6-Amino-5-chloro-2-cyclopyrimidine-4-carboxylic acid

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 2007 to 07 March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 4 April 1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

EC Number:

617-769-9

Cas Number:

858956-08-8

Molecular formula:

C8H8ClN3O2

IUPAC Name:

6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: at least 8 weeks
- Weight at study initiation: 228 - 279 g (males), 164 - 218 g (females)
- Housing: individually in stainless steel, wire-mesh cages suspended above cage boards
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days with the exception of cannulated rats which were quarantined for at least 3 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The target radioactive dose was 30 μCi/250 g animal. The 14C-test substance was diluted with test substance to the appropriate specific activity for the selected dose level. The test substance, 14C-test substance and dose vehicle (0.5% methylcellulose) were weighed into a vial and mixed to a clear solution or homogeneous suspension. Dose preparations were prepared and stored refrigerated until use.

Duration and frequency of treatment / exposure:

single oral administration

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day

Remarks:

pilot material balance study and main pharmacokinetics study

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

main pharmacokinetics study and metabolite profile study

No. of animals per sex per dose / concentration:

1 (pilot material balance study)
4 (main pharmacokinetics study ) + 1 additional rat per sex dosed at 500 mg/kg bw/day for analytical method development (metabolite profile)
3 (metabolite profile study)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were based on the results of a toxicity study.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (absorption, distribution, excretion)
- Tissues and body fluids sampled: 14C exhaled volatiles and 14CO2, urine, faeces, blood, cage wash, residual feed (pilot material balance study), plasma and red blood cells (RBC) (main pharmacokinetics study)
- Time and frequency of sampling: 14C exhaled volatiles and 14CO2 for 48 h at 24 h intervals (e.g. 0-24 and 24-48 h), urine and feces were collected at 24 h intervals for a total of 168 h, cage wash was collected throughout the study, blood samples (plasma and RBC) were collected pre-dose and at 5, 15 and 30 minutes, 1, 2, 4, 8, 12, 24, and 30 h post dose
- From how many animals: 1 rat per sex and dose (pilot material balance study), 4 animals per sex and dose (main pharmacokinetics study)
- Other: The following tissues were collected for analyses at the end of the study: blood (plasma and RBC were analyzed separately for 14C content), fat liver, kidney, muscle, heart, lung, testes, ovaries, uterus, bone and bone marrow (analyzed separately for 14C content), brain, spleen, adrenals, pituitary, gastro-intestinal tract and contents (analyzed separately for 14C content), pancreas, skin sample, thyroid, thymus, bladder (urine in the bladder at the time of sacrifice was aspirated and placed in the terminal urine sample vial) and remaining carcass (pilot material balance study). Liquid scintillation counting was used for quantitation of radioactive residues.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: plasma (control and 500 mg/kg bw/day dose groups at 0.5 h timepoint), urine and feces ( 25 mg/kg bw/day dose group at 0-24 h intervals)
- Time and frequency of sampling: plasma was collected 0.5 h after test substance administration (metabolite profile study), urine and feces was collected at 24 h intervals for a total of 168 h (pilot material balance study)
- From how many animals: 3 samples per sex and dose (metabolite profile study), 1 sample per sex and dose (pilot material balance study)
- Method types for identification: quantification of radioactivity in plasma, urine, and feces was performed by HPLC with in-line radioactivity (LC/ARC). Observed radiochemical chromatographic peaks were identified by performing peak fractionation and LC/MS. Each of the prepared samples was also analyzed by LSC to verify adequate extraction recoveries

Clinical Observations and Mortality
Cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats were conducted at least once daily throughout the study.

(For further details on study design please refer to table 1 in the "Any other information and methods incl. tables" section.)

Statistics:

Group data are represented as a mean ± SD.

Results and discussion

Preliminary studies:

The test substance was rapidly eliminated after single oral gavage administration. No 14C residues were detected in exhaled breath. These results suggest that the position of the radiolabeled carbon was stable to metabolic biotransformation. Excretion occurred via the urine and feces in approximately equal proportions and was substantially complete within the first 24 h after dosing. Tissue 14C residues, with the exception of 0.039% in the male rat carcass, were below the limit of detection by 168 h after dosing. (For details on Toxikokinetic parameters please refer to table 4 and 5 in the "Any other information on results incl. tables" section.)

Main ADME results

Main ADME results 1

Type:

absorption

Results:

The results indicated rapid absorption of the test substance.

Main ADME results 2

Type:

distribution

Results:

Test substance is predominantely found in plasma. Tissue 14C residues, with the exception of male rat carcass ( 0.039%), were below the limit of detection by 168 h after dosing.

