Molecular docking and oxidation kinetics of 3-phenyl coumarin derivatives by human CYP2A13.

CYP2A13 enzyme is expressed in human extrahepatic tissues, while CYP2A6 is a hepatic enzyme. Reactions catalysed by CYP2A13 activate tobacco-specific nitrosamines and some other toxic xenobiotics in lungs.To compare oxidation characteristics and substrate-enzyme active site interactions in CYP2A13 vs CYP2A6, we evaluated CYP2A13 mediated oxidation characteristics of 23 coumarin derivatives and modelled their interactions at the enzyme active site.CYP2A13 did not oxidise six coumarin derivatives to corresponding fluorescent 7-hydroxycoumarins. The Km-values of the other coumarins varied 0.85-97 µM, Vmax-values of the oxidation reaction varied 0.25-60 min-1, and intrinsic clearance varied 26-6190 kL/min*mol CYP2A13). Km of 6-chloro-3-(3-hydroxyphenyl)-coumarin was 0.85 (0.55-1.15 95% confidence limit) µM and Vmax 0.25 (0.23-0.26) min-1, whereas Km of 6-hydroxy-3-(3-hydroxyphenyl)-coumarin was 10.9 (9.9-11.8) µM and Vmax 60 (58-63) min-1. Docking analyses demonstrated that 6-chloro or 6-methoxy and 3-(3-hydroxyphenyl) or 3-(4-trifluoromethylphenyl) substituents of coumarin increased affinity to CYP2A13, whereas 3-triazole or 3-(3-acetate phenyl) or 3-(4-acetate phenyl) substituents decreased it.The active site of CYP2A13 accepts more diversified types of coumarin substrates than the hepatic CYP2A6 enzyme. New sensitive and convenient profluorescent CYP2A13 substrates were identified, such as 6-chloro-3-(3-hydroxyphenyl)-coumarin having high affinity and 6-hydroxy-3-(3-hydroxyphenyl)-coumarin with high intrinsic clearance.

In vitro sulfonation of 7-hydroxycoumarin derivatives in liver cytosol of human and six animal species.

Sulfonation is an important high affinity elimination pathway for phenolic compounds.In this study sulfonation of 7-hydroxycoumarin and 13 its derivatives were evaluated in liver cytosols of human and six animal species. 7-hydroxycoumarin and its derivatives are strongly fluorescent, and their sulfate conjugates are nonfluorescent at excitation 405 nm and emission 460 nm. A convenient fluorescence based kinetic assay of sulfonation was established.The sulfonation rate of most of the 7-hydroxycoumarin derivatives was low in liver cytosol of human and pig, whereas it was high with most compounds in dog and intermediate in rat, mouse, rabbit, and sheep. Sulfonation of the 7-hydroxycoumarin derivatives followed Michaelis-Menten kinetics with Km values of 0.1-12 µM, Vmax of 0.005-1.7 µmol/(min * g protein) and intrinsic clearance (Vmax/Km) of 0.004-1.9 L/(min * g cytosolic protein).Fluorescence based measurement of sulfonation of 7-hydroxycoumarin derivatives provides a sensitive and convenient high-throughput assay to determine sulfonation rate in different species and tissues and can be applied to evaluate sulfonation kinetics and inhibition.

Coumarin-Based Profluorescent and Fluorescent Substrates for Determining Xenobiotic-Metabolizing Enzyme Activities In Vitro.

in vivo methods, such as spectrophotometric, fluorometric, mass spectrometric,and radioactivity-based techniques. In fluorescence-based assays, the reaction produces a fluorescentproduct from a nonfluorescent substrate or vice versa. Fluorescence-based enzyme assays areusually highly sensitive and specific, allowing measurements on small specimens of tissues withlow enzyme activities. Fluorescence assays are also amenable to miniaturization of the reactionmixtures and can thus be done in high throughput. 7-Hydroxycoumarin and its derivatives arewidely used as fluorophores due to their desirable photophysical properties. They possess a large -conjugated system with electron-rich and charge transfer properties. This conjugated structure leadsto applications of 7-hydroxycoumarins as fluorescent sensors for biological activities. We describe inthis review historical highlights and current use of coumarins and their derivatives in evaluatingactivities of the major types of xenobiotic-metabolizing enzyme systems. Traditionally, coumarinsubstrates have been used to measure oxidative activities of cytochrome P450 (CYP) enzymes. For thispurpose, profluorescent coumarins are very sensitive, but generally lack selectivity for individual CYPforms. With the aid of molecular modeling, we have recently described several new coumarin-basedsubstrates for measuring activities of CYP and conjugating enzymes with improved selectivity.

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes.

Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion. The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

Metabolism of Scoparone in Experimental Animals and Humans.

