Department of Biological Science, The Royal Danish School of
Pharmacy, Growth Hormone Biology, Novo Nordisk A/S
The absorption enhancer, didecanoylphosphatidylcholine (DDPC),
improves the nasal absorption of human growth hormone in rabbits. We elucidated the uptake and the metabolism of
1,2-di[1-14C]decanoyl-L-3-phosphatidylcholine
and
1,2-didecanoyl-L-3-phosphatidyl[N-methyl-3H]choline
in rabbit nasal mucosa in vivo. One minute after nasal application of DDPC, 4.4-7.5% of the applied dose was found absorbed into the mucosa. The retained radioactivity left the tissue in <2 hr.
The lipophilic tissue extract revealed that, at t = 1 min, only 1.1-1.4% of the applied dose was found as intact DDPC in the nasal mucosa. The other labeled compounds were decanoic acid, DDPC
reacylated with endogenous fatty acids, a neutral lipid, and very small
amounts of lyso-DDPC and phosphatidylethanolamine. The water-soluble
metabolites revealed formation of phosphorylcholine, glycerophosphorylcholine, cytidinediphosphatecholine, and a slight amount of choline. The detection of these metabolites suggests that
DDPC was rapidly cleared from the nasal mucosa, partly by degradation
by phospholipases. In addition, data illustrated reutilization of DDPC
metabolites in the formation of cytidinediphosphatecholine and
phosphatidylethanolamine, together with DDPC being reacylated with
endogenous fatty acids. The rapid formation of decanoic acid raises the
possibility that this acid may contribute significantly to the
drug-enhancing properties of DDPC.
 |
Introduction |
Due to improved biotechnology, the accessibility
of biologically active peptides to the pharmaceutical industry has
increased considerably. However, a limiting factor in the development
of peptide drugs is the relative ineffectiveness when given perorally. Almost all peptide drugs are parenterally administered, although parenterally administered peptide drugs are often connected with low
patient compliance. As alternatives to these routes, several nonparenteral routes have been investigated for peptide drug delivery (e.g. nasal, buccal, rectal, vaginal, pulmonal, and
transdermal). Among these routes, Aungst et al. (1)
considered the nasal route to be the most promising. The
i.n.1
delivering of peptide drugs offers several advantages
(e.g. easy application, fast absorption, avoidance of
hepatic first-pass metabolism, and destruction of the peptide in the
gastrointestinal tract) (2-4). A common important drawback of this
route is, however, that the mucosa permeability decreases for higher
molecular weight compounds (5). Thus, i.n. application of large,
hydrophilic drugs (such as polypeptides and proteins) shows a very low
bioavailability (5), and coadministration of absorption enhancers is
often required to achieve therapeutic blood levels (6, 7). Different types of absorption enhancers have been investigated, such as bile
salts, chelating agents, and fatty acids (8). The phospholipid DDPC has
shown absorption-enhancing properties in the nasal absorption of
insulin and growth hormone (9, 10). However, many of the potential
absorption enhancers have been associated with toxic effects on the
epithelial barrier (11-13). Agerholm et al. (11) suggested
that the major transport route of human growth hormone after i.n.
administration to rabbits in vivo formulated with DDPC and
-CD was transcellular through lethally damaged ciliated cells. Vermehren et al. (10) showed that the absorption-enhancing
effect of DDPC on growth hormone was not time-dependent within 3 hr
when growth hormone and DDPC was separately administered i.n. to
rabbits in vivo. This may indicate an irreversible effect of
DDPC on the nasal membrane (10). Lysophospholipids, which are unique
surfactants, have been attributed to membrane effects [such as cell
fusion and lysis (13)], and other potential absorption enhancers
(e.g. polyoxyethylene-9 lauryl ether) and certain bile salts
have been attributed to chronic erosion of the mucous membrane (12).
For clinical application of these compounds in nasal drug formulations, it is most important to know the mechanism of action of the enhancers and with that the potential adverse effects.
The aim of the present study was to study the fate of radiolabeled DDPC
in the nasal mucosa of rabbits, thereby contributing to the elucidation
of the mechanism of action and the toxic effect of DDPC in the
absorption enhancement of peptide drugs.
| |
Materials and Methods |
Radiolabeled DDPC (14C-DDPC) (specific activity: 67 mCi/mmol) and 3H-DDPC (specific activity: 85 Ci/mmol) were
from Amersham International (Buckinghamshire, UK). The initial
radiochemical purity was 
95%. Novo Nordisk A/S (Bagsvaerd, Denmark)
delivered the unlabeled DDPC powder. Lipid standards were obtained from
Sigma Chemical Co. (St. Louis, MO) TLC plates (silica gel 60 and silica
gel 60 W) and organic solvents were obtained from Merck (Darmstadt,
Germany). For autoradiography, Kodak Safety Film was used.
