Involvement of Aldehyde Oxidase (AOXs) in Drug Metabolism: Early Prediction and Coping Strategies

Aldehyde oxidases (AOXs) are a class of molybdenum-containing flavoprotein enzymes involved in phase I metabolism of drugs. Conventional drugs are mainly metabolized by cytochrome P450 (CYP450) enzymes. In the design of novel drug compounds, medicinal chemists often incorporate nitrogen-containing aromatic heterocyclic structures to reduce CYP450-mediated metabolism and enhance the stability of the designed compounds. However, such modifications may result in the metabolism of these compounds by AOX. Over the past two decades, AOX has received increasing attention in the pharmaceutical industry. This can be ascribed primarily to an increase in the proportion of potential AOX substrates in new drug development and a growing number of reported failures in clinical trials of new drugs arising from the neglect of AOX-mediated metabolism during the early stages of development. Given the significant species-specific differences in AOX homologs, there is currently a lack of suitable preclinical animal models capable of simulating human metabolism. Moreover, attempts to predict the metabolism of compounds by AOX in humans based on in vitro–in vivo extrapolation have also been plagued by low accuracy. In this article, we present an overview of the role and importance of AOX in drug metabolism; the classification, identification, and characterization of AOX substrates; and strategies adopted to address the problem of AOX-mediated metabolism encountered in R&D. Thus, the aim is to identify approaches that could be used to enhance the druggability of candidates in new drug development.

Role and importance of aldehyde oxidases in drug metabolism

Although Pryde et al. have reported that only 13% of approved small-molecule drugs listed in the DrugBank database are potential AOX substrates, this proportion rises to 45% in the Prous Integrity source, which also includes new drugs in research and development ^[1]. Moreover, considering certain specific drug types (e.g., kinase-targeted drugs), the percentage of potential substrates for AOX can be as high as 56% ^[1]. Studies have also shown that in addition to traditional small-molecule drugs, PROTAC molecules may also be metabolized by AOX ^[2].

Given that >50% of drugs on the market are primarily metabolized by CYP450, preclinical studies of drugs remain focused mostly on CYP450-mediated metabolism and CYP450 reaction-phenotyping. However, studies designed to assess the metabolic activity of CYP450 enzymes in routine drug screening may overlook the metabolism attributable to non-CYP enzymes, particularly AOX. Differences in research on the respective activities of CYP450 and AOX enzymes are manifested in the selection of incubation systems and experimental conditions. CYP450 is found in liver microsomes, while AOX is distributed in the cell cytosol. Moreover, CYP450 activity is dependent on the assistance of the cofactor NADPH, while AOX-mediated catalysis occurs independently of coenzymes.

AOX is also characterized by distinct interspecies differences. In humans, AOX1 is the only active isoform and is mainly found in the liver, kidney, and gastrointestinal tract. Among the different commonly used preclinical animal models, rodents have both the AOX1 and AOX3 isoforms in the liver, whereas cynomolgus macaques and guinea pigs merely have the AOX1 isoform. Notably, in dogs, the liver lacks AOX gene expression. Moreover, AOX1 isoform is also absent from other tissues in dogs ^[3], with the only two expressed isoforms (AOX4 and AOX2) being confined to the lacrimal glands and nasal mucosa (Table 1). Consequently, there is a high probability that selecting unsuitable animal models (e.g., dog models) for drugs metabolized by AOX would overlook any activity attributable to this enzyme.

Table 1. Subtype expression of aldehyde oxidases in humans and common preclinical animal models ^[3]

A failure to consider the effects of AOX in preclinical studies is typically associated with a series of unpredictable risks when drugs that can serve as AOX substrates enter clinical testing. These include excessively low drug bioavailability due to the rapid clearance by AOX, the generation of toxic metabolites by AOX-mediated metabolism, the potential drug-drug interactions of AOX metabolites, or inconsistencies between humans and animals regarding the proportions of metabolites that require supplementary metabolite safety testing (Table 2) ^[4]. Consequently, early identification of drugs that can be metabolized by AOX would contribute to minimizing the difficulties encountered during the latter stages of drug development.

