The method of obtaining an anti-tumor effect in mammals


(57) Abstract:

The invention relates to medicine. The method is based on the introduction of the active therapeutic compound in the reduced form (e.g. 5-substituted uracil), which is converted in the liver cells by aldehyde oxidase in bioactive dietoterapia drug or metabolite. It is shown that the prodrug is effective in the treatment of diseases, and the drugs, and have numerous benefits and are denied many side effects. 15 C. p. F.-ly, 9 Il., 7 table.

The present invention relates to prodrugs, turning the liver, and more specifically refers to the treatment of diseases with the use of prodrugs, which is transformed by the enzyme(s) of the liver to the active medication. It also relates to a method for establishing the usefulness of prodrugs for the treatment of disease.

Iodosuccinimide (IUGR) was synthesized as protivoopujolevoe tools in 1959 Century, Prusoff, (Biochem. Biophys. Acta, 1959, 32, 295 - 296) and was the first analogue of thymidine, is used clinically as anothermessage funds (Archs. Ophthalmol., 1962, 68, 235 - 239). Toxicity iodosuccinimide the systems use limited its clinical use. Youpii cancer (J. Radiation Oncology Biol. Phys, 1984, 10, 1399 - 1406). The degree of radiosensibility directly depends on the number of substituted thymidine into the DNA of its analogues (Clin. Pharmacol, Ther. , 1988, 44, 369 - 375). Some success had intrahepatic infusion iodosuccinimide with subsequent irradiation in the treatment of tumor cells of liver Cancer. Res. 1989, 49, 6437 - 6442).

When attempting to develop selective anti-herpes virus on the basis of a wider range of substrate specificity timedancing herpes virus compared with the encoded thymidine kinase man was synthesized 5-iodine-2-pyrimidine-deoxyribose (Ipdr), which differs from IUGR exography and in position 4 of the base. It was found that IPT has strong activity against herpes viruses HSV-1 and HSV-2 in cell cultures and against HSV-2 in mice (Antimicrob. Agents Chemother, 1989, 33, 340 - 344). This tool was not toxic nor for neinfizirovanne cells or mice when administered orally in the doses (Antimocrob. Agents Chemither, 1989, 33, 340 - 344). As Ipdr and Iudr structurally similar, was tested the possibility of transforming Ipdr in IUGR. Previously it was shown that IPT cannot be turned into Iudr by xanthine oxidase (Antimicrob. Agents Chemother., 1989, 33, 340 - 344).

U.S. patent No. 4 895 937 discloses nucleoside 1-(2-is the actual content of U.S. patent No. 4 895 937 included in the consideration.


IUdR: iodosuccinimide (Iudr)

FUdR: ftordezoksiuridin (Fudr)

IPdR: 5-iodine-2-pyrimidinecarboxylic (Ipdr)

HSV: herpes virus

HPLC: high performance liquid chromatography (HPLC)

IU: iodouracil (PS)

EPdR: 5-ethinyl-2-pyrimidinecarboxylic (Apdr)

IP: 5-iodine-2-pyrimidinone (FE)

BPdR: 5-bromo-2-pyrimidinecarboxylic (Bpdr)

MPdR: 5-methyl-2-pyrimidinecarboxylic (Mpdr)

EtPdR: 5-ethyl-2-pyrimidinecarboxylic (ICPDR)

BudR: 5-bromosuccinimide (Budr)

HBV: hepatitis B virus

FU: 5 - fluorouracil (FU)

FP: 5-fluoro-2-pyrimidinone (AF)

ddI: dideoxyinosine (ddI)

ddG: digitoxigenin (FGD)

DHPG: ganciclovir (9-/1,3-dihydroxy-2-propoxy)methyl/guanine) DHPG)

ACV: (S)-N-/N-(5-amino-5-carboxy-1-oxobutyl)-L-cysteinyl/-D - valine (ACV)

D4T: 21,31-dideoxy-21,31- didehydrothymidine (DT)

AZT: 31-azido-31-deoxythymidine (AZT)

The purpose of the present invention is to provide preparations for use as prodrugs that are converted in the liver of mammals in the biologically active substance, particularly a biologically active substance, which is believed to exhibit its biological activity in petcareforum, content substituted in position 5 equivalents of the Da, especially Ipdr, for use as prodrugs, which is transformed in the liver mammalian (especially human liver) in situ into the corresponding biologically active substituted in position 5 derivatives RDL.

Another aim of the present invention is to use such substituted in position 5 equivalents of the Da for the treatment related with liver disease, especially as radiosensibility for the treatment of hepatic cell carcinoma.

Another aim of the present invention is to provide drugs other than substituted in position 5 analogues of the Da, for use as prodrugs, which is transformed in the liver of mammals in the biologically active substance, particularly a biologically active substance, which is believed exerts its biological effects in the liver or which cannot be administered orally.

Another aim of the present invention is to provide such drugs other than substituted in position 5 analogues of the Da, which are analogues substituted in position 5 pyrimidinone, especially foreminem.

Another aim of the present invention is to provide such drugs ex is active nucleosides or bases, nucleosides, other than the RDL and uracil, preferably guanosine analogues, cytidine, inosine or thymine.

Another aim of the present invention is to provide improvements in the treatment of disease.

Another aim of the present invention is to provide a method of establishing the usefulness of prodrugs for the treatment of disease.

Another aim of the present invention is to provide a method for determining preorasenesti prodrug enzyme of the liver in a biologically active substance acting on any cell in the body.

Another aim of the present invention is to provide a method of treating disease using prodrugs.

Another aim of the present invention is to provide a method for the synthesis of chemical compounds with the help of the enzyme aldehides.

The above objectives as well as other goals, purposes and advantages, satisfy the present invention.

