Br. J. clin. Pharmac. (1977), 4, 193-200
THE METABOLISM, DISTRIBUTION AND ELIMINATION OF CHLORPHENTERMINE IN MAN A.H. BECKETT & P.M. BELANGER* Department of Pharmacy, Chelsea College, University of London, London SW3 6LX
I A gas-liquid chromatography procedure for the determination of chlorphentermine (I), N-hydroxychlorphentermine (II) and a,o-dimethyl-a-nitro-,-44-chlorophenyl)ethane (IV) in urine has been developed. Also methods are reported to determine conjugated II and the total N-oxidized metabolites of I, i.e. II, conjugated II, a,a-dimethyl-a-nitroso-3-(4chlorophenyl)ethane (III) and IV in urine. 2 The synthesis of ,ct-dimethyl-a-nitroso-f-(4-chlorophenyl)ethane (III) and its properties are reported. 3 The kinetics of urinary excretion of I and its metabolic products after the oral administration of I to a human subject on separate occasions have been studied. Under normal conditions of urinary pH, metabolism by N-oxidation was the main elimination route of I; acidifying the urine increased the urinary excretion of unchanged I at the expense of the N-oxidized products. 4 The importance of the N-oxidation metabolic route in the distribution of chlorphentermine (I) in man is discussed. Introduction In man, the half-life of the anorectic drug chlorphentermine (I), Figure 1, is more than double that of the non-chlorinated analogue phentermine (Beckett & Brookes, 1971); an average elimination half-life of 41 h has been reported after monitoring whole blood concentration of I in man (Jun & Triggs, 1970). Chlorphentermine (I), but not phentermine, accumulated in the brain, lung, adrenal and spleen tissues when administered to rat and mice (Dubnick, Leeson, Leverett, Morgan & Philipps, 1963; Dubnick, Towne, Hartignan & Philipps, 1968; Liilman, Rossen & Seiler, 1973; Seiler & Wassermann, 1974). The accumulation process has been related to the interactions of I with the tissue phospholipids (Lullman, LUllman-Rauch & Wassermann, 1973; Schmien, Seiler & Wassermann, 1974; Seiler & Wassermann, 1974) and in biochemical and histopathological effects of I after repeated administration (Lillmann, LuallmanRauch & Wassermann, 1973; Lullmann et al., 1973; Karabelnik, Zbinder & Baumgartner, 1974; Lulllmann-Rauch, 1975). Pulmonary hypertension has been observed in patients after chronic treatment with I (Lillman et al., 1973 and references cited therein). Chlorphentermine (I) is N-oxidized metabolically to N-hydroxychlorphentermine (II), ci,a-di* Present address: Ecole de Pharmacie, Universite Laval, Qu6bec GIK 7P4, Canada.
methyl-a-nitroso--44-chlorophenyl)ethane (III) and a,o-dimethyl-a-nitro-(3-(4-chlorophenyl)ethane (IV) in vitro in animals and in vivo in man (Beckett & Belanger, 1974a); conjugated II was also excreted in the urine of a human subject given I. Since completing the present work, Caldwell, K6ster, Smith & Williams (1975) reported the N-oxidation of labelled [C14] -chlorphentermine (I) using radiochemical techniques for the assay of unchanged I, N-hydroxychlorphentermine (II), conjugated II and the nitro analogue (IV) in the bulked urine samples of three days of two human subjects under normal conditions of urinary pH after oral dosage. The total recovery of I and the above metabolic products in the urine of these two subjects was 43 and 44% of the administered dose respectively. The metabolism, distribution and elimination of chlorphentermine (I) and its metabolic products in man is now reported. Methods
Compounds and reagents
Chlorphentermine (I) hydrochloride and phenmetrazine hydrochloride were kindly provided by William R. Warner & Co. Ltd and Boehringer respectively. The following compounds and
A.H. BECKETT & P.M. BELANGER
194
CH3 CI ClCH-;2- ¢-R
microsomal fractions (Lindeke, Cho, Thomas & Michelson, 1973). The base of II was obtained by adding the oxalate salt to phosphate buffer at pH 6-8 and extracted with ether. Above pH 9 peaks corresponding to at,a-dimethyl-a-nitroso-3(4-chlorophenyl)ethane (III) and to a,a-dimethyla - nitro - , - (4 - chlorophenyl)ethane (IV) were obtained upon analysis of the organic extract; the latter increasing with increasing the pH and the time of contact. ca,a- Dimethyl-x-n itroso-3-(4-chlorophenyl)ethane (III) was prepared by oxidation of I using the method described for the preparation of a,o-dimethyl-a-nitroso-3-phenylethane from phentermine (Beckett & Belanger, 1 974b). Chlorphentermine (I, 0.02 mol) yielded III (850 mg, yield 20%) as a blue oil. G.l.c. analysis of an ethereal solution of this oil showed the presence of IV in addition to III; the compound IV was precipitated out on cooling a petroleum ether solution of this oil. Trials to crystallize III as a dimer failed. The structure of III was proved as follows: the p.m.r. spectrum of the monomeric form (blue) showed 'T 2.7-3 (quartet, A2 B2 system, 4H, Ar) 6.9 (s, 2H, CH2) 8.5 (s, 6H, C4CH3)2); the mass spectrum (g.c. -m.s.) gave m/e 125 (100%) 167 (38%) 127 (30%) 55 (22%) 169 (14%) 115 (11%) 126 (10%) 89 (10%) 91 (9%); compound III was readily oxidized to the nitro compound (IV) upon shaking with an aqueous alkaline solution. All the above characteristics are similar to those described earlier for an analogous C-nitroso compound (Beckett & B6langer, 1974b).
C-R~~~~~~~~~~~~~~~~~~~~
OH3 I
R
Chlorphentermine (a,a:dimethyl-4-
NH2
chlorophenethylamine)
OH N-Hydroxychlorphentermine
11 N
'H
i1 N=o
(N-hydroxy-aa,-dimethyl-4-chloro-
phenethylamine)
,a-Dimethyla-nitroso-g-(4chlorophenyl)ethane
a
IV NAO cx,ca-Dimethyl-a-nitro-#3-(4-chloro0 phenyl)ethane Figure 1 Structural formulae of chlorphentermine and its N-oxidized metabolites.
