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Comparison of the metabolism and elimination of pyrilamine maleate in the rat, mouse and female rhesus monkey
Comp. Biochem Physiol. Vol. 107C, No. 1, pp. 159-164, 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved
Pergamon
Comparison of the metabolism and elimination of pyrilamine maleate in the rat, mouse and female rhesus monkey C. L. Holder,* W. Slikker, Jrt and H. C. Thompson, Jr* *Divisions of Chemistry; and 1-Reproductive and Developmental Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, U.S.A.
Elimination and metabolic profiles of the O-glucuronide conjugated products of pyrilamine and their nonconjugated O-dealkylated and N-desmethyl pyrilamine products were determined after the oral administration of (14C)-pyrilamine maleate to Fischer 344 rats, B6C3F1 mice and female rhesus monkeys by stomach tube or i.v. The total cumulative urinary and fecal pyrilamine products were determined. The conjugated pyrilamine metabolites, isolated and identified were the glucuronide products of O-dealkylated pyrilamine and ring-hydroxylated pyrilamine, and the nonconjugated metabolites were predominately the N-desmethylpyrilamine and O-dealkylated pyrilamine and their ring-hydroxylated products. Statistically significant differences were observed in the percentages of the conjugated and nonconjugated metabolites of pyrilamine excreted by the three species studied. Key words: Pyrilamine; N-desmethyl pyrilamine; Glucuronide products; O-dealkylated pyrilamine. Comp. Biochem. Physiol. 107C, 159-164, 1994.
Introduction Pyrilamine maleate, an ethylenediamine-type antihistamine, has wide-spread use in over-thecounter antihistamine products such as cough syrups, allergy medications (Haley, 1983), pain relievers and sleep-aids (Neurath et al., 1977). Since Lijinsky et al. (1980) reported that methapyrilene hydrochloride was a potent rat hepatocarcinogen, certain antihistamines have undergone comprehensive animal studies (Jackson and Blackwell, 1988; Habs et al., 1986). Metabolism studies with pyrilamine have been conducted using primary hepatocyte cultures (Probst and Neal, 1982), Sprague-Dawley (Witiak and Lewis, 1978) and Fischer 344 rats (Kelly et al., 1992; Thompson et al., 1987) and fungi (Cerniglia et al., 1988). Consequently, methapyrilene was removed from the U.S. market and pyrilamine maleate, a structurally similar analogue, was marketed as its substitute
(Lijinsky, 1984). Since toxicological studies in various species were not available (Kelly and Slikker, 1987), studies to evaluate the acute and sub-acute toxicity, genotoxicity and potential carcinogenicity of pyrilamine maleate were initiated at the National Center for Toxicological Research (NCTR). The toxicity and mutagenicity testing of pyrilamine maleate indicated that the drug only tested positive for chemically-induced unscheduled DNA synthesis in adult rat hepatocyte studies (Hansen et al., 1987). The genotoxic potential of methapyrilene hydrochloride and other structurally similar antihistamines using the hepatocyte/DNA repair assay were also examined (Budroe et al., 1984). Determination of the relationship between chemical structure and biochemical reaction in vivo in reference to a positive indicator such as methapyrilene required a more extensive metabolism and elimination study of pyrilamine maleate in at least Correspondence to: C. L. Holder, Division of Chemistry, three species of animals. HFT-230 FDA/National Center for Toxicological ReIn this paper we report state-ofsearch, Jefferson, AR 72079, U.S.A. the-art separation, structure identification, and Received 30 June 1993; accepted 20 August 1993. 159
N-desmethylpyrilamine
VI:
N,N-didesmethylpyrilamine
H--HIN~CH2~CH2-~CH2 " = ~
VII:
CH3~N~CH2~CH ~ H
V: Pyrilamine N-oxide
O I CH3~N~CH2~CH ~ CH3
.L = .
N-desmethyl O-dealkylated pyrilamine
3
X
(F~.N
X
~1t
~
~
I'~/""'~N
CH3
[: O-dealkylated Pyrilamine O-Glucuronide
H/
Jl~°~\ ) -o--/"~-X-c. N CH CH NtCH3 --~k.J/-= - - , - - 2- =--~ k/H
HO"\OH
.
~
~].._~../h--u~' H: Pyrilamine O-Glucuronide ROH
COOH
COOH
HO
I CH30.~I--CH2~N~CH2 --CH2~NSCH3
V-O o,
Fig. 1. Structures of pyri]amine and its nonconjugated and ¢onjugat~ metabolitcs.
O--CH3
Ill:
O-dealkylated pyrilamine
Pyrilamine I
N~CH2~'-O--CH
CH3~. N ~ C H 2 ~ C H 2 ~ N ~ C H 2 - - - ~ O H
IV:
VIII:
CH3~N~CH2~CH 2 CH3
-t
o
m
Metabolism of pyrilamine maleate chromatographic techniques used to determine the complete metabolism and elimination profile of pyrilamine in Fischer 344 rats, B6C3F1 mice, and female rhesus monkeys.