The present invention relates to a method of establishing the usefulness of prodrugs for the treatment of diseases, for example, mammalian, preferably human, which consists in determining whether or not a non-toxic prodrug to turn into liver cells, in vitro, hepatic fermentability, it is an effective prodrug for the treatment of disease.

The establishment and use of prodrugs that are effective against diseases associated with the liver, and tumor diseases, is the subject of much interest.

The present invention also relates to a method for treatment of diseases in animals, which consists in the introduction of an animal, preferably human, pharmaceutically effective amount of non-toxic analogue of the nucleoside or analog base of the nucleoside, or its salt, or ester, either individually or in a mixture with a pharmaceutically acceptable carrier, and the analog prodrug can turn into liver cells by angelidakis in the active drug or other useful metabolite.

Fig. 1 shows the HPLC chromatogram of turning Ipdr in IUGR the homogenate of the liver. Ipdr cultivated in the homogenate of rat liver. For control reactions of the supernatant solution is boiled for 5 minutes to inactivate all enzymes before use. Conditions of the analysis are described below, except that 60 ál of 100 000 g for 60 min the supernatant solution (equivalent to approximately 0.5 mg protein/ml) used in the reaction volume of 1500 ml. Aliquots (300 μl) select Ei 37oC. the retention Time of 9.5 min for Ipdr (A), 8,0 min for Iudr (B) and 6.5 min for iodouracil (C), respectively.

Fig. 2 shows the survival rate of mice who were given daily oral doses of 100 mg/kg of fluorouracil and perpiration within 5 days.

Fig. 3 shows the survival rate of mice who were given orally with different doses of fluorouracil and perpiration.

Fig. 4 shows the survival rate of mice, first injected with leukemia cells, then treated orally with different concentrations of fluorouracil and perpiration.

Fig. 5 shows the change in mass of a tumor of the colon treated with different doses of fluorouracil and ftorpirimidinu for some time. All figures masses are given in comparison with the initial check digit.

Fig. 6 shows a relatively low inclusion Ipdr in tissue Timnah hairless mice, such as bone marrow and intestine, and plateau substitution of thymidine is 250 mg/kg/day.

Fig. 7 shows comparative substitution Ipdr in liver tissues and tumors Timnah hairless mice between liver tissue and metabolic tumor, and inclusion into the fabric of pecheninka female transgenic mice, ekspressiruyushchikh antigen of SV 40 large tumor, causing various cancers, and processed by ftorpirimidinam mice lived longer and had less weight gain than the control.

Fig. 9 shows the results of processing ftorpirimidinam male transgenic mice, ekspressiruyushchikh antigen of SV 40 large tumor, causing various cancers, and processed by ftorpirimidinam mice had less weight gain and lived longer than the control.

The present invention is based on the discovery that the prodrug is activated by aldehyde oxidase in vitro and in vivo and become active drugs or metabolites, achieving high selectivity and therapeutic index. The above described the following reaction scheme:

The prodrug to the active drug or metabolite

One of the circuits of the above reaction scheme is the following:

< / BR>
in which R - I, F, Br, Cl, H, -CH3, -OR1, CF3, NO2, -SR1, -CH=CR2R3, -C CR2or-N=N+-n-, R1- C1-C5-alkyl, preferably alkyl having one carbon atom, R2and R3, independently of one another, hydrogen, C1-C5-alkyl or halogen, UB>-O-CH(CH2OH), substituted or unsubstituted alkyl, aryl, cycloalkyl, cicourel or any other group that has a size which does not impede the steric effect of aldehides liver. It is shown that these residues do not interfere with the desired action aldehides liver.

Other examples of reaction schemes the following:

< / BR>
< / BR>
< / BR>
in which R' is defined above.

The choice of prodrugs that can be tested or used in accordance with the present invention, is limited only by the structure of the active drug, obtained by the action of aldehides liver. Representative examples of useful prodrugs include nucleoside analogues and analogues of the bases of the nucleosides. In this situation, transforming the prodrug is converted into a product that is toxic or otherwise treated such as to show the desired biological activity (such as radiosensibility), only in relation to growing cells (such as cancer cells) or can replicate the virus, but not toxic or at least less toxic, or does not show the same biological action, or shows less of the same biological effect on nerazruschayuschego cells.

1-nicotine amide, 4-aminoantipyrine and methotrexate and their analogues. The finding of the authors of the invention is the oxidation of substituted in position 5 pyrimidinones in their Wratislavia or originalia twins by aldehyde oxidase person or rats, which leads to a completely new category of substrates on which the enzyme acts. This discovery also allows you to develop drugs, which is transformed in the liver, which can suppress and destroy cancer cells, viruses, parasites and other unwanted microbial pathogens.

To offer a prodrug useful in the present invention, it is only necessary to choose the bioactive compound, such as a connection exhibiting the desired biological effect, which contains ketogroup in the structure and which wish to form in situ in the liver of an animal, particularly a mammal, most preferably a human. Any such bioactive compound with geography in the structure is a potential candidate for the present invention. When this bioactive compound is selected, it can be a simple in vitro assays to determine whether the corresponding prodrug okinaka connection corresponding to the desired bioactive compound, but with ketogroup in restored form. This is a potential prodrug then subjected to analysis in vitro, similar to the analysis described in examples 2 and 3 to determine whether it can serve as a substrate for aldehides liver. If the prodrug is converted in a predetermined bioactive compound oxidase liver, it is a prodrug can be considered as corresponding to the present invention and may be used in accordance with the present invention.

