![Metabolism of [IC ]Methamphetamine in Man, the Guinea Pig and the Rat](http://data.docslib.org/img/0c05d8dfdc59b0c3ba880db1b3d8eaba-1.webp)
Biochem. J. (1972) 129, 11-22 11 Printed in Great Britain
Metabolism of [IC ]Methamphetamine in Man, the Guinea Pig and the Rat
By J. CALDWELL, L. G. DRING and R. T. WILLIAMS Department of Biochemistry, St. Mary's Hospital Medical School, London W.2, U.K. (Received 2 March 1972)
1. The metabolites of (±)-2-methylamino-1-phenyl[1-14C]propane ([14C]methamphet- amine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% ofthe 14C was excreted in the urine in 4 days. The urine ofthe first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15 %). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxy- norephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82 % of the dose of '4C (45mg/kg) was excreted in the urine and 2-3 % in the faeces in 3-4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31 % ofdose), 4-hydroxynorephedrine (16 %) and unchanged drug (11 %). Minor metabo- lites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the 14C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19 % on the higher dose and about 1 % on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.
Methamphetamine (2-methylamino-1-phenylprop- hydroxylation and demethylation are significant re- ane) is regarded as dangerous from the point of view actions. All three species also excrete norephedrine of drug dependence and its use has been restricted derivatives after dosage with the drug, the rat produc- since 1968 to hospitals for therapeutic purposes ing 4-hydroxynorephedrine [2-amino-1-(4'-hydroxy- (Editorial, 1968). However, little work has been done phenyl)propan-l-ol], the guinea pig, norephedrine on its metabolism. Richter (1938) reported that it was (2-amino-1-phenylpropan-1-ol), and man, both. excreted unchanged by man, but more recently Norephedrine and its 4-hydroxy derivative have been Cartoni & de Stefano (1963) and Beckett & Rowland implicated as false transmitters at nerve endings and (1965) have shown that it is excreted partly un- may be involved in the undesirable effects of the changed and partly as amphetamine (2-amino-i- chronic intake of the amphetamines (see Brodie et al., phenylpropane). In the dog, Axelrod (1954) found 1970). Evidence is also produced that in the guinea that it was largely demethylated to amphetamine and pig at least, the production of norephedrine is dose- converted to some extent into 4-hydroxyamphet- dependent. Part of the work has been briefly reported amine [2-amino-1-(4'-hydroxyphenyl)propane]. (Caldwell et al., 1971). In the present paper we show that in the rat and guinea pig, (±)-methamphetamine is extensively Materials and Methods metabolized and that there are marked differences between these two species in the way in which they Compounds metabolize the drug. In the rat the main metabolic reaction is aromatic hydroxylation, as was found to (±)-Amphetamine sulphate, m.p. 302°C (decomp.), be the case with amphetamine (Dring et al., 1970), (±)-4-hydroxyamphetamine hydrobromide (Pared- whereas in the guinea pig the main reaction is de- rine), m.p. 190-1920C, and (±)-norephedrine hydro- methylation followed by side-chain degradation. In chloride, m.p. 190-1940C, were gifts from Smith, man, much more of the drug is excreted unchanged Kline and French Laboratories, Philadelphia, Pa., than in the rat and guinea pig, but both aromatic U.S.A. (±)-4-Hydroxymethamphetamine sulphate, Vol. 129 12 J. CALDWELL, L. G. DRING AND R. T. WILLIAMS m.p. 296°C (decomp.), was the gift from Knoll A.G., hydrochloride in water was administered to rats by Ludwigshafen-am-Rhein, West Germany; (±)-meth- stomach tube or intraperitoneal injection and to amphetamine hydrochloride, m.p. 132-134°C, was guinea pigs by intraperitoneal injection. All doses are purchased from K & K Laboratories, Plainview, expressed as methamphetamine hydrochloride, which N.Y., U.S.A.; (±)-4-hydroxyephedrine hydro- contains 80% of methamphetamine. The animals chloride, m.p. 188-189°C, was from R. N. Emanuel, were kept singly in suitable metabolism cages and Wembley, Middx., U.K. (±)-4-Hydroxynorephedrine urine and faeces were collected daily. was a sample prepared by Dring et al. (1970). The other known compounds used were purchased and Radiochemical techniques suitably purified. Synthesis of [14C]methamphetamine [(±)-2-methyl- The 14C in urine and faeces was determined as amino-l-phenyl[1-_4Cjpropane]. [carboxy-14C]Ben- described by Bridges et al. (1967) with the Packard zoic acid (m.p. 121°C; 3.6,uCi/mg) was prepared Tri-Carb liquid-scintillation spectrometers (models from Ba14CO3 (The Radiochemical Centre, Amer- 3214, 3320 and 3375). Scans of paper and thin-layer sham, Bucks., U.K.) as described by Dauben et al. chromatograms were made with a Packard radio- (1947). This was converted into [carbonyl-'4C]benzoyl chromatogram scanner (model 7200), and identifica- chloride as described by Calvin et al. (1949). The tions of 14C peaks were made by comparison with chloride (2.08g) was added drop by drop to NH3 authentic compounds (see Table 1). The 14C was (sp.gr. 0.88; 10ml) cooled in ice. The [carbonyl-'4C]- measured from the chromatogram scans and also by benzamide which separated was recrystallized from cutting paper chromatograms into 1 cm segments and water (yield 1.42g; m.p. 129°C). The amide was inti- counting the radioactivity of these with scintillator mately mixed with phosphorus pentoxide (3g) and fluid in the scintillation counter. With t.l.c. plates, heated under reduced pressure, whereby benzo['4C]- 0.5cm-wide sections of the absorbent were scraped nitrile (0.88g) distilled at 162°C/20mmHg and was into scintillation vials, scintillator fluid was added