Preclinical Safety, Novartis Pharma Ltd. (A.E.M.V., M.C.S., R.M.J.,
V.P.); and
Department of Pharmacology and Toxicology, University of
Arizona (R.L.F., K.B.)
Lung biotransformation of the immunosuppressants, cyclosporin A
(CSA), the hydroxyethyl derivative SDZ IMM 125 (IMM), and the
methylcarbonate derivative SDZ SCP 764 (SCP), was demonstrated in
slices from human and rat. The major biotransformation pathway for CSA
and IMM (0.1-10 µM) was hydroxylation at amino acid 1 to form AM1 or
IMM1, while for SCP it was an esterase cleavage of the methylcarbonate
group to form AM1 in both species. The initial rate (0-1 hr) of human
total metabolite formation increased proportionally with substrate
concentration. AM1 formation was five times greater from SCP, an
esterase pathway, than CSA, an oxidative pathway which was inhibited
(50%) by ketoconazole. At 24 hr human lung CSA metabolite formation
was greater than IMM (3-fold) or SCP (2-fold), whereas rat lung and
liver and human bronchial epithelial cell SCP metabolite formation
generally exceeded CSA or IMM metabolism.
CSA biotransformation is expected to occur throughout the human lung as
demonstrated by the similar metabolite profile and extent of metabolism
by slices derived from five different regions. The scaling of slice
total metabolism to organ metabolism revealed that initially lung CSA
metabolite formation would be equal to liver but with time liver
metabolism would exceed lung for human and rat.
This study has demonstrated that human and rat lung are metabolically
active, exhibiting oxidative and esterase pathways toward cyclosporin
derivatives. The lung will play an important role in this metabolism,
particularly when administered via inhalation; however, the
liver will also be a major organ involved in the total clearance of
these compounds.
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Introduction |
Bronchial asthma,
hypersensitivity pneumonitis, and
lung allograft rejection are generally characterized by an infiltration of inflammatory cells. The involvement of T lymphocytes in the late
asthmatic response is suggested by the increased number of activated
CD4+ T cells in broncho-alveolar lavage fluid and bronchial
biopsy specimens from asthmatic subjects (1). Therapeutic intervention has included the regulation of T-cell derived cytokine production, which in turn affects eosinophil and mast cell survival and
infiltration. Lung allograft recipients often develop progressive
bronchiolitis obliterans, for which the cause is unknown and which is
treated with the same immunosuppressive agents as acute rejection (2).
The immunosuppressant cyclosporin A (CSA)1
is effective through its action of inhibiting T-cell activation and
cytokine production in animal models of hypersensitivity (3-5). Human
mononuclear cells derived from patients with bronchial asthma were less
responsive in the presence of CSA to interleukin-2 or
phytohemagglutinin stimulation (6, 7). Additionally, significant
decreases in the production of granulocyte/macrophage
colony-stimulating factor and interleukin-5 resulted in a decreased
eosinophil activity and proliferation (7). The clinical benefit of CSA
therapy has been shown in patients with severe chronic bronchial asthma by an improvement of pulmonary function, as monitored by the peak expiratory flow rate, and it has been associated with a decrease in
serum interleukin-2 receptor levels (8, 9).
Administration of CSA directly to the lung as an aerosol would increase
the local immunosuppression within the lung and decrease CSA systemic
exposure, as well as reduce the risk of developing renal and liver side
effects. Data to support the improved efficacy of aerosolized CSA and
safety was shown for the prevention of lung allograft rejection in rats
and dogs (10, 11). Delivery of aerosolized CSA in humans has been shown
with single and double-lung allograft recipients exhibiting acute
persistent graft rejection and bronchiolitis obliterans (12).
Two additional cyclosporins that exhibit a similar potency to CSA and a
potentially wider safety margin include the hydroxyethyl derivative,
SDZ IMM 125 (IMM), and the methylcarbonate derivative, SDZ SCP 764 (SCP) (fig. 1). IMM differs from CSA in that it is less
dependent on biotransformation for elimination and displays fewer
adverse effects on kidney function (13, 14). The design of SCP
anticipates a local immunosuppressive effect of SCP followed by its
degradation via a metabolic ester cleavage to form the non-immunosuppressive metabolite AM1 of CSA, such that systemic levels
of SCP would be low.
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Fig. 1.
Structures of CSA, IMM and SCP.