While substituted in position 5 equivalents da preferred, and as shown, are converted into the corresponding substituted compounds of the RDL in vivo, also found that a fragment of desoxyribose is not necessary for specificity of substrate and that foroperation also turns into fluorouracil by illegitimizes in vitro as well as in vivo. As most of nucleosides and bases, nucleosides contains ketogroup in their formulas and many structurally similar to the structure of uracil, expect bioactive compounds which are analogs of nucleosides or bases, nucleosides, are the first candidates in bioactive compounds, d is evidenziato in such compounds. Thus, in addition to analogs of uracil and uridine can be used as bases for the formation of prodrugs in accordance with the present invention the other analogues of nucleosides and bases, nucleosides, such as analogs of cytidine, guanosine, 6-azauridine and 6-azaguanine. Examples of bioactive compounds in this category include FGD, DGPG, ACV, ddI, DT and ET.

Additional examples of commercially available analogues of pyrimidines and purines, which can serve as a bioactive drugs to develop appropriate prodrugs in accordance with the present invention are listed in tables 1 and 2.

Table 1. Analogues of pyrimidine:






1-Amylobarbitone acid






















4-Aminocrotonate acid























Barbituric acid






5-Bromo-2,3'-digesic is/BR> 5-Bromocrotonate acid












































































5-Ethyl-2'-de is R>















5-Ogorodova acid























5-Nitrobarbituric acid


5-Microanatomy acid

5-Nitrosodibutylamine acid














2-Thiouracil-5-carboxylic acid










AramilTM< / BR>
Table 2. Analogues of purine








2-Amino-6-( -carboxyethyl)mercaptopurine




























KorditsepinTM< / BR>


2', 3'-Dideoxyadenosine

2', 3'-Dideoxyuridine
























6 - (Hydroxyethylamino)purine







6-the urin

N-(- Isopentenyl)adenosine
















































2,6,8-Trioxymethylene, cytidine, guanosine, 8-azaguanine or 6-azauridine counterparts, are the following connections:

< / BR>
< / BR>
< / BR>
in which R - I, F, Br, Cl, H, -CH3, -OR', -CF3, NO2, SR', -CH=CR2R3, -CCR2or-N=N+-N-, R1is alkyl with 1 to 5 carbon atoms, preferably one carbon atom, R2and R3, independently of one another, hydrogen, C1-C5-alkyl or halogen, and R'is hydrogen, the residue of sugar, such as ribose or deoxyribose, a group-CH2-O-CH2-CH2OH, -CH2-O-CH(CH2OH), substituted or unsubstituted alkyl, aryl, cycloalkyl, cicourel or any other desired balance, the dimensions of which do not sterically hinder the action of aldehides liver. It is shown that these residues do not interfere with the desired action aldehides liver on these connections. Preferred groups of formula-CH= CR2R3are-CH= CF2and-CH=CH2. Sugar residues preferably have the formula:

< / BR>
< / BR>
< / BR>
< / BR>
Ester analogues of sugars used in the invention include esters in which H group HOCH2analogue substituted by a group-COR4where decarbonising the radical R4selected from hydrogen, normally lcil (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (e.g. phenyl, possibly substituted with halogen, C1-C4-alkyl or-alkoxygroup); substituted dihydropyridines (for example, N-methyldihydromorphine); sulphonate esters such as alkyl - or aralkylamines (for example, methanesulfonyl); sulfate esters; amino acid esters (e.g. L-poured or L-isoleucyl) and mono-, di - or trifosfatnogo ethers.

Also included in the limits of such esters used in the invention are esters produced from polyfunctional acids containing more than one carboxypropyl, for example dicarboxylic acids HO2C(CH2)nCO2H where n=1-10 (e.g., succinic acid) or phosphoric acid. Methods of obtaining such esters are well known.

With regard to the above-described esters, unless otherwise stated, any present alkyl residue mainly containing 1 to 16 carbon atoms, preferably 1 to 4 carbon atoms, and may contain one or more double bonds. Any aryl residue present in such esters, mainly contains phenyl.

In particular the esters may be C1-C8-alkalemia esters, nasalcrom or iodine), saturated or unsaturated C1-C6-alkyl, saturated or unsaturated C1-C6-alkoxygroup, the nitro-group or trifluoromethyl.

Pharmaceutically acceptable salts of the above-described analogs include salts made from pharmaceutically acceptable organic acids and bases. Examples of suitable acids include hydrochloric, Hydrobromic, sulphuric, nitric, perchloro, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, p-toluensulfonate, tartaric, acetic, citric, methansulfonate, formic, benzoic, malonic, naphthalene-2-sulphonic and benzosulfimide acid.

The term "analog" (or "active ingredient") includes the analog as well as its esters or salts.

Salt produced from appropriate bases include melodramatycheskye (for example, sodium), alkaline earth (for example, magnesium), ammonium and NR+4(where R is C1-C4-alkyl) salts.

The selection of bioactive compounds for which the prodrug can be formed in accordance with the present invention is not limited to analogues of nucleosides or bases, nucleosides, factual is evidenziato liver in this connection. This can be set by a simple analysis discussed above.

The term "Bioactive compound" or "biologically active substance" is considered to include compounds that regulate any aspect of the metabolism of the animal, which it is introduced, or that the connection invades the body of the animal to which it is typed. The term includes, without limitation, antidepressants, antibiotics, medicines regulating the blood pressure, analgesics antitumor agents, antiviral agents, etc.

