BG106438A - Radiopharmaceutical products and method for their preparation - Google Patents

Abstract

These products can be used for pulmonary scintography or for therapy. They comprise a polysaccharide and sequestering group of formulae R-NH-, R-N=, and formula in which R is a hydrocarbonic or aromatic group comprising at least one atom of sulphur, and R1 is an atom of hydrogen or an alkyl grouping such as methyl, said sequestering group forming a chelate type complex with a radioactive metal such as technetium. 20 claims

Description

The present invention relates to radiopharmaceutical products that can be used for diagnosis or therapy and to a method for their preparation.

In particular, it relates to radiopharmaceutical products obtained, for example, from a suspension of radiolabelled particles used in particular for pulmonary scintigraphy, for example, to establish a diagnosis when pulmonary embolism is suspected.

According to the present invention, the products are used in the form of particles, which preferably have a spherical shape and sizes in the range from 10 to 100 μΜ. In practice, since the capillaries of the lungs have a diameter of about 7 μΜ, after intravenous injection, the particles remain blocked in the capillaries, which makes it possible to detect abnormalities of blood perfusion in the lungs.

Obviously, these products must meet a number of pharmaceutical requirements. In particular, they must have an adequate rate of degradation in vivo, which must be slow enough to allow images to be taken, such as with a gamma-ray camera for at least one hour, but at the same time fast enough so that not to cause continuous obstruction of the capillaries of the lungs, which may cause small blood clots. In addition, these products should not be toxic to the body, they must be adapted for sterilization, for example in an autoclave or by irradiation, should they be easily labeled with radioactive metal and be packaged? in the form of a stable marking whale.

BACKGROUND OF THE INVENTION

French patent application FR-A-2 273 516, filed in. 1975 from the PHARMACIA AKTIEBOLAG Company, based in Sweden, describes the use of amylopectin microspheres crosslinked with epichlorohydrin and labeled with a simple ^99m Tc mixture for pulmonary perfusion scintigraphy. These particles have some disadvantages, in fact only the hydroxyl groups of the amylopectin used can allow the labeling of this mixture and unfortunately they form only weak bonds with technetium and do not allow for a stable labeling. In addition, the preparation method described uses many solvents and emulsifiers that are difficult to remove from the resulting particles. Moreover, in this particular type, the exact networking speed cannot be accurately measured or controlled.

Furthermore, this document does not accurately describe a kit compatible with routine use in nuclear medicine, in fact for human injection preparations, some manipulation is required, such as tin application, sterile flask, centrifugation, suspension reconstitution, etc. which is incompatible with the sterility requirement.

Finally, the resulting solutions are unstable and the epichlorohydrin used for cross-linking is considered to be very toxic and mutagenic.

The inventors point to other disadvantages of these microparticles in comparative Examples 1 and 2 below.

For example, the French patent application FR-A-2 285 857, filed in 1975 by PHARMACIA FINE CHEMICALS AB Company, based in Sweden, describes the use of polysaccharide particles bound to different binders and labeled with radioactive isotopes. The particles comprise chelating groups bonded by covalent bonds to which radioactive nuclei in the form of chelate-type complexes are attached, which are generally composed of at least four, preferably at least five to eight, cyclic nuclei of 5 to 6 groups, including metal and two metal-coordinating atoms. A polysaccharide is a chemically cross-linked polysaccharide, for example by epichlorohydrin or epibromhydrin. After separation of the labeling azide, these particles have the same problems as those previously mentioned for the particles described in FR-A-2 273 516. Furthermore, this document does not provide any examples of technetium labeled. Moreover, the labeling process involves heating to 100 ° C in the presence of a radioactive element, washing and drying after labeling, which are not fully compatible with the idea of the labeling kit mentioned above for sterility in use.

Even if the aforementioned labeling method allows the particles to be labeled in a relatively stable manner, it is not possible to obtain a labeling kit that is pharmaceutically acceptable, especially since it is

includes © pichlorhydrin, and © is readily applicable in nuclear medicine.

The microspheres described in these two patent applications do not meet the pharmaceutical requirements and cannot be used. In addition, they have never been used for pulmonary scintigraphy. This product was abandoned.

Since 1975, many studies of improved radiopharmaceuticals have focused on albumin-based products and their derivatives. These blood products must actually meet the pharmaceutical requirements and may be used in particular for pulmonary scintigraphy. These are the products now used in nuclear medicine.

For example, in 1975, M.A. Davis, in Radiopharmaceuticals NY, 1975, pages 267 to 281 describes radioactive particles intended for lung perfusion testing. The particles described in this document are macro-aggregates of radio-iodinated serum albumin ( ¹³¹ 1-MAA) or microspheres of denatured human serum albumin labeled with technetium ( ^99m TcNAM). The ^99spg Tc-HAM microspheres are preferred because of the uniformity of the particle size in the interval mainly between 40 and 50 μΜ. In addition, this document describes the main characteristic requirements for such radiopharmaceutical particles.

Source of information - Guiraud “Macro-aggregates and radioactive microspheres” Radiopharmaceuticals, 1997, 519 describes albumin macroaggregates (MAA) and human serum albumin microspheres. Describes the labeling of such micro-aggregates and microparticles with technetium 99m by dissolving tin chloride. It is also noted that the optimum microparticle size is 15 ± 5 µm. Organic starch microspheres are indicated.