reagents were used: AnalaR ether, freshly distilled,
benzophenone, p-chlorobenzoic acid, diphenylmethylcyanide and trifluoroacetic anhydride from B.D.H. Limited, acetonitrile (Fisons), redistilled over phosphorus pentoxide and kept over molecular sieve at room temperature, a,a-dimethyl - a - nitro - B- (4 - chlorophenyl)ethane (IV), prepared as described before (Beckett & Belanger, 1975), N ,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA, Pierce Chemical Co., U.S.A.) and potassium permanganate (Ralph N. Emanuel Limited). All the drugs and their metabolic products were checked for purity by gas-liquid chromatography. N-Hydroxychlorphentermine (II) was prepared by reduction of a,a-dimethyl-a-nitro-f-4-chlorophenyl)ethane (IV, Beckett & Belanger, 1975) with zinc dust at pH 7 according to the same procedure described for the preparation of N-hydroxyphentermine from a,ct-dimethyl-anitro-j-phenylethane (Beckett & Belanger, 1 974b). a.,a-Di methyl-a-nitro-f-(4-chlorophenyl)ethane (IV, 0.01 mol) yielded N-hydroxychlorphentermine (II,N - hydroxy - a,a - dimethyl - 4- chlorophenethylamine, 0.0025 mol, yield 25%). The oxalate salt of II which precipitated upon addition of a saturated solution of oxalic acid in dry ether to the ethereal solution of II was recrystallized from ethanol: m.p. 176-80 (Found: C,53.8; H,6.2; N,5.8. C22H3oN206C12 requires C,54.1; H,6.2; N,5.7%), p.m.r. (DMSO) T 2.6-2.9 (quartet, A2 B2 system, 4 H, Ar) 7.2 (s, 2 H, CH2) 8.95 (s, 6 H, C4CH3)2) and 4 H at r 2.2 which disappeared upon addition of D2 0, mass spectrum (solid probe) m/e 74 (100%) 32 (33%) 125 (26%) 56 (26%) 58 (16%) 42 (11%) 127 (8%). Compound II has been recently identified after in vitro incubation of chlorphentermine (I) with hepatic
Studies in man A male subject (age 27 years), under normal (pH 6 to 7.3) conditions or with acidic urine (pH 5.0 ± 0.2), was given on separate widely spaced occasions, an oral dose of chlorphentermine (I) hydrochloride (75 mg) in 60-100 ml aqueous solutions. The general procedure adopted for diet and collection of urine was similar to that previously described by Beckett & Rowland (1965). The induction and maintenance of acidic urine was as described by Beckett & Brookes (1967). Urine samples were extracted and analysed by gas-iquid chromatography as described below. The analytical instruments and the columns used for gas-liquid chromatography were described elsewhere (Beckett & Belanger, 1975).
Determination of chlorphentermine (I) in urine as its trifluoroacetyl (TFA) derivation The urine (5 ml) was rendered alkaline by addition of NaOH (10%, 1 ml), phenmetrazine hydro-
CHLORPHENTERMINE IN MAN
chloride (22 Mg/ml, 1 ml) aqueous solution added as reference standard and the mixture extracted with ether (3 x 10 ml with 20 min shaking period each time). A portion (2-4 Ml) of the concentrated extract was chromatographed on g.l.c. column A (1 50 ) after adding trifluoroacetic anhydride (5 ,l); the amounts of I determined as its
trifluoroacetyl (TFA) derivative. Determination of N-hydroxychlorphentermine (II) in urine as its trimethylsilyl derivative (TMS)
Tlhe pH of the urine sample (5 ml) was adjusted to 7-7.5 after the addition of benzophenone solution (12 ,g/ml in water, 1 ml) as reference standard and the mixture extracted with ether (3 x 10 ml with 20 min shaking period each time). The combined ethereal extracts were evaporated to dryness at room temperature; dry acetonitrile (1O ml) and the silylating reagent BSTFA [N,Obis4trimethylsilyl)-trifluoroacetamide, 5 M1] were added to the residue and the mixture was allowed to stand at room temperature for 1-2 min. An aliquot (2-5 ml) of the organic solution was chromatographed on g.l.c. column A (1 50°) and the amount of N-hydroxychlorphentermine (II) was determined as its trimethylsilyl (TMS)
derivative. Determination of conjugated N-hydroxychlorphentermine (II) in urine The pH of the urine sample (5 ml) was adjusted to 1.5-2.0 by the addition of HCI (IN, 1 ml) and the mixture kept at 370 for 12-16 h with shaking; then the total amount of II, i.e. free plus liberated by acid hydrolysis (conjugated), was determined as described above. The amount of conjugated II was estimated by subtracting the amount of II determined after acid hydrolysis from that determined without acid-treatment (as above). Mild acid hydrolysis liberates hydroxylamines
from their conjugates (Beckett & Belanger, 1974a; Belanger, 1975). Determination of total N-oxidized metabolites in the urine The pH of the urine sample (5 ml) was adjusted to 7-7.5, potassium permanganate solution (10% in water, 0.5 ml) added and the mixture was shaken at room temperature for 20 min, then extracted with ether (3 x 10 ml with 20 min shaking period each). Diphenylmethylcyanide solution (8.2 ,g/ml in ether, 1 ml) as reference standard was added to the bulked ethereal extract which was then concentrated and a portion (2-4 Ml) chromatographed on column A (1900) to determine a, a- dimethyl - c - nitro -f3- (4-chlorophenyl)ethane (IV). [Hydroxylamino, free and conjugated, and C-nitroso compounds are oxidized to the nitro compound under these conditions (Beckett & Belanger, 1974a, 1974b, 1975).] In one experiment, the urine sample was oxidized under the same conditions for various periods of time (10, 20, 30 and 60 min). Calibration curves
Standard solutions of chlorphentermine (I, 0.1-40 jg/ml) hydrochloride, N-hydroxychlorphentermine (II, 0.5-15 Mg/ml) oxalate and a,a-dimethyl - at - nitro - 3 - (4 - chlorophenyl)ethane (IV,. 0.3-50 mg/ml) were freshly prepared in water and added to control urine (5 ml). Calibration curves based on the peak height ratio of compounds I (as TFA), II (as TMS) and IV to their g.l.c. reference standard using the methods described above were obtained from sixteen points representing eight different concentrations (in the range described above) in duplicate analysis. The data were subjected to linear regression analysis to give the appropriate calibration factors.
Table 1 G.l.c. characteristics of chlorphentermine, its metabolic products and related derivatives. The following standards were used: phenmetrazine (as TFA, column A, 1500, Rt 15.8 min), diphenylmethylcyanide (column A, 1900, Rt 8.8 min) and benzophenone (column A, 1500, Rt 21 min) G.l.c. Rt values (min) Column A
Compound
Chlorphentermine (I) N-Hydroxychlorphentermine (II)
Derivative TFA
a,a-dimethyl-a-nitro-3-(4-chlorphenyl)ethane (lll) p-Chlorobenzoic acid TFA: trifluoroacetyl derivative.
195
TMS TMS
TMS: trimethylsilyl derivative.
1500 8.8 8.7 17.8 4.0
1900
4.5
Rt: retention time.
Column B Column D 120° 130" 14.5 13.5
15.4 15.4
196
A.H. BECKETT & P.M. BELANGER
Oxidation of N-hydroxychlorphentermine (II) in urine with potassium permanganate Freshly prepared solutions of N-hydroxychlorphentermine (II, 7-22,ug/ml, 1 ml) oxalate in water were added to control urine (4 ml) at pH 7-8, potassium permanganate (10% in water, 0.5 ml) added and the mixtures were shaken at room temperature for 20 min. Then the mixtures were extracted with ether and analysed for their content of the hydroxylamine (II) and the nitro (IV) compounds according to the methods described above. Investigation for p-chlorobenzoic acid in urine
The urine sample (5 ml) was hydrolysed under acid conditions (HCI 6N, 1 ml) overnight at 370 and the mixture extracted with ether (3 x 10 ml). The bulked organic extract evaporated to dryness, acetonitrile (10 ,l) and the silylating reagent BSTFA (5 ,l) added to the residue. After a period of 1 to 2 min, a portion (2-4 Ml) was chromatographed on g.l.c. column A (150°). Under these conditions of analysis, p-chlorobenzoic acid added to control urine gave a peak (Table 1) identified as its trimethylsilyl derivative by g.l.c.-mass spectra. Investigation for conjugated chlorphentermine (I) in urine
The urine sample (5 ml) was hydrolysed with HCI (2N, 1 ml) for 4 h at room temperature, then the amount of I determined by the method described above and compared with that determined without acid treatment.
