CN108403692B - Application of the hydrochloric acid Atipamezole as non-specificity P450 enzyme inhibitor - Google Patents
Application of the hydrochloric acid Atipamezole as non-specificity P450 enzyme inhibitor Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Abstract
The present invention relates to the new opplication of hydrochloric acid Atipamezole, specially application of the hydrochloric acid Atipamezole as non-specificity P450 enzyme inhibitor.Hydrochloric acid Atipamezole does not have significant difference to the rejection ability of each isodynamic enzyme, is especially significantly stronger than 1-ABT to the inhibiting effect of CYP2C9, and species variation is small.The present invention is using veterinary drug hydrochloric acid Atipamezole as a new generation's P450 enzyme nonspecific inhibitor, it can be not only used for outside animal body, In vivo study, it can also be used for human vitronectin system research, it and since the inhibiting effect of hydrochloric acid Atipamezole is drug prototype is not metabolite, preincubate need not be carried out when carrying out in vitro study as instrument medicine, it need not be administered in advance when In vivo study, there is the clear superiority for substituting existing classical tool medicine 1-ABT.
Description
Technical field
The present invention relates to the new opplication of veterinary drug hydrochloric acid Atipamezole, specially hydrochloric acid Atipamezole is non-specific as the second generation
The application of property P450 enzyme inhibitor.
Background technique
The reset mode of drug in vivo mainly includes two kinds of approach of metabolism and excretion, and wherein metabolic conversion relies primarily on medicine
Object is metabolized enzymatic.Drug metabolic enzyme includes I phase and II phase metabolic enzyme, and wherein I phase metabolic enzyme is main metabolic enzyme, and P450
Enzyme (CYP enzyme) is main I phase metabolic enzyme, and P450 enzyme is a superfamily, wherein again same comprising multiple responsible drug metabolisms
Work enzyme.Whether drug is very crucial one of ADME property through P450 enzyme metabolite clearance, determines that can it patent medicine and potential
Potential applicability in clinical practice.Therefore in drug discovery early stage, using nonspecific P450 enzyme inhibitor as tool drug
It can determine the metabolite clearance compound contribution in total body clearance in vivo that P450 enzyme mediates, can determine whether that compound is according to result
The no interaction that can occur based on P450 enzyme, whether be easy to appear crowd be metabolized variation and whether need further evaluate and
Exploitation.The prerequisite of non-specific P450 enzyme inhibitor as instrument medicine is their ability to non-specifically inhibit all
P450 isodynamic enzyme plays the role of chemical method and knocks out P450 gene.
1- amino benzotriazole (1-aminobenzotriazole, 1-ABT) is classical non-specificity based on mechanism
P450 enzyme inhibitor.By after P450 enzyme metabolic activation, product can covalently be tied 1-ABT itself with the prosthetic heme group of P450 enzyme
It closes, leads to the reduction of P450 enzymatic activity non-specificity.It is right largely studies have shown that 1-ABT can be applied in vitro and in vivo studies
The P450 enzyme of people and common experimental animal (rat, rabbit, cavy etc.) all has non-specific inhibiting effect.Conventional is external
Experimental method is that 1-ABT the and NADPH preincubate 15- of high concentration (1mM) is first added in hepatomicrosome or liver cell system
After 30min, the probe substrate of each P450 isodynamic enzyme is added.A large amount of experiment in vitro prove, to CYP after 1-ABT (1mM) preincubate
Multiple isodynamic enzymes have obvious inhibiting effect in enzyme, especially to CYP2A6 and 3A4, can almost completely inhibit its work
With；But not etc. (19-58%) to the rejection ability power of other several isodynamic enzymes, in addition to the inhibition to CYP2C19 and CYP2E1
Effect be significantly stronger than other than its specific inhibitor, to other several isodynamic enzymes include CYP1A2, CYP2B6, CYP2C8,
The inhibiting effect of CYP2C9 and CYP2D6 is below respective specific inhibitor, especially very to the inhibiting effect of CYP2C9
It is weak, such as in rat liver microsomes system, 50 μM of 1-ABT is only capable of 60% inhibition CYP2C dependence
The 4- hydroxylating of cyclophosphamide, the 1-ABT of 1mM is only capable of inhibiting 40% or so double chlorine in people's hepatomicrosome
Fragrant acid hydroxylating.Since the substrate of CYP2C9 mostly contains carboxylic acid group, and 1-ABT lacks carboxylic acid negative charge and explains portion
Divide reason, therefore 1-ABT is apparently higher than Diclofenac to the rejection ability of the CYP2C9 S- neodicoumarin hydroxylating mediated.By
In the substrate that many clinical medicines are all CYP2C9, this defect of 1-ABT may obviously underestimate the effect of CYP2C9, thus
Cause the deviation of result.There is the inhibiting effect intensity to each isodynamic enzyme as classical P450 enzyme non-specific inhibitors in 1-ABT
It obviously differs, problem especially weaker to the rejection ability of CYP2C9, usually needs to consider experiment carefully in practical application
As a result, being the early stage that drug metabolism Mechanism Study and compound metabolism remove property when necessary with other inhibitor use in conjunction
Evaluation brings great uncertainty.
