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Drug Metabolism: The Body’s Defense against Chemical Attack Andrew V. Stachulski* Ultrafine UFC Ltd., Synergy House, Guildhall Close, Manchester Science Park, Manchester M15 6SY, UK;
[email protected] Martin S. Lennard Section of Molecular Pharmacology and Pharmacogenetics, Royal Hallamshire Hospital, University of Sheffield, Sheffield S10 2JF, UK
Our bodies are exposed daily to a vast range of chemicals present in foods, in the air we breathe, and in the medicines we take for illness. Most of these chemicals are not required for normal biological function and may even be harmful. Nature has evolved an array of enzyme-catalyzed processes to rid our bodies of these “invading” substances, commonly referred to as xenobiotics (literally meaning “foreign bodies”). Much of the recent focus has been on drugs, and the study of the biological fate of xenobiotics has become known as drug metabolism. Historically, our understanding of drug metabolism began with Keller’s isolation in 1842 of hippuric acid (N-benzoylglycine) from the urine of patients who had ingested benzoic acid (1). We shall see later that this conversion is a typical “phase 2” reaction. Drug Metabolism—The Basics With the exception of very polar substances, which may be directly excreted via the kidneys into the urine, most medicines are quite lipid-soluble and are reabsorbed from the kidney tubule back into the bloodstream. Subsequently, these compounds undergo metabolism, generating more polar species, which can avoid renal reabsorption and be excreted into the urine (2). It is common, but misleading, to think of drug metabolism as purely a detoxifying or deactivating process. Metabolites may be similar in pharmacological activity to the parent, as for example nortriptyline, the N-demethylated metabolite of the antidepressant amitriptyline. By contrast, a metabolite may have life-threatening toxicity, as exemplified by the common painkiller paracetamol, which is partly metabolized to a highly reactive species that damages the liver in overdose. NHBu t H OH
O
O
OH Hydroxybufuralol (Aliphatic CH hydroxylation)
Benzo[a]pyrene-4,5-epoxide (Epoxidation) HO O
H Cl
N
HO
O
O
Hydroxychlorzoxazone (Aryl CH hydroxylation)
Ph
NMe Ph
NHMe
Des-N -methylmethadone (N -demethylation)
Dextrorphan (O -demethylation)
Figure 1. Typical CYP metabolic products. (Chemically modified sites emboldened.)
The Chemistry of Drug Metabolism The chemical pathways by which drugs are metabolized are extremely varied. However, they are now divided into phase 1 and phase 2 reactions, which involve fundamentally different chemistries.
Phase 1 Metabolism These reactions are ones of functionalization, where a polar grouping is added to or exposed on the molecule. By far the most important phase 1 pathway is oxidation, although other reactions, notably reduction and hydrolysis, do occur. The cytochrome P450 monooxygenases (CYPs) are the enzymes largely responsible for drug oxidation. While the initial reaction, namely the insertion of a high-energy oxygen atom into a single C–H or multiple C–C bond, is the same for all substrates, the end result may be quite different. Figure 1 shows some typical CYP oxidative products. Phase 2 Metabolism These reactions are ones of conjugation in which a polar group, possibly formed in a phase 1 reaction, reacts with an endogenous molecule such as glucuronic acid to form a watersoluble product suitable for excretion. Drugs already possessing a suitable functional group may undergo conjugation directly. In some cases, a phase 2 metabolite can be more pharmacologically active than the parent drug. The Biochemistry of Drug Metabolism
Sites of Metabolism The enzymes that metabolize drugs are present mainly in the liver, though significant metabolism may take place elsewhere, for example in the intestines, lung, or brain. When a drug is administered orally, metabolism in the liver may be so efficient that little or none of the drug enters the systemic blood circulation. This is termed the “first pass” effect and is a major consideration for the pharmaceutical industry during new drug development. Drugs undergoing significant “first pass” metabolism may need to be given by a non-oral route, such as intravenous administration. Phase 1 Enzymes Enzymes catalyzing phase 1 reactions include the CYPs (see above), flavin monooxygenases, aldehyde oxidases, and epoxide hydrolases. The most intensively studied and arguably most important enzymes are the CYPs (cytochromes P450), named for their ability to absorb light at 450 nm when in
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CYP 3 A 4 Family
Epoxide
P450
Individual isoform
hydrolase OH
O
Subfamily
Figure 2. Cytochrome nomenclature. A gene “family” (CYP3) exhibits greater than 40% identity in expressed amino acid sequence among its members; a “subfamily” (CYP3A), 70% or greater.
