Fate of the anthelmintic, phenothiazine, in man

Article abstract of DOI:10.1080/00498250210143038

1. Radiolabelled [³⁵S]-phenothiazine has been administered orally to two healthy adult male volunteers (6 mg kg^-1 body weight). Faeces were the major route of excretion of radioactivity (68%), the remainder being eliminated via the urine (32%) with an estimated urinary half-life (biphasic) of 6-16 h. Over the 5 days of the study a complete recovery of radioactivity was achieved. 2. From urinary data, it was shown that metabolism occurred via ring carbon oxidation to form phenothiazone and thionol and via ring sulphur oxidation to form phenothiazine sulphoxide. The majority of urinary material (92%) was present in the form of conjugates of phenothiazine and phenothiazone. Only unchanged phenothiazine was detected in the faeces. Phenothiazine sulphoxide was reduced to phenothiazine during incubation with faecal homogenates.

Full text of DOI:10.1080/00498250210143038

xenobiotica,
vol.
no.
32, 9, 771±782
2002,
Fate of the anthelmintic, phenothiazine, in man
{
S. C. MITCHELL *, P. KESTELL , G. B. STEVENTON and
{
}
}
Biological Chemistry, Division of Biomedical Sciences, Faculty of Medicine, Imperial
R. H. WARING
{
College, South Kensington, London SW7 2AZ, UK.
{
Private Bag 92019, Auckland, New Zealand.
Cancer Research Laboratory, Auckland Medical School, University of Auckland,
§Department of Pharmacy, King’s College London, Franklin-Wilkins Building, 150
Stamford Street, London SE1 8WA, UK.
}
School of Biosciences, University of Birmingham, PO Box 363, Edgbaston, Birmingham
B15 2TT, UK
Received 13 December 2001
1. Radiolabelled [35S]-phe¡nothiazine has been administered orally to two healthy
1
adult male volunteers (6mg kg body weight). Faeces were the major route of excretion
of radioactivity (68%), the remainder being eliminated via the urine (32%) with an
estimated urinary half-life (biphasic) of 6±16 h. Over the 5 days of the study a complete
recovery of radioactivity was achieved.
2. From urinary data, it was shown that metabolism occurred via ring carbon
oxidation to form phenothiazone and thionol and via ring sulphur oxidation to form
phenothiazine sulphoxide. The majority of urinary material (92%) was present in the form
of conjugates of phenothiazine and phenothiazone. Only unchanged phenothiazine was
detected in the faeces. Phenothiazine sulphoxide was reduced to phenothiazine during
incubation with faecal homogenates.
Introduction
The synthesis of phenothiazine (thiodiphenylamine, dibenzothiazine) was ®rst
published in 1883 during researches into the chemical structure of methylene blue
and its derivatives (Bernthsen 1883, 1885). It remained a chemical curiosity for 50
years until it was shown to possess several biological properties (antifungal,
bacteriocidal, insecticidal, vermifugal) and then quickly became regarded as the
panacea for parasitic infestation. It was the ®rst of the `modern’ anthelmintics and
was extensively used during the Second World War enabling the provision of
many tons of desperately needed infection-free food and sheep intestines (catgut
sutures/ligatures). However, its potential adverse reactions including haemolytic
anaemia, dermatitis, photosensitization and neuromuscular incoordination
(Mitchell 1994), coupled with the development of more potent agents, have now
limited its use. Nevertheless, low-dose phenothiazine has been shown to be active
against strains of nematodes that have become resistant to more recently developed
products and this property, together with its relative cheapness, still secures it a
place on the world scene (Kelly et al. 1981, Lyons et al. 1993). Indeed, it is quoted
as the `drug of choice’ for treatment of caecal nematode (Heterakis gallinae)
infestations in poultry (Jacobs et al. 1997).
* Author for correspondence; e-mail: s.c.mitchell@ic.ac.uk
Xenobiotica ISSN 0049±8254 print/ISSN 1366±5928 online
2002 Taylor & Francis Ltd
#
http://www.tandf.co.uk/journals
DOI: 10.1080/00498250210143038

