Abstract
Methanol was widely used as a substrate-delivering solvent in in vitro metabolic stability screenings. Its interaction with enzyme activities, particularly those of cytochrome P450s, has been investigated extensively in the past. Little was known about the interaction of methanol, whether direct or indirect, with substrates. The present study provided data for the first time to show that use of methanol may result in the formation of artifacts, which could mislead the metabolic stability information. The disappearance of LAQ094, metaraminol, and (−)-isoproterenol following 1-h incubation with human liver microsomes was 73, 85, and 66%, respectively, in the presence of 1% methanol, but was only 3, 15, and 24%, respectively, in the absence of organic solvent. The dramatically increased instability in the presence of methanol of these three compounds, each with 1,2-diamino or 1,2-amino hydroxy functional groups, was due to the formation of [M + 12] products resulting from condensation reaction of the substrates with formaldehyde. Formaldehyde was formed from methanol by human liver microsomal enzymes with an apparentKm of 35 mM and aVmax of 7.9 nmol/min/mg of protein. The concentration of formaldehyde reached as high as 600 μM following a 60-min incubation. The [M + 12] products were characterized as five-membered heterocycles by liquid chromatography and tandem mass spectrometry analysis. Inclusion of 10 mM glutathione prevented the formation of such artifacts and is therefore suggested for future in vitro screenings. Our study also documented the novel finding of enzyme-dependent conversion of NADPH to nicotinamide in microsomal incubations.
In vitro metabolic stability screening of compounds using liver microsomes or postmitochondrial S9 fractions are commonly used in the pharmaceutical industry in the early selection of drug candidates, in an effort to better the chance of finding drug leads with desirable pharmacokinetic and metabolic properties. Fast chromatography coupled with mass spectrometric detection (LC/MS1) is typically used to monitor the disappearance of compounds as a result of incubation (Van Breemen et al., 1998; Korfmacher et al., 1999) without detailed investigation of the structure of metabolites in screening mode.
One of the variables in the in vitro incubations is the selection of organic solvent, which is used to dissolve compounds and deliver them to the incubation systems. Commonly used organic solvents are methanol, ethanol, dimethyl sulfoxide (DMSO), acetonitrile, and acetone. The selection of organic solvents are governed by their ability to solubilize the compounds and their effect on the activity of various cytochrome P450 isoforms (Kawalek and Andrews, 1980; Chauret et al., 1998; Hickman et al., 1998; Busby et al., 1999), either inhibitory (Chauret et al., 1998; Busby et al., 1999; Tang et al., 2000) or stimulatory (Palamanda et al., 2000; Tang et al., 2000). Methanol and acetonitrile are considered as the better alternatives in light of their less severe inhibition effect against various P450 isoforms (Chauret et al., 1998; Hickman et al., 1998; Busby et al., 1999) when present at low levels.
In the present study, we report that the apparent in vitro metabolic instability of a certain class of compounds could be dramatically augmented by the presence of methanol solvent. LAQ094, metaraminol, and (−)-isoproterenol (Fig. 1), each with 1,2-diamino or 1,2-amino hydroxy functional groups, were selected to illustrate those effects. The structures of the unusual products from these three compounds were characterized and the nature of the methanol solvent effect on metabolic stability was investigated. The kinetics of formaldehyde formation from methanol in human liver microsomes was characterized.
Experimental Procedures
Materials.
Metaraminol, (−)-isoproterenol hydrochloride salt,d4-methanol, formaldehyde, glutathione, nicotinamide, and NADPH were purchased from Sigma (St. Louis, MO). LAQ094 was synthesized by Novartis Pharmaceuticals Corporation (Summit, NJ). 4-Amino-3-penten-2-one (Fluoral-P) was obtained from Acros Organics (Fairlawn, NJ). Human liver microsomes (pool of 10 male subjects) were purchased from Xenotech (Kansas City, KS). All other reagents are of the highest grade commercially available.
Human Liver Microsomal Incubations.
