BIOMEDICAL CHROMATOGRAPHY, VOL. 6,50-52 (1992)
Isolation of Clobazam-Orosomucoid Complexes from Patients’ Sera Michael Treuheit and H. Brian Haisall* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, USA
Orosomucoid, a member of the lipocalin family, may function in the in uiuo transport of lipophilic compounds such as basic and neutral drugs. We describe the identification of 7-chloro-l-metbyl-l,5-benzodiazepine-2,4dione (clobazam) bound to the serum orosomucoid from individuals actively taking this tranquillizer. This suggests not only that other endogenous factors limit access to the benzodiazepine binding site on human serum albumin, but also that the differential binding of benzodiazepines and their metabolites by orosomucoid should be considered in determining therapeutic doses, particularly in the acute phase response.
INTRODUCTION
EXPERIMENTAL
a,-Acid glycoprotein, orosomucoid (OMD), is generally considered to be a member of the lipocalin family, which functions either in the transport of lipophilic compounds or as receptors for them (Clark et ul., 1984; Pevsner et af., 1988). Numerous basic and neutral drugs have been shown to bind to isolated OMD. These have recently been thoroughly reviewed by Kremer et al. (1988). These studies, and other inferential data, have led to the widespread thesis that OMD is the major carrier of basic drugs, and, to a lesser extent, neutral drugs, in uiuo. However, there is a paucity of data that directly demonstrate that these drugs or other compounds are bound to OMD in serum. Human serum albumin (HSA) has also been shown to be a major binding protein for many neutral and acidic drugs in serum. Numerous studies (Kober and Sjoholm, 1979; Johnson et af., 1979; Jones et al., 1988) have indicated that the neutral benzodiazepines are mainly bound to a single class of binding sites on HSA. However, Miiller et af. (1983) and Kornguth et al. (1981) have also indicated by competitive binding assays that diazepam, the more notable member of the benzodiazepine family, displaces imipramine and chlorpromazine from OMD. Thus, although HSA may have a greater binding capacity than OMD, especially for acidic and neutral drugs, OMD may have a role in the plasma binding of these compounds, a role whose importance would increase during the acute phase response (Miiller et al., 1983). The approach taken here was to attempt to isolate drug-OMD complexes from a patient in treatment. OMD from individual donors, isolated in a rapid single step, has been previously used for the extraction and identification of bound ligands (Treuheit and Halsall, 1991). This communication, in a continuing effort to define endogeneous ligands bound to serum OMD, describes the isolation and identification of the parent compound 7-chloro-l-methyl-l,5-benzodiazepine-2,4-dione (clobazam) bound to the serum OMD from individuals that were actively taking this tranquillizer.
Materials. All reagent grade chemicals and high performance liquid chromatographic (HPLC) grade solvents were purchased from Fisher Scientific, Pittsburgh, PA. HPLC grade water was purified in our laboratory.
* Author
to whom correspondence should be addressed.
0269-3879/92/010050-03 $05.00
01992 by John Wiley & Sons, Ltd.
