ES2361831T3 - TEST AGENTS TO EVALUATE THE PHARMACOLOGICAL EFFECT OF DRUGS, AND PROCEDURE AND REAGENTS FOR DRUG SCREENING WHICH PRESENT AN EXCELLENT ADMINISTRATION EFFECT AND / OR A LITTLE SECONDARY EFFECT OF DRUGS / RECEIVING OTHERS IN ORDER THEREOF. - Google Patents

Abstract

Use of any of [1- 13 C] DOPA or benzoyl-arginyl- [1- 13 C] alanine to evaluate the pharmacological effect of a medicament containing a pharmaceutical agent comprising an enzyme, an enzyme inhibitor or a receptor ligand or a prodrug of the pharmaceutical agent.

Description

Technical field

The present invention relates to a diagnostic reagent for evaluating the pharmacological effect of a medicament, and a method and a reagent for selecting pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of Pharmaceutical agents to select one or ones that have high drug efficacy and / or little side effect.

Background of the invention

The pharmacological effect of a medication has been determined based on the change in clinical conditions (improvement or occurrence of a side effect).

Determination based on clinical conditions is only possible after a medication is administered for a certain period of time.

Blood level monitoring has also been used in which the pharmacological effect of a drug is indirectly evaluated by determining whether the blood level of a pharmaceutical agent derived from the drug is within the therapeutic range or the safety interval obtained from empirical way. However, this procedure only has an effect with the previous condition that there is a good correlation between the pharmacological effect and the blood level to some extent and that the therapeutic interval or the safety interval is clearly defined (“Hyojunyakurigaku” (Standard Textbook of Pharmacology) 5th edition, edited by Akio Ebihara (1997) p. 31), and also the procedure is only indirect as the procedure to evaluate a pharmacological effect.

An objective of the present invention is to evaluate the pharmacological effect of a medicament by providing a diagnostic reagent to accurately determine the degree of potentiation in a short period of time.

or inhibition of the function of a target (for example, a bioenzyme or receptor) for the medicament after administration of the medicament.

In the screening of therapeutic compounds, a compound having a therapeutic effect as high and a side effect as low as possible is selected. Most medications exert pharmacological effects by inhibiting or enhancing the function of their targets (for example, bioenzymes or receptors). Therefore, the pharmacological effect of a medication can be assessed by determining the degree of inhibition or potentiation of the function of a target for the medication.

At present, the degree of inhibition or potentiation of the function of a target is evaluated in vitro. In recent years, the most common has been an in vitro test system called "HTS" (high performance screening) that allows the treatment of a large number of samples. This procedure uses a high density plate such as a 96-well, 384-well or 1,536-well plate, uses techniques such as advantageous automation, and can evaluate the pharmacological effect on a large number of samples of up to hundreds of thousands at a speed. high.

However, a result provided in an in vitro evaluation does not always coincide with a result provided when a medication is administered to an individual. US Patent No. 5,625,125 relates to the evaluation of PLA2 inhibitors in transgenic mice that have a pathogenic phenotype.

The only way to know the result of administering a medication to an individual is to observe the change in clinical conditions (improvement or occurrence of a side effect) in the individual.

Therefore, an objective of the present invention is to provide a method and reagent for the screening of pharmaceutical agents and / or prodrugs thereof to select one or ones that have high drug efficacy and / or little side effect evaluating the degree of inhibition of a target in an entire organism and in real time what is impossible to carry out by any prior art technique.

Description of the invention

Most medications exert pharmacological effects by inhibiting or enhancing the function of their targets (for example, bioenzymes or receptors). For example, a drug containing an enzyme exerts the pharmacological effect by potentiating an enzymatic reaction in vivo, a drug containing an enzyme inhibitor exerts the pharmacological effect by inhibiting an enzymatic reaction in vivo, and a drug containing a receptor ligand exerts the effect. pharmacological enhancing or inhibiting the function of a receptor by binding to the receptor, thereby enhancing or inhibiting a related in vivo enzyme reaction. Therefore, the pharmacological effect of a medicament can be assessed by determining the degree of potentiation or inhibition of the function of a target for the medicament after administration of the medicament.

The present invention provides the use of any of [1-13C] DOPA or benzoyl-arginyl- [1-13C] alanine to evaluate the pharmacological effect of a medicament containing a pharmaceutical agent comprising an enzyme, an enzyme inhibitor or a ligand of receptor or a prodrug of the pharmaceutical agent.

The diagnostic reagent of the present invention can be used to evaluate the pharmacological effect of a medicament, the method comprising the steps consisting of:

administer the medication and diagnostic reagent to a non-human subject;

collect a biological sample of the subject at least once;

measure the amount of [1-13C] DOPA or benzoyl-arginyl [1-13C] alanine or a metabolite thereof in the biological sample; Y

evaluate the pharmacological effect of the medicine based on the value obtained in the measurement stage.

The medicament can be administered to the subject one or more times simultaneously with or before administration of the diagnostic reagent of the present invention.

In the above procedure, the level of 13C in the CO2 exhaled in the subject can be measured.

As used herein, the term "medicament containing an enzyme" refers to a pharmaceutical preparation that can produce pharmaceutical efficacy by enhancing an enzymatic reaction in vivo after administration of the enzyme. Examples of the drug containing an enzyme include preparations containing each streptokinase, streptodornase, hyaluronidase, urokinase, lysozyme, amylase, sugary pepsin, diastase, pancreatin or the like.

The term "enzyme inhibitor" refers to a substance that can bind to a bioenzyme (that is, an enzyme in a living organism) to slow or stop the reaction rate (see "Yakubutsu Taisha Jiten" (Dictionary of Drug Metabolisms), edited by Ikuo Yamamoto, (1996) p. 241). Examples of enzyme inhibitors include acarbose (glu-glucosidase inhibitor) which is used as an antidiabetic agent; an HMG-CoA reductase inhibitor such as simvastatin, atorvastatin, pravastatin, fluvastatin and cerivastatin that are used as agents against hyperlipidemia; an angiotensin converting enzyme inhibitor such as enalapril maleate, alacepril, imidapril hydrochloride, quinapril hydrochloride, captopril and cilazapril that are used as antihypertensive agents; methotrexate (folate reductase inhibitor) and fradozol hydrochloride (aromatase inhibitor) that are used as antineoplastic agents; an HIV protease inhibitor such as saquinavir mesylate, zidovudine (an inhibitor of viral reverse transcriptase); foscarnet (DNA polymerase inhibitor) that are used as anti-AIDS agents; and a neuraminidase inhibitor such as zanamivir hydrate that is used as an anti-flu agent.

The term "receptor ligand" refers to a substance that can specifically bind to a receptor protein in the cell membrane or in a cell. Upon binding of a receptor ligand to a receptor, a specific biological reaction occurs for the combination of the receptor and the receptor ligand. Naturally occurring receptor ligands in a living organism include a hormone, a neurotransmitter and the like. In the case where the naturally occurring receptor ligand is administered in a living organism as a pharmaceutical agent, it is used as a stimulant or agonist to enhance a biological reaction caused by the binding of the receptor ligand to its receptor. Alternatively, the receptor ligand can also be used as an antagonist or blocker to inhibit or block such a biological reaction. Specific examples of receptor ligand include histamine H2 receptor blockers that include lafutidine, famotidine, cimetidine and ranitidine hydrochloride and which are used as therapeutic agents for peptic ulcer; hormonal agents including estradiol and derivatives thereof, estriol and derivatives thereof, progesterone and derivatives thereof and testosterone and derivatives thereof;  receptor inhibitors that include tolazoline, ifenprodil and nicergoline that are used as vasodilators; and  receptor blockers that include alprenolol hydrochloride and bufetolol hydrochloride that are used as therapeutic agents for angina pectoris.

The term "prodrug" refers to a substance that is pharmacologically inactive in nature but can be converted into a substance that has a pharmacological effect through an enzymatic or non-enzymatic reaction in vivo (see "Yakubutsu Taisha Jiten" (the Dictionary of Drug Metabolisms), edited by Ikuo Yamamoto, (1996) p. 377). Examples of prodrug include sulindac which can be converted by reduction into a form of sulfur that has antiphlogistic and analgesic effects; loxoprofen that can become a form of trans-OH that has antiphlogistic and analgesic effects; nabumetone that can be converted into 6-methoxy-2-naphthylacetic acid that has antiphlogistic and analgesic effects; and indomethacinfarnesyl that can become indomethacin that has antiphlogistic and analgesic effects.

The term "pharmacological effect of a medicine" refers to an effect that is produced by enhancing or

5 inhibiting the function of a target (a bioenzyme or a receptor), including drug efficacy and a side effect. Drug efficacy refers to an improvement in the pathological state produced by the prolonged potentiation or inhibition of the function of a target (a bioenzyme or a receptor), and a side effect means a detrimental effect produced by such prolonged potentiation or inhibition.

10 Therefore, “evaluation of a pharmacological effect of a medicine” refers to the evaluation of whether a potentiation or inhibition of the function of a target for a medicine is sufficiently achieved to improve the pathological state, or the evaluation of whether causes an enhancement or inhibition of the function of a target for a drug that can produce a side effect.

The term "substrate for an enzyme" refers to a substance that can undergo a change by direct binding to the enzyme. Examples of the enzyme and its substrate include diastase and cyclodextrin; Glu-glucosidase and maltose; pancreatin and a protein; HMG-CoA reductase and HMG-CoA; and aromatase and androgen.

The expression "different enzyme that may present an alteration in activity by coupling to the effect of

"An enzyme" refers to a different enzyme that can constitute a coupling reaction system with the enzyme, a different enzyme that can be activated by the enzyme, or the like.

Examples of the enzyme, the different enzyme that may exhibit an alteration in activity by coupling to the effect of the enzyme and the substrate for the different enzyme are shown in the table below. 25 Table 1:

(1) Enzymes

(2) Different enzymes that may present an alteration in activity due to the coupling to the effect of (1) (3) Substrates for (2)

Enterokinase

 Trypsin Peptide

Trypsin

Carboxypeptidase A Peptide

The expression "enzyme that can present an alteration in activity by coupling to the effect of a

"Enzyme inhibitor for the other enzyme" refers to an enzyme that may constitute a coupling reaction system with the other enzyme that is directly inhibited by the enzyme inhibitor so that the enzyme itself is not inhibited by the enzyme inhibitor but rather causes the alteration in its activity, or similar.

