WO2009094741A1 - Radiopharmaceutical drug and pharmaceutical composition for the treatment and diagnosis of cancer - Google Patents

Radiopharmaceutical drug and pharmaceutical composition for the treatment and diagnosis of cancer Download PDF

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WO2009094741A1
WO2009094741A1 PCT/BR2009/000039 BR2009000039W WO2009094741A1 WO 2009094741 A1 WO2009094741 A1 WO 2009094741A1 BR 2009000039 W BR2009000039 W BR 2009000039W WO 2009094741 A1 WO2009094741 A1 WO 2009094741A1
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mag
pharmaceutical compositions
derivative
tumor
radiopharmacy
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WO2009094741A8 (en
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Valbert Nascimento Cardoso
André Luis BRANCO DE BARROS
Ricardo José Alves
Mônica Cristina de OLIVEIRA
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Universidade Federal De Minas Gerais - Ufmg
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0491—Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a radiopharmaceutical and its pharmaceutical compositions, containing a combination of a radioactive component and a chemical component (organic ligand), which may also contain pharmaceutically and pharmacologically acceptable excipients.
  • a radiopharmaceutical and its pharmaceutical compositions containing a combination of a radioactive component and a chemical component (organic ligand), which may also contain pharmaceutically and pharmacologically acceptable excipients.
  • compositions are characterized by containing a combination of a radionuclide, Tecnecio-99m, and a chemical component (organic ligand) that has affinity for tumor cells, especially in early stages, favoring treatment and increasing the chances of cure of the disease.
  • a radionuclide Tecnecio-99m
  • a chemical component organic ligand
  • Biopsy for example, may not be representative of the entire tumor because of its heterogeneity, they may be difficult to perform depending on the depth of the tumor and, moreover, there may be differences between the primary tumor and metastases (KENNY, LM; ABOAGYE, EO; PRICE, PM Positron emission tomography imaging of cell proliferation in oncology (Clinical Oncology, v. 16, pp. 176-185, 2004).
  • scintigraphy is capable of detecting biochemical and physiological changes in the organ studied, thus identifying the disease in its early stage.
  • the possibility of early diagnosis of neoplasic cells will favor the therapy, increasing the survival of patients and even enabling their cure.
  • the body image is obtained by capturing the radioactivity emitted by the administered radiotracer.
  • Radiopharmaceuticals are administered intravenously and the diagnosis is based on biochemical and physiological alterations of the organ under examination, without the need for anatomic alterations, providing the diagnosis of the disease at an early stage (CONTI, PS; LILIEN, DL; HAWLEY, K.; KEPPLER, J .; GRAFTON, ST; BADING, JR PET and [ 18 F] -FDG in oncology: A clinical uptake.Nuclear Medicine and Biology, v. 23, pp. 717-735, 1996 THRALL, JH; ZIESSMAN, HA Nuclear Medicine 2. Rio de Janeiro: Guanabara Koogan, 2003. p. Radiopharmaceuticals are radioactive compounds used in the diagnosis and treatment of diseases.
  • Radiopharmaceuticals In nuclear medicine about 95% of radiopharmaceuticals are used for diagnostic purposes. Most of these radiopharmaceuticals are a combination of a radioactive component (radionuclide), which allows external detection of the organ organism's physiology and / or anatomy under examination, and a chemical component (organ ligand), which is responsible for targeting in the body. These nouns have no pharmacological action as they are administered in extremely low doses. Radionuclide must emit radiation that will be easily detectable by a nuclear instrument and the dose of radiation to the patient should be minimal (SAHA, GB Fundamentals of nuclear pharmacy. 4.ed. New York: Springer-Verlag, 1998a. 358 p. ; THRALL; THRALL, JH; ZIESSMAN, HA Nuclear Medicine 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p.
  • radionuclides used in nuclear medicine such as: iodine-131 (131I), indio-111 (HlIn), Talio-201 (20 ITl), Fl ⁇ or-18 (18F), Technecio-99m (99mTc), Galio -67 (67Ga), among others (SAHA, GB Fundamentals of nuclear pharmacy. 4.ed. New York: Springer-Verlag, 1998a. 358 p.).
  • tumor imaging agent 2- [18F] fluoro-2-deoxy-D-glucose ([18F] FDG)
  • CELEN S.
  • GROOT T.
  • BALZARINI J .
  • VUNCKY K .
  • TERWINGHE C. Synthesis and evaluation of a 99m Tc-MAMA-propyl-thymidine complex as a potential probe for in vivo visualization of tumor cell proliferation with SPECT Nuclear Medicine and Biology, v. 34. pp. 283-291 , 2007).
  • the fluorine-18 is produced in cyclotron, has a physical half-life of 109 minutes and decays by positron emission ( ⁇ + radiation) which when interacting with a middle electron annihilates generating two 511 keV gamma photons at an angle of 180 ° , which will be detected by positron emission tomography (PET) - (THRALL, JH; ZIESSMAN, HA Nuclear Medicine. 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p. 408).
  • PET positron emission tomography
  • [18F] FDG is a D-glucose analog as shown in Figure 4, and its application is based on the increased glycolytic activity of malignant tumor cells (CONTI, PS; LILIEN, DL; HAWLEY, K .; KEPPLER, J GRAFTON, ST; BADING, JR PET and [ 18 F] -FDG in oncology: A clinical uptake. Nuclear Medicine and Biology, v. 23, pp. 717-735, 1996).
  • the second step in glycolysis would be the isomerization of glucose-6-phosphate to fructose 6-phosphate by the enzyme phosphohexose isomerase, but for the formation of fructose-6 phosphate, the presence of oxygen in C-2 is required. at 18FDG.
  • phosphorylated [18F] FDG cannot proceed in the normal metabolic pathway of glucose, being retained within the cell in proportion to the glycolytic activity of this cell (PAUWELS, EKJ; RIBEIRO, MJ; STOOT, JH; MCCREADY, VR; BOURGUIGNON, M .; MAZIERE, B. FDG accumulation and tumor biology., Nuclear Medicine and Biology, v. 25, pp. 317- 322,1998 .; BOS, R.; HOEVEN, JJM; WALL, E.
  • Tecnecio-99m is the most widely used radionuclide in nuclear medicine because it has the ideal physical and chemical properties for a radioisotope for scintigraphic imaging, such as: 6.01 hour physical half-life, low energy gamma emission (140 (keV), high availability of the radioisotope from a Molibdenio-99 / Tecnecio-99m (99Mo / 99mTc) generator system and relatively low cost (JURISSON, S.; BERNING, D .; JIA, W .; DANGSHE, M. Coordination compounds in nuclear medicine, Chemical Reviews, v. 93, no. 3, pp. 1137-1156, 1993.
  • the technetium is a transition metal of the VII B family and has an atom number 43, and may exist in eight oxidation states (-I to +7).
  • the stability of these transition states depends on the type of binding and the chemical environment.
  • the +7 and +4 states are more stable and are represented in oxides, sulfides, halides and pertechnets (DEWANJEE, MK The chemistry of 99m Tc-labeled radiopharmaceuticals. Seminars in Nuclear Medicine, v. 20, no. 1, p. 5 -27, 1990; SAHA, GB Fundamentals of nuclear pharmacy, 4.ed. New York: Springer-Verlag, 1998a, 358 p).
  • the pertechnetate ion, 99mTcO4 - has a +7 oxidation state to 99mTc, which makes it a non-reactive species and unable to bind to any compound, requiring the reduction of the technician from the +7 state to a smaller oxidation state.
  • Tin chloride II SnCl 2 • 2H 2 O
  • ALOTNIK Physico-chemical concepts in the preparation of radiopharmacy technetium. In: SAMPSON, CB Textbook of radiopharmacy theory and practice V.3 Gordon and Breach Science Publishers SA, 1990. Chapter 3, pp.
  • the reduced 99mTc is a chemically reactive species and combines with a wide variety of chelating agents.
  • the chelating agent is usually electron donor and forms a covalent bond coordinated with the reduced Tc-99m.
  • MAG 3 mercaptoacetylglycylglycylglycine, and an N3S ligand, which when labeled with Tecnecio-99m has a radiochemical purity greater than 90%
  • ROBLES et al, 2003 Is capable of forming a stable complex and negatively charged with technetium (V ANBILLOEN, HP; Bormans, G, Roo, MJ;. Verbruggen, AN Complexes of technetium-99m with tetrapeptides, a new class of 99m Tc-labeled agents Nuclear Medicine and Biology, v. 22, no. 3, pp.
  • the first step in the formation of the 99mTc-MAG 3 radiopharmaceutical is the formation of a 99mTc complex with a weak binder in aqueous medium. This complex then reacts with a second ligand (MAG 3 ), which forms a more stable complex. Strong binders such as MAG 3 are less soluble in aqueous media and require heating or long time to dissolve.
  • the 99mTc-tartrate or 99mTc-gluconate complex is prepared by reducing 99mTcO4 - with stannous ion in the presence of sodium tartrate or sodium gluconate, followed by heating with MAG 3 , resulting in 99mTc-MAG 3 , as outlined in Figure 6.
  • 99mTc-MAG 3 is used as a marker of renal tubular function. It has a high binding to plasma proteins, about 90%, which gives it a small glomerular filtration rate (less than 3%). Following intravenous administration, its blood clearance is rapid and biphasic and is rapidly excreted by renal tubular secretion (FRITZBERG, AR; KASINA, S.; ESHIMA, D .; JOHNSON, DL. Synthesis and biological evaluation of technetium-99m MAG 3 as Hippuran replacement Journal of Nuclear Medicine, v. 27, no. 1, pp. 111-116, 1986; ESHIMA, D .; TAYLOR, A.
  • US5245018 patent application “Process for preparing a radiopharmaceutical composition” describes a process for preparing a radiopharmaceutical composition comprising 99m technetium, MAG 3 , water soluble reducing and stabilizing agent which removes any nuisance from the local diagnosis and decreases technician exposure to radiation during operation.
  • the pharmaceutical composition described in this report is distinct and has not been claimed in US5245018.
  • Patent application US5986074 "Metal chelates as pharmaceutical imaging agents", processes of making such and uses thereof, describes the process of obtaining and pharmaceutical composition of chelated metals, such as the Tecnecio-99m, for corresponding binders for diagnosis. of various diseases.
  • chelated metals such as the Tecnecio-99m
  • the pharmaceutical composition described in this specification is quite different from the cited patent application and has therefore not been claimed.
  • the present invention proposes to obtain a composition containing a combination of a radionuclide, Tecnecio-99m, and a chemical component (organic ligand) that has affinity for tumor cells, capable of detecting tumors mainly in early stages, which it even favors treatment and greatly increases the chances of cure of the disease.
  • the present invention is characterized by the use of chemically coupled organic binders to sugars, which increase their affinity for tumor cells. These ligands are complexed to Tecnecio-99m, being able to detect tumors mainly in early stages, favoring treatment and increasing the chances of cure.
  • the formulation of the invention is characterized by the use of pharmaceutically acceptable excipients combined to the mixture obtained product derived from the MAG 3 bound technetium-99m and / or other radionuclides.
  • Formulations may be prepared with an excipient or mixtures thereof.
  • excipients include water for injectables, sterile saline, phosphate buffered solutions, Ringer's solution, dextrose solution, Hank's solution, biocompatible saline solutions whether or not containing polyethylene glycol.
  • Non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglyceride may also be used.
  • Other useful formulations include agents capable of increasing viscosity, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients may also contain minor amounts of additives, such as substances that increase isotonicity and chemical stability of substances or buffers.
  • buffers include phosphate buffer, bicarbonate buffer and Tris buffer
  • preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol.
  • Standard formulations may be liquid or solid.
  • the excipient may include dextrose, human serum albumin, preservatives, etc., to which water or sterile saline may be added prior to administration.
  • the present invention is further characterized by the preparation of controlled release systems containing the obtained product of TEC3-99m coupled MAG 3 and / or other radionuclides.
  • Satisfactory controlled release systems include, but are not limited to, cyclodextrins, biocompatible polymers. , biodegradable polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems.
  • Figure 1 Biodistribution profile of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in the urinary system.
  • Figure 2 - Biodistribution profile of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in blood.
  • Figure 3 Biodistribution profile of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in the liver.
  • FIG. 4 Biodistribution profile of "" 1 Tc-MAG 3 and “" 1 Tc-MAG 3 -G in the heart.
  • Figure 5 Biodistribution profile of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in the lung.
  • Bz-MAG 3 (9) and Bz-MAG 3 -G QO) were labeled using amber vial containing 1 mg of compound to be labeled, 40 mg of sodium tartrate and 0.1 mL of HCl. 0.25 ⁇ L / L containing 0.2 g of stannous chloride (SnCl 2 • H 2 O). The pH was adjusted to a range of 6 to 8 with a 0.2mL / L sodium hydroxide solution. The vial was sealed and a vacuum was made to prevent tin oxidation. To the flask was added 0.1 ml of sodium pertechnetate solution with 37 MBq activity. The vial with the solution was kept in a boiling water bath for 10 to 40 minutes. After this time the solution was left at room temperature for 10 to 30 minutes for cooling.
  • the labeling yield for 1 "Tc-MAG 3 and 1 " Tc-MAG 3 -G were determined based on the values obtained by thin layer chromatography (CCD). From the data obtained by CCD using acetone as the eluent, it was possible to determine the percentage of " 1 TcO 4 ' (6.02 ⁇ 1.15), that is, of the atoms of technetium that have not been reduced. Using saline as an eluent, the percentage of approximately 40.28 ⁇ 4.62 of " 1 TcO 2" was found , this percentage being relative to the hydrolyzed Tecnecio-99m atoms. Therefore, the two products tested initially did not show satisfactory labeling yield as shown in table 1.
  • the recommended labeling yield of a product for biodistribution studies should be greater than 90% (THEOBALD, AE Physico - chemical concepts in the preparation of radiopharmaceutical technetium.In: SAMPSON, CB. Textbook of radiopharmacy theory and practice.V.3 Gordon and Breach Science Publishers SA, 1990. Chap. 7, pp. 115-148).
  • Radiochemical impurities result in poor quality images due to high background radiation around tissues and blood, and expose the patient to an unnecessary dose of radiation (SAHA, GB Fundamentals of nuclear pharmacy. 4.ed. New York: Springer - Verlag, 1998a, 358 P. THRALL, JH; ZIESSMAN, HA Nuclear Medicine 2.
  • the radiochemical purity was evaluated by silica gel 60 thin layer chromatography (CCD). Initially, the CCDs of the impurities commonly found in the "" 1 TcO 2 and "" 1 TcO 4 " labeling processes were performed. 0.9% saline and acetone as eluents. "" 1 TcO 2 was obtained by treating sodium pertechnetate (Na " m TcO 4 ) with a reducing agent (stannous chloride) for 5 to 30 minutes. One drop of the solution was applied to a CCD strip for chromatography with the eluents already mentioned. Pertechnetate chromatography was performed directly with the eluted solution of the "Mo /" m Tc generator.
  • Each CCD plate was prepared in dimensions 10 cm long by 1 cm wide. After elution of each solvent, the plates were dried, cut into 1 cm length fragments each and then radioactivity was determined on a gamma counter. After defining the R / values of the above impurities, chromatographs of the synthesized products (1) and (2) labeled with " m Tc" were also performed using CCD (silica gel 60) strips and, as eluents, acetone and 0.9% saline. The solutions containing the labeled products were column purified using magnesium silicate (Florisil 60-100 mesh) as a stationary phase and initially acetone as a mobile phase for the removal of excess pertechnetate, followed by the use of 0.9 saline solution.