~~
~
Sera. 50 mL of blood was obtained from healthy donors who had taken 10 mg clobazam 12 h previously. Blood collection was by venipuncture into a 50 mL glass syringe (Becton and Dickinson). The blood was allowed to coagulate in groundglass stoppered centrifuge tubes (Fisher) for serum collection. Immunopurification of OMD. An immunoaffinity column (50 mL) was made using the monoclonal antibody (MAb) G9. The epitope for GS) includes residues 157-168, a sequence that is specific for OMD and contains no cross-reacting glycan chains. The G9 was coupled to Sepharose-4B-CL (Sigma) using carbonyldiimidizole (Wilchek et nl., 1984), forming the affinity matrix. 10mL of the all-glass prepared serum were applied to the G9 affinity column equilibrated with the washing buffer of 0.01 M phosphate and 0.1 M NaCl, pH 7.5. After washing with six column volumes of washing buffer, the bound OMD was eluted from the affinity column using 0.01 M phosphate and 1M NaCl, pH 7.5 (eluting buffer). All column connections were made with Teflon tubing. Reversed phase (C18)extraction of ligands. The OMD solution (50 mL) from the immunoaffinity column was loaded through the solvent reservoir directly onto a semipreparative Vydac CIRcolumn (218 TP 510, 10 mm x 250 mm) (The Separations Group, Hesperia, CA) with a defined maximum capacity for protein of 5 mg. Once loaded, the column was connected to a Kratos HPLC system, consisting of Kratos 400 dual pumps, a Kratos 783 detector and a SpectraPhysics 4290 integrator, and equilibrated with HPLC grade water containing 0.1% trifluoroacetic acid (TFA). The column was eluted with a 30 min gradient of 0-1oc)% methanol containing 0.1% TFA. The detector was set at 225 nm and the flow rate was 1.5 mL/min with an aufs of 1.00 for all separations. Buffer blanks. The immunoaffinity column was washed with 300mL of the washing buffer. The column was eluted with the eluting buffer and 50 mL (one column volume) collected. Received 18 April 1W1 Accepted 30 April 1991
OROSOMUCOID LlGANDS
51
This material was also loaded through the solvent reservoir directly onto the Vydac CI8column, and the latter eluted using the same gradient as above. Mass spectrometry. Mass spectral data were obtained on a Kratos MS 80 high resolution mass spectrometer. The pooled cuts from the several runs required to process the entire 50 mL blood sample were used.
RESULTS AND DISCUSSION
There appears to be considerable evidence that the benzodiazepines as a class bind to HSA. However, the work of Muller et al. (1983) and others (Kornguth et af., 1981; Abel et al., 1979; Grossman et al., 1982; Schley and Mueller-Oerlinghausen, 1983) indicates that OMD may be responsible for considerable serum binding and transport of this drug, as it is for many others (Kremer el al., 1988). If OMD does bind clobazam in uiuo, then the observation that the free fraction of those drugs that binds to OMD in uitro is reduced in the acute phase (Piafsky et al., 1978) may have some significance in determining therapeutic doses. The purpose here was to provide direct evidence for the binding of clobazam to OMD in uiuo. Indeed, this would be the first such report for any drug-OMD interaction. It is suggested that the single-step isolation of OMD maintains the protein at its physiological pH and ionic strength until elution, allowing for the isolation of endogenous ligands. lmmunoaffinity chromatography of OMD-drug complexes on a monoclonal antibody column has shown that it is possible to isolate some of these intact, depending on the association-dissociation kinetics of OMD and the drug (lvaneic, 1988). This offered the possibility that clobazam would also remain largely bound to OMD during the isolation procedure if the binding kinetics were favourable. A possible complication was that plasticizers have been shown to displace ligands bound to OMD (Piafsky and Borgi, 1976). However, the single-step isolation procedure had the additional advantage that it minimizes contact with the plasticizers commonly found in laboratory equipment and solvents (lshida et al., 1980). The OMD isolated from the affinity column was loaded directly onto a C,, HPLC column through the solvent reservoir. Ligands bound to this OMD were isolated and collected using a steep methanol (with 0.1% TFA) gradient. (It should be noted that the recovery at this point may not be quantitative because losses during the washing steps required for the immunochromatography are unknown). The chromatogram in Fig. 1 is the reversed phase HPLC profile of the OMD from a donor who had taken 10mg of clobazam 12 h previously. The peak at 35.48 was collected and concentrated for high resolution mass spectrometric (MS) analysis, other peaks being components of the buffer blank and a phthalate found associated with the OMD of smokers (Treuheit and Halsall, 1991). Figure 2 represents the mass spectrum for this compound. The parent ion had an exact mass of 300.07, which is identical to that of clobazam (CI6Hl3O2N2C1). Clobazam is metabolized to, principally, N desmethylclobazam, which has similar pharmacological properties to clobazam, but at a lower level of activity
4
B
Figure 1. The reversed phase HPLC profile of the OMD obtained from a female donor who had taken 10 mg of clobazam 12 h previously. Elution used a gradient of 0-100% methanol with 0.1% TFA in 30 min on a Vydac 218 TP510 ClS column. Detection was at 225 nm with an aufs of 1.OO.
