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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2013

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Study performed on male Sprague-Dawley rats by either oral (n = 6) or intravenous (n = 6) administration of the test substance and monitorization of plasma and tissue concentrations by an LC-ESI-MS system to determine main toxicokinetics parameters.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chengdu Mansite Pharmaceutical Co. Ltd. (Chengdu, China)
- Purity > 98.0%
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Laboratory Animal Center of Wenzhou Medical College (Wenzhou, China)
- Weight at study initiation: 200–220 g
- Housing: housed at Wenzhou Medical College Laboratory Animal Research Center, under controlled conditions.
- Diet: prohibited for 12 h before the experiment.
- Water: ad libitum.
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 1
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): natural light–dark cycle

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: After fasting for 12 h, six rats were given a dose of 2.0 mg/kg NHDC via the sublingual vein, and the other six rats were administered with the dose of 20 mg/kg NHDC orally.
Duration and frequency of treatment / exposure:
Single dose administration, observation period 24h.
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
oral administration.
Dose / conc.:
2 mg/kg bw/day (nominal)
Remarks:
administered via the sublingual vein.
No. of animals per sex per dose:
6 males per dose.
Control animals:
no
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood; heart, liver, brain, lung, kidney, stomach and spleen.
- Time and frequency of sampling: Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0, 0.083, 0.167, 0.333, 0.5, 0.833, 1.333, 2.0, 2.667 and 3.333 h after intravenous administration and at 0, 0.083, 0.167, 0.333, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 24 h after oral administration. The samples were immediately centrifuged at 3000 g for 5 min. The plasma obtained (100 mL) was stored at 20 C until analysis.
- Three groups of 2 rats, which had been administered the test item intravenously, were euthanized by decapitation at 5 min, 0.5 and 1 h after dosing. Tissues, including the heart, liver, brain, lung, kidney, stomach and spleen were dissected and washed with cold saline. The tissues were then weighed and homogenized in cold saline solution (500 mg/mL). The obtained tissue homogenates were immediately stored at 20ºC until analysis.
- Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 mL of the IS working solution (8.0 mg/mL) was added to 100 mL of collected plasma sample followed by the addition of 200 mL acetonitrile. The tubes were mixed by vortex for 0.5 min. After centrifugation at 14 900g for 10 min, the supernatant (10 µL) was injected into the LC-ESI-MS system for analysis.

- Analytical methods: A sensitive, simple and specific LC-ESI-MS method for the determination of NHDC in rat biological samples was developed and validated over the concentration range of 10–3000 ng/mL NHDC. Lower limit of quantification (LLOQ) for NHDC was 10 ng/mL. Mean recovery of NHDC from plasma and tissues was better than 80.3%. Coefficient of variation of intra-day and inter-day precision were both less than 15%. Method description: Biological samples were processed with one-step protein precipitation. Rutin was chosen as the internal standard (IS). Chromatographical separation was achieved on an SB-C18 (2.1 mm 150 mm, 5 mm) column with acetonitrile–0.1% formic acid in water as the mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in negative ion mode; selected ion monitoring mode was used for quantification using target fragment ions m/z 611.4 for NHDC and m/z 609.1 for IS.
Statistics:
Pharmacokinetic analyses and plasma concentration versus time data were analyzed by DAS software (Version 2.0, Drug Clinical Research Center of Shanghai University of T.C.M and Shanghai BioGuider Medicinal Technology Co., Ltd., China). Pharmacokinetic parameters were calculated by using the non-compartmental model.

Results and discussion

Main ADME resultsopen allclose all
Type:
absorption
Results:
Fast, in 0.2h plasma concentrations start to increase.
Type:
distribution
Results:
Fast and wide, Cmax 800-1100ng/mL, can cross blood-brain barrier.
Type:
excretion
Results:
Short residence time. Elimination after 24h, clearance rate 7.4 L/h/kg.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
For oral administration, the plasma NHDC concentrations increased very quickly within 0.2 h, which suggests fast absorption.
Details on distribution in tissues:
The maximum plasma concentration (Cmax) of NHDC ranged from 801 to 1100 ng/mL, while the half-life (t1/2) was 1.0 ± 0.2 h. Results indicated that NHDC underwent a rapid and wide distribution into tissues within the time period examined. It was merely 5 min after i.v. administration that NHDC had already reached its Cmax in most of tissues, including the brain, showing that NHDC could effectively cross the blood–brain barrier.
Details on excretion:
Residence time of NHDC in vivo was very short, both for oral and i.v. administration. Plasma concentrations gradually decreased until 24 h. The clearance rate was 7.4 ± 2.5 L/h/kg.
Toxicokinetic parametersopen allclose all
Key result
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 1.0 ± 0.2 h
Key result
Test no.:
#1
Toxicokinetic parameters:
Cmax: 980.3±255.2
Key result
Test no.:
#1
Toxicokinetic parameters:
Tmax: 0.167
Key result
Test no.:
#1
Toxicokinetic parameters:
AUC: 2558.7±697.1

Metabolite characterisation studies

Metabolites identified:
not specified

Bioaccessibility

Bioaccessibility testing results:
The absolute bioavailability of the test item was observed to be 21.8%.

Any other information on results incl. tables

Table 1. Single-compartmental pharmacokinetic parameters following oral dose (20 mg/kg) and intravenous administration (2 mg/kg; n=6). 

Phramacokinetic parameters

Unit

Oral Values

(mean ± SD)

I.V. Values

(mean ± SD)

Half-life (t1/2)

H

1.0 ± 0.2

0.46 ± 0.10

Peak concentration (Cmax)

µg/L·h

 

980.3 ± 255.2

 

2125.9 ± 596.3

Time to peak concentration (Tmax)

h

 

0.167 ± 0

 

0.083 ± 0

Area under concentration–time curve (AUC0-t)

µg/L·h

 

2558.7 ± 697.1

 

1204.5 ± 384.7

AUC0-

µg/L·h

 

2750.6 ± 786.2

 

1215.2 ± 389.9

Apparent volume of distribution (V)

L/kg

 

72.9 ± 18.5

 

1.2 ± 0.49

Clearance (CL)

 

L/h/kg

7.4 ± 2.5

 

1.8 ± 0.6

Mean residence time (MRT0-t)

h

 

5.1 ± 0.9

 

0.57 ± 0.07

MRT0-

h

7.2 ± 1.8

0.60 ± 0.08

Bioavailability (F)

%

21.8

 

F = [(AUCp.o)·(Dosei.v)]/[(AUCi.v)·(Dosep.o)]·100%.

Applicant's summary and conclusion

Conclusions:
The test item undergoes rapid absorption (bioavailability 21.8%), rapid and wide distribution but short residence time, and rapid elimination (around 24h) after oral administration. Based on the available data, the test item shows no potential for bioaccumulation.
Executive summary:

A toxicokinetic study was performed on male Sprague-Dawley rats by either oral (n = 6, 20 mg/kg bw) or intravenous (n = 6, 2 mg/kg bw) administration of the test substance and monitorization of plasma and tissue concentrations by an LC-ESI-MS system. Under test conditions, the test item undergoes rapid absorption (bioavailability 21.8%), rapid and wide distribution but short residence time, and rapid elimination (around 24h) after oral administration, showing no signs of potential bioaccumulation.