  • magnesium silicate Florisil 60-100 mesh
  • mice Aliquots of the eluted fractions from the column containing 370 KBq of "" 1 Tc-MAG 3 or "" 1 Tc-MAG 3 -G were injected intravenously into male SWISS mice weighing 25 to 30 grams. Twenty animals divided into 4 groups of 5 mice each were used. Following administration of the labeled products, the mice were intraperitoneally anesthetized with 40 mg / kg and 5 mg / kg Ketamine / Xylazine solution, respectively. At 5, 15, 30 and 60 minutes the animals were sacrificed. Liver, bacchus, kidneys, stomach, heart, lung, blood and bladder were removed, weighed and placed in a well gamma counter for determination of radioactivity.
  • a dose standard containing the same activity and the same volume of the injected dose was used.
  • the radioactivity contained in each organ studied was divided by the mass of this organ, thus obtaining the measurement in counting per minute per tissue gram (cpm / g).
  • This procedure aimed to standardize the experiments in order to avoid the interference of different organ masses.
  • the cpm value obtained for the dose standard was considered as 100%, obtaining the% dose / g ratio at the end.
  • MAG 3 shows that during the experiments, the organs with the highest radioactivity uptake were the kidneys and the bladder. This shows renal elimination of the " 1 Tc-MAG 3" product .
  • Commercially available MAG 3 is known to have high affinity for the urinary system: kidneys and bladder (FRITZBERG, AR; KASINA, S.; ESHIMA, D .; JOHNSON, DL. Synthesis and biological evaluation of technetium. -99m MAG 3 as a Hippuran replacement.Journal of Nuclear Medicine, v. 27, no. 1, pp. 111-116, 1986; ROBLES, BR; HERRERA, J .; CABALLERO, J .; OTERO, M.
  • Biodistribution of the saccharide derivative ( 9m Tc-MAG 3 -G) is characterized by faster renal elimination for this complex.
  • Table 4 and Figure 1 show that at 5 and 15 min after administration a statistically higher renal excretion was observed when compared to that observed for " m Tc- MAG 3 . This can probably be explained by the fact that glucose contributed to increase the water solubility of the molecule favoring renal elimination.
  • Literature data show that water-soluble molecules are eliminated faster (RANG, HP; DALE, MM; RITTER, JM; MOORE, PK. Cellular mechanisms: cell proliferation and apoptosis. In: Pharmacology. 5. ed. Rio de Janeiro: Elsevier, 2004 (Chapter 5, pp. 77-89).
  • Ehrlich's tumor cells were implanted intraperitoneally into male SWISS mice weighing 25 to 30 g in order to produce an ascites tumor focus. Eight animals were used for this experiment after 8 days of implant were anesthetized intraperitoneally with a solution of Ketamine / Xylazine at a dose of 40 mg / kg and 5 mg / kg, respectively. Then the animals were sacrificed. Ascites fluid containing viable tumor cells was removed and transferred to a sterile 50 ml Falcon tube. The ascites liquid was centrifuged at 3000 times the gravitational force for 5 minutes and the precipitate was resuspended with 0.9% saline. This process was repeated and then the cells were stained with 0.4% Trypan solution and counted under a 400-fold magnification with the aid of a Newbawer chamber, determining the concentration of the solution containing the tumor cells.
  • the solution containing the Ehrlich tumor cells was centrifuged again and the supernatant (saline) was removed.
  • the pellet containing the cells was resuspended tumor, using the same volume of supernatant was removed with medium containing fetal calf serum for freezing bo vino inactivated at 57 0 C for 1 hour and dimetilsulf ⁇ xido the ratio of 9: 1.
  • the cells were aliquoted into 1.5 mL cryovials with a concentration of 10 8 cells / mL and subsequently stored in nitrogen.
  • mice were kept in a bioterium for 15 to 20 days without food and water restriction, radiolabelled derivatives (1) and (2) were then administered.
  • the mice were anesthetized intraperitoneally with Ketamine / Xylazine solution in 40 mg / kg and 5 mg / kg respectively, and sacrificed at 5, 30, 12, and 240 minutes after administration: liver, bacchus, kidneys, stomach, heart, lung, blood, bladder, tumor (hind paw right) and control muscle (right hind paw) were removed, weighed and placed in a well gamma counter for as well as the dose pattern for the experiment.
  • Ehrlich tumor cells are transplantable cells from rapidly developing female mouse mammary adenocarcinoma. Such cells have been used as a model in the study of several substantia, as therapeutic agents and diagnostic agents (SEGURA, JA; BARBERO, L; G .; MARQUEZ. J. Ehrlich ascites tumor imbalances splenic cell populations and responsiveness of T cells to Staphylococcus aureus enterotoxin B. stimulation Immunology Letters, v. 74, pp. 111-115, 2000; Oloris, SCS; DAGLI, MLZ; WAR, JL Effect of ⁇ -carotene on the development of solid Ehrlich tumor in mice. , v. 71, pp.
  • Radioactivity present in the tumor tissue (right hind paw) and muscle tissue (left posterior paw) was also evaluated. In this way, it was possible to express the results in the form of the target / non-target relationship, assuming that the left hind paw muscle was considered the non-target region.
  • the data in Table 7 showed a ratio higher than 2.0 for all investigated times. This suggests that 9m Tc-MAG 3 -G was captured by 100% more tumors than the "" Tc-MAG 3 1.
  • Substances that have a target / non-target ratio greater than 1.5 may be considered as potential diagnostic agents (PHILLIPS, WT Delivery of gamma-imaging agents. 37, pp. 13-32, 1999). Therefore, the data obtained suggest that the " 1 Tc-MAG 3 -G" complex can be used for tumor identification.
  • the region of interest (ROI) in the right (target) and left (non-target) hind paws was selected and it was thus possible to determine the area and activity present in each ROI.
  • the target / non-target relations corrected by the area of each region were obtained. This ratio for " 1 Tc-MAG 3 -G remained close to two and for"" 1 Tc-MAG 3 close to 1, as shown in Table 8, being statistically different by the Tukey-Kramer test.
  • Results were expressed as mean ⁇ standard deviation. The values were evaluated by the t test (p> 0.05).
  • Results were expressed as mean ⁇ standard deviation. The values were evaluated by the t test (p> 0.05).

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Abstract

This invention reports radiopharmaceutical and pharmaceutical compositions containing a combination of a radioactive component and a chemical component (organic ligand), which may also contain pharmaceutical and pharmacologically acceptable excipients. These pharmaceutical compositions are characterized by containing a combination of a radionuclide, technetium-99m, and a chemical component (organic ligand) showing affinity for tumor cells, especially in the early stages, thereby favoring the treatment and increasing the chances of curing the disease.

Description

"RADIOFARMACO E SUA COMPOSICAO FARMACEUTICA PARA TRATAMENTO E DIAGNOSTICO DE CANCER" "RADIOFARMACO AND ITS PHARMACEUTICAL COMPOSITION FOR TREATMENT AND CANCER DIAGNOSIS"
A presente invencao relata urn radiofarmaco e suas composicδes farmaceuticas, contendo uma combinacao de um componente radioativo e um componente quimico (ligante organico), podendo tambem conter excipientes farmaceutica e farmacologicamente aceitaveis.The present invention relates to a radiopharmaceutical and its pharmaceutical compositions, containing a combination of a radioactive component and a chemical component (organic ligand), which may also contain pharmaceutically and pharmacologically acceptable excipients.
Essas composicδes farmaceuticas caracterizam-se por conter uma combinacao de um radionuclideo, o Tecnecio-99m, e um componente quimico (ligante organico) que apresenta afinidade por celulas tumorais, principalmente em estagios iniciais, favorecendo o tratamento e aumentando as chances de cura da doenca. Estado da tecnica:These pharmaceutical compositions are characterized by containing a combination of a radionuclide, Tecnecio-99m, and a chemical component (organic ligand) that has affinity for tumor cells, especially in early stages, favoring treatment and increasing the chances of cure of the disease. . State of the art:
A proliferacao celular desordenada e fundamental para o desenvolvimento do cancer. Para a determinacao dessa proliferacao sao utilizadas tecnicas de diagnόstico que necessitam de amostras de tecido para a sua realizacao, como: imuno histoquimica, citometria de fluxo e contagem do nύmero de mitoses visiveis. No entanto, estas tecnicas apresentam algumas limitacόes. A biόpsia, por exemplo, pode nao ser representativa de todo o tumor devido a heterogeneidade deste, elas podem ser dificeis de serem realizadas dependendo da profundidade do tumor e, alem disso, pode haver diferencas entre o tumor primario e metastases (KENNY, L. M.; ABOAGYE, E. O.; PRICE, P. M. Positron emission tomography imaging of cell proliferation in oncology. Clinical Oncology, v. 16, p. 176-185, 2004).Disordered cell proliferation is critical to cancer development. In order to determine this proliferation, diagnostic techniques are used that require tissue samples to perform them, such as: immunohistochemistry, flow cytometry and counting the number of visible mitoses. However, these techniques have some limitations. Biopsy, for example, may not be representative of the entire tumor because of its heterogeneity, they may be difficult to perform depending on the depth of the tumor and, moreover, there may be differences between the primary tumor and metastases (KENNY, LM; ABOAGYE, EO; PRICE, PM Positron emission tomography imaging of cell proliferation in oncology (Clinical Oncology, v. 16, pp. 176-185, 2004).
Outros meios de avaliacao da proliferacao tumoral se baseiam em mudancas anatδmicas observadas em exames clinicos, raios-X, ultra-sonografϊa, tomografia computadorizada e ressonancia magnetica nuclear, que permitem avaliar alteracδes no tamanho do tumor. Essas mudancas, no entanto, podem tardar semanas ou meses para serem observadas e avaliadas, dificultando o tratamento (SHIELDS, A. F.; GRIERSON, J. R.; DOHMEN, B. M.; MACHULLA, H.; STAYANOFF, J. C; OBRADOVICH, J. E. Imaging proliferation in vivo with [F- 18] FLT and positron emission tomography. Nature Medicine, v. 4, n. 11, p. 1334-1336, 1998.). A medicina nuclear vem se tornando uma ferramenta poderosa para a deteccSo de diversas doencas, como o cancer. Enquanto as demais tecnicas avaliam alteracδes anatδmicas, a cintilografϊa e capaz de detectar alteracδes bioquimicas e fisiolόgicas no όrgao estudado, e assim, identifϊcar a doenca em seu estagio inicial. A possibilidade do diagnόstico precoce de celulas neoplasicas ira favorecer a terapeutica, aumentando a sobrevida dos pacientes e, ate, possibilitando sua cura. Nesta tecnica, a imagem do corpo e obtida atraves da captacao da radioatividade emitida pelo radiotracador administrado. Os radiofarmacos sao administrados, em sua maioria, por via endovenosa e o diagnόstico e baseado em alteracδes bioquimicas e fϊsiolόgicas do όrgao em exame, sem a necessidade de alteracδes anatόmicas, fornecendo o diagnόstico da doenca em um estagio inicial (CONTI, P. S.; LILIEN, D. L.; HAWLEY, K.; KEPPLER, J.; GRAFTON, S. T.; BADING, J. R. PET and [18F]-FDG in oncology: A clinical uptake. Nuclear Medicine and Biology, v. 23, p. 717-735, 1996; THRALL, J. H.; ZIESSMAN, H. A. Medicina Nuclear. 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p. 408). Radiofarmacos sao compostos radioativos usados no diagnόstico e no tratamento de doencas. Em medicina nuclear cerca de 95% dos radiofarmacos sao utilizados para fins de diagnόsticos. A maioria destes radiofarmacos e uma combinacao de um componente radioativo (radionuclideo), que permite a deteccao externa da fisiologia e/ou anatomia do όrgao em exame, e um componente quimico (ligante organico), que e responsavel pelo direcionamento no organismo. Essas substantias nao apresentam acao farmacolόgica, pois sao administradas em doses extremamente baixas. O radionuclideo deve emitir uma radiacao que sera facilmente detectavel por um instrumento nuclear e a dose dessa radiacao para o paciente devera ser minima (SAHA, G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer-Verlag, 1998a. 358 p.; THRALL; THRALL, J. H.; ZIESSMAN, H. A. Medicina Nuclear. 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p. 408).Other means of assessing tumor proliferation are based on anatomical changes observed in clinical examinations, X-rays, ultrasonography, computed tomography and nuclear magnetic resonance, which allow to evaluate changes in tumor size. These changes, however, may take weeks or months to observe and evaluate, making treatment difficult (SHIELDS, AF; GRIERSON, JR; DOHMEN, BM; MACHULLA, H.; STAYANOFF, J. C; OBRADOVICH, JE Imaging proliferation in vivo with [F-18] FLT and positron emission tomography (Nature Medicine, v. 4, no. 11, pp. 1334-1336, 1998). Nuclear medicine has become a powerful tool for detecting various diseases, such as cancer. While the other techniques evaluate anatomical changes, scintigraphy is capable of detecting biochemical and physiological changes in the organ studied, thus identifying the disease in its early stage. The possibility of early diagnosis of neoplasic cells will favor the therapy, increasing the survival of patients and even enabling their cure. In this technique, the body image is obtained by capturing the radioactivity emitted by the administered radiotracer. Most radiopharmaceuticals are administered intravenously and the diagnosis is based on biochemical and physiological alterations of the organ under examination, without the need for anatomic alterations, providing the diagnosis of the disease at an early stage (CONTI, PS; LILIEN, DL; HAWLEY, K.; KEPPLER, J .; GRAFTON, ST; BADING, JR PET and [ 18 F] -FDG in oncology: A clinical uptake.Nuclear Medicine and Biology, v. 23, pp. 717-735, 1996 THRALL, JH; ZIESSMAN, HA Nuclear Medicine 2. Rio de Janeiro: Guanabara Koogan, 2003. p. Radiopharmaceuticals are radioactive compounds used in the diagnosis and treatment of diseases. In nuclear medicine about 95% of radiopharmaceuticals are used for diagnostic purposes. Most of these radiopharmaceuticals are a combination of a radioactive component (radionuclide), which allows external detection of the organ organism's physiology and / or anatomy under examination, and a chemical component (organ ligand), which is responsible for targeting in the body. These nouns have no pharmacological action as they are administered in extremely low doses. Radionuclide must emit radiation that will be easily detectable by a nuclear instrument and the dose of radiation to the patient should be minimal (SAHA, GB Fundamentals of nuclear pharmacy. 4.ed. New York: Springer-Verlag, 1998a. 358 p. ; THRALL; THRALL, JH; ZIESSMAN, HA Nuclear Medicine 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p.
Existe uma grande variedade de radionuclideos usados em medicina nuclear como: iodo-131(131I), indio-111 (H lIn), Talio-201 (20 ITl), Flύor-18 (18F), Tecnecio- 99m (99mTc), Galio-67 (67Ga), entre outros (SAHA, G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer-Verlag, 1998a. 358 p.).There are a wide variety of radionuclides used in nuclear medicine such as: iodine-131 (131I), indio-111 (HlIn), Talio-201 (20 ITl), Flύor-18 (18F), Technecio-99m (99mTc), Galio -67 (67Ga), among others (SAHA, GB Fundamentals of nuclear pharmacy. 4.ed. New York: Springer-Verlag, 1998a. 358 p.).