Incubation mixtures contained a final concentration of 0.1 M potassium phosphate buffer (pH 7.4), 5 mM MgCl2, 1 mM EDTA, 1.0 mg of human liver microsomal protein per milliliter, 20 μM substrate, and 1 mM NADPH in a total volume of 0.5 ml. The substrate was delivered with 1% (final concentration, v/v) methanol, DMSO, ord4-MeOH, in some cases with subsequent evaporation of the organic solvent, or addition of glutathione (10 mM). The reactions were initiated by the addition of NADPH after a 5-min preincubation at 37°C, and terminated by mixing with an equal volume of cold acetonitrile after a 60-min incubation at 37°C. The zero time point was prepared by mixing the incubates with an equal volume of cold acetonitrile before the addition of NADPH. The incubates were mixed by vortexing and centrifuged to precipitate the proteins. The supernatants were analyzed for disappearance of substrates and structural characterization of metabolites.
Reaction with Formaldehyde.
Incubation mixture containing 0.1 M potassium phosphate buffer (pH 7.4), 20 μM substrate, and 1% formaldehyde (v/v) were kept at room temperature for 2 h with gentle shaking and then analyzed by HPLC with UV and mass spectrometric detection immediately.
Measurement of Substrate Disappearance by HPLC with UV or Mass Spectrometric Detection.
Disappearance of LAQ094 following human liver microsomal incubations was carried out by HPLC analysis with UV detection at 247 nm. The HPLC system consisted of a Waters Alliance 2690 Separations Module equipped with a 996 photodiode array detector. A Metachem Metasil Basic C18 column (5 μm, 4.6 × 150 mm) preceded by a 0.5-μm frit and the corresponding guard column was used. Separation was carried out with solvent A (10 mM ammonium acetate with 0.05% formic acid, pH 3.5) and solvent B (acetonitrile) with a linear gradient at a flow rate of 1 ml/min. The mobile phase was initially kept at 100% A for 5 min, ramped to 30% B over 15 min, and then to 95% B over 1 min and kept at that condition for 3 min. The total run time was 30 min. The disappearance of LAQ094 was assessed by comparing the peak areas from the zero time point and the 60-min incubations.
Disappearance of metaraminol and (−)-isoproterenol following microsomal incubations was carried out by LC/MS/MS analysis. The HPLC system consisted of Shimadzu LC-10AD pumps (Columbia, MA), a Lee Visco mixer with 10-μl dead space, and a Leap Technologies HTS-PAL autosampler (Carrboro, NC) with a 25-μl sample loop. Separation was achieved with a Keystone betasil C18 column (5 μm, 2.0 × 50 mm) with a flow rate of 0.3 ml/min and a linear gradient with 10 mM ammonium acetate containing 0.1% formic acid, pH 3.5 (solvent A), and methanol (solvent B). The mobile phase was initially kept at 5% B for 1 min, ramped to 25% B over 2.5 min, and then to 5% B over 0.5 min. The total run time was 7 min. A PE Sciex API3000 mass spectrometer (Concord, Ontario, Canada) with TurboIonSpray interface in positive ionization mode was used for detection. The instrument was operated at a probe temperature of 375°C, ion spray voltage of 4500 V, orifice voltage of 30 V, and ring voltage of 200 V. A collision energy of 25 eV was used for MS/MS experiments with a nitrogen collision gas setting of 4. (−)-Isoproterenol and metaraminol were monitored as the transitions of m/z 212.2 to 152.2 andm/z 168.2 to 150.2, respectively, with a dwell time of 0.2 s each. The disappearance of substrates was assessed by comparing peak areas from the zero time point and the 60-min incubations.
Structural Characterization of Metabolites by LC/MS/MS Analysis.
LAQ094 and its metabolites were characterized by LC/MS/MS analysis. The HPLC system used was as described above. The HPLC effluent was diverted to waste during the first 4 min of each run to protect the mass spectrometer from nonvolatile salts. Thereafter, the effluent was split to deliver ∼200 μl/min to the mass spectrometer for optimum sensitivity. Mass spectrometric conditions were as described above. Full scan spectra were typically obtained from 100 to 650m/z using a 0.2-amu step size.