For therapeutic treatment of diseases associated with the liver, particularly preferred prodrugs of the present invention due to the fact that aldehyde oxidase was found exclusively in the liver. Thus, a particular advantage of the prodrugs of the present invention is the elimination or reduction of side effects that will occur with the systemic administration of bioactive compounds, although the effect I wish to have only in the liver. Many compounds, such as IUGR, useful in the treatment of cell-proliferation, have significant systemic toxicity, limiting their clinical use. The corresponding prodrug usually will not have readouts metabolism in the desired bioactive compounds. For example, substituted in position 5 pyrimidinone predecessors Iudr or fluorouracil are not substrates for timedancing and timeintervals person and essentially non-toxic. In the treatment of liver cancer, for example, Ipdr will turn into liver aldehyde oxidase in IUGR, which is then preferably will be perceived by cancer cells of the liver to a substantial distribution Iudr other tissues. Thus, therapeutic index is substituted in position 5 derivative Da for primary liver cancer or metastases liver cancer will be a lot better than their partners RDL.

Along with the treatment of diseases associated with the liver, there are other cases where the introduction of prodrugs will be preferred in comparison with the introduction of bioactive compounds formed from such prodrugs. For example, many drugs cannot be entered pills for different reasons. For example, fluorouracil, known anticancer drug, you cannot type pills. Stabilisation is a prodrug, is converted into illegitimate liver in the fu. Torpedinidae you can enter the patient orally. As shown in the examples below, torpedinidae on the and But used as radio sensibilizators. However, their effectiveness is limited by their cytotoxicity and catabolism to free bases with subsequent dihalogenoalkane (Clin. Pharmacol, Ther., 1988, 44, 369 - 375). As Ipdr and its analogues is really non-toxic and are not substrates for timeintervals, using Ipdr and its analogues instead Iudr or But will allow you to bypass the difficulties of toxicity and decay associated with radiation therapy.

"Diseases associated with the liver include viral hepatitis, such as hepatitis A, hepatitis B, hepatitis C, hepatitis D; hepatoma; cancer metastases or liver; infection cytomegaly virus or other viruses, parasitic infections, such as schistosomiasis, clonorchis, fascioliasis, opisthorchiasis; and infection by flukes and worms, microbes, such as fungal or bacterial infections, such as Paracoccidioides brasiliensis, and other liver disease such as cirrhosis or rejection liver transplant.

The metabolic condition or disease also respond to treatment with the selective use of prodrugs, turning into active connection with liver aldehyde oxidase. Medication may be such that target receptors in or on the liver cells or vnepechenochnykh the Asia drugs acting on receptors, can be created by introduction of the corresponding prodrug to a subject or in the preparation of aldehides in vitro.

Thus, prodrugs of drugs acting on distant places, such as the heart or brain, can also be used in accordance with the present invention. Further, if the liver cells lack the target receptors, it reduces the potential for undesirable toxicity, due to its high concentration in the liver. The use of prodrugs, turning the liver enzyme in the active drug has a wide application.

Depending on the level of enzyme activity in relation to the prodrug is possible to achieve a slow rate of formation or slow excretion of active drug in the body. The compounds given in the examples are a fast metabolism, but they can be modified to slow the metabolism. This should lead to less frequent reception prodrugs compared with medication, with all the attendant benefits associated with the condition of the patient and the constancy of the dose. If the metabolism of the active drug is quite slow, entirely new classes of compounds can be used in therapy, kotlety 5 below, you can see the nature of the group R of the pyrimidine substrate significantly affects the rate of conversion. Thus, it is clear that it is possible to offer a prodrug with a predetermined rate of metabolism.

An example of a drug that can be improved due to the slow transformation in vivo is closterium drug suramin. Effective anti-cancer dose metastases prostate cancer is very close to the dose that causes paralysis, which was random and poorly detected during testing. Medication in the form of balls, as it was found much less effective. As a consequence, patients requiring such treatment, was hospitalized, was continuously observed and subjected to infusion to maintain narrow range of effective and acceptable concentrations. When using aldehyde or other proletarienne form. becoming an aldehyde oxidase in the active suramin, it became possible to give the patient the medication in the form of balls without having a permanent hospitalization.

As mentioned above, the orally acceptable prodrugs can be used instead of the active product, unacceptable oral. The prodrug may differ from active what sesivumelo, less irritable ability or toxic to the digestive system. Imbibing, the prodrug is converted into the active drug by the liver aldehyde oxidase, thereby bypassing the digestive tract. The advantage of oral administration over parenteral obvious to all senior scientists.

Regardless of the route of administration of any medication has a limited period of existence, due to renal clearance, enzymatic decomposition and too large or too small serum protein binding, etc., the Application of prodrugs enhances the development of drugs, balancing such issues.

The number of analogue described above for use in the present invention will vary not only from individual selected connection, but also on the route of administration, the nature treatable condition, age and condition of the patient and will ultimately be determined by the experience of the attending physician or veterinarian. In General, however, a suitable dose will be in the range 1 - 100 mg/kg of body weight per day, preferably 2 to 50 mg/kg/day, most preferably 2 to 10 mg/kg/day.

The desired dose can usually be represented by e or more podos a day.

Similar, described above, is usually administered in a single dose, for example, containing 0.5 - 50 mg, preferably 20 to 1000 mg, most preferably 50 to 700 mg of active ingredient in a unit dosage form.

Ideally the active ingredient should be administered to achieve peak plasma concentrations of the active ingredient, 1 to 75 microns, preferably 2 to 50 μm, most preferably 3 to 30 μm. This can be achieved, for example, intravenous injection of a 0.1 to 5% solution of prodrugs, possibly in physiological solution, or by the introduction in the form of a ball containing 0.1 - 50 mg/kg of the active ingredient.

According to the assessment of the various prodrugs can be used in different dosage. Further, the treatment of diseases of various tissues or organs also require different dosing. These dosages are easily definable by an experienced employee of the known methods.

It is possible that, for use in therapy, similar to that described above can be introduced in the form of the raw chemical, but preferably the presence of the prodrug in a mixture with a pharmaceutically acceptable carrier in the form of a pharmaceutical preparation.

