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Department of Pharmaceutics, University of Florida, Gainesville, Florida
(Received May 31, 2005; accepted October 25, 2005)
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Abstract |
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90%) was present in the stomach, intestines, and intestinal contents, suggesting biliary excretion of MF and its metabolites. Radiochromatography showed that most radioactivity was associated with MET1, MET2, and MET 3. Fractionation of the high-performance liquid chromatography eluate (MET1-5) revealed that only MF [relative binding affinity (RBA) 2900] and MET2 (RBA 700) had appreciable glucocorticoid receptor binding affinity. These results suggest that MF undergoes distinct extrahepatic metabolism but generates active metabolites that might be in part responsible for the systemic side effects of MF.
; Prakash and Benfield, 1998). Recently, MF has been approved in Europe and the United States as an oral inhalation powder, in the treatment of mild-to-moderate persistent asthma (Bernstein et al., 1999; Affrime et al., 2000a; Nayak et al., 2000; Sharpe and Jarvis, 2001). There is very limited information on the pharmacokinetics of MF after an oral inhalation. Affrime et al. (2000a) reported the pharmacokinetics, after a single-dose administration of 400 µg of MF given intravenously, by a metered dose inhaler (MDI) and by a dry powder inhaler (DPI). After administration by MDI, MF was undetectable in plasma, and after DPI administration, plasma concentrations of MF were also extremely low and close to the limit of quantification (50 pg/ml). The mean bioavailability of MF after DPI was estimated to be 0.96% (Affrime et al., 2000a). Minimal systemic exposure to MF was also seen after multiple-dose administration by DPI with mean Cmax values close to or below the limit of quantification (50 pg/ml) after a 200 to 800 µg/day dose administered by DPI for 4 to 52 weeks (Bernstein et al., 1999; Affrime et al., 2000b). These initial studies on the pharmacokinetics of MF suggest that the systemic levels and bioavailability of MF are extremely low after an oral inhalation when compared with other inhaled glucocorticoids. The reason for these observations might be that MF exhibits a more efficient systemic clearance either via extrahepatic metabolism or through low pulmonary availability. Recent studies (Derks et al., 2005) have shown that the systemic availability of MF might not be as low as suggested previously. Also, the drug shows considerable systemic effects as manifested by the suppression of the hypothalamic-pituitary-adrenal axis (Fardon et al., 2004), although original reports claimed minimal cortisol suppression (Affrime et al., 2000b). A further complication is that the formation of active MF metabolites (Isogai et al., 1993) might be responsible, in part, for the observed systemic effects despite the low levels reported by Affrime et al. (2000b). Because of these controversies, we were interested in further evaluating pharmacokinetic and pharmacodynamic properties of MF, which included hepatic and extrahepatic metabolism, and the assessment of the bioactivity of formed metabolites.
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). Other studies have suggested that MF undergoes hydroxylation at the 6-position to form 6ß-hydroxy-MF, hydrolysis of the furoate ester, and the substitution of the C-21 chlorine with a hydroxyl group (Onrust and Lamb, 1998; Affrime et al., 2000a; Crim et al., 2001). The above-mentioned suggested routes of metabolism have not been confirmed, and there is no evidence of a single major metabolite (Sharpe and Jarvis, 2001). Two reports by Teng et al. (2003a,b) on the metabolism of unlabeled MF in liver and intestinal microsomes described the formation of one metabolite (tentatively identified as 6ß-hydroxy-MF), while mentioning that other parallel and subsequent metabolic pathways could be involved. These studies also reported MF to be stable in plasma and lung tissue. To be able to capture the overall metabolism of MF in a more complete fashion, we decided to evaluate the hepatic and extra-hepatic metabolism of MF in vitro as well as in vivo, using a radiolabeled tracer, and to assess the biological activity of the whole metabolic spectrum rather than performing such studies for just one single metabolite. These studies should be able to confirm whether extrahepatic metabolism might explain the low systemic levels after inhalation of MF, give information about the generated metabolites and their pharmacological activity, and give information on the biodistribution and elimination of the drug.