At present, these 99 ^mg Tc-labeled human serum albumin microspheres and microspheres are incomparably used in nuclear medicine. However, they have several disadvantages. For example, the variability and quality of lots of human albumin sometimes make it difficult to prepare diagnostic kits containing particles that can vary in size and number. But one of the biggest disadvantages is their human origin, which can pose serious problems for potentially fatal HIV, hepatitis or Creutzfeld-Jacob disease.

Therefore, of particular interest is the possibility of having non-human-labeled ^99m Tc microspheres to ensure perfect safety.

With this in mind, the recently released document by A.S. Parkins, Nuclear Medicine Communications 1999, 20,1-3 describes ways to replace blood-derived radiopharmacological products. In particular, reference is made to the use of materials recombinants, synthetic polymers and polypeptides. But no polysaccharides are mentioned in this news source.

SUMMARY OF THE INVENTION

It is an object of the present invention to avoid the disadvantages of the products mentioned above prior art, creating a radiopharmaceutical product that is readily ^labelable, for example with ^99mt, Tc, which has a very good lung 'capture, as demonstrated by the inventors in rats; non-toxic, easy biodegradation, easy sterilization and fit to be packaged as a kit ready for

labeled ©, stable and meeting the pharmaceutical requirements for this type of product. These and other advantages will become apparent from the following description.

The radiopharmaceutical product according to the present invention is characterized in that it contains a polysaccharide formed by binders to be bound to the polysaccharide by covalent bonds and selected from the groups of formula -R-NH-, R-N = and R-N-N =

R 'in which R' is a hydrocarbon or aromatic group containing at least one sulfur atom and R 'is a hydrogen atom or an alkyl group, for example methyl, said linking groups form a chelate type complex with radioactive metal selected from technetium, rhenium, copper, yttrium , erbium, gallium and samarium.

The alkyl groups used for R 'may be linear or branched and preferably have 1 to 5 carbon atoms.

According to the present invention, the polysaccharide may be soluble or in the form of microparticles. According to the present invention, the polysaccharide may be selected, for example, from natural starch, cellulose or cross-linked amylopectin.

Natural starch may, for example, be corn starch.

The polysaccharide may be in the form of microparticles, for example in the form of microspheres.

The present invention shows that the modified cellulose according to the present invention exhibits very good pulmonary captivity and a lower elimination rate than starch. Therefore, the modified cellulose according to the present invention can also be used for radiotherapy, for example by rhenium, copper or one of the aforementioned metals, since it fulfills the requirement of radiotherapy for the use of microparticles

with a longer half-life.

According to the present invention, the linking groups may be selected from, for example, the groups of formula:

p

in which R ¹ , R ² , R ³ , R ⁴ and R ⁵ each independently are hydrogen atoms, saturated or unsaturated hydrocarbon groups, carboxyl groups or aromatic groups is an aromatic nucleus optionally containing one or more heteroatoms and η is integer a number between 1 and 5.

For example, they can be selected from the groups of formula:

= Ν - NH-C> - \ s

SCH ₃ ^s == N - N - - C

I ^SCH 3

CH ₃

S 1 = N-NH-C; -

- ^X NH - With _A

N -

S

NHCH ₃

u According to the present invention, microparticles, for example, in the form of microspheres, may have trammaters in the range of 0.01 to 100 μΜ, preferably 10 to 50 μΜ, for diagnosis by pulmonary scintigraphy and

I sizes from 0.1 to 5 μΜ for therapy.

According to the present invention, the amount of! the linking groups may be from 0.1 to 50% relative to

The I moieties of the saccharide in the polysaccharide, preferably from 2 to 15%.

and? According to the present invention in the radiopharmaceutical! product, especially when used for diagnostics, the radioactive metal may be ^99m Tc or gallium-67.

This may be the case, for example, when a radiopharmaceutical is used for pulmonary scintigraphy.

According to the present invention in the radiopharmaceutical product, in particular when used for therapy, the radioactive metal may be rhenium186 or 188, copper-64 or 67, yttrium-90, erbium-169 or samarium-153.

According to the present invention said radiopharmaceutical product may be in the form of a suspension of microspheres in a physiologically acceptable liquid or lyophilized.

The present invention also relates to a method for preparing a radiopharmaceutical product according to the invention, which comprises the following steps:

a) a polysaccharide such as the one mentioned above is subjected to oxidation controlled by periodate,

b) the oxidized polysaccharide is reacted with a compound containing a primary amine functional group or a hydrazine of formula R-NH ₂ or

R - - - - - NH ₂

R '

c) in which R is a hydrocarbon or aromatic group containing at least one sulfur atom so as to covalently bind to the polysaccharide via linking groups of metals of formula R-NH-, RN = or R-NH-N =, and R 'is hydrogen an atom or an alkyl group such as methyl,

d) the polysaccharide containing the linking groups is reacted with a radioactive metal salt selected from technetium, rhenium, copper, yttrium, erbium and samarium.

Oxidation controlled by periodate may be, for example, that described in C.L. Mehltretter, “Methods in Carbohydrate Chemestry”, vol. IV, 1964, used in particular for the oxidation of starch, dextrin or cellulose. It is used in the examples below.