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Materials and Methods Chemicals and supplies The antihistamine, pyrilamine maleate, was obtained from Hexagon Laboratories Inc. (Bronx, NY). The C14-pyrilamine maleate was >99% radiochemically pure and was obtained from Southwest Foundation for Research and Education (San Antonio, TX). The structural identity and purity of this antihistamine were verified by HPLC, mass spectrometry and nuclear magnetic resonance spectrometry (NMR). High pressure liquid chromatography (HPLC) The HPLC was performed with an analytical column (Supelco LC-18; 25cm x 4.6mm id) using a C18 reverse phase packing. Typically, the mobile phase was (A) aqueous buffer (B) methanol using a gradient program from A to B. The aqueous buffer (A) was 0.5/aM aqueous ammonium acetate (pH 7.5) and methanol (B) over a 20 min gradient program (A to B over 10 min and B from 10 to 20 min, then reequilibrating the C18 column back to A condition from 20 to 25 min). The mobile phase was pumped at 1 ml/min using a Spectra Physics Model 8700 solvent delivery system. The sample was injected into the HPLC system via a Model 7125 injector (Rheodyne) equipped with a 50/zl loop. A FLO-One Beta Model IC radiochemical detector and a Waters Model 440 ultraviolet detector, operated at 254 nm, were used in series to detect pyrilamine and its metabolites. Dose administration and sample collection The animals were housed in stainless steel cages as described by Thompson et al. (1987). The Fischer 344 rats and B6C3FI mice (male and female) were given 2, 5 and 10 mg of dose by gavage in a volume of deionized water (1 ml or less). The female rhesus monkeys were dosed using a stomach tube or i.v. (7 and 0.7 mg/kg) in a 2 ml volume of saline solution on the basis of mg per kg of body weight. The animals were allowed free access to water and feed prior to dosing and throughout the experiment. All urinary and fecal samples were collected before and after dosing at timed intervals and stored at - 2 0 ° C until needed for analysis by HPLC.
Results and Discussion An HPLC-thermospray mass spectrometric (TSMS) method was developed for the separation and identification of pyrilamine and its
"
/
"m
IV
_;
mI
°
77' Time (minutes)
Fig. 2. HPLC-Ultraviolet chromatogram at 2.0 A U F S and at 254nm for 24hr. F-344 rat urine. (I: O-dealkylated pyrilamine O-glucuronide, II: Pyrilamine O-glucuronide, III: O--dealkylated N-desmethylpyrilamine, IV: O-dealkylated pyrilamine, V: Pyrilamine N-oxide, VI: N,Ndidesmethylpyrilamine, V|I: N--desmethyl pyrilamine, VIII: Pyrilamine).
metabolites (Korfmacher et ai., 1990). The metabolites of pyrilamine were identified as the O-glucuronide products of O-dealkylated pyrilamine (I) and pyrilamine (II), O-dealkylated N-desmethylpyrilamine (III), O-dealkylated pyrilamine (IV), N-oxide of pyrilamine (V), N,N-didesmethylpyrilamine (VI) and Ndesmethylpyrilamine (VII) (Fig. 1). The O-glucuronide conjugated products were predominately excreted in the urine of the three species of animals (Fischer 344 rats, B6C3FI mice, and female rhesus monkeys), while the nonconjugated compounds of pyrilamine were excreted via the biliary tract within the feces. A typical HPLC chromatogram of a 24hr rat urine sample is illustrated in Fig. 2. The conjugated O-glucuronide metabolites of O-dealkylated pyrilamine (I) and pyrilamine (II) were the major metabolites, and the nonTable 1. HPLC retention times (tR) for pyrilamine and the metabolites reported in this report Compounds O-Dcalkylated pyrilamine O-Glucuronide, I Pyrilamine O-glucuronide, II O-Dealkylated N-desmethyl pyrilamine, II1 O-Dea|kylated pyrilamine, IV Pyrilamine N-oxide, V N,N-Didesmethylpyrilamine, V1 N-Desmethylpyrilamine, VII Pyrilamine, VIII
HPLC retention (t R) time (min) 9.3 rain 9.8 rain 11.6 min 12.0 rain 12.8 rain 13.6 rain 14.8 rain 16.1 rain
C. L. Holder et al.
162
Table 2. Percent (%) elimination of I+C-pyrilamine maleate and its metabolites in urine and fecal samples from Fischer 344 rats
Table 3. Percent (%) elimination of t4C-pyrilamine maleate and its metabolites in urine and fecal samples from BrC3FI mice
Dose level(s) (rag)
Dose level(s) (rag)
Dose administration Oral/Gavage
2 5 10
Total r4C-pyrilamine recovered (%) Male 92.6 + 15.1 94.4 _+7.9 91.9 + 8.7
Gavage Gavage Gavage
Female 93.1 + 5.1 99.5 + 4.5 97.6 _+ 1.9
conjugated metabolites of pyrilamine (peaks III-VII) were the minor metabolites (Table 1). The H P L C procedure used in determining metabolism and elimination profiles of pyrilamine and its biological products worked well for the substrates collected from the rats, mice and monkeys (urine and feces). Results of the elimination of the C 14-radioactivity of the pyrilamine administered to male and female Fischer 344 rats and BrC3F1 mice (2, 5 and 10mg) are shown in Table 2. A typical HPLC chromatogram of 24 hr mouse urine illustrates the major conjugated metabolites of pyrilamine and the minor nonconjugated metabolites of pyrilamine as shown by Fig. 3. The amount of the C14 radiolabeled compounds from the urine and feces of male Fischer 344 rats at the 2, 5 and 10mg dose levels (shown in Table 2) was 92.6 + 15.1, 94.4 _ 7.9, and 91.9 _ 8.7% of total dose, respectively. The male rat urine contained O-glucuronide conjugates of O-dealkylated and ring-hydroxylated pyrilamine as the major metabolites excreted in the urine. This was a distribution of approximately 70% of the total dose administered that was eliminated as the
~IPLC+UV(25~Im); ----Urlne(24hr.)