and converted into [carbonyl-14C]benzaldehyde (0.77g) the radioactivity was then counted in the spectro- by the method of Stephen (1925). The aldehyde in meter. benzene (lOml) was treated with nitroethane (4ml) as Guinea-pig faeces were homogenized with 2vol. of described by Blackburn & Burghard (1965) to give water in a Waring Blendor. The homogenate (20ml) 2-nitro-1-phenyl[1-4C]prop-l-ene (0.89g) as an oil, was made alkaline with 10M-NaOH and an equal which was converted without further purification volume of H202 solution (100vol) added with a few into I-phenyl[1-14C]propan-2-one (0.34g) by the drops of 2-ethylhexanol to control foaming. The general method of Pearl & Beyer (1951). mixture was kept at room temperature for 3 days The phenylpropan-2-one in ethanol (30ml) and an until decolorized, and was then assayed for 14C by ethanolic solution of methylamine (20ml; 33 %, w/w) scintillation counting for radioactivity. The un- was placed in the reaction vessel of a hydrogenator. bleached homogenate was centrifuged and the radio- Palladium-charcoal (10%; 2g) was added and the active compounds present in the supernatant were mixture hydrogenated at 310kN/m2 (451bf/in2) for separated by paper chromatography and t.l.c. and 24h. The catalyst was filtered, and excess of methyl- determined as described above. amine and ethanol were removed from the filtrate by Isotope-dilution procedures distillation in vacuo. The residual oil was then treated with a dry saturated ethereal solution (5ml) of HCI Amphetamine, 4-hydroxyamphetamine, benzyl gas. White crystals (0.42g) of (±)-2-methylamino- methyl ketone (1-phenylpropan-2-one), 4-hydroxy- 1-phenyl[1-14C]propane hydrochloride separated on norephedrine, hippuric acid, benzoic acid and 4- standing. It had m.p. 134-1350C and specific radio- hydroxybenzoic acid were assayed as described by activity 2.36,uCi/mg [Woodruff et al. (1940) give the Dring et al. (1970). About one-tenth of the urine m.p. of (±)methamphetamine hydrochloride as collected was used for the isotope dilutions described 135-136°C]. On chromatography in solvents A, B below. and C (see Table 1) it showed a single spot of RF 0.56, Methamphetamine. (+)-Methamphetamine hydro- 0.93 and 0.80 respectively. The radiochemical yield chloride (1 g) was dissolved in the urine and the solu- based on the Ba14CO3 used initially was 10%, and the tion adjusted to pH 14 with 1OM-NaOH. The solution radiochemical purity was >99 %, as measured by was extracted with diethyl ether (3 x 100ml), the ex- isotope dilution with the tosyl derivative (see below). tract was dried (with anhydrous Na2SO4) and evapor- ated to dryness and the residue was dissolved in Animals 2.5M-NaOH (20ml). Toluene-p-sulphonyl chloride (1.75g) in acetone (6ml) was added and the mixture Female Wistar albino rats weighing 220+15g and boiled under reflux for 20min. The solution was female Duncan-Hartley albino guinea pigs weighing poured on ice (lOg) and the solid toluenesulphonyl 250± 15g were used. The (±)-[L14Cjmethamphetamine derivative filtered and recrystallized from aq. 1972 SPECIES VARIATIONS IN METHAMPHETAMINE METABOLISM 13 ethanol to constant specific radioactivity. The This was filtered, washed with 1 M-NaOH and then N-methyl-N-toluene-p-sulphonylamphetamine had water and recrystallized to constant specific m.p. 82-84°C and appears to be a new compound. An radioactivity from aq. ethanol (50%, v/v). The NO- authentic sample was prepared by standard dibenzoyl derivative of 4-hydroxynorephedrine had methods and was obtained as a white crystalline m.p. 162-1630C (Dring et a!., 1970). powder, m.p. 82-84°C, from aq. ethanol (Found: Methods ofidentification C, 67.1; H, 7.05; N, 4.6; S, 10.3; C17H21N02S re- quires C, 67.1; H, 6.9; N, 4.6; S, 10.6%). Paper and thin-layer chromatography. The RF 4-Hydroxymethamphetamine [1-(4'-hydroxyphen- values of the relevant compounds on paper and thin- yl)-2-methylaminopropane]. The urine and an equal layer chromatograms are given in Table 1. volume of lOM-HCl were boiled under reflux for Gas-liquid chromatography. This technique was 2h. After cooling, the free base 4-hydroxymeth- used to analyse the excreta for methamphetamine and amphetamine (1 g) was added to the solution, which its possible basic metabolites, amphetamine, ephed- was adjusted to pH9.5-10 with lOM-NaOH. The rine and norephedrine. These compounds were resulting solution was saturated with solid NaCl and analysed as N-acetyl derivatives, which were less extracted with ether (3 xSOml). After drying (with volatile than the bases and showed better resolution anhydrous Na2SO4), the extract was evaporated to (see Xnggard, 1970). dryness and the residue dissolved in 2.5M-NaOH Urine (3 ml) was diluted to Sml withwater and made (20ml). Toluene-p-sulphonyl chloride (1.75g) in alkaline (pH 14) with IOM-NaOH and then saturated acetone (6ml) was added and the mixture boiled under with solid NaCl. The mixture was then extracted with reflux for 20min. The product was poured on ice ether (2 x lOml), the extract dried over anhydrous (lOg) and the oil which separated was extracted with Na2SO4 and evaporated to a small volume in a stream ethyl acetate (3 x 20ml). The extracts were washed in of N2. The residue was treated with pyridine (O.5 ml) turn with I M-HCI, 1 M-NaOH and water, and the and acetic anhydride (0.5ml) and kept at room tem- solvent was removed on a rotary evaporator. The perature for 24h. The reagents were removed on a residue was recrystallized to constant specific radio- rotary evaporator and the residue was dissolved in activity from aq. ethanol and had m.p. 108-1 10°C. ethyl acetate (1 ml). Portions (101L) of this solution The authentic NO-di(toluene-p-sulphonyl) derivative were injected into the g.l.c. column. For g.l.c., an of 