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pharmacokinetics and tolerability of nsc23925b, a novel p-glycoprotein inhibitor: preclinical study in mice and rats - clear polycarbonate tube

by:Cailong     2019-08-11
pharmacokinetics and tolerability of nsc23925b, a novel p-glycoprotein inhibitor: preclinical study in mice and rats  -  clear polycarbonate tube
P-over-expressionglycoprotein (Pgp)
Increase multi-drug resistance (MDR)
In cancer, this greatly hinders the satisfactory clinical treatment and results of cancer patients.
Due to unknown drug generation dynamics, although in vitro results are promising, it is still difficult to achieve the use of Pgp inhibitors in clinical settings to overcome drug resistance.
The purpose of our current pre-clinical study is to study the drug generation dynamics and tolerance of NSC23925b, a new and effective P-
A protein inhibitor in rodents.
Study on plasma drug generation dynamics of single administration
Dose NSC23925b alone or in combination with paclitaxel or polybenic in male mice and sd-Dawley rats.
In addition, it is necessary (CYP450)
In vitro test was performed with NSC23925b.
Finally, the maximum tolerance dose (MTD)
The content of NSC23925b was determined.
The NSC23925b showed a good kinetic profile after abdominal/intravenous injection (I. P. /I. V. )
Injection alone or in combination with chemotherapy drugs.
When co-used, the plasma drug generation kinetic features of chemotherapy drugs are not affected
Administration with NSC23925b.
All animals tolerate I. P. /I. V.
Management of NSC23925b.
In addition, NSC23925b did not inhibit the enzyme activity of human cyp 50.
Our results suggest that the Pgp inhibitor NSC23925b exhibits encouraging clinical prodrug kinetic features and limited toxicity in vivo.
NSC23925b is likely to treat cancer patients with multi-drug resistance in the future.
NSC23925b is split (
Molecular mass: 384. 9u2009g/mol)
Synthesized by Chengdu chempartners Co. , Ltd.
Purity> 99%.
NSC23925b was originally dissolved in 5% px (DMSO)
And then dilute to concentration in normal saline as required.
The final carrier concentration is 1-5% acetone and 95-99% saline. Paclitaxel (6u2009mg/mL)
And more than a star (2u2009mg/mL)
Provided by Teva Pharmaceutical Company (
PA)
And APP Pharmaceutical (Schaumburg, IL), respectively.
Dilute two chemotherapy drugs with normal saline for injection.
From Sigma-Aldrich (St. Louis, MO).
Get clinical saline from Jiling Dubang pharmaceutical company. , Ltd.
Based on available data from previous relevant studies, the dose levels of NSC23925b, paclitaxel, and polybenic were determined.
Specific doses of each chemical are described in the specific experiments below.
All other reagents used in this study are provided by the Pharmacy Laboratory (Shanghai, China).
Data for processing and disposing of instructions and any available security information are kept in the Archives of the pharmaceutical laboratory.
Mice and sd-Dawley rats (SD rats)
Order from Shanghai SLAC experimental animal Co. , Ltd. Ltd (Shanghai, China).
All animals do not have specific pathogens and are about 6-8 weeks old when they arrive at the pharmacy laboratory.
The mice were 18-22 μg, and the rats were 200-2 50 μg after receiving it.
Health checks were performed on each animal, including assessment of fur, limbs, and holes.
It was also checked to see if there were any signs of abnormality in each animal's posture or movements.
The animals are locked in a pharmaceutical laboratory in a transparent polycarbonate plastic cage.
The adaptation time of animals to the environment is not less than seven days.
Observe every day signs of adverse health in animals and general reactions to treatment.
The procedure applied to animals in this study has been approved by the MIT General Hospital Research Animal Care Subcommittee (
Yy Agreement No. 203n000121)
Committee on Institutional Animal Care and Use.
These methods are carried out in accordance with the approved guidelines.
For humanitarian reasons, any dying animal is combined with inhalation for euthanasia, and then the cervical spine is dislocated.
All surviving animals placed in the study were euthanasia at the end of the scheduled study.
Additional animals obtained for this study, but not placed in the study, were returned to pharmacy vivarium for other purposes such as training programs.
In male mice and SD rats, the drug generation dynamics studies of NSC23925b, paclitaxel and polybenic were performed.
Specifically, 5. 00u2009mg/kg (
Dose concentration: 0. 50u2009mg/mL)and 2. 50u2009mg/kg (
Dose concentration: 0. 25u2009mg/mL)
NSC23925b was administered via abdominal and intravenous administration (I. P. and I. V. )
Injection separately; 5. 00u2009mg/kg (
Dose concentration: 0. 50u2009mg/mL)
Paclitaxel was administered to all mice through I. V. ; and 2. 00u2009mg/kg (
Dose concentration: 0. 20u2009mg/mL)
The mouse and the 1. 00u2009mg/kg (
Dose concentration: 0. 10u2009mg/mL)
The rats were injected with the I. V.
After administration, each group collected blood samples at different time points.
Time point of I. P.
The administration Group is in advance
Dose and post-dose (
15, 30 minutes, then 1, 2, 4, 6, 8, and 24 hours)
Testing chemicals; for the I. V.
Administration Group, time point
Dose and post-dose (
5, 15, 30 minutes, then 1, 2, 4, 6, 8, 24 hours)
Test agent.