Results The retention times of the trifluoroacetyl deriva-
tive of chlorphentermine (I), the trimethylsilyl derivatives of N-hydroxychlorphentermine (II) and p-chlorobenzoic acid and the nitro compound (IV) are listed in Table 1. G.l.c. column A separated I from its metabolic products and was used for the quantitative analysis (see experimental) of I, II and IV excreted in the urine of a subject given I. No peak from the control urine interfered with these analyses under the conditions described. Chlorphentermine (I) and N-hydroxychlorphentermine (II) were analysed as the TFA and TMS derivatives respectively; conversion of II to its TMS derivative in acetonitrile was complete within 30 s and the product was stable for at least 30 min at room temperature (Belanger, 1975). Potassium permanganate oxidized quantitatively the hydroxylamino and C-nitroso compounds of phentermine to the nitro compound in aqueous solution and in in vitro experiments (Beckett & Belanger, 1975). The strength of the solution of oxidant (KMnO4 1%) used in the present studies was found unsatisfactory probably because of the presence of oxidizable substances in the urine. However, oxidizing the urine from a subject given chlorphentermine (I) with a more concentrated solution of potassium permanganate (10%) according to the method described in the experimental section followed by extraction and analysis of the extracts led to complete disappearance of the peaks corresponding to the hydroxylamine (II) and the nitroso (III) compounds to give that of the nitro (IV) compound; conjugated II was also oxidized to IV under the same conditions. Under these conditions, chlorphentermine (I) and the nitro compound (IV) were not oxidized; thus IV is the final oxidation product of free and conjugated II and of III. Time study experiments showed that the oxidation of II was complete after 10 min and that prolonging the oxidation time did not increase the recovery of IV. Known amounts of II
Table 2 Potassium permanganate oxidation of N-hydroxychlorphentermine (Il) to a,cc-dimethylat-nitro-f-(4-chlorophenyl)ethane (IV) in control urine. The oxidation time was 20 min
N-hydroxychlorphentermine (II) added
Amount of I V recovered
(Mg)
(M9)
7.14 7.14 7.14 7.14
4.4 4.3 4.9 4.4
9.3 9.3
6.0 6.1 7.2 7.8 10.5
11.4 11.4 22.0
% formation of IV 61.0 60.5
68.6 61.6 64.5
Mean 64.3
t
65.6 63.4 68.6 57.3 3.9 (s.d.)
.D&P_\*jkv:-r,012'slxi%|}
CHLORPHENTERMINE IN MAN
.,%.
197
4
'ri
t-F
I1 r
.,
* i
...+:
...
WP
32
Figure 2(a) The urinary excretion and (b) semi-logarithmic plot of the excretion of chlorphentermine (c), N-hydroxychlorphentermine (o), conjugated N-hydroxychlorphentermine (AN) and total N-oxidized metabolites (A) after oral administration of chlorphentermine (75 mg) to a subject with normal urine conditions. were therefore added to control urine and oxidized with potassium permanganate as described in the general procedure. The results are listed in Table 2. Under these conditions, a mean recovery of 64% was obtained upon oxidation of various concentrations of II in urine; no unchanged II or the nitroso compound (III) could be recovered and p-chlorobenzoic acid was not formed under these conditions. Oxidation using this procedure gave linear calibration graphs. Therefore, the calibration factor was corrected to give 100% recovery of II and the results obtained through this method are considered to be an estimate of the total N-oxidized metabolites.
Urinary excretion of chlorphentermine and its metabolites under normal conditions of urine production
After an oral dose of chlorphentermine (I), the maximum rate of excretion of unchanged drug was
reached within 1-2 h (Figure 2) and was maintained for up to 10 h under normal urinary pH indicating storage in tissue as proposed by others (Dubnick et al., 1968; Jun, Hlynka & Triggs, 1969; Jun & Triggs, 1970). 17 and 47% of the dose of I was excreted as unchanged drug and N-oxidized metabolites respectively in urine over a period of 48 h (Table 3). The low recovery of chlorphentermine under these conditions is in agreement with that of others (Jun & Triggs, 1968; Beckett & Brookes, 1 971 ). The curves of the excretion of the total N-oxidized metabolites suggests rapid and extensive biotransformation of I in the gut or the liver during the 'first pass' (Figure 2). The major metabolic products of I excreted in urine were N-hydroxychlorphentermine (II) and its conjugate. The difference between the amounts of total N-oxidized metabolites and the added amounts of free and conjugated II was equivalent to 18% of the dose (Table 3) and indicates the amount of nitroso (III) and nitro (IV) compounds
A.H. BECKETT & P.M. BELANGER
198
a 3
.-
O E _-E
510
f
I-1
I a- 5.5
-
CtQ) 4.5
c
E 0)
56
K
a) CU
48 c
0 a)
40F
0
x
0
E0) 32 a)
0.1
c
0 24 a)
16 _
a) I
I
0
4
8
16
12
20
24
28
32
0
4
8
12
16
20
24
28
Time (h) Figure 3(a)
The urinary excretion and (b) semi-logarithmic plot of the excretion of chlorphentermine (c),
Nhydroxychlorphentermine (o), conjugated N-hydroxychlorphentermine (A) and total N-oxidi,ed metabolites (-) after oral administration of chlorphentermine (75 mg as HCI) to a subject under acidic urine control. 70-
60 50
-40-
x
a)30
-
0
-o20
Time (h) Figure 4 Cumulative urinary excretion of chlorphentermine (% dose) and the total N-oxidized metabolites under normal (open symbols) and acidic (dark symbols) urine control. o and 0: experimental points for chlorphentermine; A and A: experimental points for total N-oxidized metabolites.
32
CHLORPHENTERMINE IN MAN
present in the urine. Neither p-chlorobenzoic acid, free or conjugated, nor conjugated I were excreted in the urine although the latter has been identified in the urine of animals dosed with I (Dubnick et al., 1968; Koster, Caldwell & Smith, 1974) but not that of man (Caldwell et al., 1975).
199
metabolic products excreted in the urine under the above conditions, was 13 h.
Discussion The importance of N-oxidation metabolic route in the distribution of chlorphentermine
Urinary excretion of chlorphentermine and its metabolites under acidic urine control
Chlorphentermine (I) is extensively metabolized by N-oxidation in man. Under normal conditions of urine pH, 75% of the excreted dose of I were N-oxidized metabolites. Acidifying the urine increased the urinary excretion of the drug and decreased the amount of metabolism; 25% of the administered dose of I was excreted as N-oxidized metabolites when the urine was maintained acid (pH 5.0 ± 0.2). The kinetics of excretion of I and its metabolic products under normal and acidic conditions of urine gave curves indicating that I and its excretion products had similar distribution characteristics (Figures 2 and 3); the ratio of the total N-oxidized metabolites to unchanged I (Table 3) at various time intervals did not vary significantly. N-Oxygenated metabolites have been detected in the plasma and red blood cell of a subject given a single dose of I (Beckett & Belanger, 1974a). N-Oxidized metabolic products have been involved in the pharmacology, toxicology and pharmacokinetics of 'amphetamines' and biogenic amines (Beckett, 1973; Ross, Gosztonyi & Renyi, 1974; Gorrod & Jenner, 1975). Hydroxylamines are much weaker bases (3.0 pK units) than their parent amines and consequently much more of these non-ionized forms exist at physiological pH values; hydroxylamines are powerful nucleophilic agents in their non-ionized form.