Table 2. Challenges and difficulties in drug development are posed due to the involvement of AOX in metabolism ^[4]

Types and structural characterization of aldehyde oxidase substrates

AOX can metabolize an extensive range of substrates and catalyzes three main types of reactions, namely, oxidation, reduction, and hydrolysis.

(1) Oxidation

In its oxidative role, AOX catalyzes the oxidation of aldehydes to yield the corresponding carboxylic acids (hence the name “aldehyde oxidase”). Although aldehyde functional groups are rarely present in the structures of common drugs, aldehydes are often produced as intermediate metabolites by the action of other drug-metabolizing enzymes such as CYP450 or monoamine oxidases.

Given their frequent use as structural scaffolds in drug development and design processes, aromatic nitrogen-containing heterocycles are the most common and valued substrates of AOX-mediated oxidation. Although there are instances in which oxidation occurs on carbons at distant positions (e.g., quinoline and pyrazine), the majority of oxidation sites in these compounds are located on heterocyclic carbons adjacent to nitrogen. Furthermore, certain properties have been established to render compounds more susceptible to AOX oxidation. These include the following:

1. Number of nitrogen atoms: Heterocycles containing larger numbers of nitrogen atoms are more likely to be oxidized. For example, pyrimidine oxidation is more common than pyridine oxidation.
2. Size of the nitrogen-containing aromatic heterocyclic ring: Among monoheterocyclic rings, for example, a six-membered ring is more susceptible to oxidation by AOX than a five-membered ring.
3. Electron-absorbing or electron-donating nature of functional groups within the ring: In general, electron-accepting and electron-donating groups, respectively, increase and reduce the probability of AOX-catalyzed oxidation. However, certain electron-donating groups may promote the binding of compounds to AOX, thereby increasing the likelihood of AOX oxidation.

In this context, Manevski et al. have concluded that compounds containing pyrimidine, quinoline, and purine structures are highly likely to serve as AOX substrates. Accordingly, in drug metabolism studies, priority should be given to investigating the potential AOX metabolism of such compounds ^[4].

(2) Reduction

In contrast to its oxidative activity, AOX can also catalyze the reduction of nitro compounds, nitrogen oxides, sulfoxides, isoxazoles, isothiazoles, nitrites, and hydroxamic acids. In particular, nitro compounds are reduced to hydroxylamines and primary amines, which may result in a series of adverse drug reactions when bioactivated. For example, in response to AOX reduction, nimesulide may be further oxidized by CYP450 and subsequently bind to GSH, causing hepatotoxicity.

(3) Hydrolysis

The clinical failure of the drug GDC-0834 (a BTK inhibitor candidate) revealed for the first time that AOX can also catalyze the hydrolysis of compounds. Based on the analyses of the structure-activity relationships in AOX-mediated amide hydrolysis, it has been inferred that AOX-catalyzed hydrolysis is a more widespread phenomenon than hitherto believed and that aromatic amides are notably susceptible to hydrolysis by AOX.

Identification and characterization of AOX-mediated metabolism

A summary of the classification and common structures of AOX substrates can provide valuable guidance in the initial determination and prediction of whether a compound will be metabolized by AOX. In this regard, attention should also be given to inconsistent results obtained in specific in vitro studies and species-specific differences in the results of animal pharmacokinetic studies (Figure 1). Collectively, an assessment of these factors can contribute to confirming the involvement or non-involvement of aldehyde oxidases in drug metabolism.

In vitro studies mainly involve assessing the stability of compounds in liver microsomes and hepatocytes. If the rate of drug elimination in hepatocytes is found to be substantially higher than that detected in liver microsomes, or if the test compound is still metabolized in liver microsomes incubated in the absence of NADPH, the involvement of non-CYP enzymes (including AOX) in metabolism should be suspected.