Results of these studies would be able to support or argue against the pharmacokinetic reports by Affrime et al. (2000b). Distinct extra-hepatic metabolism would support the pharmacokinetic findings by Affrime et al. (2000b) of low systemic MF levels and reduced systemic side effects (in the absence of active metabolites). Assuming extrahepatic metabolism, the existence of active metabolites might explain the reported cortisol suppression (Fardon et al., 2004) despite low MF levels. In the second case, the lack of extrahepatic metabolism and the formation of only minor amounts of active metabolites would argue against the reported pharmacokinetic profile of MF and the possibility that MF does not differ from other inhaled glucocorticoids with respect to safety issues.
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Materials and Methods |
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The use of Sprague-Dawley rats (Harlan, Indianapolis, IN) was approved by the local Institutional Animal Care and Use Committee at the University of Florida (Gainesville, FL). Approval was obtained from the Institutional Review Board, University of Florida, for the use of human lung and liver tissues.
Stability of MF in Plasma. The stability of MF in fresh rat plasma was assessed by incubating the plasma spiked with MF, at a concentration of 6.2 µM, at 37°C in a thermostatically controlled water bath. Experiments were conducted in triplicates. Aliquots (200 µl) were removed at regular intervals up to 72 h into prechilled tubes, and the samples were precipitated with 600 µl of methanol. Blank rat plasma was used in a control experiment. Identical stability studies were performed using fresh human plasma also. The samples were stored at –20°C and were analyzed by HPLC within 48 h of storage.
The precipitated plasma samples were vortex mixed and centrifuged at 10,000 rpm for 5 min and 100 µl of the supernatant was injected directly for HPLC-UV analysis. An LDC/Milton Roy CM4000 multiple solvent delivery system (Milton Roy Company, Rochester, NY) using a Milton Roy SM 4000 programmable wavelength detector set at 
max 254 nm, a CR-3A Chromatopac integrator (Shimadzu, Kyoto, Japan), and an automatic injector (PerkinElmer Life and Analytical Sciences, Boston, MA) was used as the HPLC system. Chromatographic separation of the analytes was achieved on Waters (Milford, MA) 5-µm symmetry RP-18 (150 x 4.6 mm i.d.) column preceded with a guard column (10 x 4.6 mm i.d., filled with reverse phase precolumn material) using a mobile phase of 65:35% (v/v) methanol/water at a flow rate of 1 ml.
Calibration curves based on MF peak areas over the concentration range of 0.125 to 5.0 µg/ml using unweighted least-squares linear regression analysis resulted in r2 values of at least 0.999. The limit of quantification of the assay was 0.125 µg/ml. The good reproducibility of the system allowed quantification without an internal standard since potential internal standards cross-eluted with either the endogenous interferences or the degradation products formed.
Because of the lack of suitable standards for the degradation products, quantification was based on the assumption that their molar absorptivities were identical to that of MF. Results for MF and degradation products are presented as micromolar concentration and expressed as percentage of MF starting concentrations in the graphical representations.
Metabolism of MF in Liver and Lung. Preparation of Homogenizing Buffer (1.15% KCl in 50 mM K2HPO4, pH 7.4). A total of 870 mg of dipotassium hydrogen orthophosphate (K2HPO4) and 1.15 g of potassium chloride (KCl) were dissolved in 100 ml of deionized water, and the pH was adjusted with 20% (v/v) orthophosphoric acid to 7.4. The buffer was chilled to 4°C before use.
Preparation of NADPH Generating System. NADPH generating system, which was composed of cofactor salts NADP (2 mM), glucose 6-phosphate (8 mM), nicotinamide (200 mM), and magnesium chloride (200 mM), was prepared in the homogenizing buffer. This cofactor solution was incubated at 37°C for 15 min to allow generation of NADPH and then was used immediately.