To solve this critical issue, as can the stronger non-specificity P450 enzyme inhibitor substitution 1-ABT of discovery effect will
It is significantly.
Hydrochloric acid Atipamezole is a with glyoxaline structure2Adrenoceptor antagonists can quickly and effectively restore
The anesthesia and calmness of animal mitigate adverse reaction caused by isoflurane or Patients Under Ketamine Anesthesia.Clinically commonly use hydrochloric acid Atipamezole
Calmness or bradycardia caused by the animals such as antagonism Medetomidine anesthetized cat, dog, pig, while hydrochloric acid Atipamezole can effectively delay
Solve adverse reaction caused by Dexmedetomidine-midazolam-ketamine combined anesthesia.
Summary of the invention
The problem to be solved in the present invention is to be found by screening to the preferable P450 enzyme of each isodynamic enzyme inhibiting effect of P450
Non-specific inhibitors provide better instrument medicine as second generation instrument medicine, for drug screening and drug metabolism Mechanism Study.
The present invention has found in screening drug interaction potential, hydrochloric acid Atipamezole to each major isoenzyme of P450 enzyme all
It significantly inhibits, and inhibition strength is higher, effect is substantially better than classical similar tools medicine 1-ABT, can be used as
P450 enzyme nonspecific inhibitor of new generation.
The present invention is by the liver microsomes incubation system of three kinds (rat, dog and people), studying hydrochloric acid in vitro
Atipamezole finds that hydrochloric acid Atipamezole is bright as P450 enzyme nonspecific inhibitor to the inhibiting effect of P450 major isoenzyme
It is aobvious to be better than classical similar tools medicine 1-ABT, it shows:
1, hydrochloric acid Atipamezole is non-specific good to the inhibiting effect no significant difference of each major isoenzyme of P450 enzyme；Institute
Stating P450 isodynamic enzyme includes CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4；
2, hydrochloric acid Atipamezole can carry out kind extrapolation to the inhibiting effect of P450 enzyme without obvious species variation；
3, IC of the hydrochloric acid Atipamezole to P450 enzyme inhibition50Value is significantly lower than 1-ABT, and rejection ability is strong, using agent
It is small to measure (concentration)；
4, the physicochemical property of hydrochloric acid Atipamezole is good, solubility is good, and stability is good, no obvious toxic-side effects, external internal
Using safe and reliable；
5, hydrochloric acid Atipamezole is non-time dependent to the inhibiting effect of metabolic enzyme, during preincubate be added and not
After NADPH starting reaction is added, hydrochloric acid Atipamezole does not substantially change the inhibiting effect of each CYP isodynamic enzyme, prompts main
It is that drug prototype hydrochloric acid Atipamezole plays inhibiting effect.Preincubate need not be carried out when in vitro study, greatly simplify experimental implementation
Process.
