One way to minimise systemic side effects of drugs is to design molecules, soft drugs, in such a way that they are metabolically inactivated rapidly after having acted on their pharmacological target. Hydrolases (esterases, peptidases, lipases, glycosidases, etc.) are enzymes well suited to use for drug inactivation since they are ubiquitously distributed. Insertion of ester bonds susceptible to enzymatic cleavage may represent one approach to make the action of a drug more restricted to the site of application. The present study describes the chemical synthesis of fourteen model compounds comprising a bicyclic aromatic unit connected by an ester-containing bridge to another aromatic ring. Initial attempts to define a) the tissue selectivity of the hydrolytic metabolism and b) the molecular structural factors affecting the rate of enzymatic ester cleavage are presented. The data show that human and rat liver fractions were more active than human duodenal mucosa and human blood leukocytes at hydrolysing the compounds. The rank order of the compounds was, however, very similar in the different biological systems. Commercially available pig liver carboxyl esterase and cholesterol esterase both reasonably well predict the rank order in the tissue fractions.
        
Title: Reversible fatty acid conjugation of budesonide. Novel mechanism for prolonged retention of topically applied steroid in airway tissue Miller-Larsson A, Mattsson H, Hjertberg E, Dahlback M, Tunek A, Brattsand R Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 26:623, 1998 : PubMed
A high airway concentration might be required for the antiasthmatic efficacy of inhaled glucocorticosteroids (GCS). The topical uptake and retention of GCS in airway tissue were compared for GCS of the inhaled type [budesonide (BUD), fluticasone propionate (FP), and beclomethasone dipropionate (BDP)] and of the noninhaled type (dexamethasone and hydrocortisone). 3H-labeled GCS solutions were administered into rat airways by either perfusion of trachea in vivo, intratracheal instillation, or inhalation. Radioactivity was determined in the airway tissue, lung parenchyma, and plasma 20 min to 24 hr after exposure. Ethanol extracts of exposed tracheas were analyzed by HPLC. Exposed tracheas were also incubated in vitro in buffer, and the released radioactivity was analyzed by HPLC. BUD, FP, and BDP were equally well taken up into the airway tissue; their uptake was 25-130 times greater than that of dexamethasone and hydrocortisone. BUD was shown to form very lipophilic intracellular fatty acid esters (at carbon 21) in the airway and lung tissue after topical application. In large airways 20 min after administration, approximately 70-80% of retained BUD was conjugated. BUD stored in esterified form in the tissue was retained in large airways for a prolonged time, compared with FP and BDP, which do not form such conjugates. The fatty acid conjugation of BUD is reversible in vivo; BUD conjugates are gradually hydrolyzed and free BUD is regenerated. This reversible conjugation may improve airway selectivity, as well as prolong the local anti-inflammatory action of BUD in the airways and might be one explanation for why BUD is efficacious in the treatment of mild asthma when inhaled once daily.
        
Title: Pharmacologic importance of the reversible fatty acid conjugation of budesonide studied in a rat cell line In vitro Wieslander E, Delander EL, Jarkelid L, Hjertberg E, Tunek A, Brattsand R Ref: American Journal of Respiratory Cellular & Molecular Biology, 19:477, 1998 : PubMed
Functional implications of the recently described fatty acid conjugation of budesonide (BUD) (Tunek, A., K. Sjodin, and G. Hallstrom, Drug Metabol. Dispos. 1997;25:1311-1317; Miller-Larson, A., E. Hjertberg, H. Mattsson, M. Dahlback, A. Tunek, and R. Brattsand, Am. J. Respir. Crit. Care Med. 1997;155:A353 [Abstr.]) were studied in a rat cell line, Rat1, transfected with the activation protein-1 (AP-1)-controlled regulatory element (TRE) driving the reporter gene beta-galactosidase. TRE is downregulated by glucocorticosteroids (GCS) through interaction with the AP-1 complex. BUD was compared to fluticasone propionate (FP), a potent glucocorticosteroid that does not form fatty acid conjugates. The kinetics and metabolism of the GCS were studied after incubation of either 3H-BUD or 3H-FP with transfected Rat1 cells. Up to 20% of added BUD was taken up into the cells over 24 h. The great majority of the intracellular radioactivity (80-90%) consisted of lipophilic BUD conjugates. After removing extracellular 3H-GCS, the outflow of radioactivity was studied. Only free BUD and not fatty acid conjugates was detected extracellularly, suggesting that hydrolysis of the conjugates was required to release BUD from the cell. During 165 min, less BUD (about 65% of totally incorporated) was released than FP (more than 90%). In the functional studies, FP was about six times more potent than BUD in downregulating TRE after 24 h continuous exposure. However, after a 6-h pulse of GCS, the effect of BUD persisted unchanged 18 h later, whereas FP had almost lost its efficacy (P < 0.05 between the drugs). In addition, the reversible conjugation process of BUD resulted in transferable GCS effects. Medium containing released BUD from previously loaded cells mediated nearly the same downregulatory effect after addition to naive cells as did continuous treatment. No such transferable effect was seen for FP. In conclusion, the reversible fatty acid conjugation of BUD resulted in prolonged cellular retention and anti-inflammatory activity after pulse exposure in this in vitro system. This fatty acid conjugation mechanism appears to add to the beneficial pharmacologic profile of BUD.
        
