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Global Drug Metabolism, Pharmacia, Kalamazoo, Michigan
(Received March 6, 2003; accepted April 2, 2003)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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26%) by coincubation with clindamycin (100 µM). Thus, it is concluded
that CYP3A4 appears to account for the largest proportion of the observed P450
catalytic clindamycin S-oxidase activity in vitro, and this activity
may be extrapolated to the in vivo condition.
). The
lincosamide antibiotics seem to be most useful against the bacteria classified
as Gram-positive cocci. In addition, clindamycin is helpful against protozoans
such as Toxoplasma and Mycoplasma as well as many anaerobic
bacteria (Luft and Remington,
1988; Dannemann et al.,
1991; Mazur et al.,
1999).
In humans, absorption of clindamycin is rapid and virtually complete (90%)
following oral administration (DeHaan et
al., 1972; Metzler et al.,
1973). Concentrations of clindamycin in the serum increase
linearly with increased dose, and levels exceed the minimum inhibitory
concentration for most indicated organisms for at least 6 h following
administration of the recommended dose. Clindamycin is widely distributed
throughout the body and has an average biological half-life of 2.4 h. The
major bioactive metabolites excreted in urine and feces are clindamycin
sulfoxide and N-desmethylclindamycin
(Seaberg et al., 1984;
Flaherty et al., 1988;
Gatti et al., 1998).
To date, there are no published reports that comprehensively describe the metabolic pathways associated with clindamycin clearance in humans. Identification of the drug-metabolizing enzymes responsible for the biotransformation of clindamycin and the interindividual differences in the expression and catalytic activities of those enzymes may help explain and/or predict population variability in the metabolic clearance of clindamycin. In vitro methodologies using human liver tissue have been developed to aid in the prediction of possible variation in metabolic clearance in vivo and drug-drug interactions for a variety of drugs. The specific objectives of the current study were to: 1) duplicate the major clindamycin metabolites observed in vivo using appropriate in vitro systems, 2) characterize the drug hepatic enzyme(s) responsible for metabolite formation, and 3) assess the drug-drug interaction potential of clindamycin toward select human P4501 enzyme activities.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Microsomes. Human livers were acquired from the International
Institute for the Advancement of Medicine (Exton, PA). Liver microsomal
protein isolation and the specific catalytic activity of individual enzymes of
P450 were determined as previously described
(Wienkers et al., 1996). For
FMO heat liability experiments, incubations were performed as described above,
except that microsomal preparations were preincubated with or without NADPH at
50°C for 2 min. Microsomes from a baculovirus-insect cell line expressing
CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9(*1), CYP2C9(*3), CYP2C19, CYP2C18,
CYP2D6(*1), CYP2E1, CYP3A4, CYP3A5, CYP4A11, FMO1, FMO3, and FMO5 were
purchased from BD Gentest (Woburn, MA). AntiCYP3A4 inhibitory antibodies and
control sera were purchased from BD Gentest. Microsomes from human ileum and
jejunum were purchased from Tissue Transformation Technology (Edison, NJ).
Incubation Conditions. A typical incubation (final volume 0.1 ml) consisted of 0.1 to 0.3 mg/ml microsomal protein in 100 mM potassium phosphate buffer (pH 7.4). Stock solutions of clindamycin were prepared in sterile water. The drug was added to each incubation well, followed by the assay buffer containing microsomes. Reactions were started by the addition of NADPH (1 mM final concentration) and continued for 30 min at 37°C. For control incubations, NADPH was omitted. Reactions were terminated upon addition of 50 µl of acetonitrile (ACN), after which samples were vortex mixed and centrifuged for 10 min at 1750g. The subsequent supernatants were injected directly from the 96-well plate, in a refrigerated plate holder using radio-HPLC analysis.
