CS208162B2 - Method of making the macroagregates of the technicium-99m,albumine and tin ion - Google Patents

Abstract

1403384 Radioactive materials E R SQUIBB & SONS Inc 20 July 1972 [20 July 1971] 34062/72 Heading G6R A radioactive composition for clinical diagnosis comprises macroaggregates of size 5-100 microns containing technetium -99 m, denatured albumin or denatured globulin, e.g. fibrinogen, and stannous tin. To prepare the composition, stannous chloride, iodide, or bromide is added to an acidified solution of human serum albumin, the pH is adjusted to the isoelectric point to form the macroaggregates, supernatant liquid is removed, the residue is resuspended in water, saline solution, or a solution containing dextrose or a sulphonate, and Tc- 99 m solution is added. The composition may be injected into the body and the amount of aggregates retained in the lungs, liver, spleen, or bone marrow measured by scintiscanning.

Description

(54) Method of production of technetium-99m macroaggregates _? albumin and tin ions

The present invention relates to a process for the production of technetium-OOm, albumin and tin ions by binding radioactive tecbium-99m to human blood albumin.

Preparations of human blood albumin to which radioactive iodine is bound have already been used to investigate diverse areas of the human body. Technetium | m-99im has also been widely used in recent years as an indicator component of albumin. A variety of techniques have been used or proposed by which technetium-99m can be bound to albumin instead of radioactive iodine.

The applicability of techuecia-99m in scintigraphic detection methods is due both to its physical and chemical properties. The short half-life of techinecia-goim and the fact that it does not emit beta-radiation result in exposed tissue being exposed to low radiation doses only. The half-life of tec brown a-99m is about 6 hours. Piotc can be administered high activity, reducing the time required for examination.

As mentioned above, tetecium can be prepared in a variety of chemistry and can be pre-complexed with proteins. Use of teichnecla-99m in the form of complexes with maicroaggregates

Human blood albumin for lung testing has already been described.

Μ M, Gwyčher and E, O. Field on the use of technetium-99im labeled albumin aggregates in lung sointigraphy in Int. J. Appl. Order. and Isotcpes 17, 485-486 (1966)] described a method of labeling albumin with a desert. elevated concentrations of ferrous chloride and .alpha.-rbic acid.

A different approach to the problem has been described by T. Depaoli, A. 1-Ia.ger and JO Nholi.ni in the article "Albumin Macroaggregates Labeled with ^99m Tc", published in Inter. J. Αιρρί. Order. and Isotojpes, 17, 5'51-554 (1936).

In this paper, the following criteria for applicable albumin microaggregates are published:

1. it must be sterile and must not contain pyregenes;

2. their unbound radioactivity shall not exceed 5% of the total radioactivity present;

3. less than 10% of the particles are less than 5 micrometres in the largest diameter and none of the particles is larger than ТОО. micrometers; the average particle size shall be between 10 and 50 micrometer;

4. have a lay specific activity that the required dose of radioactivity can be bound to less than 1.5 mg of albumin.

All of these methods have the main disadvantage of requiring trained personnel and special equipment to obtain a radioactive element complex with an allbum.in ¹ m. Attempts have been made to simplify these methods of preparation, but each of them developed so far. The process requires a number of mixing and heating steps.

The macroaggregates produced according to the invention comprise human blood albumin aggregates.

The above-mentioned drawbacks are not the same as the method of producing macromaggregates of techinecia-99m, albumin and tin ions according to the invention, which comprises adding stannous halogeinide to the aqueous solution of denatured albumin, adjusting the pH of the resulting reaction mixture to 4.5-6 k. to obtain albumin macroaggregates, then an aqueous solution of technetium-99m is added to mm.

In the process according to the invention, the pH of the reaction mixture is preferably adjusted with an inorganic base.

Preferably, the albumin macroaggregate solution is dehydrated prior to addition of the aqueous technetium-99m solution.

An advantage of the invention is that they are _(with him · eliíminovány drawbacks described above. The inventive method comprises adding stannous halide · (e.g., stannous chloride, iodide or bromide, stannous címatého-) к acidified flowable human blood albumin, adjusting the pH of the mixture to iisoelektrický albumin point to form macro-aggregates, separating the formed supernatant from the formed macro-aggregates, re-suspending the formed macro-aggregates in a solution suitable for injection, which may optionally be dehydrated, adding teChinec? -99im to the resulting drying, and eliminating the macro-aggregates component.

Preferably, the macrographer min- ¹ comprises albumin particles having a PO size of up to 100 microns, preferably 30 to 50 micrometers.

The macroaggregates of the invention are formed using human blood albumin, which is denatured. Denatured albumin can be prepared by any of the known methods.