The site of primary metabolite formation and amino acid number within
the cyclic undecapeptide structure is denoted. The location of the
tritium radionuclide was the same for each compound.
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The biotransformation of CSA, IMM, and SCP by human lung in comparison
with rat lung was achieved using lung slices derived from each species.
The lung is a heterogeneous organ composed of many distinct cell types,
with the distribution of the xenobiotic metabolizing enzymes primarily
located in the epithelial bronchial cells, Clara and ciliated
bronchiolar cells, alveolar type II cells, and, to a lesser degree, the
endothelial cells. Several cytochrome P-450 families have been
identified in human and rat lung, as well as esterases, epoxide
hydrolase, glutathione S-transferases, glucuronyl transferases,
sulfotransferases, and acetyl transferases (15-19). In general, the
levels and activities of these enzymes are up to 10-fold lower,
particularly for human, as compared with liver (20, 21).
Lung slices were selected as the in vitro system to readily
compare human lung biotransformation with lung biotransformation of the
rat because slices closely resemble the in vivo spatial arrangement and functional heterogeneity of the intact lung. This study
demonstrates lung biotransformation of CSA and its derivatives in both
species with a comparison of human bronchial epithelial cells and liver
metabolism.
| |
Materials and Methods |
Chemicals.
[3H]CSA [Mebmt-
-3H]cyclosporin A with a
specific activity of 10.7 Ci/mmol was obtained from Amersham
(Buckinghamshire, UK). [3H]SDZ IMM 125, [2-hydroxy-ethyl-D-Ser8][Mebmt--3H]cyclosporin with
a specific activity of 7.6 Ci/mmol, [3H]SDZ SCP 764, [8
-methylcarbonate][Mebmt--3H]cyclosporin with a
specific activity of 3.8 Ci/mmol, and unlabeled CSA, IMM, and SCP were
prepared at Sandoz Pharma Ltd. (Basel, Switzerland). The radiochemical
purity of each compound was checked by HPLC and was greater than 98%
for CSA and IMM, and 96% for SCP. Tissue culture media components
including Glutamax were purchased from Gibco (Grand Island, NY and
Basel, Switzerland), and the Mito+ serum extender and NuSerum from
Collaborative Biomedical Products/Becton Dickinson (Heidelberg,
Germany). The protease inhibitors aprotinin and AEBSF were purchased
from Boehringer Mannheim (Rotkreuz, Switzerland) and Calbiochem
(Lucerne, Switzerland), respectively. A low melting agarose
(25-30°C) was obtained from Serva (Heidelberg, Germany). The Cell
Death Detection Elisa Kit was obtained from Boehringer Mannheim
(Mannheim, Germany), and ketoconazole was obtained from Janssen Biotech
(Olen, Belgium). All other reagents used were of the highest grade
available and were purchased from commercial sources.
Human Lung Slice Preparations.
Human lungs (N = 6), obtained through the Association
of Human Tissue Users (Tucson, AZ) from accident donors after clinical death, were maintained in ice-cold Belzer's University of Wisconsin solution (table 1). Each lung was inflated with agar and
the slices prepared according to the method of Stefaniak et
al. (22) with the modifications of Fisher et al. (23).
After inflation, 2.5 cm slabs were cut across the middle portion of the
lungs (region 1), and for two lungs slabs were cut from five regions,
which were then cored transversely (fig. 2). The slices
(~400-500 µm thickness and 35 mg wet weight in the presence of
agar, and ~3 mg protein) were prepared in ice-cold oxygenated (95%
O2:5% CO2) V-7 preservation solution, using
the Vitron tissue slicer (Tucson, AZ) and incubated as described by
Fisher et al. (23).
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Fig. 2.
Schematic of core preparation from human
lung regions.
From five regions of two human lungs, slabs (2.5 cm) were prepared and
then cored (10 mm diameter).
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Rat Lung and Liver Slice Preparations.