Study Design.
A radiolabeled DDPC solution (3H-DDPC and
14C-DDPC), including 0.1% unlabeled DDPC in Krebs-Ringer
buffer, was prepared by sonication. After sonication, a sample of the
DDPC solution was incubated for 2 hr in Krebs-Ringer buffer at
37°C. Subsequently, the stability of this DDPC was evaluated by TLC
and found 98-99% intact. Thus, a dose of 50 µl contained 50 µg
unlabeled DDPC, 2,727,300 dpm 3H-DDPC (final specific
activity: 10 mCi/mmol), and 4,284,000 dpm 14C-DDPC (final
specific activity: 22 mCi/mmol).
Twenty-five New Zealand White rabbits weighing 2.5-3.5 kg (Novo
Nordisk A/S) were divided into five test groups, with five rabbits in
each group. Animals were anesthetized with pentobarbital (30 mg/kg iv)
immediately before the experiment started. At t = 0, groups 1-5 were dosed intranasally with 50 µl test preparation in
one nostril. At t = 1, 15, 30, 60, and 120 min, the
animals were killed by 200 mg pentobarbital (intravenously; five
animals at each time point), and the nasal mucosas were isolated as
described by Carstens et al. (14). The isolation procedure
took ~5 min before the mucosas were placed on ice. This period may
have influence on the metabolite profile. The isolated mucosas were
washed 5 times in a total of 5 ml of ice-cold 0.9% NaCl.
Autoradiography of the intact mucosas was done to evaluate the
localization of the radioactivity. The mucosas were placed on a board.
The film was fixed firmly on the mucosas and exposed at 
80°C in the
dark for 1 week. The mucosas and rinsing water from the first wash of
each mucosa were analyzed for DDPC and DDPC metabolites.
Lipid Analysis.
The nasal mucosas were homogenized and extracted with
chloroform/methanol (2:1) and 0.92% CaCl solution to derive lipid and aqueous fractions. Radioactivity in both fractions was determined.
Internal standards were added to the lipid fraction:
1-decanoylglycerophosphorylcholine, decanoic acid, MAG, DAG, PE,
cholesteryl ester, and DDPC. The metabolites in the lipid fraction were
separated by TLC on silica gel 60, and the plates were developed for 72 min in a solvent system of chloroform/methanol/ammonia/water
(120:80:10:5). The plates were dried and then rotated 90° and
developed for 90 min in a solvent system of
chloroform/acetone/methanol/acetic acid/water (100:40:30:20:12). After
the second development, the TLC plates were exposed for autoradiography
for 1 week at 80°C.
Internal standards were added to the aqueous fraction: CHO, P-CHO,
CDP-CHO, and GPC. Metabolites in the aqueous fraction were separated by
TLC on silica gel 60 W. Plates were developed for 3-4 hr in a solvent
system of 0.5% NaCl/ethanol/methanol/concentrated ammonium hydroxide
(50:30:20:5) and were then exposed for autoradiography for 1 week at
80°C. Radioactivity was visualized by autoradiography, and the
silica gel of these areas were scraped for quantitative measurement of
the radioactivity by liquid scintillation counting (Packard 1900 TR
liquid scintillation analyzer).
Data are presented as means ± SE (N = 5).
| |
Results |
Autoradiography of intact mucosas showed that the amount of total
radioactivity associated with the tissue was largest at t = 1 min and smallest at t = 120 min,
and that the dosing was reproducible (data not shown). 203,049 3H-dpm ± 10,883 3H-dpm and 187,861 14C-dpm ± 15,201 14C-dpm were absorbed at
t = 1 min (fig. 1), which
corresponded to an absorption of 7.5 ± 0.4% and 4.4 ± 0.4% of the applied dose, respectively (fig.
2). At t = 1 min,
9.1 ± 2.0% of the applied 3H-dose and 6.1 ± 1.3% of the applied 14C-dose was found in the mucosal wash
water (N = 5, data not shown). Preliminary studies of
dosing-colored DDPC solutions showed pronounced accumulation of color
in the nasal conchae. Besides this, a majority of the dose was probably
swallowed or to a minor degree lost by sneezing. At t = 1 min, only 1.4 ± 0.3% 3H-dpm and 1.1 ± 0.2%
14C-dpm of the applied dose corresponded to intact DDPC
(fig. 2). This indicated that only a small part of the applied DDPC was associated with the nasal epithelium as intact DDPC. Total
radioactivity and radioactivity of intact DDPC in the tissue decreased
with time (figs. 1 and 2). Two hours after dosing, only 4.1 ± 1.1% 3H-radioactivity and 0.2 ± 0.0%
14C-radioactivity of the applied dose was
tissue-associated, and only 0.02 ± 0.00% 3H- and
14C-radioactivity corresponded to intact DDPC (fig. 2).
