parenterally in the animal body is known for diagnostic and treatment purposes, the materials heretofore used for such purposes have been relatively insoluble, very finely divided irregular or spherical particles which, when used, lodge in the body and remain there during substantially the entire life of the radioisotope. Such particles, for example, are shown in US. Pats. 3,334,050 and 3,147,225. While these are very useful for certain purposes where long-continued radioisotopic treatment, for example, is desirable and advantageous, there are other areas in which their use is less desirable and in some instances may be contraindicated. Irregular macroaggregates of human serum albumin, labeled with radionuclides, have been used for diagnostic purposes. So far as is known, these materials cannot be prepared in narrow ranges of particle size and are prepared in particulate form directly in the solution in which they are to be used; they cannot be dried and sized or otherwise treated, and then resuspended.
SUMMARY OF THE INVENTION The present invention provides means to prepare certain physiologically acceptable, parenterally metabolizable materials in the spherical form, in highly pure, substantially 3,663,687 Patented May 16, 1972 undenatured condition which can be administered parenterally as a solid without injury to the organism. These spherical materials can be labeled or tagged with radiochemicals possessing desirable radioactive emissions, to form homogeneous radioactive parenterally metabolizable spheres. The desirable radioactive emissions of the spherules are useful for diagnotic, prophylactic and therapeutic purposes. The invention also contemplates the provision of a process for making such spherules and their concomitant or subsequent treatment of modify their solubility characteristics without bringing about denaturization which would prevent their absorption in the body.
A particularly valuable feature of the invention is that it provides spherules of closely controlled, predetermined size and shape. Up to now, one has been restricted to particulate materials which are either spherical but are inert and non-disappearing in the body, or which do dissolve in the body but whose size distribution and, very particularly, whose wide variety of shapes make it impossible to tell exactly how they will travel in the blood vessels.
The new spherules can be injected into a vessel carrying body fluids. By virtue of their size and shape control on knows that when the fluid stream reaches an area where vessel diameter is reduced, e.g. a capillary bed, the microspherules will lodge there quantitatively. Thus, by using microspherules of say 20 microns in diameter, and injecting them into a large vessel, one can discover where the diameter of that vessel, or of a remote downstream vessel, first falls to 20 microns. Moreover, because the spherules disappear in the body, they do not permanently block the vessel where they lodge. Thus, one can use them routinely to study the progress of diseases or of treatments where vessel diameter or constriction is involved.
Likewise, when one already knows the diameter of the vessels in a given organ, or more particularly when one knows their minimum diameter (eg. the lung or liver), one can now inject suitably sized spherules into a suitable vessel which will lead to that organ. The new spherules will lodge in that organ quantitatively and are there detected. The size and shape of the organ are then apparent from the distribution of the spherules within it. There is no interference from odd shaped or odd sized particles which have either traversed the organ because of substandard size or which have failed to reach it because of being aggregated into too large agglomerates.
Another significant advantage of the invention is that the microspherules dissolve in the body over a range from relatively few minutes to hours, days or even weeks. For example, by using the process described herein in which the shortest heat treatment at about C. is employed, histological studies show that microspherules are made which begin to dissolve in about 15 minutes after injection. Thus, the conditions under which the spherules are made by the process of the invention constitute a heat treatment which changes the normal solubility of the spherules. Sterilization using heat also is a. heat treatment within the scope of the invention. Using conditions of longer heating at higher temperatures, e.g. to a maximum of 18 hours at C., the beads can be made to dissolve in body fluids in periods ranging from several hours to several weeks and up to six months or more. The heat treatment is stopped before denaturization of the protein, which would result in complete insolubility of the microspherules. Depending on ones needs (e.g. a single study, a study after a time lapse, or several consecutive studies), material tailored to the purpose is available. It will be apparent that empirical methods, based on the disclosure herein, will be useful to determine the exact time and temperature to be used to prepare spherules which dissolve in body fluids at the particular time desired. ill; will be understood that the spherules do not dissolve instantly at the expiration of the times noted, but begin to become soluble and gradually become completely soluble over the periods set forth herein. This is shown by histological studies, wherein it is found that the spherules become increasingly diifuse with the passage of time.
The heat treatment apparently causes some crosslinking of the protein, which alters the normal solubility of the protein. Chemical crosslinking with agents which produce parenterally acceptable crosslinking also can be used for the purposes of the invention.
