Cyanocobalamin

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment. GLP study.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2005

Materials and methods

Objective of study:

toxicokinetics

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Cyanocobalamin

EC Number:

200-680-0

EC Name:

Cyanocobalamin

Cas Number:

68-19-9

Molecular formula:

C63H88CoN14O14P

IUPAC Name:

cyanocobalamin

Details on test material:

- Name of test material (as cited in study report): Cyanocobalamin
- Analytical purity: 96.74%

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

intravenous

Vehicle:

unchanged (no vehicle)

Details on exposure:

Cyanocobalamin was administered intravenously as a slow bolus injection via the tail vein using either an ordinary needle or a butterfly needle at a rate of 3 ml/min. The volume administered for each dose was calculated based on the most recent bodyweight for each animal and was 0.1, 0.5, 2.5 and 10 ml/kg, respectively.

Duration and frequency of treatment / exposure:

182 days (26 weeks).

Doses / concentrations

Remarks:

Doses / Concentrations:
1, 5, 25 and 100 mg/kg.

No. of animals per sex per dose / concentration:

24 males and 24 females altogether.

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

None.

Details on dosing and sampling:

PHARMACOKINETIC STUDY
- Tissues and body fluids sampled: blood samples (0.5 ml per time point) were collected from the jugular vein or the orbital sinus with animals under CO2/O2 anaesthesia.
- Time and frequency of sampling: animals were sampled in two different schedules, as follows: six rats (three male and three female rats) from each dose group were sampled before dosing and at 10, 20 and 100 min. Another six rats (three male and three female rats) from each dose group were sampled at 3, 15, 40 and 180 min after completion of the injection. A similar procedure was performed on days 1, 85 (week 13) and 182 (week 26) of treatment.
- Other: Cyanocobalamin plasma concentrations: sample deproteinization was performed by the addition of 800 µl methanol to 50-100 µl plasma, vortexing and refrigeration for 45 min and lyophilization of the resulting supernatant fraction, which was then suspended in 200 µl HPLC mobile phase A (100 mmol/l sodium phosphate monobasic; 5% methanol) prior to analysis. An aliquot (10-100 µl) was injected into an HPLC system using a 4.5 mm x 150 mm C8 reverse-phase column. The column was eluted over 12 min at 1.0 ml/min using a linear gradient of mobile phase A to 100% methanol (mobile phase B) at room temperature. Cyanocobalamin was monitored at 361 nm and exhibited a retention time of 11.7 min.

Results and discussion

Any other information on results incl. tables

Plasma concentrations: the time-course of plasma concentrations after single administration (day 1) of different doses (1, 5, 25 and 100 mg/kg) of cyanocobalamin was similar to the time-course of plasma concentrations observed after 85 and 182 days of daily dosing.

C_maxand AUC: values of C_max and AUC increased proportionally to the dose of cyanocobalamin. No changes in the C_max and AUC were observed in relation to the duration of treatment, indicating that there was no accumulation of cyanocobalamin in plasma.

t_1/2and K_el : the t_1/2 and K_el ranged from 20 to 30 min and 0.024 -0.031 /min, respectively, after single administration for different dose levels. A similar range was observed for both parameters at 85 days and 182 days.

Cl and Vd_ss: the Cl and Vd_ss showed similar results among the different doses after single administration of cyanocobalamin. However, after multiple doses, Cl and Vd_ss exhibited a modest tendency to increase in parallel with the dose.

AUC_0-∞: it was observed a negative and significant relationship between dose AUC_0-∞/dose. This finding is compatible with a possible saturation of tubular reabsorption.

Table 1. Pharmacokinetic data determined on first day of intravenous administration of cyanocobalamin in rats.