One method for preparing denatured albumin involves heating and alkylating normal blood albumin and then basifying it. Nolrimary human blood albumin is used when the indication tests are performed in humans. Where the tests are carried out, for example, with cows, dogs or rats, the appropriate animal serum is used. Normal blood albumin is heated to a temperature of 55 to 95 ^° C, preferably a temperature of 75 to ° C, for 15 to 60 minutes depending on the amount of albumin used. Larger amounts of albumin require longer heating. Using some inorganic acid such as sulfuric, hydrochloric, nitric, some organic bases such as sodium acetate, acetate buffer solution, some inorganic bases such as alkali metal hydroxide, especially sodium hydroxide, or an alkali metal hydrocarbonate such as sodium hydrogen carbonate The pH of the albumin is adjusted to a value of 4.5 to 6, preferably 5.3. This causes the deinatarain albumin to precipitate.

It should be noted that albumin can be denatured by adjusting the pH to about · 8, then using the acid to adjust the pH to 4.5 to 6, or adjusting the pH to olkoto 2, then using the base to reach the pH The pH change is generally accompanied by heating to ensure an adequate degree of denaturation.

Another way to prepare albumin is by heating the albumin emulsion in an oil, such as cottonseed oil, separating, arranging the size, and drying the formed aggregates, which are generally known to those skilled in the art as microsferences. More specific guidelines for the preparation of -mV cross-conferences can be found in "Radiology ¹ Clinics of Nopth America" 8, 233 (1'969) and "Interinatiohal

Journal of Applied Radiatioii and Isotopes, 21, 155 (1970); both methods of preparation. they are supported by literary references.

When preparing albumin · method according to the invention is preferred to use blood ксн merčního human albumin ^1, which contains preservatives and foaming agents Pirotte. The pH of this material is adjusted to a value of 1 to 3, preferably a value of 1.5 to 2, by adding a strong inorganic acid, for example sulfuric acid, or acidic acid.

Then, stannous halide dissolved in hydrochloric acid is added to the mixture. The ratio of stannous ions to albumin should be in the range of 1: 2 to 1:20. Particularly exceptional results were obtained when the ratio of stannous ions to albumin was about 1: 5.

When denatured human blood albumin has been prepared, the necessary amount of stannous halide is added in an acidic medium such as hydrochloric acid; The pH of this solution should be 1 to 2, preferably 1.1 to 1.3.

The pH of the solution is then adjusted to an isoelectric point of denatured albumin, which is about 5.3; however, this value may optionally be in the range 4.8 to 5.8.

This will create macro aggregates. In order to ensure the stability of these maicroaggregates, these solutions are heated for a period of from 10 to 45 ml, preferably 10 to 20 minutes. The temperature used is 75 to 85 ° C, preferably 78 to 81. Deň: 32 ° C.

The macroaggregates thus formed are washed with distilled water, a salt solution or a reducing agent such as a sultanoic acid solution. The particles are then resuspended in water for injection, in a saline solution or in a solution containing a suitable reducing agent, such as dextrose or a sulfoic acid salt.

The macroaggregates may also optionally be dehydrated, which increases the stability of the tin ion. A preferred method of dehydration is lyophilization, although other standard methods may be used.

Although albumin is used to describe the preparation of the maicroagiregates of the invention, globulins, preferably alpha-, beta- or gamma-globulin with fibrinogen, may also be used.

Up to this stage, the preparation of the macroaggregates is carried out in such a way that sterile and non-pyrogenic macroaggregates are obtained which are thus in a form suitable for injection. Dosage units are prepared to deliver that a single intravenous injection, administered to a mammal with a body weight of about 70 kg, delivers about 3.7.107 to 1.85. 108 with ¹ technetium-99im, preferably about ДИ. 108 s'1 technetium-99m.

In carrying out the method according to the invention, they are supplied to the technician by using pre-prepared macro aggregates. Then, the necessary amount of technetium-99m from the gel generator, for example the generator described in U.S. Pat. No. 3,369,121, must be added thereto.

To the solution is added the desired amount of tetrachloride-99m in aqueous solution, preferably in saline as predetermined by the physician, meaning the desired radioactivity, which is a decisive factor for the amount of technetium -99m added. Measuring the amount of teehnec ^: .alpha.-99im is not practical as it is present in varying quantities, depending on the activity of the generator and the time the generator was last eluted. The reaction proceeds equally well when the macro aggregates are added to the ecium-9 9 m solution.

The process according to the invention is explained in more detail in the following by way of example. All temperatures are in degrees Celsius.

Example 1

Preparation of unlabeled albuimm macroaggregate

Mix 0.4 ml of 25% human blood albumin (containing 100 mg of albumin), 0.9 ml of benzyl alcohol, 2 ml of propyleinglyicol, 2 drops of antifoam and 75 nulls of water for injections and mix. The pH was adjusted to 1.75 by addition of 0.1 N HCl; 100 mg of stannous chloride dihydrate is added and the simes are mixed. While stirring, 20 ml of 0.5 M acetic buffer (pH = 5.1) is then added to form the albumin mono-aggregates, and the mixture is heated on a water-bath. a temperature of 80 ° C (± 15 ° C) with continuous stirring for 40 minutes.