Em oncologia, o agente de imagem tumoral mais usado e o 2-[18F]fluoro-2- desoxi-D-glicose ([18F]FDG) (CELEN, S.; GROOT, T.; BALZARINI, J.; VUNCKY, K.; TERWINGHE, C. Synthesis and evaluation of a 99mTc-MAMA-propyl-thymidine complex as a potential probe for in vivo visualization of tumor cell proliferation with SPECT. Nuclear Medicine and Biology, v. 34. p. 283-291, 2007).In oncology, the most commonly used tumor imaging agent is 2- [18F] fluoro-2-deoxy-D-glucose ([18F] FDG) (CELEN, S.; GROOT, T.; BALZARINI, J .; VUNCKY, K .; TERWINGHE, C. Synthesis and evaluation of a 99m Tc-MAMA-propyl-thymidine complex as a potential probe for in vivo visualization of tumor cell proliferation with SPECT Nuclear Medicine and Biology, v. 34. pp. 283-291 , 2007).
O flύor-18 e produzido em ciclotron, tern meia-vida fisica de 109 minutos e decai por emissao de positron (radiacao β+) que ao interagir com um eletron do meio se aniquila gerando dois fόtons gama de 511 keV num angulo de 180°, que serao detectados por tomografϊa por emissao de positron (PET)-( THRALL, J. H.; ZIESSMAN, H. A. Medicina Nuclear. 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p. 408). O [18F]FDG e um analogo da D-glicose, como mostrado na figura 4, e sua aplicacao baseia-se na maior atividade glicolitica das celulas tumorais malignas (CONTI, P. S.; LILIEN, D. L.; HAWLEY, K.; KEPPLER, J.; GRAFTON, S. T.; BADING, J. R. PET and [18F]-FDG in oncology: A clinical uptake. Nuclear Medicine and Biology, v. 23, p. 717-735, 1996).O [18F]FDG penetra na celula por difusao facilitada atraves dos transportadores para glicose (GLUT-I), que, conforme ja explicitado, tambem apresentam maior expressao em celulas tumorais (SCHIBLI, R.; DUMAS, C; PETRIG, J.; SPADOLA, L.; SCAPOZZA, L.; GARCIA-GARAYOA, E.; SCHUBIGER, P. A. Synthesis and in vitro characterization of organometallic rhenium and technetium glucose complexes against GLUT-I e hexokinase. Bioconjugate Chemistry, v. 16, p. 105-112, 2005., (LOVE, C; TOMAS, M. B.; TRONCO, G. G.; PALESTRO, C. J. FDG PET of infection and inflamation. Radiographics, v. 25, n. 5, p. 1357-1368, 2005.), e fosforilado pela enzima hexoquinase, como ocorre normalmente com a D-glicose, gerando o [18F]FDG-6-fosfato (GU et al, 2006). O segundo passo na glicόlise seria a isomerizacao da glicose-6-fosfato a frutose 6-fosfato pela enzima fosfo- hexose isomerase, porem para a formacao da frutose-6 fosfato, e necessario a presenca do oxigenio em C-2, que esta ausente no 18FDG.The fluorine-18 is produced in cyclotron, has a physical half-life of 109 minutes and decays by positron emission (β + radiation) which when interacting with a middle electron annihilates generating two 511 keV gamma photons at an angle of 180 ° , which will be detected by positron emission tomography (PET) - (THRALL, JH; ZIESSMAN, HA Nuclear Medicine. 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p. 408). [18F] FDG is a D-glucose analog as shown in Figure 4, and its application is based on the increased glycolytic activity of malignant tumor cells (CONTI, PS; LILIEN, DL; HAWLEY, K .; KEPPLER, J GRAFTON, ST; BADING, JR PET and [ 18 F] -FDG in oncology: A clinical uptake. Nuclear Medicine and Biology, v. 23, pp. 717-735, 1996). [18F] FDG penetrates the cell by facilitated diffusion through glucose transporters (GLUT-I), which, as already explained, also show greater expression in tumor cells (SCHIBLI, R.; DUMAS, C; PETRIG, J .; SPADOLA, L .; SCAPOZZA, L GARCIA-GARAYOA, E. SCHUBIGER, PA Synthesis and in vitro characterization of organometallic rhenium and technetium glucose complexes against GLUT-I and hexokinase Bioconjugate Chemistry, v. 16, pp. 105-112, 2005. (LOVE, C; TOMAS, MB; TRUNCO, GG; PALESTRO, CJ FDG PET of infection and inflammation. Radiographics, v. 25, no. 5, pp. 1357-1368, 2005.), and phosphorylated by the hexokinase enzyme, as normally occurs with to Dg licose, generating [18F] FDG-6-phosphate (GU et al, 2006). The second step in glycolysis would be the isomerization of glucose-6-phosphate to fructose 6-phosphate by the enzyme phosphohexose isomerase, but for the formation of fructose-6 phosphate, the presence of oxygen in C-2 is required. at 18FDG.
Portanto, a partir desse ponto, o [18F]FDG fosforilado nao pode prosseguir no caminho metabόlico normal da glicose, ficando retido no interior da celula de maneira proporcional a atividade glicolitica desta celula (PAUWELS, E. K. J.; RIBEIRO, M. J.; STOOT, J. H.; MCCREADY, V. R.; BOURGUIGNON, M.; MAZIERE, B. FDG accumulation and tumor biology. Nuclear Medicine and Biology, v. 25, p. 317- 322,1998.; BOS, R.; HOEVEN, J. J. M.; WALL, E.; GROEP, P.; DIEST, P. J.; COMANS, E. F. J.; SEMENZA, G. L. Biological correlates of 18fiuorodesoxyglucose uptake in human breast cancer measured by positron emission tomography. Journal of Clinical Oncology, v. 20, n. 2, p. 379-387, 2002).Therefore, from this point on, phosphorylated [18F] FDG cannot proceed in the normal metabolic pathway of glucose, being retained within the cell in proportion to the glycolytic activity of this cell (PAUWELS, EKJ; RIBEIRO, MJ; STOOT, JH; MCCREADY, VR; BOURGUIGNON, M .; MAZIERE, B. FDG accumulation and tumor biology., Nuclear Medicine and Biology, v. 25, pp. 317- 322,1998 .; BOS, R.; HOEVEN, JJM; WALL, E. ; GROEP, P.; DIEST, PJ; COMANS, EFJ; SEMENZA, GL Biological correlates of 18 fluorodeoxyglucose uptake in human breast cancer measured by positron emission tomography.Journal of Clinical Oncology, v. 20, no. 2, p. 387, 2002).
O uso do [18F]FDG na rotina clinica e, ainda, limitado devido a sua baixa disponibilidade e o custo relativamente alto, tanto em sua producao (ciclotron) como na deteccao das imagens (camara PET), alem da necessidade de uma sintese rapida devido ao baixo tempo de meia-vida fisica do flύor-18 (109 minutos). Assim, seria de grande valia o desenvolvimento de agentes de diagnόstico baseados em isόtopos emissores de radiacao gama, direcionados a producao de imagens em camaras de cintilacao convencionais (CHEN, X.; LI, L.; LIU, F.; LIU, B. Synthesis and biological of technetium-99m-labeled deoxyglucose derivatives as imaging agents for tumor. Bioorganic and Medicinal Chemistry Letters, v. 16, p. 5503-5506, 2006.).The use of [18F] FDG in the clinical routine is also limited due to its low availability and the relatively high cost, both in its production (cyclotron) and in the detection of images (PET camera), besides the necessity of a quick synthesis. due to the low physical half-life of floror-18 (109 minutes). Thus, the development of diagnostic agents based on gamma-emitting isotopes for imaging in conventional scintillation cameras (CHEN, X .; LI, L .; LIU, F .; LIU, B.) would be of great value. Synthesis and biological of technetium-99m-labeled deoxyglucose derivatives as imaging agents for tumor. Bioorganic and Medicinal Chemistry Letters, v. 16, p. 5503-5506, 2006.).
O Tecnecio-99m e o radionuclideo mais usado em medicina nuclear, por apresentar propriedades fisicas e quimicas ideais para um radioisόtopo destinado a producao de imagens cintilograficas, tais como: meia-vida fisica de 6,01 horas, emissao gama de baixa energia (140 keV), alta disponibilidade do radioisόtopo a partir de um sistema gerador de Molibdenio-99/Tecnecio-99m (99Mo/99mTc), alem de apresentar um custo relativamente baixo (JURISSON, S.; BERNING, D.; JIA, W.; DANGSHE, M. Coordination compounds in nuclear medicine. Chemical Reviews, v. 93, n. 3, p. 1137- 1156, 1993.; JONES, A. G. Technetium in nuclear medicine. Radiochimica Acta, v. 70/71, p. 289-297, 1995; MARQUES, F. L. N.; OKAMOTO, M. R. Y.; BUCHPIGUEL, C. A. Alguns aspectos sobre geradores e radiofarmacos de Tecnecio- 99m e seus controles de qualidade. Radiologia Brasileira, v. 34, n. 4, p. 233-239, 2001; YANG, D. J.; KIM, C; SCHECHTER, N. R.; AZHDARINIA, A.; YU, D.; OH, C, BRYANT, J. L.; WON, J.; KIM, E. E.; PODOLOFF, D. A. Imaging with 99mTc ECDG targeted at the multifunctional glucose transport system: feasibility study with rodents. Radiology, p. 465-473, 2003).Tecnecio-99m is the most widely used radionuclide in nuclear medicine because it has the ideal physical and chemical properties for a radioisotope for scintigraphic imaging, such as: 6.01 hour physical half-life, low energy gamma emission (140 (keV), high availability of the radioisotope from a Molibdenio-99 / Tecnecio-99m (99Mo / 99mTc) generator system and relatively low cost (JURISSON, S.; BERNING, D .; JIA, W .; DANGSHE, M. Coordination compounds in nuclear medicine, Chemical Reviews, v. 93, no. 3, pp. 1137-1156, 1993. JONES, AG Technetium in nuclear medicine Radiochimica Acta, v. 70/71, p. -297, 1995; MARQUES, FLN; OKAMOTO, MRY; BUCHPIGUEL, CA Some Aspects of Tecnecio-99m Generators and Radiopharmaceuticals and Their Quality Controls Radiologia Brasileira, v. 34, no. 4, pp. 233-239, 2001 ; YANG, DJ; KIM, C; SCHECHTER, NR; AZHDARINIA, A.; YU, D .; OH, C, BRYANT, JL; WON, J.; KIM, EE; PODOLOFF, DA Imaging with 99m Tc ECDG targe ted at the multifunctional glucose transport system: feasibility study with rodents. Radiology, p. 465-473, 2003).
O tecnecio e um metal de transicao da familia VII B e tern nύmero atomico 43, podendo existir em oito estados de oxidacao (-I a +7). A estabilidade desses estados de transicao depende do tipo de ligacao e do ambiente quimico. Os estados +7 e +4 sao mais estaveis e sao representados em όxidos, sulfetos, haletos e pertecnetatos (DEWANJEE, M. K. The chemistry of 99mTc-labeled radiopharmaceuticals. Seminars in Nuclear Medicine, v. 20, n. 1, p. 5-27, 1990; SAHA, G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer- Verlag, 1998a. 358 p). O ion pertecnetato, 99mTcO4 -, tern estado de oxidacao +7 para o 99mTc, isso o torna uma especie nao reativa e incapaz de ligar a algum composto, sendo necessaria a reducao do tecnecio, do estado +7 para um estado de oxidacao menor. O cloreto de estanho II (SnCl2 • 2H2O) e o agente redutor mais comum usado na preparacao de compostos ligados ao 99mTc (NOWOTNIK, D. P. Physico-chemical concepts in the preparation of technetium radiopharmaceuticals. In: SAMPSON, C. B. Textbook of radiopharmacy theory and practice. V.3. Gordon and Breach Science Publishers S.A., 1990. Cap. 3, p. 53-72; 1990; SAHA, G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer- Verlag, 1998a. 358 p). O 99mTc reduzido e uma especie quimicamente reativa e combina com uma grande variedade de agentes quelantes. O agente quelante geralmente e doador de eletrons e forma uma ligacao covalente coordenada com o Tc-99m reduzido. (SAHA,The technetium is a transition metal of the VII B family and has an atom number 43, and may exist in eight oxidation states (-I to +7). The stability of these transition states depends on the type of binding and the chemical environment. The +7 and +4 states are more stable and are represented in oxides, sulfides, halides and pertechnets (DEWANJEE, MK The chemistry of 99m Tc-labeled radiopharmaceuticals. Seminars in Nuclear Medicine, v. 20, no. 1, p. 5 -27, 1990; SAHA, GB Fundamentals of nuclear pharmacy, 4.ed. New York: Springer-Verlag, 1998a, 358 p). The pertechnetate ion, 99mTcO4 -, has a +7 oxidation state to 99mTc, which makes it a non-reactive species and unable to bind to any compound, requiring the reduction of the technician from the +7 state to a smaller oxidation state. Tin chloride II (SnCl 2 • 2H 2 O) is the most common reducing agent used in the preparation of 99mTc-bound compounds (NOWOTNIK, DP. Physico-chemical concepts in the preparation of radiopharmacy technetium. In: SAMPSON, CB Textbook of radiopharmacy theory and practice V.3 Gordon and Breach Science Publishers SA, 1990. Chapter 3, pp. 53-72; 1990; SAHA, GB Fundamentals of nuclear pharmacy 4.ed.New York: Springer-Verlag, 1998a. 358 p). The reduced 99mTc is a chemically reactive species and combines with a wide variety of chelating agents. The chelating agent is usually electron donor and forms a covalent bond coordinated with the reduced Tc-99m. (SAHA,
G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer-Verlag, 1998a. 358 p).G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer-Verlag, 1998a. 358 p).
Desse modo, a alternativa mais viavel seria a producao de substantias que pudessem ser marcadas com Tecnecio-99m, contribuindo para a reducao dos custos e, desta forma, tornando a tecnica acessivel a todos. Existem muitos compostos capazes de se ligar ao Tecnecio-99m, sendo usados em medicina nuclear para avaliar a funcao e/ou a morfologia de um όrgao, determinando o estado patolόgico do paciente. Sao exemplos desses compostos o 99mTc-HMPAO (marcador cerebral), 99mTc- MAG3 (marcador renal), 99mTc-ECD (marcador para imagem cerebral), 99mTc-HIDA (marcador adequado para imagens do sistema hepatobiliar), 99mTc-DMSA (agente de marcacao renal). (JURISSON, S.; BERNING, D.; JIA, W.; DANGSHE, M. Coordination compounds in nuclear medicine. Chemical Reviews, v. 93, n. 3, p. 1137-1156, 1993).Thus, the most viable alternative would be the production of substantives that could be marked with Technecio-99m, contributing to the reduction of costs and thus making the technique accessible to all. There are many compounds capable of binding to Tecnecio-99m, being used in nuclear medicine to evaluate the function and / or morphology of an organ, determining the pathological state of the patient. Examples of such compounds are 99mTc-HMPAO (brain marker), 99mTc-MAG 3 (renal marker), 99mTc-ECD (brain imaging marker), 99mTc-HIDA (suitable marker for hepatobiliary system imaging), 99mTc-DMSA (agent renal labeling). (JURISSON, S .; BERNING, D .; JIA, W.; DANGSHE, M. Coordination compounds in nuclear medicine. Chemical Reviews, v. 93, no. 3, p. 1137-1156, 1993).