OH
Figure 4. Benzo[a]pyrene metabolism.
HO HO
O
OH
CO2H O H O
OH O
P
UDPGT O O
P
R-OH (substrate) O
Uridine
10
O
O
6
H 7 8
O
O
4'
CO2H O
HO HO
OR
+
UDP
OH
Figure 3. Metabolic hydroxylation sites of warfarin. The (S) enantiomer is shown.
Figure 5. The glucuronidation reaction. Note the α to β inversion at C-1 of the sugar.
the reduced form and complexed to carbon monoxide. The endogenous (natural) role of the CYPs involves the regulation of steroids, eicosanoids, and other hormones by oxidative processes (3). CYPs are heme-containing enzymes incorporating a bound protoporphyrin IX ligand that is responsible for their oxidative chemistry. The reaction mechanism is partly understood (4 ) and involves sequential binding of the substrate and dioxygen to the iron atom, which undergoes a series of cyclic oxidation–reduction reactions prior to product formation. Present in all species, CYP450 represents a superfamily of several hundred members, arranged in multigene gene families and subfamilies according to their amino acid sequence (Fig. 2). In humans, five subfamilies appear to be primarily involved in drug metabolism, namely CYPs 1A, 2C, 2D, 2E, and 3A. CYP isoforms exhibit a varying, but overlapping, substrate specificity (see the later Case Studies): a given drug could be a substrate for more than one CYP isoform, while a single form of CYP may be capable of metabolizing a range of drugs. For instance, CYP2D6 metabolizes many widely used drugs, such as antidepressants, antischizophrenic agents, cardiovascular drugs, pain killers, and even drugs of abuse, for example, “ecstasy”. The various contributions of CYP isoforms to the metabolism of a single drug are illustrated by the anti-blood-coagulant warfarin (in the USA, coumadin) (Fig. 3). When administered, warfarin is given as a racemic mixture, and there are important differences in the metabolism of the two enantiomers. (S)-Warfarin is metabolized more rapidly than the (R) form, the major pathway being 7-hydroxylation. CYP2C9 is almost entirely responsible for this reaction. In contrast, (R)-Warfarin is mainly metabolized via 6- and 8-hydroxylation, pathways that are catalyzed predominantly by CYPs 1A1 and 1A2. The 4′- and 10-hydroxy derivatives have also been characterized. Much less commonly, CYP and other enzymes act in reductive roles (5). Substrates include azo and nitro compounds, as well as some epoxides.
Hydrolysis and hydration reactions of drugs are also well known, occurring in the plasma as well as in the liver and mediated by esterases, amidases, and epoxide hydrolases. The epoxide substrate for the last enzyme may itself have been generated by an oxidative phase 1 process, which is exemplified by the two-step conversion of polycyclic aromatic hydrocarbons to diols (Fig. 4).