Examples of the enzyme inhibitor, the enzyme that is directly inhibited by the enzyme inhibitor and its substrate,

35 and the enzyme that can present an alteration in activity by coupling to the effect of the enzyme inhibitor and its substrate are shown in the table below.

Table 2:

(1) Enzymatic inhibitors

(2) Enzymes directly inhibited by (1) (3) Substrates for (2) (4) Enzymes that may present an alteration in activity due to the coupling to the effect of (1) but which is not directly inhibited by (1) (5) Substrates for (4)

Methotrexate

Folate reductase Folate  Thymidylate synthase dUMP

Ozagrel Sodium

Thromboxane synthetase Prostaglandin I2 Prostaglandin I synthetase Prostaglandin H2

40

Examples of the enzyme that may exhibit an alteration in activity by binding a ligand to a receptor and the substrate for the enzyme include those depicted in the table below.

Four. Five

Table 3:

(1) Receivers

(2) Ligands for (1) (3) Enzymes that may present an alteration in activity through the union between (1) and (2) (4) Substrates for (3)

Dopamine receptor

Bromocriptine, cabergoline Adenylate Cyclase ATP

Acetylcholine receptor

Acetylcholine chloride, betanechol chloride Adenylate Cyclase ATP

Adrenergic receptor

Norepinephrine, isoproterenol, phentolamine Adenylate Cyclase ATP

The term "marked form" refers to a state in which a compound or its metabolite has a detectable property. Examples of the labeling procedure include procedures using an isotope such as a stable isotope (for example, 13C, 15N, 2H and 18O) and a radioactive isotope (for example, 14C, 3H, 35S, 32P and

5 125I), a luminescent or fluorescent substituent (eg, fluorescein, methylcoumarylamide, calcein and rhodamine), or the like.

The term "form marked with 13C" refers to a substance that has an increase in the level of 13C compared to natural abundance. Particularly, a substance that presents an increase in the level of 13C in a specific position is desirable. Examples of the 13C-labeled form comprise non-limitingly organic acids such as 13C-labeled NaHCO3, 13C-labeled formic acid, 13C-labeled acetic acid, 13C-labeled propionic acid, 13C-labeled lactic acid, 13C-labeled butyric acid , 13C-labeled pyruvic acid and commercialized 13C-labeled benzoic acid; 13C-labeled amino acids such as 13C-labeled alanine; 13C-labeled saccharides such as 13C-labeled glucose; and marked alcohols

15 with 13C such as ethanol labeled with 13C.

The term "subject" refers to a living organism to which the diagnostic reagent of the present invention is to be administered, in which "living organism" means a living individual organism. Examples of subject include: (1) human being; (2) pets such as dogs and cats; (3) laboratory animals such as rats, mice, guinea pigs, rabbits and monkeys; or (4) other mammals.

The biological sample to be collected from the subject is, for example, breath, blood or urine.

The term "metabolite" refers to a substance that is produced in the metabolic process of a substance that is

25 administers to a living organism. Examples of the metabolites of the substances (a) to (f) above are the substances represented in the table below.

Table 4:

(1) Substances corresponding to from (a) to (e)

(2) Medicines to be evaluated by diagnostic reagents (1) (3) Metabolites of (1)

Maltose

 Acarbose Glucose, CO2

Hemiacetal of mevaltic CoA

HMG-CoA reductase inhibitor Mevalonic acid, CO2

Androgen

Aromatase inhibitor Estrogen

dUMP

 Methotrexate dTMP

Most medications exert the pharmacological effects by enhancing or inhibiting the function of their targets (for example, bioenzymes or receptors). For example, a medicine that contains an enzyme exerts the pharmacological effect by potentiating an enzymatic reaction in vivo, a medicine that contains an enzyme inhibitor exerts the pharmacological effect by inhibiting an enzymatic reaction in vivo, and a medicine that contains a ligand

The receptor exerts the pharmacological effect by enhancing or inhibiting the function of a receptor by binding to the receptor, thereby enhancing or inhibiting a related in vivo enzyme reaction. Therefore, the pharmacological effect of a medicament can be assessed by determining the degree of potentiation or inhibition of the function of a target for the medicament after administration of the medicament. The diagnostic reagent of the present invention is one to evaluate the pharmacological effect of a medicament by determining the degree of potentiation or inhibition of the function of a target.

The principle of the evaluation procedure is set out below.

1. A case in which the target is an enzyme (for a medicine containing an enzyme, an enzyme inhibitor or a prodrug thereof)

The degree of potentiation or inhibition of the function of a target by a medicament is evaluated by administering to a subject a substrate for the target, a substrate for a second enzyme involved in an enzymatic reaction that may undergo a change by potentiation or inhibition of the target function (e.g., a coupling reaction with the target) or a labeled form of any substrate as a diagnostic reagent, and then determining the increase or decrease in the amount of a metabolite derived from the diagnostic reagent or the Diagnostic reagent per se caused by potentiation or inhibition of target function. For example, in the case of a drug containing an enzyme, a substrate for a bioenzyme (a target for the drug) or a marked form of the substrate is administered to a non-human subject as a diagnostic reagent. If the amount of a metabolite derived from the diagnostic reagent increases after administration, then the drug would be considered to enhance the function of the target. Therefore, it can be confirmed that the medicine has sufficient efficacy. In the case of a medicament containing an enzyme inhibitor, for example, a substrate for an enzyme (a target for the drug) or a labeled form of the substrate is administered to a subject as a diagnostic reagent. If little metabolite derived from the reagent is detected

diagnosis, then the medication would be considered to inhibit the function of the target satisfactorily. Therefore, it can be confirmed that the medicine has sufficient drug efficacy.

2. A case in which the target is a receptor (for a medicine that contains a receptor ligand or a prodrug thereof)

The degree of potentiation or inhibition of the function of a target by a medicament is evaluated by administering to a subject a substrate for an enzymatic reaction that may undergo a change by binding the medicament to the target or a marked form of the substrate as a diagnostic reagent. , and then determining the increase or decrease in the amount of a metabolite derived from the diagnostic reagent or the diagnostic reagent per se caused by the enzymatic reaction. For example, in the case where a substrate is administered for an enzymatic reaction in vivo that can be enhanced by binding the medicament to the target or a marked form of the substrate to a subject as a diagnostic reagent, if the amount of a metabolite derived from the diagnostic reagent, then the drug would be considered to enhance the function of the target. Therefore, it can be confirmed that the medicine has sufficient efficacy. In the case where a substrate is administered for an in vivo enzymatic reaction that can be inhibited by binding the medicament to the target or a marked form of the substrate to a subject as a diagnostic reagent, for example, if little metabolite derived from the diagnostic reagent, then the medication would be considered to inhibit the function of the target completely. Therefore, it can be confirmed that the medicine has sufficient drug efficacy.

The diagnostic reagent can be formulated in an oral preparation (for example, tablet, capsule, powder, granule, solution) or in an injectable preparation that depends on the route of administration, using any one of the following substances (a) to (f ) alone or in combination with an excipient or vehicle:

(to)

 a compound that serves as a substrate for the enzyme contained in the medicament to be evaluated or for an enzyme generated from the prodrug contained in the medicament to be evaluated, or a pharmaceutically acceptable salt thereof;

(b)

 a compound that serves as a substrate for a different enzyme that can present an alteration in activity by coupling to the effect of the enzyme contained in the drug to be evaluated, or to the effect of an enzyme generated from the prodrug contained in the medicine to be evaluated, or a pharmaceutically acceptable salt thereof;

(C)

 a compound that serves as a substrate for an enzyme that is directly inhibited by the enzyme inhibitor contained in the medicament to be evaluated or by an enzyme inhibitor generated from the prodrug contained in the medicament to be evaluated, or a pharmaceutically acceptable salt of the same;

(d)

 a compound that serves as a substrate for an enzyme that can exhibit an alteration in activity by coupling to the effect of the enzyme inhibitor for the other enzyme, contained in the drug to be evaluated, or to the effect of an enzyme inhibitor generated from the prodrug contained in the medication to be evaluated, or a pharmaceutically acceptable salt thereof;

(and)

 a compound that serves as a substrate for an enzyme that can exhibit an alteration in activity by binding between a receptor and the receptor ligand contained in the medicament to be evaluated or a receptor ligand generated from the prodrug contained in the medicament to be evaluated, or a pharmaceutically acceptable salt thereof; or

(F)

 a marked form of any one of the substances (a) to (e).

With respect to substances (a) to (f), examples of the pharmaceutically acceptable salt include salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid; salts with organic acids such as acetic acid, propionic acid, succinic acid, malic acid, tartaric acid, citric acid and maleic acid, salts with alkali metals such as sodium and potassium; and salts with alkaline earth metals such as calcium.

Substances (a) to (f) may be products marketed or may be produced from starting materials marketed according to known procedures.

For example, DOPA labeled with 13C can be produced as described in production example 1 below. In the same way, DOPA marked with 14C can also be produced.

A 13C or 14C labeled peptide or a salt thereof can be synthesized using a 13C labeled amino acid

or 14C marketed according to known procedures, for example according to the procedure described in "Jikkenkagakukouza 22" (volume 22 of A Course in Experimental Chemistry), Organic Synthesis IV, edited by the Chemical Society of Japan, published by Maruzen (1992). An example of the production process is illustrated below.

A 13C-labeled amino acid is dissolved in hydrogen chloride / methanol and then refluxed. The resulting methyl ester is suspended in dichloromethane, and then triethylamine is added dropwise thereto while stirring under ice cooling conditions. To the solution are added N-benzoyl amino acid, HOBt (1-hydroxy-1H-benzotriazole · H2O) and dichloromethane. A solution of WSC (1-ethyl-3- (3dimethylaminopropyl) -carbodiimide · HCl) in dichloromethane is added to the mixed solution and then stirred. After concentration, the solution is extracted with ethyl acetate, washed with 1 N hydrochloric acid, 5% NaHCO3 and water, dried over magnesium sulfate, concentrated until dry, then optionally saponified, thus providing a labeled peptide. with 13C.