Given the significant species-specific differences regarding the isoform types and activities of AOX among different animals, comparisons of the results (metabolite identification) of in vivo studies conducted with different preclinical animal models can also aid in determining whether AOX is involved in metabolism. For example, given that the AOX1 isoform is absent in dogs, the detection of a certain metabolite of a compound in other animals (e.g., mice or monkeys) but not in dogs could serve as evidence to indicate that the compound may be metabolized by AOX.

Further verification of whether a compound is metabolized by AOX can be performed by selecting either liver S9 (without added cofactors) or cytosol systems for incubation. The involvement or non-involvement of AOX in metabolism can be determined by comparing the rates of compound metabolism in the presence or absence of an AOX inhibitor. Commonly used inhibitors in this regard include the highly selective hydralazine and the most potent raloxifene. Alternatively, the generation of oxidation products can be directly assessed by incubation with a recombinant AOX.

Figure 1. Flowchart for the identification and validation of drug metabolism by aldehyde oxidases ^[5]

Coping strategies during the development of drugs involving AOX metabolism

Candidate drug compounds as substrates of AOX are often characterized by rapid clearance and extremely low bioavailability in clinical trials, also inducing issues relating to toxicity and metabolite safety. Accordingly, modification of compound structure to minimize the extent of AOX catalysis remains the mainstream strategy in the field of medicinal chemistry. However, in this context, it is necessary to determine the circumstances under which attention should be paid to AOX metabolism and the need for drug optimization. In this regard, Deepak et al. have proposed the application of a preliminary decision tree (Figure 2) ^[5], the principal steps of which are outlined below.

1. For compounds metabolized by multiple enzymes, including AOX, reaction phenotyping is required to determine the contribution of AOX to overall metabolism. Drug development may proceed if AOX is not the primary metabolizing enzyme.
2. In cases in which AOX is the only or primary enzyme involved in metabolizing a compound, modification of the site in the chemical structure to which AOX binds should be considered. If the implicated motif is a pharmacophore that cannot be readily modified or replaced, the compound may be initially classified as a low, moderate, or high AOX clearance type with reference to the semi-quantitative scale developed by Zientek et al. based on known AOX substrates ^[6].

• For compounds classified as having moderate to high clearance, modification of the compound structure to terminate or reduce the rate of AOX metabolism must still be considered.
• For compounds classified as having low clearance, methods for reducing the rate of AOX metabolism may be adopted, or drug development may be continued.

Figure 2. A decision tree designed to reduce the risks posed by the involvement of AOX in the metabolism of new drugs ^[5]

The key question is to find effective ways of structurally modifying a drug to inhibit or reduce AOX metabolism. To this end, some studies have described approaches that can be adopted to effectively modify the structure of compounds, examples of which are as follows ^[4].

(1) Termination of AOX metabolism

1. Blocking of oxidation sites: Sites susceptible to oxidation and metabolism by AOX can be blocked by substitution with methoxy groups, difluoromethyl groups, or fluorine atoms. The successful introduction of amino groups has also been reported, although the application of this method should be carefully considered, as the introduction of arylamine structures can enhance the risk of adverse drug reactions.
2. Substitution of oxidized carbon atoms with heteroatoms: The conversions of a pyridine to a pyridazine and isoquinoline to cinnoline are two successful examples of this type of substitution; however, the addition of new heteroatoms may lead to a shift in metabolic soft spot rather than an inhibition of AOX metabolism.
3. Removal of nitrogen atoms within the benzene ring or heterocycle: Nitrogen-free heterocycles would unlikely serve as substrates for AOX. Accordingly, modification involving the elimination of nitrogen would effectively inhibit the AOX metabolism of compounds and also contribute to enhancing their lipophilicity, which would, in turn, increase the likelihood of metabolism by CYP450.
4. Changing the ring size: The oxidative activity of aldehyde oxidases primarily occurs in six-membered ring structures. In a monocyclic structure, changing the six-membered ring to a five-membered ring facilitates the inhibition of AOX metabolism. In the case of fused-ring structures, the situation is a little more complex, in that oxidation by AOX can occur in a polycyclic ring formed by the fusion of two six-membered rings or the fusion of one five-membered ring and one six-membered ring; however, oxidation by AOX rarely occurs in a polycyclic ring formed by the fusion of two five-membered rings.
5. Saturation of aromatic heterocycles: The conversion of an aromatic heterocycle to an aliphatic heterocycle generally has the effect of terminating AOX metabolism. However, if CYP450 can metabolize the aliphatic heterocycle to yield iminium ions, AOX may still oxidize these ions.

