Registration Dossier

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:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Metabolism of 3,5-Dimethylpyrazole-C14 in the Rat
Author:
Smith D.L., Forist A.A. & Gerritsen G.C.
Year:
1965
Bibliographic source:
The Journal of Pharmacology and Experimental Therapeutics Vol. 150, No. 2, p316-321

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
3,5-dimethylpyrazole-C14 has been orally administrated to rats to characterise its metabolism.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Test substance: 3,5-Dimethylpyrazole
Purity: not specified
Specific details on test material used for the study:
- Specific activity of test material: 130µc/mmol.
- The test material was prepared from acetone-2-C14 via 2,4-pentanedione-2,4-C14 using standard methods (Horning, 1955; Schreibre, 1951).
- Chemical and radiochemical purity was established by its infrared spectrum, melting point and paper and tin-layer chromatography.
Radiolabelling:
yes
Remarks:
C14

Test animals

Species:
rat
Strain:
other: C.D.
Details on species / strain selection:
Source: Charles River
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Weight at study initiation: 140g
- Housing: In cages designed to collect and separate the animals excrement.
- Fasting period before study: 16 hr
- Glucose-primed rats were used as described in Dulin (1962).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
methylcellulose
Remarks:
sterile 0.25% solution
Doses / concentrations
Dose / conc.:
25 other: mg/kg
Remarks:
(1.19 x 10^7 dpm/rat), samples were within 1.4% of each other.
Control animals:
not specified
Positive control:
No
Details on study design:
The metabolism of 3,5-dimethylpyrazole was investigated by orally exposing rats and anayising their urine in order to identify any metabolites.

Treatment:
- Rats received corn oil (0.2 ml/rat) immediately prior to dosing with the test material.
- Rats received β-diethylaminoethyl diphenylpropylacetate hydrochloride (40 mg/kg), injected intraperitoneally, 30 minutes prior to treatment with the test material.
- Rat received the test item by oral administration at 25 mg/kg

Details on dosing and sampling:
Hypoglycemic Activity Sampling:
- Tissues and body fluids sampled: urine.
- Time and frequency of sampling: 2 hours after administration.
- Method type(s) for identification: Liquid scintillation counting, radiochromoatogram and both paper and thin-layer chromatography.

Radioactive Samples:
- Apparatus: Liquid scintillation spectrometer, Packard Tri-Carb, Models 314EX2A abd 314X.
- Counts were performed in duplicate.
- The procedure followed is the one set out in Herberg (1960).

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
3,5-dimethylpyrazole is absorbed completely from the gatroinestinal tract.
Details on excretion:
Essentially all of the oral dose (95%) is found in the urine within 24 hours. About 60% is excreted during the first 3 hours.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
3,5-dimethylpyrazole is completely converted (>99%) to 4 metabolites. Two metabolites together with their conjugated forms account for about 98% of the oral dose of the test item.

The metabolites were identified as follows along with their average excreted percentages:
5-methylpyrazole-3-carboxylic acid (13.1 ± 3.8),
conjugated 5-methylpyrazole-3-carboxylic acid (13.6 ± 4.4),
conjugated 4-hydroxy-3,5-dimethylpyrazole (70.8 ± 5.5),
the identity of the 4th is unknown (1.3 ± 1.9).

Any other information on results incl. tables

Distribution of metabolites of 3,5-dimethylpyrazole-C14 in the urine of fasted rats

Both paper and thin-layer chromatography showed essentially no unchanged 3, 5-dimethylpyrazole in the urine. The Rf values of the metabolites are presented in table 1. Based on the radioactive intensity of the 4 zones, in 5 fasted rats the average percentages excreted (±S.D.) as metabolites designated as A, B, C, and D were 13.1 ± 3.8, 13.6 ± 4.4, 70.8 ± 5.5, and 1.3 ± 1.9, respectively (table 2). Three of the fasted rats did not excrete a detectable amount of metabolite D.