Tissue Metabolism. S9 fractions of rat and human lung and liver were used for studying the metabolism of MF. Human tissue samples were obtained from the Molecular Tissue Bank at the University of Florida. These samples were obtained by the tissue bank from surgical procedures (liver and lung resection, nonsmokers), but further information was not obtainable from the Molecular Tissue Bank. Samples were stored at –70°C. Rat tissues were obtained by sacrificing the rats by decapitation and removing the lung and liver tissue immediately. The tissues were then rinsed in ice-cold homogenizing buffer, blotted dry, and weighed.
The lung and liver tissue were homogenized in ice-cold homogenizing buffer to obtain 20 and 40% (w/v) tissue, respectively. The lung and liver tissue homogenates were centrifuged at 4°C at 10,000g for 45 min to obtain the S9 fraction for lung and liver. S9 fractions were preincubated with equal volumes of cofactor solution and incubated in a shaker bath to equilibrate the mixture to 37°C. Unlabeled MF was added to initiate the reaction; the final drug concentration was 4.8 µM and the final tissue concentration was 10% (w/v) for lung and 20% (w/v) for liver. The lung and liver S9 fractions were incubated at 37°C for up to 24 h under atmospheric air. Drug-free S9 fraction (lung or liver) was used in a control experiment. Serial samples of 250 µl were withdrawn at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, or 240 min, and 24 h into prechilled tubes. The samples were precipitated with 250 µl of acetonitrile and analyzed by HPLC. The precipitated samples of lung and liver homogenates were centrifuged at 10,000 rpm for 5 min. One hundred microliters of the supernatant was injected directly for HPLC analysis. The chromatographic conditions for analysis of samples from S9 studies was identical to those mentioned earlier for analysis of the plasma samples. However, a mobile phase composition of 50:50% (v/v) acetonitrile/water was used for the analysis of the S9 samples.
Results for MF and metabolites are presented as micromolar concentration and expressed as percentage of MF starting concentrations in graphical representations. Semilogarithmic plots of percentage of MF remaining versus time were used to determine the apparent disappearance rate constant of MF. The disappearance half-life was determined using the equation t1/2 = 0.693/kdiss, where kdiss is the apparent disappearance rate constant for that compound.
Metabolism in Liver and Lung Tissue Using Labeled [1,2-3H]MF. An identical parallel incubation was performed simultaneously with 5 ml of lung and liver S9 fraction in which [1,2-3H]MF (concentration <0.125 µg/ml) was added in addition to the unlabeled MF (2.5 µg/ml). The homogenizing buffer was also spiked with identical concentrations of labeled and unlabeled MF and was used in a control experiment. The samples containing labeled MF were also incubated as mentioned in the previous section. Aliquots (250 µl) were withdrawn every 20 min for the first 2 h and finally at 24 h. The samples were withdrawn into prechilled tubes and were precipitated with 250 µl of acetonitrile to terminate the reaction. The samples were immediately analyzed by HPLC using the conditions mentioned for the analysis of unlabeled MF. One-minute fractions were collected using a fraction collector and transferred to individual scintillation vials, and counted using a liquid scintillation counter (LS 5000 TD; Beckman Coulter, Fullerton, CA) for the presence of radioactivity. The total counts present for MF at time 0 were considered as 100%. The percentages of metabolites formed were estimated as the ratio of dpm for metabolite to dpm of parent drug at time 0.
In Vivo Metabolism and Distribution of MF. A total of 5 µCi of [1,2-3H]MF per 250 mg of body weight, in normal saline, was administered by injection into the tail vein of male Sprague-Dawley rats. The animals were sacrificed by decapitation after 2 h. The abdominal and thoracic cavities were opened immediately and blood from the heart was withdrawn. Urine was collected by puncturing the urinary bladder. The organs (Table 1) were dissected, rinsed with ice-cold normal saline, blotted, weighed, and then homogenized in methanol. A 1-cm2 area of skin and the vastus lateralis muscle from the right leg were resected and homogenized without being rinsed. The contents of the stomach and the intestines (both small and large) were flushed out of the organs before homogenization and collected. Thirty milliliters of methanol was used to homogenize the liver and 20 ml was used to homogenize the other tissues. One milliliter of the homogenates was transferred to scintillation vials and 10 ml of the scintillation cocktail was added. The scintillation vials were read in a liquid scintillation counter (LS 5000 TD; Beckman Coulter).