A still further feature of the invention is one particularly useful in the preparation and handling of the microspherules. They can be isolated from liquid suspension and obtained as an easily handled, dry powder. This is very useful for metering, dispensing, storing and shipping. There is no need to maintain colloidal suspensions at critical pHs, temperatures or ionic strength. When desired for use, the microspherules are resuspended, for example in a pharmaceutical extending medium, quite simply by shaking or by treatment with ultrasonics, with, if desired, a suitable wetting agent. A particularly valuable feature of the ability to dry and resuspend the microspherules is that they can be very conveniently classified in the dry state, for example by sieving, into as fine a size distribution range as one desires. Thus one can now perform diagnoses or blood flow studies, etc. which have been impossible heretofore, because sufiiciently homogeneous, parenterally acceptable, dissolving media have not been known.
The spherules of the invention comprise a physiologically acceptable, solid, substantially water-insoluble (at body temperature) material which can be metabolized or degraded, apparently by the enzymes or other metabolic mechanisms in the parenteral body fluids, such as blood, serum, plasma, lymph and the like, in a manner which does not form toxic residues. When so metabolized or degraded the substances formed are solubilized, and this process and the solution of the resulting products in body fluids is what is meant herein by the term soluble in body fluids.
Suitable materials for the spherules of the invention are physiologically acceptable proteinaceous substances such as albumin, gelatin, hemoglobin and the like.
The spherules are usually prepared in a state in which the sizes of the particles are closely controlled by sorting techniques so as to be in a narrow size range adapted to the specific use. Particles thus segregated into narrow ranges can be from about /2 to 1000 microns in average diameter and preferably the size ranges chosen do not vary more than about plus or minus 20 percent from the mean.
Spherules from /2 to 60 microns in diameter are most useful for diagnostic purposes. Larger spherules, even up to l millimeter in diameter, can be used for certain therapeutic purposes. Being uniform in their dimensions, spheroidal or spherular particles are more easily controlled with respect to radioisotope content and time of elimination from the body. Particularly, they are preferred because, by matching the diameter of the spherules to the size of the body passages, e.g. arteries, capillaries, etc., one can predict their route through a healthy body and determine where they should lodge with high accuracy.
To make the spherules of the invention, a convenient method consists in forming a sol by dispersing the suitable protein as heretofore described in warm water, then causing the vehicle to gel into tiny spheroids or spherules. While these gelled spherules are prevented from coalescing, water is removed from them, and the particles are dried to a free-flowing, unagglomer'ated form. These spherules may be somewhat porous, but this does not affect their utility for the purposes described herein. The essentially cold water-insoluble spherules are then subjected to heat treatment to modify their solubility and 4 screened or otherwise graded. They can be soaked in water at 37 C. for at least 30 minutes without dissolving. Usually the particles are completely insoluble in water at or below this temperature. In physiological fluids such as blood serum, however, they soon begin to be broken down and eventually are completely solubilized in periods ranging from 15 minutes to about four weeks.
Thus, for example, it has been found that by dispersing a solution of albumin, such as human serum albumin, e.g. by stirring into a warm, inert fluid which is immiscible with the solution of albumin and in which the albumin itself is not soluble, small spherules of the albumin are formed. The speed of stirring, use of bafiles and the like controls the size of the particles obtained; empirical methods are used to establish parameters of dispersion to yield spheroidal particles of any particular size. Alternatively, and preferably for continuous production, tiny droplets of the aqueous liquid are injected through a small orifice into a moving stream of the warm, inert fluid. The water is removed from the albumin solution through the medium of the warmed, inert liquid, so that dry, practically perfectly round, free-flowing tiny spherules of albumin are obtained. These spherules are from 1 to 500 microns or even up to a millimeter in diameter and can be obtained through the process in very narrow, predetermined size distribution ranges. Although their normal solubility has been altered, they are not completely denatured, and can be administered parenterally in the animal organism. When so administered, it is found that they are converted to soluble form.
When albumin is used in this invention, a very convenient starting material is the Normal Serum Albumin (human) which is an article of commerce. It is available in aqueous solutions of several concentrations.
The albumin referred to herein is broadly any of the several natural proteins which are so described. Such albumins include those of egg, blood serum, milk and the like, as obtained from various animal species. For the purpose of this invention, the preferred albumins are animal albumins from blood serum, human serum albumin, and in general, for eventual use in a given animal organism, albumin obtained from the serum of that organism.