According to the present invention, a compound containing a primary amine functional group may correspond to formula NH ₂ - (CH ₂ ) n-SH, with η being an integer from 1 to 5 and may further include a step of reducing this compound with sodium borohydride, between steps (b) and (c).

According to the present invention, the compound attached to the polysaccharide may correspond, for example, to one of the following formulas:

NH

ΝΗ -, --- NH - SCH

SCH ₃

CH

NH

NH.

NH

NHCH

NH

NH -NH --- CH ₂ CH = CH _:

NH

SH

NH.

According to the present invention, the amount of binding groups fixed to the polysaccharide can be adjusted by controlling the oxidation rate of the polysaccharide in step (a) mentioned above. This degree of oxidation of the polysaccharide may be, for example, between 10 and 50%. The amount of the linking group may for example be between 2 and 15%.

In order to carry out the polysaccharide labeling of the present invention, for example, with ^99m Tc, the two-step transformation method is used.

This method can be represented as consisting of conducting a controlled oxidation of a polysaccharide with a periodate in the first step. Each unit of oxidised glucose form two aldehyde groups in neighboring positions following the indicated below diagram of the ^{chi:-chemical} reaction:

natural glucoside monomer oxidized glucoside monomer

The oxidation level of the polysaccharide can be varied and easily adjusted, in fact the yield from this oxidation reaction is very close to 100% and the oxidation rate can be calculated from the amounts of periodate added. Usually an oxidation rate of less than 50% is used so that the structure of the macromolecule is only slightly modified. A real oxidation rate in the range of 1 to 100% can be easily determined colorimetrically.

In the second embodiment of the present invention, the oxidized polysaccharide is reacted with a molecule containing an amine or hydrazine group of the general formula RNH ₂ or RNHNH ₂ and a chelating group capable of binding to technetium is formed. This produces a Schiff-based ligand or thiosemicarbazone.

This second stage can be summarized as follows:

R • \ ^ °

With VR - NH ₂

H aldehyde group - amine or hydrazine

I

R —— With ^N R

H ' functionalized aldehyde group where:

.R = NR ₁ (C = S) SR ₂ (Schiff bases of dithiocarbazate),

2. R = NR ₁ (C = S) NR ₂ R ₃ (thiopolycarbazones)

3. P = aromatic group (aromatic Schiff bases)

4. R = alkyl group (alkyl Schiff bases); in this case the Schiff bases are not stable and a second reduction step of the C = N bond with borohydride is carried out to stabilize them, and then an amine C-NHR bond is formed.

According to the present invention, step (c) may consist, for example, of contacting polysaccharide microspheres containing linking groups with, for example, a solution of pertechnetate ^99n, TcO ₄ in the presence of a reducing agent, for example tin chloride.

According to the present invention, microparticles, for example microspheres, such as corn starch or starch based on cross-linked amylopectin, can be oxidized, then linked to a molecule containing an amine or hydrazine functional group, for example Bmethyl, dithiocarbazate. These particles modified in this way can be easily labeled with, for example, ^99m Tc.

Thus, the present invention produces particular microparticles obtained, for example, from starch particles, which do not have the disadvantages inherent in the above-mentioned albumin. In addition, starch has been described as an excipient in the pharmacopoeia. Therefore, it is easily accessible and at a low cost.

The microparticles of the present invention also have the advantage of being readily sterilized, for example by irradiation, and can be produced in the form of a label-ready kit.

In addition, the inventors demonstrate according to the present invention that the rate of lung purification can be varied according to the oxidation level of the microparticles used according to the present invention, which is not possible with, for example, human albumin microspheres.

Another advantage of the present invention is related to the simplification of the operation of the procedure: the mode of.:.

the reaction is very mild. reactions at ambient temperature, in aqueous medium, in almost quantitative yields. Additionally, coupling reactions, for example with technetium, are quantitative; they are carried out at room temperature and without final purification, which makes it possible to adapt to the sterility and simple preparation requirements required for the use of technetium kits in a hospital setting.

The present invention also relates to a diagnostic kit that can be used, for example, for pulmonary scintigraphy. This kit consists of:

a first flask containing a polysaccharide according to the present invention which is formed by coupling groups attached to the polysaccharide by covalent bonds and selected from the groups of formula R-NH-, R-N = and ρμ ._. | ψ. "Ν =

R 'in which R is a hydrocarbon or aromatic group containing at least one sulfur atom and in which R' is a hydrogen atom or an alkyl group such as methyl.

According to the present invention, the polysaccharide may for example be in the form of microcrystals, for example in the form of microspheres, the microparticles being able to be in lyophilized form or in suspension in a pharmaceutically acceptable liquid.

The kit of the present invention may further comprise a second flask containing tin chloride preferably in a lyophilized form or also when the polysaccharide is in a lyophilized form, for example in the form of microparticles in the first flask, said flask may further comprise lyophilized tin chloride.

The kits of the present invention have been stable for at least twelve months, as shown in the examples below.

Therefore, the radiopharmaceutical product of the present invention has all the properties necessary for use as a radiopharmaceutical application, for example pulmonary perfusion scintigraphy or radiotherapy.

Other advantages will be apparent from the following examples related to the present invention.