(Fielding and Hoffmann, 1979). The terminal half-life of elimination of the parent compound is about 18 h, and for the N-desmethylclobazam about 40 h (Rupp et al., 1979). At the time of blood sampling the ratio of unchanged clobazam to this metabolite was about 2 : 1, with a total free fraction of about 15%, therefore providing an adequate amount of both the drug and the metabolite for analysis. However, no separate peak was identified as the latter. These results demonstrate that clobazam and perhaps other benzodiazepines are bound to OMD in uiuo. That this occurs in the presence of a huge excess of HSA suggests that other endogenous factors limit access to the drug binding site on HSA, making it less effective as a receptor. In addition, OMD is an acute phase reactant whose levels may vary up to four times normal levels in disease (Kremer et al., 1988), making consideration of it a necessity in determining therapeutic doses of the drugs that bind to it.
Figure 2. The high resolution mass spectrum of the peak with a retention time of 35.48. The exact mass of 300.07 for the parent ion for this peak identifies it as clobazam.
52
M. TREUHEIT AND H. B. HALSALL
REFERENCES Abel, J. G., Sellers, E. M., Naranjo, C. A., Shaw, B. N., Kader, D. and Romach, M. K. (1979). Clin. Pharmacol. Ther. 26, 247. Clark, A. J.. Clissold, P. M., Shawi, R. A., Beattie, P. and Bishop, J. (1984). €MBO J. 3, 1045. Fielding, S. and Hoffrnann, 1. (1979). Brit. J. Clin. Pharmacol. 7, 7s-15s. Grossman, S. H., Davis, D., Kitchell, B. B., Shand, D. G. and Routledge, P. A. (1982) Clin. Pharmacol. Ther. 31, 350. Ishida, M., Suyama, K. and Adachi, S. (1980). J. Chromatogr. 189, 421. Ivancic, J. (1988). Ph.D. Thesis, University of Cincinnati, Cincinnati, OH. Johnson, R. F.. Schenker, S., Roberts, R. K., Desmond, P. V. and Wilkinson, G. R. (1979) J. Pharm. Sci. 66, 1320. Jones, D. R., Hall, S. D., Jackson, E. K., Branch, R. A. and Wilkinson, G . R. (1988). J. Pharm. Exp. Ther. 245, 816. Kober, A. and Sjoholm, I. (1979). Biochem. Pharm. 28, 1037.
Kornguth, M. L., Hutchins, L. G . and Etchelman, B. S. (1981). Biochem. Pharm. 30,2435. Kremer, J. M., Wilting, J. and Janssen, L. H. (1988). Pharm. Rev. 40, 1. Muller, W. E . , Stillbauer, A. E. and El-Garnel, S. (1983).J. Pharm. Pharmacol. 35, 684. Pevsner, J., Reed, R. R., Feinstein. P. G. and Snyder, S. H. (1988). Science 241, 336. Piafsky, K. M. and Borgb, 0. (1976). lancet 2, 963. Piafsky, K. M., BorgB, O., Odar-Cederlof, I., Johansson, C. and Sjoqvist, F. (1978). New f n g l . J. Med. 299, 1435. Rupp, W., Badian, M., Christ, O., Hadju, P., Kulkarni, R. D., Taeuber, K., Uihlein, M., Bender, R. and Vanderbeke, 0. (1979) Brit. J. Clin. Pharmacol. 7, 51s-57s. Schley, A. J. and Mueller-Oerlinghausen, B. (1983). Pharmacopsychiatry16,82. Treuheit, M. and Halsall, H. B. (1991). Chromatographia 31, 63. Wilchek, M., Miron, T. and Kohn, i. (1984). Meth. Enz. 104, 3.