The present invention can be not only used for animal body using veterinary drug hydrochloric acid Atipamezole as a new generation's P450 enzyme nonspecific inhibitor
Outside, In vivo study, it can also be used to which human vitronectin system research, inhibiting effect are substantially better than existing classical tool medicine 1-ABT.
Medical compounds is removed in vitro using hydrochloric acid Atipamezole screening study P450 enzyme the present invention also provides a kind of
Medical compounds and hydrochloric acid Atipamezole to be measured is added in the method for effect in liver microsomes incubation system, be not added hydrochloric acid Ah
For U.S. azoles as negative control, the external clearance rate (CL of medical compounds more to be measuredint) ratio, according to formula contribution degree
(fm)=[1-CLInt+ Atipamezole/CLInt- Atipamezole] × 100% judges the contribution that P450 enzyme removes medical compounds In vitro metabolism.
Wherein, concentration of the hydrochloric acid Atipamezole in liver microsomes incubation system is preferably 10~50 μM, and more preferably 20~30 μM.
A method of using hydrochloric acid Atipamezole screening study P450 enzyme to scavenging effect in medical compounds body, simultaneously
Medical compounds to be measured and hydrochloric acid Atipamezole are taken orally, not take hydrochloric acid Atipamezole as negative control, medicine more to be measured
Drug prototype exposed amount (AUC) changes ratio in compounds body, according to formula contribution degree (fm)=[1-AUCAtipamezole/
AUC+ Atipamezole] × 100% judges contribution of the P450 enzyme to metabolite clearance in medical compounds body.Wherein, hydrochloric acid Atipamezole is given
The oral dose of rat is preferably 2~4mg/kg, more preferably 3mg/kg.
In above-mentioned technical proposal, hydrochloric acid Atipamezole is determined as the tool drug of nonspecific P450 enzyme inhibitor
Contribution of the metabolite clearance that P450 enzyme mediates in medical compounds total body clearance, provides for drug screening and drug metabolism mechanism
Method and foundation.
Detailed description of the invention
Fig. 1 is hydrochloric acid Atipamezole to the time dependent inhibiting effect of CYP1A2；
Fig. 2 is hydrochloric acid Atipamezole to the time dependent inhibiting effect of CYP2B6；
Fig. 3 is hydrochloric acid Atipamezole to the time dependent inhibiting effect of CYP2C8；
Fig. 4 is hydrochloric acid Atipamezole to the time dependent inhibiting effect of CYP2C9；
Fig. 5 is hydrochloric acid Atipamezole to the time dependent inhibiting effect of CYP2C19；
Fig. 6 is hydrochloric acid Atipamezole to the time dependent inhibiting effect of CYP2D6；
Fig. 7 is hydrochloric acid Atipamezole to the time dependent inhibiting effect of CYP3A4.
Specific embodiment
The present invention is by vitro in the liver microsomes incubation system of three kinds (rat, dog and people), using P450
Isodynamic enzyme probe substrate method research hydrochloric acid Atipamezole finds hydrochloric acid Atipamezole pair to the inhibiting effect of P450 major isoenzyme
The substrate utilization reaction that three kind P450 major isoenzymes mediate has apparent inhibiting effect.The results show that hydrochloric acid Ah replacing
U.S. azoles makees the probe substrate metabolism that CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are mediated
With there is inhibiting effect, 1-ABT especially is significantly stronger than to the inhibiting effect of CYP2C9, and without apparent species variation.
The external drug interaction model research that application time of the present invention relies on also found that hydrochloric acid Atipamezole is to metabolism
The inhibiting effect of enzyme be it is non-time dependent, be added and be added without during preincubate NADPH starting reaction after, hydrochloric acid Ah replacing
U.S. azoles does not substantially change the inhibiting effect of each CYP isodynamic enzyme, prompts to be mainly that drug prototype hydrochloric acid Atipamezole plays suppression
Production is used.