Main ADME results 3

Type:

metabolism

Results:

No biotransformation was evident under the condition of single dose administration at 25 or 500 mg/kg bw in plasma urine and feces.

Main ADME results 4

Type:

excretion

Results:

Radioactivity was not detected in respired breath (exhaled volatiles or CO2). Excretion occurred in both the urine and feces and was substantially complete in the first 24 h after dosing.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The results indicated rapid absorption with peak concentrations in plasma at 0.4 to 1 h after dosing. The mean Tmax values in red blood cells were 0.3 to 1 h. The peak concentrations in red blood cells were 0.33 to 0.48 fractionally lower than those observed in plasma indicating very limited potential for binding within red blood cells. The red blood cell concentration data were insufficient for calculation of pharmacokinetic parameters. The mean peak concentrations in plasma (3.8-5.0 μg equiv/g at the low dose and 57.3-61.6 μg equiv/g at the high dose, respectively) were approximately proportional to the 20-fold increase in dose from 25 to 500 mg/kg bw. Near direct proportionality was observed for the AUCINF values suggesting linear first-order kinetic processes for uptake and elimination. (For details on Toxikokinetic parameters please refer to table 4 and 5 in the "Any other information on results incl. tables" section.)

Details on distribution in tissues:

The peak concentrations in red blood cells were 0.33 to 0.48 fractionally lower than those observed in plasma indicating very limited potential for binding within red blood cells. The test substance is distributed within plasma. By 168 h after dosing, 14C residues were not detected in any of the various tissues collected to evaluate tissue distribution. One exception was the male rat carcass which contained 0.039% of the dose. Radioactivity detected in cage wash and residual feed was 1.4% and 0.4% of the administered dose for the male and female rat, respectively. The overall material balance was 69.9% for the single male rat and 107.9% for the single female rat. (Please refer to table 2 in the "Any other information on results incl. tables" section.)

Details on excretion:

Radioactivity was not detected in respired breath as either exhaled volatiles or CO2, and thus collection was stopped at 48 h. Excretion occurred in both the urine and feces and was substantially complete in the first 24 h after dosing. By 168 h after dosing, the individual male and female rat urine contained 36.3% and 55.8%, respectively; and the percentages were approximately equal to the 32.1% and 51.7% excreted in the feces. Radioactivity detected in cage wash and residual feed was 1.4% and 0.4% of the administered dose for the male and female rat, respectively. The overall material balance was 69.9% for the single male rat and 107.9% for the single female rat. In plasma, the terminal elimination half-lives were 5.6 to 5.7 h for male and female rats with no difference between the low (25 mg/kg bw) or high (500 mg/kg bw) dose levels. Plasma 14C residues were quantifiable for up to 30 h after dosing. (Please refer to table 2, 3, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15 and 16 in the "Any other information on results incl. tables" section.)

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Toxicokinetic parameters:

half-life 1st: Plasma: 5.6 h in males and 5.7 h in females (25 and 500 mg/kg bw)

Remarks:

The half-lives were unchanged with the 20-fold increase in dose.

Toxicokinetic parameters 2

Key result

Toxicokinetic parameters:

Cmax: Plasma: 3.8 and 5.0 μg equiv/g in male and female rats, respectively (25 mg/kg bw dose) 57.3 and 61.6 μg equiv/g for male and female rats, respectively (500 mg/kg bw dose)

Toxicokinetic parameters 3

Key result

Toxicokinetic parameters:

Cmax: Red blood cells: 1.3 and 2.0 μg equiv/g in males and females, respectively (25 mg/kg bw/day) 27.2 and 28.7 in males and females, respectively (500 mg/kg bw/day)

Toxicokinetic parameters 4

Key result

Toxicokinetic parameters:

Cmax: RBC/Cmax plasma ratios: 0.33 and 0.4 in males and females, respectively (25 mg/kg bw/day) 0.48 and 0.47 in males and females, respectively (500 mg/kg bw/day)

Remarks:

(very limited potential for uptake and binding in the red blood cell)

Toxicokinetic parameters 5

Key result

Toxicokinetic parameters:

AUC: Plasma: 7.0 and 9.0 hr*μg/g in males and females, respectively (25 mg/kg bw dose) 150.8 and 168.4 hr*μg/g in males and females, respectively (500 mg/kg bw dose)

Toxicokinetic parameters 6

Key result

Toxicokinetic parameters:

Tmax: Plasma: 0.5 h and 0.4 h in males and females, respectively (25 mg/kg bw dose) 6.0 h and 1.0 h in males and females, respectively (500 mg/kg bw dose)

Toxicokinetic parameters 7

Toxicokinetic parameters:

Tmax: Red blood cells: 0.5 h and 0.3 h in males and females, respectively (25 mg/kg bw dose) 6.0 h and 1.0 h in males and females, respectively (500 mg/kg bw dose)

Metabolite characterisation studies

Metabolites identified:

no

Details on metabolites:

No metabolite was evident under the condition of single dose administration of 25 or 500 mg/kg bw in plasma at 0.5 h timepoint and urine or feces 0-24 h time interval (pilot study). Only test substance, as parent chemical was confirmed in all 3 matrices by radiochemical and mass spectral analysis. Biotransformation of the test substance was absent in rats under conditions of single oral gavage at the 25 and 500 mg/kg bw doses. One expected metabolite, 5-chloro-2-cyclopropyl-pyrimidin-4-ylamine, was not observed. This metabolite has been quantified in rat plasma at Day 56 of a 90-day toxicity study by dietary administration at concentrations corresponding to 100 to 945 mg/kg bw/day for male rats and 129 to 1268 mg/kg bw/day in female rats. Although not identified under conditions of the current study, the metabolite 5-chloro-2-cyclopropyl-pyrimidin-4-ylamine appears to be formed by de-carboxylation of the test substance upon repeated daily uptake of the test substance. Under the conditions of this study, no metabolism of the test substance was observed.

Any other information on results incl. tables

 Table 2: Material balance for rats administered a single oral dose of¹⁴C-test substance (25 mg/kg bw)

		Percent of dose
Sample	Hour	Male	Female
		101M	102F
urine	168 h	36.304	55.761
feces	168 h	32.145	51.688
exhaled a	48 h	<LOD	<LOD
cage wash	168 h	1.209	0.358
residual feed	168 h	0.197	0.076
tissues	168 h	<LOD	<LOD
carcass	168 h	0.039	<LOD
	Total	69.9	107.9

a Includes data from 3 traps (CO2, volatiles, and water).

 LOD limit of detection

LOQ limit of quantitation

Table 3: Percent recovery in excreta of rats administered a single oral dose of¹⁴C-test substance (25 mg/kg bw)

Percent of dose				Cumulative percent of dose a
Male Female		Male	Female	Male	Female
Sample	Hour	101M	102F	101M	102F
urine	Pre-dose	<LOD	<LOD	0	0
urine	24 h	35.8876	53.7113	35.888	53.711
urine	48 h	0.2835	1.5093	36.171	55.221
urine	72 h	0.0893	0.5271	36.260	55.748
urine	96 h	0.0184	0.0086	36.279	55.756
urine	120 h	0.0048	0.0025	36.284	55.759
urine	144 h	0.0164	0.0016	36.300	55.760
urine	168 h	0.0041	0.0008	36.304	55.761
feces	Pre-dose	<LOD	<LOD	0	0
feces	24 h	31.1368	47.9955	31.137	47.996
feces	48 h	0.84	3.6031	31.977	51.599
feces	72 h	0.1249	0.0804	32.102	51.679
feces	96 h	0.014	0.0068	32.116	51.686
feces	120 h	0.0062	0.0023	32.122	51.688
feces	144 h	0.0176	<LOQ	32.140	51.688
feces	168 h	0.0054	<LOD	32.145	51.688

a Pre-dose time point assigned zero hour and concentration for graphical representation

 LOD limit of detection

LOQ limit of quantitation

Table 4: Concentration (μg equiv/g) in plasma and red blood cells following a 25 or 500 mg/kg bw single oral dose of¹⁴C-test substance

