Further evidence of extensive pulmonary first-pass ester hydrolysis after airways administration in rats

Article abstract of DOI:10.1211/146080800128735485

The aim of this study was to synthesize an ester with low lipophilicity, and to use this ester to further investigate pre-absorptive pulmonary first-pass metabolism. Hexanoic acid phenethyl ester was synthesized by reacting 2-phenylethanol with hexanoyl chloride. Pre-absorptive first-pass metabolism was assessed by comparing the areas under the blood concentration-time curves after intra-arterial administration of the hexanoic acid phenethyl ester with those after intratracheal instillation. Hexanoic acid phenethyl ester experienced extensive first-pass metabolism (53% of the absorbed dose) before or during absorption. This and earlier data suggests that the extent of this first-pass extraction is dependent on the physicochemical properties of the ester and in particular whether a compound experiences diffusion-limited absorption. Pre-absorptive pulmonary first-pass metabolism of compounds whose absorption iS diffusion-rate limited may be extensive even when pulmonary enzyme expression is low. This has consequences for the systemic delivery of drugs and in particular esters via the lungs.

Full text of DOI:10.1211/146080800128735485

Pharm. Pharmacol. Commun. 2000, 6: 441±445
Received May 11, 2000
Accepted July 7, 2000
# 2000 Pharm. Pharmacol. Commun.
Further Evidence of Extensive Pulmonary First-pass Ester
Hydrolysis after Airways Administration in Rats
PAUL A. DICKINSON, PETER C. SEVILLE AND GLYN TAYLOR
The Welsh School of Pharmacy, Cardiff University, Cathays Park, Cardiff CF10 3XF, UK
Abstract
The aim of this study was to synthesize an ester with low lipophilicity, and to use this ester
to further investigate pre-absorptive pulmonary ®rst-pass metabolism.
Hexanoic acid phenethyl ester was synthesized by reacting 2-phenylethanol with hex-
anoyl chloride. Pre-absorptive ®rst-pass metabolism was assessed by comparing the areas
under the blood concentration±time curves after intra-arterial administration of the hex-
anoic acid phenethyl ester with those after intratracheal instillation. Hexanoic acid phen-
ethyl ester experienced extensive ®rst-pass metabolism (53% of the absorbed dose) before
or during absorption. This and earlier data suggests that the extent of this ®rst-pass
extraction is dependent on the physicochemical properties of the ester and in particular
whether a compound experiences diffusion-limited absorption.
Pre-absorptive pulmonary ®rst-pass metabolism of compounds whose absorption is
diffusion-rate limited may be extensive even when pulmonary enzyme expression is low.
This has consequences for the systemic delivery of drugs and in particular esters via the
lungs.
Using a series of biphenylacetic acid esters we
recently demonstrated extensive pre-absorptive
pulmonary ®rst-pass metabolism (after airways
administration) but little post-absorptive ®rst-pass
metabolism (after intra-vascular presentation)
(Dickinson & Taylor 1998). The in-vitro enzyme
kinetics predicted the extent of post-absorptive
®rst-pass metabolism, however they did not predict
the pre-absorptive ®rst-pass metabolism. The pre-
absorptive metabolism was substantially greater
than predicted by the in-vitro enzyme kinetics and
in the inverse rank order for the esters investigated
(Dickinson & Taylor 1998). Reasons for this lack
of correlation between the in-vitro enzyme kinetics
and pre-absorptive ®rst-pass metabolism, include
the possibility that the esters may have encountered
the esterases at a site where blood ¯ow was much
less than that of the whole organ (i.e. the well-
stirred model did not apply), or that a distribution-
limited rather than a perfusion-limited kinetic
model applied. That is, the esters investigated were
all highly lipophilic and therefore may have
exhibited poor permeability across the pulmonary
mucosa, their absorption, therefore, being diffu-
sion-limited rather than perfusion-limited.
The aim of this study was to investigate the pre-
absorptive pulmonary ®rst-pass metabolism of a less
lipophilic ester to elucidate the reasons for the
observed pre-absorptive pulmonary ®rst-pass meta-
bolism. An ester of 2-phenylethanol was chosen for
investigation as 2-phenylethanol has a similar struc-
ture to some commonly used b-agonists.