O MAG3, mercaptoacetilglicilglicilglicina, e um ligante N3S, que ao ser marcado com Tecnecio-99m apresenta uma pureza radioquimica superior a 90% (ROBLES et al, 2003). E capaz de formar um complexo estavel, e carregado negativamente com tecnecio (V ANBILLOEN, H. P.; BORMANS, G. N.; ROO, M. J.; VERBRUGGEN, A. N. Complexes of technetium-99m with tetrapeptides, a new class of 99mTc-labeled agents. Nuclear Medicine and Biology, v. 22, n. 3, p. 325-338, 1995; YAMAMURA, N.; MAGATA, Y.; ARANO, Y.; KAWAGUCHI, T.; OGAWA, K.; KONISHI, J.; SAJI, H. Technetium-99m-labeled medium-chain fatty acid analogues metabolized by α-oxidation: radiopharmaceutical for assessing liver function. Bioconjugate Chemistry, v. 10, n. 3, p. 489-495, 1999; OKARVI, S. M. Synthesis, radiolabeling and in vitro and in vivo characterization of a technetium-99m-labeled alpha-M2 peptide as a tumor imaging agent. Journal Peptide Research, v. 63, p. 460- 468, 2004). O primeiro passo para a formacao do radiofarmaco 99mTc- MAG3, consiste na formacao de um complexo do 99mTc com um ligante fraco em meio aquoso. Esse complexo reage, em seguida, com um segundo ligante (MAG3), que forma um complexo mais estavel. Ligantes fortes, como o MAG3, sao menos solύveis em meio aquoso e necessitam de aquecimento ou longo tempo para dissolverem. Inicialmente e preparado o complexo 99mTc-tartarato ou 99mTc-gluconato por reducao do 99mTcO4 - com ion estanoso em presenca de tartarato de sόdio ou gluconate de sόdio, seguido de aquecimento com MAG3, resultando no 99mTc- MAG3, como esquematizado na fϊgura 6 (BORMANS, G.; CLEYNHENS, B.; ADRIAENS, P.; VANBILLOEN, H.; ROO, M.; VERBRUGGEN, P. Investigation of the labeling characteristics of 99mTc-mercaptoacetyltriglycine. Nuclear Medicine and Biology, v. 22, n. 3, p. 339-349, 1995; SAHA, G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer- Verlag, 1998a. 358 p).MAG 3 , mercaptoacetylglycylglycylglycine, and an N3S ligand, which when labeled with Tecnecio-99m has a radiochemical purity greater than 90% (ROBLES et al, 2003). Is capable of forming a stable complex and negatively charged with technetium (V ANBILLOEN, HP; Bormans, G, Roo, MJ;. Verbruggen, AN Complexes of technetium-99m with tetrapeptides, a new class of 99m Tc-labeled agents Nuclear Medicine and Biology, v. 22, no. 3, pp. 325-338, 1995; YAMAMURA, N.; MAGATA, Y .; ARANO, Y .; KAWAGUCHI, T .; OGAWA, K .; KONISHI, J .; SAJI H. Technetium-99m-labeled medium-chain fatty acid analogues metabolized by α-oxidation: radiopharmaceutical for assessing liver function Bioconjugate Chemistry, v. 10, no. 3, pp 489-495, 1999; OKARVI, SM Synthesis, radiolabeling and in vitro and in vivo characterization of a technetium-99m-labeled alpha-M2 peptide as a tumor imaging agent (Journal Peptide Research, v. 63, p. 460-468, 2004). The first step in the formation of the 99mTc-MAG 3 radiopharmaceutical is the formation of a 99mTc complex with a weak binder in aqueous medium. This complex then reacts with a second ligand (MAG 3 ), which forms a more stable complex. Strong binders such as MAG 3 are less soluble in aqueous media and require heating or long time to dissolve. Initially, the 99mTc-tartrate or 99mTc-gluconate complex is prepared by reducing 99mTcO4 - with stannous ion in the presence of sodium tartrate or sodium gluconate, followed by heating with MAG 3 , resulting in 99mTc-MAG 3 , as outlined in Figure 6. (BORMANS, G .; CLEYNHENS, B .; ADRIAENS, P .; VANBILLOEN, H .; ROO, M .; VERBRUGGEN, P. Investigation of the labeling characteristics of 99m Tc-mercaptoacetyltriglycine. Nuclear Medicine and Biology, v. 22, No. 3, pp. 339-349, 1995; SAHA, GB Fundamentals of nuclear pharmacy, 4.ed. New York: Springer-Verlag, 1998a, 358 p).
O 99mTc- MAG3 e usado como marcador da funcao tubular renal. Apresenta alta ligacao a proteinas plasmaticas, cerca de 90%, que confere a ele uma pequena taxa de filtracao glomerular (menor que 3%). Apόs administracao por via intravenosa, seu clearance sanguineo e rapido e bifasico, sendo rapidamente excretado por secrecao tubular renal (FRITZBERG, A. R.; KASINA, S.; ESHIMA, D.; JOHNSON, D. L. Synthesis and biological evaluation of technetium-99m MAG3 as a Hippuran replacement. Journal of Nuclear Medicine, v. 27, n. 1, p. 111-116, 1986; ESHIMA, D.; TAYLOR, A. Technetium-99m (99mTc) Mercaptoacetyltriglycine: Update on the new 99mTc renal tubular function agent. Seminars in Nuclear Medicine, v. 22, n. 2, p. 61-73, 1992; SAHA, G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer- Verlag, 1998a. 358 p; THRALL, J. H.; ZIESSMAN, H. A. Medicina Nuclear. 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p. 408).99mTc-MAG 3 is used as a marker of renal tubular function. It has a high binding to plasma proteins, about 90%, which gives it a small glomerular filtration rate (less than 3%). Following intravenous administration, its blood clearance is rapid and biphasic and is rapidly excreted by renal tubular secretion (FRITZBERG, AR; KASINA, S.; ESHIMA, D .; JOHNSON, DL. Synthesis and biological evaluation of technetium-99m MAG 3 as Hippuran replacement Journal of Nuclear Medicine, v. 27, no. 1, pp. 111-116, 1986; ESHIMA, D .; TAYLOR, A. Technetium-99m ( 99m Tc) Mercaptoacetyltriglycine: Update on the new 99m Tc renal tubular function agent Seminars in Nuclear Medicine, v. 22, no. 2, pp. 61-73, 1992; SAHA, GB Fundamentals of nuclear pharmacy 4.ed.New York: Springer-Verlag, 1998a.358 p; , JH; ZIESSMAN, HA Nuclear Medicine, 2. ed., Rio de Janeiro: Guanabara Koogan, 2003. p.
Com base no exposto e valendo-se do metabolismo acelerado das celulas tumorais, que as tornam mais avidas por glicose, realizou-se a sfntese em oito etapas de β-D-glicopiranosideo de 4-N-[N3-(benzoilmercaptoacetil)glicilglicilglicil]aminofenila, um derivado da D-glicose que complexado ao Tecnecio-99m seria capaz de detectar tumores, em estagios iniciais favorecendo o tratamento e aumentando as chances de cura.Based on the above and taking advantage of the accelerated metabolism of the tumor cells, which make them more avid by glucose, the 4-N- [N 3 - (benzoylmercaptoacetyl) glycylglycol glycyl β-D-glycopyranoside synthesis was performed. ] Aminophenyl, a D-glucose derivative that complexed to Tecnecio-99m would be able to detect tumors at early stages favoring treatment and increasing the chances of cure.
Foram relatados, em alguns estudos, a sintese de (S-EOE- MAG3-β-alanine: S- (1-ethoxy ethyl) mercaptoacetyltriglycyl -β-alanine and CCK4:Trp-Met-Asp-Phe-NH2) atraves de sucessivas condensacόes de aminoacidos ativados em resinas quimicas de pureza elevada e o desenvolvimento de metodo de complexacao do Tecnecio usando pH fisiolόgico e temperatura controlados. Alem disso, estudos farmacolόgicos in vitro e in vivo demonstraram a afinidade especifica do composto obtido por receptores CCKB que sao expressos preferencialmente por algumas celulas tumorais do trato digestivo. (HAFID BELHADJ-TAHAR, JEAN-PAUL ESQUERRE AND YVON COULAIS. "An easy-to-use imaging tool and radiopharmaceutical agent derived from CCK4 for internal radiotherapy": Synthesis and assessment of an original biovector. Nuclear Instruments and Methods in Physics Research Section.)Synthesis of (S-EOE-MAG 3 -β-alanine: S- (1-ethoxy ethyl) mercaptoacetyltriglycyl -β-alanine and CCK4: Trp-Met-Asp-Phe-NH2) has been reported in some studies. successive condensations of activated amino acids in high purity chemical resins and the development of the Tecnecio complexation method using controlled physiological pH and temperature. In addition, in vitro and in vivo pharmacological studies have demonstrated the specific affinity of the compound obtained by CCKB receptors which are preferentially expressed by some digestive tract tumor cells. (HAFID BELHADJ-TAHAR, JEAN-PAUL LEFT AND YVON COULAIS. "An easy-to-use imaging tool and radiopharmaceutical agent derived from CCK4 for internal radiotherapy ": Synthesis and assessment of an original biovector. Nuclear Instruments and Methods in Physics Research Section.)
O desenvolvimento de urn radiofarmaco analogo do peptideo aM2, o Mercaptoacetiltriglicina (MAG)3-derivado do peptideo aM2 com Tecnecio-99m, foi realizado para o diagnόstico do cancer de mama. Estudos farmacolόgicos de biodistribuicao in vivo demonstram a afinidade do composto obtido por celulas tumorals da regiao da mama. (S. M. OKARVI. "Synthesis, radiolabeling and in vitro and in vivo characterization of a technetium-99m labeled alpha-M2 peptide as a tumor imaging agent". Journal of Peptide Research).Development of an analogous radiopharmaceutical of the aM2 peptide, Mercaptoacetyltriglycine (MAG) 3-derived from the aM2 peptide with Technecio-99m, was performed for the diagnosis of breast cancer. Pharmacological studies of in vivo biodistribution demonstrate the affinity of the compound obtained by breast region tumor cells. (S. M. OKARVI. "Synthesis, radiolabeling and in vitro and in vivo characterization of a technetium-99m labeled alpha-M2 peptide as a tumor imaging agent." Journal of Peptide Research).
Alguns estudos descrevem a sintese e as caracteristicas biolόgicas do Tecnecio-Some studies describe the synthesis and biological characteristics of the
99m triamida derivados do mercaptoacetiltriglicina (MAG3). (S. M. OKARVI, P.99m triamide derived from mercaptoacetyltriglycine (MAG 3 ). (SM OKARVI, P.
ADRIAENS, A. M. VERBRUGGEN. "Synthesis and biological characteristics of theADRIAENS, A. M. VERBRUGGEN. "Synthesis and biological characteristics of the
Technetium-99m triamide derivatives of mercaptoacetyltriglycine (MAG3)". Journal of Labelled Compounds and Radiopharmaceuticals).Technetium-99m triamide derivatives of mercaptoacetyltriglycine (MAG 3 ) ". Journal of Labeled Compounds and Radiopharmaceuticals).
Outros estudos descrevem a sintese de Benzoyl-mercaptoacetylglycylglycyl- glycine-mannosyldextran (Bz MAG3-mannosyl-dextran) e o uso do composto obtido como radiofarmaco para diagnόstico de celulas malignas no tecido reticuloendotelial. (DAVID R. VERA, ANNE M. WALLACE AND CARL K. HOHA. "99mTc] MAG3- mannosyl-dextran: a receptor-binding radiopharmaceutical for sentinel node detection". Nuclear Medicine and Biology).Other studies describe the synthesis of Benzoyl-mercaptoacetylglycylglycylglycine-mannosyldextran (Bz MAG 3 -mannosyl-dextran) and the use of the compound obtained as a radiopharmaceutical for the diagnosis of malignant cells in reticuloendothelial tissue. (DAVID R. VERA, ANNE M. WALLACE AND CARL K. HOHA. "99mTc] MAG 3 - mannosyl-dextran: a radiopharmaceutical receptor-binding for sentinel node detection." Nuclear Medicine and Biology.
O pedido de patente sob o nύmero US5245018, "Process for preparing a radiopharmaceutical composition", descreve um processo de preparo de uma composicao radiofarmaceutica, compreendendo tecnecio 99m, MAG3, agente redutor e estabilizante solύveis em agua, a qual remove qualquer incόmodo oriundo do diagnόstico local e diminui a exposicao do tecnico a radiacao durante a operacao. Contudo, a composicao farmaceutica descrita no presente relatόrio e distinta e nao foi reivindicada no pedido de patente US5245018.US5245018 patent application "Process for preparing a radiopharmaceutical composition" describes a process for preparing a radiopharmaceutical composition comprising 99m technetium, MAG 3 , water soluble reducing and stabilizing agent which removes any nuisance from the local diagnosis and decreases technician exposure to radiation during operation. However, the pharmaceutical composition described in this report is distinct and has not been claimed in US5245018.
O pedido de patente de nύmero US5840273, "Technetium-99m complexes for use as radiopharmaceuticals", descreve uma composicao radiofarmaceutica compreendendo complexo de tecnecio 99-m para exame de funcao renal, bem como o metodo de exame usando a composicao. Dessa forma, a composicao da presente invencao (caracterizada por descrever um ligante organico que complexado ao Tecnecio-99m seria capaz de detectar tumores em estagios iniciais) e diferente da descrita na patente supra citada.US5840273, "Technetium-99m complexes for use as radiopharmaceuticals", describes a radiopharmaceutical composition comprising technetium 99-m complex for renal function examination, as well as the method of examination using the composition. Thus, the composition of the present invention (characterized in that it describes an organic binder that is complexed to the Tecnecio-99m would be able to detect tumors at early stages) and different from that described in the above cited patent.
O pedido de patente sob o nύmero US5986074, "Metal chelates as pharmaceutical imaging agents", processes of making such and uses thereof, descreve o processo de obtencao e composicao farmaceutica de metais quelatos, como o Tecnecio- 99m, para ligantes correspondentes destinado ao diagnόstico de diversas doencas. Entretanto, a composicao farmaceutica descrita no presente relatόrio descritivo e bem diferente do pedido de patente citado e, portanto, nao foi reivindicado.Patent application US5986074, "Metal chelates as pharmaceutical imaging agents", processes of making such and uses thereof, describes the process of obtaining and pharmaceutical composition of chelated metals, such as the Tecnecio-99m, for corresponding binders for diagnosis. of various diseases. However, the pharmaceutical composition described in this specification is quite different from the cited patent application and has therefore not been claimed.
Nao foi encontrada no estado da tecnica nenhuma patente contendo composicao de radiofarmaco derivado de MAG-3 capaz de detectar tumores ou que tenham grande avidez por celulas tumorals em estagios iniciais.No patent containing MAG-3-derived radiopharmaceutical composition capable of detecting tumors or having great avidity for tumor cells in early stages has not been found in the prior art.
Para solucionar tal problema a presente invencao propδe a obtencao de composicao contendo uma combinacao de um radionuclideo, o Tecnecio-99m, e um componente quimico (ligante organico) que apresenta afinidade por celulas tumorais, capaz de detectar tumores principalmente em estagios iniciais, o que inclusive favorece o tratamento e aumenta enormemente as chances de cura da doen9a.To solve such problem the present invention proposes to obtain a composition containing a combination of a radionuclide, Tecnecio-99m, and a chemical component (organic ligand) that has affinity for tumor cells, capable of detecting tumors mainly in early stages, which it even favors treatment and greatly increases the chances of cure of the disease.