Male Wistar rats (N = 5, ~250 g) obtained from Iffa
Credo (Lyon, France) were anesthetized with Isofluran (Abbott Labs,
Cham, Switzerland) and the lung and liver excised. The lung was
inflated with pre-warmed 0.8% agar in EBSS (pH 7.4) and submerged in
ice-cold EBSS-medium for 5-10 min to facilitate the gelling of the
agar. Lung and liver slices (8 mm diameter) were prepared in ice-cold oxygenated EBSS-medium supplemented with 25 mM HEPES, MEM-essential and
non-essential amino acids, BME-vitamins, 10 mM glucose, 10 mM fructose,
10 mM mannitol, 5 mM glutathione, 0.2 mM adenosine, and 0.1 mM AEBSF
using the Krumdieck Tissue Slicer (Alabama Research, Munford, AL). The
lung slices in the presence of agar were 200-400 µm in thickness,
12.1 ± 2.5 mg wet weight, 0.9 ± 0.7 mg slice protein; the
liver slices were 250 ± 25 µm in thickness, 13.2 ± 0.9 mg
wet weight, 1.8 ± 1.2 mg slice protein. The rat slices were
maintained at 37°C in a humidified incubator with circulating 5%
CO2-air mixture in roller cultures (1 slice/insert) in 2 ml of the EBSS slicing buffer (pH 7.4) additionally fortified with 2 mM
Glutamax, 0.2% Mito+ serum extender, 1 µM dexamethasone, 25 mM
nicotinamide, 10 nM glucagon, 0.1 mM 
-aminolevulinic acid, 0.3 µM
aprotinin, 2.5% antibiotic/antimycotic solution, 10% NuSerum, and
glucose (25 mM) in place of fructose and mannitol.
Human Bronchial Epithelial Cells.
Normal human bronchial/tracheal epithelial cells were obtained from
Clonetics (San Diego, CA). The cells were cultured in 24-well Falcon
culture plates in bronchial epithelial growth medium (BEGM, Clonetics)
supplemented with retinoic acid and bovine pituitary extract and
maintained at 37°C in a humidified 5% CO2-air mixture.
Biotransformation.
Lung biotransformation was evaluated by HPLC from slice incubations of
0.1, 1, or 10 µM [3H]CSA (1 µCi/ml for 0.1 µM, or
3.2 µCi/ml), [3H]IMM (0.8 µCi/ml for 0.1 µM or 3.8 µCi/ml), or [3H]SCP (0.38 µCi/ml for 0.1 µM or 3.8 µCi/ml). The biotransformation of [3H]CSA (1 µM) was
also evaluated in the presence of ketoconazole (2 µM) in human lung
slices, which was added 30 min prior to CSA. All compounds were
dissolved in dimethylsulfoxide which had a final culture concentration
of 0.1%, or 0.2% for the ketoconazole studies. The stability of CSA,
IMM, and SCP was assessed in the absence of a slice for the duration of
the culture period.
Sample collection and extraction from slices were made following the
method described by Vickers et al. (24). For the human bronchial epithelial cells, the medium was collected and the cells scraped in the presence of methanol (0.5 ml).
All fractions were pooled to a final volume of 1 ml containing 30%
methanol. Prior to HPLC analysis the sample proteins were pelleted by
centrifugation (60,000g) for 10 min at 25°C, and the supernatant collected for injection (200 µl). Slice protein was determined by the method of Bradford (25) using bovine immunoglobulin as the standard.
HPLC analysis of samples derived from [3H]CSA and
[3H]IMM incubations followed the conditions previously
published (24, 26). The samples derived from [3H]SCP
incubations were analyzed following the CSA HPLC conditions (24).
Sample radioactivity was monitored on-line with a Berthold LB 507A
radioactivity monitor (Berthold, Wildbad, Germany). The cyclosporin
derivatives and metabolites were identified according to the known
retention times of parent and reference metabolites and by comparison
with biological reference samples. Quantification of the cyclosporin
derivative and the metabolites was achieved by integration of the peak
areas using the Hewlett Packard or Kontron data system.
The stability of CSA was 98% after 24 hr and 93% after 48 hr of
incubation, while the stability of IMM was 99% after 24 and 98% after
48 hr of incubation in the absence of a slice. The stability of SCP was
93% after 24 hr, with 4% of the radioactivity co-eluting with AM1,
and 88% after 48 hr with 9% co-eluting with the AM1 peak, which was
subtracted from AM1 formation.
Viability.
The slice viability of control and CSA, IMM, and SCP (0.1, 1, and 10 µM) exposed human lung slices was determined by slice protein
synthesis and ATP content as described by Fisher et al. (27).