Characterization of metabolites of metaraminol and (−)-isoproterenol was carried out on the same LC/MS system used for the assessment of their disappearance, but with modified HPLC conditions. A YMC ODS-AQ C18 column (5 μm, 2.1 × 150 mm) with a flow rate of 0.3 ml/min and a linear gradient with 10 mM ammonium acetate containing 0.1% formic acid, pH 3.5 (solvent A), and methanol (solvent B) were used. The mobile phase was initially kept at 100% A for 4 min, ramped to 22% B over 7 min, and then to 95% B over 5 min. The total run time was 34 min.
Measurement of Formaldehyde Formation in Human Liver Microsomal Incubations.
Formaldehyde formation from methanol in human liver microsomal incubations was measured according to literature (Wojciechowski and Fall, 1996) with modifications. Fluoral-P (2.5 mg/ml) was added to the incubation mixture to react with formaldehyde to form a stable, fluorescent compound, 3,5-diacetyl-1,4-dihydro-2,6-dimethylpyridine, which was quantitated fluorometrically (excitation at 410 nm and emission at 510 nm) on a Spectra Max Plus fluorescence plate reader (Molecular Devices, Sunnyvale, CA). This assay method had a limit of quantitation of 10 μM formaldehyde. Initial human liver microsomal incubations with 1% methanol (245 mM) were carried out for 0, 5, 10, 15, 30, and 60 min, under the conditions specified previously. Subsequent 60-min incubations with methanol concentrations of 2.45, 6.13, 24.5, 61.3, 245, and 735 mM were carried out to characterize the formaldehyde formation kinetics.
Kinetic Data Analysis.
Michaelis-Menten parameters (Km,Vmax) were estimated by nonlinear curve fitting using the Scientist program (Micromath Scientific Software, Salt Lake City, UT) to the following equation: v =Vmax*[S]/(Km+ [S]), where v is the initial rate of formaldehyde formation and [S] is the methanol concentration.
Results
Dependence of Metabolic Stability of LAQ094, Metaraminol, and (−)-Isoproterenol on Methanol in Liver Microsomal Incubations.
Following a 60-min incubation of LAQ094 with human liver microsomes, when methanol (1% v/v) was used as the substrate-delivering solvent, the concentration of LAQ094 decreased by 73% with the concurrent formation of a product eluting at 18.1 min (Fig.2A). The disappearance of LAQ094 was however minimal (∼3%) when methanol was avoided in the incubation (Fig. 2B), or when DMSO was used as the alternative substrate-delivering solvent (Fig. 2C). Incubation with heat-inactivated liver microsomes did not lead to significant substrate depletion or product formation (Fig. 2D). Inclusion of 10 mM glutathione effectively prevented the instability of LAQ094 in the presence of methanol solvent (Fig. 2E). Reaction of 1% (v/v) formaldehyde with LAQ094 for 2 h at room temperature led to the complete disappearance of LAQ094, and the concurrent formation of a product with the same retention time (18.1 min, Fig. 2F) as that from the enzymatic incubation in the presence of methanol.
Incubation of metaraminol and (−)-isoproterenol with human liver microsomes for 60 min led to 85 and 66% substrate disappearance (Table1), respectively, when methanol (1% v/v) was used in the incubations. The disappearance in the absence of any organic solvent was, however, only 15 and 24%, respectively, for metaraminol and (−)-isoproterenol (Table 1).
Characterization of the Metabolites of LAQ094, Metaraminol, and (−)-Isoproterenol.