Materials and Methods
All reagents were analytical grade, except aceto-
nitrile which was HPLC grade. 2-Phenylethanol
and Pluronic F-68 were obtained from Sigma
(Poole, UK). Acetonitrile, triethylamine, petroleum
ether, ethyl acetate and tri¯uoroacetic acid were
purchased from Fisher Chemicals (Loughborough,
UK). Hexanoyl chloride, octanoyl chloride and
dichloromethane were obtained from Aldrich
Chemical Company (Poole, UK). Magnesium sul-
phate was purchased from Acros Organics (Geel,
Belgium). 1-(p-Tolyl) ethanol was from Fluka
Chemika (Buchs, Switzerland). The research
Correspondence: P. A. Dickinson, The Welsh School of
Pharmacy, Cardiff University, Cathays Park, Cardiff CF10
3XF, UK.
E-Mail: dickinsonpa@cardiff.ac.uk

442
PAUL A. DICKINSON ET AL
adhered to the Code of Practice for the Housing and
Care of Animals Used in Scienti®c Procedures
(HMSO publication HC107) and the Universities
Federation for Animal Welfare Guidelines.
the tracheal bifurcation. The dose was then instilled
over a 20-s period during which time the rat was
inclined at an angle of 35ꢀ to ensure retention and
distribution of the dose within the lung. Intra-
1
arterial doses (44Á4 mg kg 1 and 8Á9 mL kg ) were
administered as 10-min zero-order infusions using
a syringe pump, via the carotid artery. Separate
groups of four rats each were used for each dosing
route.
Blood samples (250 mL) were collected from the
femoral artery, at selected times up to 90 min after
dosing, and immediately added to micro-
centrifugation vials containing internal standard in
acetonitrile (375 mL) and stored at 4±8ꢀC until
analysis.
Synthesis of 2-phenylethanol±hexanoic acid ester
model prodrug
Dry dichloromethane (60 mL) was added to 2-
phenylethanol (5Á0 g, 40Á9 mmol) and triethylamine
(5Á6 mL, 40Á9 mmol). The solution was stirred
rapidly over ice (0ꢀC). A solution of hexanoyl
chloride (5Á7 mL, 40Á9 mmol) in dry dichloro-
methane (30 mL) was slowly added dropwise and
then the reaction was maintained at 0±5ꢀC with
stirring under nitrogen for 3Á5 h. The reactants were
concentrated under reduced pressure. Ethyl acetate
(100 mL) was added to dilute the residue (pre-
cipitation of triethylammonium chloride occurs).
The ethyl acetate layer was washed with water
(4 6 50 mL). The combined aqueous layers were
washed with ethyl acetate (3 6 50 mL). The
majority of the product was found in the initial
ethyl acetate layer, which was dried over anhydrous
magnesium sulphate and concentrated under
reduced pressure. This yielded a yellow liquid. The
progress of the reaction was monitored by thin-
layer chromatography, using petroleum ether±ethyl
acetate (12 : 1 v=v) as the mobile phase, giving a
spot at RF 0Á64 that stained green on heating with
vanillin dip.
After intratracheal dosing the lungs were
removed at the end of the experiment to determine
the amount of hexanoic acid phenethyl ester and
2-phenylethanol remaining.
Assay of hexanoic acid phenethyl ester and
2-phenylethanol
2-Phenylethanol and 2-phenylethanol prodrug
concentrations were determined by isocratic
reverse-phase HPLC using a Fluo®x column at
ambient temperature, with detection at 213 nm;
Promis II Injection System (Spark Holland,
Emmen, Netherlands); Constametric 3000 solvent
delivery system (Laboratory Data Control, Stone,
UK); Spectrasystem UV1000 detector (Thermo-
quest, Withenshaw, UK); Berthold Version 1Á65
integration software (Herts, UK). 2-Phenylethanol
was assayed using a mobile phase consisting of
acetonitrile±0Á1% tri¯uoroacetic acid (20 : 80 v=v)
Determination of hexanoic acid phenethyl ester
pre-absorptive pulmonary ®rst-pass metabolism
Male Wistar rats, 271±330 g, were anaesthetized
1
by intra-peritoneal administration of 0Á26 mg kg
1
at a ¯ow rate of 1 mL min . 2-Phenylethanol
1
fentanyl citrate, 8Á3 mg kg ¯uanisone (Hypnorm;
eluted with a retention time of approximately
10Á5 min. 1-( p-Tolyl) ethanol, used as an internal
standard, eluted with a retention time of approxi-
mately 19Á5 min. Hexanoic acid phenethyl ester
was assayed using acetonitrile±0Á1% tri¯uoroacetic
acid (60 : 40 v=v) as the mobile phase at a ¯ow rate
Janssen Animal Health, High Wycombe, UK) and
1
4Á16 mg kg midazolam (Hypnovel; Roche Pro-
duct Ltd, Welwyn Garden City, UK). Anaesthesia
was maintained by the intra-peritoneal administra-
1
tion of 0Á087 mg kg
fentanyl citrate and
1
2Á8 mg kg ¯uanisone every 30 min. In all rats the
trachea was cannulated with polythene tubing
(2Á08 mm o.d.). The femoral artery and, where
necessary, the left carotid artery were cannulated
with polythene tubing.