This means that the DDPC taken up at t = 1 min was
nearly 100% decomposed or disappeared within 2 hr.
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|
Fig. 1.
Catabolism of 3H-DDPC and
14C-DDPC in the nasal mucosa in vivo.
At t = 0, 25 rabbits were dosed with 2.5-4 * 106 dpm + 50 µg of unlabeled DDPC in 50 µl. Mean
values ± SEM (N = 5). It took 5 min to prepare
the mucosas, and these 5 min are not included on the time axes. *,
3H-dpm;  , 14C-dpm.
|
|
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Fig. 2.
Catabolism of 3H-total,
14C-total, 3H-DDPC, and 14C-DDPC in
the nasal mucosa in percentage of injected dosis.
100% 3H = 2,727,300 dpm and 100%
14C = 4,284,000 dpm. Mean values ± SEM
(N = 5). *, 3H-total; ,
3H-DDPC;  , 14C-total;
(  ), 14C-DDPC.
|
|
At the start of the experimental period (t = 1 min),
the 14C radioactivity of the lipid fraction as evaluated by
TLC was mainly associated with decanoic acid (35.3 ± 0.4%),
intact DDPC (29.6 ± 3.6%), PC (mainly DDPC reacylated with
endogenous fatty acids) (18.4 ± 2.5%), and neutral lipid
(11.8 ± 1.0%) (fig. 3). Only a
negligible amount of 14C-dpm was found in the aqueous
fraction. The detection of these metabolites may indicate degradation
of DDPC mainly by PLA1/PLA2, and reacylation.
Within 2 hr, the occurrence of decanoic acid and DDPC decreased by 30%
and 19%, respectively, whereas the percentage of PC (18.4 ± 2.5%, t = 1 min) was doubled. The increase in DDPC reacylation was supported by an increasing
3H-/14C-ratio in the elongated PC spot on the
TLC plate (figs. 4 and 5). This ratio increased from 1.8 ± 0.1 to 10.2 ± 2.5 (fig. 4), whereas the
3H-/14C-ratio in the DDPC spot held a constant
value of 0.64. In the lipid extract, there was initially found a
minimal occurrence of lyso-DDPC, which disappeared with time (fig. 3).
The percentage of a neutral metabolite
which comigrated with MAG, DAG,
TAG, and cholesteryl estersincreased from 11.8 ± 1.0% to
28.6 ± 4.7% during the experimental time (fig. 3). The formation
of radiolabeled neutral lipids can be caused by reacylation with
decanoic acid and/or the activity of PLC and PLD + phosphatidic
acid phosphatase. Figure 6 shows the
distribution of the 3H-labeled metabolites in the nasal
tissue as obtained from the lipid and aqueous extracts each analyzed by
TLC. At the start of the experiment (t = 1 min), P-CHO
and GPC constituted 32% and 12%, respectively, of the
3H-labeled, water-soluble compounds. Both metabolites
decreased gradually during the 2-hr period (fig. 6). In addition,
CDP-CHO and small amounts of CHO were also detected in the aqueous
extract, suggesting reutilization of CHO and P-CHO for PC synthesis.
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|
Fig. 5.
Autoradiography of a TLC plate with
localization of the 14C-labeled metabolites in the lipid
fraction.
|
|
| |
Discussion |
Initially, the nasal mucosa accumulated 7.5 ± 0.4%
3H-dpm and 4.4 ± 0.4% 14C-dpm of the
applied dose (figs. 1 and 2). The wash water contained 9.1 ± 2.0% and 6.1 ± 1.3% of the applied 3H- and
14C-doses, respectively. The dose of DDPC was applied to
the nasal mucosa. A minor amount of the dose may also have been lost to the nasal conchae, and the continuous ciliary motion probably transported a major part of the enhancer solution toward the throat. A
minor amount of the DDPC solution may also have been lost by sneezing.
Within 2 hr, the radioactivity in the tissue decreased to 4.1 ± 1.1% and 0.2 ± 0.0% of the applied dose for 3H and
14C, respectively. The decrease of radioactivity with time
suggested that DDPC and especially 14C-labeled DDPC
metabolites left the nasal mucosa to enter the circulation or the nasal
mucus. The pronounced metabolism of DDPC in the tissue and the rich
vascularization of the tissue may be a reason for the fast
disappearance of tissue-associated DDPC. The removal of
14C-radioactivity (label in the fatty acids) was faster,
compared with the removal of 3H-radioactivity (label in
CHO) (figs. 1 and 2). This indicated an extensive deacylation of DDPC.
