JP2003508455A - Radiopharmaceutical products and methods for their preparation - Google Patents

Abstract

(57) Summary The present invention relates to radiopharmaceutical products and methods for their preparation. The products can be used for pulmonary scintigraphy or therapy. They are polysaccharides and the formulas R-NH-, R-N =, and the following formula: wherein R is a hydrocarbon or aromatic group comprising at least one sulfur atom, and R 'is A hydrogen atom, an alkyl group, for example, a methyl group], wherein the metal ion-blocking group forms a chelate-type complex with a radioactive metal, for example, technetium. I do. Embedded image

Description

Detailed Description of the Invention

The present invention relates to radiopharmaceutical products that can be used for diagnosis or therapy, and methods for their preparation. In particular, it relates to a radiopharmaceutical product formed from a suspension of radioisotope-labeled particles that can be used to establish a diagnosis when pulmonary embolism is suspected, especially for lung scintigraphy. In this application, the product is preferably in the form of particles and is in the form of particles, which are large, in the range 10-100 μm. In fact, since lung capillaries have a diameter of approximately 7 μm, the particles remain blocked in the capillaries after their intravenous infusion, which allows visualization of abnormalities of pulmonary blood perfusion.

Obviously, those products should meet a certain number of pharmaceutical regulations. In particular,
They are slow enough to allow visualization by a γ-ray camera for a minimum of about 1 hour, but also fast enough in vivo not to induce permanent destruction of pulmonary capillaries which causes small thrombosis. Should have an appropriate decomposition rate at. Furthermore, their products should not be toxic to organisms, should be sterilized by autoclaving or irradiation, should be easily labeled with radioactive metals, and should be in the form of a stable labeling kit. Should be able to be packaged.

Prior art : For example, the application of French patent FR-A-2273516, filed in 1975 by the PHARMACIA AKTIEBOLAG Company in Sweden, is reticulated with epichlorohydrin and for lung perfusion scintigraphy. Describes the use of amylopectin microspheres labeled by simple mixing with ^99m Tc. Those particles offer some inconvenience. In fact, the hydroxyl groups of the amylopectin used can allow the labeling of this mixture, but unfortunately they only form weak bonds with technetium and do not allow stable labeling. Furthermore, the preparation method described uses many solvents and emulsifiers which are difficult to remove from the particles prepared. Moreover, the exact rate of reticulation cannot be accurately measured or adjusted based on this particle type.

Furthermore, this document does not describe a kit that is compatible with normal use in nuclear medicine. In fact, for injectable preparations for humans, some manipulations are required, such as adding tin to the flask, centrifuging, resuspending the suspension, etc., which is not compatible with sterilization requirements. Finally, the resulting solution is unstable and the epichlorohydrin used for reticulation is recognized as being highly toxic and a mutagen. The present inventor has shown other drawbacks of those fine particles in Comparative Examples 1 and 2 below.

The application of French patent FR-A-2285857 filed in 1975 by PHARMACIA FINE CHEMICALA AB Company in Swedel is a polysaccharide linked to different sequestering agents and labeled with the aid of radioactive epitopes. It describes the use of particles. Said particles are such that the radioactive nucleus is mainly composed of at least 4, and preferably at least 5-8, ring nuclei and 2 metal coordination atoms, which have 5-6 groups encapsulating the metal. A chelating machine bound by a covalent bond bound in the form of a chelate-type complex.

Polysaccharides are polysaccharides that have been chemically reticulated, for example by epichlorohydrin or epibromohydrin. Aside from labeling, those particles are FR-A-22735.
It provides the same problems as previously mentioned for the particles described in No. 16. Furthermore, this document does not give any example of labeling with technetium. In addition, the labeling method comprises heating to 100 ° C. in the presence of radioactive elements, washing and labeling followed by drying, which method is completely incompatible with the labeling kit and the sterilization rules used.

Even though the labeling method described allows the labeling of particles in a relatively stable manner, it does not allow the preparation of a pharmaceutically acceptable labeling kit as it contains epichlorohydrin, and Make it unusable in nuclear medicine services. Therefore, the microspheres described in those two patent applications are not in compliance with pharmaceutical regulations and they cannot be utilized. Moreover, they have never been used for lung scintigraphy. This type of product has since been abandoned.

Much research that has been done since 1975 to improve new radiopharmaceutical products has focused on products based on albumin-serum and its derivatives. Their blood products correspond, in fact, to pharmaceutical regulations and can be used in particular for lung scintigraphy. They are the products currently used in nuclear medicine. For example, in 1975, in the document "Radiopharmaceuticals NY", 1975, p.267-281, MA Davis described radioactive particles intended for studies of pulmonary perfusion. The particles described in this document are radioiodinated serum albumin microaggregates ( ¹³¹ I-MAA) or technetium labeled denatured human serum albumin microspheres ( ^99m Tc-HAM). ^99m Tc-HAM microspheres are substantially 40 ~
Preferred due to their uniformity of particle size of 50 μm. In addition, this document describes the general characteristics required for such radiopharmaceutical particles.

G. Guiraud “Macro-aggregates and radioactive microspheres”, Radiophar
The paper of maceuticals, 1997, 519 describes micro-aggregates (MAA) of albumin and microspheres of human serum albumin. It describes the labeling of such micro-aggregates and microspheres with technetium 99m by a solution of stannous chloride. It is also noted that the optimal size for microspheres is 15 ± 5 μm.
It refers to organic microspheres of starch.