The invention also relates to pharmaceutical preparations containing similar, you described is Kimi and/or prophylactic ingredients. The carrier(s) must be acceptable in the sense that it must be compatible with other ingredients of the drug and harmless for the recipient.

Pharmaceutical drugs include drugs that are suitable for oral, rectal, nasal, local (including transbukkalno, sublingual, and transdermal), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or preparations suitable for administration by inhalation or insufflate. Drugs can usually be presented in discrete dosage units and may be prepared by any methods well known in pharmacy. All methods include the stage of mixing the active ingredient with liquid carriers or finely powdered solid carriers or both, and then stage, if necessary, shaping the product into the desired product. Encapsulating chemical in liposomes or vesicles can also be used, if indicated for delivery purposes or stabilization.

Pharmaceutical preparations suitable for oral administration can usually be provided in the form of discrete units such as capsules, sachets or tablets, containing a certain number omponent can also be in the form of balls, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binders, lubricants, dezintegriruetsja funds or wetting means. Tablets can be coated with known methods. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or Alexiou, or can be dried products mixed with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspendresume tools, emulsifying agents, non-aqueous media (including edible oils), or preservatives.

The active component may also be included in parenteral drugs (eg, injection, such as injection loading dose or continuous infusion) with a single dosage form, such as ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. Drugs can have such forms as suspensions, solutions or emulsions in oily or aqueous carriers, and may contain tools such as suspendida, stabilizing and/or despergiruemaya sterile solid or by lyophilization from solution, for mixing with a suitable carrier, such as sterile, free of pyrogenic substances with water, before use.

Pharmaceutical preparations suitable for rectal injection with a solid carrier, most preferably represented by suppositories with a single dose. Suitable carriers include cocoa butter and other commonly used materials. Suppositories are usually obtained by mixing the active component with the softened or melted carrier with the receiving cooling and molding.

Drugs suitable for vaginal insertion, can be represented by pessaries, tampons, creams, gels, pastes, foams or razbryzgivayuschie means containing in addition to the active component known carriers.

Intranasal application of the active component is used as a liquid spray solution or dispersible powder or drops. Drops can be prepared from aqueous or nonaqueous basis, containing one or more dispersants, solubilizers or suspendisse agent. Liquid spray solutions is usually served in an aerosol package.

For administration by inhalation, the active ingredient obyknovenny packaging may contain conventional propellant, such as DICHLORODIFLUOROMETHANE, Trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of an aerosol under pressure dosing unit can be determined by using the valve for supplying a measured quantity.

Alternatively, for administration by inhalation or insufflate active component may be in the form of a dry powder, such as powder (active) compound and a suitable powder base, such as lactose or starch. In the case of powder preparation of unit dosage form can be, for example, capsules or cartridges, or gelatin or bubble wrap, from which the powder is administered using a nebulizer or insufflator.

If desired, the described preparations can be modified to slow the selection of the active component.

Pharmaceutical preparations for use in accordance with the invention can also contain other active ingredients such as antimicrobial drugs or preservatives.

Pharmaceutical preparations for use in accordance with the invention may also contain inert components, such as dehumidifiers, substances, providing ease of processing lesofat in a mixture with other therapeutic means, for example, other anti-infective agents. In particular, the compounds of formula I can be used in combination with known anti-virus tools, such as adrenalised or interferon.

The invention, therefore, in an additional aspect provides a method of using analogues described above, with other therapeutically active means, in particular means against hepatitis B.

If the active component is generated from the prodrug, is anti-cancer, other cancer, or immunomodulatory features can be used together with him, also any other compatible combination of drugs, whether their synergistic activity, additional or separate.

Combinations discussed above, can generally be represented in the form of a pharmaceutical preparation, and thus, the use of pharmaceutical preparations containing a combination as defined above together with a pharmaceutically acceptable carrier for her, it constitutes an additional aspect of the invention.

Individual components of such combinations can be entered either sequentially or simultaneously in separate or objem therapeutic tool for the same disease, for example, active against the same virus dose of each compound may be the same or different from the dose when similar used separately. The appropriate dose can be easily estimated by an experienced employee.

The aldehyde oxidase secreted from the liver and can be immobilized on a solid phase for easy separation of the enzyme catalyst from the reaction mixture. Target bioactive optically active compounds are then separated from the compounds predecessors and regenerate. Methods of purification of the enzyme is well known and can be used any suitable. Methods of immobilization of the enzyme on the solid phase, whether this adsorption, trapping, chemical binding or holding for a semi-permeable membrane, is also well-known.

The contents of all references cited in this application are included in the description of the invention as references. The invention is described with the following examples illustrate the invention but without limiting it.


Example 1. Drugs cloth

Tissue rat liver was washed with cooled to the temperature of the ice to 1.15% KCl get wet and dry. The tissue is then homogenized in a homogenizer in 1.15% KCl solution, extracted 3 times choicest g for 10 min at 4oC. Forming a supernatant solution is filtered through Miracles (similar to cheese cloth), and then subjected to dialysis overnight against the background of 50 mm buffer Tris-HCl pH 7.5 and maintained at -80oC before use. The rat hepatocytes obtained by perfusion technique, cells are extracted with 10 mm phosphate buffer pH 7.5, containing 1 M KCl, and cialiswhat 4 hours on a background of 50 mm buffer Tris-HCl with a pH of 7.5.