Radio-HPLC. Analytical separation of clindamycin and its metabolites was achieved using a HPLC system equipped with a PerkinElmer Series 200 pump and autosampler (PerkinElmer Instruments, Norwalk, CT) equipped with a chilled sample tray maintained at 4°C. The analytical column was a reverse-phase Zorbax C-18 (250 x 4.6 mm, 5-µm particle size) (Agilent Technologies Inc., Wilmington, DE). The mobile phase consisted of A (water/methanol/acetic acid, 90:10:0.02%) and B (methanol/water/acetic acid 90:10:0.02%). Chromatographic separation was achieved using a gradient run with a flow rate of 1 ml/min 100% A to 100% B over 15 min. Quantitation of clindamycin and its metabolites was performed using a FLO-ONE/[B]eta Series A500 flow-through radioactivity detector (PerkinElmer Life Sciences), and peak areas were integrated with Windows-based Radio-HPLC Workstation software (FLO-ONE/[D]ata for Windows). Ultima-Flo-M liquid scintillant was introduced postcolumn at a rate of 2.5 ml/min. Rates of formation of the clindamycin sulfoxide metabolite were determined from the fractional conversions of [14C]clindamycin apparent from the radiochromatogram. Peaks were identified using retention time comparison with authentic clindamycin and clindamycin sulfoxide standards. In addition, all standards were chromatographed and structure was confirmed using LC/MS.
LC/APCI/MS and Metabolite Confirmation. The identities of the primary in vitro clindamycin metabolites were confirmed using an LCQ ion-trap (Thermo Finnigan, San Jose, CA) operated in positive-ion APCI mode. The APCI vaporizer temperature was 450°C, and the discharge current and spray voltage were set at 5 µA and 4.5 kV, respectively. Nitrogen (99.9% pure; AGA, Maumee, OH) was employed as a drying gas at a sheath pressure of 80 psi and auxiliary flow rate of 20 ml/min, and the heated capillary was set at 250°C. Analytical separation of clindamycin and its metabolite was accomplished using the HPLC conditions described above. Under these conditions, authentic standards of clindamycin and the clindamycin sulfoxide derivative were characterized by retention time, molecular ion, and fragmentation pattern. The collision energy used was 1.3 and 1.6 V for clindamycin and clindamycin sulfoxide, respectively.
In Vitro Enzyme Kinetics. For kinetic studies in human liver microsomes, clindamycin concentrations ranged from 10 to 500 µM, with 0.2 mg/ml microsomal protein in each incubation. All other conditions were as already described. Analysis of clindamycin S-oxidation was performed using a PerkinElmerSciex API 150 single quadrupole mass spectrometer connected to PerkinElmer Series 200 micropumps and autosampler. Ions generated by electrospray ionization were detected using selected-ion monitoring in positive-ion mode. Clindamycin sulfoxide formation was detected by analysis of [M + H]+ molecular ion at m/z 441, with carbamazepine as internal standard (m/z 237). Analytical separation was accomplished using a Waters symmetry C8 2.1 mm x 150 mm column with mobile phase A (20 mM ammonium acetate, pH 4.5) and B (100% acetonitrile) delivered at 0.3 ml/min at an initial ratio of 80% A:20% B with a linear gradient to 90% B over the first 2 min, held for another 1.5 min, then back to initial conditions for re-equilibration over the next 4.5 min. Data were analyzed using Sciex Analyst version 1.2 software, and kinetic parameters for clindamycin S-oxidation by CYP3A4 were estimated by nonlinear regression analysis (Michaelis-Menten equation) with GraphPad PRISM 3.0 software (GraphPad Software, Inc., San Diego, CA).
Incubations with Recombinant Human P450s. The metabolism of clindamycin was examined in microsomes prepared from a baculovirus-insect cell line expressing for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9(*1), CYP2C9(*3), CYP2C19, CYP2C18, CYP2D6 (*1), CYP2E1, CYP3A4, CYP3A5, and CYP4A11. The incubations were conducted in a manner essentially as described above, with 100 µM [14C]clindamycin and equivalent P450 concentrations (10 pmol) of each P450 enzyme in 100 mM potassium phosphate buffer, pH 7.4. Incubations identical to those described above, containing clindamycin (1–1000 µM), were conducted using recombinant expressed CYP3A4 to determine the Km and Vmax for clindamycin metabolism.