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Phase 2 Enzymes Phase 2 processes are mediated by a diverse group of transferase enzymes including glucuronosyl transferases, sulfotransferases, and glutathione-S-transferases. The historical example of hippuric acid formation cited above is the result of the action of an amino acid transferase. Just as the existence of a large family of CYP isoforms has been recognized, there is now strong evidence that the phase 2 enzymes, notably the uridine diphosphate glucuronosyl transferases (UDPGTs), occur in multiple forms (6 ).These UDPGTs require uridine diphosphate glucuronic acid (UDPGA) as a cofactor, which is derived enzymically from glucose and is ubiquitous in body tissues. Responsible for the conjugation of a range of endogenous molecules, glucuronidation is the most common phase 2 reaction, probably because of the abundance of UDPGA (Fig. 5). Although N-, S-, and C-glucuronides are all known, Oglucuronidation of aryl, alkyl, and acyl hydroxy groups is a particularly common reaction. Indeed, it would be unusual for a relatively lipid-soluble phenolic drug not to form at least some glucuronide conjugate in vivo. Phenols are the most common substrates for sulfotransferases, although some alcohols (especially steroidal) and amines may also be sulfated. The donor molecule is 3′-phosphoadenosine-5′-phosphosulfate (PAPS) (1). (NOTE: Numbered structures are shown in the Box.) Compared to UDPGA, PAPS has limited availability so that the sulfation capacity of the body is low and easily saturated. Many electrophilic compounds, for example halides, epoxides, and enones, are detoxified by conjugation with the sulfur-containing tripeptide glutathione, γ-(S )-glutamyl-(S )-
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Chemistry for Everyone Table 1. Some Substrates and Inhibitors of Human CYPs
cysteinylglycine (GSH) (2). This can occur spontaneously or through catalysis by glutathione-S-transferase. It is believed that a major role of glutathione is to act as a scavenger, protecting vital cellular constituents from damage by potentially toxic metabolites (see the paracetamol case study below). GSH conjugation is also important in the catabolism of endogenous compounds. For example, in the reaction of leukotriene A4 (LTA4) epoxide with GSH, higher members of the LT series are generated. Clinical and Industrial Implications The catalytic activities of the drug-metabolizing enzymes, particularly those of the CYPs, vary widely between species and between individuals. Because of this differing ability to eliminate drugs by metabolism, the concentration of a drug in the blood and at the tissue where it acts may range widely from one person to another given the same dose. Thus metabolism is an important determinant of the way our bodies react to drugs. Many factors in both our lifestyle and our genetic makeup play a role. One example of particular clinical importance is the case where the metabolism of one drug is inhibited by another being taken at the same time (7 ). This may lead to a decrease in the efficiency of the liver in eliminating the drug from the body, and in turn increases the blood concentration of the drug whose metabolism has been inhibited. In some cases, serious or life-threatening toxicity may then occur (see the terfenadine case study). Genetic influences on drug metabolism may be equally important (8). The function of a number of enzymes, including pseudocholinesterase, N-acetyltransferase, S-methyltransferase, and some CYP isoforms, is under genetic control. A proportion of the human population possesses malfunctioning or nonfunctioning genes. An individual who inherits one faulty gene from each parent is likely to lack or have much less of a particular enzyme, preventing effective metabolism of certain drugs. The most widely researched polymorphism affects CYP2D6. About one in twelve people of the Caucasian population (far fewer in other ethnic groups) inherits a non-
Isoform
Substrate
Inhibitor
CYP1A2
Caffeine, phenacetin
Furafylline Sulfaphenazole
CYP2C9
Tolbutamide, (S)-warfarin
CYP2C19
S-Mephenytoin
CYP2D6
Metoprolol, debrisoquine, dextromethorphan, bufuralol
Quinidine
CYP2E1
Chlorzoxazone, p-nitrophenol
Diethyldithiocarbamate
CYP3A
Testosterone, erythromycin, nifedipine, lignocaine, midazolam
Ketoconazole, troleandomycin
functional enzyme and is termed a “poor metabolizer”. With some drugs, poor metabolizers, because of their deficiency in CYP2D6, may achieve blood levels many times higher than the rest of the population. Thus, poor metabolizers may be at a much higher risk of toxic side-effects when taking drugs metabolized by CYP2D6, many of which are in widespread use. For these and other reasons, the national bodies regulating the licensing of new drugs advise or stipulate that the pharmaceutical industry gain as much information as possible and as early as possible on the metabolism of a new drug that is being developed. Studying metabolism prior to human administration helps with: 1. The isolation and identification of metabolites for subsequent pharmacological screening. 2. The identification of potentially toxic metabolites. 3. The characterization of any species differences in metabolism to aid the selection of animal models for toxicological testing. 4. The prediction of how a drug will be eliminated when given to humans. 5. The prediction of drug–drug interactions and thus the prevention of their occurrence when the drug is marketed. 6. The prediction of the influence of genetic and environmental factors on in vivo fate.