II
.=
°
II it,' ulL.,v
.l'
2 5 10
Dose administration Oral/Gavage Gavage Gavage Garage
Total I+C-pyrilamine recovered (%) Male 93.5 + 2.3 98.4 + 9.5 99.6 + 5.1
Female 93.4 _+9.8 87.1 + 5.6 99.1 + 7.7
conjugated metabolites of pyrilamine. The remaining 30% was eliminated as the nonconjugated metabolites of pyrilamine (O-dealkylated N-desmethylpyrilamine, O-dealkylated pyrilamine, N-oxide of pyrilamine, N,N-didesmethylpyrilamine and N-desmethylpyrilamine products). The amounts recovered in the urine and feces of female Fischer 344 rats at the 2, 5 and 10 mg dose levels shown in Table 2 were 93.1 _ 5.1, 9 9 . 5 _ 4.2, and 97.6 + 1.9% of total administered dose, respectively. The elimination of conjugated and nonconjugated pyrilamine metabolites via the urine of animals for both male and female rats dosed at the 10 mg level in the first 2 4 h r were 63.5___8.5% and 36.5 ___4.1%, respectively. The C-14 radiolabeled compounds found in the urine and feces of BrC3F1 male mice dosed at the 2, 5 and 10 mg levels (shown in Table 3) were 93.5 __+2.3, 98.4 ___9.5 and 99.6 ___5.1% of the total administered dose, respectively. The amount of pyrilamine and its metabolites recovered from the urine and feces of female B6C3F1 mice (Table 3) dosed at 2, 5 and 10 mg levels was 93.4 + 9.8, 87.1 _ 5.6 and 99.1 + 7.7% of the total administered dose, respectively. The contribution of the conjugated and nonconjugated pyrilamine metabolites eliminated via the urine from the male and female mice at the 10 mg dose level in the first 24 hr was 94.1 ± 1.0 and 5.9 + 0.6% of the total administered dose, respectively. This data indicates that the male and female mice essentially excreted pyrilamine as O-glucuronide conjugates (93%) for the three dose levels, while the male and female Fischer 344 rats only excreted the conjugated metabolites at about 66% for the three dose levels. The results of the dose accountability of the pyrilamine in the female Rhesus monkeys dosed with a stomach tube or i.v. at levels of 7 and
Table 4. Percent (%) elimination of 14C-pyrilamine maleate and its metabolites in urine and fecal samples from female rhesus monkeys Time (minutes)
Fig. 3. HPLC-Ultraviolet c h r o m a t o g r a m at 2.0 A U F S and at 2 5 4 n m for 24hr. B6C3F1 Urine. (l: O-dealkylated pyrilamine O-glucuronide, II: Pyrilamine O-glucuronide, III: O-dealkylated N-desmethylpyrilamine, IV: O-dealkylated pyrilamine, V: Pyrilamine N-oxide, VI: N , N didesmethylpyrilamine, VII: N-desmethyl pyrilamine).
Dose level(s) (mg) 0.7 7 0.7 7
Dose administration stomach tube/i.v, Stomach tube Stomach tube i.v. i.v.
Total ~4C-pyrilamine recovered (%) 66.3 + 20.5 68.4 + 13.6 80.5 + 18.3 91.5 __+10.9
Metabolism of pyrilamine maleate °°"
163
Conclusion
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This report describes the elimination of the major conjugated metabolites of pyrilamine as the O-glucuronide of pyrilamine and the O-glucuronide of O-dealkylated pyrilamine for the three species of animals studied (rat, mouse, and monkey). The B6C3F1 mice eliminated the pyrilamine O-glucuronide conjugated metabolites at the l0 mg level at 94.1% of the total dose. The Fischer 344 rats eliminated the O-glucuronide conjugated metabolites of pyrilamine at the l0 mg dose level at 63.5%, and the female rhesus monkeys eliminated these O-glucuronide conjugated metabolites of pyrilamine at the 7.0 mg dose level at 84.2% of the total dose, respectively.
Time (minutes) Fig. 4. HPLC-Ultraviolet chromatogram at 2.0 AUFS and at 254 nm for 24 hr. Rhesus monkey urine. (I: O-deaikylated pyrilamine O-glucuronide, II: pyrilamine O-glucuronide, III: O-dealkylated N-desmethylpyrilamine, IV: O-dealkylated pyrilamine, V: pyrilamine N-oxide, VI: N,Ndidesmethylpyrilamine, VII: N-desmethyl pyrilamine).