772
S. C. Mitchell et al.
Despite its wide-scale application amongst many animal species, investigations
of the fate of phenothiazine have been limited (Mitchell 1982) and data for man are
scant. This is surprising, since vast amounts of the compound have been used and
exposure in man, through accidental ingestion, inhalation and potential dermal
absorption, has been inevitable. The few studies undertaken (DeEds et al. 1938,
DeEds and Thomas 1941, 1942) have shown the presence of red/purple quinoidal
compounds in human urine after its relatively harsh chemical treatment and
suggest extensive metabolism, but no advantage has been taken of the use of
radiolabelled material. Any estimate of the persistence of phenothiazine within
man necessarily requires the inclusion of all metabolic products, whether known or
not, and would be aided greatly by the incorporation of a readily quanti®able
marker within the parent molecule. The present paper describes the fate of [35S]-
phenothiazine in two adult men.
Materials and methods
Chemicals
Phenothiazine was obtained from Sigma-Aldrich Ltd (Dorset, UK) and puri®ed by recrystallization
8
from diethyl ether (Smith 1938) then aqueous ethanol‡to give light tan crystals, m.p. 181±183 C, UV
absorption peaks at 254 and 317 nm, mass spectrum M m/z 199 (mol. wt 199) (Shine and Mach 1965,
McLa€erty and Stau€er 1989). Radiolabelled phenothiazine was synthesized by re¯uxing elementary
[
35S]-sulphur (Nycomed-Amersham, Amersham, UK) with diphenylamine in the molar ratio of 2:1.3
8
respectively, for 1h at 180 C in 1,2-dichlorobenzene under an atmosphere of nitrogen, 1% (w/w) iodine
being added as a catalyst (Lannung 1941, Massie 1954, Co€ey 1978). The evolved radioactive hydrogen
m
sulphide was trapped in an aqueous solution of FeCl3 (1 ). The solvent was removed by distillation, the
excess diphenylamine precipitated in anhydrous ethereal solution by dry HCl gas and the phenothiazine
recrystal¡lized twice from aqueous ethanol to give light yellow crystals of speci®c activity 189.4
1
8
mCi mol , radiochemical purity >98% (TLC), mixed m.p. 181±183 C, with spectral properties as
above.
Phenothiazine sulphoxide (phenothiazine-5-oxide) was prepared by oxidizing phenothiazine with 3-
chloroperbenzoic acid in cold dry acetone (Beckett et al. 1975) to give light green crystals, m.p. 247±
¡
24‡8 C, UV absorption peaks 229, 271, 303 and 336 nm, IR sharp band at 1070 cm 1, mass spectrum
8
M
m/z 215 (mol. wt 215) (McLa€erty and Stau€er 1989). Phenothiazone (phenothiaz-3-one) was
synthesized by the oxidation of phenothiazine with ferric chloride in hot water (Pummerer and Gabner
1913, Houston et al. 1949a). The crude residue was extracted with hot ethanol and the crystalline
product obtained by chilling (Granick et al. 1940). Further puri®cation by elution from aluminium
oxide columns with chloroform, followed by preparative thin-layer chromatography (TLC) (2.0 mm
8
silica gel G) in carbon tetrachloride/acetone, 4/1 (v/v), gave red crystals, ‡m.p. 161±163 C, UV/vis.
absorption peaks at 236, 258, 272, 368 and 508 nm, mass spectrum M m/z 213 (mol. wt 213)
(McLa€erty and Stau€er 1989). Thionol (7-hydroxyphenothiaz-3-one) was prepared by re¯uxing
8
phenothiazine with 80% (v/v) sulphuric acid at 160±165 C for 6 h, the residue being poured into excess
iced-water and ®ltered (Houston et al. 1949b, Granick and Michaelis 1947). The crude product was
puri®ed by elution with 10% (v/v) aqueous NH4OH from aluminium oxide columns, followed by
preparative TLC (2.0 mm silica gel G) in carbon tetrachloride/acetone, 4/1 (v/v), to give r‡ed/purple
8
crystals, m.p. >300 C, UV/vis. absorption peaks at 232, 276 and 592 nm, mass spectrum M m/z 229
(mol. wt 229).
Human excretion study
Two healthy male volunteers (aged 27 and 28 years, weight 70.5 and 74 kg, height 1.85 and 1.80 m)
were given a single oral dose of [35S]-phenothiazine (423 mg (40.3 Ci) and 444mg (42.3 Ci); dose-rate
m
m
6 mg kg¡ body weight) in a gelatin capsule with water (100 ml) during the early morning at least 2 h
1
after a light breakfast. The subjects were non-smokers and no alcohol or medication was permitted for 1
week before or during the study. Urine was collected for known intervals for the ®rst 0±24h and then
7
daily for the following 4 days, all samples being stored in darkened bottles at 70 C until analysis.
Faeces were collected daily for 0±5 days directly into lyophilization containers (plastic drums) and then
8
7
stored at 70 C until processing. Ethical approval for the study was obtained from the local ethics
committee, following consultation with the local radiation protection panel.
8