Realizou-se a marcacao dos compostos BenzoilmercaptoacetilglicilglicilglicinaLabeling of the compounds Benzoylmercaptoacetylglycylglycine
(1) e β-D-glicopiranosideo de 4-jV-|W3-(benzoil- mercaptoacetil)glicilglicilglicil]aminofenila (2), formando complexos com Tecnecio- 99m como o 99mTc- MAG3 £3} e o 99mTc- MAG3-G £4}_respectivamente, que serao utilizados nos estudos de biodistribuicao.(1) and 4-JV-β-D-glycopyranoside 3 - (benzoyl mercaptoacetyl) glycylglycylglycyl] aminophenyl (2), forming complexes with Tecnecio-99m such as 99m Tc-MAG 3 £ 3} and 99m Tc MAG 3 -G £ 4} respectively, which will be used in biodistribution studies.
Foram realizados os estudos de biodistribuicao de ""1Tc-MAG3-G £4), um derivado da D-glicose, inicialmente em camundongos SWISS, machos, 25-3Og, sadios, utilizando o 99mTc-MAG3 {3} como padrao. Os estudos demonstraram que (4) e (3) apresentam padrδes de biodistribuicao diferentes, observando-se o perfil de clearance bifasico para o (3) que e ausente em (4). Em seguida, realizaram-se os estudos de biodistribuicao de (4) em camundongos com tumor de Erhlich implantados na pata posterior direita, novamente utilizando-se o (3) como padrao. O derivado (4) apresentou concentracao cerca de duas vezes maior no tumor em relacao a pata posterior esquerda (controle) para todos os tempos analisados (5, 30, 120 e 240 minutos), o que o credencia como um promissor marcador tumoral. TaI caracteristica nao foi observada para o (3).MAG3 Biodistribution studies of " 1 Tc-MAG 3 -G £ 4), a D-glucose derivative, were initially performed in healthy male SWISS 25-3Og mice using 99m Tc-MAG 3 {3} as a standard. Studies have shown that (4) and (3) have different biodistribution patterns, observing the biphasic clearance profile for (3) which is absent in (4). Subsequently, biodistribution studies of (4) were performed on Erhlich tumor-bearing mice implanted in the right hind paw, again using (3) as standard. Derivative (4) presented about twice the concentration in the tumor in relation to the left posterior paw (control) for all analyzed times (5, 30, 120 and 240 minutes), which accredits it as a promising tumor marker. Such characteristic was not observed for (3) .MAG 3
Nesse sentido, a presente invencao caracteriza-se pelo uso de ligantes organicos acoplados quimicamente a aςύcares, que aumentam sua afinidade por celulas tumorais. Esses ligantes sao complexados ao Tecnecio-99m, tornando-se capaz de detectar tumores principalmente em estagios iniciais, favorecendo o tratamento e aumentando as chances de cura.In this sense, the present invention is characterized by the use of chemically coupled organic binders to sugars, which increase their affinity for tumor cells. These ligands are complexed to Tecnecio-99m, being able to detect tumors mainly in early stages, favoring treatment and increasing the chances of cure.
A formulacao da presente invencao caracteriza-se pelo uso da mistura de excipientes aceitaveis farmaceuticamente combinados ao produto obtido, derivado de MAG3 acoplado ao Tecnecio-99m e/ou outros radionuclideos. Podem ser preparadas formulacόes com um excipiente ou misturas desses. Exemplos de excipientes incluem agua para iηjetaveis, solucao salina esteril, solucδes tamponadas com fosfato, a solucao de Ringer, solucao de dextrose, a solucao de Hank, solucόes salinas biocompativeis contendo ou nao polietileno glicol. Veiculos nao aquosos, como όleos fixos, όleo de sesame, etil-oleato, ou triglicerideo tambem podem ser usados. Outras formulacόes ύteis incluem agentes capazes de aumentar a viscosidade, como carboximetilcelulose de sόdio, sorbitol, ou dextran.The formulation of the invention is characterized by the use of pharmaceutically acceptable excipients combined to the mixture obtained product derived from the MAG 3 bound technetium-99m and / or other radionuclides. Formulations may be prepared with an excipient or mixtures thereof. Examples of excipients include water for injectables, sterile saline, phosphate buffered solutions, Ringer's solution, dextrose solution, Hank's solution, biocompatible saline solutions whether or not containing polyethylene glycol. Non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglyceride may also be used. Other useful formulations include agents capable of increasing viscosity, such as sodium carboxymethylcellulose, sorbitol, or dextran.
Os excipientes tambem podem conter quantidades menores de aditivos, como substantias que aumentam isotonicidade e estabilidade quimica de substantia ou tampόes. Exemplos de tampδes incluem tampao fosfato, tampao bicarbonato e tampao Tris, enquanto exemplos de conservantes incluem timerosal, m - ou o-cresol, formalina e alcool benzilico.. As formulacδes padrδes podem ser liquidas ou sόlidas. Desta forma, uma formulacao nao-liquida, o excipiente pode incluir dextrose, albumina de soro humano, conservantes, etc, na qual agua ou solucao salina esteril possa ser acrescentada antes da administracao.Excipients may also contain minor amounts of additives, such as substances that increase isotonicity and chemical stability of substances or buffers. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations may be liquid or solid. Thus, a non-liquid formulation, the excipient may include dextrose, human serum albumin, preservatives, etc., to which water or sterile saline may be added prior to administration.
A presente invencao, caracteriza-se ainda pela preparacao de sistemas de liberacao controlada contendo o produto obtido derivado de MAG3 acoplado ao Tecnecio-99m e/ou outros radionuclideos Os sistemas de liberacao controlada satisfatόrios incluem, mas nao sao limitados as ciclodextrinas, polimeros biocompativeis, polimeros biodegradaveis, outras matrizes polimericas, capsulas, microcapsulas, microparticulas, preparacδes de bolus, bombas osmόticas, dispositivos de difusao, lipossomas, lipoesferas, e sistemas de administracao transdermicos. Lista de figuras: Figura 1: Perfil de biodistribuicao do ""1Tc-MAG3 e do ""1Tc-MAG3-G no sistema urinario.The present invention is further characterized by the preparation of controlled release systems containing the obtained product of TEC3-99m coupled MAG 3 and / or other radionuclides. Satisfactory controlled release systems include, but are not limited to, cyclodextrins, biocompatible polymers. , biodegradable polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. List of figures: Figure 1: Biodistribution profile of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in the urinary system.
Figura 2: - Perfil de biodistribuicao do ""1Tc-MAG3 e do ""1Tc-MAG3-G no sangue. Figura 3: Perfil de biodistribuicao do ""1Tc-MAG3 e do ""1Tc-MAG3-G no fϊgado.Figure 2: - Biodistribution profile of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in blood. Figure 3: Biodistribution profile of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in the liver.
Figure 4: Perfil de biodistribuicao do ""1Tc-MAG3 e do ""1Tc-MAG3-G no coracao. Figura 5: Perfil de biodistribuicao do ""1Tc-MAG3 e do ""1Tc-MAG3-G no pulmao.Figure 4: Biodistribution profile of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in the heart. Figure 5: Biodistribution profile of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in the lung.
Figura 6 - Imagens cintilograficas de ""1Tc-MAG3-G em todos os tempos estudados, onde se nota maior concentracao do produto testado na pata posterior direita (PPD) em relacao a esquerda. A presente invencao pode ser melhor entendida atraves dos seguintes exemplos, porem nao limitantes:Figure 6 - Scintigraphic images of " 1 Tc-MAG 3 -G" in all studied times, where there is a higher concentration of the tested product in the right hind paw (PPD) in relation to the left. The present invention may be better understood by the following, but not limiting examples:
EXEMPLO 1- Rendimento de marcacao e purificacao de ""1Tc-MAG3 e "111Tc- MAG3-GEXAMPLE 1- Marking and Purification Yield of " 1 Tc-MAG 3 and" 111 Tc-MAG 3 -G
Realizou-se a marcacao de Bz-MAG3 (9) e de Bz-MAG3-G QO) utilizando-se frasco ambar contendo 1 mg do composto a ser marcado, 40 mg de tartarato de sόdio e 0,1 mL de HCl 0,25 moL/L contendo 0,2 g de cloreto estanoso (SnCl2 • H2O). Ajustou- se o pH para uma faixa de 6 a 8 com uma solucao de hidrόxido de sόdio 0,2 moL/L.O frasco foi lacrado e realizou-se vacuo para evitar a oxidacao do estanho. Ao frasco adicionou-se 0,1 mL de solucao de pertecnetato de sόdio com atividade de 37 MBq. O frasco com a solucao foi mantido em banho-maria fervente por 10 a 40 minutos. Apόs este periodo a solucao foi deixada a temperatura ambiente por 10 a 30 minutos para resfriamento.Bz-MAG 3 (9) and Bz-MAG 3 -G QO) were labeled using amber vial containing 1 mg of compound to be labeled, 40 mg of sodium tartrate and 0.1 mL of HCl. 0.25 µL / L containing 0.2 g of stannous chloride (SnCl 2 • H 2 O). The pH was adjusted to a range of 6 to 8 with a 0.2mL / L sodium hydroxide solution. The vial was sealed and a vacuum was made to prevent tin oxidation. To the flask was added 0.1 ml of sodium pertechnetate solution with 37 MBq activity. The vial with the solution was kept in a boiling water bath for 10 to 40 minutes. After this time the solution was left at room temperature for 10 to 30 minutes for cooling.
O ""1Tc se complexa com os derivados Benzoilmercaptoacetilglicilglicilglicina (1) e β-D-glicopiranosideo de 4-N-[N3-(benzoil- mercaptoacetil)glicilglicilglicil]aminofenila (2) atraves dos tres atomos de nitrogenio e do atomo de enxofre, que e desprotegido apόs o aquecimento (WINNARD, P. Jr; CHANG, F.; RUSCKOWSKI, M.; MARDIROSSIAN, G.; HNATOWICH, D. J. Preparation and use of NHS-MAG3 for Technetium-99m labelinf of DNA. Nuclear Medicine & Biology, v. 24, n. 5, p. 425-432, 1997), fornecendo o ""1Tc-MAG3 e o ""1Tc-MAG3-G que serao utilizados nos estudos de biodistribuicao." 1 Tc" is complexed with the 4-N- [N 3- [benzoyl mercaptoacetyl) glycylglycylglycyl] aminophenyl (2) benzoylmercaptoacetylglycylglycylglycine derivatives (2) through the three nitrogen atoms and the atom of sulfur, which is unprotected after heating (WINNARD, P. Jr; CHANG, F .; RUSCKOWSKI, M .; MARDIROSSIAN, G .; HNATOWICH, DJ. Preparation and use of NHS-MAG 3 for Technetium-99m labelinf of DNA. Nuclear Medicine & Biology, v. 24, no. 5, pp. 425-432, 1997), providing the "" 1 Tc-MAG 3 and the "" 1 Tc-MAG 3 -G that will be used in the biodistribution studies.
O rendimento de marcacao para o ""1Tc-MAG3 e o ""1Tc-MAG3-G foram determinados com base nos valores obtidos por cromatografia em camada delgada (CCD). Pelos dados obtidos por CCD, utilizando acetona como eluente, foi possivel determinar o percentual de ""1TcO4 ' (6,02 ± 1,15), ou seja, dos atomos de tecnecio que nao foram reduzidos. Utilizando-se salina como eluente, encontrou-se o percentual de aproximadamente 40,28 ± 4,62 de ""1TcO2, sendo este percentual relativo aos atomos de Tecnecio-99m hidrolizados. Portanto, os dois produtos testados, inicialmente, nao apresentaram rendimento satisfatόrio de marcacao como mostrado na tabela 1. De acordo com a literatura, o rendimento de marcacao recomendado de um produto para estudos de biodistribuicao deve ser superior a 90% (THEOBALD, A. E. Physico- chemical concepts in the preparation of technetium radiopharmaceuticals. In: SAMPSON, C. B. Textbook of radiopharmacy theory and practice. V.3. Gordon and Breach Science Publishers S. A., 1990. Cap. 7, p. 115-148). Impurezas radioquimicas resultam em imagens de baixa qualidade devido a alta radiacao de fundo ao redor dos tecidos e no sangue, alem de expor o paciente a uma dose desnecessaria de radiacao (SAHA, G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer- Verlag, 1998a. 358 p; THRALL, J. H.; ZIESSMAN, H. A. Medicina Nuclear. 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p. 408). Desta forma, para atender os objetivos propostos tornou-se necessario a purificacao dos produtos marcados, ou seja, a retirada das impurezas radioquimicas ""1TcO4- e ""1TcO2.The labeling yield for 1 "Tc-MAG 3 and 1 " Tc-MAG 3 -G were determined based on the values obtained by thin layer chromatography (CCD). From the data obtained by CCD using acetone as the eluent, it was possible to determine the percentage of " 1 TcO 4 ' (6.02 ± 1.15), that is, of the atoms of technetium that have not been reduced. Using saline as an eluent, the percentage of approximately 40.28 ± 4.62 of " 1 TcO 2" was found , this percentage being relative to the hydrolyzed Tecnecio-99m atoms. Therefore, the two products tested initially did not show satisfactory labeling yield as shown in table 1. According to the literature, the recommended labeling yield of a product for biodistribution studies should be greater than 90% (THEOBALD, AE Physico - chemical concepts in the preparation of radiopharmaceutical technetium.In: SAMPSON, CB. Textbook of radiopharmacy theory and practice.V.3 Gordon and Breach Science Publishers SA, 1990. Chap. 7, pp. 115-148). Radiochemical impurities result in poor quality images due to high background radiation around tissues and blood, and expose the patient to an unnecessary dose of radiation (SAHA, GB Fundamentals of nuclear pharmacy. 4.ed. New York: Springer - Verlag, 1998a, 358 P. THRALL, JH; ZIESSMAN, HA Nuclear Medicine 2. Ed Rio de Janeiro: Guanabara Koogan, 2003. 408). Thus, in order to meet the proposed objectives, it was necessary to purify the labeled products, ie, the removal of " 1 TcO 4 - and"" 1 TcO 2 radiochemical impurities.
A pureza radioquimica foi avaliada por cromatografia em camada delgada (CCD) em silica gel 60. Inicialmente, realizaram-se as CCD das impurezas comumente encontradas nos processos de marcacao, ""1TcO2 e ""1TcO4 ", empregando-se solucao salina a 0,9% e acetona como eluentes. O ""1TcO2 foi obtido tratando-se o pertecnetato de sόdio (Na"mTcO4) com um agente redutor (cloreto estanoso) por 5 a 30 minutos. Aplicou-se uma gota da solucao em uma tira de CCD para a realizacao da cromatografia com os eluentes ja citados. A cromatografia com pertecnetato foi realizada diretamente com a solucao eluida do gerador "Mo/"mTc.The radiochemical purity was evaluated by silica gel 60 thin layer chromatography (CCD). Initially, the CCDs of the impurities commonly found in the "" 1 TcO 2 and "" 1 TcO 4 " labeling processes were performed. 0.9% saline and acetone as eluents. "" 1 TcO 2 was obtained by treating sodium pertechnetate (Na " m TcO 4 ) with a reducing agent (stannous chloride) for 5 to 30 minutes. One drop of the solution was applied to a CCD strip for chromatography with the eluents already mentioned. Pertechnetate chromatography was performed directly with the eluted solution of the "Mo /" m Tc generator.