Since rejection ability of the hydrochloric acid Atipamezole to each isodynamic enzyme does not have significant difference, especially to the inhibition of CYP2C9
Effect is significantly stronger than 1-ABT, and species variation is small, can be not only used for outside animal body, In vivo study, it can also be used to which human vitronectin system is ground
Study carefully；And since the inhibiting effect of hydrochloric acid Atipamezole is drug prototype be not metabolite, it need not when carrying out in vitro study
Preincubate is carried out, when In vivo study need not be administered in advance, have the clear superiority for substituting existing classical tool medicine 1-ABT.Cause
This, hydrochloric acid Atipamezole can be used as a new generation's P450 enzyme nonspecific inhibitor.
Tool drug of the hydrochloric acid Atipamezole as nonspecific P450 enzyme inhibitor can be used in measuring P450 enzyme Jie
Contribution of the metabolite clearance led in medical compounds total body clearance, be drug screening and drug metabolism mechanism providing method and according to
According to.
The present invention is using hydrochloric acid Atipamezole screening study P450 enzyme to the method packet of the external scavenging effect of medical compounds
It includes, medical compounds and hydrochloric acid Atipamezole to be measured is added in liver microsomes incubation system, hydrochloric acid Atipamezole work is not added
For negative control.External clearance rate (the CL of medical compounds more to be measuredint) ratio, according to formula contribution degree (fm)=[1-
CLInt+ Atipamezole/CLInt- Atipamezole] × 100% judges the contribution that P450 enzyme removes medical compounds In vitro metabolism.In the present invention,
The type of small molecule drug compound to be measured is not particularly limited, any kind of compound could be used for of the invention small point
In sub- medical compounds screening.
Wherein, in liver microsomes incubation system, protein concentration is preferably 0.1~1mg/ml, more preferably 0.5mg/ml；To
Surveying concentration of the medical compounds in liver microsomes incubation system is preferably 1~2 μM；Hydrochloric acid Atipamezole is in liver microsomes incubation
Concentration in system is preferably 10~50 μM, and more preferably 20~30 μM.The present invention, which is preferably started with NADPH, to react, described
The concentration of NADPH is preferably 1~2mM.Ice acetonitrile is added preferably in the incubation system and terminates reaction by the present invention.The present invention is incubated
The temperature for educating reaction is preferably 36~38 DEG C, and more preferably 37 DEG C；The time of the incubation reaction is preferably 10~60min, more
Preferably 20~40min.When reaction terminating, the external clearance rate of medical compounds is measured.
External clearance rate calculation method of the present invention is preferred are as follows:
Concentration when the zero of each medical compounds to be measured is set as 100%, the medical compounds concentration of different incubation time points
Medical compounds is obtained compared with concentration when zero in the remaining percentage at each time point, by each time point residue hundred of medical compounds
The natural logrithm and the linear recurrence of incubation time for dividing ratio obtain slope (- k), obtain hydrochloric acid Atipamezole micro- by formula (1)
The T being metabolized in plastochondria1/2(min)；
T1/2=-0.693/k (1)
Extrapolation is carried out to hepatomicrosome data by Well StierredModel and obtains medical compounds in different genera liver
Intrinsic clearance CL in microsomeint(mL·min-1·kg-1) (formula 2)
Wherein, in formula (2), X is the common scale factor of different genera (g liver weight/kg weight), such as people=20；Beasle dog
=25；Rat=45.
The ratio variation of medical compounds to be evaluated In vitro metabolism clearance rate after hydrochloric acid Atipamezole is not added in adduction is calculated,
According to formula contribution degree (fm)=[1-CLInt+ Atipamezole/CLInt- Atipamezole] × 100% judges P450 enzyme to medical compounds external generation
Thank to the contribution of removing.
In the present invention, the probe substrate of each isodynamic enzyme of P450 is added in liver microsomes incubation system, be added hydrochloric acid Ah
It is incubated for, is incubated with NADPH starting reaction hydrochloric acid Atipamezole is not added as negative control as positive control for U.S. azoles
After educating, each probe substrate external intrinsic clearance (CL after hydrochloric acid Atipamezole is not added in adduction is calculatedint) ratio, e.g.,
CLInt+ Atipamezole/CLInt- Atipamezole< 0.2, then prove that hydrochloric acid Atipamezole inhibits P450 enzymatic activity substantially, the system is reliable.