Phenothiazine in man
773
Faecal metabolism
The possible contribution to metabolism by the lower gut contents was investigated by the separate
incubation of phenothiazine, phenothiazine sulphoxide, phenothiaz-3-one and thionol (50 mg) with
8
aqueous human faecal homogenate (2 ml) in sealed tubes under nitrogen at 37 C for 48h (Scheline
1966). The solutions were then lyophilized, extracted with methanol and analysed by TLC and HPLC
as described below. Incubates with phosphate bu€er (0.1 m, pH 7.4) instead of homogenates were
included as controls.
Quanti®cation of radioactivity
Aliquots of urine and HPLC eluant (100 l±1.0ml) and areas of silica from thin-layer plates were
m
added directly to vials containing scintillation ¯uid (`Ecoscint’, National Diagnostics, NJ, USA) and
analysed by liquid scintillation spectrometry using a Packard model 385 scintillation counter (Ambac
Industries, Inc., Downers Green, IL, USA), eciency being determined by automatic external
standardization and previously prepared quench-correction curves. Faeces were lyophilized, ground
to a mixed powder, weighed and triplicate samples (about 100mg) combusted in oxygen (Harvey
Biological Material Oxidizer, ICN Tracer Labs, High Wycombe, Surrey, UK), the [35SO2] so
produced being trapped in an alkaline scintillation mixture containing 2-phenylethylamine and then
analysed as above.
Chromatography
Thin-layer chromatography (TLC) was carried out in nitrogen ¯ushed tanks at 4 C in the absence
8
of light on plates coated with silica gel G (20 20 cm, 0.4 mm thick; Merck, Darmstadt, Germany)
£
using solvents A (chloroform/acetone, 9/2 v/v), B (carbon tetrachloride/acetone, 4/1 v/v) and C (hexane/
acetone, 3/2 v/v). Phenothiazine and its derivatives were detected under UV light and by spraying the
dried plates with either (1) 50% v/v aqueous H2SO4, (2) 1% (w/v) I2 in methanol, (3) 0.8% w/v HgI2 in
ethanol (with gentle warming), (4) 5% (w/v) AgNO3 in 10% (v/v) aqueous NH4OH or (5) 1% w/v PdCl2
m
in 60 ml 0.1 HCl plus 40ml ethanol (Elliot 1959).
High-performance liquid chromatography (HPLC) was undertaken using a Perkin-Elmer LC
sample processor ISS200 (PerkinElmer Analytical Instruments, Buckinghamshire, UK) connected to
a Dionex quaternary gradient pump (Dionex Ltd, Camberley, Surrey, UK) in line with an LDC
SpectroMonitor 5000 photodiode array detector (LDC/Milton Roy, Riviera Beach, FL, USA) and a
fraction collector (LKB Bromma; Pharmacia LKB, Piscataway, NJ, USA). Data acquisition was by
Chromquest chromatography software (ThermoFinnigan, Hemel Hempstead, Hertfordshire, UK). A
m
£
reverse phase Zorbax Rx-C8 (5 , 250 4 6 mm) analytical column (Phenomenex UK Ltd, Maccles-
:
®eld, Chesh¡ire, UK) was used together with a gradient elution programme at an overall solvent ¯ow rate
of 1ml min 1. The mobile phase consisted of aqueous acetic acid (0.1% v/v) with its acetonitrile
component increasing linearly from an initial 5% to 90% over 15 min, thereafter rapidly returning to 5%
during the next 3min and remaining at these proportions for another 7 min (a total run time of 25 min).
Investigation of metabolites
Urine (0±24 h) was examined both neat (concentrated by lyophilization where necessary) and
following incubation with a -glucuronidase/sulphatase mixture (type H-1 from Helix pomatia,
-
Sigma-Aldrich) in acetate bu€er (equal vol.; 0.05 , pH 5) at 37 C for 18 h (to hydrolyse any conjugates
m
present). Incubations with standard phenolphthalein glucuronide or p-nitrophenol sulphate con®rmed
8
activity of this enzyme preparation. Lyophilized powdered faeces (0±48h) were extracted with cold
¡
methanol (3 1 ml g 1). The combined extracts were then ®ltered and decreased in volume by rotary
£
evaporation at room temperature under reduced pressure. The extracted faecal residues were lyophil-
ized and combusted as described above to detect any residual radioactivity. Known amounts of
§
[ S]-phenothiazine added to control faeces and then extracted gave recoveries of 88 6 4 2%
and the results obtained were corrected for this.
35
… ˆ †
n
6
:
:
m
These ¯uids (urine, incubates, extracts) were examined, as spots (10±50 l) or streaks (100±500 l),
by TLC in the solvent systems A, B and C. On some plates where urine spots had been used (as opposed
m
m
to streaks), once the non-conjugated material had been removed, cold HCl (9.7 ; about 100 l) was
applied to the origin, and the plates dried and rerun at an angle of 90 in the same solvent to give a two-
m
8
dimensional separation. When developed and dried, the thin-layer plates were scanned directly for
radioactivity using a Berthold Series 272 computerized radiochromatogram scanner (Berthold Instru-
ments, Inc., Aliquippa, PA, USA) or otherwise cut into 0.3-cm strips and analysed for radioactivity as
described above. Quanti®cation was also undertaken by adding the relevant eluant fractions obtained
from HPLC examination to scintillation ¯uid and counting as above. Authentic metabolites, dissolved
in urine or methanolic extracts of faeces, were co-chromatographed by both TLC and HPLC to provide
provisional identi®cation. Radioactive areas removed from preparative plates (2 mm thick) were
extracted with methanol, concentrated, redissolved in minimum quantities of methanol and, after
®ltration, examined by UV and mass spectral techniques. Similarly, the collection of relevant HPLC