Example 2. Conditions analysis

In Standard conditions the analysis of the reaction mixture contained 50 mm Tris-HCl with pH 7.5, 1 mm EDF, 180 μm Ipdr (or its analogues) and approximately 0.01 mg supernatant protein solution homogenate tissue (obtained at 10 000 g) in a final volume of 500 ml and kept at 37oC for 10 minutes, unless otherwise stated. At the end of the incubation were removed 300 μl of the reaction mixture, was mixed with 630 μl of acetonitrile and stirred. Precipitated protein was removed by centrifugation and the supernatant solution liofilizirovanny to dryness. The samples were restored to the original volume of the aliquot buffer mobile phase HPLC and analyzed on a column of PP-18 Altech. Ipdr, IUGR and iodouracil was determined at a wavelength of UV absorption 230 nm, Ipdr also was determined at a wavelength of 335 nm. Mobile phase was a mixture of 10% acetonitrile /90% mm acetate elicin known concentrations.

Example 3. The conversion of 5-iodine-2-pyrimidinone-2'-deoxyribose (Ipdr) 5-iodosuccinimide (Iudr)

To study the transformation Ipdr in IUGR the liver enzyme metabolites Ipdr analyzed after incubation with the supernatant solution homogenate of rat liver, using the technique of HPLC reversed phase. Ipdr and IUGR can be detected at the absorption wavelength of 230 nm, but only Ipdr can be detected at 335 nm. To limit phosphorylytic splitting Iudr to iodouracil timeintegrated (Radiation Oncology Biol. Phis., 1984, 10, 1399 - 1406) the analysis used the buffer is Tris-HCl. As shown in Fig. 1, there is a time dependence of the transformation Ipdr in IUGR and IUGR is the only product produced Ipdr. Identification Iudr confirmed by retention times on the column C-18 (8 min compared with 9.5 min for Ipdr) and UV spectrum as well as spectroscopy nuclear magnetic resonance (results not shown).

Example 4. Activity properties "Ipdr oxidase

Activity Ipdr oxidase does not require exogenous cofactors, it is much smaller in extracts of kidney and spleen than in the liver (table 3) and was not detected in extracts of lungs or intestines of rats. The human liver contains a similar number is a recreational activity in comparison with the extract of the entire liver, suggesting that the oxidation Ipdr occurs mainly in these cells. In an attempt to localize the activity of the enzyme of conversion Ipdr used fractional differential centrifugation. For each fraction was performed by analysis of enzyme activity. The only faction that showed the enzyme activity, has emerged as the supernatant solution at 100 000 g for 60 minutes. This means that the enzyme is soluble fraction cytosol. To further purify the used column chromatography on DEAE-cellulose column chromatography on Sepharose blue and centrifugation with a density gradient of glycerol in the same order. Activity Ipdr oxidase increased in 380 times compared with the original crude extract of rat liver. Partially purified enzyme catalyzes the synthesis Iudr speed 3.8 ámol / min on milligram of protein at 37oC in these conditions. The average molecular weight of this enzyme as a rat, and human, as defined by centrifugation with a gradient of 20 to 40% glycerol is approximately 280,000. As was found, nor cofactor or divalent cation is not required for the enzyme from rat liver or liver enzyme human.

PI a number of well-known oxido-reductase, which has a similar ability to oxidize the carbon atom in the neighboring amino group to the carbonyl, has been studied for the ability to catalyze oxidation Ipdr. The xanthine oxidase (Boeringer the Mannheim), isolated from cow's milk and catalytic oxidation gipoksantina in xanthine, not turned Ipdr in IUGR. Oxidase system mixed functions obtained from liver microsomes of rats (and active) to a wide range of substrates depends on NADPH. However, this microsome fraction from rat liver showed no activity Ipdr oxidase, regardless of whether it's been NADPH or not. Alcoholdehydrogenase (Sigma) and alcoholecstasy (Sigma) are located in the soluble fraction of extracts of liver cells, but the conversion Ipdr in Iudr not detected with purified preparations of each enzyme under the same conditions in which they turn natural substrates efficiently. Sarcosinates (Boeringer the Mannheim), which catalyzes the conversion of N'-methylglycine in glycine, has no activity Ipdr-oxidase. Fenilalaningidroksilazy, urokinase, cystathionine -- lease, L-glutamate dehydrogenase, cystathionase and some other oxidoreductase were excluded on the basis of abstractcontainer analysis, JV is chem, 1976, 64, 341-350; J. Biol. Chem 1979, 254, 843-851; J. Biol. Chem., 1970, 245, 528-537; Science, 1955, 121, 603-604, Eur. J. Biochem., 1971, 20, 269-275).

As the aldehyde oxidase has the same molecular weight is in the cytosolic fraction of liver cells and has a broad specificity for substrates (J. Biol. Chem, 1962, 237, 922-928), it is considered that it is the enzyme "Ipdr-oxidase", the one responsible for turning Ipdr in IUGR. Hepatic aldehyde oxidase, which catalyzes the oxidation of various aldehydes to the corresponding acids, also turns N'-nicotine amide (Sigma) in N-methyl-2-pyridone-5-carboxamide and N'-methyl-4-pyridone-5-carboxamide (J. Bil., Chem., 1962, 237, 922-928; J. Biol. Chem., 1964, 239, 2022-2035; Arohs of Biochem and Biophy, 1971, 145, 27-34; Arohs of Biochem and Biophy, 1971, 145, 35-42; J. Biol. Chem., 1973, 248, 2580-2587; Biochemistry, 1982, 21, 3561-3568; J. Biol. Chem 1981, 256, 3479-3486). Activity aldehides, as was expected, stimulates potassium ferricyanide and Tris buffer, but not MgCl2(J. Biol, Chem, 1973, 248, 2580-2587). This enzyme can inhibit 2-mercaptoethanol, dithiothreitol and other thiol substances (J. Biol. Chem, 1964, 239, 2022-2035). No inhibition by cysteine at 5 mm, but at 50 mm was observed a strong inhibition of enzyme activity (J. Biol, Chem, 1973, 248, 2580-2587). Cations of divalent metals, such as Cu++, Zn++and Fe++, vizinada or formaldehyde (J. Biol. Chem, 1964, 239, 2022-2035); Archs of Biochem and Biophy, 1971, 145, 35-42; J. Biol. Chem 1981, 256, 3479-3486). Therefore, the number of compounds have been tested for their effect on the oxidation Ipdr in IUGR, and it was found that the profile of inhibition of compounds with activity Ipdr oxidase (table 4) substantially identical to the characteristic patterns of aldehides. Further, at all stages of the purification of the aldehyde oxidase could not be separated from the activity "Ipdr-oxidase".