4-hydroxymethamphetamine was prepared by F & M model 402 apparatus (Hewlett-Packard Inc., standard methods. It formed a white crystalline pow- Pasadena, Calif., U.S.A.) with flame-ionization der of the monohydrate, m.p. 108-110°C, from detector was used. The glass column [1.83m (6ft); aqueous ethanol (Found: C, 59.0; H, 5.7; N, 3.1; internal diam., 3mm] was packed with AW-DMCS- S, 12.9; C24H2705NS2,H20 requires C, 58.7; H, 5.9; treated Chromosorb G (100-120mesh) coated with N, 2.9; S, 13.0%). SE-30 (3 %, w/w). Theconditionsused were: injection- Norephedrine. The base hydrochloride (1 g) was port temperature, 240°C; N2, air and H2 pressures, dissolved in urine and the solution was adjusted to 275, 165 and 14OkN/m2 (40, 24 and 201bf/in2) pH 14 with IOM-NaOH, saturated with NaCl and respectively. The temperature-programming facility extracted with ether (3 x lOOml). After drying (with was used with an initial column temperature of 1 50°C anhydrous Na2SO4) the extract was evaporated to and after a delay of 8min the temperature was raised dryness and the residue dissolved in 1 M-NaOH by 5°C/min to an upper limit of 185°C, which was (lOml) and benzoylated with benzoyl chloride (5ml). maintained. The retention times were: N-acetyl- NO-Dibenzoylnorephedrine was filtered, washed amphetamine, 7.5; N-acetylmethamphetamine, 10.6; with 1 M-NaOH and then water and recrystallized to NO-diacetylnorephedrine, 14.1; and NO-diacetyl- constant specific radioactivity from aq. 50% (v/v) ephedrine, 15.75min. ethanol. It had m.p. 166°C (see Heilbron & Bunbury, G.l.c.-mass spectrometric identification of nor- 1953). ephedrine in guinea-pig urine. A Varian Aerograph 4-Hydroxynorephedrine. Because of the large vol- 1700 gas chromatograph in conjunction with a umes involved, the estimation of 4-hydroxynor- Varian MAT CH5 mass spectrometer was used. The ephedrine in human urine had to be modified from chromatographic column was SE-30 (3%, w/w) on that of Dring et al. (1970). 4-Hydroxynorephedrine Chromosorb W AW-DMCS in a glass column hydrochloride (1 g) was dissolved in the urine and [1.5m (5ft); internal diam. 2mm]. The column 0.2vol. of lOM-HCl added. The mixture was refluxed temperature was kept at 185°C for 2min after in- for 2h and evaporated to dryness under low pressure. jection of a sample, then programmed to rise at The residue was extracted with methanol (lOml) and 8°C/min to a maximum temperature of 250°C. The the solution filtered. The methanol was removed temperatures were: injector-port, 220°C; detector, and the residue treated with 2.5M-NaOH (15ml) and 250°C; the interface, 265°C; the Biemann-Watson benzoyl chloride (5ml). The mixture was shaken for type molecular separator, 240°C; the line-of-sight 10min and then kept until the derivative solidified. inlet line, 200°C; the source, 180°C. The electron- Vol. 129 14 J. CALDWELL, L. G. DRING AND R. T. WILLIAMS
Table 1. RF values and colour reactions ofmethamphetamine and related compounds The solvents used were: A, 3-methylbutan-1-ol-2-methylbutan-2-ol-water-formic acid (5:5:10:2, by vol.) (Alleva, 1963); B, butan-1-ol saturated with 1.5M-NH3-ammonium carbonate buffer (pH 9.5) (Fewster & Hall, 1951); C, methanol-chloroform (1:1, v/v) (Noirfailise, 1966); D, methanol-acetone (1:1, v/v) (Moerman, 1964). Solvents A and B were used for descending chromatography on Whatman no. 1 paper and solvents C and D for t.l.c. on plates (20cm x 20cm or 5cm x 20cm) spread with 0.25mm-thick alumina G (E. Merck A.-G., Darmstadt, Germany) and silica gel G respectively, which were heated at 120°C for 1 h before use. The solvents were allowed to run from the origin to 35cm on paper and 12cm on thin layers. Colour reactions were those described by Dring et al. (1970). RF Paper Thin layyer Colour with - diazotized Compound Solvent A B C D nitroaniline Methamphetamine (2-methylamino- 0.57 0.92 0.81 0.24 None 1-phenylpropane) (I) Amphetamine (2-amino-1-phenylpropane) 0.51 0.88 0.45 0.59 Pale pink (IV) 4-Hydroxyamphetamine [2-amino-1-(4'- 0.32 0.79 0.39 Purple hydroxyphenyl)propane] (III) 4-Hydroxymethamphetamine [1-(4'- 0.35 0.82 0.64 Purple hydroxyphenyl)-2-methylamino- propane] (II) Ephedrine (2-methylamino-1-phenyl- 0.52 0.86 0.70 0.30 Pale pink propan-l-ol) 4-Hydroxyephedrine [1-(4'-hydroxy- 0.17 0.80 0.15 Purple phenyl)-2-methylaminopropan-l-ol] Norephedrine (2-amino-1-phenyl- 0.39 0.82 0.18 0.67 Pale pink propan-1-ol) (VII) 4-Hydroxynorephedrine [2-amino-i- 0.17 0.64 0.07 - Purple (4'-hydroxyphenyl)propan-1-ol] (VI) Benzoic acid (VIII) 0.93 0.33 0.00 None Hippuric acid 0.82 0.20 None Benzoyl glucuronide 0.60 0.09 None Benzyl methyl ketoxime 0.86 0.88 0.88 - None beam energy was 70eV, and the helium carrier-gas Materials from urine extracts. Guinea pigs flow rate was 12ml/min at 275kN/m2 (401bf/in2). (250± 15g) were injected intraperitoneally with [I4C]- Samples of 1 ,ul of standards (1 mg/ml in ethyl methamphetamine hydrochloride (45mg/kg; 1.7,uCi). acetate) and 1 ,l of acetylated urine extracts in ethyl A sample (lOml) of the first 24h urine was saturated acetate (see below) were injected on to the column. with NaCl and adjusted to pH 12 with 2.5M-NaOH. (±)-N-Acetylamphetamine, m.p. 66-670C, was pre- It was then extracted with ether (3 x lOml), and the pared as described by Hey (1930). extract was treated with pyridine (0.5ml) and acetic (±)-Methamphetamine or (±)-norephedrine as anhydride (0.5ml) and an ethyl acetate solution of hydrochlorides (5mg) in water (lOml) were treated the product prepared as described above. A blank with 2.5M-NaOH (2ml). The solution was saturated solution was prepared in the same way from the urine with NaCl and extracted with ether (3 x lOml). of a control animal. Pyridine (0.5ml) and acetic anhydride (0.5ml) were Acetylated amphetamine, methamphetamine and added to the ether and the mixture was kept at room norephedrine each gave a single peak of retention temperature overnight. The solution was then evapor- time 2.0, 2.6 and 4.25min respectively, as recorded ated under reduced pressure and the oily acetyl by the total ion-current monitor of the mass spectro- derivative was dissolved in ethyl acetate (5 ml). G.l.c. meter. The mass spectrum of each was recorded (see of this solution showed that the acetyl derivatives of Table 2), and it was clear that amphetamine and methamphetamine and norephedrine were essentially methamphetamine formed N-monoacetyl deriva- pure. tives, and norephedrine the NO-diacetyl derivative. 1972 SPECIES VARIATIONS IN METHAMPHETAM1NE METABOLISM 15