An oligosaccharide or polysaccharide labeled with 13C or 14C or a salt thereof can be synthesized according to any procedure described in the literature (Carbohydrate Research, 176 (1988), 107-115; Japanese Patent Application Publication No. 3-91496; patent No. 4649108; Journal of Chromatography, 336 (1984), 368-373; Japanese Patent Application Publication No. 8-291). In this case, 13C or 14C labeled forms of the above compounds can be synthesized starting from, for example, a commercialized 13C or 14C labeled maltooligosaccharide or a 13C or 14C labeled maltooligosaccharide produced by known procedures.

For example, galactosyloligosaccharide can be produced, which is one of the oligosaccharides having a modified non-reducing terminal end, adding lactose to an oligosaccharide, treating the resulting mixture with lactase and separating the desired product from the reaction mixture by column chromatography (publications Japanese Patent Application No. 4-209277, No. 8-196289 and No. 10-316697). In this case, 13C or 14C labeled galactosyloligosaccharide can be produced using a 13C or 14C labeled oligosaccharide, and galactosyloligosaccharide can be produced having a 13C or 14C labeled galactosyl group using a 13C- or 14C-galactosyl labeled lactic acid, for example .

The excipient or vehicle can be any one that is conventionally used in the art and that is pharmaceutically acceptable. The type and composition of the excipient or vehicle may be appropriately varied depending on the route and the method of administration. For example, water can be used as a liquid vehicle. As a solid carrier, a cellulose derivative such as hydroxypropylcellulose, a salt with an organic acid such as magnesium stearate can be used. For an injectable preparation, sterile water, physiological saline and various types of buffers are generally preferred. A lyophilized preparation may also be possible, which may be used as an oral preparation or may be administered by dissolving in a solvent suitable for injection such as a solution for intravenous administration (eg, sterile water, physiological saline solution and an electrolyte solution) before utilization.

The content of any one of the above substances (a) to (f) (hereinafter referred to as "a major component") in the pharmaceutical preparation may vary depending on the type of the pharmaceutical preparation, but is generally 1 100% by weight, preferably 50 to 100% by weight. For example, in an injectable preparation, the main component can generally be added in a content of 1 to 40% by weight. In a capsule, tablet, granule or powder preparation, the content of the main component in the preparation is approximately 10 to 100% by weight, preferably 50 to 100% by weight, the rest being a vehicle.

The dose amount of the diagnostic reagent of the present invention should be an amount sufficient to allow the detection of the main component or its metabolite in a biological sample of a subject, and may be varied depending on the age and body weight of the subject and the intended purpose of the test. For example, the unit dose amount is approximately 1 to 1,000 mg / kg body weight for an adult human being.

In the test with the diagnostic reagent of the present invention, the diagnostic reagent of the present invention (for example, orally) can be administered to a subject after or simultaneously with the administration (for example, orally) of a medicament. The amount of migration in the blood or the amount of excretion outside the body (for example, the amount of excretion in the urine or breath) is measured after a predetermined period of time, or the integral or time course (pending onset , change in slope, peak time, etc.) of the amount of migration in the blood or the amount of excretion outside the body for a predetermined period of time after administration with respect to the main component or its metabolite. Based on the measurement data, the pharmacological effect of the medicine can be determined.

The measurement procedure is appropriately selected depending on the nature or type of material to be tested (eg, blood, urine, breath) and the substance to be administered (main component), and includes colorimetry, fluorometry , mass spectrometry, NMR (nuclear magnetic resonance), HPLC, gas chromatography, gas chromatography - mass spectrometry (GC-MS), photoelectric acoustic spectroscopy, GM counter procedure, liquid scintillation, solid scintillation, autoradiography, procedure with ionization chamber and the like.

Specifically, to determine the amount of migration in the blood, the extracted blood can be used for measurement without any treatment or it can undergo some treatment (eg isolation, pretreatment) before the measurement. For example, the determination can be carried out by administering a compound labeled with a fluorescent residue as a diagnostic reagent, extracting the blood after a predetermined period of time, preparing the serum or plasma from the blood, and then comparing the fluorescence intensity in the serum and plasma In the case where a compound labeled with a substituent that is absorbed somewhere in the ultraviolet or visible region is administered as a diagnostic reagent, the determination can be carried out by extracting the blood after a predetermined period of time, and then measuring the absorption in the ultraviolet or visible region of a serum or plasma sample from the blood with an absorber or similar. Alternatively, absorption in the ultraviolet or visible region of the blood can be measured externally in the skin without extracting blood.

In the case where the urine is used as a sample, the extracted urine can be used for measurement without any treatment or it can be subjected to some treatment (for example, isolation, pretreatment) before the measurement. For example, the determination can be carried out by administering a compound labeled with a fluorescent residue as a diagnostic reagent, extracting the urine after a predetermined period of time, and then comparing the fluorescence intensity in the urine. In the case where a compound labeled with a substituent that is absorbed somewhere in the ultraviolet or visible region is administered as a diagnostic reagent, the determination can be carried out by extracting the urine after a predetermined period of time, and measuring the absorption in the ultraviolet or visible region of the urine with an absorber or similar.

To determine the amount of excretion in the breath using, for example, a compound labeled with 13C as a diagnostic reagent, the determination can be carried out by gas chromatography - mass spectrometry (GC-MS), infrared spectroscopy, mass spectrometry , photoelectric acoustic spectroscopy, NMR (nuclear magnetic resonance) or the like in 13CO2. In the case where a compound labeled with 14C is used as a diagnostic reagent, the breath, either without any treatment or after entrapment of CO2 in a solvent, can be measured with a GM counter, a liquid scintillation counter or a solid scintillation counter or by autoradiography, ionization chamber procedure or the like.

As stated above, most medications exert the pharmacological effects by enhancing

or inhibiting the function of their targets (bioenzymes or receptors). For example, a pharmaceutical agent comprising an enzyme can exert the pharmacological effect by potentiating an enzymatic reaction in vivo, a pharmaceutical agent comprising an enzymatic inhibitor or a prodrug of the pharmaceutical agent can exert the pharmacological effect by inhibiting an enzymatic reaction in vivo, and a Pharmaceutical agent comprising a receptor ligand or a prodrug of the pharmaceutical agent can exert the pharmacological effect by enhancing or inhibiting the function of a receptor by binding the ligand to the receptor, thereby enhancing or inhibiting a related in vivo enzyme reaction. Therefore, the pharmacological effect of a medicament can be assessed by determining the degree of potentiation or inhibition of a function of a target for a pharmaceutical agent or its prodrug after administration of the pharmaceutical agent or prodrug. Applying this principle, screening for high drug efficacy and / or little side effect between pharmaceutical agents and / or prodrugs thereof is made possible.

The present invention provides a method for screening pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of pharmaceutical agents to select one or ones that have high drug efficacy and / or little effect. secondary, the procedure comprising the steps that consist of:

selecting a pharmaceutical agent or prodrug to be evaluated from pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of the pharmaceutical agents

administer to a non-human subject the pharmaceutical agent or prodrug to be evaluated and a reagent comprising any one of the following substances (a) to (f):

(to)

 a compound that serves as a substrate for the enzyme that is included in the pharmaceutical agent to be evaluated or for an enzyme generated from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof;

(b)

 a compound that serves as a substrate for a different enzyme that may exhibit an alteration in activity by coupling to the effect of the enzyme that is included in the pharmaceutical agent to be evaluated,

or to the effect of an enzyme generated from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof;

(C)

 a compound that serves as a substrate for an enzyme that is directly inhibited by the enzyme inhibitor that is included in the pharmaceutical agent to be evaluated or by an enzyme inhibitor generated from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof ;

(d)

 a compound that serves as a substrate for an enzyme that can present an alteration in activity by the

coupling to the effect of the enzyme inhibitor for the other enzyme, which is included in the pharmaceutical agent to be evaluated, or to the effect of an enzyme inhibitor generated from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof;

(and)

 a compound that serves as a substrate for an enzyme that can exhibit an alteration in activity by binding between a receptor and the receptor ligand that is included in the pharmaceutical agent to be evaluated or a receptor ligand generated from the prodrug that to be evaluated, or a pharmaceutically acceptable salt thereof; or

(F)

 a marked form of any one of the substances (a) to (e);

collect a biological sample of the subject at least once;

measure the amount of any one of the above substances (a) to (f) or a metabolite thereof in the biological sample; Y

evaluate the pharmacological effect of the pharmaceutical agent or prodrug to be evaluated based on the value obtained in the measurement stage. The pharmaceutical agent and / or prodrug to be screened according to this procedure preferably has such a superior drug efficacy, such a minor side effect,

or a drug efficacy as superior and as minor a side effect as possible.

The pharmaceutical agent and / or prodrug to be evaluated can be administered to the subject one or more times simultaneously with or before administration of the reagent comprising any one of the substances (a) to (f).

The present invention also provides a reagent for use in the screening of pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of the pharmaceutical agents for one or those having a high drug efficacy and / or a small side effect, in which the reagent comprises [1-13C] DOPA for screening a pharmaceutical agent directed to DOPA decarboxylase having a high medication effect and / or a prodrug thereof.

The reagent of the present invention can be used in the method of screening pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of pharmaceutical agents to select one or ones that have high drug efficacy and / or little side effect, the procedure comprising the steps of:

selecting a pharmaceutical agent or prodrug to be evaluated from pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of the pharmaceutical agents;

administer to a non-human subject the pharmaceutical agent or prodrug to be evaluated and the reagent;

collect a biological sample of the subject at least once;

measure the amount of [1-13C] DOPA or benzoyl-arginyl- [1-13C] alanine or a metabolite thereof in the biological sample; Y

evaluate the pharmacological effect of the pharmaceutical agent or prodrug to be evaluated based on the value obtained in the measurement stage. The pharmaceutical agent and / or prodrug to be screened according to this procedure preferably has such a superior drug efficacy, such a minor side effect,

or a drug efficacy as superior and as minor a side effect as possible.