Decanoic acid, which is the product of a deacylation process and
initially was the predominant 14C-labeled metabolite, is
relatively water-soluble and may have left the tissue via
the blood or via the nasal mucus. The amount of this
metabolite in the wash water (used for washing the mucosas immediately
after isolating) supported this, as 90% of 14C
radioactivity in the first milliliter of washing water was decanoic acid (data not shown). From t = 1 min to
t = 120 min, the amount of
14C-radioactivity in the wash water decreased by 98%,
suggesting that the majority left the mucosa via the blood
(data not shown). The more slower decrease in 3H
radioactivity may suggest that the choline moiety was reused for PC
synthesis by the salvage pathway (15).
When isolating the mucosas 1 min after nasal application, the
radioactivity corresponding to intact DDPC was only 18-25% of the
total radioactivity in the tissue (fig. 2), suggesting a very rapid
catabolism. A rapid catabolism of DDPC was also seen in an in
vitro study in rabbit nasal mucosa (16). Like this, the in
vivo metabolism products constituted ~59% 14C-dpm
and 72% 3H-dpm of the total radioactivity retained in the
tissue at t = 1 min, and half of the detected
3H-labeled metabolites were found to be water-soluble. In
contrast, lung perfusions with surfactant DPPC for 2 hr in rats
resulted in only 13% of water-soluble metabolites (17). This
difference may in part be due to internalization of surfactant in
endosomes, together with surfactant protein A that may hinder the
accessibility of DPPC for the metabolizing enzymes. DDPC is most likely
absorbed as monomers into the plasma membrane and may be accessible for the degrading enzymes. Lamellar bodies in the nose (18) indicate the
occurrence of surfactant depots. The mechanisms for internalization and
reutilization of DPPC seem to be relatively unspecific (19). However,
it may seem likely that these mechanisms concern DPPC to a much
greater extent than DDPC. DPPC may be taken up by the cells
via endocytosis to far greater extent than DDPC, thereby escaping the rapid metabolism seen for DDPC. Short-chain PC will rapidly incorporate into phospholipid membranes, as opposed to PC
molecules containing the long-chain fatty acids (20). The predominant
occurrence of decanoic acid indicated activity of PLA1,
PLA2, or phospholipase B. A number of previous studies
indicated metabolism of exogenous PC catalyzed by phospholipase A
(21-23). It is well known that short-chain phospholipids are better
substrates than long-chain phospholipids for different phospholipases
(24, 25). A recent study (26) showed a DDPC-induced increase in prostaglandin synthesis in rabbit nasal mucosa, probably related to a
cytotoxic-mediated increase in PLA2 activity.
A form of PLA2 is activated and secreted by tissue injury
(27), and human nasal lavage contain a PLA2, which activity
selectively increases in allergic subjects subsequent to nasal
challenge with antigen (28). These PLA2's may thus have
been involved in formation of the 14C-labeled decanoic
acid. During a PLA-induced degradation of DDPC, a simultaneous
formation of lyso-DDPC was expected. In this study, only extremely
small amounts of lyso-DDPC were detected, which is in agreement with
other studies on PC metabolism (21, 29). A low content of lyso-DDPC may
be due to a high lyso-DDPC turnover. The turnover of lyso-PC has been
reported to be 30- to 80-fold faster than the PC turnover (21). The
occurrence of GPC at t = 1 min (11.9 ± 3.6%) may
be caused by combined PLA1/PLA2 action. Formation of GPC has also been found in other studies of metabolism of
extracellular PC (21, 22, 29). The decrease in GPC with time may be due
to reacylation or GPC hydrolysis catalyzed by phosphohydrolases
generating P-CHO and glycerol (30). At t = 1 min,
considerable amounts of DDPC were remodeled with fatty acids of
different chain length seen as an elongated PC spot on TLC (23.8 ± 2.4% of total 3H-radioactivity) (figs. 5 and 6).