Table 2. Results from mass spectra of the various compounds G.l.c.-mass spectrometry was carried out on acetyl derivatives of amphetamine, methamphetamine and nor- ephedrine and on extracts of guinea-pig urine after dosing with ['4C]methamphetamine (see the text). Values of mle are given, with relative intensities in parentheses. Only characteristic major peaks are given, together with the parent ion. Retention times are given in brackets after the name of the compound or fraction of urine examined. N.D., Not detected. N-Acetylamphetamine N-Acetylmethamphetamine NO-Diacetylnorephedrine [2.Omin] [2.6min] [4.25min] 177 (2.5) 191 (2.2) 235 (0.3) 118 (27) 100 (72) 175 (9) 86 (42) 58 (17) 86 (23) 44 (100) 43 (52) 69 (100) 43 (88) 44 (36) 43 (39) Peak 1 [2.Omin] Peak 2 [4.25min] 177 (2.8) 235 (0.8) 118 (29) 175 (11) 86 (45) N.D. 86 (59) 44 (100) 69 (100) 43 (84) 44 (79) 43 (37) Probable nature of peaks in the mass spectrum N-Acetylamphetamine mle ... 177 (parent ion) 118 86 44 Structure ... NH-CO-CH3 NH-CO-CH3 NH2 11 11 C6Hs-CH2-CH-CH3 C6H5-CH=CH-CH3 CHt CH3 CHt-CH3 N-Acetylmethamphetamine m/e ... 191 (parent ion) 100 58 + + Structure ... CH3-N-CO-CH3 CH3-N-CO-CH3 CH3-NH I 11 11 C6Hs-CH2-CH-CH3 CH-CH3 CH-CH3 NO-Diacetylnorephedrine mle ... 235 (parent ion) 175 86 69 + Structure ... NH-CO-CH3 NH-CO-CH3 NH-CO--CH3 N=-CH 11 + >0 C6Hj-CH-CH-CH3 C6Hs-C;H=-CCH3 CH-CH3 HC==CH O-CO-CH3
The mass spectrum of N-acetylamphetamine agreed their mass spectra. The urine extract did not show a well with that found by ALnggArd (1970). The control peak corresponding to N-acetylmethamphetamine, guinea-pig urine extract produced no interfering thus suggesting that the unchanged drug was not peaks in the region of the peaks of the three amines. present in the urine in significant amounts. The urine extract from the dosed guinea pigs gave two G.I.c. ofbenzyl methyl ketoxime. The F & M model peaks of retention time 2.0 (peak 1; see Table 2) and 402 gas chromatograph was used. The glass column 4.25min (peak 2; see Table 2), which correspond to [1.2m (4ft)] was packed with W-98 (3.8%, w/w) on the acetyl derivatives of amphetamine and nor- Diatoport S (100-120 mesh). The conditions were: ephedrine respectively, and this was confirmed by injection-port temperature, 120°C; column tempera- Vol. 129 16 J. CALDWELL, L. G. DRING AND R. T. WILLIAMS ture 90°C; N2 pressure, 275kN/m2 (40lbf/in2); air peak of RF 0.43 gave a pale-pink colour with diazo- pressure, 165kN/m2 (241bf/in2) ;H2pressure, l4OkN/- tized nitroaniline and corresponded to amphetamine, m2 (201bf/in). With this system retention times whose identity was confirmed by g.l.c. The material were 2.0min for benzyl methyl ketone and 8.0min at RF 0.8 gave no colour with diazotized nitroaniline for benzyl methyl ketoxime. The ketoxime was and corresponded to methamphetamine, also con- prepared as described by Hey (1930). firmed by g.l.c. The material of RF 0.0 was an acidic Urine (lOml) before and after hydrolysis substance whose identity is at present unknown. with ,B-glucuronidase (Ketodase; Warner-Chilcott, The 14C-labelled peak of RF 0.89 in solvent A, Eastleigh, Hants., U.K.) (see Dring et al., 1970) was which is suspected to be hippuric acid and benzoic adjusted to pH7.4 with a few drops of 1 M-NaOH or acid, was too small to measure accurately. Labelled 1 M-HCI as appropriate and extracted with ethyl benzoic acid in the urine was therefore measured by acetate (3 x 15 ml). The extracts were evaporated to isotope dilution directly on the original urine as dryness at low temperature on a rotary evaporator. described by Dring et al. (1970). Isotope dilution was The residue was dissolved in ethyl acetate (0.2- also used to analyse for 4-hydroxynorephedrine, 0.4ml) and a 3,ul portion was injected on to the 4-hydroxymethamphetamine and 4-hydroxybenzoic column. Control extracts were prepared from normal acid. urine and no interfering material was found when they Guinea-pig urine. The urine was chromatographed were chromatographed. This method would detect in solvent A as described above and gave two broad the ketoxime, if it occurred, down to 1 % of the dose peaks whose centres had RF 0.38 and 0.79. of methamphetamine. The peak of RF 0.38 gave a pale-pink colour with diazotizedp-nitroaniline but was unaffected by treat- ment with 3-glucuronidase. The peak was eluted with Identification ofmetabolites water and re-chromatographed on thin layers in solvent C, and was thus resolved into four peaks of Rat urine. The urine (1 ml) was chromatographed RF 0.0, 0.20, 0.47 and 0.79. The first was an acidic on paper in solvent A (Table 1) and the chromatogram unknown compound -and the succeeding peaks in scanned for 14C. Three broad peaks whose centres order corresponded to norephedrine, amphetamine had RF 0.0, 0.3 and 0.49 and a minor peak of RF 0.89 and methamphetamine. were found. Colour tests showed that the three broad The urine was chromatographed on paper in sol- peaks contained respectively glucuronides, phenolic vent B (Table 1). It gave four