The pharmaceutical agent and / or prodrug to be evaluated can be administered to the subject one or more times simultaneously with or before administration of the reagent of the present invention.

In the screening procedure and the reagent of the present invention, in the case where the reagent comprises a 13C-labeled form of any one of substances (a) to (e), the level of 13C of exhaled CO2 from the subject.

In the present specification, the term "pharmaceutical agent comprising an enzyme" refers to a pharmaceutical preparation that can exert the pharmacological effect by enhancing an enzymatic reaction in vivo after administration of the enzyme. Examples of a pharmaceutical agent comprising an enzyme include those preparations comprising each a streptokinase, streptodornase, hyaluronidase, urokinase, lysozyme, amylase, sugary pepsin, diastase, pancreatin or the like.

The term "enzyme inhibitor" is as defined above.

The term "receptor ligand" is as defined above.

The term "prodrug" is as defined above.

5 The term "pharmacological effect of a pharmaceutical agent or a prodrug thereof" refers to an effect that is produced by potentiating or inhibiting the function of a target (a bioenzyme or a receptor), including drug efficacy and a side effect. Drug efficacy means an improvement in the pathological state produced by prolonged potentiation or inhibition of the function of a target (a bioenzyme or a receptor), and a side effect means a detrimental effect produced by such prolonged potentiation or inhibition.

Therefore, "evaluation of a pharmacological effect of a pharmaceutical agent or a prodrug thereof" refers to the evaluation of whether potentiation or inhibition of the function of a target is sufficiently achieved so that

15 the pharmaceutical agent or prodrug improves the pathological state, or the evaluation of whether the potentiation or inhibition of the function of a target for a medicament that can produce a side effect is prevented.

The term "substrate for an enzyme" is as defined above.

The "different enzyme that can present an alteration in activity by coupling to the effect of an enzyme" is as defined above.

Examples of the enzyme, the different enzyme that may exhibit an alteration in activity by coupling to the effect of the enzyme and the substrate for the different enzyme are shown in Table 1 above.

The expression "enzyme that can exhibit an alteration in activity by coupling to the effect of an enzyme inhibitor" is as defined above.

Examples of the enzyme inhibitor, the enzyme directly inhibited by the enzyme inhibitor and its substrate, and the enzyme that can exhibit an alteration in activity by coupling to the effect of the enzyme inhibitor and its substrate are those shown in Table 2 above.

Examples of the enzyme that can present an alteration in activity by binding the ligand to the receptor and the substrate for the enzyme are those shown in Table 3 above.

35 The expression "marked form" is as defined above.

The expression "shape marked with 13C" is as defined above.

The term "subject" is as defined above.

The term "metabolite" refers to a substance that is produced in the metabolic process of a substance that is administered to a living organism. Examples of the metabolites of the substances (a) to (f) above include the substances represented in Table 5 below.

45 Table 5:

(1) Substances corresponding to from (a) to (e)

(2) Pharmaceutical agents screened with (1) (3) Metabolites of (1)

Maltose

 Acarbose Glucose, CO2

Hemiacetal of mevaltic CoA

HMG-CoA reductase inhibitor Mevalonic acid, CO2

Androgen

Aromatase inhibitor Estrogen

dUMP

 Methotrexate dTMP

Most pharmaceutical agents and their prodrugs produce the pharmacological effects by enhancing or inhibiting the function of their targets (bioenzymes or receptors). For example, a pharmaceutical agent comprising an enzyme may have the pharmacological effect by potentiating an enzymatic reaction in vivo, a pharmaceutical agent comprising an enzymatic inhibitor or an enzyme inhibitor generated from a prodrug may have the pharmacological effect by inhibiting an enzymatic reaction in in vivo, and a pharmaceutical agent comprising a receptor ligand or a receptor ligand generated from a prodrug can have the pharmacological effect by enhancing or inhibiting the function of a receptor by binding to the receptor, thereby enhancing or inhibiting an enzymatic reaction. in vivo related. Therefore, the pharmacological effect of a pharmaceutical agent can be evaluated by determining the degree of potentiation or inhibition of the function of a target for the pharmaceutical agent or its prodrug after administration of the pharmaceutical agent or prodrug. The screening process and reagent of the present invention are for the screening of pharmaceutical agents and / or prodrugs of

the same to select one or ones that have high drug efficacy and / or little side effect by evaluating the pharmacological effect of a medicine by determining the degree of potentiation or inhibition of the function of a target.

The principle of evaluation procedures is as follows.

one.

 A case in which the target is an enzyme (for the evaluation of an enzyme, an enzyme inhibitor or a prodrug thereof)

The degree of potentiation or inhibition of the function of a target for an enzyme, an enzyme inhibitor or a prodrug thereof is evaluated by administering to a subject a substrate for the target, a substrate for a second enzyme involved in an enzymatic reaction that can undergo a change by potentiating or inhibiting the function of the target (for example, a coupling reaction with the target) or a marked form of any substrate as a screening reagent, and then determining the increase or decrease in the amount of a metabolite derived from the diagnostic reagent or the diagnostic reagent per se caused by the potentiation or inhibition of the target function. For example, in the case where an enzyme is to be evaluated, a substrate for a bioenzyme (a target) or a marked form of the substrate is administered to a subject as a screening reagent. If the amount of a metabolite derived from the screening reagent increases, then the bioenzyme function would be considered to be enhanced. Therefore, it can be confirmed that the enzyme to be evaluated has sufficient drug efficacy. In the case where an enzyme inhibitor is to be evaluated, for example, a substrate for an enzyme (a target) or a labeled form of the substrate is administered to a subject as a screening reagent. If little metabolite derived from the screening reagent is detected, then the enzyme inhibitor would be considered to inhibit the function of the target satisfactorily. Therefore, it can be confirmed that the enzyme inhibitor has sufficient drug efficacy.

2.

 A case in which the target is a receptor (for the evaluation of a receptor ligand or a prodrug thereof)

The degree of potentiation or inhibition of the function of a target by a receptor ligand or its prodrug is evaluated by administering to a subject a substrate for an enzymatic reaction that may undergo a change by binding the receptor ligand or its prodrug to the target. or a marked form of the substrate as a screening reagent, and then determining the increase or decrease in the amount of a metabolite derived from the screening reagent or the screening reagent per se caused by the enzymatic reaction. For example, in the case where a substrate is administered for an enzymatic reaction in vivo that can be enhanced by binding the receptor ligand or its prodrug to the target or a labeled form of the substrate to a subject as a screening reagent, if the amount of a metabolite derived from the screening reagent increases, then the receptor ligand or its prodrug would be considered to enhance the function of the target. Therefore, it can be confirmed that the receptor ligand or its prodrug has sufficient efficacy. In the case where a substrate is administered for an enzymatic reaction in vivo that can be inhibited by binding the receptor ligand or its prodrug to the target

or a marked form of the substrate to a subject as a screening reagent, for example, if little metabolite derived from the screening reagent is detected, then the receptor ligand or its prodrug would be considered to inhibit the function of the target completely. Therefore, it can be confirmed that the receptor ligand or its prodrug has sufficient drug efficacy.

The screening reagent may be formulated in an oral preparation (e.g., tablet, capsule, powder, granule, solution) or in an injectable preparation depending on the route of administration, using any one of the following substances (a) to (f) alone or in combination with an excipient or vehicle:

(to)

 a compound that serves as a substrate for the enzyme that is included in the pharmaceutical agent to be evaluated or for an enzyme generated from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof;

(b)

 a compound that serves as a substrate for a different enzyme that may exhibit an alteration in activity by coupling to the effect of the enzyme that is included in the pharmaceutical agent to be evaluated,

or to the effect of an enzyme generated from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof;

(C)

 a compound that serves as a substrate for an enzyme that is directly inhibited by the enzyme inhibitor that is included in the pharmaceutical agent to be evaluated or by an enzyme inhibitor generated from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof ;

(d)

 a compound that serves as a substrate for an enzyme that can exhibit an alteration in activity by coupling to the effect of the enzyme inhibitor for the other enzyme, which is included in the pharmaceutical agent to be evaluated, or to the effect of a generated enzyme inhibitor from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof;

(and)

 a compound that serves as a substrate for an enzyme that can exhibit an alteration in activity by binding between a receptor and the receptor ligand that is included in the pharmaceutical agent to be evaluated or a receptor ligand generated from the prodrug that to be evaluated, or a pharmaceutically acceptable salt thereof; or

(F)

 a marked form of any one of the substances (a) to (e).

With respect to substances (a) to (f), examples of the pharmaceutically acceptable salt include salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid; salts with organic acids such as acetic acid, propionic acid, succinic acid, malic acid, tartaric acid, citric acid and maleic acid; salts with alkali metals such as sodium and potassium; and salts with alkaline earth metals such as calcium.

Substances (a) to (f) can be marketed products or can be produced from commercially available starting materials according to known procedures. The procedures are as described above.

The excipient or vehicle can be any one that is conventionally used in the art and that is pharmaceutically acceptable. The type and composition of the excipient or vehicle may be appropriately varied depending on the route and the method of administration. For example, water can be used as a liquid vehicle. As a solid carrier, a cellulose derivative such as hydroxypropylcellulose, a salt with an organic acid such as magnesium stearate can be used. For an injectable preparation, sterile water, physiological saline and various types of buffers are generally preferred. A lyophilized preparation may also be possible, which may be used as an oral preparation or may be administered by dissolving in a solvent suitable for injection such as a solution for intravenous administration (eg, sterile water, physiological saline solution and an electrolyte solution) before utilization.

The content of any one of the above substances (a) to (f) (hereinafter referred to as "a major component") in the pharmaceutical preparation may vary depending on the type of the pharmaceutical preparation, but is generally 1 100% by weight, preferably 50 to 100% by weight. For example, in an injectable preparation, the main component can generally be added in a content of 1 to 40% by weight. In a capsule, tablet, granule or powder preparation, the content of the main component in the preparation is approximately 10 to 100% by weight, preferably 50 to 100% by weight, the rest being a vehicle.