Acidic urine minimizes tubular reabsorption of amines, thus avoiding further metabolism and increases urinary excretion and allows the maximum urinary recovery of basic drugs. Under these conditions, the excretion rates of an amine drug and its basic metabolites are proportional to their plasma concentration (Beckett, 1966; Beckett, Salmon & Mitchard, 1969). Under acidic urine control, the above subject excreted 96% of the administered dose of chlorphentermine within 36 h (Table 3). Thus, chlorphentermine (I) administered to humans in solution is completely absorbed and urinary excretion and metabolism are the two major routes of elimination of I. The recoveries of unchanged drug and total N-oxidized metabolites represented 72 and 24% of the dose respectively. The maximum excretion rates of I and its metabolic products were reached with 2-3 h (Figure 3). The ratio of the total N-oxidized metabolites to unchanged drug was drastically decreased when the urine was kept acidic (pH 5.0 ± 0.2) compared to that under normal urine production (pH 5.7-7.1) (Table 3). From the cumulative excretion plot (Figure 4), the half-life of the total drug moiety, i.e. unchanged I plus the
Table 3 Comparative urinary excretion of chlorphentermine and its metabolites in man over a period of 48 h after an oral dose of chlorphentermine (75 mg as HCI) under acidic and normal urinary pH control
% dose excreted Product
Chlorphentermine (I) N-Hydroxychlorphentermine (II) Free Conj ugated Total N-oxidized metabolites Total recovery Ratio of total N-oxidized metabolites to unchanged drug
Acid urine control Time (hi 12 24 36
12
Normal urine Time (hi 24 36
48
30.0
54.0
72.0
9.0
11.4
14.4
16.7
7.7
6.8 15.6 46.0
11.0 7.8 20.0 74.0
12.0 9.0 24.0 96.0
6.0 8.5 23.0 32.0
8.7 10.2 32.0 43.0
11.0 14.4 42.0 56.0
13.0 16.3 47.0 64.0
0.5
0.4
0.3
2.6
2.7
2.9
2.8
200
A.H. BECKETT & P.M. BELANGER
References BECKETT, A.H. (1966). Drug distribution and metabolism. Dansk Tidskr. Farm., 40, 197-223. BECKETT, A.H.(1973). The importance of the hydroxylamine metabolic route in pharmacology, toxicology and pharmacokinetics. In Frontiers in catecholamines research, ed: Usdin, E. & Snyder, S., pp. 139-143, Oxford: Pergamon. BECKETT, A.H. & BELANGER, P.M. (1974a). Metabolism of chlorphentermine and phentermine in man to yield hydroxylamino-, C-nitroso- and nitro compounds. J. Pharm. Pharmac., 26, 205-206. BECKETT, A.H. & BELANGER, P.M. (1974b). The identification of three new in vitro metabolic products after liver microsomal incubation of phentermine.
Xenobiotica, 4, 509-519. BECKETT, A.H. & BtLANGER, P.M. (1975). The identification and analysis of the metabolic products of mephentermine. J. Pharm. Pharmac., 27, 928-932. BECKETT, A.H. & BROOKES, L.G. (1967). The absorption and urinary excretion in man of fenfluramine and its main metabolite. J. Pharm. Pharmac., 19, 41-52. BECKETT, A.H. & BROOKES, L.G. (1971). The metabolism and urinary excretion in man of phentermine, and the influence of N-methyl and p-chloro-substitution. J. Pharm. Pharmac., 23, 288-294. BECKETT, A.H. & ROWLAND, M. (1965). Urinary excretion kinetics of amphetamine in man. J. Pharm. Pharmac., 17, 628-639. BECKETT, A.H., SALMON, J.A. & MITCHARD, M. (1969). The relation between blood levels and urinary excretion of amphetamine under controlled acidic and under fluctuating urinary pH values using ['4C] amphetamine. J. Pharm. Pharmac., 21, 251-258. BtLANGER, P.M. (1975). The metabolism and distribution of phentermine and some related compounds. Ph.D. Thesis, University of London. CALDWELL, J., KOSTER, U., SMITH, R.L. & WILLIAMS, R.T. (1975). Species variations in the N-oxidation of chlorphentermine. Biochem. Pharmac.,
24, 2225-2232. DUBNICK, B., LEESON, G.A., LEVERETT, R., MORGAN, D.F. & PHILIPPS, G.E. (1963). Sympathomimetic properties of chlorphentermine: metabolism, metabolic effects, interaction with reserpine and biogenic amines. J. Pharmac. exp. Ther., 140, 85-92. DUBNICK, B., TOWNE, C.A., HARTIGNAN, J.M. & PHILIPPS, G.E. (1968). Distribution and metabolism of chlorphentermine-C1 in rats and mice. Biochem.
Pharmac., 17, 1243-1251. GORROD, J.W. & JENNER, P. (1975). Metabolic N-oxidation products of aliphatic amines as potential
mediators in amine pharmacology. Int. J. clin. Pharnwc. Biopharm., 12 180-185. JUN, H.W., HLYNKA, J.N. & TRIGGS, E.J. (1969). The pharmacokinetics of chlorphentermine in man. Can. J. pharm. Sci., 4, 27-32. JUN, H.W. & TRIGGS, E.J. (1968). The quantitative determination of chlorphentermine in urine by gasliquid chromatography. Can. J. pharm. Sci., 3, 73-74. JUN, H.W. & TRIGGS, E.J. (1970). Blood levels of chlorphentermine in man. J. pharm. Sci., 59, 306-309. KARABELNICK, D., ZBINDEN, G. & BAUMGARTNER,
E. (1974). Drug induced foam cell reactions in rats I. Histopathologic and cytochemical observations after treatment with chlorphentermine, RMi 10.393 and Ro 4-4318. Tox. Appl. Pharmac., 27, 395407. KOSTER, W., CALDWELL, J. & SMITH, R.L. (1974). Species variation in the N-oxidation of chlorphentermine. Biochem. Soc. Trans., 2, 881-882. LINDEKE, B., CHO, A.K., THOMAS, T.L. & MICHELSON, L. (1973). Microsomal N-hydroxylation of phenylalkylamines. Identification of Nhydroxylated phenylalkylamines as their trimethylsilyl derivatives by GS/MS. Acta Pharm. Suecica, 10, 493-506. LULLMANN, H., LULLMANN-RAUCH, R. & WASSERMANN, 0. (1973). Drug-induced phospholipidoses. Germ. Med., 3,128-1 38. LULLMANN, H., ROSSEN, E. & SEILER, K.-U. (1973). The pharmacokinetics of phentermine and chlorphentermine in chronically treated rats. J. Pharm. Pharmac., 25, 239-243. LULLMANN-RAUCH, R. (1975). Chlorphentermineinduced cytoplasmic inclusions in peripheral blood cells of rats and guinea pigs. Tox. Appl. Pharmac., 32, 32-39. ROSS, S.B., GOSZTONYI, T. & RENYI, A.L. (1974). Long-term effects of N-hydroxy-4-chloroamphetamine on the level and accumulation of 5-hydroxytryptamine in the rat brain. Eur. J. Pharmac., 28, 222-224. SCHMIEN, R., SEILER, K.-U. & WASSERMANN, 0. (1974). Drug-induced phospholipidosis I. Lipid composition and chlorphentermine content of rat lung tissue and alveolar macrophages after chronic treatment. Naunyn-Schmiedebergs Arch. Pharmac., 283, 331-334. SEILER, K.-U. & WASSERMANN, 0. (1974). Evidence for an unusual distnrbution of chlorphentermine in vivo; an autoradiographic study in mice. NaunynSchmiedebergs Arch. Pharmac., 282, 113-122.