Human bronchial epithelial cell, rat liver, and lung slice viability
was determined by the leakage of histone-associated DNA fragments,
mono- and oligonucleosomes using the Cell Death Detection ELISA Kit
from Boehringer. Aliquots (100 µl) of slice and cell homogenate
prepared in 500 µl EBSS and diluted to 100 ng protein/100 µl with
incubation buffer were added to the microtiter plates. The medium (100 µl) was analyzed straight. The per cent leakage of DNA fragments was
calculated from the optical density of medium over the total (slice or
cell homogenate plus medium).
| |
Results |
Metabolite Patterns.
The biotransformation of [3H]CSA, [3H]IMM,
and [3H]SCP at 0.1, 1, and 10 µM was investigated in
human lung slices, human bronchial epithelial cells, and rat lung and
liver slices. The major metabolite formed by human lung slices from all
three compounds was a hydroxylated metabolite at amino acid 1 (fig.
3). This metabolite has been previously characterized in
human liver slices as AM1 for CSA and IMM1 for IMM. (24, 26). The other
known CSA primary liver metabolites, hydroxylation at amino acid 9 to
form AM9, and the N-demethylated metabolite at amino acid 4, AM4N, were also formed but in very low amounts. For IMM, the analogous
primary metabolites were more extensively formed.
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Fig. 3.
Human lung slice biotransformation of CSA,
IMM, and SCP.
Typical HPLC radiochromatograms of metabolite profiles from human lung
slice cultures incubated with 0.1 µM [3H]CSA 24 hr, 1 µM [3H]IMM 24 hr, and 1 µM [3H]SCP 48 hr. Equal volumes of culture extracts (200 µl) were injected containing about 1 × 105 dpm for 0.1 µM and 1 × 106 dpm for 1 and 10 µM substrate concentrations. The
scale of the radiochromatograms represent 600 mv for CSA and 200 mv for
IMM with a full scale corresponding to 3000 mv, and 500 mv for SCP with
a full scale of 1500 mv.
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The human bronchial epithelial cells metabolized each compound;
however, low amounts of the primary metabolites of CSA and IMM were
evident, indicating that these metabolites were rapidly converted to
further secondary metabolites. For SCP the major metabolite formed was
AM1 (data not shown).
The metabolite profile of CSA and IMM from rat lung slices revealed low
amounts of the primary hydroxylated and N-demethylated metabolites, the highest metabolite formed being AM1 and IMM1, respectively. Liver slices produced all the primary metabolites as
described previously (24, 26). For SCP the major metabolite formed by
rat lung and liver slices was AM1 (fig. 4).
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Fig. 4.
Rat lung and liver slice biotransformation
of CSA and SCP.
Typical HPLC radiochromatograms of metabolite profiles from 24-hr rat
lung slice (RLuS) and liver slice (RLS) cultures incubated with 1 µM
[3H]CSA and 10 µM [3H]SCP. Equal volumes
of culture extracts were injected containing about 1 × 106 dpm. The scale of the radiochromatograms represent 500 mv for CSA with a full scale corresponding to 1500 mv, and 200 mv for RLuS and 1420 mv for RLS of SCP with a full scale of 2000 mv.
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The metabolism of CSA by both human and rat lung resulted in the
formation of several polar peaks. These peaks have not been identified,
and there is a difference in the production depending on the tissue
studied. In the lung, the appearance of these polar metabolites occurs
within the first hour of culture, while in the liver the polar peaks
appear after several hours of culture time.
Biotransformation.
The initial rate (0-1 hr) of CSA total metabolite and AM1 formation by
human lung slices increased proportionally from 0.1 to 10 µM
substrate concentrations. The rate of AM1 formation derived from SCP
was in general five times greater than that derived from CSA, and AM1
formation represented 13-85% of the SCP metabolites compared with 7%
or less from CSA. The rate of IMM metabolite formation was generally
lower than CSA (table 2). Lung slice metabolite
formation was linear from 1-48 hr with total metabolite formation
paralleling AM1 formation in the CSA cultures. The rapid rate of
initial total metabolite formation from 0-1 hr was evident in both the
human and rat lung slices (fig. 5) and not in rat liver
slices. Metabolite formation of IMM and SCP was also linear with the
time (data not shown).
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Fig. 5.
Time course of CSA metabolite
formation.
Total CSA metabolite (TM) and AM1 formation from human lung slice
cultures incubated with 1 µM [3H]CSA from 0 to 48 hr.