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Glucocorticoid Receptor Binding Assay Experiments in Rat Lung Cytosol. As described earlier, in the metabolism experiments, liver S9 fractions were spiked only with unlabeled MF and incubated at 37°C in a shaker bath for 2 h. The 2-h samples were analyzed by HPLC-UV analysis and the metabolites were isolated by collecting the fractions corresponding to the retention times of the metabolites. The fraction corresponding to the elution of MF (denoted by MFfrac) in the samples was also collected. These fractions were then evaporated under vacuum and reconstituted in methanol. The concentrations of the MFfrac and the metabolites in the fractions were calculated from percentage conversion of MF into the metabolite data obtained from the previous section. The dilutions for MF (from a stock of 1 mg/ml), MFfrac, and the metabolites were prepared in methanol. Dexamethasone (DEXA) was used as a reference compound for the receptor binding experiments. The concentration ranges for the compounds in the study were 0.01 to 100 nM for MF, MET1, and MET3; 0.01 to 30 nM for MFfrac and MET2; 0.01 to 50 nM for MET4 and MET5; and 0.01 to 1000 nM for DEXA.
A previously described method (Hochhaus and Moellmann, 1990), with slight modifications, was used for performing the competition assays. Sprague-Dawley rats were anesthetized, using a cocktail mixture of ketamine, xylazine, and acepromazine (3:3:1 v/v), and were decapitated. The lungs were removed and homogenized in 8 volumes of ice-cold incubation buffer (10 mM Tris/HCl, 10 mM sodium molybdate, 2 mM 1,4-dithioerythritol). The homogenate was incubated with 5% (w/v) charcoal suspension (in deionized water) for 10 min. The homogenate was then centrifuged for 20 min at 40,000 g in the J2 rotor of a Beckman Coulter centrifuge to obtain the cytosol. Fresh cytosol was prepared and used for all the individual experiments.
A final tracer concentration of 10 nM 3H-labeled dexamethasone solution was used as a tracer, based on previous saturation binding experiments performed in our laboratory (data not shown). Ten microliters of the dilutions of the test compound in methanol was added to prechilled tubes. Blank methanol was used for the determination of total binding. Nonspecific binding was determined after addition of 10 µl of 100 µM unlabeled DEXA (10 µM in the final incubation mixture). A total of 10 µl of 100 nM 3H-dexamethsone solution (10 nM in final incubation mixture) was then added. Then, 80 µl of the lung cytosol was added, and the tubes were vortexed and incubated at 4°C for 24 h. After the incubation, 100 µl of 5% (w/v) charcoal suspension (in water) was added to the tubes to remove the excess unbound radioactivity. The tubes were vortexed and 150 µl of the supernatant was transferred to the scintillation vials. Five milliliters of the scintillation cocktail was added and the scintillation vials were read in the liquid scintillation counter. Control experiments were also conducted in which receptor binding assays were performed on fractions collected during HPLC-UV analysis of blank S9 fractions and on fractions collected during blank mobile phase runs.
The data obtained were fitted by SCIENTIST (MicroMath Inc., Salt Lake City, UT) using the following Emax model to obtain the estimates of Bmax and IC50.
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The IC50 obtained for dexamethasone (IC50,dex) was used to calculate the relative binding affinity of the test compound (RBAtest) from its IC50 value (IC50,test) as:
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Results |
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), the molecular masses ([MH+]) of MF, D1, D2, and D3 were determined to be 521, 485, 467, and 503, respectively. The polarity sequence of MF and its degradation products were in the order D1 > MF > D2 > D3. The decline and formation of MF and D1, D2, and D3 is shown in Fig. 1A. The degradation half-life of MF as calculated from the terminal phase of the concentration-time curve was found to be 85 h.