Suitable inert liquids for the process of making the spherules of the invention include vegetable oils, for example cottonseed oil, corn oil, olive oil and the like; low melting animal fats; mineral oils, particularly those having boiling points above about C.; inert hydrocarbons, halogenated hydrocarbons and the like. The function of the inert liquid is to remove water from the protein and to cause gelling, and it will be apparent that various solvents can be used to accomplish this end.
The function of the spherules of the invention is to follow the path of blood flow in the living animal body. To do this, one administers them, in a suitable pharmaceutical extending medium, into a convenient blood vessel. The blood vessel chosen is one which will convey the spherules to the organ or part of the anatomy being investigated or treated. Because of their regular spherical shape and because of their very close size regularity the particles will follow a regular path through the vascular system. A particularly useful feature, made possible by the extremely fine control one can exert over their size distribution, is that they can be made to traverse some blood vessels (i.e. those which are larger than the particles) but become trapped temporarily in others (i.e. those smaller in diameter than the particles). In this way one can investigate fluid flow in the healthy body, the dis eased body in which the flow pattern is abnormal, and in the repaired body, for example where a diseased state has been treated by medical or surgical techniques.
In order to detect the particles in the living body, they are made radioactive. Their position can then be detected by means of external radiation detectors. This obviates the need to explore the body physically or to take tissue samples. They are conveniently made radioactive by reacting them with radio-nuclides which are isotopes of chemical elements which will reactwith the material of which the particles are made. For example, a wide range of elements is known to react with albumin. However, for control over the radiation dose received by the patient, and because they are simply and conveniently detected and assayed by contemporary radiation detection equipment, two radio-elements, radio-iodine and radio-technetium, are currently preferred.
For use in humans, spherules of human serum albumin can be marked with radio-iodine by reaction with an aqueous solution of Nal A suspension of spherules in a carrier-free aqueous solution of NaI or Nal is digested for several hours at a mildly elevated temperature, for example four hours at 40 C. The spherules react with the iodine and become radio-active. They are recovered by filtration and are Washed thoroughly to remove traces of unreacted radio-iodine. A modification of this procedure is to perform the reaction in mildly oxidizing conditions, for example in the presence of hydrogen peroxide. In these conditions it is found that more radio-iodine is taken up by the spherules.
Similarly, microspherules of human serum albumin can be reacted with radio-technetium. The isotope Tc is an especially convenient one for this purpose. It is readily available from commercially-supplied generators, and its radiation characteristics (a low energy gamma ray and a short half-life are very favorable for diagnostic nuclear medicine. Its gamma radiation is very efficiently detected by contemporary equipment, and its short half-life greatly reduces the total radiation dose to the patient.
Albumin microspherules are reacted with technetium in the presence of ferric iron and a reducing agent. A convenient method is to elute a commercial Tc generator to obtain a solution of radioactive sodium pertechnetate. After pH adjustment to about 4.5 and the addition of the iron (e. g. ferric chloride) and reducing agent (e.g. ascorbic acid), the microspherules are suspended in the solution. The pH is readjusted to'2.5, and the suspension is agitated for a few minutes at room or elevated temperature. The microspherules react with the technetium and become radioactive. They are filtered off, or recovered by centrifugation, and washed with water to remove traces of unreacted radio-technetium. After drying, they are obtained as a free-flowing powder, which can be resuspended, when desired for use, in a suitable pharmaceutical extending medium.
For use in diagnostic procedures, a suspension of the particles of the invention, such as microspherules of albumin reacted with a radionuclide, are suspended in a pharmaceutical extending medium suitable for parenteral administration. This may be e.g. physiological saline, or dextran or gelatin solutions. A quantity of such a composition containing the desired amount of radioactivity, e.g. one millicurie, is injected e.g. intravenously into the animal body. The material thus injected circulates throughout the body in the blood stream and, because of the selected particle size, will lodge in a particular, predetermined organ, e. g. the lung. Radiation detectors, or autoradiography, may then be employed to visualize the organ. Because the microspherical particles remain substantially intact for a short time in the animal organism, a period of time ranging up to several days is available for such diagnostic procedure. Thereafter, the body enzymes begin to attack the material, causing it to become solubilized and absorbed. Theradioisotope or its decay product, is, however, swept away from the localized area in the blood stream and excreted, generally by the kidneys.