The dose amount of the screening reagent of the present invention should be an amount sufficient to allow the detection of the main component or its metabolite in a biological sample of a subject, and may be varied depending on the age and body weight of the subject and the intended purpose of the test. For example, the unit dose amount is approximately 1 to 1,000 mg / kg body weight for an adult human being.

In screening with the reagent of the present invention, the reagent of the present invention (for example, orally) may be administered to a non-human subject after or simultaneously with the administration (eg, oral) of the pharmaceutical agent or its prodrug It will be evaluated. The amount of migration in the blood or the amount of excretion outside the body (for example, the amount of excretion in the urine or breath) is measured after a predetermined period of time, or the integral or the course of time (pending beginning, change of slope, peak time, etc.) in the amount of migration in the blood or the amount of excretion outside the body for a predetermined period of time after administration with respect to the main component

or its metabolite. The pharmacological effect of the pharmaceutical agent or its prodrug to be evaluated can be determined based on the measurement data.

The measurement procedure is appropriately selected depending on the nature or type of the material to be tested (for example, blood, urine, breath) and the substance to be administered (main component), and includes colorimetry, fluorometry, mass spectrometry, NMR (nuclear magnetic resonance), HPLC, gas chromatography, gas chromatography - mass spectrometry (GC-MS), photoelectric acoustic spectroscopy, GM counter procedure, liquid scintillation, solid scintillation, autoradiography, procedure in ionization chamber and the like.

Specifically, to determine the amount of migration in the blood, the extracted blood can be used for measurement without any treatment or it can undergo some treatment (eg isolation, pretreatment) before the measurement. For example, the determination can be carried out by administering a compound labeled with a fluorescent residue as a reagent, extracting the blood after a predetermined period of time, preparing the serum or plasma from the blood, and then comparing the fluorescence intensity in the serum or plasma. In the case where a compound labeled with a substituent that has an absorption somewhere in the ultraviolet region or visible as a reagent is administered, the determination can be carried out by extracting the blood after a predetermined period of time, and then measuring the absorption in the ultraviolet or visible region of a blood serum or plasma sample with an absorber or similar. Alternatively, absorption in the ultraviolet or visible region of the blood can be measured externally in the skin without extracting blood.

In the case where the urine is used as a sample, the urine withdrawn for measurement can be used without any treatment or it can be subjected to some treatment (eg isolation, pretreatment) before the measurement. For example, the determination can be carried out by administering a compound labeled with a fluorescent residue as a reagent, extracting the urine after a predetermined period of time, and then comparing the fluorescence intensity in the urine. In the case where a compound labeled with a substituent that has an absorption somewhere in the ultraviolet region or visible as a reagent is administered, the determination can be carried out by extracting the urine after a predetermined period of time, and measuring the absorption in the ultraviolet or visible region of the urine with a spectrophotometer or similar.

To determine the amount of excretion in the breath using, for example, a compound labeled with 13C as a reagent, the determination can be carried out by gas chromatography-mass spectrometry (GCMS), infrared spectroscopy, mass spectroscopy, photoelectric acoustic spectroscopy , NMR (nuclear magnetic resonance) or the like in 13CO2. In the case where a compound labeled with 14C is used as a reagent, the breath, either without any treatment or after entrapment of CO2 in a solvent, can be measured with a GM counter, a liquid scintillation counter or a counter solid scintillation or by autoradiography, an ionization chamber procedure or the like.

This specification includes part or all of the content disclosed in the descriptive reports and / or drawings of Japanese patent applications No. 2002-44526 and No. 2002-44791 which are priority documents of the present application.

Brief description of the drawings

Figure 1 shows the results of 1H-NMR and 13C-NMR spectrometric analysis for L- [1-13C] DOPA;

Figure 2 shows the 1H-NMR, 13C-NMR and ESI-MS spectra for Glc- [U-13C] Glc;

Figure 3 shows the results of HPLC and 1H-NMR and 13C-NMR spectrometric analysis for Bz-Arg- [113C] Ala;

Figure 4 shows the 1H-NMR, 13C-NMR and ESI-MS spectra for [U-13C] -cyclodextrin;

Figure 5 shows the time course of the breath test of [1-13C] DOPA;

Figure 6 shows the comparison of the values at the 20 minute time point in the breath test of glucosyl- [U-13C] glucose;

Figure 7 shows the comparison of the values at the 20 minute time point in the breath test of Bz-Arg- [1-13C] Ala;

Figure 8 shows the comparison of the values at the 20 minute time point in the breath test of [U13C] -cyclodextrin; Y

Figure 9 shows the comparison of the values at the 10 minute time point in the breath test of [1,2-13C] ornithine.

Best way to practice the invention

Hereinafter, the present invention will be described more specifically with reference to the examples. However, the examples are provided by way of illustration and not limiting the scope of the invention. The rats used in the experiments (male Wistar, 7 weeks old) were acquired from Nippon Charles River.

[Production example 1] Production of L- [1-13C] DOPA

H2O (10 ml) and Ac2O (6.88 g, 64 mmol) were added to [1-13C] glycine (MassTrace; 2.44 g, 32 mmol) and stirred at room temperature for 20 minutes. The mixture was allowed to stand in a refrigerator overnight, and the precipitated crystals were filtered off and washed with cold water (3 ml). The filtrate was concentrated under reduced pressure and then H2O (2.5 ml) was added, and the resulting mixture was dissolved by heating under reflux. The solution was allowed to stand in a refrigerator overnight, the precipitated crystals were filtered off, washed with cold water (1 ml) and then dried under reduced pressure at 100 ° C giving AcNHCH213CO2H as colorless fine needle crystals.

3,4-Dihydroxybenzaldehyde (1.5 g, 10.8 mmol), AcNHCH213CO2H (1.18 g, 10 mmol) and AcONa (2.46 g, 30 mmol) were mixed thoroughly and then added to AcO2 (10 ml) The resulting solution was stirred at 120 ° C for 5 hours, and then allowed to stand at room temperature overnight. The reaction solution was added to ice water (500 ml). The precipitated yellow powder was filtered off, washed with water (100 ml) and hot water (20 ml) and then air dried. Purification by silica gel column chromatography (hexane-EtOH = 1: 1) gave [carbonyl-13C] 2-methyl-4- (3,4-diacetoxybenzal) -5-oxzolone (1.76 g, 58% ) as yellow crystals.

[Carbonyl-13C] 2-methyl-4- (3,4-diacetoxybenzal) -5-oxzolone (1.31 g, 4.3 mmol) and AcONa (1.6 g, 19.5 mmol) were added to MeOH (12 ml) and refluxed for 5 hours. The reaction solution was purified by silica gel column chromatography (CHCl3-MeOH = 10: 1, AcOEt-MeOH = 40: 1) to provide a pale yellow amorphous solid (1.02 g, 64%). The pale yellow amorphous solid was triturated with a mixed solution of acetone-CHCl3 and then recrystallized to provide [carbonyl-13C] methyl ace-acetoamide-3,4-dihydroxycinnamate.

To a solution of methyl [carbonyl-13C] -acetoamide-3,4-dihydroxycinnamate (0.618 g, 2.45 mmol) in a mixed solution of EtOH: PhH (4: 1) (12 mL) in a stream of hydrogen was added a solution that had been prepared by dissolving s, s-DIOP (249 mg, 0.5 mmol) and [RhCl (cyclooctene) 2] 2 (123 mg, 0.25 mmol) in PhH (2 ml) and stirring the solution at room temperature for 15 minutes in a stream of nitrogen. The resulting solution was stirred at room temperature for 24 hours, the solvent was distilled off under reduced pressure. Purification by silica gel column chromatography (CHCl3-MeOH = 10: 1) gave [carbonyl-13C] N-acetyl-3,4-dihydroxyphenylalanine methyl ester as a reddish brown amorphous solid (0.5 g, 80 %).

[Carbonyl-13C] N-acetyl-3,4-dihydroxyphenylalanine methyl ester (0.435 g, 1.71 mmol) was added to 10% HCl (4 mL) and stirred at 120 ° C for 3 hours. After distillation under reduced pressure 10% HCl, H2O (5 ml) was added to the residue, dried under reduced pressure, H2O (5 ml) was further added and then dried under reduced pressure given an amorphous solid. EtOH (6 ml) was added to the amorphous solid and dissolved therein, 25% aqueous NH3 was added dropwise with ice cooling to adjust the pH to 6.5. The solution was allowed to stand in a refrigerator overnight. The precipitated crystals were filtered off, washed with EtOH and Et2O and then dried under reduced pressure to give a pale brown powder (316 mg, 93%). The pale brown powder was added to H2O (7 ml) containing activated carbon and a trace amount of NaHSO3, heated to reflux and then decolorized. The filtrate was allowed to stand in a refrigerator for 3 days giving colorless prismatic crystals (238 mg, 70%).

Identification data (1 H-NMR and 13 C-NMR spectra) for L- [1-13 C] DOPA are shown in Figures 1 (a) and 1 (b).

[Production example 2] Production of glucosyl- [U-13C] glucose (hereinafter referred to as "Glc- [U-13C] Glc")

Benzyl alcohol (66.7 ml, 645 mmol) and 4.5 N hydrogen chloride / dioxane (9.6 ml, 43.0 mmol) were added to D-glucose-U-13C6 (5.00 g, 26, 87 mmol, SHOKO) and then heated to 80 ° C while stirring. After 50 minutes, the oil bath solution was removed, allowed to cool to room temperature, ether (1.0 1) was added and then cooled with ice. The precipitate produced was filtered off, washed with ether and then dried under reduced pressure (amount produced: 4.52 g,% yield: 60.9%).