Currently, ^99m Tc labeled human serum albumin macro-aggregates and microspheres are by far the most utilized in nuclear medicine. For example, the availability and quality of a range of human albumins makes it difficult to prepare diagnostic kits containing particles that vary in size and number. However, one of the major inconveniences is HIV, pneumonia or Creutzfeld-Ja.
They are of human origin with severe problems of potentially fatal contamination of cob disease.

It is therefore of great interest to have ^99m Tc-labeled microspheres which are not of human origin, in order to ensure complete safety. In this regard, AC Perkins, Nuclecar Medicine Communications, 1999
The most recent papers, 20, 20, 1-3 describe means for displacing radiopharmaceutical products obtained from blood. In particular, it refers to the use of recombinant materials, synthetic polymers and polypeptides. However, this document does not mention polysaccharides.

Description of the invention : The object of the present invention is to show the very good lung acquisition shown by the inventor for rats (
captation) can be easily labeled with ^99m Tc, easily biodegradable,
By providing a radiopharmaceutical product that can be easily sterilized and packaged as an easy kit for labeling, and by meeting the pharmaceutical regulations for this type of product, the inconvenience mentioned above in the prior art To overcome that. Their advantages and others will be apparent from the following description.

The radiopharmaceutical product of the invention, which comprises a polysaccharide, is covalently attached to the polysaccharide and has the formula R-NH-, R-N =, and the following formula:

[Chemical 8]

[Wherein R is a hydrocarbon or aromatic group comprising at least one sulfur atom, and R ′ is a hydrogen atom, an alkyl group, for example a methyl group] Characterized by having a sequestering group selected, wherein the sequestering group forms a chelate-type complex with a radioactive metal selected from technetium, rhenium, copper, yttrium, erbium, gallium and samarium. Be attached.

The alkyl groups that can be used for R ′ can be linear or branched and preferably have 1 to 5 carbon atoms. According to the invention, the polysaccharide can be soluble or present in the form of microspheres. According to the invention, the polysaccharide is a natural starch, cellulose or reticulate.
d) It may be selected from amylopectin. The natural starch may be corn starch. The polysaccharide can be present in the form of microparticles, for example in the form of microspheres.

The inventors have also shown that the modified celluloses of the invention provide very good lung acquisition and a slower elimination rate than starch. Therefore, the modified celluloses of the present invention can also be used for radiotherapy by labeling with rhenium, copper or the metals mentioned above. Because it addresses the need for radiotherapy with microparticles that have longer half-lives. According to the present invention, the sequestering group has the following formula:

[Chemical 9] [In the formula, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are independently a hydrogen atom, a saturated or unsaturated hydrocarbon group, a carboxyl group or an aromatic group,

[Chemical 10] Is an aromatic nucleus which may contain one or several heteroatoms, and n is an integer from 1 to 5].

[0017]   For example, they have the following formula:

[Chemical 11] Is selected from the groups represented by

According to the invention, the microparticles in the form of microspheres are 0.01 to 100 μm, preferably 10 to 50 μm for diagnosis by lung scintigraphy, and 0.1 to 5 μm for therapy.
Can be of any size. According to the invention, the level of sequestration can be 0.1 to 50%, preferably 2 to 15% compared to the sugar pattern in the polysaccharide. According to the invention, in the radiopharmaceutical product, especially when it is used for diagnosis, the radiometal may be ^99m Tc or gallium-67.

This is the case when the radiopharmaceutical product is used for lung scintigraphy. According to the invention, in the radiopharmaceutical product, especially when it is used for therapy, the radiometal is rhenium-186 or 188, copper-64 or 67, yttrium-90, erbium-169 or samarium-. It can be 153. According to the invention, the radiopharmaceutical product can be present in a physiologically acceptable liquid, in the form of a suspension of microspheres or in lyophilized form.

The invention also provides a process for the preparation of the radiopharmaceutical product of the invention, wherein said process comprises the following steps: (a) a polysaccharide, such as those mentioned above. Subject to periodate-controlled oxidation, (b) primary amine function or formula R-NH ₂ or formula:

[0021]

[Chemical 12]

[Wherein R is a hydrocarbon comprising at least one sulfur atom for covalently linking the sequestering group of formula R-NH-, RN = or R-NH-N = to the polysaccharide or An aromatic group, and R'is a hydrogen atom, or an alkyl or methyl group], and a compound containing hydrazine represented by the formula (c) containing the sequestering group. The polysaccharide consisting of technetium, rhenium,
Reacting with a salt of a radioactive metal selected from copper, yttrium, erbium, and samarium.

Periodate regulated oxidation has particular application to the oxidation of starch, dextran or cellulose, CL Mehltretter, “Methods in Carbohydrate Chemis.
... try ", Vol IV , may be oxidized as described in 1964 which may, for example according to the present invention used in the examples given below, the compound containing a primary amine function has the formula NH ₂ - (CH ₂ ) n−SH (
Where n is an integer of 1-5), and between steps (b) and (c) there may be included a supplementary step of reducing this compound with sodium borohydride.

[0024]   According to the present invention, the compound attached to the polysaccharide may be, for example, of the formula:

[Chemical 13] It is possible to correspond to one formula shown by.