Cada placa de CCD foi preparada nas dimensδes de 10 cm de comprimento por 1 cm de largura. Apόs a eluicao de cada solvente, as placas foram secadas, cortadas em fragmentos de 1 cm de comprimento cada e, em seguida, a radioatividade foi determinada em um contador gama. Apόs definidos os valores de R/ das impurezas acima citadas, realizaram-se as cromatografϊas dos produtos sintetizados (1) e (2) marcados com "mTc, tambem utilizando tiras de CCD (silica gel 60) e, como eluentes, acetona e solucao salina a 0,9%. As solucδes contendo os produtos marcados foram purificadas por coluna utilizando silicato de magnesio (Florisil 60-100 mesh) como fase estacionaria e, inicialmente, acetona como fase mόvel para a remocao do pertecnetato em excesso, seguida pela utilizacao de solucao salina a 0,9% para a retirada do produto marcado, fϊcando o ""1TcO2 retido na coluna. Retirou-se 30 aliquotas de, aproximadamente, 0,25 mL com cada eluente e contou-se a radioatividade destas aliquotas, determinando as fracόes com maior atividade, nas quais o produto eluido estaria em maior concentracao. Os resultados mostraram que as impurezas radioquimicas foram removidas (tabela 2).Each CCD plate was prepared in dimensions 10 cm long by 1 cm wide. After elution of each solvent, the plates were dried, cut into 1 cm length fragments each and then radioactivity was determined on a gamma counter. After defining the R / values of the above impurities, chromatographs of the synthesized products (1) and (2) labeled with " m Tc" were also performed using CCD (silica gel 60) strips and, as eluents, acetone and 0.9% saline. The solutions containing the labeled products were column purified using magnesium silicate (Florisil 60-100 mesh) as a stationary phase and initially acetone as a mobile phase for the removal of excess pertechnetate, followed by the use of 0.9 saline solution. % for the removal of the labeled product by "" retaining the 1 TcO 2 retained in the column. Thirty aliquots of approximately 0.25 mL were removed with each eluent and the radioactivity of these aliquots was counted, determining the fractions with the highest activity, in which the eluted product would be in the highest concentration. Results showed that radiochemical impurities were removed (table 2).
Figure imgf000013_0001
Figure imgf000013_0002
EXEMPLO 2- Estudos de biodistribuicao de ""1Tc-MAG3 e ""1Tc-MAG3-G em animais sadios:
Figure imgf000013_0001
Figure imgf000013_0002
EXAMPLE 2- Biodistribution studies of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G in healthy animals:
Aliquotas das fracδes eluidas da coluna contendo 370 KBq de ""1Tc-MAG3 ou ""1Tc-MAG3-G foram iηjetadas por via endovenosa, em camundongos SWISS, machos, pesando de 25 a 30 gramas. Utilizou-se 20 animais divididos em 4 grupos de 5 camundongos cada. Apόs a administracao dos produtos marcados os camundongos foram anestesiados, por via intraperitoneal, com solucao de Ketamine/Xylazine em dose de 40 mg/Kg e 5 mg/Kg, respectivamente. Nos tempos de 5, 15, 3O e 60 minutos os animais foram sacrificados. Figado, baco, rins, estδmago, coracao, pulmao, sangue e bexiga foram retirados, pesados e colocados em contador gama de poco para determinacao da radioatividade. Para correcao do decaimento radioativo do Tecnecio-99m utilizou-se um padrao de dose contendo a mesma atividade e o mesmo volume da dose injetada nos animais. A radioatividade contida em cada όrgao estudado foi dividida pela massa desse όrgao, conseguindo-se, assim, a medida em contagem por minuto por grama tecidual (cpm/g). Este procedimento teve como finalidade a padronizacao dos experimentos, de maneira a evitar a interferencia das diferentes massas dos όrgaos. O valor em cpm obtido para o padrao de dose foi considerado como 100%, obtendo-se ao final a relacao %dose/g.Aliquots of the eluted fractions from the column containing 370 KBq of "" 1 Tc-MAG 3 or "" 1 Tc-MAG 3 -G were injected intravenously into male SWISS mice weighing 25 to 30 grams. Twenty animals divided into 4 groups of 5 mice each were used. Following administration of the labeled products, the mice were intraperitoneally anesthetized with 40 mg / kg and 5 mg / kg Ketamine / Xylazine solution, respectively. At 5, 15, 30 and 60 minutes the animals were sacrificed. Liver, bacchus, kidneys, stomach, heart, lung, blood and bladder were removed, weighed and placed in a well gamma counter for determination of radioactivity. To correct the radioactive decay of the Tecnecio-99m, a dose standard containing the same activity and the same volume of the injected dose was used. The radioactivity contained in each organ studied was divided by the mass of this organ, thus obtaining the measurement in counting per minute per tissue gram (cpm / g). This procedure aimed to standardize the experiments in order to avoid the interference of different organ masses. The cpm value obtained for the dose standard was considered as 100%, obtaining the% dose / g ratio at the end.
Observa-se pela tabela 3 que no decorrer dos experimentos os όrgaos que apresentaram maior captacao de radioatividade foram os rins e a bexiga. Isso evidencia eliminacao renal do produto ""1Tc-MAG3. Sabe-se, pela literatura, que o MAG3 que se encontra disponivel comercialmente apresenta alta afinidade pelo sistema urinario: rins e bexiga (FRITZBERG, A. R.; KASINA, S.; ESHIMA, D.; JOHNSON, D. L. Synthesis and biological evaluation of technetium-99m MAG3 as a Hippuran replacement. Journal of Nuclear Medicine, v. 27, n. 1, p. 111-116, 1986; ROBLES, B. R.; HERRERA, J.; CABALLERO, J.; OTERO, M. Purifϊcaciόn del benzoil-mercaptoacetil triglicina complementado con evaluaciones biolόgicas. Alasbimn Journal, v. 5, n. 21, 2003). Portanto, os dados obtidos de biodistribuicao do complexo ""1Tc-MAG3 sintetizado nesse trabalho encontram-se respaldados pela literatura.Table 3 shows that during the experiments, the organs with the highest radioactivity uptake were the kidneys and the bladder. This shows renal elimination of the " 1 Tc-MAG 3" product . Commercially available MAG 3 is known to have high affinity for the urinary system: kidneys and bladder (FRITZBERG, AR; KASINA, S.; ESHIMA, D .; JOHNSON, DL. Synthesis and biological evaluation of technetium. -99m MAG 3 as a Hippuran replacement.Journal of Nuclear Medicine, v. 27, no. 1, pp. 111-116, 1986; ROBLES, BR; HERRERA, J .; CABALLERO, J .; OTERO, M. Purifcaciόn del benzoyl mercaptoacetyl triglycine supplemented with biological evaluations (Alasbimn Journal, v. 5, no 21, 2003). Therefore, the data obtained from the biodistribution of the " 1 Tc-MAG 3 " complex synthesized in this work are supported by the literature.
Tabela 3 - Biodistribuicao ""1Tc-MAG3 em animais sadios (%dose/g).Table 3 - Biodistribution "" 1 Tc-MAG 3 in healthy animals (% dose / g).
Tecido 5 min 15 min 30 min 60 minFabric 5 min 15 min 30 min 60 min
Figado 5,78 ± 0,99 5,28 ± 0,54 14,29 ± 1,68 10,54 ± 1,09Liver 5.78 ± 0.99 5.28 ± 0.54 14.29 ± 1.68 10.54 ± 1.09
Baco 1,63 ± 0,25 1,85 ± 0,05 3,85 ± 0,50 3,18 ± 0,17Bacchus 1.63 ± 0.25 1.85 ± 0.05 3.85 ± 0.50 3.18 ± 0.17
Rins 6,99 ± 1,64 6,06 ± 0,45 13,74 ± 1,96 9,03 ± 1,66Kidneys 6.99 ± 1.64 6.06 ± 0.45 13.74 ± 1.96 9.03 ± 1.66
Estόmago 0,69 ± 0,12 0,91 ± 0,13 2,43 ± 0,33 2,06 ± 0,29Stomach 0.69 ± 0.12 0.91 ± 0.13 2.43 ± 0.33 2.06 ± 0.29
Coracao 1,34 ± 0,14 1,00 ± 0,14 2,34 ± 0,27 1,78 ± 0,24Heart 1.34 ± 0.14 1.00 ± 0.14 2.34 ± 0.27 1.78 ± 0.24
Pulmao 2,96 ± 0,44 2,37 ± 0,34 7,91 ± 0,70 5,41 ± 0,34Lung 2.96 ± 0.44 2.37 ± 0.34 7.91 ± 0.70 5.41 ± 0.34
Sangue 2,86 ± 0,15 1,60 ± 0,28 3,27 ± 0,21 1,98 ± 0,25Blood 2.86 ± 0.15 1.60 ± 0.28 3.27 ± 0.21 1.98 ± 0.25
Bexiga 27,70 ± 2,27 33,99 ± 3,84 85,25 ± 3,75 90,63 ± 6,41Bladder 27.70 ± 2.27 33.99 ± 3.84 85.25 ± 3.75 90.63 ± 6.41
Os resultados foram expressos como media ± desvio padrao («=5).Results were expressed as mean ± standard deviation (≤ = 5).
A biodistribuicao do derivado sacaridico ( 9mTc-MAG3-G) caracteriza-se por uma eliminacao renal mais rapida para este complexo. Observa-se pela tabela 4 e pela figura 1 que nos tempos de 5 e 15 min apόs a administracao foi verificado uma excrecao renal estatisticamente superior quando comparada com aquela observada para o "mTc- MAG3. Isso provavelmente pode ser explicado pelo fato da glicose ter contribuido para aumentar a hidrossolubilidade da molecula favorecendo a eliminacao renal. Dados da literatura mostram que moleculas hidrosolύveis sao eliminadas mais rapidamente (RANG, H. P.; DALE, M. M.; RITTER, J. M.; MOORE, P. K. Mecanismos celulares: proliferacao celular e apoptose. In: Farmacologia. 5. ed. Rio de Janeiro: Elsevier, 2004. Cap. 5, p. 77-89).Biodistribution of the saccharide derivative ( 9m Tc-MAG 3 -G) is characterized by faster renal elimination for this complex. Table 4 and Figure 1 show that at 5 and 15 min after administration a statistically higher renal excretion was observed when compared to that observed for " m Tc- MAG 3 . This can probably be explained by the fact that glucose contributed to increase the water solubility of the molecule favoring renal elimination. Literature data show that water-soluble molecules are eliminated faster (RANG, HP; DALE, MM; RITTER, JM; MOORE, PK. Cellular mechanisms: cell proliferation and apoptosis. In: Pharmacology. 5. ed. Rio de Janeiro: Elsevier, 2004 (Chapter 5, pp. 77-89).
Tabela 4 - Biodistribuicao "1Tc-MAG3-G em animais sadios (%dose/g).Table 4 - Biodistribution " 1 Tc-MAG 3 -G in healthy animals (% dose / g).
Tecido 5 min 15 min 30 min 60 minFabric 5 min 15 min 30 min 60 min
Figado 8,77 ± 1,22 5,29 ± 0,68 2,33 ± 0,17 1,82 ± 0,15Liver 8.77 ± 1.22 5.29 ± 0.68 2.33 ± 0.17 1.82 ± 0.15
Baco 0,99 ± 0,14 0,97 ± 0,05 0,42 ± 0,05 0,33 ± 0,04Bacchus 0.99 ± 0.14 0.97 ± 0.05 0.42 ± 0.05 0.33 ± 0.04
Rins 22,97 ± 3,91 8,06 ± 1,39 2,95 ± 0,41 1,24 ± 0,16Kidneys 22.97 ± 3.91 8.06 ± 1.39 2.95 ± 0.41 1.24 ± 0.16
Estδmago 1,74 ± 0,14 1,46 ± 0,20 0,77 ± 0,10 0,51 ± 0,06Stomach 1.74 ± 0.14 1.46 ± 0.20 0.77 ± 0.10 0.51 ± 0.06
Coracao 1,66 ± 0,24 1,06 ± 0,13 0,53 ± 0,07 0,34 ± 0,04Heart 1.66 ± 0.24 1.06 ± 0.13 0.53 ± 0.07 0.34 ± 0.04
Pulmao 2,11 ± 0,27 1,70 ± 0,20 0,75 ± 0,12 0,63 ± 0,08Lung 2.11 ± 0.27 1.70 ± 0.20 0.75 ± 0.12 0.63 ± 0.08
Sangue 3,28 ± 0,21 1,80 ± 0,21 0,76 ± 0,10 0,57 ± 0,06Blood 3.28 ± 0.21 1.80 ± 0.21 0.76 ± 0.10 0.57 ± 0.06
Bexiga 41,17 ± 5,40 71,30 ± 5,85 95,54 ± 3,05 94,24 ± 4,19Bladder 41.17 ± 5.40 71.30 ± 5.85 95.54 ± 3.05 94.24 ± 4.19
Os resultados foram expressos como media ± desvio padrao (n=5).Results were expressed as mean ± standard deviation (n = 5).