(2) Reduction in the rate of AOX metabolism

1. Adjusting the electronic properties of nitrogen-containing heterocycles: The rate of AOX metabolic activity can be reduced by rearrangement of the positions of heteroatoms or substituent groups in ring structures or by introducing new electron-withdrawing or electron-donating groups without directly blocking the site of metabolism.
2. Increasing the steric hindrance at the site of metabolism: Introducing a larger substituent group near the oxidized site increases the steric hindrance at the site of metabolism, thereby reducing the rate of AOX metabolism.
3. Exploiting the kinetic isotope effect of deuterium: Deuterium is an isotope of hydrogen, and given that C-D bonds are more difficult to break than C-H bonds and that C-H bond breakage is the rate-limiting step in AOX-mediated oxidation, the deuterium isotope effect can be utilized to attenuate AOX metabolism;
4. Modifying the distant substituent groups to weaken AOX binding: Substituents distant from the site of metabolism can be modified to interfere with the binding of AOX to compounds or interactions with the molybdopterin cofactor.

In addition to the strategies designed to inhibit or reduce AOX metabolism, a further approach that can contribute to advances in drug development entails exploiting the rapid metabolism by AOX. If the desired potency is retained in the metabolite generated via AOX catalysis, it may be feasible to adopt a prodrug design strategy to transform the disadvantage of the original compound into an advantage. Figure 3 shows an example of a prodrug metabolized by AOX. Whereas the bioavailability of orally administered penciclovir is typically only 5%, penciclovir derived from the conversion of the prodrug famciclovir has a bioavailability of approximately 77%, which can be attributed to the rapid absorption and esterase and AOX metabolism characteristics of the prodrug.

Figure 3. An example illustrating the adoption of a prodrug modification strategy for AOX substrates ^[4]

By using the comprehensive drug-metabolizing enzyme reaction phenotyping platform available at WuXi AppTec DMPK, it is possible to determine whether AOX is actively involved in drug metabolism via chemical inhibition and recombinant AOX incubation and to identify sites of metabolism combined with metabolite identification. This approach can provide a basis for the structural optimization and modification of candidate drug compounds.

Concluding remarks

The lack of success encountered in multiple drug clinical trials conducted in recent decades has been ascribed to a failure to sufficiently consider the involvement of AOX in drug metabolism, and for long, the activity of AOX has been viewed as a significant impediment to drug development. Moreover, AOX is also characterized by notable species-specific differences, which accordingly present difficulties in selecting suitable animal models that can be used to simulate in vivo human metabolism. Nevertheless, the cumulative findings of studies performed to date have yielded valuable insights that provide a sound basis for predicting AOX metabolic activity and designing strategies that can be adopted to minimize the undesirable effects of this enzyme. In drug screening, it is recommended that the involvement or non-involvement of AOX in metabolism be identified and verified based on early assessments of the drug structure and analysis of the abnormal findings in liver microsomal and hepatocyte stability assays and animal studies. If a drug is confirmed to be metabolized by AOX, a series of remedial measures, including the optimization of drug structure and prodrug strategies can be adopted to inhibit, reduce, or favorably exploit the metabolic activity of AOX for the advancement of drug development.

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References:

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[2] Goracci L, Desantis J, Valeri A, Castellani B, Eleuteri M, Cruciani G. Understanding the Metabolism of Proteolysis Targeting Chimeras (PROTACs): The Next Step toward Pharmaceutical Applications. J Med Chem. 2020 Oct 22;63(20):11615-11638. doi: 11.1021/acs.jmedchem.0c00793. Epub 2020 Oct 7. PMID: 33026811; PMCID: PMC8015227.

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Authors: Shiyan Chen, Ruixing Li, Jing Jin

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