The present invention is using hydrochloric acid Atipamezole screening study P450 enzyme to the method for scavenging effect in medical compounds body, packet
It includes: while medical compounds to be measured and hydrochloric acid Atipamezole are taken orally, not take hydrochloric acid Atipamezole as negative control, compare
Drug exposure changes ratio in medical compounds body to be measured, judges tribute of the P450 enzyme to metabolite clearance in medical compounds body
It offers.
The present invention preferably applies in the medical compounds body to be measured in pharmacokinetic methods measurement different time sections
Drug exposure (AUC).According to formula contribution degree (fm)=[1-AUCAtipamezole/AUC+ Atipamezole] × 100% judges P450 enzyme to medicine
The contribution of metabolite clearance in compounds body.
In the present invention, oral object can be such as rat or dog animal, or people.The present invention is according to drug to be measured
The oral dose that the oral recommended dose setting of compound adapts to.Oral dose of the hydrochloric acid Atipamezole of the present invention to rat
Preferably 2~4mg/kg, more preferably 3mg/kg.
To make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment to the present invention into
Row detailed description, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Inhibiting effect of the hydrochloric acid Atipamezole to each isodynamic enzyme of CYP
Each same work of CYP shown in table 1 is added in rat, dog and people's hepatomicrosome solution (protein concentration 0.5mg/ml) respectively
The hydrochloric acid of the probe substrate of enzyme and 0~100 μM of series of concentrations (0,0.091,0.412,1.23,3.7,11.1,33.3,100 μM)
After Atipamezole preincubate 5min, NADPH (final concentration of 1mM) starting reaction, 37 DEG C of common incubations is added, incubation time is shown in Table
1.After the completion of incubation, ice acetonitrile is added and terminates reaction, high speed centrifugation after vortex takes supernatant LC-MS/MS to measure each probe substrate
The production quantity of metabolite.Logarithm will be converted into various concentration hydrochloric acid Atipamezole, under each concentration metabolite production quantity with
Blank control group is fitted, and then obtain hydrochloric acid Atipamezole to each kind P450 compared to ratio is obtained using Prism software
Half-inhibitory concentration (the IC of major isoenzyme50)。
Concentration and incubation time of the probe substrate of 1 each hypotype of CYP enzyme of table in liver microsomes incubation liquid
Comparative example 1
Inhibiting effect of the 1-ABT to each isodynamic enzyme of CYP
It is each same that CYP shown in table 1 in embodiment 1 is added in rat liver microsomes liquid solution (protein concentration 0.5mg/ml) respectively
The 1-ABT of the probe substrate and 0~10mM series of concentrations (0,0.91,4.12,12.3,71.111,333,1000 μM) of work enzyme is pre-
After being incubated for 5min, NADPH (final concentration of 1mM) starting reaction is added, 37 DEG C are incubated for jointly, and incubation time is shown in Table 1.It is incubated for and completes
Afterwards, ice acetonitrile is added and terminates reaction, high speed centrifugation after vortex takes supernatant to measure each probe substrate metabolite with LC-MS/MS
Production quantity.It will be converted into logarithm in various concentration 1-ABT, the ratio that metabolite production quantity and blank control group are compared under each concentration
Value, is fitted, and then obtain 1-ABT to the half-inhibitory concentration of each kind P450 major isoenzyme using Prism software
(IC50)。
The inhibiting effect of 2 hydrochloric acid Atipamezole of table and 1-ABT to each kind P450 major isoenzyme
As can be seen from Table 2, hydrochloric acid Atipamezole reacts the substrate utilization that three kind P450 major isoenzymes mediate
There are apparent inhibiting effect, IC50Value is significantly lower than 1-ABT；1-ABT especially is significantly stronger than to the inhibiting effect of CYP2C9,
And without apparent species variation.