Recent advances in the understanding of CYP enzymes in particular enable scientists to predict from laboratory studies how drugs are likely to be metabolized in vivo. A systematic approach has been developed using extracts of human liver (now usually obtained during surgery to remove a liver tumor) and genetically engineered human CYPs to determine the specific isoform responsible for a particular reaction and to establish whether a drug is a significant inhibitor or inducer of a particular CYP isoform. This approach utilizes a range of chemical probes that are selectively metabolized by or inhibit the metabolism of the major CYP isoforms involved in human drug metabolism (Table 1). The Synthetic Chemist’s Contribution The organic chemist can play a valuable role in the synthesis of drug substrates, metabolites, and inhibitors needed for the experimental studies. Authentic samples of the metabolites of these chemical probes are required so that
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the rate of their formation can be determined and used as an index of CYP activity. For instance, the beta-blocker bufuralol (3), never marketed, is an excellent substrate for CYP2D6 and is converted to the 1′-hydroxy product (first structure in Fig. 1). An efficient synthesis of this metabolite has been devised (9). Originally designed as an antiasthmatic agent, the xanthine analogue furafylline 4 has proved to be a highly selective inhibitor of CYP1A2 and has also been readily synthesized. Because glucuronidation is an important route of drug metabolism, there is a growing demand for reference samples of glucuronides of both new and existing drugs (e.g., various steroids), both for analytical purposes and to be evaluated for pharmacological activity in their own right. Organic chemists have therefore gained much experience in glucuronide synthesis, and the topic has been reviewed (10, 11). Until recently, traditional Koenigs–Knorr synthesis via the bromosugar (5) was by far the commonest route to glucuronides. However, this approach may give low yields or complete failure, especially with phenols. Alternative intermediates such as the 1-hydroxysugar (6) and imidate (7) (12), both activated by Lewis acids, are now often being successfully used. Molecular Modeling In a parallel approach to these experimental methods, considerable effort has been put into developing molecular models of the active sites of CYP isoforms. These take the form of either templates based on structure–metabolism relationships or models based on protein structure (homology models). Most progress has been made on models for CYP2D6, whose known substrates are all lipophilic bases. From early models, it was suggested that the amino group of the drug forms an ionic bond to a carboxylic acid amino acid residue at a distance 5–7 Å from the site of catalytic attack. Subsequent modeling and experimental studies have broadly confirmed these predictions and have identified a number of amino acid residues on the surface of the active-site cavity, which may be important for substrate binding (13). Molecular models of CYP are relatively unsophisticated, and the goal is to refine them aided by data from site-directed mutagenesis and genetically engineered enzyme studies. These data may allow an accurate prediction of the substrates and inhibitors of CYPs early in drug discovery. Case Studies The following case studies illustrate the clinical significance of drug metabolism.
Paracetamol Paracetamol (acetaminophen: in the USA, Tylenol) (8) is widely used as a mild analgesic and antipyretic agent. Although the drug is very safe at recommended doses, it can cause severe, often fatal, liver damage in overdose. More than 90% of 8 is metabolized as the sulfate and glucuronide at normal therapeutic doses (Fig. 6). About 5% of 8 is converted by CYP oxidation to the quinoneimine (9) (NAPQI), a potent alkylating agent. Small amounts of 9 can be safely detoxified by conjugation with 352
OSO3 H OH
GSH 2
HNCOCH3 OH Via N -hydroxylation or direct oxidation
O
Cys-Gly CH3CONH
Glu
OH HNCOCH3 8
HO2C O O
OH OH OH
HNCOCH3 9
Cell macromolecule
NHCOCH3
HNCOCH3
Figure 6. Metabolism of paracetamol in humans.
GSH (2), followed by excretion of the resulting mercapturic acid conjugate. If 8 is taken in overdose, the body’s supply of 2 is rapidly depleted. As a result, NAPQI is free to bind covalently to liver cell constituents, with possibly fatal results from as little as 10 g (20 tablets) of 8. Since 1973, mechanism-based antidotes for paracetamol have become available, namely, N-acetyl-L-cysteine and Lmethionine, which augment the body’s supply of GSH and are independently able to detoxify 9. They prevent liver damage if taken within 10–12 hours of overdose. This is a striking example of how an understanding of drug metabolism has led to the development of efficient antidotes for the treatment of overdose.