peaks, three sharp ones substances and amines. The minor peak corre- of RF 0.10, 0.18 and 0.28 corresponding to benzoyl sponded in RF value to hippuric acid and benzoic acid glucuronide (positive naphtharesorcinol test), hip- (see Table 1). Hydrolysis of the urine with HCI or puric acid (p-dimethylaminobenzaldehyde test) and /3-glucuronidase caused the peak of RFO.O to dis- benzoic acid (Bromophenol Blue test), and a broad appear and gave an equivalent increase in the peak of one of RF 0.82. The peak of RF 0.10 disappeared on RF 0.3. The peaks at RF 0.49 and 0.89 were unaffected treatment with /-glucuronidase and that at RF 0.28 by acid or ,B-glucuronidase. correspondingly increased. On hydrolysis with HCI, The material of RF 0.0 described above was eluted the peak at RF 0.10 disappeared and that at RF 0.18 with water and after hydrolysis with ,B-glucuronidase decreased, with a corresponding increase in that at was subjected to t.l.c. in solvent C. Three radioactive RF 0.28. The broad peak of RF 0.82 in solvent B was peaks of RF 0.04, 0.40 and 0.65 were found, all of eluted with water and re-chromatographed on thin which gave an intense purple colour for phenols with layers in solvent C. It gave the same pattern of four diazotizedp-nitroaniline. These RF values correspond peaks as did the original material ofRF 0.38 in solvent to 4-hydroxynorephedrine, 4-hydroxyamphetamine A re-chromatographed in solvent C, and therefore and 4-hydroxymethamphetamine. These compounds consisted of the unknown, norephedrine, amphet- were determined by liquid-scintillation counting for amine and methamphetamine. The presence ofbenzyl radioactivity after scraping 0.5 cm sections of the methyl ketone and norephedrine was shown by iso- absorbent from the t.l.c. plate as described above. tope dilution, and that ofmethamphetamine, amphet- The peak of RF 0.3 in solvent A was eluted with amine and norephedrine by g.l.c. The supernatant water and re-chromatographed as described above in obtained by centrifuging aqueous homogenates of solvent C. Again three peaks of RF 0.04, 0.40 and 0.65 guinea-pig faeces was examined for metabolites in were obtained, corresponding to the above three the same way as was urine. phenols released by P-glucuronidase from the peak At a dose of 10mg/kg, chromatography of the of RF 0.0 in solvent A. These were determined as urine in solvent A gave two "C-labelled peaks, described above. RF 0.38 and 0.79, neither of which was affected by The peak of RF 0.49 in solvent A was eluted with ,B-glucuronidase. The peak of RF 0.38 was decreased water and re-chromatographed in solvent C to yield on acid hydrolysis, indicating a volatile metabolite(s). three radioactive peaks of RF 0.0, 0.43 and 0.80. The In solvent B, four "C-labelled peaks were seen, with 1972 SPECIES VARIATIONS IN METHAMPHETAMINE METABOLISM 17
Rp 0.1 (benzoyl glucuronide), 0.28 (hippuric acid), in solvent D. These correspond to methamphetamine, 0.38 (benzoic acid) and 0.82. The peak at RF 0.82 amphetamine and an unknown amine respectively. contained insufficient radioactivity for elution, so The extracts of acidified urine contained 5-8 % of the compounds suspected of being under this peak dose and showed one 14C-labelled peak of RF 0.79 on (methamphetamine, amphetamine and norephedrine) paper in solvent A (Table 1). This corresponded to were assayed by isotope dilution directly on the urine, hippuric acid. In solvent B, however, two peaks were as was also benzyl methyl ketone, before and after found, of RF 0.26 (major) and 0.86 (very minor), acid hydrolysis. The amine metabolites were also which corresponded to hippuric acid and an unknown sought by g.l.c., as was benzyl methyl ketoxime, be- acid respectively. Hippuric acid was confirmed by fore and after ,B-glucuronidase hydrolysis. isotope dilution. Human urine. The urine (50ml) was adjusted to Other metabolites, which were not extracted by the pH I with 1OM-HCl or to pH 14 with 1OM-NaOH, above procedure, were assayed by isotope dilution then was saturated with solid NaCl and extracted directly on the urine or after acid hydrolysis where with ether (3 x 75 ml). The extracts were dried over necessary. anhydrous Na2SO4 and then evaporated under re- duced pressure. The residue was dissolved in ethanol Results (2ml) and the solution used for radiochromato- graphy. The excretion of 14C by rats, guinea pigs and The extracts ofalkaline urine contained 21-30 % of humans after the administration of [14C]meth- the 14C dose and when chromatographed on thin amphetamine hydrochloride is shown in Table 3. In layers in solvent C (Table 1) gave three radioactive the rat some 84% ofthe dose of45mg/kg is accounted peaks, a large one at RF 0.83 and two minor ones at for in 4 days after oral administration, 82% in the RF 0.47 and 0.99. Three similar peaks of RF 0.22 urine and 2% in the faeces. On intraperitoneal injec- (major), 0.60 (minor) and 0.93 (minor) were obtained tion, the radioactivity was more rapidly excreted,
Table 3. Excretion of 14C in man, rat and guinea pig after [14C]methamphetamine administration (+)-['4C]Methamphetamine hydrochloride was administered dissolved in water. The dose of radioactivity was 3,uCi to man and 1.5 or 1.7,uCi to rats and guinea pigs. The values are percentages of the dose. Where three animals were used average values to the nearest two numbers are quoted, with ranges in parentheses. Where two animals were used, the individual results are quoted. The doses and routes of administration are indicated in appropriate places in the table. Urine and faeces were collected daily and their 14C contents determined by scin- tillation counting for radioactivity as described in the text. The two human subjects are referred to as C and D and only their urine was examined.