Use three animals at each point in time.
Collect about 800 µμ l of blood through the orbital vein (
Collect sources of sufficient volume of venous blood)
Take out from each anesthesia mouse with isofluoride and collect 200 µμ l of blood from the sleeve implanted into the jugular vein.
When blood samples were taken for the first time, the catheter was implanted.
Next, transfer the blood to a prepared test tube containing the heparin sodium inhibitor and mix briefly through a mild reversal of three to four times.
Record the blood collection target and actual time relative to the time of administration.
Put the sample on wet ice immediately after collection.
Within 1 hour after collection, plasma was separated by centrifuge at 4,000 rpm at 4 °c for 10 minutes
The plasma is protected from light and stored at-80 °c until it is passed through liquid chromatography-
Mass/mass spectrometry (LC-MS/MS)
For quantification.
The standard is prepared with blank mouse plasma, which is in a test tube containing heparin sodium moderator with a nominal concentration range of 2-1,000 ng/mL (NSC23925b), 2. 50-1,000 ng/mL (paclitaxel)
, And 1-1,000 ng/mL (doxorubicin).
Use data from the standard to generate calibration curves for each analysis batch. Aliquots of 10.
Supplement 0 μ l samples, calibration curve samples and QC samples with 100 μ l precipitation (
A mixture of acetone and acetone (30:70)).
After 3 minutes of rotation, centrifuge at 12,000 rpm for 3 minutes and 75 minutes.
The supernatant of 00 μ l was transferred to 96-
Well microplates with equal volume of water.
10 μ l mixture solution for LC-injectionMS/MS analysis.
Specific instrument conditions for LC-
MS/MS Bioanalysis of NSC23925b, paclitaxel and polybenic is available.
The estimation of the kinetic parameters is shown in, and.
Understanding potential drugs
Drug Interaction of NSC23925b, HLM-LC-
The inhibition test of MS cyp 50 was carried out.
This analysis uses a drug cocktail.
Probe substrate specific to each homogase with HLM and LC-MS detection.
The test products and reference inhibitors were dissolved in acetone of 40% and acetone of 60% respectively (ACN)mixture.
Specific 0.
Add 20 ml HLM, test compound set, and reference inhibitor solution to the specified 96-
Polypropylene board.
The substrate is then added to the holes that each contain a compound/HLM solution to start the reaction. The drug-
The probe substrate concentration and the estimated individual CYP enzyme concentration were shown from HLM.
Nucleotide phosphate (NADPH)cofactor (66.
In 10 ml of 100 mM phosphate buffer, pH 7. 4)
Added to each well.
After incubation for 3A4 for 5 min;
10 min for 1A2, 2C8, 2C9 and 2d4;
In the incubator at 37 °c, 45 min of 2cca, the protein was modified by adding 120 μ l ACN, and the reaction was terminated.
The solution was centrifuge at 3,700 rpm for 30 min at 4 °c and the supernatant was transferred to LC-MS analysis.
The concentration of metabolites is determined from the standard curve using commercially available metabolites standards.
Compound in triplicate.
Half maximum inhibition concentration (IC)
The value is determined by using Prism 5 to map the formation of metabolites on the logarithmic concentration of the compound. 0 software (
GraphPad SoftwareSan Diego, California).
The MTD test consists of observation study and main study.
A targeted study to determine the maximum tolerable dose (MTD)
NSC23925b, one of each animal (
1 female mouse, 1 male mouse, 1 female mouse, 1 male mouse)
Use every I. V. and I. P.
Injection volume level.
Select the starting dose from the fixed dose level (
5, 50, 500, 2000 mg/kg)
As a dose that is expected to produce significant toxicity.
Feed and observe the first animal.
The time, extent, and duration/recovery time of symptoms were recorded in detail.
The animals were then observed twice on the administration day and then changed to daily observation.
The observation lasted at least 14 days.
If the first animal dies or has an injury, the second animal receives a medium dose of both doses and vice versa.
When a decision is made on the initial dose of the main study, the aiming study is completed.
During the main study, a total of five animals of one sex were used at each dose level.
The five animals consist of an animal in an observation study taken at a selected dose level and an additional four animals.
Each group received one of the fixed doses of I. V. and I. P.
Based on AIM Research.
If the first animal dies or has an injury, the second animal receives a medium dose of both doses and vice versa.
Animals fasted overnight before administration.
Following the pre-fasting, the animals weigh and calculate the dose according to the weight.
After the animal is given a dose of 10 mL/kg, the food is held for another 3-4 hours.
Shortly before the application of the test substance, the individual weight of the animal was determined, and twice a week thereafter.
Calculate and record weight changes.
Animals injected with normal saline were the control group for weight change.
The results are expressed in mean ± sd and 95% confidence intervals.
Perform data collection using Analyst version 1.
From the applied biological system.
All the kinetic parameters of NSC23925b, paclitaxel and polybenic after I. V. or I. P.
Calculation of administration of mice/Rats based on plasma concentrations obtained by non-mice/rats in this study
Partition analysis of WinNonLin software (Version 5.
01, Pharsight, Inc.
Mountain View, California).
Data was also analyzed using Prism 5. 0 software.
Using two independent methods to determine statistical significance
Student ttests and one-
Analysis of variance of independent data.
Will . . . . . . The difference
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