Values represent data from one human lung.
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The biotransformation of CSA by the human lung slices was CYP
dependent, as shown by slices derived from region 1 of two human lungs
and incubated in the presence of [3H]CSA (1 µM) and
ketoconazole (2 µM). Formation of AM1 was inhibited ~50% in the
presence of ketoconazole at 2 and 4 hr post-[3H]CSA
addition compared with slices exposed to [3H]CSA only. By
24 hr of culture, AM1 formation in the presence of ketoconazole was
still decreased as much as 25% compared with CSA alone (data not
shown).
The mean extent of CSA total metabolite formation and AM1 formation by
the human lung slices, the HBEC, the rat lung, and liver slices
increased proportionally when the substrate concentrations were
increased from 0.1 to 10 µM (table 3). In accordance
with a greater rate of CSA metabolite formation by the human lung was a
larger amount of CSA total metabolite formation at 24 and 48 hr
compared with IMM and SCP. IMM metabolite formation represented about
25% of CSA at 10 µM. The extent of SCP metabolite formation was
lower than CSA (~50%) and higher than IMM metabolite formation (fig.
6).
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Fig. 6.
A comparison of three cyclosporin
derivatives.
Total metabolite (TM) formation by human lung slices from 10 µM
[3H]CSA, [3H]IMM and [3H]SCP
after 1, 24, and 48 hr of incubation. Values represent the mean ± SD from the same three lungs.
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The importance of the involvement of an esterase reaction in the
formation of AM1 from SCP versus an oxidative pathway from CSA was apparent in the human bronchial epithelial cells. SCP total
metabolite formation generally exceeded CSA metabolite formation twofold. AM1 formation from SCP was about 13-fold greater at 0.1 µM
and 17-fold greater at 10 µM substrate concentrations compared with
CSA AM1 formation. These results could indicate that the esterases were
more prominent than the oxidative enzymes in the human
bronchial-derived cells as compared with the human lung slices (table
3).
In the rat, the mean initial rate (1 hr) of CSA (1 µM) total
metabolite formation by lung slices was 452 ± 134 pmol/hr/mg slice protein, which was 14 times greater than CSA total metabolite formation by liver slices, 32 ± 4 pmol/hr/mg slice protein. The extent of CSA total metabolite formation increased proportionally from
0.1 to 10 µM, and was about twofold greater in the lung than in the
liver; however, the extent of AM1 formation was greater in the liver,
7-fold at 0.1 µM and 1.3-fold at 1 and 10 µM. IMM metabolite
formation by the rat lung and liver slices was generally comparable and
lower than CSA. Biotransformation of SCP by rat liver slices exceeded
lung slice metabolism and CSA liver metabolism. The main metabolite of
SCP, AM1, represented up to 63% of the total metabolites in lung and
up to 73% in liver (table 3).
Lung Regions.
The regional biotransformation of [3H]CSA (10 µM, 2, 24, and 48 hr) was investigated in slices derived from different
regions of two human lungs, as shown in fig. 2. The metabolite profile was the same in all regions and similar to the radiochromatogram shown
for region 1 in fig. 3, demonstrating that the primary hydroxylated and
N-demethylated metabolites and several secondary metabolites would be formed throughout the lung. The extent of total metabolite formation was also similar for the various regions. The viability of
the slices from the different lung regions, as assessed by protein
synthesis and ATP levels at 24 and 48 hr, were also comparable (data
not shown).
Upscaling.
The relative contribution of the lung first-pass effect of
[3H]CSA as compared with that of the liver was estimated
by scaling the initial rate of total metabolite formation (µmol/mg
slice protein) in the slices (mg wet weight) to the whole organ, based on the weight of the organ (µmol total metabolite/organ). Lung tissue
represents about 0.7% of the body weight in man and 0.6% in the rat,
while liver represents 2-5% of the body weight in man and 3.5% in
the rat (28-30). Scaling of the 24-hr total metabolite formation data
was done to determine the relative contribution of the lung to the
total biotransformation of [3H]CSA (systemic and
presystemic metabolism) as compared with that of the liver.