(Received March 5, 1976)
THE METABOLISM, DISTRIBUTION AND ELIMINATION OF CHLORPHENTERMINE IN MAN A.H. BECKETT & P.M. BELANGER* Department of Pharmacy, Chelsea College, University of London, London SW3 6LX
I A gas-liquid chromatography procedure for the determination of chlorphentermine (I), N-hydroxychlorphentermine (II) and a,o-dimethyl-a-nitro-,-44-chlorophenyl)ethane (IV) in urine has been developed. Also methods are reported to determine conjugated II and the total N-oxidized metabolites of I, i.e. II, conjugated II, a,a-dimethyl-a-nitroso-3-(4chlorophenyl)ethane (III) and IV in urine. 2 The synthesis of ,ct-dimethyl-a-nitroso-f-(4-chlorophenyl)ethane (III) and its properties are reported. 3 The kinetics of urinary excretion of I and its metabolic products after the oral administration of I to a human subject on separate occasions have been studied. Under normal conditions of urinary pH, metabolism by N-oxidation was the main elimination route of I; acidifying the urine increased the urinary excretion of unchanged I at the expense of the N-oxidized products. 4 The importance of the N-oxidation metabolic route in the distribution of chlorphentermine (I) in man is discussed. Introduction In man, the half-life of the anorectic drug chlorphentermine (I), Figure 1, is more than double that of the non-chlorinated analogue phentermine (Beckett & Brookes, 1971); an average elimination half-life of 41 h has been reported after monitoring whole blood concentration of I in man (Jun & Triggs, 1970). Chlorphentermine (I), but not phentermine, accumulated in the brain, lung, adrenal and spleen tissues when administered to rat and mice (Dubnick, Leeson, Leverett, Morgan & Philipps, 1963; Dubnick, Towne, Hartignan & Philipps, 1968; Liilman, Rossen & Seiler, 1973; Seiler & Wassermann, 1974). The accumulation process has been related to the interactions of I with the tissue phospholipids (Lullman, LUllman-Rauch & Wassermann, 1973; Schmien, Seiler & Wassermann, 1974; Seiler & Wassermann, 1974) and in biochemical and histopathological effects of I after repeated administration (Lillmann, LuallmanRauch & Wassermann, 1973; Lullmann et al., 1973; Karabelnik, Zbinder & Baumgartner, 1974; Lulllmann-Rauch, 1975). Pulmonary hypertension has been observed in patients after chronic treatment with I (Lillman et al., 1973 and references cited therein). Chlorphentermine (I) is N-oxidized metabolically to N-hydroxychlorphentermine (II), ci,a-di* Present address: Ecole de Pharmacie, Universite Laval, Qu6bec GIK 7P4, Canada.
methyl-a-nitroso--44-chlorophenyl)ethane (III) and a,o-dimethyl-a-nitro-(3-(4-chlorophenyl)ethane (IV) in vitro in animals and in vivo in man (Beckett & Belanger, 1974a); conjugated II was also excreted in the urine of a human subject given I. Since completing the present work, Caldwell, K6ster, Smith & Williams (1975) reported the N-oxidation of labelled [C14] -chlorphentermine (I) using radiochemical techniques for the assay of unchanged I, N-hydroxychlorphentermine (II), conjugated II and the nitro analogue (IV) in the bulked urine samples of three days of two human subjects under normal conditions of urinary pH after oral dosage. The total recovery of I and the above metabolic products in the urine of these two subjects was 43 and 44% of the administered dose respectively. The metabolism, distribution and elimination of chlorphentermine (I) and its metabolic products in man is now reported. Methods
Compounds and reagents
Chlorphentermine (I) hydrochloride and phenmetrazine hydrochloride were kindly provided by William R. Warner & Co. Ltd and Boehringer respectively. The following compounds and
A.H. BECKETT & P.M. BELANGER
194
CH3 CI ClCH-;2- ¢-R
microsomal fractions (Lindeke, Cho, Thomas & Michelson, 1973). The base of II was obtained by adding the oxalate salt to phosphate buffer at pH 6-8 and extracted with ether. Above pH 9 peaks corresponding to at,a-dimethyl-a-nitroso-3(4-chlorophenyl)ethane (III) and to a,a-dimethyla - nitro - , - (4 - chlorophenyl)ethane (IV) were obtained upon analysis of the organic extract; the latter increasing with increasing the pH and the time of contact. ca,a- Dimethyl-x-n itroso-3-(4-chlorophenyl)ethane (III) was prepared by oxidation of I using the method described for the preparation of a,o-dimethyl-a-nitroso-3-phenylethane from phentermine (Beckett & Belanger, 1 974b). Chlorphentermine (I, 0.02 mol) yielded III (850 mg, yield 20%) as a blue oil. G.l.c. analysis of an ethereal solution of this oil showed the presence of IV in addition to III; the compound IV was precipitated out on cooling a petroleum ether solution of this oil. Trials to crystallize III as a dimer failed. The structure of III was proved as follows: the p.m.r. spectrum of the monomeric form (blue) showed 'T 2.7-3 (quartet, A2 B2 system, 4H, Ar) 6.9 (s, 2H, CH2) 8.5 (s, 6H, C4CH3)2); the mass spectrum (g.c. -m.s.) gave m/e 125 (100%) 167 (38%) 127 (30%) 55 (22%) 169 (14%) 115 (11%) 126 (10%) 89 (10%) 91 (9%); compound III was readily oxidized to the nitro compound (IV) upon shaking with an aqueous alkaline solution. All the above characteristics are similar to those described earlier for an analogous C-nitroso compound (Beckett & B6langer, 1974b).
C-R~~~~~~~~~~~~~~~~~~~~
OH3 I
R
Chlorphentermine (a,a:dimethyl-4-
NH2
chlorophenethylamine)
OH N-Hydroxychlorphentermine
11 N
'H
i1 N=o
(N-hydroxy-aa,-dimethyl-4-chloro-
phenethylamine)
,a-Dimethyla-nitroso-g-(4chlorophenyl)ethane
a
IV NAO cx,ca-Dimethyl-a-nitro-#3-(4-chloro0 phenyl)ethane Figure 1 Structural formulae of chlorphentermine and its N-oxidized metabolites.