774
S. C. Mitchell et al.
eluant fractions, with their lyophilization and reconstitution in methanol, provided further samples for
spectral analysis.
Spectrometry
UV and visible spectra were recorded in methanolic solution on a Pye-Unicam SP 1800 UV
spectrophotometer (Pye-Unicam, Cambridge, UK). IR spectra were measured using a potassium
bromide disc and a Pye-Unicam SP200 IR spectrophotometer (Pye-Unicam). Electron impact mass
spectrometry was carried out on a Kratos MS80 instrument (Kratos Ltd, Manchester, UK) with Kratos
D555 (data generator) computerized display and printout facilities. The sample was inserted directly
8
into the ionization chamber at 70 eV with a source temperature of 200 C.
Results
Excretion study
The studies showed similar results for both subjects. About one-third of the
administered radioactivity was recovered from the urine, with the majority (about
26% dose, about 84% of that in the 0±5-day urine) being voided during the ®rst
day. The portion of the dose that was absorbed appeared to be so quite rapidly;
with 9% of the radioactive dose (about 29% of that in the 0±5-day urine) being
detected in the urine within 0±3 h. The remaining two-thirds of the radioactive
dose was recovered from the faeces over 3±4 days with excretion tapering o€
towards the end of the study. Overall, excellent total recoveries (98.4 and 100.4%)
were achieved (table 1).
Metabolism
Separate examination by TLC of the complete 0±24-h urine collected from
each volunteer showed the presence of ®ve areas of radioactivity at various
distances along the plate from the origin to the solvent front. This pattern was
identical for both subjects. For identi®cation purposes the RF obtained in all three
TLC solvents systems are given in the order A, B and C.
Table 1. Excretion of radioactivity by two adult male subjects
following the oral administration of
(6 mg/kg body weight).
[
35S]-phenothiazine
Subject 1
Subject 2
Period
Day 1
Urine
Faeces
Urine
Faeces
0±3 h
3±6 h
6±9 h
9±12h
12±18 h
18±24 h
8.4
8.0
3.3
2.0
2.7
1.3
25.7
3.6
0.9
0.2
0.1
10.2
7.2
4.8
2.0
1.8
1.2
27.2
4.2
1.1
0.1
0.1
Day 1
19.6
32.0
14.0
4.2
28.8
29.8
6.7
0.3
0.1
Day 2
Day 3
Day 4
Day 5
0.1
Subtotal
Total
30.5
69.9
32.7
98.4
65.7
100.4