According to the present invention, the level of sequestering groups immobilized on the polysaccharide can be adjusted by adjusting the level of oxidation of the polysaccharide in step (a) mentioned above. This level of oxidation of the polysaccharide can be 10-50%. For example, the level of sequestering groups can be 2-15%. Therefore, in order to allow labeling of the polysaccharides of the invention with ^99m Tc, we used a two-step conversion method. The method may be provided as comprising the controlled oxidation of the polysaccharide with periodate in the first step. Thus, each unit of oxidized glucose produces two aldehyde groups at adjacent positions according to the chemistry given below.

[0026]

[Chemical 14]

The level of oxidation of polysaccharides is variable and can be easily regulated. In fact, the yield of this oxidation reaction is close to 100%, and the level of oxidation can be calculated by the amount of periodate added. Generally, oxidation levels below 50% are used to only slightly modify the structure of macromolecules. True oxidation levels in the range of 1-100% can be easily determined by colorimetric methods. In the second step, the oxidized polysaccharide is reacted with a molecule containing an amine or hydrazine group having the general formula RNH ₂ or RNHNH ₂ to form a chelating group capable of sequestering technetium. Therefore, a Schiff base type ligand or thiosemicarbozone can be obtained.

[0028]   This second stage can be summarized as follows:

[Chemical 15]

Here, 1. R = NR ₁ (C = S) SNR ₂ (Schiff base obtained from dithiocarbazate), R = NR ₁ (C = S) NR ₂ R ₃ (thiosemicarbazone), 3. R = aromatic group (aromatic Schiff base), 4. R = alkyl group (alkyl Schiff base); in this case the Schiff base is unstable and to stabilize it undergoes a second reduction step of the C = N bond with borohydride, and then an amine A C-NHR bond is obtained.

According to the invention, the step (c) is a microsphere of a polysaccharide comprising the sequestering groups, pertechnetate ^99m TcO ₄ ^- of a solution, a reducing agent, for example stannous chloride Contacting in the presence of. According to the invention, microparticles, such as microspheres, such as corn starch or reticulated amylopectin starch, are thus oxidized and then converted into molecules containing amine or hydrazine functions, such as S-methyldithiocarbazate. Can be combined. Those particles modified by this means can be easily labeled with eg ^99m Tc.

Accordingly, the present invention specifically provides microparticles prepared from starch particles that do not provide the inconvenience of albumin referred to above. In addition, starch is described as a pharmaceutically pharmaceutically excipient. It is readily available and low cost. The microparticles of the invention also have the advantage that they can be easily sterilized by irradiation and can be processed in the form of easy kits for labeling. Furthermore, the present inventors have shown in accordance with the present invention that the rate of lung clearance can be modified according to the level of oxidation of the microparticles used in the present invention, which is not possible with, for example, human albumin microspheres.

Another advantage of the present invention is the simplicity of the process operation; very simple reaction conditions; reaction in aqueous medium at ambient temperature; semi-quantitative yield. Furthermore, sequestration reactions with eg technetium are quantitative; they are necessary due to the sterilization and simplicity of preparation necessary for the use of technetiumized kits in room and in hospital settings. Occurs without final purification to allow good compatibility. The invention also provides diagnostic kits that can be used, for example, for lung scintigraphy. This kit is covalently attached to a polysaccharide and has the formula R-NH-, R
-N = and the following formula:

[0033]

[Chemical 16]

Wherein R is a hydrocarbon or aromatic group comprising at least one sulfur atom and R ′ is a hydrogen atom, an alkyl group such as a methyl group It comprises a first flask containing a polysaccharide which is provided with a sequestering group of choice. According to the invention, the polysaccharide can be present, for example, in the form of microparticles, for example in the form of microspheres, said microparticles being in lyophilized form or in suspension in a pharmaceutically acceptable liquid. Can exist in a form.

The kit of the invention further comprises a second flask containing stannous chloride, preferably in lyophilized form, or the polysaccharide is in lyophilized form, eg in the form of microparticles. The first flask, if present in a flask, can further include lyophilized stannous chloride. The kit of the present invention is stable for at least 12 months, as shown in the example below. Thus, the radiopharmaceutical product of the present invention provides all amounts needed for use, eg radiopharmaceutical use, eg for scintigraphy of lung perfusion, or for radiotherapy. Other advantages will also become apparent upon reading the following examples relating to the present invention.

[0036] Example Example 1. A suspension of 10 g of corn starch from the Pharmacopoeia, pre-sieved on a 10-40 μm sieve, is prepared, containing about 10% water, ie 0.055 mol glucose in 100 ml water. Add 0.0168 mol, or 3.6 g of sodium periodate (0.3 equivalents) (NaIO ₄ ) dissolved in 100 ml of water. The suspension is then stirred at room temperature for 18 hours. The solution is filtered and the oxidized starch is taken up with 20 ml of water 5 times and with acetone.
Rinse twice with 50 ml. Vacuum dried starch and 10g oxidized starch at 30%
(Yield = 100%).

A suspension of 10 g of oxidized starch at 30% was added to 60% water / ethanol (2: 1 by volume).
Prepare in ml. Next, 0.1 equivalent (0.1 x 0.055 x 2), or 0.011 mol (1
.34G) of S- methyl dithiocarbazate the _{(NH 2 NH (C = S} ) SCH 3) (M = 122), dissolved in ethanol 10 ml. The suspension is stirred at room temperature for 18 hours. Then the solution is filtered and the denatured starch is washed 3 times with 20 ml of ethanol,
Then, vacuum drying is performed.