Example 6. The substrate specificity

Several analogues of 2-pyrimidinetrione were tested for transformation in their deoxyuridine twins. The Michaelis constant K for Ipdr in the reaction at pH 7.5 and pH 9.5 to 150 μm and 87 μm, respectively. To 5-ethinyl-2-pyrimidinecarboxylic (Apdr) in the reaction at pH 7.5 and pH of 9.5 is 77 μm and 46 μm, respectively. However, the relative Vmaxfor Ipdr in the reaction at pH 7.5 and pH 9.5 to the same. 5-Iodine-2-pyrimidinone (PI), aglycone Ipdr was an excellent substrate for aldehides. Synthetic substrates for aldehides was better than natural substrates, N'-nicotine amide and acetaldehyde, judging by the strength of the inhibiting their oxidation reactions Ipdr (table 4). The reaction rate of the enzyme of the liver with various analogues Proxyremote (Mpdr) or 5-ethyl-2-pyrimidinecarboxylic (ICPDR) (table 5). Electronegative substituents in position 5, as it turned out, increase the activity of the substrate in this oxidation reaction.

Example 7. The toxicity of fluorouracil and perpiration

BDFI mice were administered a daily oral dose of either 50, 75 or 100 mg/kg of fluorouracil either 100, 150 or 200 mg/kg of perpiration. The survival rates of each are presented in table 6 and Fig. 2 and 3. The toxicity of perpiration significantly less at higher doses.

Example 8. The effect of fluorouracil and perpiration on cell leukemia

The mice were injected 100000 cell leukemia R-R to cause leukemia. These cells resistant to Adriamycin. These leukemic mice daily oral gave 25 and 50 mg/kg of fluorouracil and 50 and 100 mg/kg of perpiration and measured time of survival. The results are shown in table 7 and Fig. 4. Survival time was the same when used as fluorouracil and perpiration.

Example 9. The effect of fluorouracil and perpiration on colon cancer

The mice were injected cells 33 of the colon, the part is not treated, the rest were given daily oral 25 and 50 mg/kg of fluorouracil and 50 and 100 mg/kg of perpiration and measured within framerate comparable to fluorouracil and perpiration

Example 10. The impact of inclusion Ipdr in tissue Timnah hairless mice

Mice were administered daily oral doses of 0, 100, 250 and 500 mg/kg the Percent substitution of thymidine as an indication of the inclusion Ipdr in the bone marrow, the tissue of the intestines and liver were analyzed by known methods. As shown in Fig. 6, a relatively small number of thymidine is replaced Ipdr in the bone marrow and intestines, and plateau substitution of thymidine occurs when the dose level of 250 mg/kg/day. Enable Ipdr in the liver was not found. The results presented in Fig. 6 are the average percentage of substitution of thymidine standard error. 11 is greater than or equal to 3 for each dose. These results show that there is no significant substitution of thymidine on Iudr in the liver and a very small percentage of substitution found in the bone marrow and gut. These results establish that the introduction of IPT as prodrugs in accordance with the present invention suitable for the treatment of mammals, including humans.

Example 11. The inclusion Ipdr in tissue Timnah hairless mice having metastasis tumor

Mice were administered daily doses of 0, 100, 250 and 500 mg/kg to determine the substitution of thymidine into liver tissue and tumors. The results, shown in Fig. 7, the tumor N = 2). While a very small percentage of inclusions were found in normal liver, a higher percentage of substitution found in the tumor, showing that the use of IPT as prodrugs for the treatment of tumors will result, as you can expect good results.

Examples 12. Treatment of transgenic mice ftorpirimidinam

Transgenic mice, obtained by the method Sepulveda (Cancer Research 1989, 49, 6108-6117), was administered the drug, starting with nine weeks after birth, separately for males and females, and processing group received 100 mg/kg twice a day once a week. Fig. 8 and 9 show the change in body weight during the 9 - 17 weeks for females and males. Both females and males treated, show a significant decrease in the gain in weight after 13 weeks, correlating with increased longevity. Treated females outlive control rats dying from complications of cancer on the 16th week. These data suggest that the use of perpiration as a means of treating tumor in accordance with the present invention gives, as expected, good results.

The above-cited references included in the review regardless of whether specific or not. Priority injunction of this invention, experienced employee can appreciate that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without deviation from the spirit and scope of the invention and without undue experimentation.

While this invention is described in connection with a particular case it should be understood that additional modifications. This proposal intends to cover any changes to the application or adaptation of the invention following, in General, the principles of this invention and including such departures from the disclosed content of which fall within a known and customary practice this field of knowledge and that can be attributed to the essential features set forth in the framework of the attached claims.

1. The method of obtaining an anti-tumor effect in a mammal, which includes the introduction of an active compound, which is a bioactive ticketstream uridine, uracil or a basic analogue of uracil, characterized in that the active compound is administered in restored form compounds having a structure corresponding to the structure of the active compounds, except that ketogroup in the structure Aracinovo ring is Oh to bioactive dietoterapia active compounds.