The products formed from LAQ094, metaraminol, and (−)-isoproterenol were characterized by LC/MS analysis. Incubation of LAQ094 with human liver microsomes in the presence of 1% methanol led to the formation of only one major metabolite eluting at 18.1 min (Fig. 2A). The protonated molecular ion of this metabolite was observed atm/z 212, 12 amu higher than that of LAQ094 (m/z200, 17.5 min). Comparison of their MS/MS product ion spectra (Fig.3) showed that the product ion (m/z 129) corresponding to the amino chloropyridyl moiety in LAQ094 is retained in the metabolite. The major product ion (m/z 183) from LAQ094 was consistent with the loss of NH3 molecule, whereas that from the metabolite was consistent with the loss of NH=CH2 molecule, suggesting the existence in the metabolite molecule a methylene group connecting the terminal amino group. Based on this information, the metabolite was proposed as a 1,3-tetrahydroimidazole derivative (Fig.3). When d4-MeOH was used as the solvent, the molecular ion of the metabolite was observed at m/z 214 (data not shown). The major product ion (m/z 183) from thed2-labeled metabolite resulted from the loss of NH=CD2 molecule (data not shown), indicating that the methylene group connecting the two nitrogen atoms originated from methanol. The reaction of LAQ094 with formaldehyde formed a product (Fig. 2E) with retention time, molecular ion, and MS/MS product ion spectrum identical to those of the metabolite, thus further supporting the structural assignment of the unusual metabolite from LAQ094.
Similar results were obtained following microsomal incubations of metaraminol. Total ion chromatogram (TIC) (Fig.4A) of the incubates showed three peaks. The extracted ion chromatogram (XIC) of metaraminol (Fig. 4B) showed a weak peak at 7.5 min, which does not correspond to any of the three peaks in TIC trace. The two peaks at 8.1 and 10.3 min in TIC trace appeared to be metabolites each with 12 amu mass addition (see XIC in Fig. 4C). These two metabolites had MS/MS product ion spectra (Fig.5, bottom) identical to each other and their major fragments m/z 162, 147, 145,127,121, and 117 are each 12 amu higher than the fragments m/z 150, 135, 133, 115, 109, and 105 from metaraminol (Fig. 5, top). Based on the mass spectral information, its formation dependence on methanol solvent, and analogy to LAQ094 findings, the [M + 12] metabolites of metaraminol were proposed as isomeric tetrahydrooxazole derivatives (Fig. 5). The reaction of metaraminol with 1% formaldehyde produced the same two products with MS/MS product ion spectra (data not shown) and retention times identical to those of the metabolites, thus further supporting the structural assignments. The third peak at 8.5 min in the TIC trace had a molecular ion of m/z 123. Its product ion mass spectra showed fragments at m/z 123 (100, protonated parent ion),m/z 106 (6.3, loss of NH3), 96 (6.5, loss of HCN), 80 (63.4, loss of CO=NH) and 78 (12.8, loss of HCO-NH2) and was, therefore, proposed as nicotinamide resulting from enzymatic breakdown of NADPH. The product ion mass spectra of synthetic nicotinamide were identical to this metabolite, thus further supporting the structural assignment. The formation of nicotinamide was enzyme-dependent since a 60-min incubation with heat-inactivated microsomes did not lead to its formation (data not shown).
Incubation of (−)-isoproterenol (m/z 212) with liver microsomes in the presence of methanol also produced two [M + 12] metabolites (m/z 224). TIC (Fig.6A) of the incubates showed three peaks, two of them eluting at 9.5 and 10.2 min correspond to two metabolites each with 12 amu mass addition (see XIC in Fig. 6C), and the third one eluting at 8.6 min was nicotinamide. The XIC of (−)-isoproterenol (Fig. 6B) showed a weak peak at 9.7 min. The two metabolites had molecular ion as well as MS/MS product ion spectra (Fig.7, bottom) identical to each other. The two major fragments m/z 164 and 206 are each 12 amu higher than the fragments m/z 152 and 194 from (−)-isoproterenol (Fig. 7, top). Based on the mass spectral information, its formation dependence on methanol solvent, and analogy to LAQ094 findings, the two [M + 12] metabolites of (−)-isoproterenol were also proposed as tetrahydrooxazole derivatives (Fig. 7). The reaction of (−)-isoproterenol with 1% formaldehyde produced the same two products with identical MS/MS product ion spectra (data not shown) and retention times to that of the metabolites, thus further supporting the structural assignments.