reagents were used: AnalaR ether, freshly distilled,
benzophenone, p-chlorobenzoic acid, diphenylmethylcyanide and trifluoroacetic anhydride from B.D.H. Limited, acetonitrile (Fisons), redistilled over phosphorus pentoxide and kept over molecular sieve at room temperature, a,a-dimethyl - a - nitro - B- (4 - chlorophenyl)ethane (IV), prepared as described before (Beckett & Belanger, 1975), N ,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA, Pierce Chemical Co., U.S.A.) and potassium permanganate (Ralph N. Emanuel Limited). All the drugs and their metabolic products were checked for purity by gas-liquid chromatography. N-Hydroxychlorphentermine (II) was prepared by reduction of a,a-dimethyl-a-nitro-f-4-chlorophenyl)ethane (IV, Beckett & Belanger, 1975) with zinc dust at pH 7 according to the same procedure described for the preparation of N-hydroxyphentermine from a,ct-dimethyl-anitro-j-phenylethane (Beckett & Belanger, 1 974b). a.,a-Di methyl-a-nitro-f-(4-chlorophenyl)ethane (IV, 0.01 mol) yielded N-hydroxychlorphentermine (II,N - hydroxy - a,a - dimethyl - 4- chlorophenethylamine, 0.0025 mol, yield 25%). The oxalate salt of II which precipitated upon addition of a saturated solution of oxalic acid in dry ether to the ethereal solution of II was recrystallized from ethanol: m.p. 176-80 (Found: C,53.8; H,6.2; N,5.8. C22H3oN206C12 requires C,54.1; H,6.2; N,5.7%), p.m.r. (DMSO) T 2.6-2.9 (quartet, A2 B2 system, 4 H, Ar) 7.2 (s, 2 H, CH2) 8.95 (s, 6 H, C4CH3)2) and 4 H at r 2.2 which disappeared upon addition of D2 0, mass spectrum (solid probe) m/e 74 (100%) 32 (33%) 125 (26%) 56 (26%) 58 (16%) 42 (11%) 127 (8%). Compound II has been recently identified after in vitro incubation of chlorphentermine (I) with hepatic
Studies in man A male subject (age 27 years), under normal (pH 6 to 7.3) conditions or with acidic urine (pH 5.0 ± 0.2), was given on separate widely spaced occasions, an oral dose of chlorphentermine (I) hydrochloride (75 mg) in 60-100 ml aqueous solutions. The general procedure adopted for diet and collection of urine was similar to that previously described by Beckett & Rowland (1965). The induction and maintenance of acidic urine was as described by Beckett & Brookes (1967). Urine samples were extracted and analysed by gas-iquid chromatography as described below. The analytical instruments and the columns used for gas-liquid chromatography were described elsewhere (Beckett & Belanger, 1975).
Determination of chlorphentermine (I) in urine as its trifluoroacetyl (TFA) derivation The urine (5 ml) was rendered alkaline by addition of NaOH (10%, 1 ml), phenmetrazine hydro-
CHLORPHENTERMINE IN MAN
chloride (22 Mg/ml, 1 ml) aqueous solution added as reference standard and the mixture extracted with ether (3 x 10 ml with 20 min shaking period each time). A portion (2-4 Ml) of the concentrated extract was chromatographed on g.l.c. column A (1 50 ) after adding trifluoroacetic anhydride (5 ,l); the amounts of I determined as its
trifluoroacetyl (TFA) derivative. Determination of N-hydroxychlorphentermine (II) in urine as its trimethylsilyl derivative (TMS)
Tlhe pH of the urine sample (5 ml) was adjusted to 7-7.5 after the addition of benzophenone solution (12 ,g/ml in water, 1 ml) as reference standard and the mixture extracted with ether (3 x 10 ml with 20 min shaking period each time). The combined ethereal extracts were evaporated to dryness at room temperature; dry acetonitrile (1O ml) and the silylating reagent BSTFA [N,Obis4trimethylsilyl)-trifluoroacetamide, 5 M1] were added to the residue and the mixture was allowed to stand at room temperature for 1-2 min. An aliquot (2-5 ml) of the organic solution was chromatographed on g.l.c. column A (1 50°) and the amount of N-hydroxychlorphentermine (II) was determined as its trimethylsilyl (TMS)
derivative. Determination of conjugated N-hydroxychlorphentermine (II) in urine The pH of the urine sample (5 ml) was adjusted to 1.5-2.0 by the addition of HCI (IN, 1 ml) and the mixture kept at 370 for 12-16 h with shaking; then the total amount of II, i.e. free plus liberated by acid hydrolysis (conjugated), was determined as described above. The amount of conjugated II was estimated by subtracting the amount of II determined after acid hydrolysis from that determined without acid-treatment (as above). Mild acid hydrolysis liberates hydroxylamines
from their conjugates (Beckett & Belanger, 1974a; Belanger, 1975). Determination of total N-oxidized metabolites in the urine The pH of the urine sample (5 ml) was adjusted to 7-7.5, potassium permanganate solution (10% in water, 0.5 ml) added and the mixture was shaken at room temperature for 20 min, then extracted with ether (3 x 10 ml with 20 min shaking period each). Diphenylmethylcyanide solution (8.2 ,g/ml in ether, 1 ml) as reference standard was added to the bulked ethereal extract which was then concentrated and a portion (2-4 Ml) chromatographed on column A (1900) to determine a, a- dimethyl - c - nitro -f3- (4-chlorophenyl)ethane (IV). [Hydroxylamino, free and conjugated, and C-nitroso compounds are oxidized to the nitro compound under these conditions (Beckett & Belanger, 1974a, 1974b, 1975).] In one experiment, the urine sample was oxidized under the same conditions for various periods of time (10, 20, 30 and 60 min). Calibration curves
Standard solutions of chlorphentermine (I, 0.1-40 jg/ml) hydrochloride, N-hydroxychlorphentermine (II, 0.5-15 Mg/ml) oxalate and a,a-dimethyl - at - nitro - 3 - (4 - chlorophenyl)ethane (IV,. 0.3-50 mg/ml) were freshly prepared in water and added to control urine (5 ml). Calibration curves based on the peak height ratio of compounds I (as TFA), II (as TMS) and IV to their g.l.c. reference standard using the methods described above were obtained from sixteen points representing eight different concentrations (in the range described above) in duplicate analysis. The data were subjected to linear regression analysis to give the appropriate calibration factors.
Table 1 G.l.c. characteristics of chlorphentermine, its metabolic products and related derivatives. The following standards were used: phenmetrazine (as TFA, column A, 1500, Rt 15.8 min), diphenylmethylcyanide (column A, 1900, Rt 8.8 min) and benzophenone (column A, 1500, Rt 21 min) G.l.c. Rt values (min) Column A
Compound
Chlorphentermine (I) N-Hydroxychlorphentermine (II)
Derivative TFA
a,a-dimethyl-a-nitro-3-(4-chlorphenyl)ethane (lll) p-Chlorobenzoic acid TFA: trifluoroacetyl derivative.
195
TMS TMS
TMS: trimethylsilyl derivative.
1500 8.8 8.7 17.8 4.0
1900
4.5
Rt: retention time.
Column B Column D 120° 130" 14.5 13.5
15.4 15.4
196
A.H. BECKETT & P.M. BELANGER
Oxidation of N-hydroxychlorphentermine (II) in urine with potassium permanganate Freshly prepared solutions of N-hydroxychlorphentermine (II, 7-22,ug/ml, 1 ml) oxalate in water were added to control urine (4 ml) at pH 7-8, potassium permanganate (10% in water, 0.5 ml) added and the mixtures were shaken at room temperature for 20 min. Then the mixtures were extracted with ether and analysed for their content of the hydroxylamine (II) and the nitro (IV) compounds according to the methods described above. Investigation for p-chlorobenzoic acid in urine
The urine sample (5 ml) was hydrolysed under acid conditions (HCI 6N, 1 ml) overnight at 370 and the mixture extracted with ether (3 x 10 ml). The bulked organic extract evaporated to dryness, acetonitrile (10 ,l) and the silylating reagent BSTFA (5 ,l) added to the residue. After a period of 1 to 2 min, a portion (2-4 Ml) was chromatographed on g.l.c. column A (150°). Under these conditions of analysis, p-chlorobenzoic acid added to control urine gave a peak (Table 1) identified as its trimethylsilyl derivative by g.l.c.-mass spectra. Investigation for conjugated chlorphentermine (I) in urine
The urine sample (5 ml) was hydrolysed with HCI (2N, 1 ml) for 4 h at room temperature, then the amount of I determined by the method described above and compared with that determined without acid treatment.