Dosage ... Oral (45mg/kg) Intraperitoneal (45mg/kg) Time Species (days) Urine Faeces Total Urine Faeces Total Rat 1 47 (35-58) 0.6 (0.0-1.8) 48 76 (69, 81) 1.9 (2.0, 1.9) 78 2 74(68-79) 1.6(0.7-3.0) 76 79 (74, 83) 2.2(2.3, 2.1) 81 3 81 (77-86) 1.9 (1.3-3.3) 83 81(77, 84) 2.9 (3.1, 2.8) 84 4 82 (78-87) 2.0(1.4-3.5) 84 Dosage ... Intraperitoneal (45mg/kg) Intraperitoneal (10mg/kg) Guinea pig 1 65(39-81) 18.1(5.1-28) 83 81 (76-84) 2.7 (2.1-3.5) 84 2 68(43-86) 18.4(5.5-29) 87 83 (77-87) 2.7 (2.1-3.5) 86 3 69(43-86) 18.4(5.5-29) 87 4 69(44-87) 18.4(5.5-29) 88 Dosage ... Oral (20mg/person) C D Man 1 69 55 2 90 78 3 94 86 - 4 96 88 Vol. 129 18 J. CALDWELL, L. G. DRING AND R. T. WILLIAMS since 70-80 % of the dose was excreted in the urine in with glucuronic acid, since the free phenols are re- 24h, whereas after oral administration only about leased by ,B-glucuronidase. The benzoic acid output 47 % was excreted in this time and 2 days were re- is small (3.5 %) and norephedrine was not detected. quired for 70-80 % to be excreted in the urine. In the Most of the 14C excreted is accounted for by these guinea pig two dosages were used, both injected metabolites, except for about 4% of the dose, which intraperitoneally. At the higher dose of 45mg/kg, appears to be an unknown acidic substance. 88% of the 14C was excreted in 4 days, 69% in the urine and 18% in the faeces, but at the lower dosage Methamphetamine metabolism in the guinea pig of 10mg/kg, the urinary excretion was greater, at (Table 5) 83 % in 2 days, only about 3 % appearing in the faeces. The drug was injected intraperitoneally into guinea In the two human subjects, whose informed consent pigs at two dosages, namely 10 and 45mg/kg. At the was obtained, about 90% of the administered 14C higher dosage there was an appreciable faecal excre- was found in the urine in 4 days, most of it being tion, amounting to nearly 18 % ofthe dose. The quan- excreted in the first 2 days. tity ofunchanged drug excreted in the urine was quite small, amounting to only 2% at the higher dosage metabolism in the rat and 0.4% at the lower. Much more amphetamine Methamphetamine was excreted than unchanged drug. In fact the The metabolites of methamphetamine in urine amphetamine excretion was greater in the faeces after oral administration to the rat are shown in (10 %) than in the urine (3 %) on the higher dosage. Table 4. About one-tenth of the dose is excreted At the dosage of 45mg/kg, the amines were found in unchanged and some 3 % appears as amphetamine. the free state in guinea-pig faeces. Abdel Aziz et al. The main metabolites are the phenols, 4-hydroxy- (1971) have suggested that for a compound to be methamphetamine (31 %), 4-hydroxynorephedrine excreted in the bile in significant amounts in the (16%) and 4-hydroxyamphetamine (6%). These guinea pig it should have a molecular weight greater phenols are excreted mainly as conjugates, probably than 400. The metabolites ofmethamphetamine in the
Table 4. Metabolites of [14C]methamphetamine in the urine of the rat Three female Wistar albino rats (20010g) were given oral doses of 45mg of (±)-[14C]methamphetamine hydrochloride/kg body wt. in water. The urine and faeces were collected daily for 2 days. The urines were analysed as described in the text. The faeces contained only about 2 % of the dose (see Table 2) and were not ex- amined further. The results are expressed as averages for the three animals with the ranges in parentheses. N.D., Not detected. % of dose Metabolite Day 1 Day 2 Total Methamphetamine (I) 10.5 (5.3-17.9) 0.5 (0.0-0.8) 11.0 Amphetamine (IV) 2.0(0.7-2.6) 1.1 (0.8-1.4) 3.1 4-Hydroxymethamphetamine (II) Free 2.9 (0.9-6.4) 0.0 Conjugated 12.7 (3.8-23.7) 15.8 (10.2-22.4) Total 15.6 (10.2-24.6) 15.8 (10.2-22.4) 31.4 4-Hydroxyamphetamine (III) Free 1.0 (0.4-1.9) 0.0 Conjugated 2.8 (1.1-3.7) 2.1 (1.8-2.3) Total 3.8 (3.0-4.2) 2.1 (1.8-2.3) 5.9 Norephedrine (VII) N.D. N.D. 4-Hydroxynorephedrine (VI) Free 1.7 (1.3-2.3) 0.0 Conjugated 7.7 (2.0-14.5) 6.4 (3.0-10.1) Total 9.4(4.3-16.1) 6.4 (3.0-10.1) 15.8 Benzoic acid (free +conjugated) (VIII) 2.4 (0.7-4.1) 1.1 (0.7-1.8) 3.5 Unknown 3.9 (2.9-5.0) 0.0 3.9 Total of above metabolites 47.4 (35.3-59.3) 26.9 (18.2-33.0) 74.3 Total '4C in urine examined 46.8 (35.2-58.2) 26.9 (18.2-33.0) 73.7 1972 SPECIES VARIATIONS lN METHAMPHETAMINE METABOLISM 19