Kinetics of Formaldehyde Formation by Human Liver Microsomes.
The conversion of methanol to formaldehyde by human liver microsomes was NADPH-dependent (data not shown), and was linear for at least 60 min at 245 mM methanol and 1 mg/ml protein (data not shown). Formaldehyde was formed up to 600 μM. The reaction followed a typical Michaelis-Menten kinetics (Fig. 8), with an apparent Km of 35 mM and aVmax of 7.9 nmol/min/mg of protein.
Discussion
The current study showed that the apparent in vitro metabolic instability of compounds with 1,2-diamino or 1,2-amino hydroxy moieties could be artificially augmented by the presence of methanol, a common solvent used to dissolve and deliver compounds into the incubation systems. Investigation of the mechanism of instability of a few new chemical entities revealed unusual metabolites with 12 amu mass addition. Three compounds, LAQ094, metaraminol, and (−)-isoproterenol, were chosen in the present study to illustrate such a methanol solvent effect.
Mechanism of [M + 12] Metabolites Formation.
Methanol or other organic solvents are typically used at 1% (v/v) or less in microsomal or S9 incubations, as a balance of compound solubility and the solvent inhibition of P450 activity considerations. Because of the small molecular weight of methanol, 1% (v/v) corresponds to 245 mM in concentration. It was well known that methanol can be oxidized to formaldehyde by liver enzymes (Dawidek-Pietryka et al., 1998). The current study showed that formaldehyde formed by human liver microsomes in 1 h reached a concentration as high as 600 μM. It was shown previously (Yin et al., 1996) that formaldehyde readily reacts with ethylene 1,2-diamino and ethanolamine to yield tetrahydroimidazole and tetrahydrooxazole derivatives, respectively. By analogy, LAQ094, metaraminol, and (−)-isoproterenol, each containing 1,2-diamino or 1,2-amino hydroxy functional groups, can react with formaldehyde to form heterocyclic products (Fig.9) that have molecular weights 12 amu higher than their respective reactants. Previous work (Yin et al., 1996) showed glutathione could readily react with formaldehyde to form a thiohemiacetal intermediate that is further converted to a stable six-membered heterocyclic ring. When 10 mM glutathione was supplemented in the microsomal incubations, it appeared all the formaldehyde formed from methanol was effectively trapped and no [M + 12] metabolite from LAQ094 was produced (Fig. 2E). It is, therefore, suggested to include glutathione in the incubation system for future in vitro screening to avoid misleading results, when methanol is used as the substrate-delivering solvent. Previous work (Koppel et al., 1991; Yin et al., 1996) showed that primary amines could also react with formaldehyde to form imine products with 12 amu mass addition. Further investigation is necessary to examine whether other structural moieties can also lead to the formation of [M + 12] products in the presence of formaldehyde.
Metabolism of LAQ094, Metaraminol, and (−)-Isoproterenol by Human Liver Microsomes.
Based on HPLC chromatography and MS analysis, the [M + 12] metabolite was the only detectable metabolite from LAQ094 in the presence of methanol. This compound is very stable during the 60-min incubation in the absence of methanol. The trace amount of [M + 12] metabolite peak observed in the incubations with DMSO or no solvent might be due to the incomplete evaporation of methanol solvent, which was used for the preparation of stock solution.