Phenothiazine in man
775
Figure 1. Structure of the anthelmintic, phenothiazine, and its oxidized metabolites, phenothiazone
(phenothiaz-3-one), phenothiazine sulphoxide (phenothiazine-5-oxide) and thionol (7-hydro-
xyphenothiaz-3-one).
Area 1 (RF 0.0, 0.0, 0.0) (conjugated material)
The majority of radioactivity within the urine (91.0, 92.3%) remained on the
TLC plate origin, where the urine sample had been applied, in all of the solvent
systems examined. Two-dimensional chromatography following in situ acid
hydrolysis of the material on the origin, and examination of urine following
enzyme hydrolysis, both demonstrated no radioactivity at the TLC plate origin
but displayed concomitant increases in phenothiazone (area 4) and phenothiazine
(area 5). This general pattern was con®rmed by HPLC examination.
Cold butanol extraction of powdered lyophilized urine provided a radioactive
product that remained on the TLC origin and was unstable in concentrations of
m
cold HCl >0.5 , decomposing to yield phenothiazine and glucuronic acid only, as
detected by TLC (Elliot 1959). This conjugate was tentatively assigned the
structure of phenothiazine N-glucuronide (Mitchell and Waring 1979). Rapid
cooling of concentrated urine (by lyophilization) provided a crude white precipi-
tate that turned purple on heating in air. This precipitate provided a radioactive
m
product that remained on the TLC origin and decomposed in warm HCl (9.7 ) to
yield phenothiazone and inorganic¡sulphate (Mitchell and Waring 1978). Infrared
analysis (bands at 1100±1200 cm 1, sulphur-oxygen bond vibrations, covalent
sulphate) (Bellamy 1954, Avram and Mateescu 1972) and a mass spectrum (inter
alia, mass peak at m/z 278, M‡-16) were consistent with the tentative assignment
of this conjugate as leucophenothiazone sulphate (Collier et al. 1943, Clare 1962,
Mitchell and Waring 1979).
Area 2 (RF 0.26, 0.19, 0.42) (thionol)
This radioactive metabolite co-chromatographed with authentic thionol (7-
hydroxyphenothiaz-3-one) in all TLC solvent systems and by HPLC (RT 5.8 min)
following methanolic elution. The pink/purple tint was decolourized by covering

776
S. C. Mitchell et al.
the TLC spot with a saturated solution of sodium dithionite. This reduces thionol
to leucothionol (3,7-dihydroxyphenothiazine) thereby removing the quinoidal
chromophore. Authentic thionol behaved in an identical manner. Absorption
maxima were recorded at 232, 276 and 592 nm and mass spectral analysis gave a
molecular ion at m/z 229 and a fragmentation pattern identical to the authentic
compound (table 2).
Area 3 (RF 0.35, 0.13, 0.37) (phenothiazine sulphoxide)
Co-chromatography of this radioactive excretion product was obtained with
phenothiazine sulphoxide (phenothiazine-5-oxide) in all TLC solvent systems and
by HPLC (RT 4.0 min) following methanolic elution. Characteristic ultraviolet
absorption maxima were observed at 229, 271, 303 and 336 nm. The molecular ion
obtained for this metabolite was at m/z 215 with a fragmentation pattern identical
to the authentic compound (McLa€erty and Stau€er 1989, Sutherland et al. 2001)
(table 2).
Area 4 (RF 0.87, 0.77, 0.75) (phenothiazone)
This radioactive compound was shown clearly to co-chromatograph with
authentic phenothiazone (phenothiaz-3-one) in all three TLC solvent systems
employed and also by HPLC (RT 7.4 min) when eluted and examined. Absorption
maxima were recorded at 236, 258, 272, 368 and 508 nm and mass spectral analysis
of this material gave a molecular ion at m/z 213 and fragment ions identical to those
obtained from the authentic compound (McLa€erty and Stau€er 1989, Sutherland
et al. 2001) (table 2).
Area 5 (RF 0.96, 0.87, 0.91) (phenothiazine)
Co-chromatography with authentic phenothiazine was achieved for this radio-
active metabolite in all three solvent systems used for TLC and also by HPLC (RT
13.7 min) following elution and rechromatography. Distinctive colorations,
produced by authentic phenothiazine, were also noticed with various TLC
reagents; green with ammoniacal AgNO3, blue with PdCl2 and brown with I2
and HgI2. UV absorption maxima were recorded at 254 and 317 nm. Mass spectral
examination gave a molecular ion at m/z 199 and an expected splitting pattern
characteristic of this molecule (Shine and Mach 1965, McLa€erty and Stau€er
1989) (table 2).
For both subjects, the majority of radioactivity present within the urine was in
the form of conjugated material (91.0 and 92.3% of the total radioactivity in the 0±
24 h urine corresponding to 23.4 and 25.1% of the administered radioactive dose).
Non-conjugated metabolites together accounted for <10% (9.0 and 7.7% in urine;
2.3 and 2.1% dose), with phenothiazine sulphoxide being the most prominent
(table 3).
The only radioactive component found in methanolic extracts of faeces showed
chromatographic and spectral properties identical to those of the parent com-
pound, phenothiazine (table 2). Methanolic extraction, although ecient in
35
§
removing [ S]-phenothiazine added to control faeces, only removed 82 4 5 2%
:
:

Phenothiazine in man
777

778
S. C. Mitchell et al.
Table 3. Excretion of metabolites in urine (0±24 h) following the ingestion of [35S]-phenothiazine
(6 mg/kg) by two male subjects
Percentage radioactivity
in urine (0-24 h)
Percentage of the
administered radioactive dose
Metabolite
Subject 1
Subject 2
Subject 1
Subject 2
Phenothiazine
2.4
4.0
2.1
0.5
1.9
3.3
1.5
1.0
0.6
1.0
0.6
0.1
0.5
0.9
0.4
0.3
Phenothiazine sulphoxide
Phenothiazone
Thionol
Phenothiazine conjugate*
Phenothiazone conjugate**
53.2
37.8
62.2
30.1
13.7
9.7
16.9
8.2
100.0
100.0
25.7
27.2
The conjugates have been tentatively assigned the structures of *phenothiazine N-glucuronide and
**leucophenothiazone sulphate.
of the radioactivity associated with the faeces. This meant that the chemical
identity of around 10% (9.1%, 10.3%) of the radioactive material within the faeces
(0±48 h) obtained from both subjects was unaccounted for; probably remaining
bound to the cellulose and other polyphenolic components of the faeces. Incuba-
tion of metabolites with faecal homogenates produced no chemical alterations to
phenothiazine or its derivatives except for the almost quantitative (90%+) reduc-
tion of phenothiazine sulphoxide to phenothiazine (data not shown).
Discussion
In agreement with the present results for man, other workers have reported low
urinary recoveries following oral administration; 5 and 10% for sheep (Lipson
1940, Swales and Collier 1940), 14% for the horse (Swales et al. 1942), 23% for the
rabbit (Benham 1945) and 25% for the cow (Guyton et al. 1976). In addition, traces
of large doses of phenothiazine have been shown to remain within calves for
between 5 and 15 days (Sundukov 1963) and in sheep for 14±16 days (Dobryina
1963). However, the proportion of the dose that does enter the body appears to be
absorbed quickly from the upper gastrointestinal tract and then rapidly voided in
the urine. The greatest rate of urinary excretion of a single dose of phenothiazine
has previously been shown to occur within 0±9 h in the cow, sheep and horse
(Swales and Collier 1940, Swales et al. 1942, Ellison and Todd 1957) and this is
consistent with the ®ndings presented here for man. Simple graphical plotting
of the present 0±24-h urinary data was suggestive of a biphasic excretion pattern
with an initial half-life of around 6 h (0±12 h period) and a latter half-life of 16 h
(12±24-h period). However, it must be appreciated that there are only a few data
points during this ®rst day, and there are only two subjects. Recalculated urinary
data from the literature gives half-life values ranging from 11 to 18 h for both the
sheep and the horse (Swales and Collier 1940, Swales et al. 1942).
The delay in excretion via the gastrointestinal tract is indicative of a decreased
transit time through the gut, perhaps assisted by enterohepatic cycling (DeEds and
Thomas 1941, Davey and Innes 1942, Mitchell 1982). This cycling may be a
mechanism that prolongs its residence and assists its mode of action as an
anthelmintic. Local e€ects of phenothiazine on the gut may also produce a