	Cyanocobalamin
	1 mg/kg	5 mg/kg	25 mg/kg	100 mg/kg
n	6	6	6	6
Cmax(µg/mL)	6.0 ± 2.5	22.9 ± 6.10	128.8 ± 15.50	439.1 ± 60.2
tmax(min)	5.3 ± 3.6	6.2 ± 5.2	3.0*	3.0*
AUC0-t(µg/ml per min)	119.0 ± 33.70	705.7 ± 224.9	3034 ± 5540	11 490 ± 1526
AUC0-∞(µg/mL per min)	137.1 ± 36.40	764.7 ± 243.8	3690 ± 9120	11 684 ± 1704
t1/2(min)	23.0 ± 10.3	36.8 ± 13.6	46.8 ± 26.0	29.0 ± 6.1
Kel(/min)	0.039 ± 0.027	0.022 ± 0.009	0.019 ± 0.010	0.025 ± 0.005
Cl (ml/min)	7.8 ± 2.4	6.9 ± 1.6	7.1 ± 1.7	8.7 ± 1.3
Vdss(mL)	247.3 ± 85.10	356.5 ± 131.4	467.6 ± 291.2	335.6 ± 46.7
MRT (min)	33.2 ± 12.1	53.0 ± 19.5	69.8 ± 43.7	39.6 ± 6.0

Data are the mean ±SD. The sample size (n) represents the number of plasma concentration–time curves for cyanocobalamin obtained by sampling two rats at different times.

*All animals showed the same value. The first blood sample after cyanocobalamin administration was 3 min and parameters were estimated by noncompartmental methods.

AUC_0–t, area under the plasma concentration–time curve from time zero to the last sampling time;AUC_0-∞, area under the plasma concentration-time curve from time zero to infinity; t_1/2, plasma terminal half-life; k_el, terminal elimination rate constant; Cl, total body clearance; Vd_SS, distribution at steady state; MRT, mean residence time.

Table 2. Pharmacokinetic data determined after 85 days (week 13) of intravenous administration of cyanocobalamin in rats.

	Cyanocobalamin
	1 mg/kg	5 mg/kg	25 mg/kg	100 mg/kg
n	6	6	6	6
Cmax(µg/ml)	9.3 ± 2.5	34.6 ± 13.4	126.2 ± 20.70	426.4 ± 48.8
tmax(min)	3.0*	3.0*	3.0*	3.0*
AUC0-t(µg/mL per min)	198.1 ± 30.60	773.3 ± 154.6	3175 ± 3600	10 928 ± 1,681
AUC0-∞(µg/ml per min)	227.5 ± 34.20	811.1 ± 174.5	3202 ± 3620	11 020 ± 1,729
t1/2(min)	20.4 ± 7.90	28.9 ± 13.8	26.3 ± 0.80	25.6 ± 2.9
Kel(/min)	0.039 ± 0.015	0.027 ± 0.009	0.026 ± 0.001	0.027 ± 0.003
Cl (mL/min)	4.5 ± 0.7	6.4 ± 1.4	7.9 ± 0.8	9.3 ± 1.6
Vdss(mL)	138.9 ± 22.70	250.3 ± 45.50	261.1 ± 26.90	314.5 ± 29.9
MRT (min)	31.7 ± 7.30	40.7 ± 13.1	33.1 ± 0.80	34.2 ± 2.6

Data are the mean ±SD. The sample size (n) represents the number of plasma concentration–time curves for cyanocobalamin obtained by sampling two rats at different times.

*All animals showed the same value. The first blood sample after cyanocobalamin administration was 3 min and parameters were estimated by noncompartmental methods.

AUC_0–t, area under the plasma concentration–time curve from time zero to the last sampling time;AUC_0-∞, area under the plasma concentration-time curve from time zero to infinity; t_1/2, plasma terminal half-life; k_el, terminal elimination rate constant; Cl, total body clearance; Vd_SS, distribution at steady state; MRT, mean residence time.

Table 3. Pharmacokinetic data determined after 182 days (week 26) of intravenous administration of cyanocobalamin in rats.