Examples

Example 1

A suspension of 10 g of pharmacopoeial corn starch is prepared, pre-screened between 10 and 40 µm, containing about 10% water, which represents about 0.055 mol of glucose in 100 ml of water. 0.0168 mol of sodium periodate (0.3 eq (NalO ₄ )) was added, representing 3.6 g dissolved in 100 ml of water. The suspension was then stirred for 18 hours at room temperature. The solution was filtered and the oxidized starch was washed 5 times. with 20 ml of water and then twice with 50 ml of acetone The starch is dried in vacuo to give 10 g of starch, oxidized 30% (yield = 100%).

A suspension of 10 g of starch, 30% oxidized in 60 ml of water / ethanol 2: 1 (volume ratio) was obtained. 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 mol of S-methyl dithiocarbazate (NH ₂ NH (C = S) SCH ₃ ), M = 122, i. 1.34 g dissolved in 10 ml of ethanol. The suspension was stirred for 18 hours at room temperature. The solution was then filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo. This gives about 10 g of modified starch. Elemental powder analysis showed a sulfur content of 5.4%, which corresponds to a 7% binding rate of S-methyl dithiocarbazate (DTCZ) (7 units of dithiocarbazate per 100 theoretical aldehyde functional groups, ie 14 units of dithiocarbazate per 100 units of glucose ). Therefore, the yield of blocking is 70%. This gives 10 g of starch oxidized 30% and bound to DTCZ 7%.

^99pg labeling reaction Tc, mg of starch modified in this way is placed in a penicillin type flask. Then 4 ml of physiological serum was added and then 10 μΓ SnCI ₂ , 2H ₂ O (20 ml solution of 0.5 mg / ml in HCl 0.1 N). Then a 1 ml solution of ^99m Tc (5mc) was added. The solution was stirred for 15 minutes and radiochemical purity (RCP) was monitored, filtering 1 ml of the solution through a 0.22 μΜ microporous filter, then washing the filter with 2 ml of physiological serum. The labeled microspheres are retained on the filter until the radioactive impurities bind to the microspheres present in the filtrate. Radioactive purity corresponds to:

RCP = (filter activity / total activity) x100.

It is equal to 98.9%.

Example 2

Modification of starch

Proceed as in Example 1 using 0.2 eq periodate to conduct the oxidation reaction. Obtained starch oxidized 20%.

Proceed in the same manner as in Example 1 for the coupling reaction to give a starch oxidized 20% bound to DTCZ 7%.

^Marking reaction with ^99gt1 Tc,

Proceed as in Example 1. Radiochemical purity (RCP) is 99%.

Example 3

Modification of starch

Proceed as in Example 1 using 0.1 eq periodate to conduct the oxidation reaction. Obtained starch oxidized 10%.

The procedure was followed in the same way as in Example 1 for the coupling reaction to give a starch oxidized 10% bound to DTCZ 7%.

^99t Tc Marking Reaction.

Proceed as in Example 1. Radiochemical purity (RCP) is 98.8%.

Example 4

Proceed as in Example 1 to give 10 g of starch oxidized 30%. A suspension of 10 g of starch oxidized 30% is obtained in 60 ml of a water / ethanol mixture (2/1 volume ratio). 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 mol of M-methyl-B-methyl dithiocarbazate (NH ₂ N (C = S) (C = S) SCH ₃ ), M-136, i. 1.50 g dissolved in 10 ml of ethanol. The suspension was stirred for 18 hours at room temperature. Next

the solution was filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo. This gives about 10 g of modified starch. Elemental powder analysis shows a sulfur content of 5%, which corresponds to a 6.5% binding rate of N-methyl S-methyl dithiocarbazate (6.5 units of dithiocarbazate per 100 theoretical aldehyde functional groups i.e. 13 units of dithiocarbazate per 100 units of glucose ). Therefore, the binding is 65%.

^99t Tc Marking Reaction.

Proceed as in Example 1.

RCPe 95%.

Example 5

Modification of starch

Proceed as in Example 1 to give 10 g of starch oxidized 30%. A suspension of 10 g of starch oxidized 30% is obtained in 60 ml of a water / ethanol mixture (2/1 volume ratio). 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 mol of 4-phenyl 3-thiosemicarbazide (NH ₂ NH (C = S) NH (C ₆ H ₅ ), M = 167, i.e. 1.83 g dissolved in 10 ml ethanol. The suspension is stirred for 18 hours The solution was then filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo to give about 10 g of modified starch. Elemental analysis of the powder showed a sulfur content of 3.16%, which corresponds to an 8% degree of 4-phenyl bonding

3-thiosemicarbazone (8 units of thiopolucarbazide per 100 theoretical aldehyde functional groups, ie 16 units of thiosemicarbazone per 100 units of glucose). Therefore, the binding yield is 80%.

^99t Tc Marking Reaction.

Proceed as in Example 1.

The RCP is 98%.

Example 6

Modification of starch

Proceed as in Example 1 to give 10 g of starch oxidized 30%. A suspension of 10 g of starch oxidized 30% is obtained in 60 ml of a water / ethanol mixture (2/1 volume ratio). 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 moles of 4-methyl 3-thiosemicarbazide (NH ₂ NH) (C = S) NH (CH ₃ ), M = 105; 1.15 g dissolved in 10 ml ethanol. The suspension was shaken for 18 hours at room temperature. The solution was then filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo. This gives about 10 g of modified starch. Elemental powder analysis showed a sulfur content of 2.9%, corresponding to a 7.3% binding rate of 4-methyl 3-thiosemicarbazone (7.3 units of thiosemicarbazide per 100 theoretical aldehyde functional groups, ie 14.6 units of thiosemicarbazone per 100 units of glucose) . Therefore, the binding yield is 73%.