For both man and rat, the initial lung biotransformation of
[3H]CSA just after administration (first-pass effect) is
predicted to exceed that of liver, whereas total liver
biotransformation of [3H]CSA is predicted to exceed that
of the lung. In man, lung CSA initial metabolite formation was
predominate, 1.5-fold greater at 1 µM, as compared with that of
liver, but liver total metabolite formation was 8-fold greater at 1 µM, as compared with the lung. In the rat, the initial metabolism
formation of [3H]CSA was 1.8-fold higher in the lung
compared with the liver, but the total extent of liver CSA metabolite
formation was about 4-fold greater at 1 µM [3H]CSA than
the lung biotransformation of CSA (table 4).
Viability Parameters.
Protein synthesis in the human lung slices was linear over the 48-hr
culture, virtually doubling between 24 and 48 hr (fig. 7). No significant effects on protein synthesis were
caused by CSA, IMM or SCP exposure. ATP levels in lung slices derived
from different humans ranged from 12 to 38 nmoles ATP/mg slice protein and remained stable throughout the 48-hr culture period. There were no
significant differences in the lung slice ATP content by CSA, IMM, or
SCP treatment (data not shown).
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Fig. 7.
Viability of human lung slices.
Protein synthesis in human lung slices from three human lungs after 1, 24, and 48 hr of culture. Data are expressed as dpm [3H]leucine incorporated/mg protein.
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The viability of the human bronchial epithelial cells and rat lung and
liver slices was characterized by the leakage of histone-associated DNA
fragments, mono- and oligo-nucleosomes into the medium. There were no
significant increases resulting from CSA, IMM, or SCP treatment (data
not shown).
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Discussion |
This study demonstrates that both human and rat lungs would
metabolize cyclosporin A, its hydroxyethyl derivative IMM, and the
ester-derivative SCP. Precision-cut lung slices were used to facilitate
the comparison of compound biotransformation in human and rat lungs and
rat liver.
The biotransformation pathway of the cyclosporins CSA and IMM in the
lung was similar to that of liver in that the primary hydroxylated and
N-demethylated metabolites previously reported for liver
slices were also produced by lung slices of both human and rat (24,
26). Differences, however, in the proportion of the primary metabolites
formed by the lung slices as compared with the liver slices were
evident. The hydroxylated metabolite at amino acid 1 (AM1 for CSA and
IMM1 for IMM) was clearly the major primary metabolite formed by human
lung, while only small amounts of the hydroxylated metabolite at amino
acid 9 (AM9 for CSA) and the N-demethylated metabolite (AM4N
for CSA and IMM4N for IMM) were formed. Both human and rat lung slices
revealed a rapid formation of polar peaks from CSA which are usually
evident in liver slices at later incubation times (25). The structures of these polar peaks have not been elucidated. We have also found these
metabolites in lung and liver microsomal incubations from human and
rat, but not in incubations from dead slices (data not reported) or
stability samples, indicating that the polar peaks represent
metabolites.
The initial rapid rate of total metabolite formation in human and rat
lung slices as compared with that in liver is partly a result of the
more rapid formation of the polar peaks. Additionally, the more
permeable nature of lung tissue could lead to a more rapid
accessibility of the compound to the xenobiotic metabolizing enzymes,
yielding a more rapid initial rate of metabolism. Metabolite formation
was linear with the culture time, indicating that the metabolic
functionality and viability of the slices was good.
CSA metabolite formation in lung slices was generally greater for CSA
than IMM as reported for human and rat liver (26). Formation of the
primary hydroxylated and N-demethylated metabolites of CSA
and IMM are known to be CYP3A dependent in human liver (31, 32). The
partial inhibition of AM1 formation from CSA by the CYP inhibitor
ketoconazole indicates the involvement of CYP enzymes in human lung
biotransformation of CSA.
The major biotransformation pathway of SCP was cleavage of the ester
group to form the nonimmunosuppressive metabolite AM1. The design of
SCP predicted a local immunosuppressive effect in the lung, followed by
a rapid degradation via esterases, so that the systemic
exposure to the parent compound would be low. Esterase cleavage was the
predominant pathway in the lung of both human and rat and in rat liver.
The rate and extent of AM1 formation from SCP far exceeded AM1
formation from CSA in all the in vitro systems investigated
in this study, further indicating differences in the enzymes involved.