Dry acetonitrile (35.0 ml) and dry DMF (7.0 ml) were added to U-13C-Glc-OBzl (4.49 g, 16.26 mmol) under an argon atmosphere and dissolved therein. Benzaldehyde-dimethylacetal (3.17 ml, 21.14 mmol) and p-toluenesulfonic acid monohydrate (618 mg, 3.25 mmol) were added to the solution and then stirred at room temperature. After 2.5 hours, another part of the benzaldehyde-dimethylacetal (245 μl, 1.63 mmol) was added. After 4.5 hours, saturated aqueous sodium bicarbonate was added to neutralize the solution and then concentrated under reduced pressure to remove acetonitrile. The solution was extracted with ethyl acetate, the organic phases were combined, and then washed successively with water and saturated saline. The solution was dried over anhydrous magnesium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in pyridine (100 ml) and cooled on ice. Anhydrous acetic acid (50 ml, 530 mmol) was added to the solution. After 30 minutes, the ice bath solution was removed, and then stirred at room temperature. After 14 hours, the solution was concentrated under reduced pressure, the residue was added with toluene and then azeotropically distilled. This procedure was repeated three times. The residue was purified by silica gel column chromatography to give 4,6-O-benzylidene-2,3-di-O-acetyl-U-13C-Glc-Obzl (amount produced: 3.31 g,% yield : 45.4%).

4,6-O-benzylidene-2,3-di-O-acetyl-U-13C-Glc-OBzl (1.65 g, 3.68 mmol) was dissolved in dry acetonitrile (33.0 ml), and he added dimethylamineborane (1.08 g, 18.4 mmol) dissolved in an argon atmosphere. The reaction mixture was cooled to -40 ° C, and boron trifluoride etherate (2.33 ml, 18.4 mmol) was added thereto. The reaction mixture was gradually heated to -15 ° C for 6 hours and poured into saturated aqueous sodium bicarbonate to neutralize the solution. After concentration under reduced pressure to remove acetonitrile, the solution was extracted with ethyl acetate, the organic phases were combined and washed successively with water and saturated saline. The solution was dried over anhydrous magnesium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 2,3-di-Oacetyl-6-O-benzyl-U-13C-Glc-Obzl (amount produced: 973 mg,% yield: 58.7%) .

2,3-di-O-acetyl-6-O-benzyl-U-13C-Glc-OBzl (191 mg, 0.424 mmol) and 2,3,4,6-tetra-O-benzyl-Glc-OC were dissolved (= NH) -CCl3 (290 mg, 0.424 mmol) in dry chloroform (10.0 ml) and then stirred together with 4A molecular sieve (500 mg) at room temperature for 15 minutes under an argon atmosphere. The reaction mixture was cooled to -60 ° C, boron trifluoride etherate (107 µl, 0.848 mmol) was added thereto, and the mixture was gradually heated to -10 ° C for 3 hours. The reaction mixture was diluted with ethyl acetate and filtered to remove molecular sieve 4A. The resulting solution was washed with ethyl acetate, the filtrate and washings were combined and then washed successively with saturated aqueous sodium bicarbonate, water and saturated saline. The solution was dried over anhydrous magnesium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (amount produced: 254 mg,% yield: 61.5%). Following this procedure, two more condensation reactions were carried out, giving 1.01 g of 2 ', 3', 4 ', 6'-tetra-O-benzyl-Glc- (1-4) -2.3 -di-O-acetyl-6-O-benzyl-U-13C-Glc-OBzl out of a total of 967 mg of 2,3-di-O-acetyl-6-O-benzyl-U-13C-Glc-OBzl .

2 ', 3', 4 ', 6'-tetra-O-benzyl-Glc- (1-4) -2,3-di-O-acetyl-6-O-benzyl-U-13C-Glc was dissolved -OBzl (229 mg, 0.235 mmol) in dry methanol (10.0 ml), a 5.18 M solution of sodium methoxide in methanol (9.1 μl, 47.0 μmol) was added thereto and stirred at room temperature. After 4 hours, another part of a 5.18 M solution of sodium methoxide in methanol (4.6 μl, 23.8 μmol) was added and stirred at room temperature for an additional hour. The reaction solution was added with Amberlyst 15 for neutralization and then filtered. The resin was washed with methanol, the filtrate and washings were combined and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (amount produced: 182 mg,% yield: 87.2%). The deacetylation reaction was carried out again according to this procedure giving 852 mg of 2 ', 3', 4 ', 6'-tetra-Obencil-Glc- (1-4) -6-O-benzyl-U- 13C-Glc-OBzl of a total of 1.01 g of 2 ', 3', 4 ', 6'-tetra-O-benzyl-Glc- (1-4) -2,3-di-Oacetyl-6 -O-benzyl-U-13C-Glc-OBzl.

2 ', 3', 4 ', 6'-tetra-O-benzyl-Glc- (α1-4) -6-O-benzyl-U-13C-Glc-OBzl (180 mg, 0.202 mmol) was dissolved in methanol (30.0 ml) -water (6.0 ml), palladium black (20 mg) was added thereto, and then purged with hydrogen. After 1 hour, the solution was heated to 35 ° C and purged with hydrogen. Water (4.0 ml) was added after 2 hours, and another part of water (20.0 ml) was added after 4 hours. After 6 hours, the solution was filtered, and the catalyst was washed with water. The filtrate and washings were combined, concentrated under reduced pressure to remove methanol and then lyophilized. The residue was dissolved in water (30 ml), palladium black (20 mg) was added thereto, and the solution was heated to 35 ° C and purged with hydrogen. After 6 hours, the solution was filtered, and the catalyst was washed with water. The filtrate and washings were combined, concentrated under reduced pressure to reduce the volume of liquid, and then lyophilized. The residue was purified by HPLC (TSK-Gel Amide-80) (amount produced: 61.5 mg,% yield: 87.6%). The reduction reaction was carried out again according to this procedure giving 290 mg of Glc- [U-13C] Glc of a total of 850 mg of 2 ', 3', 4 ', 6'-tetra-O-benzyl- Glc- (1-4) -6-O-benzyl-U-13C-Glc-OBzl. Identification data (1H-NMR and 13C-NMR spectra) for Glc- [U-13C] Glc are shown in Figures 2 (a), 2 (b) and 2 (c).

[Production example 3] Production of benzoyl-arginyl- [1-13C] alanine (hereinafter referred to as "Bz-Arg- [1-13C] Ala")

[1-13C] Alanine (Masstrace, Inc.) (10.0 g, 0.111 mol) was dissolved in aqueous sodium hydroxide (111 ml), a solution of Boc2O (28.0 ml, 0.122 mol was added thereto) ) in acetone (110 ml) and triethylamine (7.71 ml, 55.5 mmol), and then stirred at room temperature overnight. The solution was concentrated under reduced pressure and saturated saline was added to a total volume of 200 ml. The solution with citric acid was added for pH adjustment to 4, saturated with common salt, and then extracted with ethyl acetate four times. The organic phases were washed with saturated saline solution twice and dried over anhydrous magnesium sulfate. The resulting solution was concentrated under reduced pressure, the residue was dissolved in diethyl ether (150 ml), cyclohexylamine (12.7 ml, 0.111 mol) was added, and then allowed to stand at room temperature for 2 hours. The precipitated crystals were filtered off, washed with diethyl ether, and then dried under reduced pressure to give Boc- [1-13C] Ala-OH · CHA.

10% aqueous citric acid (100 ml) was added to a suspension of Boc- [1-13C] Ala-OH · CHA (32.08 g, 0.111 mol) in ethyl acetate (400 ml), stirred at temperature ambient in a solution and saturated with common salt, and the organic phase was separated from the solution. The resulting solution was extracted with ethyl acetate twice, the organic phases were combined, and then washed with saturated saline twice. The solution was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give a colorless extract. The extract was dissolved in ethanolagua (9: 1) (200 ml) and cesium carbonate (19.0 g, 58.3 mmol) was added. After the foaming ceased, the solution was concentrated under reduced pressure, toluene was added to the residue and then azeotropically distilled to remove water, thereby providing a gel. The gel was suspended in DMF (200 ml), benzyl bromide (13.2 ml, 0.111 mol) was added, and stirred at room temperature for 12 hours. The solution was concentrated under reduced pressure, ethyl acetate was added to the residue, washed successively with water, 10% aqueous citric acid, saturated saline, saturated aqueous sodium bicarbonate and saturated saline, and dried over sodium sulfate. anhydrous magnesium Ethyl acetate was removed by distillation of the solution under reduced pressure to give Boc- [113C] Ala-OBzl.

4.5 N hydrogen chloride / dioxane (250 ml) was added to Boc- [1-13C] Ala-OBzl (31.27 g, 0.111 mol) and then allowed to stand at room temperature for 30 minutes. After concentration under reduced pressure, diethyl ether (200 ml) was added, and the precipitated crystals were filtered off and dried under reduced pressure to give HCl · H- [1-13C] Ala-OBzl (22.48 g).

HCl-H- [1-13C] Ala-OBzl (700 mg, 3.23 mmol), Boc-Arg (Tos) .4 / 5AcOEt.1 / 4H2O (1.63 g, 3.23 mmol) were dissolved and HOBt (459 mg, 3.39 mmol) in DMF (8 ml), WSCD (602 µl, 3.39 mmol) was added dropwise while stirring with ice cooling, and stirred with ice cooling for 30 minutes and then at room temperature for 3 hours. Ethyl acetate was added to the solution, washed successively with saturated aqueous sodium bicarbonate, water, 10% aqueous citric acid and water and dried over anhydrous magnesium sulfate. The ethyl acetate was distilled off from the solution under reduced pressure, diisopropyl ether-hexane was added to the residue, the precipitated solid was filtered and dried under reduced pressure to give Boc-Arg (Tos) - [1-13C] Ala -OBzl (1.78 g, 91%).

6 N hydrogen chloride / dioxane (30 ml) was added to Boc-Arg (Tos) - [1-13C] Ala-OBzl (1.78 g, 2.94 mmol) and stirred at room temperature for 1 hour. Ethyl acetate was added to the solution, and the solution was washed successively with saturated aqueous sodium bicarbonate, water, 10% aqueous citric acid and water and dried over anhydrous magnesium sulfate. The ethyl acetate was distilled off from the solution under reduced pressure, diethyl ether-hexane was added to the residue, and the precipitated solid was filtered off and dried under reduced pressure to give Bz-Arg (Tos) - [1- 13C] Ala-OBzl (1.63 g, 91%).