For therapeutic or prophylactic use, the products are administered as described above except that the activity is usually much higher (e.g. 50 millicuries), and the biodegradability of the particles is adjusted so as to retain the radionuclide until it has delivered the energy required for these purposes.
It will be apparent that the particular protein chosen to prepare the spherules of the invention which convey radioisotopes into predetermined, temporary location in the body is not critical.
It is only necessary that it possesses a sufiicient number of radioactive atoms, or that it can combine with enough radioactive specie to emit radiation of the desired intensity and energy; that the product be physiologically acceptable; capable of being prepared in essentially insoluble form with respect to water at 37 C. and capable of being metabolized or degraded by body fluids to soluble form.
The following specific examples will more clearly illustrate the specific embodiments of the invention. In these examples, all parts are by weight unless otherwise specified. As a practical matter, radioactive materials are dispensed in terms of their radiation level rather than by exact weight, and wherever radiation level is mentioned, this is the exact amount of radionuclide used.
Example 1 A convenient method for continuous production of spheroidal particles is the following: A 25 percent aqueous solution of human serum albumin at room temperature is passed through a number 27 needle into a stream of cottonseed oil warmed to about 50 C., moving at a rate of about 12 feet per minute. The albumin solution breaks up into droplets which are suspended in oil. The stream of droplets-in-oil is carried through a 50 ft. long tube, heated to ca. C. This dries the droplets to microspherules of about 20-50 microns in diameter. The oil and dried spherules are then cooled to about room temperature, and the spherules are collected, the oil being removed by filtration.
The spherules thus collected are washed with acetone, ether, heptane or the like to remove adherent oil. The washed spherules are graded by sieving and can then be stored under ambient conditions without change for many months.
Heat treatment of the spherules can conveniently be done in oil suspension if desired, but is also equally effective after the particles are Washed and/or graded and stored. It has been found that the effect upon the spherules is time and temperature connected; thus heating at tem peratures of the order of 70-80 C. for several hours would be equivalent to heating at a higher temperature,
say C., for one hour.
Spherules of human serum albumin heated for 40 minutes at various temperatures are altered so they are biodegraded and solubilized in body fluids to the extent of 50 percent in the periods set forth in the following table:
TABLE 1 Temperature, C.: 50% solution time 24 hours. 84 hours. 4 days. 30 days.
Example 2 A solution of serum albumin is prepared in concentration of 25 percent in water. Four milliliters of this solution are injected, conveniently through a hypodermic needle, into about 1 liter of vegetable oil (cottonseed oil) which is heated to about 3050 C. The rate of stirring determines the ultimate size of the spherular material obtained. Using a container which is greater in height than in diameter, with a 25 gauge hypodermic needle and stirring at about 500 r.p.m. with a 2- /2-inch propellertype stirrer, microspherular particles of about 10-20 microns in diameter are obtained. Stirring is continued while heating to 110 C. until the microspheres are essentially dehydrated as may be determined by removal from the mixture of a small number of spheres to determine whether or not they are still tacky. After thus drying, the particles are filtered away from the oil and washed with diethyl ether. Microspherular particles of serum albumin are obtained. They are about 10-20 microns in diameter and are an unagglomerated, free-flowing tan powder.
Somewhat higher temperatures can be employed in the process, accompanied by an increase in the time required for biodegradation and solubility. Thus, for example, the oil is heated to 135 C. and maintained there for 40 minutes while stirring. Again, free-flowing, unagglomerated particles of serum albumin are recovered. These are biodegraded and solubilized to the extent of 50 percent in about one day.
Example 3 A 2 percent aqueous solution of hemoglobin is injected into stirred cottonseed oil at 3050 C. and converted into microspherular form in the manner of Example 1. The microspherules of hemoglobin are obtained as an 7 unagglomerated, free-flowing, dark reddish-brown powder.
Labeling microspheres of the invention with radioactive isotopes can be accomplished by the methods of the following examples. After labeling, the spherules can be further treated to effect additional crosslinking, if desired.