De uma maneira geral, observa-se pelas Tabelas 3 e 4 que os niveis de radioatividade presentes no estδmago e baco, durante todo o periodo, nao ultrapassaram 7%. Este dado e de grande importancia, pois indica que o teor de impurezas radioquimicas (""1TcO4 " e "1TcO2) presentes nas preparacόes injetadas estao dentro dos limites preconizados. Sabe-se, pela literatura, que o TcO4 " uma vez presente na solucao injetada e captado preferencialmente pelo estδmago, enquanto que o TcO2 e captado pelo baςo (THRALL, J. H.; ZIESSMAN, H. A. Medicina Nuclear. 2. ed. Rio de Janeiro: Guanabara Koogan, 2003. p. 408). No sangue os resultados de biodistribuicao do ""1Tc-MAG3 mostraram um perfil de clareamento bifasico (figura 2). Este mesmo perfil encontra-se descrito na literatura para o ""1Tc-MAG3 encontrado comercialmente e disponivel para os exames de medicina nuclear (SAHA, G. B. Fundamentals of nuclear pharmacy. 4.ed. New York: Springer- Verlag, 1998a. 358 p). Esse dado corrobora para o sucesso da sintese do mercaptoacetiltriglicina (MAG3) obtida nessa invencao. Outro aspecto que merece destaque e que, quando a molecula de glicose encontra-se ligada a estrutura do MAG3, o composto ""1Tc-MAG3-G apresenta um perfil de biodistribuicao monofasico no sangue. Esses comportamentos distintos foram reproduzidos nos όrgaos que apresentam maior aporte sangϋineo, como fϊgado (figura 3), coracao (figura 4) e pulmao (figura 5). Esses resultados mostraram, de forma inequivoca, que a presenca da molecula de glicose ligada ao MAG3 modifica a biodistribuicao do mesmo. Portanto, trata-se de produtos distintos apresentando perfis de biodistribuicao caracteristicos (KIM, L; KOBAYASHI, H.; YOO, T. M.; KIM, M.; LE, N.; HAN, E.; WANG, Q-C; PASTAN, L; CARRASQUILLO, J. A.; PAIK, C. H. Lowering of pi by acylation improves the renal uptake of 99mTc-labeled anti-Tac ds Fv: effect of different acylating reagents. Nuclear Medicine and Biology, v. 29, p. 795-801, 2002; CHEN, X.; LI, L.; LIU, F.; LIU, B. Synthesis and biological of technetium-99m-labeled deoxyglucose derivatives as imaging agents for tumor. Bioorganic and Medicinal Chemistry Letters, v. 16, p. 5503- 5506, 2006).In general, it is observed from Tables 3 and 4 that the levels of radioactivity present in the stomach and bacchus during the whole period did not exceed 7%. This data is very important because it indicates that the content of radiochemical impurities ("" 1 TcO 4 " and" 1 TcO 2 ) present in the injected preparations are within the recommended limits. It is known from the literature that TcO 4 " once present in the injected solution is preferentially captured by the stomach, whereas TcO 2 is captured by the shell (THRALL, JH; ZIESSMAN, HA Nuclear Medicine. 2. ed. Rio de January: Guanabara Koogan, 2003. p. 408) In the blood the biodistribution results of the " 1 Tc-MAG 3 showed a biphasic whitening profile (Figure 2). This same profile is described in the literature for"" 1 Tc-MAG 3 commercially available and available for nuclear medicine examinations (SAHA, GB Fundamentals of nuclear pharmacy. 4.ed.New York: Springer-Verlag, 1998a. 358p.) This data corroborates the successful synthesis of Mercaptoacetyltriglycine (MAG 3 ) obtained in this invention Another noteworthy aspect is that when the glucose molecule is linked to the structure of MAG 3 , the compound " 1 Tc-MAG 3 -G has a monophasic biodistribution profile in the blood. These distinct behaviors were reproduced in the organs with the highest blood supply, such as liver (Figure 3), heart (Figure 4) and lung (Figure 5). These results have unequivocally shown that the presence of the MAG 3- linked glucose molecule modifies its biodistribution. Therefore, these are distinct products with characteristic biodistribution profiles (KIM, L; KOBAYASHI, H .; YOO, TM; KIM, M .; LE, N .; HAN, E .; WANG, QC; PASTAN, L; CARRASQUILLO, JA; PAIK, CH Lowering of pi by acylation improves renal uptake of 99m Tc-labeled anti-Tac ds Fv: effect of different acylating reagents Nuclear Medicine and Biology, v. 29, pp 795-801, 2002; CHEN, X .; LI, L .; LIU, F .; LIU, B. Synthesis and biological of technetium-99m-labeled deoxyglucose derivatives as imaging agents for tumor. Bioorganic and Medicinal Chemistry Letters, v. 16, p 5503- 5506, 2006).
Os padrδes de biodistribuicao apresentados nas tabelas acima foram avaliados pelo teste t. Valores de p < 0,05 foram considerados com significancia estatistica. EXEMPLO 3- Implantaςao do tumor Ehrlich e estudos de biodistribuicao.The biodistribution patterns presented in the tables above were evaluated by the t test. P values <0.05 were considered statistically significant. EXAMPLE 3- Ehrlich tumor implantation and biodistribution studies.
Inicialmente, celulas de tumor de Ehrlich foram implantadas, intraperitonealmente, em camundongos SWISS, machos, pesando 25 a 30 g, com o intuito de produzir urn foco de tumor ascitico. Utilizaram-se para este experimento oito animais que apόs 8 dias do implante foram anestesiados, intraperitonealmente, com solucao de Ketamine/Xylazine em dose de 40 mg/Kg e 5 mg/Kg, respectivamente. Em seguida, os animais foram sacrificados. Retirou-se o liquido ascitico contendo celulas tumorais viaveis, que foram transferidas para um tubo Falcon esteril de 50 mL. Centrifugou-se o liquido ascitico a 3000 vezes a forca gravitacional por 5 minutos e o precipitado foi ressuspendido com solucao salina a 0,9%. Esse processo foi repetido e, em seguida, as celulas foram coradas com solucao de Tripan 0,4% e contadas em microscόpio com aumento de 400 vezes com o auxilio de uma camara de Newbawer, determinando a concentracao da solucao contendo as celulas tumorais.Initially, Ehrlich's tumor cells were implanted intraperitoneally into male SWISS mice weighing 25 to 30 g in order to produce an ascites tumor focus. Eight animals were used for this experiment after 8 days of implant were anesthetized intraperitoneally with a solution of Ketamine / Xylazine at a dose of 40 mg / kg and 5 mg / kg, respectively. Then the animals were sacrificed. Ascites fluid containing viable tumor cells was removed and transferred to a sterile 50 ml Falcon tube. The ascites liquid was centrifuged at 3000 times the gravitational force for 5 minutes and the precipitate was resuspended with 0.9% saline. This process was repeated and then the cells were stained with 0.4% Trypan solution and counted under a 400-fold magnification with the aid of a Newbawer chamber, determining the concentration of the solution containing the tumor cells.
A solucao contendo as celulas de tumor de Ehrlich foi novamente centrifugada e o sobrenadante (salina) foi retirado. O sedimento contendo as celulas tumorais foi ressuspendido, utilizando o mesmo volume do sobrenadante retirado, com meio para congelamento contendo soro fetal bo vino inativado a 57 0C por 1 hora e dimetilsulfόxido na proporcao de 9:1. As celulas foram aliquotadas em criotubos de 1,5 mL com concentracao de 108 celulas/mL sendo posteriormente armazenadas em nitrogenio. Para os estudos de biodistribuicao em animais com tumor sόlido, foram utilizados 20 camundongos SWISS, machos, pesando 25 a 30 g, para cada experiment*) (Bz-MAG3 (1) e o Bz-MAG3-G (2) marcados com 99mTc divididos em 4 grupos de 5 animais cada. Implantou-se, via subcutanea, na pata posterior direita, o volume de 0,1 mL de solucao salina a 0,9% contendo, aproximadamente, 106 celulas tumorals. Os animas foram mantidos em um bioterio por 15 a 20 dias sem restricao de alimento e agua. Administraram-se, entao, os derivados (1) e (2) radiomarcados. Os camundongos foram anestesiados, por via intraperitoneal, com solucao de Ketamine/Xylazine em dose de 40 mg/Kg e 5 mg/Kg respectivamente, e sacrificados nos tempos de 5, 30, 12O e 240 minutos apόs a administracao. Figado, baco, rins, estδmago, coracao, pulmao, sangue, bexiga, tumor (pata posterior direita) e musculo controle (pata posterior direita) foram retirados, pesados e colocados em um contador gama de poco para a determinacao da radioatividade, bem como o padrao de dose relativo ao experimento.The solution containing the Ehrlich tumor cells was centrifuged again and the supernatant (saline) was removed. The pellet containing the cells was resuspended tumor, using the same volume of supernatant was removed with medium containing fetal calf serum for freezing bo vino inactivated at 57 0 C for 1 hour and dimetilsulfόxido the ratio of 9: 1. The cells were aliquoted into 1.5 mL cryovials with a concentration of 10 8 cells / mL and subsequently stored in nitrogen. For biodistribution studies in solid tumor animals, 20 male SWISS mice weighing 25 to 30 g were used for each experiment *) (Bz-MAG 3 (1) and Bz-MAG 3 -G (2) labeled with 99m Tc divided into 4 groups of 5 animals each, the volume of 0.1 ml 0.9% saline containing approximately 10 6 tumor cells was implanted subcutaneously into the right hind paw. were kept in a bioterium for 15 to 20 days without food and water restriction, radiolabelled derivatives (1) and (2) were then administered.The mice were anesthetized intraperitoneally with Ketamine / Xylazine solution in 40 mg / kg and 5 mg / kg respectively, and sacrificed at 5, 30, 12, and 240 minutes after administration: liver, bacchus, kidneys, stomach, heart, lung, blood, bladder, tumor (hind paw right) and control muscle (right hind paw) were removed, weighed and placed in a well gamma counter for as well as the dose pattern for the experiment.
Celulas de tumor de Ehrlich sao celulas transplantaveis provenientes de adenocarcinoma mamario de camundongos femeas, que se desenvolvem rapidamente. Tais celulas tern sido utilizadas como modelo no estudo de diversas substantias, como agentes terapeuticos e agentes de diagnόstico (SEGURA, J. A.; BARBERO, L; G.; MARQUEZ. J. Ehrlich ascites tumor imbalances splenic cell populations and reduces responsiveness of T cells to Staphylococcus aureus enterotoxin B stimulation. Immunology Letters, v. 74, p. 111-115, 2000; OLORIS, S. C. S.; DAGLI, M. L. Z.; GUERRA, J. L. Effect of β-carotene on the development of the solid Ehrlich tumor in mice. Life Science, v. 71, p. 717-724, 2002; SILVA, A. E.; SERAKIDES, R.; FERREIRA, E.; MORAES, J. R. C; OCARINO, N. M.; CASSALI, G. D. Efeito do hipertiroidismo no tumor de Ehrlich sόlido em camundongos femeas castradas e nao castradas. Arq. Bras. Endocrinol. Metabol., v. 48, n. 6, p. 867-874, 2004; FERREIRA, E.; SILVA, A. E.; SERAKIDES, R.; GOMES, M. G.; CASSALI, G. D. Ehrlich tumor as model to study artificial hyperthyroidism influence on breast cancer. Pathology: Research and practice, v. 203, p. 39-44, 2007).Ehrlich tumor cells are transplantable cells from rapidly developing female mouse mammary adenocarcinoma. Such cells have been used as a model in the study of several substantia, as therapeutic agents and diagnostic agents (SEGURA, JA; BARBERO, L; G .; MARQUEZ. J. Ehrlich ascites tumor imbalances splenic cell populations and responsiveness of T cells to Staphylococcus aureus enterotoxin B. stimulation Immunology Letters, v. 74, pp. 111-115, 2000; Oloris, SCS; DAGLI, MLZ; WAR, JL Effect of β-carotene on the development of solid Ehrlich tumor in mice. , v. 71, pp. 717-724, 2002; SILVA, AE; SERAKIDES, R.; FERREIRA, E .; MORAES, JR C. OCARINO, NM; CASSALI, GD Effect of hyperthyroidism on solid Ehrlich tumor in female mice. castrated and non-castrated Arq. Bras Endocrinol Metabol, v. 48, no. 6, pp. 867-874, 2004; FERREIRA, E.; SILVA, AE; SERAKIDES, R.; GOMES, MG; CASSALI, GD Ehrlich tumor as model to study artificial hyperthyroidism influence on breast cancer Pathology: Research and practice, v. 203, p. 44, 2007).
Os estudos de biodistribuicao dos compostos marcados (""1Tc-MAG3 e 99mTc- MAG3-G) em animais portadores de tumores, nao apresentaram diferencas estatisticamente significativas para o sistema urinario, quando comparado com resultados obtidos em animais sadios (Tabelas 5 e 6). Portanto, a presenca do tumor nao alterou o tropismo dos compostos marcados pelo sistema renal, mantendo o alto nivel de excrecao renal verificado para os radiofarmacos testados. Tabela 5 - Biodistribuicao no sistema urinario de ""1Tc-MAG3 em animais sadios e com tumor (%dose/g)The biodistribution studies of the labeled compounds ("" 1 Tc-MAG 3 and 99m Tc-MAG 3 -G) in tumor bearing animals did not show statistically significant differences for the urinary system when compared to results obtained in healthy animals (Tables 5 and 6). Therefore, the presence of the tumor did not alter the tropism of compounds labeled by the renal system, maintaining the high level of renal excretion observed for the radiopharmaceuticals tested. Table 5 - Urinary system biodistribution of " 1 Tc-MAG 3" in healthy and tumor animals (% dose / g)
Animais 5 min 30 minAnimals 5 min 30 min
Sadios 34,69 ± 2,30 98,99 ± 2,75Healthy 34.69 ± 2.30 98.99 ± 2.75
Tumor 40, 17 ± 6,94 96,68 ± 6,19Tumor 40, 17 ± 6.94 96.68 ± 6.19
Os resultados foram expressos como media ± desvio padrao («=5). Os valores apresentados nao sao estatisticamente diferentes (p>0,05).Results were expressed as mean ± standard deviation (≤ = 5). The values presented are not statistically different (p> 0.05).
Tabela 6 - Biodistribuicao no sistema urinario de ""1Tc-MAG3-G em animais sadios e com tumor (%dose/g)Table 6 - urinary biodistribution of "system" Tc-MAG 1 -G 3 and in healthy animals with tumors (% dose / g)
Animais 5 min 30 minAnimals 5 min 30 min
Sadios 64,14 ± 6,36 98,49 ± 3,40Healthy 64.14 ± 6.36 98.49 ± 3.40
Tumor 58,87 ± 6,61 99,66 ± 12,97Tumor 58.87 ± 6.61 99.66 ± 12.97
Os resultados foram expressos como media ± desvio padrao («=5). Os valores apresentados nao sao estatisticamente diferentes (p>0,05).Results were expressed as mean ± standard deviation (≤ = 5). The values presented are not statistically different (p> 0.05).
Avaliou-se, tambem, a radioatividade presente no tecido tumoral (pata posterior direita) e no tecido muscular (pata posterior esquerda). Desta maneira, foi possivel expressar os resultados em forma da relacao alvo/nao alvo tomando-se como premissa que, o musculo da pata posterior esquerda foi considerado a regiao nao alvo. Os dados da Tabela 7 mostraram relacao superior a 2,0 para todos os tempos investigados. Isto sugere que o 9mTc-MAG3-G foi 100% mais captado pelo tumor do que o ""1Tc-MAG3. Substantias que apresentarem relacao alvo/nao alvo maior que 1,5 (captacao 50% maior no tecido alvo) podem ser consideradas como potenciais agentes de diagnόstico (PHILLIPS, W.T. Delivery of gamma-imaging agents by liposomes. Advanced grug delivery reviews, v. 37, p. 13-32, 1999). Portanto, os dados obtidos sugerem que o complexo ""1Tc-MAG3-G pode ser empregado para a identifϊcacao de tumores.Radioactivity present in the tumor tissue (right hind paw) and muscle tissue (left posterior paw) was also evaluated. In this way, it was possible to express the results in the form of the target / non-target relationship, assuming that the left hind paw muscle was considered the non-target region. The data in Table 7 showed a ratio higher than 2.0 for all investigated times. This suggests that 9m Tc-MAG 3 -G was captured by 100% more tumors than the "" Tc-MAG 3 1. Substances that have a target / non-target ratio greater than 1.5 (50% higher uptake in the target tissue) may be considered as potential diagnostic agents (PHILLIPS, WT Delivery of gamma-imaging agents. 37, pp. 13-32, 1999). Therefore, the data obtained suggest that the " 1 Tc-MAG 3 -G" complex can be used for tumor identification.