^99t Tc Marking Reaction.

Proceed as in Example 1.

The RCP is 97%.

Example 7

Modification of starch

Proceed as in Example 1 to give 10 g of starch oxidized 30%. A suspension of 10 g of starch oxidized 30% is obtained in 60 ml of a water / ethanol mixture (2/1 volume ratio). 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 mol of 4,4-dimethyl 3-thiosemicarbazide (NH ₂ NH) (C = S) N (CH ₃ ) ₂ , M = 119; 1.30 g dissolved in 10 ml of ethanol. The suspension was stirred for 18 hours at room temperature. The solution was then filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo. This gives about 10 g of modified starch. Elemental powder analysis shows a sulfur content of 3%, which corresponds to a 7.5% coupling rate of 4,4-dimethyl 3-thiosemicarbazone (7.5 units of thiochemicarbazide per 100 theoretical aldehyde functional groups, ie 15 units of thiosemicarbazone per 100 glucose units). The yield of the binding is 75%.

^99gp Tc ^Marking Reaction.

Proceed as in Example 1. RCPe 96%.

Example 8

Modification of starch

Proceed as in Example 1 to give 10 g of starch oxidized 30%. A suspension of 10 g of starch oxidized 30% is obtained in 60 ml of a water / ethanol mixture (2/1 volume ratio). 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 mol of 4-allyl 3-thiosemicarbazide (NH ₂ NH) (C = S) NH (CH ₂ CH = CH ₂ ), M = 131, i. 1.44 g dissolved in 10 ml of ethanol. The suspension was stirred for 18 hours at room temperature. The solution was then filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo. This gives about 10 g of modified starch. Elemental powder analysis shows a sulfur content of 3%, which corresponds to a 7.5% binding rate of 4-allyl 3-thiosemicarbazone (7.5 units of thiosemicarbazide per 100 theoretical aldehyde functional groups, ie 15 units of thiosemicarbazone per 100 units of glucose) . The yield of the binding is 75%.

^99gp Tc ^Marking Reaction.

Proceed as in Example 1.

RCPe 98%.

Example 9

Modification of starch

Proceed as in Example 1 to give 10 g of starch oxidized 30%. A suspension of 10 g of starch oxidized 30% is obtained in 60 ml of a water / ethanol mixture (2/1 volume ratio). 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 mol of 3-thiosemicarbazide (NH ₂ NH) (C = S) NH ₂ , M = 91, i. 1 g dissolved in 10 ml of ethanol. The suspension was stirred for 18 hours at room temperature. The solution was then filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo. This gives about 10 g of modified starch. Elemental powder analysis showed a sulfur content of 2.9%, corresponding to a 7.3% degree of 3-thiosemicarbazone binding (7.3 units of thiosemicarbazide per 100 theoretical aldehyde functional groups, ie 14.6 units of thiosemicarbazone per 100 units of glucose). The yield of binding was 73%.

2'2

^99gp Tc ^Marking Reaction.

Proceed as in Example 1.

RCPe 95%.

Example 10

Modification of starch

Proceed as in Example 1 to give 10 g of starch oxidized 30%. A suspension of 10 g of starch oxidized 30% is obtained in 60 ml of a water / ethanol mixture (2/1 volume ratio). 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 mol of 2-aminothiophenol (C ₆ H ₄ ) (NH ₂ ) (SH), M < 125 >, i.e. 1.37 g dissolved in 10 ml of ethanol. The suspension was stirred for 18 hours at room temperature. The solution was then filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo. This gives about 10 g of modified starch. Conducting elemental powder analysis shows a sulfur content of 3%, which corresponds to a 7.5% binding rate of 2aminothiophenol (7.5 units of aminothiophenol per 100 theoretical aldehyde functional groups, ie 15 units of aminothiophenol per 100 units of glucose). The binding yield is 75%.

^99t Tc Marking Reaction.

Proceed as in Example 1.

RCPe 94%.

Example 11

Modification of starch

Proceed as in Example 1 to give 10 g of starch oxidized 30%. A suspension of 10 g of starch oxidized 30% is obtained in 60 ml of a water / ethanol mixture (2/1 volume ratio). 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 mol of 2-mercaptoethylamine (or 2-aminoethanethiol) (NH ₂ CH ₂ CH ₂ SH), M = 91, i. 1 g dissolved in 10 ml of ethanol. The suspension was stirred for 18 hours at room temperature. 0.015 moles of sodium borohydride (NaBH ₄ ) was then added to reduce the Schiff base formed to stabilize it (the non-aromatic Schiff base was not stable) and separated to react for 1 hour. The solution was then filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo. This gives about 10 g of modified starch. Elemental powder analysis showed a sulfur content of 3.4%, which corresponds to an 8.5% binding rate of 2 mercaptoethylamine (8.5 units of 2-mercaptoethylamine per 100 theoretical aldehyde functional groups, ie 17 units of 2 mercaptoethylamine per 100 units of glucose). The yield of the binding is 85%.