Anhydrous hydrogen fluoride (8.5 ml) was charged into a mixture of Bz-Arg (Tos) - [1-13C] Ala-OBzl (1.63 g, 2.74 mmol) and anisole (1.5 ml, 13.9 mmol) while stirring with methanol-dry ice bath cooling, and then stirring for 1 hour with ice cooling. The hydrogen fluoride was distilled off from the solution with ice cooling, water (20 ml) and diethyl ether (10 ml) were added to the solution and stirred, and the aqueous phase was removed and concentrated under reduced pressure. . The residue was dissolved in water (30 ml), a trace amount of the insoluble substance was filtered off, and the filtrate was purified by RP-HPLC (10 μm YMC-PAK ODS, 30 x 250 mm, 1-60 MeCN % (containing 0.1% TFA), 80 min., 20 ml / min.). After the main fractions were collected and lyophilized, the residue was dissolved in water (30 ml), exchanged in acetate with a strong ionic anion exchange resin (Muromac 1x2, form of AcO-20 ml), and then lyophilized to give Bz -Arg- [1-13C] Ala as a colorless powder (774 mg, 81%).

Identification data (1H-NMR and 13C-NMR spectra, HPLC analysis graph) for Bz-Arg- [1-13C] Ala are shown in Figures 3 (a), 3 (b) and 3 (c ).

[Production example 4] Production of [U-13C] -cyclodextrin

[U-13C] starch (Chlorella Kogyo Co., Ltd .; Algal Starch (water soluble), lot # 8031, S, U-13C: 98.6% in atoms, starch content: 93 was dissolved , 5%, 4.65 g) in 50 mM acetate buffer (pH 5.4), a concentration of 0.5% (w / v) cycloomaltodextrin glucanotransferase was added thereto (Hayashibara Biochemical Laboratories Inc.) ( 186 units). The solution was allowed to react at 40 ° C for 2 hours and 20 minutes and then treated at 95 ° C for 15 minutes to deactivate the enzyme. This procedure was repeated four times, and the resulting reaction solution was applied in Sephadex G-25 to separate fractions containing [U-13C] iclocyclodextrin. Lyophilized substance (1.4 g) was purified in 6 parts by HPLC (TSK-Gel Amide-80 column). [U-13C] -cyclodextrin fractions were lyophilized to give the product (398 mg). Identification data (1H-NMR, 13C-NMR and ESI-MS spectra) for [U-13C] -cyclodextrin are shown in Figures 4 (a), 4 (b) and 4 (c).

[Example 1] Diagnostic reagent of the pharmacological effect for a component of the antiparkinsonian agent benserazide ([1-13C] DOPA) and screening reagent to evaluate the degree of inhibition or potentiation of DOPA decarboxylase: [1-13C] DOPA (substance that to be evaluated: a component of the antiparkinsonian agent benserazide)

The pharmacological effect of benserazide is the inhibition of DOPA decarboxylase. [1-13C] DOPA is provided (see production example 1) which is a substrate for DOPA decarboxylase as a diagnostic reagent for the pharmacological effect and screening reagent. Comparison was made in the breath test of [1-13C] DOPA in rats that were administered intraperitoneally benserazide hydrochloride (Sigma) at a dose of 50 mg / kg three times (that is, in the morning and night of the day before the breath test of [1-13C] DOPA and 30 minutes before starting the breath test on the day of the test) (administration group) and rats without administration (control group). The breath test of [1-13C] DOPA was performed under anesthesia and [1-13C] DOPA was administered at a dose of 50 mg / kg through the femoral vein, and the course of time of degree was measured 13C level increase in exhaled CO2 (13C (‰)).

The rats of the administration group and the control group that had been fasting overnight under anesthesia were fixed in a supine position. The breath was collected at a rate of 100 to 300 ml / min. using a pulsation pump (variable flow pump VS-500, Shibata Kagaku Kogyo) and was introduced directly into the flow cell of a 13CO2 EX-130S analyzer (Nihon Bunko). A Perma Pure dryer (MD-050-12P, Perma Pure INC.) Was placed between the rat holder and the flow pump to remove water vapor from the breath. When the CO2 concentration was stabilized, [1-13C] DOPA was determined at a dose of 50 mg / kg through the femoral vein. The concentration of CO2 in the breath collected was maintained at 3  0.5%.

The output data of the 13CO2 analyzer was converted to AD and entered into a personal computer (Apple Power Macintosh 8500). Using the Lab VIEW (National Instruments) data processing software, 10 pieces of data were integrated and averaged every 100 ms. at intervals of 5 seconds and converted to% of 13C atoms, 13C (‰) and CO2 concentration (%). In this way, the 13C breath test was continuously carried out. Data converted in real time was presented and stored on a hard disk.

The 13C (‰) was calculated from the level of 13C in the exhaled CO2 at each time point (13Ctmin) and the level of 13C in the standard CO2 (13Cstd) according to the following equation:

image 1

The breath test results of [1-13C] DOPA were as follows: in the control group, 13C increased rapidly after administration, reached 100 ‰ at peak value two minutes after administration and then gradually decreased over time; In the administration group, 13C (‰) showed little increase after administration and was only 5 ‰ even 20 minutes after administration (Figure 5). From results, it was confirmed that the function of the target enzyme for benserazide was almost 100% inhibited under the conditions of administration of benserazide of the present example. Therefore, using [1-13C] DOPA as a diagnostic reagent for the pharmacological effect, it is possible to evaluate the pharmacological effect of benserazide. In addition, using [1-13C] DOPA as a screening reagent, it is possible to provide the method and reagent for screening a pharmaceutical agent directed to DOPA decarboxylase having a stronger effect and / or a prodrug thereof.

[Example 2] Diagnostic reagent of the pharmacological effect for the acarbose antidiabetic agent (Glc- [U-13C] Glc) and screening reagent to assess the degree of inhibition or potentiation of -glucosidase: Glc- [U-13C] Glc (substance to be evaluated: acarbose antidiabetic agent)

The pharmacological effect of acarbose is the inhibition of -glucosidase. Glc- [U-13C] Glc is provided (see production example) which is a substrate for -glucosidase as a diagnostic reagent for the pharmacological effect and screening reagent. The time course of the degree of increase in the level of 13C in exhaled CO2 (13C (‰)) after the administration of Glc- [U-13C] Glc was measured and compared in rats administered by route oral acarbose (trade name: Glucobay, Bayer Corporation) (2 mg / kg, 4 mg / kg or 10 mg / kg) simultaneously with oral administration of Glc- [U-13C] Glc (25 mg / kg) and rats a those administered Glc- [U-13C] Glc alone (acarbose group 0 mg / kg).

The rats (Wistar males, 8 weeks old) were fasted overnight on a rat holder for the microwave irradiation system without anesthesia. The breath was collected at a rate of approximately 100 to 300 ml / min. using a flow pump (variable flow pump VS-500, Shibata Kagaku Kogyo), and the concentration of CO2 in the collected breath was maintained at approximately 3%. A Perma Pure dryer (MD-050-12P, Perma Pure INC.) Was placed between the rat holder and the flow pump to remove water vapor from the breath. When the CO2 concentration was stabilized, the rats were removed from the rat holder and the rats were administered Glc- [U-13C] Glc dissolved in distilled water through the stomach using a tube adapted for oral administration (dose amount : 35 μmol / kg (5 ml / kg)). Breath samples were taken with a syringe to the 20 minute time point at 5 minute intervals. A fifteen ml sample of breath was taken by a syringe in a vacuum vial (10 ml) and sealed, and then subjected to automated analysis by GC-MS (Breath MAT) [FinniganMAT]. The 13C (‰) was calculated from 13C which is the value of 13C for the breath sample (a difference of the value of 13C for the PDB standard substance) according to the following equation:

image 1

<Measuring conditions for Breath MAT> Device: Breath MAT plus (Finnigan) Carrier gas: Measurement Ion: m / z = 44, 45, 46 The breath test results of Glc- [U-13C] Glc are as set out below: in all groups, the

13C increased linearly; in the group of 0 mg / kg (n = 2), 13C reached 72.60  9.47 ‰ in 20 minutes, while 13C reached 37.06  0.16 ‰ in the group of 2 mg / kg (n = 2), reached 26.01  0.38 ‰ in the 4 mg / kg group (n = 2) and reached 9.86  0.66 ‰ in the group of 10 mg / kg (n = 2); therefore, 13C decreased with increasing dose amount (figure 6). From the results it was confirmed that the function of the target enzyme for acarbose was inhibited in a dose-dependent manner under the acarbose administration conditions of the present example. Therefore, using Glc- [U-13C] Glc as a diagnostic reagent for the pharmacological effect, it is possible to evaluate the pharmacological effect of acarbose. In addition, using Glc- [U-13C] Glc as a screening reagent, it is possible to provide the method and reagent for screening a pharmaceutical agent targeting glucosidase having a stronger effect and / or a prodrug thereof.

[Example 3] Diagnostic reagent of the pharmacological effect for camostat mesylate antipancreatitis agent (Bz-Arg- [1-13C] Ala) and screening reagent to assess the degree of inhibition or potentiation of pancreatic protease: Bz-Arg- [1-13C] Ala (substance to be evaluated: camostat mesylate antipancreatitis agent)

The pharmacological effect of camostat mesylate is the inhibition of pancreatic protease. Bz-Arg- [113C] Ala is provided (see production example 3) which is a substrate for pancreatic protease as a diagnostic reagent for the pharmacological effect and screening reagent. The time course of the degree of increase in the level of 13C in exhaled CO2 (13C (‰)) after administration of Bz-Arg- [1-13C] Ala was measured and compared in the same manner as in the example 2 in rats given camostat mesylate orally (trade name: Foipan, Ono Pharmaceutical Co., Ltd.) (20 mg / kg) simultaneously with oral administration of Bz-Arg [1-13C] Ala (12.2 mg / kg) (administration group) and rats given Bz-Arg- [1-13C] Ala alone (control group). The breath test results of Bz-Arg- [1-13C] Ala were as follows: in both groups, 13C increased linearly up to 15 minutes after administration; twenty minutes after administration, 13C reached 41.29  7.49 ‰ in the control group (n = 3), while 13C reached 27.64  4.86 ‰ in the administration group ( n = 3); therefore, 13C decreased in the administration group (figure 7). From the results, it was confirmed that the function of the target enzyme for camostat mesylate was inhibited under the conditions of administration of camostat mesylate of the present example. Therefore, using Bz-Arg- [113C] Ala as a diagnostic reagent for the pharmacological effect, it is possible to evaluate the pharmacological effect of camostat mesylate. In addition, using Bz-Arg- [1-13C] Ala as a screening reagent, it is possible to provide the method and reagent for screening a pharmaceutical agent targeting the pancreatic protease that has a greater effect and / or a prodrug thereof.