Embodiment 2
Hydrochloric acid Atipamezole is based on time dependent inhibiting effect to CYP enzyme
Incubation is divided into two groups, and one group starts reaction group for NADPH is added, and another group does not start reaction to be added without NADPH
Group.
Be added in rat liver microsomes liquid solution (protein concentration 0.5mg/ml) and 0~100 μM of series of concentrations (0,0.091,
0.412,1.23,3.7,11.1,33.3,100 μM) hydrochloric acid Atipamezole, be added or be added without NADPH (final concentration of 1mM)
It is incubated for 30min, each isodynamic enzyme probe substrate is added according to concentration shown in table 1 later, is incubated for jointly at 37 DEG C, removes CYP2C19
The appropriate incubation time of probe substrate (S)-Mei Fen be outside 30min, the incubation time of remaining probe substrate is 10min.It has been incubated for
It terminates and reacts at rear addition ice acetonitrile, high speed centrifugation after vortex takes supernatant LC-MS/MS to measure each probe substrate metabolite
Production quantity will be converted into logarithm in various concentration hydrochloric acid Atipamezole, metabolite production quantity and blank control group under each concentration
The ratio compared is fitted using Prism software, and then obtains hydrochloric acid Atipamezole to each kind P450 major isoenzyme
Half-inhibitory concentration (IC50)。
Fig. 1~7 are hydrochloric acid Atipamezole to the time dependent inhibiting effect of CYP major isoenzyme.It can be seen by Fig. 1~7
Out, after being added and being added without NADPH starting reaction during preincubate, inhibition of the hydrochloric acid Atipamezole to each CYP isodynamic enzyme
Curve essentially coincides, and the addition of NADPH inhibits the effect of CYP enzyme not substantially change hydrochloric acid Atipamezole, illustrate hydrochloric acid Ah
It is non-time dependent, prompt mainly drug prototype hydrochloric acid Atipamezole performance inhibition for inhibiting effect of the U.S. azoles to metabolic enzyme
Effect.
Embodiment 3
Be added in the hepatomicrosome solution (protein concentration 0.5mg/ml) of rat 1 μM of ZC56 medical compounds to be evaluated,
20 μM of hydrochloric acid Atipamezole, and to be added without hydrochloric acid Atipamezole as negative control, after 37 DEG C of preincubate 5min, it is added
NADPH 1mM starting reaction is added ice acetonitrile respectively at 5,15,30,60min and terminates reaction.Medical compounds is calculated in rat
Intrinsic clearance CL in hepatomicrosomeint, calculation method is as follows:
Concentration when the zero of medical compounds to be measured is set as 100%, different incubation time point concentration are when zero compared with concentration
The remaining percentage of medical compounds is obtained, by the natural logrithm and incubation time of each time point residue percentage of medical compounds
Linear recurrence obtains slope (- k), obtains the T that Atipamezole is metabolized in microsome by formula (1)1/2(min), then by Well
StierredModel carries out extrapolation to hepatomicrosome data and obtains intrinsic clearance CL of the drug in rat liver microsomesint
(mL·min-1·kg-1) (formula 2)
T1/2=-0.693/k (1)
It wherein, is 45 than putting factor X in rat liver microsomes, hepatomicrosome protein concentration is 0.5mg/ml, hepatomicrosome
Mg/ liver weight g is empirical value 45；The naturally right of external each time point residue percentage after hydrochloric acid Atipamezole is not added in adduction in ZC56
It is respectively 0.0042 and 0.0236, T that the linear recurrence of several and incubation time, which obtains slope (- k),1/2Respectively 165min and
26.5min.ZC56 In vitro metabolism after hydrochloric acid Atipamezole is not added in adduction, which is calculated, according to formula 2 according to experimental result removes difference
For 17.0 and 106.7mLmin-1·kg-1, according to formula contribution degree (fm)=[1-CLInt+ Atipamezole/CLInt- Atipamezole] × 100%
Judge that P450 enzyme participates in the compound metabolism and removes contribution as 84%.