Thus, about 10 g of modified starch is obtained. 5 powder assay by elemental analysis
It gives a sulfur content of .4%, which is 7% of S-methyldithiocarbazate (DTC
Z) corresponding to the coupling level (7 units of dithiocarbazate for 100 theoretical aldechts, ie 14 dithiocarbazate units for 100 glucose units). Therefore, the coupling yield is 70%. Thus, 10 g of starch which is oxidized at 30% and coupled to DTCZ at 7% is obtained.

Labeling reaction with ^99m Tc : 10 mg of the starch thus modified is introduced into a penicillin type flask. Then 4 ml physiological serum, then 10 μg SnCl ₂ · 2H ₂ O (0.5 mg / ml in 0.1 N HCl).
20 μl of the solution in 1.) are added. Next, ^99m TcO4 ^- adding a solution 1ml of (5mC). The solution was stirred for 15 minutes and the radiochemical purity control (RCP) was adjusted to 0.22 μm Mill.
This is done by filtering 1 ml of the above solution on an ipore filter, then rinsing the filter with 2 ml of physiological serum. The labeled microspheres are retained on the filter as opposed to radioactive impurities not found in the microspheres that are found in the filtrate. Its radiochemical purity corresponds to: RCP = (activity on filter / total activity) × 100. That is 98.9%.

Example 2. Starch modification : Perform as in Example 1. However, 0.2 equivalent of periodate is used during the oxidation reaction. 2
0% oxidized starch is obtained. For the coupling reaction, proceed as in Example 1 and obtain a starch that is 20% oxidized and 7% coupled to DTCZ. Labeling reaction with ^99m Tc : Proceed as in Example 1. Radiochemical purity (PCP) is 99%.

Example 3. Starch modification : Perform as in Example 1. However, 0.1 equivalent of periodate is used during the oxidation reaction. 1
0% oxidized starch is obtained. For the coupling reaction, proceed as in Example 1 and obtain a starch that is 10% oxidized and 7% coupled to DTCZ. Labeling reaction with ^99m Tc : Proceed as in Example 1. The radiochemical purity (PCP) is 98.8%.

Example 4. Proceed as in Example 1 to obtain 10 g of 30% oxidized starch. Water / ethanol (
A suspension of 10 g of starch which has been oxidized at 30% is prepared in 60 ml of a mixture (2/1 volume). Next, 0.1 equivalent (0.1 × 0.055 × 2), that is, 0.011 mol (1.50 g) of N-methyl-S-methyldithiocarbazate (NH ₂ N (CH ₃ ) (C = S) SCH ₃ ) (M = 136), ethanol 1
Dissolve in 0 ml. The suspension is stirred at room temperature for 18 hours. Then the solution is filtered and the modified starch is washed 3 times with 20 ml of ethanol, and then
Vacuum dry.

Thus, about 10 g of modified starch is obtained. The powder assay by elemental analysis is
It gives a sulfur content of 5%, which corresponds to a coupling level of N-methyl S-methyldithiocarbazate of 6.5% (6.5 units of dithiocarbazate for 100 theoretical aldehydes, ie 13 dithiocarbabeat units work for 100 glucose units). Therefore, the coupling is 65%. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 95%.

Example 5. Starch modification : Proceed as in Example 1 to obtain 10 g of starch which has been oxidized at 30%. Water / ethanol (
A suspension of 10 g of starch which has been oxidized at 30% is prepared in 60 ml of a mixture (2/1 volume). Next, 0.1 equivalent (0.1 × 0.055 × 2), that is, 0.011 mol (1.83 g) of 4-phenyl 3-
Thiosemicarbazide (NH ₂ N) (C = S) NH (C ₆ H ₅ ) (M = 167) is dissolved in 10 ml of ethanol. The suspension is stirred at room temperature for 18 hours.

The solution is then filtered and the modified starch is triturated with 20 ml of ethanol.
Wash once and then vacuum dry. Therefore, about 10 g of modified starch is obtained. The powder assay by elemental analysis gives a sulfur content of 3.16%, which is 8%.
Corresponding to the coupling level of 4-phenyl 3-thiosemicarbazone of (100
8 units of thiosemicarbazide for the theoretical aldehyde, ie 16 units of thiosemicarbazone for 100 glucose units). Therefore, the coupling is 80%. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 98%.

Example 6. Starch modification : Proceed as in Example 1 to obtain 10 g of starch which has been oxidized at 30%. Water / ethanol (
A suspension of 10 g of starch which has been oxidized at 30% is prepared in 60 ml of a mixture (2/1 volume). Next, 0.1 equivalent (0.1 × 0.055 × 2), that is, 0.011 mol (1.15 g) of 4-methyl 3-thiosemicarbazide (NH ₂ NH) (C = S) NH (CH ₃ ) (M = 105), Dissolve in 10 ml of ethanol. The suspension is stirred at room temperature for 18 hours.

The solution is then filtered and the modified starch is triturated with 20 ml of ethanol.
Wash once and then vacuum dry. Therefore, about 10 g of modified starch is obtained. The powder assay by elemental analysis gives a sulfur content of 2.9%, which is 7.3%.
Corresponding to the coupling level of 4-methyl 3-thiosemicarbazone (7.3 units of thiosemicarbazide for 100 theoretical aldehydes, or 14.6 thiosemicarbazone units for 100 glucose units). Therefore, the coupling is 73%. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 97%.