^99t Tc Marking Reaction.

Proceed as in Example 1. u RCPe 95%.

Example 12

Modification of starch

Proceed as in Example 1 to give 10 g of starch oxidized 30%. A suspension of 10 g of starch oxidized 30% is obtained in 60 ml of a water / ethanol mixture (2/1 volume ratio). 0.1 eq (0.1x0.055x2) is then added, i. E. 0.011 mol of 2-amino 4-mercaptotriazole (C ₄ N ₂ H) (SH) (NH ₂ ), M = 116, i.

1.27 g dissolved in 10 ml ethanol. The suspension was stirred for 18 hours at room temperature. Then

ΗΗΗΜΙΚ

The solution was filtered and the modified starch was washed three times with 20 ml of ethanol and then dried in vacuo. This gives about 10 g of modified starch. Elemental powder analysis showed a sulfur content of 2.8%, which corresponds to a 7% coupling rate of 2-amino 4-mercaptotriazole (7 units of mercaptotriazole per 100 theoretical aldehyde functional groups, ie 14 units of mercaptotriazole per 100 units of glucose) . The yield of binding was 75%.

^99t Tc Marking Reaction,

Proceed as in Example 1.

RCPe 85%.

Comparative Example 1

Use 10 g of screened maize pharmacopoeia that has not undergone chemical transformation and perform the same procedure as in Example 1 for ^{99 mg} labeling ^, Tc.

RCPe 19%.

This example is a good demonstration of the fact that chemical modification (bonding of fixing groups) carried out according to the present invention is really necessary in order to carry out the ^99m Tc labeling. Furthermore, no permanent pulmonary capture can be achieved if the microparticles are labeled without prior chemical transformation, contrary to the product of the present invention. Therefore, these results contradict what is described in FR-A-2 273 516.

Example 13

Modification of cellulose

Proceed as in Example 1, but using screened cellulose between 10 and 40. In this way, Yogh cellulose is oxidized to 30% and bound 7% to DTCZ.

^99t Tc Marking Reaction. <

Proceed as in Example 1.

RCPe 99.1%. :

Example 14

V

Sprague Dawley rats weighing about 200 g were anesthetized with sodium thiopental and injected intravenously with various solutions of Tc-labeled microspheres according to Examples 1 to 9 and 13. 0.2 ml of solution was injected into each animal's penis vein. ie 0.2 me per animal. Then the animal is placed under a gamma ray camera and static images are taken for 3 hours. Sequential images thus obtained are obtained after taking 15,000 snapshots of a single image. Areas of interest are then manually identified to evaluate activity at different organs 15 minutes after injection. The results are given in Table I.

Table I: Results ¹

% of assets. 15 min after I.V. An example 1 An example 2 An example 3 An example 4 An example 5 Pulmonary activity% 90% 85% 80% 80% 85% Hepat. Activity% <5% <5% <5% <10% <10% Pulmonary. Half-decay 2:00 1 hour 30 min. 2:00 2:00

% of assets. 15 min after I.V. An example 6 An example 7 An example 8 An example 9 An example 13 Pulmonary activity% 85% 85% 85% 85% 90% Hepat. Activity% <5% <5% <5% <5% <5% Pulmonary. Half-decay 2:00 2:00 2:00 , 2:00 > 4 hours

This indicates that the modified microspheres exhibit very good pulmonary captivity. In addition, the rate of pulmonary removal can be modulated by varying the degree of oxidation, as shown in Example 1,2 and 3 (oxidation rates of 30, 20 and 10%).

The use of cellulose makes it possible to significantly increase the rate of separation (Example 10, half-life> 4 hours).

Comparative Example 2

Natural starch is not used in this example, but microspheres derived from amylopectin cross-linked by epichlorohydrin, as in patent FR-A-2 273 516.

Part 2.1

Preparation of crosslinked starch microspheres

Dissolve 8 g of maize amylopectin in 40 ml of a solution containing 4 g of NaOH and 0.15 g of sodium borohydride. Amylopectin is allowed to dissolve for 24 hours. An emulsion was then prepared by stirring 60 ml of running paraffin and 1.6 g of soya lecithin dissolved in 4 ml of hexane at 800 rpm. The aqueous phase was then added containing amylopectin and then 3.2 ml of epichlorohydrin. The emulsion was heated to 55 ° C for 4 hours and then allowed to stir overnight. The resulting microspheres of about 50 µm in size were washed three times with 250 ml of acetone, dehydrated and then lyophilized.

Tc-labeled ^{99 tons.}

The procedure was as in Example 1, but using 1 mg of SnCl ₂ , 2H ₂ O. The RCP was 90%.

Part 2.2

Modification of starch

Proceed as in Example 1, but using 10 g of microspheres of cross-linked amylopectin with pre-prepared epichlorohydrin. This gives 10 g of amylopectin microspheres oxidized 30% and bound 7% to DTCZ.

^99t Tc Marking Reaction.

Proceed as in Example 1. The RCP is 99%.