1
of 1 mL min . Hexanoic acid phenethyl ester
eluted with a retention time of approximately
9 min. Octanoic acid phenethyl ester, used as an
internal standard, eluted with a retention time of
approximately 13 min.
Hexanoic acid phenethyl ester was administered
intratracheally neat. The intra-arterial doses of
hexanoic acid phenethyl ester were administered as
0Á5% w=v hexanoic acid phenethyl ester, 5% w=v
Pluronic F-68 normal saline (94Á5% v=v) oil in
water microemulsions. The intratracheal doses
Pharmacokinetic analysis
A non-linear least-squares regression program
(Minim; R. D. Purves, University of Otago, New
Zealand) was used to analyse the intra-arterial 10-
min infusion and intratracheal data. The area under
the blood concentration±time curve (AUC) was
1
1
(177Á4 mg kg and 182Á9 mL kg ) were adminis-
tered by instillation from a glass syringe with a
blunt needle inserted into the trachea to the point of

PULMONARY FIRST-PASS ESTER METABOLISM
443
Table 1. Hexanoic acid phenethyl ester and 2-phenylethanol
areas under the blood concentration±time curve (AUC) after
administration of hexanoic acid phenethyl ester.
determined using the trapezoidal rule and extra-
polation to in®nity using the terminal rate constant.
The dose fraction escaping pre-absorptive (pre-
systemic) ®rst-pass metabolism (fP.p.) was calcu-
lated from (Dickinson & Taylor 1998):
Administration
route
Hexanoic acid
phenethyl ester
AUC0±? (mg min mL
2-Phenylethanol
AUC0±60
1
1
)
(mg min mL
)
fP:p: ˆ AUCi:t:=ꢁAUCi:a:  fa†
ꢁ1†
Intra-arterial
Intratracheal
0Á12Æ 0Á04
0Á04Æ 0Á01
0Á51Æ 0Á20
0Á27Æ 0Á05
Where AUCi.a. and AUCi.t. are the hexanoic acid
phenethyl ester AUC after intra-arterial and intra-
tracheal hexanoic acid phenethyl ester administra-
tion; fa is the fraction of the dose absorbed in any
form (i.e. as intact hexanoic acid phenethyl ester or
metabolized to 2-phenylethanol) after intratracheal
administration and calculated from:
Data are meanÆ s.e.m., n ˆ 4. All values are normalized to
the intra-arterial dose.
hexanoic acid phenethyl ester and 2-phenylethanol
after intra-arterial and intratracheal administration
of hexanoic acid phenethyl ester are shown in
Figure 2.
fa ˆ PE AUCi:t:=PE AUCi:a:
ꢁ2†
After intra-arterial administration of hexanoic
acid phenethyl ester the exponential decline in
hexanoic acid phenethyl ester blood concentration
was very rapid, with a half-life of less than 2 min.
The total body clearance of hexanoic acid phen-
Where PE AUCi.t. and PE AUCi.a. are the 2-
phenylethanol metabolite AUC after intratracheal
and intra-arterial administration of hexanoic acid
phenethyl ester. The pulmonary ®rst-pass extrac-
tion ratio (EP.p.) was determined from:
ethyl ester after intra-arterial administration was
(meanÆ s.e.m., n ˆ 4) and
EP:p: ˆ 1 fP:p:
ꢁ3†
1
27Á9Æ 7Á1 L h 1 kg
1
the volume of distribution was 1Á2Æ 0Á8 L kg
(meanÆ s.e.m., n ˆ 4).