Terfenadine Until very recently, the antihistamine drug terfenadine (10) (Triludan, Seldane) was one of the most frequently used treatments for allergic conditions, such as hay fever. An oral dose of 10 undergoes extensive first-pass metabolism by CYP3A4 in the intestine and liver, partly to the carboxylic acid 11; thus very little 10 reaches the blood stream. Compound 11 is responsible for almost all the pharmacological effects in vivo. While hundreds of thousands of patients have taken 10 without problems, some who have simultaneously taken the antifungal drug ketoconazole or the antibacterial agent erythromycin have experienced an abnormal heart rhythm, torsade de points, which may be fatal. This toxic effect is caused by 10 itself. Both ketoconazole and erythromycin block the CYP3A4-mediated metabolism of 10, leading to its accumulation to potentially toxic levels in the heart. In the UK, 10 is no longer available over the counter, but only on prescription. In the USA, 10 has been withdrawn from clinical use and replaced by its nontoxic active metabolite 11 (14 ), now marketed as Telfast (fexofenadine, Allegra) in both the USA and UK. Dietary Flavonoids The potential of dietary substances to act as inhibitors of CYP isoforms is illustrated by the preceding case study. A serendipitous observation was made during a drug–ethanol
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interaction study that grapefruit juice substantially impaired the elimination of the blood-pressure lowering drug nifedipine from the body. This drug is cleared by CYP3A4. Subsequently a number of interactions were reported between grapefruit juice and drugs that are CYP3A4 substrates, for example, cyclosporine (used to prevent rejection of transplanted organs), ethynyloestradiol (a contraceptive pill constituent), midazolam (a sedative), triazolam (an antianxiety drug), and terfenadine (10) (15, 16 ). Patients are now warned to avoid grapefruit juice when taking terfenadine or nifedipine. Grapefruit contains a number of flavonoids, some of which have been tested for their ability to inhibit CYP3A4 activity. Originally it was thought that either naringenin or quercitin was the active species, but now it seems likely that other flavonoids, especially derivatives of the furanocoumarin bergamottin, are the inhibitors. Recently 6′,7′-dihydroxybergamottin (12), another constituent of grapefruit juice, was identified as a CYP3A4 inhibitor (17 ), and certain related dimeric molecules may be even more potent (18). In a very recent study (19), red wine, which also contains many flavonoids, has been shown to inhibit CYP3A4 with comparable potency to grapefruit juice. From this emerging information, more consideration may have to be given to the interaction of food constituents with drug-metabolizing enzymes because of the potentially serious consequences.
Morphine-6-glucuronide People have used morphine (13), in the form of opium, as an analgesic for possibly 5,000 years (early Egypt). However, only in the last 30 years has the biological fate of 13 been characterized (20), and it has become evident that a metabolite of 13 may be a superior analgesic to the parent. With its two hydroxyl groups, 13 is already prepared for phase 2 metabolism. The major human metabolite of 13 (representing about 80% of the dose) is the 3-glucuronide, but this has no analgesic properties. Studies in cancer patients (20) found that the 6-glucuronide metabolite 14 (representing 20% of the dose) was a more potent analgesic than 13 at equimolar doses, with reduced incidence of adverse effects, notably nausea, constipation, and respiratory depression. An early synthesis of 14 from 3-acetylmorphine involved a heavy metal catalyst (21), but more recently an efficient process free of toxic reagents has been patented (22) and used to synthesize 14 in sufficient quantities for clinical studies. The Future Implications for the Medicinal Chemist In most pharmaceutical companies, drug-metabolism scientists now work