Correlation Analysis. The rates of formation of clindamycin
sulfoxide were determined across a panel of microsomes prepared from 12 human
livers, 7 human jejunum, and 6 human ileum donors and compared with the
catalytic activities previously characterized for specific P450 substrates
(Wienkers et al., 1996). For
this study the concentration of clindamycin was 50 µM, incubation
conditions and sample work-up were carried out as described under
Incubation Conditions. Coefficient of determination
(r2) for enzyme activities was determined by linear
regression analysis using the graphical/statistical program Prism 3.0
(GraphPad).
Chemical Inhibition Experiments. [14C]Clindamycin (100
µM; approximately the apparent Km for clindamycin
toward CYP3A4) was incubated at a single concentration in pooled human liver
microsomes in the presence of a panel of compounds, which interacted
selectively with various cytochrome P450 enzymes. The following P450 enzyme
substrates/inhibitors were examined for their ability to inhibit the
microsomal metabolism of clindamycin: bropirimine/CYP1A2 (200 µM),
-naphthoflavone/CYP1A2 (10 µM), coumarin/CYP2A6 (20 µM),
sulfaphenazole/CYP2C9 (5 µM), (S)-mephenytoin/CYP2C19 (200 µM),
quinidine/CYP2D6 (5 µM), ketoconazole/CYP3A (5 µM), cyclosporin A/CYP3A
(50 µM), methimazole/FMO3 (200 µM). The inhibitors were dissolved in ACN
and were added to the incubations such that the final amount of solvent was
1%. Control incubations (minus inhibitor) also contained 1% ACN.
Incubations with Anti-CYP3A4. To further study metabolism by CYP3A4, clindamycin (100 µM) was incubated with pooled human liver microsomes (0.3 mg protein/ml). Each sample was incubated with and without anti-CYP3A4 antibody (0.1 mg protein). The anti-CYP3A4 antibody was added to microsomes and placed on ice for 15 min. The incubations were then carried out in a manner identical to that of the human liver microsome incubations.
P450 Inhibition Screen. The ability of clindamycin to selectively
inhibit enzyme activity was investigated against six different recombinant
human cytochrome P450 enzyme systems (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1,
and CYP3A4). Incubations were conducted in triplicate, and each incubation
contained recombinant P450 microsomal protein (0.1–0.3 mg/ml), NADPH (1
mM final concentration), P450 marker substrate ([S] = Km),
and clindamycin at concentrations of 0 (minus inhibitor control), 1, 10, and
100 µM, in a final volume of 0.1 ml of 100 mM, pH 7.4, potassium phosphate
buffer. Incubation reactions, sample work-up, and quantitation of P450 marker
metabolite formation were conducted as previously described
(Wynalda and Wienkers,
1997).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In Vitro Kinetic Analysis. The effects of substrate concentration on the rate of S-oxidation for clindamycin were determined in three human liver microsomal preparations and are listed in Table 1. For clindamycin, formation of clindamycin sulfoxide appeared to undergo saturable kinetics. Moreover, analysis of the kinetic data using Eadie-Hofstee graphical analysis (data not shown) revealed a linear relationship between V (the rate of clindamycin sulfoxide formation) and V/[S] (the velocity divided by the substrate concentration) that suggests that a single enzyme or two enzymes with similar Km values were responsible for clindamycin S-oxidation. This observation was then substantiated through a comparison of goodness-of-fit values generated for clindamycin sulfoxide velocity data modeled to single Km and multiple Km equations using sum-of-squares nonlinear regression analysis.