Isolation of Clobazam-Orosomucoid Complexes from Patients’ Sera Michael Treuheit and H. Brian Haisall* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, USA
Orosomucoid, a member of the lipocalin family, may function in the in uiuo transport of lipophilic compounds such as basic and neutral drugs. We describe the identification of 7-chloro-l-metbyl-l,5-benzodiazepine-2,4dione (clobazam) bound to the serum orosomucoid from individuals actively taking this tranquillizer. This suggests not only that other endogenous factors limit access to the benzodiazepine binding site on human serum albumin, but also that the differential binding of benzodiazepines and their metabolites by orosomucoid should be considered in determining therapeutic doses, particularly in the acute phase response.
INTRODUCTION
EXPERIMENTAL
a,-Acid glycoprotein, orosomucoid (OMD), is generally considered to be a member of the lipocalin family, which functions either in the transport of lipophilic compounds or as receptors for them (Clark et ul., 1984; Pevsner et af., 1988). Numerous basic and neutral drugs have been shown to bind to isolated OMD. These have recently been thoroughly reviewed by Kremer et al. (1988). These studies, and other inferential data, have led to the widespread thesis that OMD is the major carrier of basic drugs, and, to a lesser extent, neutral drugs, in uiuo. However, there is a paucity of data that directly demonstrate that these drugs or other compounds are bound to OMD in serum. Human serum albumin (HSA) has also been shown to be a major binding protein for many neutral and acidic drugs in serum. Numerous studies (Kober and Sjoholm, 1979; Johnson et af., 1979; Jones et al., 1988) have indicated that the neutral benzodiazepines are mainly bound to a single class of binding sites on HSA. However, Miiller et af. (1983) and Kornguth et al. (1981) have also indicated by competitive binding assays that diazepam, the more notable member of the benzodiazepine family, displaces imipramine and chlorpromazine from OMD. Thus, although HSA may have a greater binding capacity than OMD, especially for acidic and neutral drugs, OMD may have a role in the plasma binding of these compounds, a role whose importance would increase during the acute phase response (Miiller et al., 1983). The approach taken here was to attempt to isolate drug-OMD complexes from a patient in treatment. OMD from individual donors, isolated in a rapid single step, has been previously used for the extraction and identification of bound ligands (Treuheit and Halsall, 1991). This communication, in a continuing effort to define endogeneous ligands bound to serum OMD, describes the isolation and identification of the parent compound 7-chloro-l-methyl-l,5-benzodiazepine-2,4-dione (clobazam) bound to the serum OMD from individuals that were actively taking this tranquillizer.
Materials. All reagent grade chemicals and high performance liquid chromatographic (HPLC) grade solvents were purchased from Fisher Scientific, Pittsburgh, PA. HPLC grade water was purified in our laboratory.
* Author
to whom correspondence should be addressed.
0269-3879/92/010050-03 $05.00
01992 by John Wiley & Sons, Ltd.