Table 1: Paper Chromatographic Rf valuesa

Compoundb

System

BAW

BPW

KCl

Metabolite A

0.76

0.37

0.69

Metabolite B

0.72

0.32

0.77

Hydrolysis product of metabolite B

0.76

0.37

 

Metabolite C

0.45

0.49

0.85

Hydrolysis product of metabolite C

0.61

0.77

 

Metabolite D

0.36

0.09

NRc

3,5-Dimethylpyrazole

0.80

0.82

0.79

5-Methylpyrazole-3-carboxylic acid

0.76

0.37

0.69

4-Hydroxy-3,5-dimethylpyrazole

0.61

0.77

 

aThe Rf values were generally reproducible to within 0.05 Rf unit from one chromatogram to the next, but were reproducible to within 0.01 Rf unit when replicate parallel measurements were made on the same chromatogram. The latter procedure was always used when comparing unknowns with standards.

bA, 5-methylpyrazole-3-carboxylic acid; B, conjugated 5-methylpyrazole-3-carboxylic acid; C, conjugated 4-hydroxy-3, 5-dimethylpyrazole; D, unknown.

cNR = not resolved.

Table 2: Excretion and distribution of metabolites from rats treated with 3,5 -dimethylpyrazole

Pretreatment

Percent of C14in

0 - 24 hr Urine

Percent of C14in 0 - 24 hr Urine as Metabolte:

A

B

C

D

Fasted

89.1a

7.4

18.7

70.7

3.5

Fasted

95.3b

13.3

7.9

78.8

0.0

Fasted

93.1

 

 

 

 

Fasted

101.3

14.4

17.5

65.0

3.0

Fasted

94.1c

12.2

11.6

72.9

0.0

Fasted

97.2c

18.0

12.4

66.6

0.0

Mean ± S.D.

95.0 ± 4.1

13.1 ± 3.8

13.6 ± 4.4

70.8 ± 5.5

1.3 ± 1.9

 

Corn Oil

83.1

27.0

13.2

59.0

0.8

Corn Oil

21.8

10.6

10.2

75.6

2.7

Corn Oil

52.2

13.6

11.1

72.9

2.5

Mean ± S.D.

52.3 ± 30.6

17.1 ± 8.7

11.5 ± 1.4

69.2 ± 8.7

2.0 ± 1.0

 

SKF-525A

83.5

12.8

9.7

72.5

3.5

SKF-525A

62.5

 

 

 

 

Mean ± S.D.

73.0 ± 14.8

 

 

 

 

aAn additional 4.8% was recovered in the 24- to 168-hour samples.

bAn additional 1.8% was recovered in the 24- to 168-hour samples.

cAn additional 0.3% was recovered in the 24- to 48-hour sample.

The relative areas of the radioactive peaks in the BPW and BAW systems were virtually identical, strongly suggesting that the peaks were resolved completely (for example, integration of the 4 radioactive peaks resulting from the 24-hr urine collection of 1 rat gave relative areas of 7.9, 18.9, 70.1, and 3.4 using the BAW system and 6.8, 18.4, 71.3, and 3.6 using the BPW system).

Isolation and identification of metabolites

3,5-Dimethylpyrazole is extracted quantitatively from urine into chloroform at pH 8.7; the fact that essentially no radioactivity (<0.5%) is extracted under these conditions supports the theory that very little 3,5 -dimethylpyrazole is excreted intact. Extraction data are more reliable than radiochromatographic evidence because 3,5-dimethylpyrazole sublimes rather easily. At pH 1.5, metabolites A and B were essentially quantitatively extracted into the butanol, while the very polar metabolite C remained in the urine.

Metabolite A is 5-methylpyrazole-3-carboxylic acid, the active metabolite. This was established from a comparison of Rf values in 2 paper and 2 thin-layer chromatographic systems with authentic material. This metabolite was isolated previously in crystalline form and characterized after the administration of unlabeled 3,5-dimethylpyrazole upon acid hydrolysis. The total radioactivity in the hydrolyzed sample is unchanged, but the radioactive peak corresponding to metabolite A increases and metabolite B disappears. The extraction behavior of metabolite B suggests that it is an acidic compound. Metabolite B is unaffected by Ketodase or Glusulase hydrolysis, suggesting that it is not a sulfate or glucuronide conjugate. These data and the fact that the polarity of metabolite B is very similar to 5-methylpyrazole-3-carboxylic acid in all of the thin-layer and paper chromatographic systems used suggest that metabolite B is an acid. The behavior of metabolite B suggests that it is conjugated with glycine, but so far this has not been demonstrated definitively.