Table 5. Metabolites of [14C]methamphetamine in the urine andfaeces of the guinea pig Young female Duncan-Hartley albino guinea pigs (200±10g) were used. (±)-[14C]Methamphetamine hydro- chloride in water was administered by intraperitoneal injection. Two dosages were used, 45 and 10mg/kg contain- ing 1.7 and 0.7,uCi of 14C respectively. After the larger dose both urine and faeces were examined for metabolites, but after the smaller dose only the urine was examined. The results for urine are expressed as averages for three animals with the ranges in parentheses. In the case of faeces only two animals were examined. N.D., Not detected. % of dose Day 1 after dose of 45mg/kg Day 1 after dose of 10mg/kg Metabolite Urine Faeces Total Urine Methamphetamine (I) 2.1 (1.5-3.3) 1.3 (2.0, 0.6) 3.4 0.4(0.0-1.1) Amphetamine (IV) 3.1 (0.7-6.7) 9.8 (12.0, 7.6) 12.9 3.6(1.6-4.9) Norephedrine (VII) 13.3 (5.5-21.1) 5.6 (5.8, 5.3) 18.9 1.3 (0.0-2.0) Benzoic acid (free) (VIII) 4.1 (3.1-5.6) 0.0 21.6 (12.4-26.1) Benzoylglucuronide 10.3 (7.2-13.9) 0.0 23.4(10.1-34.0) Hippuric acid 16.1 (12.2-20.3) 0.0 18.6(15.4-22.1) Total benzoic acid (VIII) 30.5 (25.0-38.5) 0.0 30.5 63.4 Benzyl methyl ketone (after 1.9 (0.7-2.3) 1.9 10.9 (0.0-18.6) hydrolysis) (V) Benzyl methyl ketoxime Before hydrolysis - N.D. After fl-glucuronidase N.D. Unknown 12.8 (6.3-22.0) 7.9 (8.5, 7.4) 20.7 - Total '4C of above metabolites 63.7 (39.0-81.6) 24.6 (28.3, 20.9) 88.3 79.8 (73.7-83.3) Total 14C in excreta examined 64.7 (39.1-81.3) 24.6 (28.3, 20.9) 89.3 81.2 (76.5-83.8)
guinea pig have molecular weights ranging from 135 but this was not detected in guinea-pig urine after (amphetamine) to 298 (benzoyl glucuronide) and it intraperitoneal injection of the drug at a dosage of appears unlikely, therefore, that the metabolites in 10mg/kg. faeces arose from biliary excretion. Probably the At the higher dosage of 45mg/kg, the guinea pig amphetamine reaches the faeces by passage through excreted a substance of RF 0.83 in solvent B. This the gut wall. The demethylation ofmethamphetamine substance occurs in the urine and faeces to an by the gut flora of the guinea pig seems to occur appreciable extent and accounts for just over 20% (J. Caldwell & G. M. Hawksworth, unpublished of the 14C dose when the dosage of the drug was work). The main metabolite of methamphetamine in 45mg/kg, but was not detectable at the dosage of the guinea pig is benzoic acid, which occurs largely 10mg/kg (Table 5). This unknown substance was conjugated with glycine and glucuronic acid. Some extractable with ether from urine adjusted to pH1 30% of the dose is excreted as benzoic acid at the with 1OM-HCI. Such ether extracts gave four radio- higher dosage, but more than double this (63 %) at active peaks of RF 0.1 (benzoyl glucuronide), 0.2 the lower dosage. Another major metabolite, amount- (hippuric acid), 0.36 (benzoic acid) and 0.83 (un- ing to nearly 20% of the dose at the higher dosage, known). The unknown thus appeared to be a weak but only about 1 % at the lower dosage, is nor- acid. It was eluted with water and the solution con- ephedrine. centrated and then subjected to t.l.c. in solvent C. Benzyl methyl ketone was also found in the urine In this solvent it showed RF 0.0, but on repeated t.l.c. after hydrolysis and this was a significant fraction of it gave two radioactive peaks at RF 0.0 and 0.9, the the dose at the lower dosage, i.e. nearly 11 %, but only latter peak increasing at the expense of the former about 2 % at the higher dosage. It would appear that peak. The unknown thus appeared to be unstable in the guinea pig the production of norephedrine, and decomposed slowly to a more stable product. benzyl methyl ketone precursor and an unknown Preliminary studies suggest that the compound of metabolite (see below) is dose-dependent. Table 5 RF 0.9 in solvent C might be a non-phenolic dihyd- also refers to benzyl methyl ketoxime, which has been roxyamphetamine, possibly a hydroxynorephedrine, suggested as a possible metabolite of amphetamine, and that it also occurs in rat urine. Vol. 129 20 J. CALDWELL, L. G. DRING AND R. T. WILLIAMS Methamphetamine metabolism in man (Table 6) man than in the guinea pig or rat, since the output of In the two male subjects used, the main metabolite the unchanged drug in man (who was also given a in urine is the unchanged drug, amounting to some very much smaller dose) is much greater than in the 18 and 27% of the dose. But at least seven other rat and guinea pig. In fact, on the basis of the 24h metabolites were found