Results The retention times of the trifluoroacetyl deriva-
tive of chlorphentermine (I), the trimethylsilyl derivatives of N-hydroxychlorphentermine (II) and p-chlorobenzoic acid and the nitro compound (IV) are listed in Table 1. G.l.c. column A separated I from its metabolic products and was used for the quantitative analysis (see experimental) of I, II and IV excreted in the urine of a subject given I. No peak from the control urine interfered with these analyses under the conditions described. Chlorphentermine (I) and N-hydroxychlorphentermine (II) were analysed as the TFA and TMS derivatives respectively; conversion of II to its TMS derivative in acetonitrile was complete within 30 s and the product was stable for at least 30 min at room temperature (Belanger, 1975). Potassium permanganate oxidized quantitatively the hydroxylamino and C-nitroso compounds of phentermine to the nitro compound in aqueous solution and in in vitro experiments (Beckett & Belanger, 1975). The strength of the solution of oxidant (KMnO4 1%) used in the present studies was found unsatisfactory probably because of the presence of oxidizable substances in the urine. However, oxidizing the urine from a subject given chlorphentermine (I) with a more concentrated solution of potassium permanganate (10%) according to the method described in the experimental section followed by extraction and analysis of the extracts led to complete disappearance of the peaks corresponding to the hydroxylamine (II) and the nitroso (III) compounds to give that of the nitro (IV) compound; conjugated II was also oxidized to IV under the same conditions. Under these conditions, chlorphentermine (I) and the nitro compound (IV) were not oxidized; thus IV is the final oxidation product of free and conjugated II and of III. Time study experiments showed that the oxidation of II was complete after 10 min and that prolonging the oxidation time did not increase the recovery of IV. Known amounts of II
Table 2 Potassium permanganate oxidation of N-hydroxychlorphentermine (Il) to a,cc-dimethylat-nitro-f-(4-chlorophenyl)ethane (IV) in control urine. The oxidation time was 20 min
N-hydroxychlorphentermine (II) added
Amount of I V recovered
(Mg)
(M9)
7.14 7.14 7.14 7.14
4.4 4.3 4.9 4.4
9.3 9.3
6.0 6.1 7.2 7.8 10.5
11.4 11.4 22.0
% formation of IV 61.0 60.5
68.6 61.6 64.5
Mean 64.3
t
65.6 63.4 68.6 57.3 3.9 (s.d.)
.D&P_\*jkv:-r,012'slxi%|}
CHLORPHENTERMINE IN MAN
.,%.
197
4
'ri
t-F
I1 r
.,
* i
...+:
...
WP
32
Figure 2(a) The urinary excretion and (b) semi-logarithmic plot of the excretion of chlorphentermine (c), N-hydroxychlorphentermine (o), conjugated N-hydroxychlorphentermine (AN) and total N-oxidized metabolites (A) after oral administration of chlorphentermine (75 mg) to a subject with normal urine conditions. were therefore added to control urine and oxidized with potassium permanganate as described in the general procedure. The results are listed in Table 2. Under these conditions, a mean recovery of 64% was obtained upon oxidation of various concentrations of II in urine; no unchanged II or the nitroso compound (III) could be recovered and p-chlorobenzoic acid was not formed under these conditions. Oxidation using this procedure gave linear calibration graphs. Therefore, the calibration factor was corrected to give 100% recovery of II and the results obtained through this method are considered to be an estimate of the total N-oxidized metabolites.
Urinary excretion of chlorphentermine and its metabolites under normal conditions of urine production
After an oral dose of chlorphentermine (I), the maximum rate of excretion of unchanged drug was
reached within 1-2 h (Figure 2) and was maintained for up to 10 h under normal urinary pH indicating storage in tissue as proposed by others (Dubnick et al., 1968; Jun, Hlynka & Triggs, 1969; Jun & Triggs, 1970). 17 and 47% of the dose of I was excreted as unchanged drug and N-oxidized metabolites respectively in urine over a period of 48 h (Table 3). The low recovery of chlorphentermine under these conditions is in agreement with that of others (Jun & Triggs, 1968; Beckett & Brookes, 1 971 ). The curves of the excretion of the total N-oxidized metabolites suggests rapid and extensive biotransformation of I in the gut or the liver during the 'first pass' (Figure 2). The major metabolic products of I excreted in urine were N-hydroxychlorphentermine (II) and its conjugate. The difference between the amounts of total N-oxidized metabolites and the added amounts of free and conjugated II was equivalent to 18% of the dose (Table 3) and indicates the amount of nitroso (III) and nitro (IV) compounds
A.H. BECKETT & P.M. BELANGER
198
a 3
.-
O E _-E
510
f
I-1
I a- 5.5
-
CtQ) 4.5
c
E 0)
56
K
a) CU
48 c
0 a)
40F
0
x
0
E0) 32 a)
0.1
c
0 24 a)
16 _
a) I
I
0
4
8
16
12
20
24
28
32
0
4
8
12
16
20
24
28
Time (h) Figure 3(a)
The urinary excretion and (b) semi-logarithmic plot of the excretion of chlorphentermine (c),
Nhydroxychlorphentermine (o), conjugated N-hydroxychlorphentermine (A) and total N-oxidi,ed metabolites (-) after oral administration of chlorphentermine (75 mg as HCI) to a subject under acidic urine control. 70-
60 50
-40-
x
a)30
-
0
-o20
Time (h) Figure 4 Cumulative urinary excretion of chlorphentermine (% dose) and the total N-oxidized metabolites under normal (open symbols) and acidic (dark symbols) urine control. o and 0: experimental points for chlorphentermine; A and A: experimental points for total N-oxidized metabolites.
32
CHLORPHENTERMINE IN MAN
present in the urine. Neither p-chlorobenzoic acid, free or conjugated, nor conjugated I were excreted in the urine although the latter has been identified in the urine of animals dosed with I (Dubnick et al., 1968; Koster, Caldwell & Smith, 1974) but not that of man (Caldwell et al., 1975).
199
metabolic products excreted in the urine under the above conditions, was 13 h.
Discussion The importance of N-oxidation metabolic route in the distribution of chlorphentermine
Urinary excretion of chlorphentermine and its metabolites under acidic urine control
Chlorphentermine (I) is extensively metabolized by N-oxidation in man. Under normal conditions of urine pH, 75% of the excreted dose of I were N-oxidized metabolites. Acidifying the urine increased the urinary excretion of the drug and decreased the amount of metabolism; 25% of the administered dose of I was excreted as N-oxidized metabolites when the urine was maintained acid (pH 5.0 ± 0.2). The kinetics of excretion of I and its metabolic products under normal and acidic conditions of urine gave curves indicating that I and its excretion products had similar distribution characteristics (Figures 2 and 3); the ratio of the total N-oxidized metabolites to unchanged I (Table 3) at various time intervals did not vary significantly. N-Oxygenated metabolites have been detected in the plasma and red blood cell of a subject given a single dose of I (Beckett & Belanger, 1974a). N-Oxidized metabolic products have been involved in the pharmacology, toxicology and pharmacokinetics of 'amphetamines' and biogenic amines (Beckett, 1973; Ross, Gosztonyi & Renyi, 1974; Gorrod & Jenner, 1975). Hydroxylamines are much weaker bases (3.0 pK units) than their parent amines and consequently much more of these non-ionized forms exist at physiological pH values; hydroxylamines are powerful nucleophilic agents in their non-ionized form.