	25 mg/kg bw				500 mg/kg bw
Hour	Male		Female		Male		Female
Plasma	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Pre-dose	NAa	NA	NA	NA	NA	NA	NA	NA
5 m	1.13	0.32	2.58	0.60	3.98	1.22	6.69	3.58
15 m	3.09	0.85	4.86	1.13	28.37	5.41	35.57	14.08
30 m	3.01	0.20	4.35	1.25	54.40	18.45	54.28	20.60
1 h	2.74	1.52	2.91	0.61	51.17	8.27	52.92	14.50
2 h	1.14	0.50	1.57	0.82	29.68	4.30	42.77	7.73
4 h	0.13	0.05	0.13	0.02	4.66	1.20	3.69	1.66
8 h	0.07	0.02	0.11	0.07	2.35	0.32	1.84	0.75
12 h	0.05	0.04	0.05	0.04	1.36	0.56	1.45	0.34
24 h	NA	NA	NA	NA	0.46	NA	0.43	NA
30 h	0.005	0.002	0.01	0.002	0.16	0.02	0.13	0.04
Red blood cells
Pre-dose	NA	NA	NA	NA	NA	NA	NA	NA
5 m	0.38	0.10	0.88	0.17	1.90	NA	2.60	1.10
15 m	1.07	0.21	1.99	0.66	11.46	2.05	14.69	5.96
30 m	0.99	0.09	1.76	0.60	25.68	8.47	23.47	9.69
1 h	0.95	0.49	1.14	0.26	23.96	4.74	24.23	7.07
2 h	0.43	0.19	0.60	0.30	13.97	2.57	19.72	4.03
4 h	0.07	NA	NA	NA	1.78	0.52	2.06	NA
8 h	0.03	NA	0.06	NA	1.07	NA	0.97	NA
12 h	0.04	NA	0.04	NA	0.73	NA	NA	NA
24 h	NA	NA	NA	NA	NA	NA	NA	NA
30 h	0.004	NA	NA	NA	NA	NA	NA	NA

a Pre-dose samples and others with mean values designated NA had individual animal concentrations that were <LOD

  NA not applicable

Table 5: Pharmacokinetic parameters in plasma and red blood cells following a 25 or 500 mg/kg bw single oral dose of¹⁴C-test substance

Parameter	25 mg/kg bw				500 mg/kg bw
Plasma	Male		Female		Male		Female
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Elimination half-life (h)	5.6	0.5	5.7	0.4	5.6	0.3	5.7	0.7
Area-under-the-curve (AUCINF, hr*µg/g)	7.0	1.4	9.0	1.9	150.8	28.7	168.4	26.2
AUCINF/dose (hr*kg*µg/g/mg)	0.3	0.1	0.4	0.1	0.3	0.1	0.3	0.1
Peak concentration (Cmax, µg eq/g)	3.8	0.9	5.0	1.2	57.3	14.2	61.6	13.0
Half peak concentration (Cmax/2, µg) a	1.9		2.5		28.7		30.8
Time of Cmax (Tmax, h)	0.5	0.4	0.4	0.1	0.6	0.3	1.0	0.7
Time of Cmax/2 (Tmax/2, hr a	1.4		1.2		2.0		2.3
Red blood cells (RBC) b
Peak concentration (µg eq/g)	1.3	0.3	2.0	0.7	27.2	6.2	28.7	4.7
Time of Cmax (Tmax, h)	0.5	0.4	0.3		0.1	0.6	0.3	1.0
Ratio (Cmax RBC)/(Cmax plasma) a	0.33		0.40		0.48		0.47

a Calculated from Mean value, SD not given.

b Only Cmax and Tmax are presented. Time course data were insufficient to calculate other kinetic parameters.

AUCINF area under the concentration vs. time curve, extrapolated to infinity

AUCINF/D AUCINF, normalized to dose

 

Table 6: Concentration (μg equiv/g) in plasma of rats administered 0 or 500 mg/kg bw single oral dose of

¹⁴C-test substance

Dose			Plasma concentration (µg equiv/g)		Plasma concentration (µM)a
(mg/kg bw)	Sex	Hour	Mean	SD	Mean	SD
0 b	Male	0.5	<LOD	NA	<LOD	NA
	Female	0.5	<LOD	NA	<LOD	NA
500 c	Male	0.5	53.4	17.6	250	82
	Female	0.5	63.3	21.7	296	102

a Calculated using MW = 213.62 g/mol

b Control sample (n =1 per sex)

c Treated samples used for metabolite profiling (n = 3 per sex)

 

Table 7: Summary of retention times for reference standards and components in plasma from rats administered¹⁴C-test substance

		Reference Standard HPLC/14Ca		Sample Summary HPLC/14C
	[M+H]	Peak Retention Time	Fraction Collection Window	Minimum Retention Time	Maximum Retention Time
	(dalton)	(minutes) (minutes)	(minutes)	(minutes) b	(minutes) b
Test substance	214	5.02	4.51-5.25	5.02	5.02
Metabolite (INLXT69)	170	N/Ac	5.26-6.00	NId	NI

a ¹⁴C retention time for radiolabeled 14C-test substance reference standard spiked into the sample matrix.

b Retention time of base peak if tailing occurred and resulted in multiple integrations.

c Not applicable since the metabolite is not ¹⁴C radiolabeled.

d Not identified in the matrix.