0.7 mg/kg are shown in Table 4. The recoveries were 68.4_+ 13.6% and 66.3_20.5% when the pyrilamine maleate sample dose was administered by stomach tube at the 7.0 and 0.7mg/kg levels, and 9 1 . 5 _ 10.9% and 80.5___ 18.3% when dosed i.v at the same levels. Pyrilamine was eliminated in the urine and feces of the rhesus monkeys in the first 24hr with a recovery of 95% of the total administered dose levels. A typical HPLC chromatogram of 24 hr monkey urine is shown in Fig. 4. The pyrilamine was eliminated from the plasma with a recovery of 95% of the total dose administered after 4 hr. The pyrilamine major metabolites identified from the urine excreted in the first 24hr from the female rhesus monkeys dosed at the 7 mg/kg level were the O-glucuronide of pyrilamine and the Oglucuronide of O-dealkyated pyrilamine (84.2 + 6.7%). The major metabolites of pyrilamine identified in urine and fecal samples from the Fischer 344 rats and BrC3FI mice at the 10mg dose level were identified as the O-glucuronide of pyrilamine and the Oglucuronide of O-dealkylated pyrilamine (63.5+2.1 and 94.1+1.0%). The minor metabolites of pyrilamine identified and reported as the nonconjugated products in urine and fecal samples from the three species of animals were pyrilamine N-oxide, Ndesmethylpyrilamine, O-dealkylated pyrilamine, O-dealkylated N-desmethylpyrilamine and N,N-didesmethylpyrilamine.
References Budroe J. D., Shaddock J. G. and Casciano D. A. (1984) A study of the potential genotoxicity of methapyrilene and related antihistamines using the hepatocyte/DNA repair system. Mutat. Res. 135, 131-137. Cerniglia C. E., Hansen E. B. Jr, Lambert K. J., Korfreacher W. A. and Miller D. W. (1988) Fungal transformation of antihistamines: metabolism of methapyrilene, tbenyldiamine and tripelennamine to Noxide and N-demethylated derivatives. Xenobiotica 18(3), 301-312. Habs M., Shubik P. and Eisenbrand G. (1986) Carcinogenicity of methapyrilene hydrochloride, mepyramine hydrochioride, thenyldiamine hydrochloride and pyribenzamin¢ hydrochloride in Sprague Dawley rats. J. Cancer Res. Clin. Oncolog. 111, 71-74. Haley T. J. (1983) Physical and biological properties of pyrilamine. J. Pharmac. Sci. 72, 3-12. Hansen E. B. Jr, Cerniglia C. E., Korfmacher W. A., Miller D. W. and Heflich R. N. (1987) Microbial transformation of the antihistamine pyrilamine maleate: formation of potential mammalian metabolites. Drug Metab. Dispos. 15, 97-106. Jackson C. D. and Blackwell B. N. (1988) Subehronic studies of doxylamine in Fischer 344 rats. Fundam. appl. Toxic. 10(2), 243-261. Kelly D. W., Holder C. L., Korfmacher W. A., Getek T. A., Lay J. O. Jr, Casciano D. A., Shaddock J. G., Duhart H. M. and Slikker W. Jr. (1992) Metabolism of mcthapyrilene by Fischer 344 rat and BrC3FI mouse hepatocytes. Xenobiotica 22(12), 1367-1381. Kelly D. W. and Slikker W. Jr. (1987) The metabolism and elimination pyrilamine maleate in the rat. Drug Metab. Disp, 15, 460-465. Korfmacber W. A., Freeman J. P., Getek T. A., Bloom J. and Holder C. L. (1990) Analysis of rat urine for metabolites of pyrilamine via high-performance liquid chromatography/tbermospray mass spectrometry and tandem mass spectrometry. Biomed. Envir. Mass Spectrom. 19, 191-201. Lijinsky W. (1984) Chronic toxicity tests of pyrilamine maleate and methapyrilene hydrochloride in F-344 rats. Fd Chem. Toxic. 22, 27-30. Lijinsky W., Rueber M. D. and Blackwell B. N. (1980) Liver tumors induced in rats by oral administration of the antihistaminic methapyrilene hydrochloride. Science 209, 817--819. Neurath (3. B., Dunger M., Pein F. G., Ambrosius D. and Schreiber O. (1977) Primary and secondary
164
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amines in the human environment. Fd Cosmet. Toxic. 15, 275-282. Probst G. S. and Neal S. B. (1982) The induction of unscheduled DNA synthesis by antihistamines in primary hepatocyte cultures. Cancer Lett. 10, 213-218. Thompson H. C. Jr, Holder C. L., Siitonen P. H., Rowland K. L., Gosnell A. B. and Cmarik J. L. (1987)
Metabolism of pyrilamine maleate in Fischer 344 rats, part 1: activity excretion profiles. J. Analyt. Toxic. II, 252-256. Witiak D. T. and Lewis N. J. 0978) Absorption, distribution, metabolism and elimination of antihistamines. In Handbook o f Experimental Pharmacology. (Edited by Rocha de Silva M.), Vol. 18, pp. 513-516. Springer, Berlin.