Example 4 Two hundred milligrams of microspherules of albumin, made according to Example 1, 2030 microns in diameter, are stirred in a solution of sodium iodide (I 0.1 mc.) containing one drop of 5 percent Tween 80 wetting agent in a total volume of ca. 2 ml. of Water. A 25 microliter assay sample of the solution, before adding the microspherules, has a radioactive counting rate of 107,840 c.p.m. The system is agitated for one hour at 40 C. At this time, a 25 microliter assay sample of the aqueous phase has a counting rate of 67,201 c.p.m. The remainder of the radioactivity is found in the microspherules. They are filtered oif, washed thoroughly with water, then with acetone. After air drying, microspherules of radioactive iodinated albumin are obtained as a free-flowing tan powder.
Example 5 One hundred milligrams of albumin microspherules are suspended in a solution containing 100 microcuries of sodium iodide (1 in ml. of 0.06 molar hydrochloric acid. Two drops of 37 percent hydrogen peroxide solution are added to the suspension, which is stirred at room temperature. After 22 hours, the microspherules are recovered by filtration, followed by water and acetone washing, and air drying. It is found by radioactive assay that they have reacted with 74.6 microcuries of radio-iodine. The radio-iodinated albumin microspherules are obtained as a free-flowing tan powder.
Example 6 Ten milligrams of albumin microspherules (ca. 30 micron diameter) are suspended in an aqueous solution of 2 millicuries of iodine-125 (as carrier-free NaI of 1 cc. total volume. The system is buffered at pH 7.4 and contains one drop of 5 percent Tween 80 as wetting agent. To this is added 250 microliters of 0.4 percent of sodium N-chloro p toluenesulfonamide (Chloramine-T) in aqueous solution. The suspension is agitated gently for 2 hours. At this time it is found by radio assay that 1.1 millicuries of I have reacted with the microspherules. After washing with water, then with acetone or ether, and drying in air, microspherules of radioactive iodinated albumin are obtained as a free-flowing tan powder.
Example 7 A suspension is made containing 50 mg. of 25-30 micron spherules in an aqueous solution consisting of a mixture of 3 ml. of 5 percent dextrose and 5 ml. of 0.1 N sodium acetate. The pH of this solution is about 5.6. Another aqueous solution is made up, consisting of 1 ml. of 0.2 percent ascorbic acid, 0.2 percent ferric chloride (as FeCl 6H O) and 1 millicurie of sodium pertechnetate (T0 The latter solution is obtained from a commercial technetium generator. The pH of this mixture is adjusted to 4.5 with 1 molar sodium hydroxide solution.
The two solutions are mixed, and the pH of the mixture is lowered to 2.5 by dropwise addition of l N hydrochloric acid. The mixture is shaken at 19 C. for 15 minutes. About 60 percent of the technetium originally present in the liquid phase is now found in the microspherules. The system is shaken for another 45 minutes. About percent of the technetium is now combined with the microspherules. After filtering, washing well with water, then with acetone or ether, and drying in air, the microspherules of technetiated albumin are isolated as a dry, free-flowing tan powder.
Other radioisotopes can be used to label the protein microspheres of the invention by procedures similar to those set forth hereinabove. For use for diagnostic or therapeutic purposes, the microspheres are suspended in an isotonic pharmaceutical extending medium (e.g. isotonic, sterile aqueous solutions such as Ringers solution) and slowly injected into the bloodstream at the selected point. In this way the labeled spherules of the invention can be used to elucidate the condition of various organs, e.g. lung condition in experimental animals.
Example 8 Two hundred microcuries of technetium (as sodium pertechnetate) are mixed with 10 cc. of phosphate buffer at pH 7.4. After adding 3 drops of 4 percent Tween 80 as wetting agent, 200 milligrams of albumin microspherules (diameter 20-30 microns) are added and suspended by agitation. One ml. of 0.1 molar sodium sulfite is added, and the agitation is continued. After a half hour, radioactive assay shows that 59 percent of the technetium has been taken up by the spherules. If the reaction is continued for 2 hours, 88 percent of the technetium is found to have reacted with the spherules. The technetiated albumin microspherules are filtered off, washed well with Water, then with acetone or ether, and, after air drying, they are isolated as a free-flowing tan powder.
Example 9 Spherules of radioactive hemoglobin are prepared by shaking together 50 milligrams of hemoglobin spherules and 10 ml. of an acetone solution of radio-ferric chloride (375 microcuries of Fe), at about 40 C. for 22 hours. Assay shows that about 26 percent of the radioactive iron has been taken up.