Previous work (Fuller et al., 1981; Wollenberg and Rummel, 1984; Causon et al., 1985) showed O-methylation andO-sulfation as the major metabolic pathways for metaraminol and (−)-isoproterenol in vivo. The present study with liver microsomes did not produce these metabolites (data not shown) since the cofactors required for methyl transferase and sulfur transferase activity were not supplemented. The two [M + 12] metabolites from metaraminol (Fig.4C) eluting at 8.1 and 10.3 min well separated from each other and had comparable abundance and identical product ion mass spectra. These two metabolites are likely cis- and trans-isomers (two sets of diastereomers), since a diastereomeric mixture of metaraminol was used in the study. The two [M + 12] metabolites from (−)-isoproterenol (Fig. 6C) eluting at 9.5 and 10.1 min also separated from each other and had comparable abundance and identical product ion mass spectra to each other. The mass spectral data suggest that they are not regioisomers, i.e., none of them is the acetal formed from the reaction of the ortho dihydroxyl functional groups with formaldehyde. The most likely reason for their chromatographic separation is the protonation of the nitrogen group resulting in cis- andtrans-isomers at the mobile phase pH of 3.5.
Enzymatic Hydrolysis of NADPH.
Enzyme-dependent conversion of NADPH to nicotinamide was observed in human liver microsomal incubations in the present study. Such an enzymatic reaction was not previously documented and the reaction mechanism warrants further investigation. Since the formation of nicotinamide is enzyme-dependent and NADPH-dependent, there is a potential that nicotinamide could be mistakenly treated as a metabolite in HPLC-UV analysis, and consequently mislead the metabolic instability information when assessed by relative peak areas.
Other Implications of Methanol Solvent Effect.
In addition to the potential interference in metabolic stability assays, methanol solvent could impact the in vitro toxicological screening. Previous study (Cunningham et al., 1990a,b) showed that a mutagenic product, bis-5,5′-(2,4,2′,4′-tetraaminotolyl)methane, was produced in the Ames/Salmonella assay from the reaction of 2,4-diaminotoluene with formaldehyde, which was produced from methanol in the presence of S9. Koppel et al. (1991) showed the formation of formaldehyde adducts from various drugs, such as amphetamine, propafenone, flecainide, β-blockers, and prilocaine, by use of methanol in the toxicological screening. They observed [M + 12] adducts resulting from reactions with formaldehyde and proposed that formaldehyde was formed by thermal dehydrogenation of methanol in the injection port of the gas chromatography.
In summary, this article demonstrated that methanol is oxidized to formaldehyde by human liver microsomes with an apparentKm of 35 mM and aVmax of 7.9 nmol/min/mg of protein. The formaldehyde thus formed may undergo condensation reactions with compounds with 1,2-diamino or 1,2-amino hydroxy functional groups. When microsomal incubations are used to assess metabolic instability by measuring the amount of parent compound remaining, this reaction can lead to an erroneous conclusion about metabolic instability. The compound may appear to be unstable, although the reason for the instability is not a direct consequence of the metabolism of the compound in question, but rather an artifact of the methanolic content of the incubation.
Acknowledgments
We thank Dr. Edwin Villhauer (Novartis Pharmaceuticals Corporation, Summit, NJ) for providing LAQ094 and Drs. Alban Allentoff, Heidi Einolf, and James Mangold (Novartis Pharmaceuticals Corporation, East Hanover, NJ) for helpful discussions.
Footnotes
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Send reprint requests to: Dr. Hequn Yin, Novartis Biomedical Research Institute, 59 Route 10, East Hanover, NJ 07936. E-mail: hequn.yin{at}pharma.novartis.com
- Abbreviations used are::
- LC/MS
- liquid chromatography and mass spectrometry
- DMSO
- dimethyl sulfoxide
- Fluoral-P
- 4-amino-3-penten-2-one
- HPLC
- high-performance liquid chromatography
- LC/MS/MS
- liquid chromatography and tandem mass spectrometry
- MS/MS
- mass spectrometry/mass spectrometry
- amu
- atomic mass unit
- TIC
- total ion chromatogram
- XIC
- extracted ion chromatogram
- LAQ094
- 2-[(5-chloro-2-pyridinyl)amino]-1,1-dimethyl-ethylamine
- Received August 9, 2000.
- Accepted October 31, 2000.
- The American Society for Pharmacology and Experimental Therapeutics