2. The method according to p. 1, characterized in that the antitumor effect is obtained in the liver of a mammal.

3. The method according to p. 2, wherein the tumor is a hepatocarcinoma.

4. The method according to p. 1, characterized in that the specified recovered form the active compound is administered orally.

5. The method according to p. 1, characterized in that the active connection can not be administered orally, and the reduced form of the compounds may be administered orally.

6. The method according to any of paragraphs.1 to 5, characterized in that the active compound has the formula

< / BR>
where R - J, F, Br, Cl, H, -CH3, -CF3, NO2, SR1, OR1, -CH=CR2R3or-N= N+= N-;

R1- C1- C5-alkyl;

R2and R3independently of one another are hydrogen, C1-C5-alkyl or halogen and R1is hydrogen, a sugar residue, -CH2-O-CH2-CH2OH, -CH2-O-CH(CH2OH), substituted or unsubstituted alkyl, aryl, cycloalkyl, cicourel or any other desired residue, and this residue to steric not interfere with the action of aldehides liver.

7. The method according to p. 6, characterized in that specified is UP>-;

R1- C1- C5-alkyl;

R2and R3independently of one another are hydrogen, C1-C5-alkyl or halogen and R1is hydrogen or similar sugar.

8. The method according to p. 7, characterized in that the active compound is 5-substituted deoxyuridine (qP) and the specified reduced form compounds corresponding to 5-substituted-2-pyrimidinetrione (qP).

9. The method according to p. 8, characterized in that said 5-Deputy is J, F, Br, Cl, -OR1, -CF3, NO2, SR1, -CH=CR2R3or-N= N+= N-;

R1- C1- C5-alkyl; R2and R3independently from each other hydrogen, C1- C5-alkyl or halogen.

10. The method according to p. 8, characterized in that the active connection is iodization, and the specified reduced form of the compound is 5-iodine-2-pyrimidinecarboxylic.

11. The method according to p. 7, characterized in that the active compound is 5-substituted uracil.

12. The method according to p. 11, characterized in that specified by the restored form of the compound is 5-fluoro-2-pyrimidinone.

13. The method according to p. 6, characterized in that decree the p. 7, wherein R1is hydrogen or the residue of sugar.

15. The method according to p. 14, wherein R1is ribose or deoxyribose.

16. The method according to p. 7, wherein R1is hydrogen or deoxyribose.


Same patents:

The invention relates to the field of pharmacy and concerns receiving means having a radioprotective effect
The invention relates to Oncology

The invention relates to medicine and relates to means for treatment of a dysbacteriosis

Antiviral agent // 2123339
The invention relates to medicine and pharmacology, and relates to means for the treatment of herpetic infections

The invention relates to compositions and methods for the reduction or regulation of inflammation and for the treatment of inflammatory diseases and other pathological disorders involving intercellular adhesion
The invention relates to medicine, in particular to the prevention and treatment of diseases caused by disturbances of carbohydrate metabolism
The invention relates to medicine, namely to "low psychiatry"

The invention relates to a new method of treatment of patients, such as people with benign prostatic hyperplasia (BPH), which includes treatment by assigning a therapeutically effective amount of an inhibitor 5- reductase in combination with blocker1- adrenergic receptor
The invention relates to medicine, Nephrology

The invention relates to the class of pyrimidine compounds, which are suitable for the treatment of diseases and disorders of the Central nervous system (CNS), for example to prevent cerebral ischemic lesions, containing their pharmaceutical compositions and to methods for their preparation

The invention relates to 1,4-disubstituted the piperazines of General formula (I), which means the group-CO - or-CH2-OCO; D - heteroaryl selected from a range including 1, 3, 5-triazinyl, pyrimidinyl and pyridinyl, possibly substituted by one or two substituents selected from a range, including mono-(C1-C6)-alkylamino, mono-(C3-C7)- alkynylamino-, di-(C1-C6)-alkylamino-,

(C1-C6)-alkyl-(C3-C7)-alkylamino and pyrrolidin-I-yl group; Raand Rbis a hydrogen atom or (C1-C3)-alkyl; n is an integer from 1 to 4; their enantiomers, racemic mixtures and their salts with pharmaceutically acceptable acids and bases
The invention relates to medicine, surgery may be used in the treatment of acute pancreatitis

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to an intermediate compound, i. e. tert.-butyl-(E)-(6-{2-[4-(4-fluorophenyl)-6-isopropyl-2-[methyl(methylsulfonyl)amino]-pyrimidine-5-yl}-(4R,6S)-2,2-dimethyl[1,3]dioxane-4-yl]acetate that can be used in synthesis of compound of the formula (IV)

eliciting inhibitory effect on activity of HMG-CoA-reductase and, therefore, can be used for preparing pharmaceutical agents for treatment, for example, hypercholesterolemia, hyperproteinemia and atherosclerosis. Also, invention relates to a method for preparing indicated intermediate compound by reaction of the new parent compound - diphenyl-[4-(4-fluorophenyl)-6-isopropyl-2-[methyl(methylsulfonyl)amino]pyrimidine-5-ylmethyl]phosphine oxide with tert.-butyl-2-[(4R,6S)-6-formyl-2,2-dimethyl-1,3-dioxane-4-yl]acetate in the presence of a strong base in simple ether or aromatic solvents or their mixtures at temperature in the range from -200C to -900C. Also, invention relates to a method for preparing of compound of the formula (IV) wherein R1 means hydrogen atom or pharmaceutically acceptable cation and to a method for preparing intermediate compounds of the formula (VI):

wherein each P1 and P2 represents independently (C1-C4)-alkyl or group:

and wherein P3 represents (C1-C8)-alkyl. Applying new intermediate compounds and proposed methods provide enhancing quality and yield of compounds.

EFFECT: improved preparing methods.

9 cl, 1 tbl, 8 ex