in tandem with chemists to discover and develop drugs that combine high potency with optimum pharmacokinetics and metabolism. Combinatorial methods of synthesis, now available for the generation of a large number of candidate compounds, will place even more importance on this symbiotic relationship. The application of in vitro models of human drug metabolism and for drug absorption from the intestine will almost certainly expand, making the process of new drug development still more cost effective. The concept of the pro-drug, which has been well reviewed (23), illustrates how chemists have taken advantage of metabolism for drug design. This approach involves the administration of a drug in a modified inactive form (the pro-drug) that can
release the active drug in vivo by direct or enzyme-catalyzed hydrolysis in the gut wall or liver. This may lead to improved absorption, more selective targeting to a specific body site, or a desirable change in the duration of action. We began by acknowledging that the body’s enzymes treat drugs, like any xenobiotics, as the invading enemy. We have also seen examples of enzymes generating metabolites with more desirable properties than the original drugs. Thus drug metabolism, as well as protecting us from toxic chemicals, can aid medicinal chemists in designing tomorrow’s drugs. It is still unclear what the endogenous function of many drug metabolizing enzymes might be. If one or more are found to play a crucial role in the disease process, chemists might be using such information in the future to design medicines that act by altering drug-metabolizing enzyme activity. Literature Cited 1. Gibson, G. G.; Skett, P. Introduction to Drug Metabolism, 2nd ed.; Chapman and Hall: London, 1994; pp 19, 20. 2. Nicholls, P. J.; Luscombe, D. K. In Introduction to the Principles of Drug Design; Smith, J.; Williams, H., Eds.; J. Wright and Sons: Bristol, 1983; p 21. 3. Lewis, D. Chem. Ind. 1997, 831. 4. Gibson, G. G.; Skett, P. Op. cit., pp 43–49. 5. Gibson, G. G.; Skett, P. Op. cit., pp 54–57. 6. Burchall, B. In Reviews in Biochemical Toxicology; Hodgson, E.; Bend, J. R.; Philpot, R. M., Eds.; Elsevier: Amsterdam, 1981; pp 1–32. 7. Gibson, G. G.; Skett, P. Op. cit., pp 78–95. 8. Lennard, M. S.; Stachulski, A. V. Chem. Br. 1997, 33(9), 25. 9. Collier, P.; Scheinmann, F.; Stachulski, A. V. J. Chem. Res. (S) 1994, 312. 10. Kaspersen, F. M.; Van Boeckel, C. A. A. Xenobiotica 1987, 17, 1451. 11. Stachulski, A. V.; Jenkins, G. N. Nat. Prod. Rep. 1998, 15, 173. 12. Fischer, B.; Nudelman, A.; Ruse, M.; Herzig, J.; Gottlieb, H. E.; Keinan, E. J. Org. Chem. 1984, 49, 4988. 13. Ellis, S. W.; Hayhurst, G. P.; Smith, G.; Lightfoot, T.; Wong, M. S. S.; Simula, A. P.; Ackland, M. J.; Sternberg, M. J. E.; Lennard, M. S.; Tucker, G. T.; Wolf, C. R. J. Biol. Chem. 1995, 270, 29055. 14. Kawai, S.; Hambalek, R. J.; Just, G. J. Org. Chem. 1994, 59, 2620. 15. Benton, R. E.; Honig, P. K.; Zamani, K.; Cantilena, L. R.; Woosley, R. L. Clin. Pharmacol. Ther. 1996, 59, 383. 16. Rau, S. E.; Bend, J. R.; Arnold, J. M. O.; Tran, L. T.; Spence, J. D.; Bailey, D. G. Clin. Pharmacol. Ther. 1997, 61, 401. 17. Edwards, D. J.; Bellevue, F. H. III; Woster, P. M. Drug Metab. Dispos. 1996, 24, 1287. 18. Fukuda, K.; Ohta, T.; Oshima, Y.; Ohashi, N.; Yoshikawa, M.; Yamazoe, Y. Pharmacogenetics 1997, 7, 391. 19. Chan, W. K.; Nguyen, L. T.; Miller, V. P.; Harris, R. Z. Life Sci. 1998, 62, 135. 20. Osborne, R.; Thompson, P.; Joel, S.; Trew, D.; Patel, N.; Slevin, M. Br. J. Clin. Pharmacol. 1992, 34, 130. 21. Yoshimura, H.; Oguri, K.; Tsukamoto, H. Chem. Pharm. Bull. 1968, 16, 2114. 22. Brown, R. T.; Lumbard, K. W.; Mayalarp, S. P.; Scheinmann, F. A Process for Making Morphine-6-glucuronide or Substituted Morphine-6-glucruonide; Int. Patent WO 93/03051, 1993 (PCT/GB 92/01449). 23. Smith, H. J. In Introduction to the Principles of Drug Design; Smith, J.; Williams, H., Eds.; J. Wright and Sons: Bristol, 1983; pp 196– 208.
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