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Incubations with Recombinant Human P450s. Of the 10 human baculovirus-insect cell-expressed P450 enzymes investigated, only CYP3A4 and, to a minor extent, CYP3A5 were able to catalyze the formation of clindamycin sulfoxide (Fig. 4). Enzyme kinetic experiments revealed that the apparent Km value for clindamycin sulfoxide metabolite formation by the recombinant CYP3A4 was consistent with the apparent Km values determined in human liver microsomes (Table 1).
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Cytochrome P450 Correlation Studies. The rates of clindamycin sulfoxide formation were determined in 12 different human liver microsomal preparations. As shown in Fig. 5A, at a clindamycin concentration of 50 µM, the rate of clindamycin sulfoxide formation correlated with CYP3A-catalyzed testosterone activity (r2 = 0.97). Moreover, the formation of this metabolite did not correlate with any other measured P450 activity in the individual livers (Table 2). Additionally, correlation studies conducted in individual intestinal microsomal preparations from human ileum (n = 6) and jejunum (n = 7) exhibited marked correlation with the measured CYP3A activity (r2 = 0.93 and r2 = 0.95, respectively) (Fig. 5, B and C).
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Cytochrome P450 Chemical Inhibition. In addition to the correlation
data, clindamycin was coincubated with the following P450 enzyme-specific
substrate/inhibitors: bropirimine (CYP1A2), -naphthoflavone (CYP1A2),
coumarin (CYP2A6), sulfaphenazole (CYP2C9), mephenytoin (CYP2C19), quinidine
(CYP2D6), ketoconazole (CYP3A), cyclosporin A (CYP3A4), and the FMO inhibitor
methimazole. The agents were examined for their ability to inhibit clindamycin
sulfoxide formation at a substrate concentration of 100 µM, as described in
Fig. 6. The data in
Fig. 6 reveal that only
inhibitors of CYP3A were able to attenuate the rate of formation of
clindamycin sulfoxide.
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Immunoinhibition Studies with Pooled Human Liver Microsomes. To
confirm the P450 reaction phenotyping results obtained with chemical
inhibitors and cDNA-expressed P450s, [14C]clindamycin (100 µM)
was incubated with pooled human liver microsomes in the absence and presence
of immunoinhibitory anti-CYP3A4 peptide antibodies. The results with
clindamycin indicated that the majority (90%) of the clindamycin
S-oxidase activity in human liver microsomes was attributable to
CYP3A4 (Fig. 7).
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Contribution of FMO in Clindamycin S-Oxidation.
Flavin-containing monooxygenases are also known to oxidize thioethers to
sulfoxides. Therefore, with incubations in human liver microsomes, the
potential involvement of FMO in clindamycin sulfoxide formation was examined.
When clindamycin (100 µM) was incubated with recombinant FMO1, FMO3, and
FMO5, no measurable clindamycin sulfoxide was formed
(Fig. 4). In addition, there
was no effect upon clindamycin sulfoxide formation following incubations with
heat-inactivated microsomes compared with control microsomes
(Fig. 6), as well as with
coincubation with the FMO inhibitor, methimazole
(Rawden et al., 2000)
(Fig. 6). Lastly, clindamycin
sulfoxide formation in human liver microsomes as a function of pH in a range
of 6 to 10 revealed a single maximum at 7.5, which further suggests sole P450
involvement (Hoskins et al.,
2001).
Effect of Clindamycin and Clindamycin Sulfoxide on Select P450
Activities. The selectivity of clindamycin to inhibit six human P450
enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2E1, CYP2D6, and CYP3A4) was evaluated
using a simple in vitro inhibition screen
(Wynalda and Wienkers, 1997).