Pergamon
Comparison of the metabolism and elimination of pyrilamine maleate in the rat, mouse and female rhesus monkey C. L. Holder,* W. Slikker, Jrt and H. C. Thompson, Jr* *Divisions of Chemistry; and 1-Reproductive and Developmental Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, U.S.A.
Elimination and metabolic profiles of the O-glucuronide conjugated products of pyrilamine and their nonconjugated O-dealkylated and N-desmethyl pyrilamine products were determined after the oral administration of (14C)-pyrilamine maleate to Fischer 344 rats, B6C3F1 mice and female rhesus monkeys by stomach tube or i.v. The total cumulative urinary and fecal pyrilamine products were determined. The conjugated pyrilamine metabolites, isolated and identified were the glucuronide products of O-dealkylated pyrilamine and ring-hydroxylated pyrilamine, and the nonconjugated metabolites were predominately the N-desmethylpyrilamine and O-dealkylated pyrilamine and their ring-hydroxylated products. Statistically significant differences were observed in the percentages of the conjugated and nonconjugated metabolites of pyrilamine excreted by the three species studied. Key words: Pyrilamine; N-desmethyl pyrilamine; Glucuronide products; O-dealkylated pyrilamine. Comp. Biochem. Physiol. 107C, 159-164, 1994.
Introduction Pyrilamine maleate, an ethylenediamine-type antihistamine, has wide-spread use in over-thecounter antihistamine products such as cough syrups, allergy medications (Haley, 1983), pain relievers and sleep-aids (Neurath et al., 1977). Since Lijinsky et al. (1980) reported that methapyrilene hydrochloride was a potent rat hepatocarcinogen, certain antihistamines have undergone comprehensive animal studies (Jackson and Blackwell, 1988; Habs et al., 1986). Metabolism studies with pyrilamine have been conducted using primary hepatocyte cultures (Probst and Neal, 1982), Sprague-Dawley (Witiak and Lewis, 1978) and Fischer 344 rats (Kelly et al., 1992; Thompson et al., 1987) and fungi (Cerniglia et al., 1988). Consequently, methapyrilene was removed from the U.S. market and pyrilamine maleate, a structurally similar analogue, was marketed as its substitute
(Lijinsky, 1984). Since toxicological studies in various species were not available (Kelly and Slikker, 1987), studies to evaluate the acute and sub-acute toxicity, genotoxicity and potential carcinogenicity of pyrilamine maleate were initiated at the National Center for Toxicological Research (NCTR). The toxicity and mutagenicity testing of pyrilamine maleate indicated that the drug only tested positive for chemically-induced unscheduled DNA synthesis in adult rat hepatocyte studies (Hansen et al., 1987). The genotoxic potential of methapyrilene hydrochloride and other structurally similar antihistamines using the hepatocyte/DNA repair assay were also examined (Budroe et al., 1984). Determination of the relationship between chemical structure and biochemical reaction in vivo in reference to a positive indicator such as methapyrilene required a more extensive metabolism and elimination study of pyrilamine maleate in at least Correspondence to: C. L. Holder, Division of Chemistry, three species of animals. HFT-230 FDA/National Center for Toxicological ReIn this paper we report state-ofsearch, Jefferson, AR 72079, U.S.A. the-art separation, structure identification, and Received 30 June 1993; accepted 20 August 1993. 159
N-desmethylpyrilamine
VI:
N,N-didesmethylpyrilamine
H--HIN~CH2~CH2-~CH2 " = ~
VII:
CH3~N~CH2~CH ~ H
V: Pyrilamine N-oxide
O I CH3~N~CH2~CH ~ CH3
.L = .
N-desmethyl O-dealkylated pyrilamine
3
X
(F~.N
X
~1t
~
~
I'~/""'~N
CH3
[: O-dealkylated Pyrilamine O-Glucuronide
H/
Jl~°~\ ) -o--/"~-X-c. N CH CH NtCH3 --~k.J/-= - - , - - 2- =--~ k/H
HO"\OH
.
~
~].._~../h--u~' H: Pyrilamine O-Glucuronide ROH
COOH
COOH
HO
I CH30.~I--CH2~N~CH2 --CH2~NSCH3
V-O o,
Fig. 1. Structures of pyri]amine and its nonconjugated and ¢onjugat~ metabolitcs.
O--CH3
Ill:
O-dealkylated pyrilamine
Pyrilamine I
N~CH2~'-O--CH
CH3~. N ~ C H 2 ~ C H 2 ~ N ~ C H 2 - - - ~ O H
IV:
VIII:
CH3~N~CH2~CH 2 CH3
-t
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Metabolism of pyrilamine maleate chromatographic techniques used to determine the complete metabolism and elimination profile of pyrilamine in Fischer 344 rats, B6C3F1 mice, and female rhesus monkeys.
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.