Metabolite C is acid-hydrolyzable, indicating that it is also a conjugate. Attempts at isolating the conjugated material were unsuccessful because of its salt-like nature; e.g. even leaching of lyophilized urine with ethanol gave poor recovery of C14. The highly polar nature of this conjugated metabolite suggests a sulfate or glucuronide conjugate, but it was resistant to both Glusulase and Ketodase hydrolysis.

Because of the difficulty encountered in the extraction of the conjugated form of metabolite C, it was decided to attempt the isolation of its hydrolysis product. Urine from 10 rats (350 ml), treated with 25 mg of unlabeled 3,5-dimethylpyrazole twice daily for 3 days, was collected, reduced in volume by lyophilization, and combined with the urine of a rat treated with labeled 3,5-dimethylpyrazole. The urine was adjusted to pH 0 with concentrated HCl and heated under reflux for 5 hours. During hydrolysis, the pH increased to 2.2. The urine was filtered, adjusted to pH 9, and filtered again (urinary precipitates were checked for radioactivity before discarding). The alkaline urine was then extracted with two 2-liter portions of ethyl acetate. The ethyl acetate solution was reduced in volume, and material which precipitated was filtered (nonradioactive). The ethyl acetate solution was evaporated to dryness and the residue, which contained 54% of the original C14, was dissolved in ethanol, applied to a silica gel column, and eluted with CHCl3:CH3OH (5:1). Tubes 30 to 50 (10 ml in each tube) contained the radioactivity corresponding to the hydrolysis product of metabolite C (and possibly metabolite D also). This material was applied to another silica gel column and was subjected to a gradient elution with CHCl3,: CH3OH, 99:1 (tubes 1-124), 97:3 tube) contained the radioactivity corresponding Tubes 290 to 355 contained the radioactivity as a single peak. Evaporation of the radioactive fractions yielded crystals, which were recrystallized twice from ethanol yielding 343 mg of colorless needles, m.p. 176 °C 179 °C. The ultraviolet spectrum has a single peak,λEtOH max = 232 mµ (εmax 4900). The nuclear magnetic resonance spectrum of this material in D2O solution exhibited only singlet absorption peaks at 2.17 ppm (methyl protons) and 4.72 ppm (exchangeable protons), having areas of 6 and 2, respectively. The absorption peak representing the vinyl hydrogen at the 4-position, present in the spectrum of 3,5-dimethylpyrazole, is lacking, having been replaced with a group with 1 exchangeable proton. The nuclear magnetic resonance data, in conjunction with the equivalent weight in glacial acetic acid and elemental analysis, can only fit the structure attatched below.

Calculated for C5H8N2O: C, 53.55; H, 7.13; N, 24.99; equivalent weight, 112. Found: C, 53.77; H, 728; N, 25.12; equivalent weight, 117. Sachs and Rohmer (1902) reported a melting point of 173.5 °C for the proposed structure. The infrared spectrum (KBr pellet) support the proposed structure, e.g., bands (cm-1) at 3400, 3250, 3130, and 2580 (NH/OH); 3000 and 2920 (CH); 1615, 1535, and 1485 (C=C/N=C); 1415 and 1385 (CH3); 1300, 1235, 1175, and 1050 (CO/CN).

Metabolite D, which is formed to the extent of about 1% of the dose, has not been isolated; this metabolite was not detected in the urine of some of the rats examined. Metabolite D is unaffected by enzymatic or acid hydrolysis.

Rate of absorption, excretion, and metabolism

The absorption of orally administered 3,5-dimethylpyrazole is essentially complete in fasted rats; 95 ± 4% of the dose is found in the urine within 24 hours (table 2).