Acidic urine minimizes tubular reabsorption of amines, thus avoiding further metabolism and increases urinary excretion and allows the maximum urinary recovery of basic drugs. Under these conditions, the excretion rates of an amine drug and its basic metabolites are proportional to their plasma concentration (Beckett, 1966; Beckett, Salmon & Mitchard, 1969). Under acidic urine control, the above subject excreted 96% of the administered dose of chlorphentermine within 36 h (Table 3). Thus, chlorphentermine (I) administered to humans in solution is completely absorbed and urinary excretion and metabolism are the two major routes of elimination of I. The recoveries of unchanged drug and total N-oxidized metabolites represented 72 and 24% of the dose respectively. The maximum excretion rates of I and its metabolic products were reached with 2-3 h (Figure 3). The ratio of the total N-oxidized metabolites to unchanged drug was drastically decreased when the urine was kept acidic (pH 5.0 ± 0.2) compared to that under normal urine production (pH 5.7-7.1) (Table 3). From the cumulative excretion plot (Figure 4), the half-life of the total drug moiety, i.e. unchanged I plus the
Table 3 Comparative urinary excretion of chlorphentermine and its metabolites in man over a period of 48 h after an oral dose of chlorphentermine (75 mg as HCI) under acidic and normal urinary pH control
% dose excreted Product
Chlorphentermine (I) N-Hydroxychlorphentermine (II) Free Conj ugated Total N-oxidized metabolites Total recovery Ratio of total N-oxidized metabolites to unchanged drug
Acid urine control Time (hi 12 24 36
12
Normal urine Time (hi 24 36
48
30.0
54.0
72.0
9.0
11.4
14.4
16.7
7.7
6.8 15.6 46.0
11.0 7.8 20.0 74.0
12.0 9.0 24.0 96.0
6.0 8.5 23.0 32.0
8.7 10.2 32.0 43.0
11.0 14.4 42.0 56.0
13.0 16.3 47.0 64.0
0.5
0.4
0.3
2.6
2.7
2.9
2.8
200
A.H. BECKETT & P.M. BELANGER
References BECKETT, A.H. (1966). Drug distribution and metabolism. Dansk Tidskr. Farm., 40, 197-223. BECKETT, A.H.(1973). The importance of the hydroxylamine metabolic route in pharmacology, toxicology and pharmacokinetics. In Frontiers in catecholamines research, ed: Usdin, E. & Snyder, S., pp. 139-143, Oxford: Pergamon. BECKETT, A.H. & BELANGER, P.M. (1974a). Metabolism of chlorphentermine and phentermine in man to yield hydroxylamino-, C-nitroso- and nitro compounds. J. Pharm. Pharmac., 26, 205-206. BECKETT, A.H. & BELANGER, P.M. (1974b). The identification of three new in vitro metabolic products after liver microsomal incubation of phentermine.
Xenobiotica, 4, 509-519. BECKETT, A.H. & BtLANGER, P.M. (1975). The identification and analysis of the metabolic products of mephentermine. J. Pharm. Pharmac., 27, 928-932. BECKETT, A.H. & BROOKES, L.G. (1967). The absorption and urinary excretion in man of fenfluramine and its main metabolite. J. Pharm. Pharmac., 19, 41-52. BECKETT, A.H. & BROOKES, L.G. (1971). The metabolism and urinary excretion in man of phentermine, and the influence of N-methyl and p-chloro-substitution. J. Pharm. Pharmac., 23, 288-294. BECKETT, A.H. & ROWLAND, M. (1965). Urinary excretion kinetics of amphetamine in man. J. Pharm. Pharmac., 17, 628-639. BECKETT, A.H., SALMON, J.A. & MITCHARD, M. (1969). The relation between blood levels and urinary excretion of amphetamine under controlled acidic and under fluctuating urinary pH values using ['4C] amphetamine. J. Pharm. Pharmac., 21, 251-258. BtLANGER, P.M. (1975). The metabolism and distribution of phentermine and some related compounds. Ph.D. Thesis, University of London. CALDWELL, J., KOSTER, U., SMITH, R.L. & WILLIAMS, R.T. (1975). Species variations in the N-oxidation of chlorphentermine. Biochem. Pharmac.,
24, 2225-2232. DUBNICK, B., LEESON, G.A., LEVERETT, R., MORGAN, D.F. & PHILIPPS, G.E. (1963). Sympathomimetic properties of chlorphentermine: metabolism, metabolic effects, interaction with reserpine and biogenic amines. J. Pharmac. exp. Ther., 140, 85-92. DUBNICK, B., TOWNE, C.A., HARTIGNAN, J.M. & PHILIPPS, G.E. (1968). Distribution and metabolism of chlorphentermine-C1 in rats and mice. Biochem.
Pharmac., 17, 1243-1251. GORROD, J.W. & JENNER, P. (1975). Metabolic N-oxidation products of aliphatic amines as potential
mediators in amine pharmacology. Int. J. clin. Pharnwc. Biopharm., 12 180-185. JUN, H.W., HLYNKA, J.N. & TRIGGS, E.J. (1969). The pharmacokinetics of chlorphentermine in man. Can. J. pharm. Sci., 4, 27-32. JUN, H.W. & TRIGGS, E.J. (1968). The quantitative determination of chlorphentermine in urine by gasliquid chromatography. Can. J. pharm. Sci., 3, 73-74. JUN, H.W. & TRIGGS, E.J. (1970). Blood levels of chlorphentermine in man. J. pharm. Sci., 59, 306-309. KARABELNICK, D., ZBINDEN, G. & BAUMGARTNER,
E. (1974). Drug induced foam cell reactions in rats I. Histopathologic and cytochemical observations after treatment with chlorphentermine, RMi 10.393 and Ro 4-4318. Tox. Appl. Pharmac., 27, 395407. KOSTER, W., CALDWELL, J. & SMITH, R.L. (1974). Species variation in the N-oxidation of chlorphentermine. Biochem. Soc. Trans., 2, 881-882. LINDEKE, B., CHO, A.K., THOMAS, T.L. & MICHELSON, L. (1973). Microsomal N-hydroxylation of phenylalkylamines. Identification of Nhydroxylated phenylalkylamines as their trimethylsilyl derivatives by GS/MS. Acta Pharm. Suecica, 10, 493-506. LULLMANN, H., LULLMANN-RAUCH, R. & WASSERMANN, 0. (1973). Drug-induced phospholipidoses. Germ. Med., 3,128-1 38. LULLMANN, H., ROSSEN, E. & SEILER, K.-U. (1973). The pharmacokinetics of phentermine and chlorphentermine in chronically treated rats. J. Pharm. Pharmac., 25, 239-243. LULLMANN-RAUCH, R. (1975). Chlorphentermineinduced cytoplasmic inclusions in peripheral blood cells of rats and guinea pigs. Tox. Appl. Pharmac., 32, 32-39. ROSS, S.B., GOSZTONYI, T. & RENYI, A.L. (1974). Long-term effects of N-hydroxy-4-chloroamphetamine on the level and accumulation of 5-hydroxytryptamine in the rat brain. Eur. J. Pharmac., 28, 222-224. SCHMIEN, R., SEILER, K.-U. & WASSERMANN, 0. (1974). Drug-induced phospholipidosis I. Lipid composition and chlorphentermine content of rat lung tissue and alveolar macrophages after chronic treatment. Naunyn-Schmiedebergs Arch. Pharmac., 283, 331-334. SEILER, K.-U. & WASSERMANN, 0. (1974). Evidence for an unusual distnrbution of chlorphentermine in vivo; an autoradiographic study in mice. NaunynSchmiedebergs Arch. Pharmac., 282, 113-122.
(Received March 5, 1976)