~~
~
Sera. 50 mL of blood was obtained from healthy donors who had taken 10 mg clobazam 12 h previously. Blood collection was by venipuncture into a 50 mL glass syringe (Becton and Dickinson). The blood was allowed to coagulate in groundglass stoppered centrifuge tubes (Fisher) for serum collection. Immunopurification of OMD. An immunoaffinity column (50 mL) was made using the monoclonal antibody (MAb) G9. The epitope for GS) includes residues 157-168, a sequence that is specific for OMD and contains no cross-reacting glycan chains. The G9 was coupled to Sepharose-4B-CL (Sigma) using carbonyldiimidizole (Wilchek et nl., 1984), forming the affinity matrix. 10mL of the all-glass prepared serum were applied to the G9 affinity column equilibrated with the washing buffer of 0.01 M phosphate and 0.1 M NaCl, pH 7.5. After washing with six column volumes of washing buffer, the bound OMD was eluted from the affinity column using 0.01 M phosphate and 1M NaCl, pH 7.5 (eluting buffer). All column connections were made with Teflon tubing. Reversed phase (C18)extraction of ligands. The OMD solution (50 mL) from the immunoaffinity column was loaded through the solvent reservoir directly onto a semipreparative Vydac CIRcolumn (218 TP 510, 10 mm x 250 mm) (The Separations Group, Hesperia, CA) with a defined maximum capacity for protein of 5 mg. Once loaded, the column was connected to a Kratos HPLC system, consisting of Kratos 400 dual pumps, a Kratos 783 detector and a SpectraPhysics 4290 integrator, and equilibrated with HPLC grade water containing 0.1% trifluoroacetic acid (TFA). The column was eluted with a 30 min gradient of 0-1oc)% methanol containing 0.1% TFA. The detector was set at 225 nm and the flow rate was 1.5 mL/min with an aufs of 1.00 for all separations. Buffer blanks. The immunoaffinity column was washed with 300mL of the washing buffer. The column was eluted with the eluting buffer and 50 mL (one column volume) collected. Received 18 April 1W1 Accepted 30 April 1991
OROSOMUCOID LlGANDS
51
This material was also loaded through the solvent reservoir directly onto the Vydac CI8column, and the latter eluted using the same gradient as above. Mass spectrometry. Mass spectral data were obtained on a Kratos MS 80 high resolution mass spectrometer. The pooled cuts from the several runs required to process the entire 50 mL blood sample were used.
RESULTS AND DISCUSSION
There appears to be considerable evidence that the benzodiazepines as a class bind to HSA. However, the work of Muller et al. (1983) and others (Kornguth et af., 1981; Abel et al., 1979; Grossman et al., 1982; Schley and Mueller-Oerlinghausen, 1983) indicates that OMD may be responsible for considerable serum binding and transport of this drug, as it is for many others (Kremer el al., 1988). If OMD does bind clobazam in uiuo, then the observation that the free fraction of those drugs that binds to OMD in uitro is reduced in the acute phase (Piafsky et al., 1978) may have some significance in determining therapeutic doses. The purpose here was to provide direct evidence for the binding of clobazam to OMD in uiuo. Indeed, this would be the first such report for any drug-OMD interaction. It is suggested that the single-step isolation of OMD maintains the protein at its physiological pH and ionic strength until elution, allowing for the isolation of endogenous ligands. lmmunoaffinity chromatography of OMD-drug complexes on a monoclonal antibody column has shown that it is possible to isolate some of these intact, depending on the association-dissociation kinetics of OMD and the drug (lvaneic, 1988). This offered the possibility that clobazam would also remain largely bound to OMD during the isolation procedure if the binding kinetics were favourable. A possible complication was that plasticizers have been shown to displace ligands bound to OMD (Piafsky and Borgi, 1976). However, the single-step isolation procedure had the additional advantage that it minimizes contact with the plasticizers commonly found in laboratory equipment and solvents (lshida et al., 1980). The OMD isolated from the affinity column was loaded directly onto a C,, HPLC column through the solvent reservoir. Ligands bound to this OMD were isolated and collected using a steep methanol (with 0.1% TFA) gradient. (It should be noted that the recovery at this point may not be quantitative because losses during the washing steps required for the immunochromatography are unknown). The chromatogram in Fig. 1 is the reversed phase HPLC profile of the OMD from a donor who had taken 10mg of clobazam 12 h previously. The peak at 35.48 was collected and concentrated for high resolution mass spectrometric (MS) analysis, other peaks being components of the buffer blank and a phthalate found associated with the OMD of smokers (Treuheit and Halsall, 1991). Figure 2 represents the mass spectrum for this compound. The parent ion had an exact mass of 300.07, which is identical to that of clobazam (CI6Hl3O2N2C1). Clobazam is metabolized to, principally, N desmethylclobazam, which has similar pharmacological properties to clobazam, but at a lower level of activity
4
B
Figure 1. The reversed phase HPLC profile of the OMD obtained from a female donor who had taken 10 mg of clobazam 12 h previously. Elution used a gradient of 0-100% methanol with 0.1% TFA in 30 min on a Vydac 218 TP510 ClS column. Detection was at 225 nm with an aufs of 1.OO.