The mixture is then centrifuged and the supernatant is discarded. For further purification of the albumin macroaggregates, water for injection is admixed to the albumin, the mixture is centrifuged again and the sulphate is again poured off. Deinaurated human blood albumin macroaggregates are further suspended in 100 µl of water for injection and cooled to about 5 ° C.

Examples 2 and 3

In these exemplary embodiments, two kinds of albumin macro aggregates are prepared

Each macroaggregate is tested to determine the degree of binding of technetium-99m to albumin and the amount of injected dose that is located in the lungs and liver of rats. Exactly 25 ml of each formulation, their composition is shown in Table 1, is injected into the throat external vein (vein-conjugate) of rats. 15 minutes after injection, rats are sacrificed and determined (percent of injected dose in lung and liver).

To determine the degree of teichnecia-99111 binding to albumin, total radioactivity of the preparation and total radioactivity of supernatant were determined by subtracting the number of pulses per unit time when measuring total preparation and supernatant after centrifugation of the macroaggregates. ’

The degree of binding (in%) of technetium-99im to albumin is determined by the following formula:

The degree of binding (%] = | number i ^'s obtained ^mp ULS \ the measurement of the number of pulses obtained by measuring isuippenatantu

x 100 number of pulses obtained by measuring the preparation

TABLE 1

Example 2 Example Suspension of macroaggregated human. blood albumin (from Example 1) 2 ml 1 ml Biftalate potassium buffer 1 ml - Technetium-99m (from generator) 2 ml 1 ml % injected dose in rat lungs 95,4 * 99,6 * % injected dose in rat liver 2.1 « 0,63 *

*) mean of three results obtained in three rats

It can be seen from these examples that the small aggregates of the invention can be readily prepared and used as a means for iscintigraphic imaging of the lung condition (lung scan).

Example 1 a d 4

0.4 ml of 25% blood albumin (containing 100 mg albumin), 50 ml of water for injections and 0.1 N HCl in an amount necessary to reach a pH of 2 are stirred (325 oscillations / inrin) for 2 minutes at 79 ° C. . 1 ml of a 2.5% tin (II) chloride solution is then added and the pH is adjusted to 5.1-5.2 with 1M acetate buffer (about 30 ml). The mixture was again heated to 79 ^° C for 20 minutes with stirring (325 kimits / min). The mixture was then centrifuged and the supernatant was poured off.

To wash the microaggregates, it is mixed with water for injection, the mixture is centrifuged again and the supernatant is discarded again. Washing is repeated and then the macroaggregates are suspended in 10 ml of water for injection and placed in the refrigerator.

. Example 1 a d 5

The procedures of Examples 2 and 3 are followed, wherein the percent localization of activity is shown in Table 2.

TABLE 2

Macro-aggregated human blood albumin 1ml

Technetium-99m (from generator) 1 µm

Water for injections 8ml% injected dose in rat lung 98 *% injected dose in rat liver 0.57

*) average of three results obtained and three rats

Example 6

Normal human blood albumin (0.75 g) is added to a mixture of hydrochloric acid (0.5 N) (20 µl) and water for injection (400 ml) and heated to 80 ° C with stirring for 30 minutes . To the cooled mixture was added a sterilizing filter over a stannous chloride solution (1%, 7 ml), followed immediately by the addition of acetate buffer (IM, 100 ml; anhydrous sodium acetate - 8.5 g; glacial acetic acid - 4 ml, water for injections). to 100 ml).

The mixture is reheated with stirring at 80% for 15 minutes, then the aggregates are allowed to settle while rinsing, 80% for 15 minutes, then the agitation and aggregates are allowed to settle.

Thereafter, the aggregates are resuspended in water for injection when the required volume has been determined by Brecan analysis to achieve the desired denatured albumin concentration of 0.75 mg / ml. According to the calculated volume of the quantities of normal human blood albumin needed к the permeating was 0.5 ^{1 * * * * *%} Solutions of the carrier.

The ampoules (preferably silicone) are filled and the contents are lyophilized to dryness, and these are filled with nitrogen before closing the ampoules.

At the time of application may have Solutions of technetium-99im activity 1,85.10 ⁷ s ^-1 to 1 ml to 7,4.10 ⁹ is ^"1 on - ml; as a rule, 1 to 3 ml of a technetium-99 µm solution having an activity of 3.7 x ^{10 7} to 3.7 ^x 10 ⁸ s- ^x per ml is added to the solution.

Claims (3)

SUBJECT OF THE INVENTION Process for the production of teichnecia-99m, albumin and tin ions macroaggregates, characterized in that tin (II) halide is added to an aqueous solution of denatured albumin, the pH of the reaction mixture is adjusted to 4.5-6 to obtain albumin macroaggregates and then added thereto aqueous technetium-99m solution. 2. The process of claim 1 wherein the pH of the reaction mixture is adjusted with an inorganic base. 3. A method according to claim 1, characterized in that the solution of the macroaggregates is dehydrated prior to the addition of the aqueous technetium-Om solution.