After intratracheal instillation, hexanoic acid
phenethyl ester had low bioavailability. The post-
peak hexanoic acid phenethyl ester blood con-
centrations declined mono-exponentially, with a
half-life of 27Á0Æ 8Á9 min (meanÆ s.e.m., n ˆ 4),
Results and Discussion
Synthesis of hexanoic acid phenethyl ester
The synthesis gave a yield of 7Á67 g equivalent to
85% hexanoic acid phenethyl ester (Figure 1).
Analysis, found: dH (CDCl3) 0Á95 (3H, m, CH3);
0
0
1Á35 (4H, m,6 CH2 and7 CH2); 1Á66 (2H, quintet, J
0
0
7Á3 Hz,5 CH2); 2Á35 (2H, t, J 7Á5 Hz, 4 CH2); 3Á00
0
(2H, t, J 7Á0 Hz,1 CH2); 4Á36 (2H, t, J 7Á1 Hz,
0
2 CH2); 7Á29 (3H, m, PhH); 7Á37 (2H, m, PhH) for
hexanoic acid phenethyl ester. The product was
greater than 95% pure by HPLC and exhibited a
retention time that would be predicted for hexanoic
acid phenethyl ester.
Determination of hexanoic acid phenethyl ester
pre-absorptive pulmonary ®rst-pass metabolism
Table 1 gives the AUC of hexanoic acid phenethyl
ester and the metabolite, 2-phenylethanol, after
administration of hexanoic acid phenethyl ester.
The geometric mean concentration±time curves of
Figure 2. Hexanoic acid phenethyl ester (PEH) and 2-phe-
nylethanol (PE) blood concentration±time pro®les after intra-
arterial 10-min infusion (d, s, respectively) and intratracheal
1
instillation (r, e, respectively) of 44Á4 mg kg
and
hexanoic acid phenethyl ester, respectively.
1
Figure 1. Structure of 2-phenylethanol and hexanoic acid
phenethyl ester.
177Á4 mg kg
Data are the geometric meanÆ s.e.m., n ˆ 4.

444
PAUL A. DICKINSON ET AL
which is signi®cantly longer than the terminal half-
life after intra-arterial administration of hexanoic
acid phenethyl ester. This indicates that the phar-
macokinetics of intratracheally administered hex-
anoic acid phenethyl ester are absorption rate-
limited.
water partition coef®cient) of the biphenylacetic
acid esters (6Á353 to 9Á527). It is probable that the
lower C log P of hexanoic acid phenethyl ester
(4Á24) explains the less extensive extraction mea-
sured and suggests that the ester physicochemical
properties, and in particular ester lipophilicity,
determines the extent of pre-absorptive ®rst-pass
metabolism. This can be rationalized by applying
a diffusion-limited model to the in-vivo data. That
is, the diffusion of the drug into the blood stream
and thus residence time in the lung tissue controls
the extent of metabolism. The maximum diffusion
rate can be expected to occur at a log P of
approximately 2 (Taylor 1990); as the log P
increases above this value the partitioning rate of
the compound from the lipophilic lung tissue into
the relatively hydrophilic blood will slow and
metabolism increase.
If diffusion-limited, rather than perfusion-
limited, metabolism is occurring this has implica-
tions for the systemic delivery of drugs, in
particular biotechnological drugs (e.g. peptides and
proteins) via the lungs. The lungs are known to
express a wide range of enzymes capable of
metabolizing biotechnological drugs (Wall &
Lanutti 1993; Forbes et al 1997). The absorption of
biotechnological drugs from the lungs is undoubt-
edly diffusion-limited; that is, the alveolar epithe-
lium presents a substantial barrier to pulmonary
absorption even for small peptides (Evans et al
1998). This suggests that even though peptidase
expression in the lungs is relatively low, extensive
®rst-pass metabolism of biotechnological drugs is
likely to occur. Although many studies have
determined the bioavailable fraction after airway
administration of biotechnological drugs (Patton
1996), the relative importance of incomplete
absorption versus ®rst-pass metabolism has not
been investigated. This is probably because of the
need to assay parent biological and metabolite
concentrations in blood if ®rst-pass metabolism is
to be determined (Dickinson & Taylor 1998).
One other explanation for the extensive pre-
absorptive pulmonary ®rst-pass metabolism
observed for the esters is the presence of p-glyco-
protein transporters at the epithelial membrane
(Abulrob et al 1999). If substrates for p-glycopro-
tein, these esters will be continually ef¯uxed back
into the pulmonary airways leading to the potential
for several ®rst-pass metabolism episodes to take
place before the esters can enter the systemic
circulation.