Example 15

The following procedure was followed as in Example 14 for testing the microspheres of cross-linked amylopectin labeled with ^99m Tc of comparative example 2. The results obtained are given in Table II.

s,

Table II

% activity 15 min after I.V. Comparative Example 2 Part 2.1 Comparative Example 2 Part 2.2 % lung activity <10% 85% % liver activity 70% <5% Pulmonary half-life - 2:00 ___________________________________________________________________________________________________________________________________________________________________________________________________I

It is seen that, contrary to FR-A-2 273 516, the microspheres of amylopectin, unmodified chemically modified, are labeled with ^99m Tc but do not exhibit pulmonary capture, undoubtedly due to the weak relationship between ^99g Tc and the microspheres. On the other hand, these microspheres chemically transformed by the process of the present invention exhibit good pulmonary Capture.

Example 16 u Starch microspheres obtained as in Example 1 (oxidized starch 30%, bound 7% with DTCZ) were used to obtain sterile labeling kits and were easily labeled with 99m _Tc

Sterilization of the microspheres and microspheres were placed in a round-bottom flask and then irradiated with a cobalt-60 source. The microspheres receive a total gamma radiation dose of 25 kGy for 20 hours. Getting whales

200 mg of sterilized microspheres were sterilized in a reactor containing 20 ml of NaCI 9/1000. The solution is

it was vented by bubbling nitrogen and then 400 ml of sterile SnCl ₂ , 2H ₂ O solution (0.5 mg / ml) in 0.1 N HCl was added. Separate 1 ml of solution for each of 20 flasks, which are then lyophilized and placed under a nitrogen atmosphere.

Thus, each flask contains:

mg modified microspheres, μΓ SnCI ₂ , 2H ₂ O mg NaCI.

A labeling reaction with ^99m Tc ml of TcO ₄ (5 me) solution was added to the flask in lyophilized form and allowed to react for 15 minutes.

Proceed as in Example 1. The RCP was 98.7%.

Determining the stability of a whale

The labeling kits obtained as described above were stored at different temperatures and then the labeling reaction was tested with ^99µg, Tc to determine stability. The results obtained are given in Table III:

Table III: RCP (%)

Storage temperature 6 months 12 months 2-8 ° C 98.5% 98.4% 25 ° C 97% 96% 41 ° C 94% 90%

A very high stability of the kit, stored at 2 to 8 ° C, is noticeable.

Example 17

Sprague Dawley rats weighing about 200 g were anesthetized with sodium thiopental and then injected intravenously with various solutions of ^Wm Tc-labeled microspheres as in Example 14. Each animal received 0.2 ml of solution in the penis vein, representing 0.2 they are an animal. The animals were sacrificed 15 minutes after injection. The measured radioactivity of each organ is then calculated to determine the percentage of activity of each organ. The results obtained are given in Table IV: ·

Table IV results% dose of blood poured 15 minutes after injection

Organ Example 1 Example 13 Blood (1 ml) 0.1% 0.2% Liver 2.2% 5.6% Kidneys 0.4% 0.4% Lungs 91% 82% Dalak 0.1% 0.1% Small intestine 1.5% 0.7% Bladder 0.1% . 1.3%

Very high pulmonary capillation is observed, while the capillation of other organs is very low for microspheres of natural starch as well as of microspheres based on cross-linked starch. The sterile kit made in the form of a kit seems to be fully suitable for use as a radiopharmaceutical for pulmonary perfusion, albumin replacement and radiotherapy.

Example 18

Cellulose oxidized 30% and bound 7% with DTCZ was used as in Example 13. The modified cellulose was labeled with rhenium 186 to illustrate the use of the present invention for therapy.

Re-labeling reaction with Re 186 mg modified cellulose was placed in a penicillin type flask. Then 2 ml of physiological serum was added and then 20 mg of citric acid and finally 1 mg of SnCl ₂ , 2H ₂ O (100 µl solution of 10 mg / ml in 0.1N HCl).

Subsequently, 0.1 ml of a solution of ReO ₄ corresponding to 2 me activity was added to the contents of the flask. The flask was heated in a water bath at 100 ° C for 30 minutes. Radiochemical purity (RCP) was determined by filtering a microporous filter as in Example 1.

The RCP is 92%.

An in vitro stability test was performed to demonstrate the stability of the relationship between rhenium 186 and the cellulose microspheres.

The mixture was incubated with HSA (human serum albumin) 20 mg / ml at 37 ° C. The following results were obtained:

Incubation time 0 2:00 6 hours 24 hours 48 hours RCP 92% 92% 91% 89% 90%

Therefore, these results demonstrate the very high labeling stability of cellulose rhenium microspheres 186 and the high stability of the microspheres themselves to HSA.

Those skilled in the art will readily understand that these results can be extrapolated to the use of rhenium 188.

Example 19

Sprague Dawley rats weighing about 200 g were anesthetized and then injected with 0.2 ml (0.1 me) solution of cellulose microspheres labeled with rhenium 186 as in Example 18.

The animals were then placed under a gamma ray camera and images were recorded for 48 hours.

The manifested activity in the zones of interest is then calculated as in Example 14.

Time after injection 1 hour 2:00 6 hours 24 hours 48 hours % lung activity 80% 85% 85% 85% 90% % liver activity <5% <5% <5% <5% <5%

Therefore, these results indicate that lung activity remains unchanged for at least 48 hours. This type of microspheres can be used for therapy.