What is claimed is:
1. Tiny, dry, free flowing, unagglomerated, storable precursor spherules for intravenous injection for radio active diagnostic or therapeutic purposes, of a predetermined size with narrow distribution range not more than about :20 percent of the selected mean, in the range of about /2 micron to 1 millimeter in diameter so as to lodge within vessels carrying body fluids at a pre-selected location, essentially composed of species-specific protein or parenterally acceptable gelatin crosslinked chemically or thermally to a predetermined degree to control solubility thereof in Water and aqueous physiological fluids, so that said spherules are resistant to solution when immersed in water at 37 C. for at least about 30 minutes and are completely metabolized parenterally in a period not less than about 15 minutes.
2. Spherules accordinng to claim 1 which are metabolized over a period of time ranging from beginning to dissolve in about 15 minutes after exposure to completely soluble in six months.
3. Spherules according to claim 1, wherein the protein is albumin.
4. Spherules according to claim 1 composed of human serum albumin.
5. Spherules according to claim 2 composed of human serum albumin.
6. Spherules according to claim 2 composed of egg albumin.
7. Spherules according to claim 2 composed of hemoglobin.
8. A parenterally injectable composition, comprising spherules according to claim 1 suspended in a pharmaceutical extending medium suitable for intravenous use.
9. A process for making spherules according to claim 1 in which an aqueous solution of the protein is converted to spherular form, gelled and dehydrated while maintaining said spherules out of contact, and treated by crosslinking as by heating at a temperature in the range of 70 to 190 C. for a period ranging from about 40 minutes to about 18 hours, to alter the normal solubility of the said protein toward parenteral body fluids without causing complete denaturization.
10. Process according to claim 9, wherein dehydration is carried out at a temperature in the range of 135 to 190 C. for about 40 minutes.
11. Process according to claim 9, wherein the protein is albumin.
12. Process according to claim 9, wherein the protein is hemoglobin.
13. Process according to claim 9, in which dehydration is carried out by heating in a vegetable oil.
14. Process according to claim 13, in which heating during dehydration is sufiicient to accomplish cross linking.
15. Process according to claim 9, in which crosslinking is effected by heating the spherules While they are in dry, free-flowing condition.
16. Process according to claim 9, in which the spherules are produced and then labeled with a radioisotope.
References Cited UNITED STATES PATENTS 3,328,257 6/ 1967 Vrancken et al. 42437 BENJAMIN R. PADGETT, Primary Examiner US. Cl. X.R.
UNliED STATES PATENT Grimes QERTEMCATE @l QQRREQ'HQN Patent No. 3 9 3 87 Dated May 1 6,- 1972 Inventor(s) Roger L. Evans It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:
References Cited should also include:
3,017,668 Sundman 39l +3, 75 Koff et a1 3,1595%5 Kidwell et al 3,202,731 Grevenstuk et al 3,3 9,7 5 La Grange 3,33M,050 Grotenhuis 33 M561? Rinfret et a1 3 1421 282 Hasegawa 3,5 5,563 Clegg et a1 Signed and sealed this 5th day of December 1972.,
EDWARDMQFLETCIER$JRQ ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents FORM PC4050 (w'ss) uscoMM-oc 80376-P69 a US. GOVERNMENT PRINTING OFFICE; 1959 O366-334.
UNHED STATES PATENT @mm QERNWQATE Gt CRRETWN Patent No. 3 3 7 Dated May 1 6, 1972 Inventor(s) Roger L. Evans It is certified that error appears in the aboveidentified patent and that said Letters Patent are hereby corrected as shown below:
References Cited should also include:
3,017,668 Sundman 3,13JI75 Koff et a1 3,159,5 5 Kidwell et a1 $202,731 Grevenstuk et a1 3,3 9,7 5 La Grange 3,33,,050 Grotenhuis 3,3 I I,6l7 Rinfret et 211 3,421,282 Hasegawa m 5,563 Clegg et al Signed and sealed this 5th day of Deoemberl972u (SEAL) Attest:
EDWARD MQFLETCHERJRQ ROBERT GOTTSCHALK Attesting Qfi'icer Commissioner of Patents FORM PO-1OSO (10-69) USCOMM-DC 60376-P69 k u,s. GOVERNMENT PRINTING OFFICE: I969 o-ass-aaa.