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Stability in Lung. Incubation of MF in rat lung S9 fractions showed no metabolic conversion until 2 h. However, incubation of MF for 24 h resulted in a degradation product, D2, that was found to be more nonpolar than the parent drug. This degradation product had retention times identical to those of the product D2 seen upon incubation of MF in plasma. Parallel control incubations of labeled MF in the incubation buffer also showed formation of D2. This indicated that the formation of D2 was a result of breakdown of MF in the homogenizing buffer and not a result of metabolism in the lung. Incubations of labeled MF in the S9 fraction of lung were also conducted, which also proved that MF was stable in lung fractions. The representative radiochemical elution profiles for MF in rat lung incubations are shown in Fig. 2. Incubation of MF in human lung S9 fractions also indicated that MF was stable with no metabolic conversion until 2 h of incubation. These results indicate that MF does not undergo significant pulmonary metabolism.
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The HPLC-UV profiles observed after incubation of unlabeled MF with S9 fraction of rat liver indicated the formation of a polar metabolite (MET2) that eluted at 5.1 min. The disappearance half-life of unlabeled MF (16 min) as determined by HPLC-UV analysis was found to be identical to the half-life of labeled MF (18.5 min) that was estimated from the radiochemical elution profiles. The metabolite MET1 (retention time between 2 and 3 min) could not be viewed using a UV-visible detector due to the presence of endogenous interferences at the same retention time. MET3, MET4, and MET5 could not be quantified using UV due to the very low concentrations of these metabolites.
Incubation of a mixture of labeled and unlabeled MF in human liver S9 fractions also showed formation of five metabolites. The retention times for these metabolites were identical to the ones formed with rat liver S9 fractions. A semilogarithmic plot of percentage dpm versus time displayed a linear relationship with a correlation coefficient of 0.9584, and the disappearance half-life of 3H-MF in the S9 fraction was found to be 0.76 h–1, with a corresponding half-life of 55 min.
The concentration-time profiles for the disappearance of MF and the appearance of the metabolites are shown in Fig. 5. The HPLC-UV profiles observed after incubation of MF with S9 fraction of human liver showed an endogenous tissue impurity eluting at around 5 min. Because of this interference, MET2 could not be observed in human liver tissues. Hence, only the decline of MF could be monitored under UV detection over time. The molecular mass ([MH+]) of MET2 was estimated to be 538 using a mass spectrometry method reported previously (Sahasranaman et al., 2004).
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In Vivo Metabolism and Disposition. The radioactivity (expressed as percentage of dose administered) in the tissues 2 h after an intravenous injection of [1,2-3H]MF in rats is given in Table 1. Radiochemical elution profiles of the intestinal contents show the formation of three metabolites that have retention times identical to those of the metabolites MET1, MET2, and MET3 (Fig. 6), which were seen upon incubation of MF with liver S9 fractions. There was no radioactivity corresponding to MF, and this is consistent with the efficient hepatic metabolism of MF that was seen in vitro. Most of the radioactivity in the intestinal contents was associated with MET1 (Fig. 6), whereas MET2 and MET3 were minor metabolites. MET4 and MET5 were either not formed or formed in too low a concentration to be seen in the intestinal contents.
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Discussion |
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,b). Our results, however, propose a different degradation pathway (Scheme 1) which was based on more detailed investigations of D1 and D2 in buffer systems. The structure of D3 has been proposed based on the structures of D1 and D2 and the molecular mass of D3. There is no conclusive evidence in this study for the conversion of D3 to D2; therefore, this pathway has been denoted by broken arrows in the scheme. D1, but not D2 (Sahasranaman et al., 2004) and, presumably, not D3 (considering similarity to D2) shows binding to the glucocorticoid receptor.