Title: Reversible formation of fatty acid esters of budesonide, an antiasthma glucocorticoid, in human lung and liver microsomes Tunek A, Sjodin K, Hallstrom G Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 25:1311, 1997 : PubMed
Microsomes from human lung and liver catalyze the formation of fatty acid esters of budesonide, a glucocorticoid used for inhalation treatment of asthma. The conjugation was dependent on coenzyme A and ATP. Addition of free fatty acids to the incubations affected the pattern of metabolites, but ester formation was observed also without such addition. Budesonide oleate, palmitate, linoleate, palmitoleate, and arachidonate were identified as metabolites. The fatty acid conjugates of budesonide were shown to be substrates for lipase in vitro, thus budesonide is regainable from the conjugates. The data suggest that an equilibrium between budesonide and these pharmacologically inactive lipoidal conjugates will be established in tissues at repeated exposure to budesonide. Since the fatty acid conjugates most likely will be retained intracellularly for a longer time than unchanged budesonide, the duration of tissue exposure to budesonide will depend partly on the rate of lipase-catalyzed hydrolysis of the conjugates. The findings in this study provide a possible explanation for the efficacy of budesonide in mild asthmatics also when inhaled once daily.
        
Title: Interactions of bambuterol with human serum cholinesterase of the genotypes EuEu (normal), EaEa (atypical) and EuEa Tunek A, Hjertberg E, Mogensen JV Ref: Biochemical Pharmacology, 41:345, 1991 : PubMed
Bambuterol, a carbamate ester prodrug of the bronchodilator terbutaline, was tested as inhibitor and substrate of human serum cholinesterases of the genotypes EuEu (the normal enzyme), EaEa (the atypical enzyme) and EuEa. The IC50 for the normal enzyme was 11 +/- 2.2 nM (mean, SD, N = 10) and for the atypical enzyme 140 +/- 6 nM (N = 13), indicating a much higher affinity of bambuterol to the normal enzyme. The heterozygotes showed a mixed behaviour; the major activity was inhibited like the normal enzyme (IC50 = 9.3 +/- 1.9 nM, N = 9), while a residual activity (10-15%) was inhibited by bambuterol like the atypical enzyme. At a bambuterol concentration of 100 nM each of the three cholinesterase genotypes responded uniquely to bambuterol; the normal enzyme was inhibited to 2.2 +/- 0.9%, the atypical enzyme to 58 +/- 4.6%, and the heterozygote to 10 +/- 1.2% of the basal activity. Bambuterol may therefore be added to the list of inhibitors useful in the genotyping of cholinesterases. Bambuterol was much less efficiently hydrolysed in serum containing the atypical cholinesterase than in serum containing the normal enzyme. The results of the hydrolysis experiments once again illustrate the difference in affinity of bambuterol to the genetic forms of cholinesterase, and also strengthen the evidence that cholinesterase is the major serum enzyme catalysing the hydrolysis of bambuterol.
        
Title: Determination of bambuterol, a prodrug of terbutaline, in plasma and urine by gas chromatography/mass spectrometry Lindberg C, Jonsson S, Paulson J, Tunek A Ref: Biomed Environ Mass Spectrom, 19:218, 1990 : PubMed
(R,S)-Bambuterol was isolated from plasma and urine by solid-phase extraction and analysed as its trimethylsilyl derivative by gas chromatography/chemical ionization mass spectrometry with ammonia as the reagent gas. Deuterium-labelled bambuterol was used as internal standard. An esterase inhibitor was added to plasma to prevent hydrolysis of bambuterol in the plasma sample. Bambuterol could be measured in plasma down to 1 nmol l-1 with a within-day variation of less than 5% (coefficient of variation). The lower limit of measurement in urine was judged to be 8 nmol l-1, a concentration at which the within-day variation was 5.4% (coefficient of variation).
        