Example 7. Starch modification : Proceed as in Example 1 to obtain 10 g of starch which has been oxidized at 30%. Water / ethanol (
A suspension of 10 g of starch which has been oxidized at 30% is prepared in 60 ml of a mixture (2/1 volume). Next, 0.1 equivalent (0.1 × 0.055 × 2), that is, 0.011 mol (1.30 g) of 4,4-dimethyl-3-thiosemicarbazide (NH ₂ NH) (C = S) N (CH ₃ ) ₂ (M = 119 ), Ethanol 10m
dissolves in l.

The suspension is stirred for 18 hours at room temperature. Then the solution is filtered and the modified starch is washed 3 times with 20 ml of ethanol and then vacuum dried.
Therefore, about 10 g of modified starch is obtained. 3% for powder assay by elemental analysis
A sulfur content of 7.5% corresponding to a coupling level of 4,4-dimethyl-3-thiosemicarbazone of 7.5% (7.5 units of thiosemicarbazide for 100 theoretical aldehydes, ie 100 glucose units). Against 15 thiosemicarbazone units work). Therefore, the coupling is 75%. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 96%.

Example 8. Starch modification : Proceed as in Example 1 to obtain 10 g of starch which has been oxidized at 30%. Water / ethanol (
A suspension of 10 g of starch which has been oxidized at 30% is prepared in 60 ml of a mixture (2/1 volume). Next, 0.1 equivalent (0.1 × 0.055 × 2), that is, 0.011 mol (1.44 g) of 4-allyl-3-thiosemicarbazide (NH ₂ NH) (C = S) NH (CH ₂ CH = CH ₂ ) (M = 131), 10 ml of ethanol
Dissolve in. The suspension is stirred at room temperature for 18 hours. Then the solution is filtered,
The modified starch is then washed 3 times with 20 ml of ethanol and then vacuum dried.

Thus, about 10 g of modified starch is obtained. Powder assay by elemental analysis 3
It gives a sulfur content of%, which corresponds to 7.5% 4-allyl-3-thiosemicarbazone (7.5 units of thiosemicarbazide for 100 theoretical aldehydes, ie 15 thios for 100 glucose units). Semicarbazone units work). Therefore, the coupling is 75%. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 98%.

Example 9. Starch modification : Proceed as in Example 1 to obtain 10 g of starch which has been oxidized at 30%. Water / ethanol (
A suspension of 10 g of starch which has been oxidized at 30% is prepared in 60 ml of a mixture (2/1 volume). Next, 0.1 equivalent (0.1 × 0.055 × 2), ie 0.011 mol (1 g) of 3-thiosemicarbazide (NH ₂ NH) (C = S) NH ₂ (M = 91) is dissolved in 10 ml of ethanol. The suspension is stirred at room temperature for 18 hours.

The solution is then filtered and the modified starch is triturated with 20 ml of ethanol.
Wash once and then vacuum dry. Therefore, about 10 g of modified starch is obtained. The powder assay by elemental analysis gives a sulfur content of 2.9%, which is 7.3%.
Corresponding to 3-thiosemicarbazone (7.3 units of thiosemicarbazide for 100 theoretical aldehydes, or 14.6 thiosemicarbazone units for 100 glucose units). Therefore, the coupling is 73%. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 95%.

Example 10. Starch modification : Proceed as in Example 1 to obtain 10 g of starch which has been oxidized at 30%. Water / ethanol (
A suspension of 10 g of starch which has been oxidized at 30% is prepared in 60 ml of a mixture (2/1 volume). Next, 0.1 equivalent (0.1 × 0.055 × 2), that is, 0.011 mol (1.37 g) of 2-aminothiophenol (C ₆ H ₄ ) (NH ₂ ) (SH) (M = 125) was dissolved in 10 ml of ethanol. To do. The suspension is stirred at room temperature for 18 hours.

The solution is then filtered and the modified starch is triturated with 20 ml of ethanol.
Wash once and then vacuum dry. Therefore, about 10 g of modified starch is obtained. The powder assay by elemental analysis gives a sulfur content of 3%, which is 7.5%.
Corresponding to 3-thiosemicarbazone (7.5 units of aminothiophenol for 100 theoretical aldehydes, or 15 aminothiophenol units for 100 glucose units). Therefore, the coupling is 75%. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 94%.

Example 11. Starch modification : Proceed as in Example 1 to obtain 10 g of starch which has been oxidized at 30%. Water / ethanol (
A suspension of 10 g of starch which has been oxidized at 30% is prepared in 60 ml of a mixture (2/1 volume). Next, 0.1 equivalent (0.1 × 0.055 × 2), that is, 0.011 mol (1 g) of 2-mercaptoethylamine (or 2-aminoethanethiol) (NH ₂ CH ₂ CH ₂ SH) (M = 91) was added to 10 ml of ethanol. Dissolve in. The suspension is stirred at room temperature for 18 hours. Then, in order to reduce the Schiff base that is formed to stabilize it, sodium borohydride (
NaBH4) 0.015 mol is added (non-aromatic Schiff bases are not stable) and it is allowed to react for 1 hour.