Even though degradation of MF is observed in plasma and lung in vitro, the relatively high stability, when compared with the hepatic processes, makes it unlikely that extrahepatic metabolism is responsible for the low systemic exposure of MF after an oral inhalation. Other events, such as the removal of the lipophilic, slow dissolving drug from the lung through mucociliary clearance, might be, in part, responsible for the low systemic levels. Very recently, the systemic bioavailability of mometasone furoate has been shown to differ between subjects with normal and reduced lung function (K. Mortimer, T. Harrison, Y. Tang, K. Wu, S. Lewis, S. Sahasranaman, G. Hochhaus, and A. Tattersfield, submitted for publication), an indication for the removal of the more centrally deposited drug in patients with lower lung function. Contrary to this, there is also some indication that the systemic exposure after MF inhalation is actually higher than originally reported (Derks et al., 2005). Therefore, it seems likely that systemic availability of MF is not being determined by extrahepatic clearance, but can be explained solely on the pharmacokinetic fate of MF in the lung.
In human plasma, the sum of MF and degradation products at 72 h did not add up to 100%. Valotis et al. (2004) suggested covalent binding of the epoxide(s), which would prevent extraction. In that paper, 9 to 16% covalent binding was found after 3 h of incubation. This binding, presumably to protein components, also explains the reduced extractable fraction found in our experiments and, therefore, supports the findings of Valotis et al. (2004). However, future studies are necessary to show whether such adducts would also be formed in vivo.
This study showed that MF is efficiently metabolized into at least five metabolites in both rat and human liver S9 fractions. There were, however, quantitative differences in the metabolites between the two species. The apparent half-life of MF in the S9 fraction of human liver was found to be 3 times greater compared with that in rat. MET1, the most polar metabolite, was the major metabolite in rat liver fractions, whereas both MET1 and MET2 were formed to an equal extent in human liver fractions. Such species differences have been noted for other glucocorticoids such as budesonide (Edsbacker et al., 1987).
The molecular mass ([MH+]) of MET2 was estimated to be 538 using a mass spectrometry method reported previously (Sahasranaman et al., 2004). Based on the molecular mass and comparing the chromatographic elution profile of MET2 to putative metabolites (Affrime et al., 2000a), MET2 is expected to be 6ß-OH-MF. This confirms other reports that have hinted at the formation of this metabolite (Zbaida et al., 1997; Affrime et al., 2000b; Teng et al., 2003a,b). Hydroxylation at the 6ß-position is a common route of metabolism among glucocorticoids, with budesonide, triamcinolone, acetonide, and flunisolide being metabolized into their respective 6ß-hydroxy derivatives (Kupfer and Partridge, 1970; Gordon and Morrison, 1978; Chaplin et al., 1980; Edsbacker et al., 1987; Jonsson et al., 1995). Using HPLC-UV detection in the present study, only the decline of MF and the formation of MET2 could be viewed. This agrees with the report by Teng et al. (2003a,b), who monitored the formation of only 6ß-OH-MF in human liver using UV and concluded that other metabolites also should be formed to explain the loss of MF. The present study clearly reveals the existence of at least five metabolites. Thus, the use of the tritium-labeled drug provided valuable information about the formation of the other metabolites as well. MET1 is not among the putative metabolites of MF that were proposed by Affrime et al. (2000a). Whether MET1 is identical to a fraction in urine reported by the same group to elute in a "very polar region" (Affrime et al., 2000a) needs further investigations. Unfortunately, we were not able to identify MET1, or MET3, MET4, and MET5, either because they could not be extracted (MET1) or because they were formed in insufficient quantities. In summary, these experiments showed the generation of a total of five MF metabolites; the identity of one (6ß-OH-MF) confirmed previous suggestions (Teng et al., 2003a,b; Davies, 2004).
In vivo experiments in rats after i.v. administration of MF revealed additional information. The data obtained 2 h postadministration showed that most of the radioactivity (90%) is present in the stomach, intestines, and the intestinal contents, whereas radioactivity in all other organs was lower and represented mainly MET1-3. This strongly suggests that MF and its metabolites are excreted through the bile. Not surprisingly, the majority of the radioactivity in the intestine was rather hydrophilic (MET1). Biliary excretion has also been reported for other glucocorticoids like triamcinolone, acetonide, and prednisolone (Kripalani et al., 1975; Mueller and Potter, 1981; Khalafallah and Jusko, 1984).