Embodiment 4
Carry out the positive control research of research system reliability simultaneously when evaluating and screening compound
Be added in the hepatomicrosome solution (protein concentration 0.5mg/ml) of rat 1 μM of mixed probe substrate (phenacetin,
Diclofenac, (S)-Mephenetoin, dextromethorphan, midazolam) and 20 μM of hydrochloric acid Atipamezole, and to be added without hydrochloric acid Ah replacing
U.S. azoles is as negative control, after 37 DEG C of preincubate 5min, NADPH 1mM starting reaction is added, adds respectively at 5,15,30,60min
Enter ice acetonitrile and terminates reaction.Calculate intrinsic clearance CL of the mixed probe substrate in rat liver microsomesint, according to formula tribute
Degree of offering (fm)=[1-CLInt+ Atipamezole/CLInt- Atipamezole] × 100% calculates P450 enzyme to the contribution degree of each probe substrate, as a result shows
Show that hydrochloric acid Atipamezole can obviously inhibit each probe substrate in the In vitro metabolism of rat liver microsomes, contribution degree fmIt is all larger than 80%
(table 3), it is believed that the system is reliable.
3 mixed probe substrate of table is not added after hydrochloric acid Atipamezole as positive control adduction and is metabolized in rat liver microsomes
Stability
Embodiment 5
Rat 10, it is randomly divided into two groups, every group 5, wherein first group of stomach-filling medical compounds ZC56 to be measured simultaneously
(2mg/kg) and hydrochloric acid Atipamezole (3mg/kg), second group of only stomach-filling ZC56 (2mg/kg).Different time points (5,15,30,
60min, 2,4,6,8,12h) blood sampling, separated plasma, using LC-MS/MS measurement plasma drug level, according to different time points medicine
Object concentration calculates separately the plasma exposure amount (AUC) of ZC56 in two groups.
After stomach-filling 12h, the plasma exposure amount (AUC) of ZC56 is respectively 158396 ± 99512 Hes in first group and second group
469937 ± 175113ng/ml*min, according to formula contribution degree (fm)=[1-AUC-Atipamezole/AUC+ Atipamezole] × 100%, judgement
P450 enzyme is 67% to the contribution of metabolite clearance in medical compounds body.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (4)
1. application of the hydrochloric acid Atipamezole as non-specificity P450 enzyme inhibitor, which is characterized in that hepatomicrosome is incubated in vitro
It educates in system, using hydrochloric acid Atipamezole screening study P450 enzyme to the external scavenging effect of medical compounds.
2. application according to claim 1, which is characterized in that the P450 enzyme includes CYP1A2, CYP2B6, CYP2C8,
CYP2C9, CYP2C19, CYP2D6 and CYP3A4.
3. a kind of method using hydrochloric acid Atipamezole screening study P450 enzyme to the external scavenging effect of medical compounds, feature
It is, medical compounds and hydrochloric acid Atipamezole to be measured is added in liver microsomes incubation system, hydrochloric acid Atipamezole is not added
As negative control, the external clearance rate CL of medical compounds more to be measuredintRatio, according to formula contribution degree (fm)=[1-
CLInt+ Atipamezole/CLInt- Atipamezole] × 100% judges the contribution that P450 enzyme removes medical compounds In vitro metabolism.
4. according to the method described in claim 3, it is characterized in that, the hydrochloric acid Atipamezole is in liver microsomes incubation system
Concentration be 10~50 μM.
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| CL2008003553A1 (en) * | 2007-12-05 | 2009-11-27 | Grindeks Jsc | Process to prepare atipamezole or 5- (2-ethyl-2,3-dihydro-1h-inden-2-yl) -1h-imidazole hydrochloride: and the intermediate compounds considered in the process |
| US8637524B2 (en) * | 2009-07-28 | 2014-01-28 | Auspex Pharmaceuticals, Inc | Pyrimidinone inhibitors of lipoprotein-associated phospholipase A2 |
| CN104274415A (en) * | 2013-07-04 | 2015-01-14 | 青岛康地恩动物药业有限公司 | Atipamezole hydrochloride-containing calming and pain easing tablet for pets, and preparation method thereof |
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