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Materials and Methods Chemicals and supplies The antihistamine, pyrilamine maleate, was obtained from Hexagon Laboratories Inc. (Bronx, NY). The C14-pyrilamine maleate was >99% radiochemically pure and was obtained from Southwest Foundation for Research and Education (San Antonio, TX). The structural identity and purity of this antihistamine were verified by HPLC, mass spectrometry and nuclear magnetic resonance spectrometry (NMR). High pressure liquid chromatography (HPLC) The HPLC was performed with an analytical column (Supelco LC-18; 25cm x 4.6mm id) using a C18 reverse phase packing. Typically, the mobile phase was (A) aqueous buffer (B) methanol using a gradient program from A to B. The aqueous buffer (A) was 0.5/aM aqueous ammonium acetate (pH 7.5) and methanol (B) over a 20 min gradient program (A to B over 10 min and B from 10 to 20 min, then reequilibrating the C18 column back to A condition from 20 to 25 min). The mobile phase was pumped at 1 ml/min using a Spectra Physics Model 8700 solvent delivery system. The sample was injected into the HPLC system via a Model 7125 injector (Rheodyne) equipped with a 50/zl loop. A FLO-One Beta Model IC radiochemical detector and a Waters Model 440 ultraviolet detector, operated at 254 nm, were used in series to detect pyrilamine and its metabolites. Dose administration and sample collection The animals were housed in stainless steel cages as described by Thompson et al. (1987). The Fischer 344 rats and B6C3FI mice (male and female) were given 2, 5 and 10 mg of dose by gavage in a volume of deionized water (1 ml or less). The female rhesus monkeys were dosed using a stomach tube or i.v. (7 and 0.7 mg/kg) in a 2 ml volume of saline solution on the basis of mg per kg of body weight. The animals were allowed free access to water and feed prior to dosing and throughout the experiment. All urinary and fecal samples were collected before and after dosing at timed intervals and stored at - 2 0 ° C until needed for analysis by HPLC.
Results and Discussion An HPLC-thermospray mass spectrometric (TSMS) method was developed for the separation and identification of pyrilamine and its
"
/
"m
IV
_;
mI
°
77' Time (minutes)
Fig. 2. HPLC-Ultraviolet chromatogram at 2.0 A U F S and at 254nm for 24hr. F-344 rat urine. (I: O-dealkylated pyrilamine O-glucuronide, II: Pyrilamine O-glucuronide, III: O--dealkylated N-desmethylpyrilamine, IV: O-dealkylated pyrilamine, V: Pyrilamine N-oxide, VI: N,Ndidesmethylpyrilamine, V|I: N--desmethyl pyrilamine, VIII: Pyrilamine).
metabolites (Korfmacher et ai., 1990). The metabolites of pyrilamine were identified as the O-glucuronide products of O-dealkylated pyrilamine (I) and pyrilamine (II), O-dealkylated N-desmethylpyrilamine (III), O-dealkylated pyrilamine (IV), N-oxide of pyrilamine (V), N,N-didesmethylpyrilamine (VI) and Ndesmethylpyrilamine (VII) (Fig. 1). The O-glucuronide conjugated products were predominately excreted in the urine of the three species of animals (Fischer 344 rats, B6C3FI mice, and female rhesus monkeys), while the nonconjugated compounds of pyrilamine were excreted via the biliary tract within the feces. A typical HPLC chromatogram of a 24hr rat urine sample is illustrated in Fig. 2. The conjugated O-glucuronide metabolites of O-dealkylated pyrilamine (I) and pyrilamine (II) were the major metabolites, and the nonTable 1. HPLC retention times (tR) for pyrilamine and the metabolites reported in this report Compounds O-Dcalkylated pyrilamine O-Glucuronide, I Pyrilamine O-glucuronide, II O-Dealkylated N-desmethyl pyrilamine, II1 O-Dea|kylated pyrilamine, IV Pyrilamine N-oxide, V N,N-Didesmethylpyrilamine, V1 N-Desmethylpyrilamine, VII Pyrilamine, VIII
HPLC retention (t R) time (min) 9.3 rain 9.8 rain 11.6 min 12.0 rain 12.8 rain 13.6 rain 14.8 rain 16.1 rain