Sequential urine collections were obtained from 2 fasted rats at 1, 2, 3, 4, 6, 8, and 24 hours after administration of 3,5-dimethylpyrazole. Semilogarithmic plots of C14 excreted vs. time give an estimation of the excretion half-life. In the 2 fasted rats an excretion half-life of 2.4 hours was obtained; i.e., an average of about 60% of the C14 is excreted in the urine during the first 3 hours. The estimated half-life of C14 excretion in rats treated with SKF-525A is 4.4 hours. The results of these excretion half-life studies are complicated by the fact that the rats were not catheterized. Each of the corn oil-treated rats voided only once during the first 8 hours; consequently, estimation of excretion half-life was impossible in this case.

The sequential urine collections from a fasted rat were chromatographed in an attempt to determine the approximate rat of excretion of each metabolite (table 3). The percentage of the excreted C14 as metabolite C in the early urine collections was found to be greater than in later ones. A semiogarithmic plot of percentage of each metabolite to be excreted vs. time gives an estimate of the excretion half-life of each metabolite. The half-life values obtained by this procedure for 1 fasted rat were 2.4, 2.0, 1.6, and 1.7 hours for metabolites A, B, C, and D, respectively; i.e., these times were required for 50% of the total of each of these metabolites to appear in the urine.

Effect of hypoglycemic blocking agents on metabolism

The effect of corn oil and SKF-525A on the metabolism of 3,5-dimethylpyrazole was studied, since these agents have been shown to inhibit the hypoglycemic activity of this compound. Specifically, it was of interest to determine whether these agents prevented the formation the active metabolite, 5-methylpyrazole-3-carboxylic acid (metabolite A). These agents, even at 10 times the doses used in these studies, inhibit the hypoglycemic activity of 3,5-dimethylpyrazole. Consequently, in order for effects on metabolism by these agents to explain inhibition of activity, a pronounced alteration of metabolism would be required. For example, metabolite A is active in the fasted rat at less than 1 mg/kg.

Table 3: Time course of metabolite excretion pattern of the fasted rat

Collection Period (hr)

Percent of Dose Excreted

Percent Excreted during Each Collection Period as Metabolite:

A

B

C

D

0 - 1

5.8

5.6

6.3

88.1

0.0

1 - 2

22.7

8.5

14.1

77.4

2.9

2 - 3

25.8

15.2

18.2

62.9

3.9

3 - 4

12.4

14.2

19.0

63.3

3.5

4 - 6

24.7

20.0

21.2

55.9

2.8

6 - 8

4.4

21.3

21.4

55.2

2.1

0 - 8

95.8

14.4

17.5

65.0

3.0

None of these agents significantly alters the pattern of metabolites excreted in 24 hours (table 2) . Since the inhibiting effects of these agents were demonstrated 2 hours after administration, the distribution of metabolites in the early urine collections was also studied.

Early metabolism, as evidenced by excretion, was also not affected significantly. The inhibition of hypoglycemic activity by these agents apparently cannot be explained by their effect on the metabolism of 3,5-dimethylpyrazole. This conclusion is supported by the recent finding that the hypoglycemic activity of the active metabolite, 5-methylpyrazole-3-carboxylic acid, is also inhibited by these agents.

Although not statistically significant, reduction of the C14 excreted in 24 hours was observed in the rats pretreated with SKF-525A (table 2). The decreased excretion from the corn oil-treated rats compared to the fasted rats is significant: .1 > P > .05 by a modified t test (Snedecor, 1956) . This may reflect trapping of the pyrazole in the gut by unabsorbed corn oil; the trapping in the gut may also account for the greater variability of C14 excretion from the corn oil-treated rats. This decreased urinary excretion in the corn oil treated rat, which probably reflects decreased absorption, is not sufficient to account for the inhibition of hypoglycemic activity by this agent.

Hypoglycemic activity of the metabolites

The hypoglycemic potency of 5-methylpyrazole-3-carboxylic acid is sufficient to account for all of the hypoglycemic activity found in the urine of 3, 5-dimethylpyrazole-treated rats. The interesting effects of this compound on in vivo and in vitro carbohydrate and lipid metabolism have been described in detail (Gerritsen and Dulin, 1965).