and thesewere, in quantitative output of '4C, the unchanged drug accounts for 30- order, 4-hydroxymethamphetamine (15 %), hippuric 40% in man, 15% in the rat and 4 and 0.5% on the acid (5 %), amphetamine (2-3 %) norephedrine (2 %), higher and lower dosages respectively in the guinea 4-hydroxynorephedrine (1-2%), 4-hydroxyamphet- pig. amine (1 %) and a precursor of benzyl methyl ketone The main metabolic reaction ofmethamphetamine (about 1 % of the dose in 24h). In addition to these in the rat is aromatic hydroxylation (about 75 % of small quantities of an unknown amine (about 1-2 %) the '4C excreted), which results in the appearance in and acid (1-3 %) were also detected. the urine of the three phenols, 4-hydroxymeth- amphetamine (II), 4-hydroxyamphetamine (III) and 4-hydroxynorephedrine (VI). In the guinea pig, how- Discussion ever, aromatic hydroxylation appears to be com- Methamphetamine(I) could beexpected to undergo pletely absent, but in man it accounts for about in the body at least three initial metabolic reactions, one-third of the 14C excreted in 24h. namely N-demethylation, aromatic hydroxylation The extent of demethylation can be assessed from and aliphatic hydroxylation at the methylene group the output of amphetamine (IV) and norephedrine next to the benzene ring. Table 7 and Scheme 1 (VII) and their hydroxy derivatives and of the pro- contain a summary ofthemetabolites ofmethamphet- ducts of degradation of the side chain, namely ben- amine in the urine of man, the guinea pig and the rat zoic acid (VIII) and benzyl methyl ketone (V). In the and an estimate of the extent of the above three reac- guinea pig, demethylation is very extensive and tions in these species. It is clear from Table 7 that there accounts for nearly all the 'IC excreted. In the rat it are wide species variations in the extent of these accounts for about 40 % and in man about 30% ofthe reactions. It would appear also that the primary total metabolites. A consequence of the demethyla- metabolic reactions are aromatic hydroxylation and tion ofmethamphetamine could be the degradation of N-demethylation and that 'p-hydroxylation' (, refer- the side chain by deamination and subsequent oxida- ring to the propane side chain) may only occur as a tion of the carbon side chain to benzoic acid. De- secondary reaction, since only norephedrine (2- methylation could thus give rise to primary amines amino-1-phenylpropan-1-ol; VII), but not ephedrine and deaminized products. Deamination of the pri- (2-methylamino-1-phenylpropan-1-ol), derivatives mary amine metabolites is extensive only in the guinea were found. pig and at a dosage of45mg/kg it accounts for nearly Methamphetamine is less readily metabolized in 50% of the 24h excretion, whereas at 10mg/kg it
Table 6. Metabolites of [14C]methamphetamine in the urine ofman Two young male subjects, C and D, took in water by mouth 20mg of (±)-['4C]methamphetamine hydrochloride containing 3,uCi. The urine excreted in the first 24h after dosing was analysed (see the text). Both subjects weighed about 70kg and the urine excreted had pH6.7. % of dose Metabolite Subject C Subject D Methamphetamine (I) 27.2 18.1 Amphetamine (IV) 2.2 3.2 Norephedrine (VII) 1.7 2.4 4-Hydroxymethamphetamine (total) (II) 14.1 15.8 4-Hydroxyamphetamine (total) (III) 1.1 1.0 4-Hydroxynorephedrine (total) (VI) 1.2 2.2 Benzyl methyl ketone (V) Before hydrolysis 0.0 0.0 After acid hydrolysis 2.1 0.8 Hippuric acid 3.8 5.6 Unknown amine 1.8 1.0 Unknown acid 0.7 3.1 Total of above metabolites 55.9 53.2 Total 14C in urine examined 68.8 54.9 1972 SPECIES VARIATIONS IN METHAMPHETAMINE METABOLISM 21
Table 7. Summary ofthe metabolites and reactions of methamphetamine % of dose Species ... Man Guinea pig Rat Dose (mg/kg) 0.29 10 45 45 Route of administration Oral Intraperitoneal Oral Metabolite in urine Methamphetamine (I) 23 0.5 3 11 Amphetamine (IV) 3 4 13 3 Norephedrine (VII) 2 1 19 0 4-Hydroxymethamphetamine (II) 15 0 0 31 4-Hydroxyamphetamine (III) 1 0 0 6 4-Hydroxynorephedrine (VI) 2 0 0 16 Benzoic acid (VIII) 5 63 31 4 Benzyl methyl ketone precursor 1 11 2 Total 52 79.5 68 71 Reactions Unchanged 23 0.5 3 11 Aromatic hydroxylation 18 0 0 53 Demethylation 14 79 64 28 f-Hydroxylation 4 1 19 16 Deamination 6 74 33 4
CR3 CH3 - ~~~~~~~~~~~~~~I HO \C 2-CH-NH-CH3 4 \ / CH2-CH-NH-CI{3 (I) (I) 4, I 1~~~~~~~~~~~~~ CR3 HO CH2-C -NH2 \ / CH2-CH-NH2 -+ \ / CH2-CO-CH3 (III) (Mr) 4, CH3 1~~~~~~~~~~~~~~~~ CR3 -O