The solution is then filtered and the modified starch is triturated with 20 ml of ethanol.
Wash once and then vacuum dry. Therefore, about 10 g of modified starch is obtained. The powder assay by elemental analysis gave a sulfur content of 3.4%, which was 8.5%.
Corresponding to 2-mercaptoethylamine (8.5 for 100 theoretical aldehydes
Unit of 2-mercaptoethylamine, ie 17 per 100 glucose units
2-Mercaptoethylamine units work). Therefore, the coupling is 85%
Is. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 95%.

Example 12. Starch modification : Proceed as in Example 1 to obtain 10 g of starch which has been oxidized at 30%. Water / ethanol (
A suspension of 10 g of starch which has been oxidized at 30% is prepared in 60 ml of a mixture (2/1 volume). Next, 0.1 equivalent (0.1 × 0.055 × 2), that is, 0.011 mol (1.27 g) of 2-amino-4-mercaptotriazole (C ₄ N ₂ H) (SH) (NH ₂ ) (M = 116) was added to ethanol. Dissolve in 10 ml. The suspension is stirred at room temperature for 18 hours.

The solution is then filtered and the modified starch is triturated with 20 ml of ethanol.
Wash once and then vacuum dry. Therefore, about 10 g of modified starch is obtained. The assay of the powder by elemental analysis gave a sulfur content of 2.8%, which corresponds to 7% of 2-amino-4-mercaptotriazole (7 units of mercaptotriazole for 100 theoretical aldehydes, i.e. 100 14 per glucose unit
The mercaptotriazole unit works). Therefore, the coupling is 75%. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 85%.

[0060]Comparative Example 1.   Using 10 g of screened corn that has not undergone chemical conversion,
The same method as in example 1,^99mLabel it by Tc.   RCP is 19%.   This example shows that the chemical modification (immobilization of sequestering groups) carried out according to the invention is ^99m A good illustration of the fact that is certainly needed to enable labeling by Tc
Is. In addition, the fine particles, in contrast to the products according to the invention, are not subject to conventional chemical transformations.
If labeled without, it is not possible to obtain permanent lung acquisition. Obey
Thus, those results are inconsistent with the results described in FR-A-2273516.

Example 13. Cellulose modification : Proceed as in Example 1. However, cellulose that has been sieved to 10 to 40 μm is used. Therefore, 10 g of cellulose are obtained which is oxidized at 30% and coupled to DTCZ at 7%. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 99.1%.

Example 14 Sprague Dawley rats weighing approximately 200 g are anesthetized with sodium thiopental and injected intravenously with different solutions of ^99m Tc-labeled microspheres according to Examples 1-9 and 13. Individual animals have 0 in the penile vein, which is 0.2 mc per animal
Receive .2 ml of solution. The animal is then placed under the γ-ray camera and a continuous still image is shot for 3 hours. Therefore, after obtaining 15,000 shots per image, a continuous image is obtained. The regions of interest are then defined manually to assess the activity present in different organs 15 minutes after injection. The results are given in Table 1 below.

[0063]

[Table 1]

It is therefore noted that denatured microspheres show very good lung acquisition. Furthermore, the oxidation levels (oxidation levels 30, 20 and 10% as shown in Examples 1, 2 and 3)
), The rate of lung elimination can be adjusted. The use of cellulose makes it possible to considerably slow down the rate of elimination (Example 10, half-life> 4 hours).

Comparative Example 2. In this example, no native starch is used, but the microspheres are FR-A-227.
Prepare from amylopectin reticulated with epichlorohydrin as in 3516. Preparation of starch reticulated microspheres : 8 g of corn amylopectin are dissolved in 40 ml of a solution containing 4 g of NaOH and 0.15 g of sodium borohydride. The amylopectin is left for 24 hours to dissolve. Next, soy lecithin 1.6 dissolved in 60 ml fluid paraffin and 4 ml hexane.
An emulsion is prepared by stirring g at 800 rpm. Then an aqueous phase containing amylopectin and then 3.2 ml epichlorohydrin are added.

The emulsion is heated to 55 ° C. for 4 hours and then left overnight with stirring. The resulting microspheres of about 50 μm size were triturated with 250 ml of acetone.
It is washed once, dried and then freeze-dried. Labeling reaction with ^99m Tc : Proceed as in Example 1. However, 1 mg of SnCl ₂ · 2H ₂ O is used. RCP is 90%.

Starch modification Proceed as in Example 1. However, 10 g of amylopectin microspheres reticulated with epichlorohydrin prepared beforehand are used. Thus, 10 g of amylopectin microspheres oxidized at 30% and coupled to 7% DTCZ are obtained. Labeling reaction with ^99m Tc : Proceed as in Example 1. RCP is 99%.

Example 15. To test the reticulated amylopectin microspheres labeled with ^99m Tc of Comparative Example 2, the same procedure is followed as in Example 14. The results obtained are given in Table II below.

[0069]

[Table 2]

[0070] Contrary to the description in Patent FR-A-2273516, not chemically modified, microspheres reticulation amylopectin has been labeled with ^99m Tc, but the ^99m Tc and microspheres Note, undoubtedly, does not provide any lung acquisition due to the weak binding between. On the other hand, those microspheres chemically transformed by the method of the invention show good lung acquisition.

Example 16. Starch microspheres prepared as in Example 1 (oxidized at 30% and DTCZ at 7%)
Lead powder) was used for the manufacture of a sterile labeling kit and is ready for labeling with ^99m Tc. Microsphere sterilization : 10 g of microspheres are introduced into a crimped flask and then irradiated with a cobalt-60 source. Microspheres receive a total dose of 25 kGy of gamma radiation over 20 hours.