The most important clinical application may be the treatment of liver cancer after injection, not intravenously, but directly into the hepatic artery (metabolic radiotherapy).

Alternatively, particles of this type may be injected subcutaneously with breast cancer. Particles migrating through the lymphatic system and may make it possible to treat sentinel nodes covered by cancer cells.

Claims (20)

A radiopharmaceutical product containing a polysaccharide with linking groups attached to the polysaccharide by covalent bonds and selected from the groups of formula -R-NH-, R-N = and RN - N in which R is a hydrocarbon or aromatic group containing at least one sulfur atom and R 'is a hydrogen atom or an alkyl or methyl group, said linking groups together with a radioactive metal selected from technetium, rhenium, copper, yttrium, erbium, gallium and samarium form a chelate complex in which. the polysaccharide is in the form of microparticles. The radiopharmaceutical product of claim 1, wherein the linking groups are selected from the groups of formula: hydrogen atoms, saturated or unsaturated hydrocarbon groups, carboxyl groups or aromatic groups Θ is an aromatic nucleus optionally containing one or more heteroatoms and η is an integer between 1 and 5. The radiopharmaceutical product of claim 1, wherein the linking groups are selected from the groups of formula: s ³ = Ν NH - - c , = N --N --C \.-t .- · ·. · -r SCH, I c? CH, s ³ 3 . = N NH - - c . = ^N --NH - C NH - ^C 6 ^H 5 SCH ₃ ^ NHCH-j The radiopharmaceutical product according to any one of claims 1 to 3, wherein the polysaccharide is selected from natural starch, cellulose and cross-linked amylopectin. The radiopharmaceutical product according to any one of claims 1 to 5, wherein the microparticles have sizes between 0.01 and 100 µm. The radiopharmaceutical product according to any one of claims 1 to 5, wherein the linking groups represent from 0.1 to 50% by weight of the polysaccharide saccharide units. A radiopharmaceutical product according to any one of claims 1 to 6 in the form of a suspension of microspheres in a physiologically acceptable liquid or in a lyophilized form. Use of a radiopharmaceutical product according to any one of claims 1 to 6, wherein the radioactive metal for the preparation of the product for ^diagnostic purposes is ⁹⁹ⁿ¹ Tc or ⁶⁷ Ga. Use of a radiopharmaceutical product according to any one of claims 1 to 6, in which the radioactive metal for the preparation of the drug is rhenium-186 or 188, copper-64 or 67, yttrium 90, ebarium 169 or samarium 153. Use of a radiopharmaceutical product according to any one of claims 1 to 6, in which the radioactive metal for producing a product intended for pulmonary scintigraphy is ^99m Tc. A method of producing a radiopharmaceutical product according to any one of claims 1 to 6, characterized in that it comprises the following steps: (a) the polysaccharide is oxidized with periodate, b) the oxidized polysaccharide is reacted with a compound containing a primary amine functional group or a hydrazine of formula R-NH ₂ or R -— N -— ΝΗ ₂ X 1. ...; T <.. R 'in which R is a hydrocarbon or aromatic group containing at least one sulfur atom so as to covalently bond to in.4. the polysaccharide of the metal-binding groups of the formula R-NH-, R-N = or R-NH-N =, and R 'is a hydrogen atom or an alkyl group or a methyl group, *' c) the polysaccharide containing linking groups interacts with a radioactive metal salt selected from technetium, rhenium, copper, yttrium, erbium and samarium. A method according to claim 11, characterized in that the compound containing a primary amine functional group is of formula NH ₂ - (CH ₂ ) _n -SH, with η being an integer * υ 4 integer from 1 to 5 and including between steps B) and (c) an additional step of reducing this compound with sodium borohydride. A method according to claim 11, characterized in that the compound attached to the oxidized polysaccharide has one of the following formulas: NH, NH C SCH ₃ NH, NH C X ^X NHC H nh ₂ S N --C I ch ₃ NH ^ - NH S 'c SGH ₃ NHCH ₃ X 3Ί A method according to any one of claims 11 to 13, characterized in that the amount of binding groups fixed to the polysaccharide is controlled by controlling the degree of oxidation of the polysaccharide in step (a). 15. The method of claim 14, wherein the oxidation rate of the polysaccharide is from 10 to 50%. A method according to claim 14, characterized in that the amount of the linking groups is from 2 to 15%. A method according to any one of claims 10 to 16, characterized in that step (c) consists of contacting the microspheres of the polysaccharide, including linking groups, with a solution of pertechnetate ^99m TCO ₄ in the presence of a reducing agent. 18. Diagnostic kit that can be used for pulmonary scintigraphy, consisting of: a first flask containing a polysaccharide with linking groups attached to said polysaccharide by covalent bonds and selected from the groups of formula R-NH-, R-N = and R.-N .. „N = R 'in which R is a hydrocarbon or aromatic group containing at least one sulfur atom and where R' is a hydrogen atom or an alkyl or methyl group, where the polysaccharides are in the form of lyophilized microparticles or in suspension in a pharmaceutically acceptable liquid. The kit of claim 18, further comprising a second flask containing tin chloride in lyophilized form. The kit of claim 18, wherein the polysaccharide is in the form of lyophilized microparticles in the first flask, said first flask also containing lyophilized tin chloride.