When the P450 enzymes were tested against clindamycin at various
concentrations (0, 1, 10, and 100 µM), only CYP3A4 testosterone hydroxylase
catalytic activity was inhibited (26% inhibition), at 100 µM
clindamycin (Table 3).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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When clindamycin S-oxidase activity was examined across human
liver, ileum, and jejunum microsomal preparations, formation of clindamycin
sulfoxide correlated with measured testosterone 6ß-hydroxylase activity,
a marker reaction for CYP3A4 (Waxman et
al., 1991; Wang et al.,
1997). In addition, the y-intercepts for each of these
correlations are near the origin, which suggests a single enzyme being
primarily responsible for the formation of metabolites
(Fig. 5). In the experiment
using microsomes expressing recombinant human P450 enzymes (CYP1A2, 2A6, 2B6,
2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5 and 4A11), only recombinant CYP3A4 and, to
a lesser degree, CYP3A5 possessed measurable clindamycin S-oxidase
activity (Fig. 4). Subsequent
kinetic analysis of the interaction between clindamycin and CYP3A4 revealed an
apparent Km value of 99 µM for the reaction
(Table 1).
The CYP enzyme-selective inhibitors bropirimine (CYP1A2;
Wynalda and Wienkers, 1997),
coumarin (CYP2A6; Pearce et al.,
1992), sulfaphenazole (CYP2C9;
Baldwin et al., 1995;
Newton et al., 1995),
(S)-mephenytoin (CYP2C19;
Wrighton et al., 1993), and
quinidine (CYP2D6; Guengerich et al.,
1986; Sai et al.,
2000) all failed to inhibit clindamycin sulfoxide formation
(Fig. 6). Interestingly,
coincubation with the CYP1A2 inhibitor -naphthoflavone
(Chang et al., 1994) results in
a slight increase in clindamycin sulfoxide formation compared with control.
However, given that clindamycin is for all intents and purposes a CYP3A4
substrate and -naphthoflavone has been demonstrated to act as an
activator of CYP3A4 oxidation (Emoto et
al., 2001; Shou et al.,
2001), this observation is not surprising. Furthermore,
ketoconazole, a potent CYP3A4 inhibitor
(Newton et al., 1995;
Sai et al., 2000), and
cyclosporin A, a known CYP3A4 substrate
(Pichard et al., 1991;
Kelly et al., 1999), both
markedly inhibited the oxidation of clindamycin, which provides additional
evidence for the involvement of CYP3A4 in the biotransformation of
clindamycin. Lastly, there was marked inhibition of clindamycin sulfoxide
formation upon pretreatment with anti-CYP3A4 antibody
(Fig. 7). In addition to
coincubations with select P450 inhibitors, incubations were also carried out
in the presence of the FMO inhibitor methimazole
(Dixit and Roche, 1984;
Rawden et al., 2000). In this
experiment, methimazole did not inhibit clindamycin sulfoxide formation
(Fig. 6).
Flavin-containing monooxygenases are known to oxygenate
heteroatom-containing compounds (Ziegler,
2002). Therefore, the potential involvement of human liver FMOs in
the S-oxidation of the clindamycin was investigated. Incubations with
human liver microsomes, verified to possess good FMO activity (data not
shown), were conducted as a function of pH (pH 6–10). A maximum for
clindamycin sulfoxide formation was observed at pH 7.5, which is
characteristic for a P450-mediated, rather than a FMO-mediated, reaction
(Grothusen et al., 1996). In
addition, purified human FMO3, the major FMO in human liver
(Cashman and Zhang, 2002), as
well as FMO1 and FMO5, did not posses clindamycin S-oxidase activity
(Fig. 4). Finally, heat-treated
(50°C, 1 min) human liver microsomes still yielded clindamycin
S-oxidation rates comparable to those of control incubations
(Fig. 6). This information,
coupled with the lack of inhibition upon coincubation with methimazole, firmly
establishes that human FMOs are not involved in the S-oxidation of
clindamycin.