 

Table 8:Summary of components identified in plasma of rats administered¹⁴C-test substance (A-male, B-female)

Component	M+H (Da) a	Peak Fraction Collection Window (min)	Dose Group b	Mass Spectral Criteria Figure
Test substance	214	4.51-5.25	A,B	Spectral and retention time match with reference standard.

a Most abundant ion in molecular ion cluster

b The structural identification was confirmed for the respective dose group: A – 601M-603M, 1F-603F.

 

Table 9: Summary of test substance parent concentrations in plasma

Time (minutes)	Subject	oncentration in plasma (µM) Test substance - Parent
30	601M	254
	602M	108
	603M	296
	Mean	219
	SD	99
30	601F	231
	602F	358
	603F	165
	Mean	251
	SD	98

 

Table 10: Summary of retention times for reference standards and components in urine from rats administered¹⁴C-test substance

		Reference Standard HPLC/^14C a		Sample Summary HPLC/^14C
	[M+H]	Peak Retention Time	Fraction Collection Window	Minimum Retention Time	Maximum Retention Time
	(dalton)n)	(minutes)	(minutes)	(minutes) b	(minutes) b
Test substance	214	5.02	4.51-5.25	5.02	5.02
Metabolite	170	N/Ac	5.26-6.00	NId	NI

a ¹⁴C retention time for radiolabeled¹⁴C-test substance reference standard spiked into the sample matrix.

b Retention time of base peak if tailing occurred and resulted in multiple integrations.

c Not applicable since the metabolite is not¹⁴C radiolabeled.

d Not identified in the matrix.

 

Table 11: Summary of components identified in urine of rats administered¹⁴C-test substance (A-male, B-female)

	[M+H] (dalton) a	Peak fraction collection window (minutes)	Dose roup b	Mass spectral criteria
Test substance	214	4.51-5.25	A, B	Spectral and retention time match with reference standard.

a Most abundant ion in molecular ion cluster

b The structural identification was confirmed in the 24-hour dose group: A – 101M, B – 102F.

 

Table 12: Summary of components identified in urine of rats administered with 14C-test substance as percent of administered dose

Identified component	^14C Retention time (min)		Component as percent of dose 500 mg/kg bw
	Minimum	Maximum	Male	Female
Test substance	5.02	5.02	35.9	53.7
Identified			35.9	53.7
Not identified			0 a	0
Total			35.9	53.7

a Zero indicates not identified (detected)

 

Table 13: Summary of retention times for reference standards and components in feces from rats administered¹⁴C-test substance

		Reference Standard HPLC/^14C a		Sample Summary HPLC/^14C
	[M+H]	Peak Retention Time	Fraction Collection Window	Minimum Retention Time	Maximum Retention Time
	(dalton) aa	(minutes)	(minutes)	(minutes) b	(minutes) b
Test substance	214	5.17	4.51-5.25	5.18	5.33
Metabolite	170	N/A c	5.26-6.00	NI d	NI

a ¹⁴C retention time for radiolabeled¹⁴C-test substance reference standard spiked into the sample matrix.

b Retention time of base peak if tailing occurred and resulted in multiple integrations.

c Not applicable since the metabolite is not¹⁴C radiolabeled.

d Not identified in the matrix.

 

Table 14: Summary of components identified in feces of rats administered¹⁴C-test substance (A-male, B-female)

	[M+H] (dalton) a	Peak fraction collection window (minutes)	Dose group b	Mass spectral criteria
Test substance	214	4.51-5.25	A, B	Spectral and retention time match with reference standard.

a Most abundant ion in molecular ion cluster

b The structural identification was confirmed in the 24-h sample dose group: A – 101M, B - 102F.

 

Table 15: Summary of components identified in feces of rats administered with¹⁴C-test substance as percent of administered dose

	14C Retention time (min)		Component as percent of dose 500 mg/kg bw
	Minimum	Maximum	Male	Female
Test substance	5.18	5.33	31.1	48.0
Identified			31.1	48.0
Not identified			0 a	0
Total			31.1	48.0

a Zero indicates not identified (detected)

 

Table 16: Summary of components quantified in urine and feces of rats administered ¹⁴C-test substance

Dose mg/kg bw	Proposed or identified component	Parent or component as percent of dose
25		Urine		Feces		Urine + Feces
		Male	Female	Male	Female	Male	Female
	Test substance	35.9	53.7	31.1	48.0	67.0	101.7
	Identified	35.9	53.7	31.1	48.0	67.0	101.7
	Not identified	0.0	0.0	0.0	0.0	0.0	0.0
	Total	35.9	53.7	31.1	48.0	67.0	101.7