C. L. Holder et al.
162
Table 2. Percent (%) elimination of I+C-pyrilamine maleate and its metabolites in urine and fecal samples from Fischer 344 rats
Table 3. Percent (%) elimination of t4C-pyrilamine maleate and its metabolites in urine and fecal samples from BrC3FI mice
Dose level(s) (rag)
Dose level(s) (rag)
Dose administration Oral/Gavage
2 5 10
Total r4C-pyrilamine recovered (%) Male 92.6 + 15.1 94.4 _+7.9 91.9 + 8.7
Gavage Gavage Gavage
Female 93.1 + 5.1 99.5 + 4.5 97.6 _+ 1.9
conjugated metabolites of pyrilamine (peaks III-VII) were the minor metabolites (Table 1). The H P L C procedure used in determining metabolism and elimination profiles of pyrilamine and its biological products worked well for the substrates collected from the rats, mice and monkeys (urine and feces). Results of the elimination of the C 14-radioactivity of the pyrilamine administered to male and female Fischer 344 rats and BrC3F1 mice (2, 5 and 10mg) are shown in Table 2. A typical HPLC chromatogram of 24 hr mouse urine illustrates the major conjugated metabolites of pyrilamine and the minor nonconjugated metabolites of pyrilamine as shown by Fig. 3. The amount of the C14 radiolabeled compounds from the urine and feces of male Fischer 344 rats at the 2, 5 and 10mg dose levels (shown in Table 2) was 92.6 + 15.1, 94.4 _ 7.9, and 91.9 _ 8.7% of total dose, respectively. The male rat urine contained O-glucuronide conjugates of O-dealkylated and ring-hydroxylated pyrilamine as the major metabolites excreted in the urine. This was a distribution of approximately 70% of the total dose administered that was eliminated as the
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Dose administration Oral/Gavage Gavage Gavage Garage
Total I+C-pyrilamine recovered (%) Male 93.5 + 2.3 98.4 + 9.5 99.6 + 5.1
Female 93.4 _+9.8 87.1 + 5.6 99.1 + 7.7
conjugated metabolites of pyrilamine. The remaining 30% was eliminated as the nonconjugated metabolites of pyrilamine (O-dealkylated N-desmethylpyrilamine, O-dealkylated pyrilamine, N-oxide of pyrilamine, N,N-didesmethylpyrilamine and N-desmethylpyrilamine products). The amounts recovered in the urine and feces of female Fischer 344 rats at the 2, 5 and 10 mg dose levels shown in Table 2 were 93.1 _ 5.1, 9 9 . 5 _ 4.2, and 97.6 + 1.9% of total administered dose, respectively. The elimination of conjugated and nonconjugated pyrilamine metabolites via the urine of animals for both male and female rats dosed at the 10 mg level in the first 2 4 h r were 63.5___8.5% and 36.5 ___4.1%, respectively. The C-14 radiolabeled compounds found in the urine and feces of BrC3F1 male mice dosed at the 2, 5 and 10 mg levels (shown in Table 3) were 93.5 __+2.3, 98.4 ___9.5 and 99.6 ___5.1% of the total administered dose, respectively. The amount of pyrilamine and its metabolites recovered from the urine and feces of female B6C3F1 mice (Table 3) dosed at 2, 5 and 10 mg levels was 93.4 + 9.8, 87.1 _ 5.6 and 99.1 + 7.7% of the total administered dose, respectively. The contribution of the conjugated and nonconjugated pyrilamine metabolites eliminated via the urine from the male and female mice at the 10 mg dose level in the first 24 hr was 94.1 ± 1.0 and 5.9 + 0.6% of the total administered dose, respectively. This data indicates that the male and female mice essentially excreted pyrilamine as O-glucuronide conjugates (93%) for the three dose levels, while the male and female Fischer 344 rats only excreted the conjugated metabolites at about 66% for the three dose levels. The results of the dose accountability of the pyrilamine in the female Rhesus monkeys dosed with a stomach tube or i.v. at levels of 7 and
Table 4. Percent (%) elimination of 14C-pyrilamine maleate and its metabolites in urine and fecal samples from female rhesus monkeys Time (minutes)
Fig. 3. HPLC-Ultraviolet c h r o m a t o g r a m at 2.0 A U F S and at 2 5 4 n m for 24hr. B6C3F1 Urine. (l: O-dealkylated pyrilamine O-glucuronide, II: Pyrilamine O-glucuronide, III: O-dealkylated N-desmethylpyrilamine, IV: O-dealkylated pyrilamine, V: Pyrilamine N-oxide, VI: N , N didesmethylpyrilamine, VII: N-desmethyl pyrilamine).
Dose level(s) (mg) 0.7 7 0.7 7
Dose administration stomach tube/i.v, Stomach tube Stomach tube i.v. i.v.
Total ~4C-pyrilamine recovered (%) 66.3 + 20.5 68.4 + 13.6 80.5 + 18.3 91.5 __+10.9
Metabolism of pyrilamine maleate °°"
163
Conclusion
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This report describes the elimination of the major conjugated metabolites of pyrilamine as the O-glucuronide of pyrilamine and the O-glucuronide of O-dealkylated pyrilamine for the three species of animals studied (rat, mouse, and monkey). The B6C3F1 mice eliminated the pyrilamine O-glucuronide conjugated metabolites at the l0 mg level at 94.1% of the total dose. The Fischer 344 rats eliminated the O-glucuronide conjugated metabolites of pyrilamine at the l0 mg dose level at 63.5%, and the female rhesus monkeys eliminated these O-glucuronide conjugated metabolites of pyrilamine at the 7.0 mg dose level at 84.2% of the total dose, respectively.
Time (minutes) Fig. 4. HPLC-Ultraviolet chromatogram at 2.0 AUFS and at 254 nm for 24 hr. Rhesus monkey urine. (I: O-deaikylated pyrilamine O-glucuronide, II: pyrilamine O-glucuronide, III: O-dealkylated N-desmethylpyrilamine, IV: O-dealkylated pyrilamine, V: pyrilamine N-oxide, VI: N,Ndidesmethylpyrilamine, VII: N-desmethyl pyrilamine).