Since the cyclosporin derivatives would be administered as an aerosol,
slices derived from various regions of the human lung indicated that
metabolism of CSA would occur throughout the lung. The CSA metabolite
profile was the same in the five regions and the extent of CSA
metabolite formation was similar out to 48 hr of culture. Lung slice
total metabolite formation was therefore scaled for the whole organ to
estimate the contribution of lung biotransformation compared with that
of liver. The scaling was also done to compare human and rat, in view
of the species differences in organ to body weight ratios and because
the 8 mm diameter slices represented a far greater proportion of rat
than human tissue. Scaling of the initial rate of metabolite formation
in the slices to the whole organ revealed that for both human and rat,
CSA metabolism by the lung would be predicted to be equal to that of
liver at the first contact with the tissue. With time, however, liver
CSA metabolism exceeded lung metabolism, 8-fold for human and 4-fold for rat, reflecting the 10-fold differences in CYP content of the
organs (20, 33).
It is not possible to predict the rate of passage of the compound from
the lung into the systemic circulation with the slice cultures, nor is
it possible to predict the proportion of compound reextracted from the
systemic circulation by the lung. Highly lipophilic compounds such as
CSA may exhibit a long lung residency, in which case lung metabolism
could contribute substantially to the clearance of the compound from
the respiratory tract (17). Additionally, the lung receives 100% of
the cardiac output as compared with 25% for the liver, increasing the
possibility for lung reextraction of such lipophilic compounds. The rat
oral and iv in vivo studies, however, revealed that liver
extraction of both CSA and IMM was greater than lung extraction. Lung
CSA levels were about 20-fold lower than liver levels after a single
oral or iv dose, and 31-fold lower following multiple oral doses, 10 mg/kg/day for 21 days (34). For the derivative IMM, lung levels were
also lower, about 4-fold, as compared with liver levels after an iv
dose for 10 days (14).
The isolated human epithelial cells used in this study were derived
from the conducting bronchial/tracheal airways and exhibited biotransformation capability toward the cyclosporin derivatives even
though esterase reactions were more prominent than oxidative in
comparison those of the lung slices. In the lung the major cytochrome
P450s identified in man include 1A1, 2A6, 2C9, 2E1, and 3A4, and in rat
they include CYP1A, CYP2B, and CYP3A (19, 21). The respiratory tract is
a more heterogenous system than the liver, composed of a wide variety
cell types, and the biotransformation enzymes are distributed
nonuniformly within each segment. Methods for the isolation and culture
of the various cell types are not developed across different species,
including human. Hence, the methodology for preparing organ slices is
ideal for investigating lung biotransformation and function in various
species. The integration of CYP-dependent pathways and conjugation
reactions in rat lung slices has been demonstrated for 7-ethoxycoumarin
(35).
In this study lung slice viability was monitored to evaluate the
culture conditions and to assess the effect of compound exposure on
lung function. Protein synthesis demonstrated a constant and cumulative
increase over the 48-hr culture period, indicating that the slices were
viable under the culture conditions. Slice ATP levels, a marker of
cellular energetics including the oxidation-reduction state of electron
carriers, such as NADH and NADPH, and for the ATP-producing machinery
and functions, remained stable throughout the 48-hr culture period.
Viability assessed by the leakage of low molecular weight DNA fragments
into the medium also proved to be a valuable method which could be used
for all eukaryotic cells.
This study has demonstrated that both human and rat lung are
metabolically active toward cyclosporin derivatives. The
biotransformation pathways for CSA and its hydroxyethyl derivative IMM,
which are known for liver, were present in the lung with hydroxylation
at amino acid 1 to form AM1 or IMM1 as the major primary metabolite formed. AM1 formation from CSA is most likely to be cytochrome P450
dependent, as demonstrated by the partial inhibition with ketoconazole.
In contrast, AM1 formation from SCP would be an esterase reaction
occurring in the lung of both rat and human as well as in the liver.
The lung will play an important role in the metabolism of each of these
cyclosporin derivatives, particularly when administered via
inhalation; however, the liver will be the major organ involved in the
clearance of these compounds.
Abbreviations used are:
CSA, cyclosporin A;
IMM, SDZ IMM 125;
SCP, SDZ SCP 764;
AEBSF, [4-(2-Aminoethyl)benzenesulfonylfluoride, HCl];
EBSS, Earle's
Balanced Salt Solution MEM, Minimum Essential Medium;
BME, Basal Medium
Eagle;
CYP, cytochrome P450;
CYP3A, cytochrome P4503A;
HBE, human
bronchial epithelial cells.
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Abstract
Introduction
Abstract