Title: Metabolism of bambuterol in rat liver microsomes: identification of hydroxylated and demethylated products by liquid chromatography mass spectrometry Lindberg C, Roos C, Tunek A, Svensson LA Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 17:311, 1989 : PubMed
The oxidative metabolism of (R,S)-bambuterol in rat liver microsomes was studied. Metabolite fractions were analyzed by thermospray LC-MS. The use of an equimolar mixture of deuterium-labeled and unlabeled bambuterol facilitated the mass spectrometric identification of the metabolites. Six metabolites, formed via hydroxylation, demethylation, and hydrolytic reactions, were identified. The demethylated metabolites were found to be chemically unstable under physiological conditions. It is likely that the complex biotransformation of bambuterol into terbutaline is one factor contributing to the long duration of action of bambuterol.
The lung uptake and biotransformation of 3H-bambuterol, a prodrug to terbutaline, were studied using isolated perfused and ventilated guinea pig lungs. 14C-Sucrose was used as an extracellular marker. The lung uptake of bambuterol was significantly (0.05 greater than or equal to P greater than or equal to 0.001) higher than that found for sucrose in single-pass perfusion experiments. High-performance liquid chromatographic (HPLC) analysis showed that 95.6 +/- 3.6% of the effluent 3H radioactivity was attributable to bambuterol. In recirculating experiments (120 min) the lung biotransformation of 3H-bambuterol (8.5 pmol/ml) was studied. Both oxidative and hydrolytic metabolism took place. The dominating metabolites were hydroxylated bambuterol and the monocarbamate derivative which is a product of hydrolysis of bambuterol. Traces of terbutaline were also formed. The results show that bambuterol has a certain affinity to lung tissue and that the drug is, to some extent, biotransformed in the guinea pig lung.
        
Title: The design and bioactivation of presystemically stable prodrugs Svensson LA, Tunek A Ref: Drug Metabolism Reviews, 19:165, 1988 : PubMed
Title: Hydrolysis of 3H-bambuterol, a carbamate prodrug of terbutaline, in blood from humans and laboratory animals in vitro Tunek A, Levin E, Svensson LA Ref: Biochemical Pharmacology, 37:3867, 1988 : PubMed
Tritiated bambuterol, a bis-dimethylcarbamate prodrug of terbutaline, was incubated in vitro with blood from both sexes of the following species: man, guinea pig, rat, mouse, dog and rabbit. The rates of hydrolysis of bambuterol to its monocarbamate derivative and further to terbutaline were measured. Large species variations were observed, e.g. blood from two of the human subjects was 15-fold more active than blood from the male rats. The rate of terbutaline formation as a function of initial bambuterol concentration was investigated in human plasma, and was found to describe a bell-shaped curve. Several pieces of evidence indicated that butyrylcholinesterase (EC 3.1.1.8) is the blood enzyme predominantly responsible for hydrolysis of bambuterol, although minor contributions from other esterases cannot be excluded. An exception may be blood from the rabbit, where the kinetics of the hydrolysis was different than in blood from the other species. The kinetics of bambuterol hydrolysis is discussed on basis of the established mechanism of carbamate interactions with cholinesterases, and the high affinity of bambuterol for butyrylcholinesterase.
        
Title: Bambuterol, a carbamate ester prodrug of terbutaline, as inhibitor of cholinesterases in human blood Tunek A, Svensson LA Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 16:759, 1988 : PubMed
Bambuterol, the bis-dimethyl carbamate prodrug of terbutaline, was tested for its potency in inhibiting cholinesterases in human blood. Preincubation of blood with bambuterol in the absence of thiocholine ester substrate was essential for obtaining maximal inhibition. The inhibition exerted by bambuterol after such preincubation was reversible and noncompetitive. Bambuterol was an extremely effective inhibitor of cholinesterase when butyrylthiocholine was used as substrate (I50 = 1.7 +/- 0.3 x 10(-8) M, N = 10) whereas it was 2400-fold less efficient in inhibiting cholinesterase with acetylthiocholine as substrate (I50 = 4.1 +/- 0.5 x 10(-5) M, N = 10). Because butyrylthiocholine is the preferred substrate for cholinesterase (EC 3.1.1.8) and acetylthiocholine for acetylcholinesterase (EC 3.1.1.7), these results indicate that bambuterol is a remarkably selective and potent inhibitor of cholinesterase.
        
Title: Poster 65. Bambuterol, a terbulaline carbamate prodrug, as substrate and inhibitor for cholinesterase in human blood Tunek A Ref: In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases, (Brzin M, Barnard EA, Sket D, Eds) De Gruyter:, 1984 : PubMed