Tabela 7 - Relacao Alvo/Nao-Alvo de ""1Tc-MAG3 e ""1Tc-MAG3-G Derivado testado 5 min 30 min 120 min 240 minTable 7 - Target / Non-Target Ratio of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G Derivative tested 5 min 30 min 120 min 240 min
""1Tc-MAG3 l,22a ± 0,14 l,22a ± 0,10 l,16a ± 0,08 l,10a ± 0,l l"" 1 Tc-MAG 3 l, 22 at ± 0.14 l, 22 at ± 0.10 l, 16 at ± 0.08 l, 10 at ± 0.11
""1Tc-MAG3-G 2,05b ± 0,25 2,22b ± 0,24 2,13b ± 0,12 2,10b ± 0,24 Os resultados foram expressos como media ± desvio padrao (w=5). Os valores foram avaliados pelo teste Tukey-Kramer. Letras diferentes indicam diferenςas estatisticamente significativa. EXEMPLO 6- Imagens cintilograficas"" 1 Tc-MAG 3 -G 2,05 b ± 0,25 2,22 b ± 0,24 2,13 b ± 0,12 2,10 b ± 0,24 Results were expressed as mean ± standard deviation (w = 5). The values were evaluated by Tukey-Kramer test. Different letters indicate statistically significant differences. EXAMPLE 6- Scintigraphic images
Para o estudo cintilografico os produtos Bz-MAG3 (I) e Bz-MAG3-G (2) foram radiomarcados com uma aliquota da solucao de pertecnetado de sόdio com atividade de 185 MBq, afim de se obter atividade suficiente para aquisicao das imagens. As imagens foram obtidas em uma gama camara, com colimador low energy high resolution (LEHR), matriz 256x256x16 e tempo de aquisicao de 600 segundos. Foram utilizados 3 animais, com tumor de Ehrlich implantado na pata posterior direita, para cada experimento (""1Tc-MAG3 e o ""1Tc-MAG3-G) e as imagens foram adquiridas nos tempos de 5, 30, 120 e 240 minutos. O estudo cintilografico foi realizado em camara de cintilacao a raios gama, comFor the scintigraphic study Bz-MAG 3 (I) and Bz-MAG 3 -G (2) products were radiolabelled with an aliquot of the 185 MBq active sodium pertechnetate solution in order to obtain sufficient activity for image acquisition. . The images were obtained in a camera range, with low energy high resolution (LEHR) collimator, 256x256x16 matrix and 600 second acquisition time. Three animals with Ehrlich's tumor implanted in the right hind paw were used for each experiment ("" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G) and the images were acquired at 5, 30, 120 and 240 minutes. The scintigraphic study was performed in a gamma-ray scintillation chamber with
3 animais por experimento (""1Tc-MAG3 e ""1Tc-MAG3-G). As imagens foram obtidas nos tempos de 5, 30, 12O e 240 minutos apόs a administracao do produto a ser testado (""1Tc-MAG3 ou ""1Tc-MAG3-G), como mostrado na fϊgura 5.3 animals per experiment ("" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G). Images were taken at 5, 30, 12O and 240 minutes after administration of the product to be tested ("" 1 Tc-MAG 3 or "" 1 Tc-MAG 3 -G), as shown in Figure 5.
Foi selecionada a regiao de interesse (ROI) nas patas posteriores direita (alvo) e esquerda (nao-alvo) e com isso foi possivel determinar a area e a atividade presente em cada ROI. Alem disso, obtiveram-se as relacόes alvo/nao alvo corrigidas pela area de cada regiao. Essa relacao para o ""1Tc-MAG3-G manteve-se prόxima a dois e para o ""1Tc-MAG3 prόxima a 1, como mostrado na Tabela 8, sendo estatisticamente diferentes pelo teste de Tukey-Kramer.The region of interest (ROI) in the right (target) and left (non-target) hind paws was selected and it was thus possible to determine the area and activity present in each ROI. In addition, the target / non-target relations corrected by the area of each region were obtained. This ratio for " 1 Tc-MAG 3 -G remained close to two and for"" 1 Tc-MAG 3 close to 1, as shown in Table 8, being statistically different by the Tukey-Kramer test.
Tabela 8 - Relacao Alvo/Nao-Alvo de ""1Tc-MAG3 e ""1Tc-MAG3-G obtida por imagens em camara de cintilacao a raios gama (%dose/cm2).Table 8 - Target / Non-Target Ratio of "" 1 Tc-MAG 3 and "" 1 Tc-MAG 3 -G obtained by gamma scintillation camera images (% dose / cm 2 ).
DerivadoDerivative
5 min 30 min 120 min 240 min testado5 min 30 min 120 min 240 min tested
""1Tc-MAG3 l,23a ± 0,02 l,09a ± 0,07 l,18a ± 0,12 l,13a ± 0,13 ""1Tc-MAG3-G l,80b ± 0,10 2,02b ± 0,09 l,95b ± 0,12 l,93b ± 0,06 Os resultados foram expressos como media ± desvio padrao (n=3). Os valores foram avaliados pelo teste Tukey-Kramer."" 1 Tc-MAG 3 l, 23 at ± 0.02 l, 09 at ± 0.07 l, 18 at ± 0.12 l, 13 at ± 0.13 "" 1 Tc-MAG 3 -G l, 80 b ± 0.10 2.02 b ± 0.09 l, 95 b ± 0.12 l, 93 b ± 0.06 Results were expressed as mean ± standard deviation (n = 3). The values were evaluated by Tukey-Kramer test.
As analises das imagens apresentaram dados que foram congruentes com os estudos de biodistribuicao (nao apresentando diferencas estatisticas pelo teste t (p>0,05)), como observado nas Tabelas 9 e 10. A coerencia dos dados foi mantida tambem para os demais όrgaos nao sendo observado sinais de impurezas radioquimicas. Tabela 9 - Relacao Alvo/ Nao Alvo apresentada pelo ""1Tc-MAG3 nos estudos de biodistribuicao (%dose/g) e imagens cintilografϊcas (%dose/cm2) Tipo de analise 5 min 30 min 120 min 240 minImage analyzes presented data that were consistent with the biodistribution studies (showing no statistical differences by the t-test (p> 0.05)), as observed in Tables 9 and 10. Data coherence was also maintained for the other organs. no signs of radiochemical impurities were observed. Table 9 - Target / Non-Target Ratio presented by " 1 Tc-MAG 3" in biodistribution studies (% dose / g) and scintigraphic images (% dose / cm 2 ) Type of analysis 5 min 30 min 120 min 240 min
BiodistribuicaoBiodistribution
1,22 ± 0,14 1,22 ± 0,10 1,16 ± 0,08 1,10 ± 0,111.22 ± 0.14 1.22 ± 0.10 1.16 ± 0.08 1.10 ± 0.11
(n=5)(n = 5)
Imagens («=3) 1,23 ± 0,02 1,09 ± 0,07 1,18 ± 0,12 1,13 ± 0,13Images ('= 3) 1.23 ± 0.02 1.09 ± 0.07 1.18 ± 0.12 1.13 ± 0.13
Os resultados foram expressos como media ± desvio padrao. Os valores foram avaliados pelo teste t (p>0,05).Results were expressed as mean ± standard deviation. The values were evaluated by the t test (p> 0.05).
Tabela 10 - Relacao Alvo/ Nao Alvo apresentada pelo ""1Tc-MAG3-G nos estudos de biodistribuicao (%dose/g) e imagens cintilograficas (%dose/cm2) Tipo de analise 5 min 30 min 120 min 240 minTable 10 - Target / Non-Target Ratio presented by " 1 Tc-MAG 3 -G" in biodistribution studies (% dose / g) and scintigraphic images (% dose / cm 2 ) Type of analysis 5 min 30 min 120 min 240 min
BiodistribuicaoBiodistribution
2,05 ± 0,25 2,22 ± 0,24 2,13 ± 0,12 2,10 ± 0,242.05 ± 0.25 2.22 ± 0.24 2.13 ± 0.12 2.10 ± 0.24
(n=5)(n = 5)
Imagens (n=3) 1,80 ± 0,10 2,02 ± 0,09 1,95 ± 0,12 1,93 ± 0,06Images (n = 3) 1.80 ± 0.10 2.02 ± 0.09 1.95 ± 0.12 1.93 ± 0.06
Os resultados foram expressos como media ± desvio padrao. Os valores foram avaliados pelo teste t (p>0,05). Results were expressed as mean ± standard deviation. The values were evaluated by the t test (p> 0.05).

Claims

REIVINDICACOES
1. RADIOFARMACOS DERIVADOS DE MAG3, caracterizadas por compreenderem a seguinte formula estrutural acoplada ao Tecnecio-99m:1. MAG 3- DERIVED RADIOPHARMACES, characterized in that they comprise the following structural formula coupled to the Tecnecio-99m:
Figure imgf000021_0001
Figure imgf000021_0001
com as possiveis substituicόes: a) Os radicals Rl por α- ou β-D-glicopiranosil, α- ou β -D-galactopiranosil, α- ou β -D-manopiranosil, α- ou β -L-fucopiranosil, 2-amino-2-desoxi- α- ou β -D- glicopiranosil, 2-acetilamino-2-desoxi α- ou β -D-glicopiranosil; b) Os radicals R2 por metil, fenil, Triclorometil, Trifluorometil, 4-nitrofenil; c) A porcao X por -OC6H4NH (orto, meta, para), ou -SC6H4NH (orto, meta, para),with possible substitutions: a) R1 radicals by α- or β-D-glycopyranosyl, α- or β-D-galactopyranosyl, α- or β-D-manopyranosyl, α- or β -L-fucopyranosyl, 2-amino -2-deoxy-α- or β-D-glycopyranosyl, 2-acetylamino-2-deoxy-α or β-D-glycopyranosyl; b) R 2 radicals by methyl, phenyl, Trichloromethyl, Trifluoromethyl, 4-nitrophenyl; (c) the X portion by -OC 6 H 4 NH (ortho, meta, para), or -SC 6 H 4 NH (ortho, meta, para),
Ou -O(CH2)nNH, -S(CH2)nNH (n = 1, Or -O (CH 2 ) n NH, -S (CH 2 ) n NH (n = 1,
2, 2,
3, 4, 5, 6), ou por -NH; 2. COMPOSiςόES FARMACEUTICAS DE RADIOFARMACO DERIVADO DE MAG3 caracterizadas por compreenderem o radiofarmaco descrito na reivindicacao 1; 3. COMPOSiςόES FARMACEUTICAS DE RADIOFARMACO DERIVADO3, 4, 5, 6), or by -NH; PHARMACEUTICAL COMPOSITIONS OF MAG 3 DERIVATIVE RADIOPharmacy comprising the radiopharmaceutical described in claim 1; 3. PHARMACEUTICAL COMPOSITIONS OF DERIVATIVE RADIOPharmacy
DE MAG3 de acordo com as reivindicacao 2 caracterizadas por compreenderem um veiculo farmaceutica e fϊsiologicamente aceitavel para uso humano como agua para iηjetaveis, solucao salina esteril, solucao tampao, mas nao limitantes;DE MAG 3 according to claims 2, characterized in that they comprise a pharmaceutically and physiologically acceptable vehicle for human use such as water for injections, sterile saline, buffer solution, but not limiting;
4. COMPOSiςόES FARMACEUTICAS DE RADIOFARMACO DERIVADO DE MAG3 de acordo com as reivindicacδes de 2-3 caracterizadas por compreenderem excipientes inertes farmaceuticamente aceitaveis tais como: amido, lactose, celulose microcristalina, hidroxipropilmetilcelulose, carboximetilcelulose, talco, estearato de magnesio, mas nao limitantes;PHARMACEUTICAL COMPOSITIONS OF MAG 3 DERIVATIVE RADIOPHARMAC according to claims 2-3 characterized in that they comprise pharmaceutically acceptable inert excipients such as starch, lactose, microcrystalline cellulose, hydroxypropyl methylcellulose, talc, non-limiting magnesium stearate;
5. COMPOSiςόES FARMACEUTICAS DE RADIOFARMACO DERIVADO DE MAG3 de acordo com as reivindicacδes 2-4, caracterizadas por serem administradas pelas vias: oral, intramuscular, intravenosa, subcutanea, transdermica ou como dispositivos que possam ser implantados ou injetados; PHARMACEUTICAL COMPOSITIONS OF MAG 3 DERIVATIVE RADIOPharmacy according to Claims 2-4, characterized in that they are administered by oral, intramuscular, intravenous, subcutaneous, transdermal or as devices which may be implanted or injected;
6. COMPOSiςόES FARMACEUTICAS DE RADIOFARMACO DERIVADO DE MAG3 de acordo com as reivindicacδes 2-5, caracterizadas por compreenderem perils de biodistribuicao caracteristicos descritos;PHARMACEUTICAL COMPOSITIONS OF MAG 3 DERIVATIVE RADIOPharmacy according to claims 2-5, characterized in that they comprise the characteristic biodistribution profiles described;
7. COMPOSiςόES FARMACEUTICAS DE RADIOF ARMACO DERIVADO DE MAG3 de acordo com as reivindicacδes 2-6, caracterizadas por apresentarem eliminacao renal mais rapida;PHARMACEUTICAL COMPOSITIONS OF MAG 3 DERIVED ARMAC RADIOF according to Claims 2-6, characterized in that they have faster renal elimination;
8. COMPOSiςόES FARMACEUTICAS DE RADIOF ARMACO DERIVADO DE MAG3 de acordo com as reivindicacδes 2-7, caracterizadas por compreenderem uma maior captacao por celulas tumorais; 9. COMPOSiςόES FARMACEUTICAS DE RADIOF ARMACO DERIVADOPHARMACEUTICAL COMPOSITIONS OF MAG 3 DERIVED ARMAC RADIOF according to claims 2-7, characterized in that they comprise increased uptake by tumor cells; 9. PHARMACEUTICAL COMPOSITIONS OF RADIOF ARMACO DERIVATIVE
DE MAG3, de acordo com as reivindicacδes 2-8, caracterizadas por compreenderem o perfil das imagens cintilograficas apresentados;DE MAG 3 according to claims 2-8, characterized in that they comprise the profile of the scintigraphic images presented;
10. COMPOSiςόES FARMACEUTICAS DE RADIOF ARMACO DERIVADO DE MAG3, caracterizadas por ser na preparacao de medicamentos para diagnόstico de processos oncolόgicos conforme descrito nas reivindicacδes de 2-PHARMACEUTICAL COMPOSITIONS OF MAG 3 DERIVATIVE ARMAC RADIOF, characterized in that it is in the preparation of medicaments for the diagnosis of cancer processes as described in the claims of 2.
4;4;
11. METODO PARA DIAGNOSTICO DE CELULAS TUMORAIS E DISTURBIOS NEOPLASICOS, caracterizado pelo fato da administracao da composicao farmaceutica, descrita na reivindicacao 2-4, por uma pessoa que necessite do tratamento;METHOD FOR TUMOR CELL DIAGNOSTIC AND NEOPLASIC DISTURBIES, characterized by the administration of the pharmaceutical composition described in claim 2-4 by a person in need of treatment;
12. METODO PARA ESTUDO DE DIAGNOSTICO DE CELULAS TUMORAIS E DISTURBIOS NEOPLASICOS caracterizado pelo fato da administracao da composicao descrita na reivindicacao 2-4; METHOD FOR STUDY DIAGNOSTIC STUDY AND NEOPLASIC DISORDERS STUDY characterized by the administration of the composition described in claim 2-4;
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Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0483704A1 (en) * 1990-10-31 1992-05-06 Nihon Medi-Physics Co., Ltd. Process for preparing a radiopharmaceutical composition
WO1999017811A1 (en) * 1997-10-08 1999-04-15 David Platt Enhancement of delivery of radioimaging and radioprotective agents
US5980861A (en) * 1996-03-12 1999-11-09 University Of Massachusetts Chelator compositions and methods of synthesis thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0483704A1 (en) * 1990-10-31 1992-05-06 Nihon Medi-Physics Co., Ltd. Process for preparing a radiopharmaceutical composition
US5980861A (en) * 1996-03-12 1999-11-09 University Of Massachusetts Chelator compositions and methods of synthesis thereof
WO1999017811A1 (en) * 1997-10-08 1999-04-15 David Platt Enhancement of delivery of radioimaging and radioprotective agents

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