Preparation of the kit : 200 mg of sterilized microspheres are introduced by aseptic means into a reactor containing 20 ml of NaCl (9/1000). The solution is degassed by nitrogen bubbling and then 400 μl of a sterile solution of SnCl ₂ .2H ₂ O at 0.5 mg / ml in 0.1 N HCl is added. Divide 1 ml of solution for each of the 20 flasks, then freeze-dry them and place under a nitrogen atmosphere. Therefore, each flask contains the following components: 10 mg denatured microspheres, 10 μg SnCl ₂ .2H ₂ O, 9 mg NaCl.

[0073] Labeling reaction with ^99m Tc: TcO ₄ of 5 ml ^- a (5mC) solution was added to the flask in a lyophilized form, and 15 minutes, it reacted. Proceed as in Example 1. RCP is 98.7%. Kit Stability Test : Labeling kits prepared as described above are stored at different temperatures and the labeling reaction with ^99m Tc is tested to assess their stability. The results obtained are given in Table III below:

[0074]

[Table 3] It shows a very high stability of the kit stored at a temperature of 2-8 ° C.

Example 17. Sprague Dawley rats weighing approximately 200 g are anesthetized with sodium thiopental and injected intravenously with different solutions of ^99m Tc-labeled microspheres according to Example 14. Each animal receives 0.2 ml of solution in the penile vein, which is 0.2 mc per animal. The animals are then killed 15 minutes after injection. The various organs are then allowed to recover, the measurements of radioactivity present in individual organs are counted, and thus the% of activity present in individual organs is determined. The results are given in Table IV below:

[0076]

[Table 4] % Of dose injected 15 minutes after injection:

Thus, for native starch and reticulated starch-based microspheres, there is a weak acquisition in other organs, indicating a very high lung acquisition. Thus, the sterile product prepared under kit form would be fully adapted for use as a radiopharmaceutical product for pulmonary perfusion that replaces albumin and for radiotherapy.

Example 18. As in Example 13, using cellulose that has been oxidized at 30% and coupled with DTCZ at 7%. The cellulose modified in this way is labeled with rhenium 186 to show the utility of the support of the invention for therapy.

Labeling reactivity with Re186 : 10 mg of modified cellulose are introduced into a penicillin type flask. Then 2 ml of physiological serum and then 20 mg of citric acid and finally 1 mg of SnCl ₂ .2H ₂ (0.
100 μl of a solution at 10 mg / ml in 1N HCl) is added. Next, ReO ₄ corresponds to the activity of 2MC ^- the solution Solution 0.1 ml, is added to the contents of the flask. The flask is heated in a water bath at 100 ° C for 30 minutes. Radiochemical purity (RCP
) Is determined by filtration on Millipore as in Example 1.

RCP = 92%. To confirm the stability of the bond between rhenium 186 and cellulose microspheres, an in vitro stability test is performed. The mixture is incubated with HAS (human serum albumin) at 20 mg / ml at 37 ° C. I get the following result:

[0081]

[Table 5]

Therefore, the results show a very high stability of the labeling of cellulose microspheres by rhenium 186 and of the microspheres themselves with respect to HAS. Those skilled in the art will readily understand that those results are extrapolated based on the utilization of rhenium 188. Example 19. Sprague Dawley rats weighing approximately 200 g are anesthetized and then injected with 0.2 ml (0.1 mc) of Re186 labeled cellulose microspheres solution as in Example 18. The animal is then placed under a gamma camera and images are recorded for 48 hours.

[0083]   The activity present in the region of interest is then calculated as in Example 14.

[Table 6]

Therefore, the results show that the activity remains blocked at the lung level for at least 48 hours. Therefore, this type of microsphere can be used for therapy. The most important clinical application may be the treatment of liver cancer after direct injection into the hepatic artery rather than intravenously (metabolic radiation therapy). Another possible application is the subcutaneous injection of particles of this type into breast cancer. Particles migrating through the lymphatic system are used by alarming lymph nodes (
processing of sentinel nodes) can be enabled.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] October 23, 2001 (2001.10.23)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

[Chemical 1] [Wherein R is a hydrocarbon or aromatic group containing at least one sulfur atom, and R'is a hydrogen atom, an alkyl or a methyl group], a metal ion selected from the group A radiopharmaceutical preparation having a sequestering group, wherein the sequestering group forms a chelate-type complex with a radioactive metal selected from technetium, rhenium, copper, yttrium, erbium, gallium and samarium. object.

[Chemical 2] [In the formula, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are independently a hydrogen atom, a saturated or unsaturated hydrocarbon group, a carboxyl group or an aromatic group,

[Chemical 3] Is an aromatic nucleus which may contain one or several heteroatoms, and n is an integer from 1 to 5]. .