Phenothiazine in man
779
decrease in gastrointestinal motility. Phenothiazine has been shown to inhibit
horse serum cholinesterase (Collier 1942, Collier and Allen 1942) and interfere
with neuromuscular transmission in the crab (Collier 1940b). This has led to the
suggestion that phenothiazine acts, in part, by paralysing the infesting helminths,
thereby causing their detachment from anchorage and promoting their passage
through and expulsion from the gut (Collier 1942, Mackie and Raeburn 1952,
Mackie 1953). Colonic impaction has been reported in horses that have received
large doses of phenothiazine but, conversely, so has diarrhoea (Habermann et al.
1941, Wolfe and Dennis 1941). The two volunteers in the present study reported
no gastrointestinal disturbances although gut irritation in man has been cited
elsewhere (Nour El-Din 1946). Persistence of phenothiazine, observed in some
animals as measured by trace amount in their urine, may be related to the relatively
large doses that these animals have received (up to 1 kg in the horse; Habermann et
al. 1941). The e€ective formation of a slow moving bolus within the gut and the
slight but constant absorption from this reservoir mimics a multiple or continuous
dosing situation which would persist for many days following the original oral
administration.
The pattern of metabolism found in man is in general agreement with that
found for other species examined and there is nothing unusual or unexpected in
the human pro®le. All of the phenothiazine metabolites identi®ed in the present
study have been previously reported (Mitchell 1982). The carbon oxidation
products, phenothiazone and thionol have been demonstrated in the urine of the
cow, dog, gerbil, horse, mouse, pig, rabbit, rat and sheep and the sulphur oxidation
product, phenothiazine sulphoxide in the cow, guinea pig and sheep (Lipson 1940,
Swales and Collier 1940, Taylor and Sanderson 1940, DeEds and Thomas 1941,
Swales et al. 1942, Collier et al. 1943, Clare 1947, 1962, Richardson and Todd
1958, Mitchell and Waring 1979, Mitchell 1980). Conjugated forms of phenothia-
zine (phenothiazine-N-glucuronide) and phenothiazone (leucophenothiazone sul-
phate) have been also previously reported in the urine from the cow, gerbil, guinea
pig, hamster, pig, rat and sheep (Lipson 1940, Swales and Collier 1940, Taylor and
Sanderson 1940, DeEds and Thomas 1941, Swales et al. 1942, Collier et al. 1943,
Clare 1947, 1962, Richardson and Todd 1958, Mitchell and Waring 1979, Mitchell
1980). The glucuronic acid conjugate of thionol appears to be absent from human
urine, unlike that of the gerbil, mouse, rabbit and rat (Benham 1945, Mitchell
1980).
Phenothiazine is the only compound that has been positively identi®ed as being
present in the faeces of cows, guinea pigs, horses, rabbits, rats and sheep,
presumably unabsorbed, although trace amounts of phenothiazone and thionol
have been found in rat faeces and phenothiazone and the sulphoxide in guinea pig
faeces (Collier 1940a, Lipson and Gordon 1940, Swales et al. 1942, Richardson
and Todd 1958, Waddell 1959, Mitchell and Waring 1979, Mitchell 1982).
Phenothiazone and phenothiazine sulphoxide have been detected in the gut of
sheep distal to the bile duct and in vitro incubation experiments with rumen
contents have shown that phenothiazine was not converted into any detectable
derivatives, except for minute traces of phenothiazone ascribed to air oxidation
(Waddell 1959), suggesting that the metabolites probably entered the enteric tract
via the bile (Clare 1947, Harpur et al. 1950a, b). Phenothiazine and thionol have
been observed in bile from one human subject but the harsh chemical detection
methods employed did not permit di€erentiation between phenothiazine and its

780
S. C. Mitchell et al.
N-glucuronide or even between the C-oxidation products (DeEds and Thomas
1941). No such metabolites made their way into the faeces to be detected in the
present study.
By analogy with its water-soluble N-substituted derivatives, the carbon and
sulphur oxidations of phenothiazine are presumably undertaken by cytochromes
P450 as these positions present favourable electromolecular environments for these
enzyme systems. Delocalization of electrons from the sulphur (and nitrogen)
moiety into the adjacent unsaturated tricyclic ring system decreases the nucleo-
philicity of this heteroatom and renders it less susceptible to oxidation by the ¯avin
monoxygenase family of enzymes. The lipophilic nature of phenothiazine
(log P…
† 3.88, log P… † 4.15; Cymerman-Craig and Warburton 1956,
octanol
heptane
m
Leo et al. 1971) and its negligible solubility in water (both 51 and 125 g
100 ml¡ quoted; Davey and Innes 1942, Cymerman-Craig and Warburton
1
1956), although presumably aiding its uptake and retention within the cuticle of
the parasite (Lazarus and Rogers 1951), prevents in vitro investigations and
precludes any attempts at intravenous studies. Investigations are therefore limited
and any useful data obtained concerning the interaction of phenothiazine with
living systems are thus welcomed.
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