(Fielding and Hoffmann, 1979). The terminal half-life of elimination of the parent compound is about 18 h, and for the N-desmethylclobazam about 40 h (Rupp et al., 1979). At the time of blood sampling the ratio of unchanged clobazam to this metabolite was about 2 : 1, with a total free fraction of about 15%, therefore providing an adequate amount of both the drug and the metabolite for analysis. However, no separate peak was identified as the latter. These results demonstrate that clobazam and perhaps other benzodiazepines are bound to OMD in uiuo. That this occurs in the presence of a huge excess of HSA suggests that other endogenous factors limit access to the drug binding site on HSA, making it less effective as a receptor. In addition, OMD is an acute phase reactant whose levels may vary up to four times normal levels in disease (Kremer et al., 1988), making consideration of it a necessity in determining therapeutic doses of the drugs that bind to it.
Figure 2. The high resolution mass spectrum of the peak with a retention time of 35.48. The exact mass of 300.07 for the parent ion for this peak identifies it as clobazam.
52
M. TREUHEIT AND H. B. HALSALL
REFERENCES Abel, J. G., Sellers, E. M., Naranjo, C. A., Shaw, B. N., Kader, D. and Romach, M. K. (1979). Clin. Pharmacol. Ther. 26, 247. Clark, A. J.. Clissold, P. M., Shawi, R. A., Beattie, P. and Bishop, J. (1984). €MBO J. 3, 1045. Fielding, S. and Hoffrnann, 1. (1979). Brit. J. Clin. Pharmacol. 7, 7s-15s. Grossman, S. H., Davis, D., Kitchell, B. B., Shand, D. G. and Routledge, P. A. (1982) Clin. Pharmacol. Ther. 31, 350. Ishida, M., Suyama, K. and Adachi, S. (1980). J. Chromatogr. 189, 421. Ivancic, J. (1988). Ph.D. Thesis, University of Cincinnati, Cincinnati, OH. Johnson, R. F.. Schenker, S., Roberts, R. K., Desmond, P. V. and Wilkinson, G. R. (1979) J. Pharm. Sci. 66, 1320. Jones, D. R., Hall, S. D., Jackson, E. K., Branch, R. A. and Wilkinson, G . R. (1988). J. Pharm. Exp. Ther. 245, 816. Kober, A. and Sjoholm, I. (1979). Biochem. Pharm. 28, 1037.
Kornguth, M. L., Hutchins, L. G . and Etchelman, B. S. (1981). Biochem. Pharm. 30,2435. Kremer, J. M., Wilting, J. and Janssen, L. H. (1988). Pharm. Rev. 40, 1. Muller, W. E . , Stillbauer, A. E. and El-Garnel, S. (1983).J. Pharm. Pharmacol. 35, 684. Pevsner, J., Reed, R. R., Feinstein. P. G. and Snyder, S. H. (1988). Science 241, 336. Piafsky, K. M. and Borgb, 0. (1976). lancet 2, 963. Piafsky, K. M., BorgB, O., Odar-Cederlof, I., Johansson, C. and Sjoqvist, F. (1978). New f n g l . J. Med. 299, 1435. Rupp, W., Badian, M., Christ, O., Hadju, P., Kulkarni, R. D., Taeuber, K., Uihlein, M., Bender, R. and Vanderbeke, 0. (1979) Brit. J. Clin. Pharmacol. 7, 51s-57s. Schley, A. J. and Mueller-Oerlinghausen, B. (1983). Pharmacopsychiatry16,82. Treuheit, M. and Halsall, H. B. (1991). Chromatographia 31, 63. Wilchek, M., Miron, T. and Kohn, i. (1984). Meth. Enz. 104, 3.