The AUC of a metabolite after administration of
the parent compound may be used as an indicator
of the dose of the parent compound absorbed or
administered (Houston 1982). Using this technique
and the 2-phenylethanol AUC0±60, the fraction of
hexanoic acid phenethyl ester absorbed (fa) after
intratracheal instillation was 0Á54 (Eqn. 2), sug-
gesting that approximately 50% of the intratracheal
dose of hexanoic acid phenethyl ester reaches the
circulation in some form. However, hexanoic acid
phenethyl ester is subject to absorption rate-limited
pharmacokinetics and therefore fa is probably
underestimated in this instance, as at 60 min hex-
anoic acid phenethyl ester was still being absor-
bed=hydrolysed after intratracheal administration.
After intra-arterial administration practically all the
hexanoic acid phenethyl ester would be eliminated
by 60 min (approx. equiv. 30 terminal half-lives).
This means that the PE AUC0±60,i.a. accounts for a
greater proportion of PE AUC0±?,i.a. than PE
AUC0±60,i.t. does for PE AUC0±?,i.t., therefore
underestimating fa. If fa is underestimated then EP.p.
will also be underestimated.
In an attempt to conclusively quantify fa, the rat
lungs were removed at the end of the experiment
and analysed for hexanoic acid phenethyl ester and
2-phenylethanol. It was found that 30Æ 6%
(meanÆ s.e.m., n ˆ 4) of the administered dose
remained in the lungs as hexanoic acid phenethyl
ester, while 11Æ 1% (meanÆ s.e.m., n ˆ 4)
remained in the lungs as 2-phenylethanol. This
indicates that fa was 0Á59. This is in close agree-
ment with the fa (0Á54) derived from the 2-phenyl-
ethanol AUC0±60 data, suggesting this method
produces a good estimate of the fraction of dose
absorbed.
Comparison of the hexanoic acid phenethyl ester
AUCi.t. with AUCi.a. indicates a pre-absorptive pul-
monary ®rst-pass extraction of 46 or 53% (Eqn. 1) for
fa ˆ 0Á54 and 0Á59, respectively. That pre-absorptive
®rst-pass metabolism occurred was strongly indi-
cated by the fact that 11% of the administered dose
remained in the lungs (equiv. 27% of the material in
the lung) as 2-phenylethanol.
An extraction ratio of 0Á53 is less than that
reported previously for aliphatic esters of bi-
phenylacetic acid (Dickinson & Taylor 1998). The
extraction ratio for biphenylacetic acid esters
ranged from 0Á83 to 0Á99 and was in the same
rank order as the C log P (computed log octanol±
In summary a model ester with low lipophilicity
has been synthesized and a blood assay developed.
The pre-absorptive pulmonary ®rst-pass metabo-
lism of the ester was approximately 50%. This,

PULMONARY FIRST-PASS ESTER METABOLISM
445
Dickinson, P. A., Taylor, G. (1998) Route dependent pulmon-
ary ®rst-pass metabolism of a series of biphenylacetic acid
esters in rats. Eur. J. Pharm. Sci. 6: 11±18
Evans, J. P., Dickinson, P. A., Farr, S. J., Kellaway, I. W.,
Tudball, N. (1998) Transport of a series of D-phenylalanine-
glycine hexapeptides across rat alveolar epithelia in vitro. J.
Drug Target. 6: 251±259
together with previously published data, suggests
that pre-absorptive pulmonary ®rst-pass metabo-
lism for compounds whose absorption is diffusion-
rate limited may be extensive even when pulmon-
ary enzyme expression is low.
Forbes, B., Campbell, L., Dickinson, P. A., Wilson, C. G.,
Gumbleton, M. (1997) Differential activity of plasma mem-
brane peptidases in primary and continuous cell cultures of
alveolar epithelium. In: Falk K. -E., Okano, T. (eds) Proc. 24th
Int. Symp. Control. Rel. Bioact. Mater. CRSInc., IL, pp 79±80
Houston, J. B. (1982) Drug metabolite kinetics. Pharmacol.
Ther. 15: 521±552
Acknowledgements
The authors are grateful to the Welsh Scheme for
the Development of Health and Social Research for
the provision of a PhD studentship (Ref: 933=1) to
Peter C. Seville.
Patton, J. S. (1996) Mechanisms of macromolecule absorption
by the lungs. Adv. Drug Del. Rev. 19: 3±36
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