A sample of 4-hydroxy-3,5-dimethylpyrazole, the hydrolysis product of metabolite C, was tested for hypoglycemic activity and was found to have insignificant hypoglycemic activity compared with 5-methylpyrazole-3-carboxylic acid. Urine from which 5-methylpyrazole-3-carboxylic acid has been completely extracted is inactive when tested for hypoglycemic activity; consequently, metabolite C, which does not extract under these conditions, must also be inactive.

Metabolites B and D have not been isolated in crystalline form, but extracts containing these metabolites were inactive in lowering blood glucose.

Applicant's summary and conclusion

Conclusions:
The metabolism of 3,5-dimethylpyrazole was investigated in a non-GLP study by orally exposing rats at 25 mg/kg and anayising their urine in order to identify any metabolites.

3,5-Dimethylpyrazole-C14 is absorbed completely from the gastrointestinal tract of fasted rats when administered as an aqueous suspension. Essentially all of the oral dose (95%) is found in the urine within 24 hours. It is converted completely (>99%) to 4 metabolites. Two metabolites, which together with their conjugated forms account for about 98% of the oral dose of 3,5-dimethylpyrazole, have been isolated in crystalline form and their structures determined. In 5 normal, fasted rats the average percentages excreted (±S.D.) as 5-methylpyrazole-3-carboxylic acid and its conjugate form were 13.1 ± 3.8 and 13.6 ± 4.4,
respectively. The major metabolite, which was excreted to the extent of 70.8 ± 5.5% in fasted rats, has been unequivocally identified as conjugated 4-hydroxy-3, 5-dimethylpyrazole; it has been isolated from hydrolyzed rat urine in its unconjugated form. An unknown metabolite, which is formed to the extent of 1.3 ± 1.9% of the dose, was not detected in the urine of some of the rats studied.

From the urinary excretion data, the half-life for the appearance of C14 in the urine of the rat was determined to be about 2.4 hours; i.e., about 60% of the C14 is excreted during the first 3 hours.

The fate of 3, 5-dimethylpyrazole in fasted rats compared to rats treated with corn oil and β-diethylaminoethyl diphenylpropylacetate hydrochloride indicates that the inhibition of hypoglycemic activity by these agents probably cannot be explained by their effect on the absorption and/or metabolism of 3, 5-dimethylpyrazole.
Executive summary:

The metabolism of 3,5-dimethylpyrazole was investigated in a non-GLP study by orally exposing rats at 25 mg/kg and anayising their urine in order to identify any metabolites.

3,5-Dimethylpyrazole-C14 is absorbed completely from the gastrointestinal tract of fasted rats when administered as an aqueous suspension. Essentially all of the oral dose (95%) is found in the urine within 24 hours. It is converted completely (>99%) to 4 metabolites. Two metabolites, which together with their conjugated forms account for about 98% of the oral dose of 3,5-dimethylpyrazole, have been isolated in crystalline form and their structures determined. In 5 normal, fasted rats the average percentages excreted (±S.D.) as 5-methylpyrazole-3-carboxylic acid and its conjugate form were 13.1 ± 3.8 and 13.6 ± 4.4,

respectively. The major metabolite, which was excreted to the extent of 70.8 ± 5.5% in fasted rats, has been unequivocally identified as conjugated 4-hydroxy-3, 5-dimethylpyrazole; it has been isolated from hydrolyzed rat urine in its unconjugated form. An unknown metabolite, which is formed to the extent of 1.3 ± 1.9% of the dose, was not detected in the urine of some of the rats studied.

From the urinary excretion data, the half-life for the appearance of C14 in the urine of the rat was determined to be about 2.4 hours; i.e., about 60% of the C14 is excreted during the first 3 hours.

The fate of 3, 5-dimethylpyrazole in fasted rats compared to rats treated with corn oil and β-diethylaminoethyl diphenylpropylacetate hydrochloride indicates that the inhibition of hypoglycemic activity by these agents probably cannot be explained by their effect on the absorption and/or metabolism of 3, 5-dimethylpyrazole.