[Example 4] Diagnostic reagent of the pharmacological effect for the digestive agent Taka-diastase ([U13C] -cyclodextrin) and screening reagent to assess the degree of inhibition or potentiation of amylase: [U13C] -cyclodextrin (substance to be to be evaluated: digestive agent Taka-diastase)

Taka-diastase is one of the amylases and corresponds to a medicine that comprises an enzyme. It has been used as a digestive agent to support the function of an amylase in a living organism (target enzyme). Therefore, the pharmacological effect of Taka-diastase is the potentiation of amylase. [U-13C] -cyclodextrin (see production example 4) which is a substrate for Taka-diastase and amylase in a living organism is provided as a diagnostic reagent for the pharmacological effect and screening reagent. The time course of the degree of increase in the level of 13C in exhaled CO2 (13C (‰)) after administration of [U-13C] -cyclodextrin was measured and compared in the same manner as in Example 2 in rats given orally [U-13C] -cyclodextrin (40 mg / kg) 5 minutes after oral administration of Taka-diastase (trade name: Takadiastasa, Sankyo Co., Ltd.) (6 mg / kg) (administration group) and rats given [U-13C] -cyclodextrin alone (control group). The breath test results of [U-13C] -cyclodextrin were as follows: in both groups, 13C increased linearly up to 30 minutes after administration; twenty minutes after administration, 13C reached 45.46  15.57 ‰ in the control group (n = 2), while 13C reached 83.66  13.87 ‰ in the administration group ( n = 2); therefore, 13C increased in the administration group (figure 8). From the results, it was confirmed that Taka-diastase enhanced the function of the target amylase enzyme under the conditions of Taka-diastase administration of the present example. Therefore, using [U-13C] -cyclodextrin as a diagnostic reagent for the pharmacological effect, it is possible to evaluate the pharmacological effect of Taka-diastase. In addition, using [U-13C] -cyclodextrin as a screening reagent, it is possible to provide the process and reagent for screening a pharmaceutical agent directed to amylase having a stronger effect and / or a prodrug thereof.

[Example 5] Diagnostic reagent of the pharmacological effect for the H1 receptor antagonist agent ([1,2-13C] ornithine) and screening reagent to assess the degree of inhibition or potentiation of the H1 receptor: [1,2-13C] Ornithine (substance to be evaluated: chlorpheniramine maleate)

The pharmacological effect of chlorpheniramine maleate is the inhibition of the H1 receptor. The time course of the degree of increase in the level of 13C in exhaled CO2 (13 C (‰)) after administration of [1,2-13C] ornithine was measured and compared in the same manner as in Example 2 in rats administered chlorpheniramine maleate (Sigma) intraperitoneally once on the day before the experiment and once on the day of the experiment (40 mg / kg each) and were additionally administered [1,2- 13C] ornithine (10 mg / kg) through the femoral vein 1 hour and 30 minutes after the last administration (administration group) and rats given [1,213C] ornithine alone through the femoral vein ( control group). The breath test results of [1,2-13C] were as follows: in the control group (n = 2), 13C increased rapidly to the 5 minute time point after administration, then increased very slowly, and reached 73.1  0.4 ‰ 10 minutes after administration; in the administration group (n = 2), 13C rapidly increased to the 5 minute time point after administration, and reached 84.7  1.9 ‰ 10 minutes after administration; therefore, 13C increased in the administration group (figure 9). From the results, the presence or absence of administration of chlorpheniramine maleate could be determined under the conditions of administration of [1,2-13C] ornithine of the present example. Therefore, using [1,2-13C] ornithine as a diagnostic reagent for the pharmacological effect, it is possible to evaluate the pharmacological effect of chlorpheniramine maleate. In addition, using [1,2-13C] ornithine as a screening reagent, it is possible to provide the method and reagent for screening a pharmaceutical agent targeting the H1 receptor that has a stronger effect and a much stronger effect, drug efficacy and / or a prodrug thereof.

Industrial applicability

It is possible to evaluate the pharmacological effect of a drug on its target regardless of the real time; This has been impossible to carry out by any prior art technique.

It is also possible to evaluate the degree of inhibition of a target in a complete organism and in real time; This has been impossible to carry out by any prior art technique. Using the results of this evaluation, it is possible to select a high drug efficacy and / or little side effect among pharmaceutical agents and / or prodrugs thereof.

Claims (12)

one.

 Use of any of [1-13C] DOPA or benzoyl-arginyl- [1-13C] alanine to evaluate the pharmacological effect of a medicinal product containing a pharmaceutical agent comprising an enzyme, an enzyme inhibitor or a receptor ligand or a prodrug of the pharmaceutical agent.

2.

 Use according to claim 1, wherein:

(i)

 [1-13C] DOPA is used to evaluate the pharmacological effects of DOPA decarboxylase inhibitors; or

(ii)

 Benzoyl-arginyl- [1-13C] alanine is used to assess the pharmacological effects of protease inhibitors.

3.

 Method of screening pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of the pharmaceutical agents for one or more of which have a high drug efficacy and / or a low side effect, the method comprising the stages that consist of:

selecting a pharmaceutical agent or prodrug to be evaluated from pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of the pharmaceutical agents; administer to a non-human subject the pharmaceutical agent or prodrug to be evaluated and a reagent comprising any of the following substances (a) to (f):

(to)

 a compound that serves as a substrate for the enzyme that is included in the pharmaceutical agent to be evaluated or for an enzyme generated from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof;

(b)

 a compound that serves as a substrate for a different enzyme that can exhibit an alteration in activity by coupling to the effect of the enzyme that is included in the pharmaceutical agent to be evaluated, or to the effect of an enzyme generated from the prodrug that to be evaluated, or a pharmaceutically acceptable salt thereof;

(C)

 a compound that serves as a substrate for an enzyme that is directly inhibited by the enzyme inhibitor that is included in the pharmaceutical agent to be evaluated or by an enzyme inhibitor generated from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof ;

(d)

 a compound that serves as a substrate for an enzyme that can exhibit an alteration in activity by coupling to the effect of the enzyme inhibitor for the other enzyme, which is included in the pharmaceutical agent to be evaluated, or to the effect of a generated enzyme inhibitor from the prodrug to be evaluated, or a pharmaceutically acceptable salt thereof;

(and)

 a compound that serves as a substrate for an enzyme that can exhibit an alteration in activity by binding between a receptor and the receptor ligand that is included in the pharmaceutical agent to be evaluated or a receptor ligand generated from the prodrug that to be evaluated, or a pharmaceutically acceptable salt thereof; or

(F)

 a marked form of any of the substances (a) to (e);

collect a biological sample of the subject at least once; measure the amount of any of the substances (a) to (f) or their metabolite in the biological sample; Y evaluate the pharmacological effect of the pharmaceutical agent or prodrug to be evaluated based on the value obtained in the measurement stage.

Four.

 Method according to claim 3, wherein the reagent comprises a 13C-labeled form of any of the substances (a) to (e) and the level of 13C in the CO2 exhaled in the subject is measured.

5.

 Method according to claim 3, wherein [1-13C] DOPA is used as a reagent to screen a pharmaceutical agent directed to DOPA decarboxylase having a high medication effect and / or a prodrug thereof.

6.

 Method according to claim 3, wherein glucosyl- [U-13C] glucose is used as a screening reagent

a pharmaceutical agent targeting -glucosidase that has a high medication effect and / or a prodrug thereof.

7.

 Method according to claim 3, wherein the benzoyl-arginyl- [1-13C] alanine is used as a reagent for screening a pharmaceutical agent directed to the protease having a high medication effect and / or a prodrug thereof.

8.

 Method according to claim 3, wherein [U-13C] -cyclodextrin is used as a reagent to screen a pharmaceutical agent directed to amylase having a high medication effect and / or a prodrug thereof.

9.

 Method according to claim 3, wherein [1,2-13C] ornithine is used as a reagent to screen a pharmaceutical agent directed at the H1 receptor that has a high medication effect and / or a prodrug thereof.

10.

 Reagent for use in the screening of pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of the pharmaceutical agents for one or some that have a high drug efficacy and / or a small side effect ,

wherein the reagent comprises [1-13C] DOPA for screening a pharmaceutical agent directed to DOPA decarboxylase having a high medication effect and / or a prodrug thereof.

eleven.

 Reagent according to claim 10, wherein the reagent is used in a screening method of pharmaceutical agents each comprising an enzyme, an enzymatic inhibitor or a receptor ligand and / or prodrugs of the pharmaceutical agents for one or more having a high drug efficacy and / or little side effect and the procedure comprising the stages consisting of:

selecting a pharmaceutical agent or prodrug to be evaluated from pharmaceutical agents each comprising an enzyme, an enzyme inhibitor or a receptor ligand and / or prodrugs of the pharmaceutical agents; administer to a non-human subject the pharmaceutical agent or prodrug to be evaluated and the reagent: collect a biological sample of the subject at least once; measure the amount of [1-13C] DOPA or a metabolite thereof in the biological sample; Y evaluate the pharmacological effect of the medicine based on the value obtained in the measurement stage. 12. Diagnostic reagent according to claim 11, wherein the level of 13C in the exhaled CO2 in the subject in the measurement stage is measured.