Remodeling increased with time, which was illustrated by the increase
in 3H-/14C-ratio (fig. 4). Acyltransferases and
transacylases may be responsible for this process (31). An increasing
percentage of a less polar metabolite was found during the experimental
time (fig. 3), and this metabolite cochromatographed with standards of
MAG, DAG, TAG, and cholesteryl esters. In vitro, only slight
amounts of MAG and diacylglycerol were formed from DDPC in nasal mucosa
(16) and from DPPC in lung tissue (22). The increase in percentage of
neutral 14C-labeled lipids in vivo may be caused
by a comparable low formation of these neutral lipids that remain in
the tissue along with a large disappearance of 14C-labeled
compounds (probably as decanoic acid) from the nasal mucosa. Thereby,
the percentage of neutral lipids will increase. Occurrence of CDP-CHO
illustrated reutilization of the choline moiety. CDP-CHO may appear by
a reversible choline phosphotransferase reaction. However, this enzyme
has a low affinity toward disaturated PC (32), and it may be more
likely that CDP-CHO was generated from P-CHO via a cytidyl
transferase reaction (33). Degradation of DDPC by PLC results in DAG
and P-CHO formation. At the start of the experiment, P-CHO accounted
for 31.6 ± 1.1%, which decreased in time (fig. 6). Decrease in
P-CHO may be connected with an increase in CDP-CHO or merely hydrolysis
by phosphatases. Alternatively, P-CHO may be a result of PLD activity
with subsequent phosphorylation of CHO. CHO could also result from
hydrolysis of P-CHO or GPC catalyzed by phosphoesterases. The present
study cannot differentiate between these catabolic pathways.
Being relatively water-soluble and possessing short-chain fatty acids,
DDPC seemed to have optimum absorption enhancer properties. The
rate of incorporation of phospholipids into membranes increases with
decreasing fatty acid chain length (20); thus, the incorporation rate
of DDPC was supposed to be high. Ott et al. (34) reported that dimyristoyl-PC after prolonged incubations was absorbed into erythrocyte membranes inducing discocyte to sphero echinocyte shape
changes. Similarly, absorption of DDPC may induce shape changes of the
epithelial cells and probably affect the cytoskeleton, as has been
found for other PCs (35). Changes in the cytoskeleton may affect the
tight junctions (36), resulting in increased paracellular
passage. This suggestion that DDPC may affect tight junctions is
supported by a decreased resistance of rabbit nasal mucosa in
vitro after application of DDPC (14, 16, 37). The i.n. application
of a large dose of 8% DDPC (0.960 mg), together with 30% -CD (3.68 mg) for 15 min in rabbits in vivo resulted in lethally
damaged cells (11). Another study showed that the absorption of
i.n.-administered growth hormone was not reduced 3 hr after the i.n.
dosing of DDPC, thereby also indicating an irreversible absorption
enhancement (10). Furthermore, 15 min incubation of isolated nasal
mucosas with 2% (35.3 mM) DDPC resulted in irreversible changes of the
electrophysiological parameters of the tissue (16, 37). These results
may indicate a toxic effect of high doses of DDPC and, with that, DDPC
may seem unqualified for use as an absorption enhancer. However, the
combination of 8% (0.960 mg) DDPC and 30% (3.68 mg) -CD as
enhancers for the nasal absorption of growth hormone in rabbits
in vivo showed a clear reversibility of the enhancing effect
after a single application. This suggested that, in this case, the
enhancing effect was not caused by irreversible damage of the
epithelial barrier, possibly due to formation of inclusion complexes
between -CD and DDPC, thus masking the toxic effect of DDPC (10).
Thus, to explain the exact mechanism and toxicity of DDPC alone or in
combination with other enhancers, further and longer studies are
required. Thus, the very rapid catabolism of DDPC with the formation of
decanoic acid raises the question of whether some of the
absorption-enhancing properties attributed to DDPC may be mediated by
decanoic acid. Medium-chain fatty acid salts have been reported to
enhance the absorption of insulin (38) and various drugs with a
molecular weight of 4,000 Da (39).
This work was supported by Novo Nordisk A/S and The Academy of
Technical Sciences.
Abbreviations used are:
i.n., intranasal;
DDPC, didecanoylphosphatidylcholine;
-CD, -cyclodextrin;
14C-DDPC, 1,2-di[1-14C]decanoyl-L-3-phosphatidylcholine;
3H-DDPC, 1,2-didecanoyl-L-3-phosphatidyl[N-methyl-3H]choline;
MAG, monoacylglycerol;
DAG, didecanoylglycerol;
CHO, choline;
P-CHO, phosphorylcholine;
CDP-CHO, cytidinediphosphatecholine;
GPC, glycerophosphorylcholine;
PC, phosphatidylcholine;
PLA1, phospholipase A1;
PLA2, phospholipase
A2;
TAG, triacylglycerol;
PLC, phospholipase C;
PLD, phospholipase D;
DPPC, dipalmitoylphosphatidylcholine.
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Abstract
Introduction
Abstract
, Lyso-DDPC; 
, DDPC;
, PC; 
, PE (phosphatidylethanolamine); 
, GPC;