The importance of pharmacokinetic drug interactions associated with human
immunodeficiency virus therapy is well documented
(Heylen and Miller, 1996) and
oftentimes occurs as a result of inhibition of CYP3A4
(Malaty and Kuper, 1999). The
human CYP3A subfamily plays a dominant role in the metabolic elimination of
many protease inhibitors; amprenavir
(Decker et al., 1998),
saquinavir (Eagling et al.,
2002), nefinavir (Lillibridge
et al., 1998), ritonavir
(Kumar et al., 1996), and
indinavir (Chiba et al., 1997),
as well as reverse transcriptase inhibitors nevirapine
(Erickson et al., 1999) and
delavirdine (Voorman et al.,
1998). Since Staphylococcus aureus is the most common
microorganism causing cutaneous and systemic infections in human
immunodeficiency virus-infected patients, it is possible for clindamycin to be
coadministered as part of a polytherapy regimen
(Styrt et al., 1997;
Manfredi et al., 2002). In
this light, understanding P450 enzyme interactions might allow physicians the
ability to better anticipate and manage each patient's response to adding
clindamycin to an established drug regimen. To gather some insight into the
potential of clindamycin as an inhibitor of cytochrome P450 enzymes,
clindamycin was screened for the ability to inhibit select P450 enzyme
catalytic activities. The current study revealed that clindamycin had a slight
inhibitory effect upon the activities of CYP3A4. Based upon the factors
governing the in vitro metabolism for clindamycin, the interaction between
clindamycin and CYP3A4 was not surprising. Moreover, the observed magnitude of
inhibition was consistent with the apparent Km determined
for clindamycin and CYP3A4 (Km = 99 µM) and previous
work described by Bohets et al.
(2000). Finally, clindamycin
lacked any inhibitory effect on the activities of other P450 enzymes (CYP1A2,
CYP2C9, CYP2C19, and CYP2E1) tested, even at high concentrations (100
µM).
Assuming first-order kinetics, liver drug metabolism is governed by the
intrinsic enzyme catalytic capacity of individual hepatocytes and the
availability of drug at the site of metabolism
(Rane et al., 1977).
Therefore, the underlying determinants for predicting a drug's potential to
inhibit a particular P450 is its [I]/Ki ratio, where [I]
is the concentration of the inhibitor at the site of metabolism and
Ki is the apparent inhibitory constant of the inhibitor
(Bertz and Granneman, 1997).
Administration of clindamycin as a single 600-mg tablet results in a plasma
Cmax of 3.08 µg/ml at about 1.5 h
(Mazur et al., 1999).
Therefore, the maximum concentration of total clindamycin (protein bound and
free) achieved in vivo would be about 7 µM (the molecular weight of
clindamycin is 424.99 atomic mass units). Using the projected
Ki value determined from the inhibition study, the
[I]/Ki ratio for the clindamycin with respect to CYP3A4 is
approximately 0.07, which suggests a nominal drug interaction with CYP3A4.
Thus, as long as the presence of clindamycin does not markedly alter the
dispositional characteristics of a second drug (i.e., the concentration of
drug available at the site of metabolism is not different in the presence or
absence of clindamycin) and the enzyme affinity constant for drug is
independent of inhibitor, the current data suggest that clindamycin should not
substantially alter the metabolism of a second drug whose clearance is
primarily mediated by CYP3A4 as well as the major hepatic P450 enzymes
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP2E1).
In conclusion, the current in vitro findings show that the antibiotic clindamycin appears to be oxidized primarily by CYP3A4 (Fig. 8). Moreover, it appears that clindamycin does not inhibit the metabolic activity of the following P450 enzymes, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP2D6, and only moderately inhibited CYP3A4. Therefore, given the poor affinity toward the human hepatic cytochrome P450 enzymes tested, clinically important interactions between clindamycin and coadministered drugs, which are metabolized by these enzymes, appear unlikely.
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Footnotes |
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Address correspondence to: Larry C. Wienkers, Global Drug Metabolism, Pharmacia Corporation, 7265-300-319, 301 Henrietta Street, Kalamazoo, MI 49007. E-mail: larry.c.wienkers{at}pharmacia.com
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