	Cyanocobalamin
	1 mg/kg	5 mg/kg	25 mg/kg	100 mg/kg
n	6	6	6	6
Cmax(µg/mL)	7.3 ± 2.7	26.6 ± 8.7	154.0 ± 23.20	445.7 ± 31.3
tmax(min)	5.3 ± 3.6	4.2 ± 2.9	3.0*	3.0*
AUC0-t(µg/mL per min)	201.2 ± 50.10	611.5 ± 64.1	3665 ± 875.7	11 518 ± 1,481
AUC0-∞(µg/mL per min)	235.2 ± 83.50	631.9 ± 73.4	3744.0 ± 985.5	11 660 ± 1,549
t1/2(min)	25.3 ± 8.90	29.7 ± 6.4	30.3 ± 7.50	28.3 ± 2.9
Kel(/min)	0.031 ± 0.013	0.024 ± 0.005	0.024 ± 0.004	0.025 ± 0.003
Cl (mL/min)	4.7 ± 1.5	8.0 ± 0.9	7.0 ± 1.5	8.7 ± 1.3
Vdss(mL)	166.4 ± 36.30	314.7 ± 37.4	251.9 ± 29.60	310.0 ± 22.5
MRT (min)	37.2 ± 9.20	39.6 ± 5.5	37.3 ± 8.40	35.9 ± 3.3

Data are the mean ±SD. The sample size (n) represents the number of plasma concentration–time curves for cyanocobalamin obtained by sampling two rats at different times.

*All animals showed the same value. The first blood sample after cyanocobalamin administration was 3 min and parameters were estimated by noncompartmental methods.

AUC_0–t, area under the plasma concentration–time curve from time zero to the last sampling time;AUC_0-∞, area under the plasma concentration-time curve from time zero to infinity; t_1/2, plasma terminal half-life; k_el, terminal elimination rate constant; Cl, total body clearance; Vd_SS, distribution at steady state; MRT, mean residence time.

Table 4. Population pharmacokinetic parameters of doses of 1-100 mg/kg, i.v., cyanocobalamin in rats.

 

PK parameters
Cl (mL/min)	6.3 ± 1.5
Vd (mL)	128.8 ± 6.500
k12(/min)	0.032 ± 0.011
k21(/min)	0.049 ± 0.012
kel* (/min)	0.049
t1/2** (min)	14.2
Goodness-of-fit
Sigma	0.11
Maximum likelihood	1625
Akaike information criteria	–3.5

 

The population pharmacokinetic (PK) modelling included data from all rats at different sampling days.

Data for clearance (Cl), volume of distribution (Vd), the transfer rate constant from the central to the peripheral compartment (k₁₂) and the transfer rate constant from the peripheral to central compartment (k₂₁) areexpressed as the mean ±SD.

*Derived fromk_el= Cl/Vd.

**Derived fromt_1/2= ln 2/k_el.

No covariate was incorporated in the final population pharmacokinetic model.

Applicant's summary and conclusion

Conclusions:

At very high prolonged doses, up to 100 mg/kg, i.v., for 26 weeks, intravascular administration of cyanocobalamin in rats follows a two-compartment kinetic model.

Executive summary:

In the present pharmacokinetic study, forty-eight rats were randomly assigned to receive 1, 5, 25 or 100 mg/kg cyanocobalamin for 182 days (26 weeks). Cyanocobalamin plasma levels were quantified by HPLC on days 1, 85 and 182 of treatment and were analysed by means of non-compartment pharmacokinetic (PK) analysis. The half-life of cyanocobalamin ranged from approximately 20 to 50 min, clearance ranged from 4.5 to 9 ml/min and the volume of distribution at steady state ranged from 140 to 470 ml. A statistically significant negative relationship existed between the dose of cyanocobalamin and the normalized area under the plasma concentration-time curve (AUC). On the basis of the study results, it can be concluded that at very high and prolonged doses (up to 100 mg/kg for 26 weeks), intravascular administration of cyanocobalamin in rats follows a two-compartiment kinetic model and cyanocobalamin undergoes extensive extravascular distribution. The negative relationship between dose and normalized AUC is compatible with possible saturation of tubular reabsorption, thus increasing renal clearance at higher doses. No evidence of toxicity was observed.