0.7 mg/kg are shown in Table 4. The recoveries were 68.4_+ 13.6% and 66.3_20.5% when the pyrilamine maleate sample dose was administered by stomach tube at the 7.0 and 0.7mg/kg levels, and 9 1 . 5 _ 10.9% and 80.5___ 18.3% when dosed i.v at the same levels. Pyrilamine was eliminated in the urine and feces of the rhesus monkeys in the first 24hr with a recovery of 95% of the total administered dose levels. A typical HPLC chromatogram of 24 hr monkey urine is shown in Fig. 4. The pyrilamine was eliminated from the plasma with a recovery of 95% of the total dose administered after 4 hr. The pyrilamine major metabolites identified from the urine excreted in the first 24hr from the female rhesus monkeys dosed at the 7 mg/kg level were the O-glucuronide of pyrilamine and the Oglucuronide of O-dealkyated pyrilamine (84.2 + 6.7%). The major metabolites of pyrilamine identified in urine and fecal samples from the Fischer 344 rats and BrC3FI mice at the 10mg dose level were identified as the O-glucuronide of pyrilamine and the Oglucuronide of O-dealkylated pyrilamine (63.5+2.1 and 94.1+1.0%). The minor metabolites of pyrilamine identified and reported as the nonconjugated products in urine and fecal samples from the three species of animals were pyrilamine N-oxide, Ndesmethylpyrilamine, O-dealkylated pyrilamine, O-dealkylated N-desmethylpyrilamine and N,N-didesmethylpyrilamine.
References Budroe J. D., Shaddock J. G. and Casciano D. A. (1984) A study of the potential genotoxicity of methapyrilene and related antihistamines using the hepatocyte/DNA repair system. Mutat. Res. 135, 131-137. Cerniglia C. E., Hansen E. B. Jr, Lambert K. J., Korfreacher W. A. and Miller D. W. (1988) Fungal transformation of antihistamines: metabolism of methapyrilene, tbenyldiamine and tripelennamine to Noxide and N-demethylated derivatives. Xenobiotica 18(3), 301-312. Habs M., Shubik P. and Eisenbrand G. (1986) Carcinogenicity of methapyrilene hydrochloride, mepyramine hydrochioride, thenyldiamine hydrochloride and pyribenzamin¢ hydrochloride in Sprague Dawley rats. J. Cancer Res. Clin. Oncolog. 111, 71-74. Haley T. J. (1983) Physical and biological properties of pyrilamine. J. Pharmac. Sci. 72, 3-12. Hansen E. B. Jr, Cerniglia C. E., Korfmacher W. A., Miller D. W. and Heflich R. N. (1987) Microbial transformation of the antihistamine pyrilamine maleate: formation of potential mammalian metabolites. Drug Metab. Dispos. 15, 97-106. Jackson C. D. and Blackwell B. N. (1988) Subehronic studies of doxylamine in Fischer 344 rats. Fundam. appl. Toxic. 10(2), 243-261. Kelly D. W., Holder C. L., Korfmacher W. A., Getek T. A., Lay J. O. Jr, Casciano D. A., Shaddock J. G., Duhart H. M. and Slikker W. Jr. (1992) Metabolism of mcthapyrilene by Fischer 344 rat and BrC3FI mouse hepatocytes. Xenobiotica 22(12), 1367-1381. Kelly D. W. and Slikker W. Jr. (1987) The metabolism and elimination pyrilamine maleate in the rat. Drug Metab. Disp, 15, 460-465. Korfmacber W. A., Freeman J. P., Getek T. A., Bloom J. and Holder C. L. (1990) Analysis of rat urine for metabolites of pyrilamine via high-performance liquid chromatography/tbermospray mass spectrometry and tandem mass spectrometry. Biomed. Envir. Mass Spectrom. 19, 191-201. Lijinsky W. (1984) Chronic toxicity tests of pyrilamine maleate and methapyrilene hydrochloride in F-344 rats. Fd Chem. Toxic. 22, 27-30. Lijinsky W., Rueber M. D. and Blackwell B. N. (1980) Liver tumors induced in rats by oral administration of the antihistaminic methapyrilene hydrochloride. Science 209, 817--819. Neurath (3. B., Dunger M., Pein F. G., Ambrosius D. and Schreiber O. (1977) Primary and secondary
164
C.L. Holder et al.
amines in the human environment. Fd Cosmet. Toxic. 15, 275-282. Probst G. S. and Neal S. B. (1982) The induction of unscheduled DNA synthesis by antihistamines in primary hepatocyte cultures. Cancer Lett. 10, 213-218. Thompson H. C. Jr, Holder C. L., Siitonen P. H., Rowland K. L., Gosnell A. B. and Cmarik J. L. (1987)
Metabolism of pyrilamine maleate in Fischer 344 rats, part 1: activity excretion profiles. J. Analyt. Toxic. II, 252-256. Witiak D. T. and Lewis N. J. 0978) Absorption, distribution, metabolism and elimination of antihistamines. In Handbook o f Experimental Pharmacology. (Edited by Rocha de Silva M.), Vol. 18, pp. 513-516. Springer, Berlin.