[Chemical 4] The radiopharmaceutical product according to claim 2, which is selected from the group represented by:

[Chemical 5] Wherein R is at least one sulfur atom for covalently bonding the sequestering group of formula R-NH-, RN = or R-NH-N = to the hydrocarbon, aromatic or aromatic group. A hydrogen atom, or R'is a hydrogen atom, or an alkyl or methyl group], and reacts a compound containing hydrazine represented by the formula (c) with the sequestering group. Polysaccharide, technetium, rhenium,
Reacting with a salt of a radioactive metal selected from copper, yttrium, erbium, and samarium;

[Chemical 6] 12. The method according to claim 11, corresponding to one of:

[Chemical 7] [Wherein R is a hydrocarbon or aromatic group containing at least one sulfur atom, and R'is a hydrogen atom, an alkyl or a methyl group], a metal ion selected from the group A kit comprising a first flask containing a polysaccharide in the form of lyophilized microparticles, which is provided with a blocking group, or in suspension in a pharmaceutically acceptable liquid.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventors Deniso and Benowa             France, F-49000 Angers, Bou             Le Bar Ugen Showman 79 F-term (reference) 4C076 CC27 EE30A EE38A EE59A                       FF63 FF68                 4C084 AA12 NA05 NA10 NA13 ZB261                       ZB262                 4C085 HH03 KA09 KA10 KB07 KB09                       KB10 KB11 KB12 LL09 LL18

Claims (20)

[Claims] 1. A radiopharmaceutical product comprising a polysaccharide in the form of microparticles, covalently attached to the polysaccharide and having the formula R-NH-, R-N = and the following formula: 1] [Wherein R is a hydrocarbon or aromatic group containing at least one sulfur atom, and R'is a hydrogen atom, an alkyl or a methyl group], a metal ion selected from the group A radiopharmaceutical preparation having a sequestering group, wherein the sequestering group forms a chelate-type complex with a radioactive metal selected from technetium, rhenium, copper, yttrium, erbium, gallium and samarium. object. 2. The sequestering group has the following formula: [Wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are independently a hydrogen atom, a saturated or unsaturated hydrocarbon group, a carboxyl group or an aromatic group, and Is an aromatic nucleus which may contain one or several heteroatoms, and n is an integer from 1 to 5]. . 3. The sequestering group is represented by the following formula: The radiopharmaceutical product according to claim 2, which is selected from the group represented by: 4. The radiopharmaceutical product according to claim 1, wherein the polysaccharide is selected from natural starch, cellulose and reticulated amylopectin. 5. The radiopharmaceutical product according to claim 1, wherein the microparticles have a size of 0.01 to 100 μm. 6. The radiopharmaceutical product according to claim 1, wherein the level of the sequestering group is 0.1 to 50% with respect to the sugar pattern of the polysaccharide. 7. Use of a radiometal according to any one of claims 1 to 6 for preparing a product intended for the diagnosis of a radiopharmaceutical product, which is ^99m Tc or ⁶⁷ Ga. 8. The radioactive metal is rhenium-186 or 188, copper-64 or 67.
, Yttrium 90, erbium 169 or samarium 153. Use of the radiopharmaceutical product according to any one of claims 1 to 6 for the preparation of a medicament. 9. Use of a radiopharmaceutical product according to any one of claims 1 to 7 for the preparation of a product intended for pulmonary scintillation, wherein the radiometal is 99mTc. 10. Radiopharmaceutical product according to claim 1, which is present in the form of a suspension of microspheres or in a lyophilized form in a physiologically acceptable liquid. 11. A method for the preparation of a radiopharmaceutical product according to any one of claims 1 to 6, comprising the steps of: (a) subjecting the polysaccharide to oxidation carried out by periodate. (B) primary amine functional group or formula R-NH ₂ or the following formula: Wherein R is at least one sulfur atom for covalently bonding the sequestering group of formula R-NH-, RN = or R-NH-N = to the hydrocarbon, aromatic or aromatic group. A hydrogen atom, or R'is a hydrogen atom, or an alkyl or methyl group], and reacts a compound containing hydrazine represented by the formula (c) with the sequestering group. Polysaccharide, technetium, rhenium,
Reacting with a salt of a radioactive metal selected from copper, yttrium, erbium, and samarium; 12. The compound containing a primary amine functional group has the formula NH ₂ — (CH ₂ ) n—S
Corresponding to H (where n is an integer of 1-5) and comprising between step (b) and (c) a supplementary step of reducing this compound with sodium borohydride. The method according to item 11. 13. The compound bound to the oxidized polysaccharide has the following formula: 12. The method according to claim 11, corresponding to one of: 14. The level of sequestrant groups immobilized on the polysaccharide is adjusted by adjusting the level of oxidation of the polysaccharide in step (a).
The method according to any one of 11 to 13. 15. The method of claim 14 wherein the polysaccharide has an oxidation level of 10-50%. 16. The level of sequestering groups is 2-15%.
The method described. 17. The step (c) comprises contacting a polysaccharide microsphere comprising a sequestering group with a solution of pertechnetate ^99m TcO ₄ ⁻ in the presence of a reducing agent. Item 17. A method according to any one of items 16 to 16. 18. A diagnostic kit that can be used for lung scintigraphy, wherein the polysaccharide is covalently attached to a polysaccharide and has the formula R-NH-, RN = and the following formula: embedded image [Wherein R is a hydrocarbon or aromatic group containing at least one sulfur atom, and R'is a hydrogen atom, an alkyl or a methyl group], a metal ion selected from the group A kit comprising a first flask containing a polysaccharide in the form of lyophilized microparticles, which is provided with a blocking group, or in suspension in a pharmaceutically acceptable liquid. 19. The kit of claim 18 which also comprises a second flask containing stannous chloride in lyophilized form. 20. The kit of claim 18, wherein the polysaccharide is present in the first flask in the form of lyophilized microspheres, the first flask also containing lyophilized stannous chloride.