MF-related radioactivity was much lower in other tissues. The amount of radioactivity associated with the brain was negligible and the least among the organs studied. This is consistent with results from other studies that show that the uptake of glucocorticoids into the brain is limited by the multidrug resistance P-glycoprotein efflux transporter at the blood-brain barrier (Karssen et al., 2002; Arya et al., 2005). Overall, these experiments clearly indicate that MF is metabolized efficiently in vivo and the metabolites are eliminated by biliary excretion into the intestinal tract.
The evaluation of the potential bioactivity of the formed metabolites was a significant part of this study, inasmuch as Isogai et al. (1993) reported that a number of potential MF metabolites showed distinct binding to the glucocorticoid receptor. Using the selected approach of assaying HPLC fractions of liver S9 fractions for glucocorticoid binding allowed us to monitor all metabolic fractions, despite not knowing their chemical identity. Among the metabolite fractions (Table 2), two of the five metabolites (MET2 and, to a smaller degree, MET3) showed measurable affinity to the glucocorticoid receptor, whereas MET1 (the most hydrophilic metabolite), MET4, and MET5 did not bind. Whereas the low affinity of MET3 and the lack of any binding of MET1, MET4, and MET5 are in agreement with reduction of glucocorticoid bioactivity through metabolism, the RBA value of MET2 (700) is greater than the RBA values of some commonly used inhaled corticosteroids like flunisolide (RBA 180), triamcinolone, and acetonide (RBA 233) and is comparable to that of budesonide (RBA 935) (Hochhaus and Moellmann, 1990; Wuerthwein et al., 1992). The strong affinity of 6ß-OH-MF is surprising because the 6ß-hydroxy derivatives of other glucocorticoids do not show significant activity toward the glucocorticoid receptor. The RBA values of 6ß-OH-budesonide and 6ß-OH-flunisolide have been reported to be 6 and less than 1, respectively (Dahlberg et al., 1984; Hochhaus and Moellmann, 1990).
In summary, the original data on MF reported low systemic availability for this compound. MF produces significant systemic effects after an oral inhalation. An 800-µg b.i.d. dose of MF administered by a metered dose inhaler for 28 days produced a 20 to 30% suppression of the hypothalamic-pituitary-adrenal axis compared with placebo (P < 0.05) (Affrime et al., 2000b). By comparison, an 880-µg b.i.d. dose of fluticasone propionate also delivered via a MDI produced a 43 to 56% suppression of the hypothalamic-pituitary-adrenal axis compared with placebo (P < 0.01) (Affrime et al., 2000b; Crim et al., 2001). In a recent study, fluticasone propionate and MF delivered via their respective DPIs showed similar dose-dependent cortisol suppressions (Fardon et al., 2004). The formation of the active metabolite, 6ß-OH-MF, might explain in part the systemic effects after MF administration of these metabolites. Recently, other studies presented in abstract form have reported a systemic availability of 5% (Derks et al., 2005). Systemic effects of MF might therefore be the result of both a higher systemic exposure than originally assumed and the presence of active metabolites. Therefore, the measurement of this metabolite is critical in obtaining a true measure of the systemic bioavailability of mometasone furoate.
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ABBREVIATIONS: MF, mometasone furoate; MDI, metered dose inhaler; DPI, dry powder inhaler; HPLC, high-performance liquid chromatography; MET, metabolite; DEXA, dexamethasone; RBA, relative binding affinity; OH, hydroxy.
1 Current affiliation: Eon Labs, Wilson, NC. 
Address correspondence to: Günther Hochhaus, Box 100 494, JHMHC, UF, College of Pharmacy, Gainesville, FL 32610. E-mail: Hochhaus{at}cop.ufl.edu
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