Scoparone, a major constituent of the Chinese herbal medicine Yin Chen Hao, expresses beneficial effects in experimental models of various diseases. The intrinsic doses and effects of scoparone are dependent on its metabolism, both in humans and animals. We evaluated in detail the metabolism of scoparone in human, mouse, rat, pig, dog, and rabbit liver microsomes in vitro and in humans in vivo. Oxidation of scoparone to isoscopoletin via 6-O-demethylation was the major metabolic pathway in liver microsomes from humans, mouse, rat, pig and dog, whereas 7-O-demethylation to scopoletin was the main reaction in rabbit. The scoparone oxidation rates in liver microsomes were 0.8 - 1.2 µmol/(min*g protein) in mouse, pig, and rabbit, 0.2 - 0.4 µmol/(min*g protein) in man and dog, and less than 0.1 µmol/(min*g) in rat. In liver microsomes of all species, isoscopoletin was oxidized to 3-[4-methoxy-ρ-(3, 6)-benzoquinone]-2-propenoate and esculetin, which was formed also in the oxidation of scopoletin. Human CYP2A13 exhibited the highest rate of isoscopoletin and scopoletin oxidation, followed by CYP1A1 and CYP1A2. Glucuronidation of isoscopoletin and scopoletin was catalyzed by the human UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, and UGT2B17. Dog was most similar to man in scoparone metabolism. Isoscopoletin glucuronide and sulfate conjugates were the major scoparone in vivo metabolites in humans, and they were completely excreted within 24 h in urine. Scoparone and its metabolites did not activate key nuclear receptors regulating CYP and UGT enzymes. These results outline comprehensively the metabolic pathways of scoparone in man and key preclinical animal species.

Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10.

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4-(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.

Comparison of In Vitro Hepatic Scoparone 7-O-Demethylation between Humans and Experimental Animals.

Scoparone is a natural bioactive compound in Chinese herbal medicines. It has numerous pharmacological actions, including liver protective, hypolipidemic, antitumor, and anti-inflammatory effects. The primary metabolism route of scoparone is O-demethylation to scopoletin or isoscopoletin catalyzed by CYP enzymes. The aims of our study were to identify the human CYP enzymes catalyzing scoparone 7-O-demethylation to scopoletin and to compare this oxidation reaction in liver microsomes among different species. A high throughput fluorescent-based assay method was developed to determine the scoparone 7-O-demethylation to scopoletin rate. The rate was 100 - 400 nmol/(min×g protein) in mouse and rabbit liver microsomes, 10 - 20 nmol/(min×g protein) in pig microsomes, 1 - 3 nmol/(min×g protein) in human and less than 1 nmol/(min×g protein) in rat liver microsomes. Human CYP1A1 (Km 13 µM and Vmax 0.8 min-1), CYP1A2 (Km 48 µM and Vmax 0.3 min-1), and CYP2A13 (Km 10 µM and Vmax 22 min-1) were the most efficient catalysts of the reaction. The CYP2A6 selective inhibitor pilocarpine and an antibody against mouse CYP2A5 inhibited scoparone 7-O-demethylation to scopoletin in rabbit, mouse, and pig liver microsomes, indicating involvement of CYP2A enzymes in the reaction. Hepatic scoparone 7-O-demethylation to scopoletin differed between species both with respect to the rate of reaction and catalyzing enzymes. These species differences need to be taken into account when testing scoparone pharmacokinetics in animals and humans.

Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives.

A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-ß-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase pathway. Five analogues produced ≥62% HSD1 inhibition at 5 µM and, furthermore, three of them produced ≥68% inhibition at 1 µM. A docking-based structure-activity relationship analysis was done to determine the molecular basis of the inhibition and the cross-reactivity of the analogues was tested against oestrogen receptor, aromatase, cytochrome P450 1A2, and monoamine oxidases. Most of the analogues are only modestly active with 17-ß-hydroxysteroid dehydrogenase 2 - a requirement for lowering effective oestradiol levels in vivo. Moreover, the analysis led to the synthesis and discovery of 3-imidazolecoumarin as a potent aromatase inhibitor. In short, coumarin core can be tailored with specific ring and polar moiety substitutions to block either the sulphatase pathway or the aromatase pathway for treating breast cancer and endometriosis.

A liquid chromatography-tandem mass spectrometry analysis of nine cytochrome P450 probe drugs and their corresponding metabolites in human serum and urine.

Cocktail phenotyping using specific probe drugs for cytochrome P450 (CYP) enzymes provides information on the real-time activity of multiple CYPs. We investigated different sample preparation techniques and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with simple protein precipitation for the analysis of nine CYP probe drugs and their metabolites in human serum and urine. Specific CYP probe drugs (melatonin, CYP1A2; nicotine, CYP2A6; bupropion, CYP2B6; repaglinide, CYP2C8; losartan, CYP2C9; omeprazole, CYP2C19 and CYP3A4; dextromethorphan, CYP2D6; chlorzoxazone, CYP2E; midazolam, CYP3A4) and their main metabolites, with the exception of 3'-hydroxyrepaglinide, were quantified in human serum and urine using the developed LC-MS/MS method. The analytical method was fully validated showing high selectivity, linearity, acceptable accuracy (85-115 %) and precision (2-19 %) and applied to a pharmacokinetic study in four healthy volunteers after oral administration of drugs given as a cocktail. All probe drugs and their metabolites (totally 19 analytes) were detected and quantified from human serum and urine over the time range of 1 to 6 h after oral administration. Therefore, the proposed method is applicable for drug interaction and CYP phenotyping studies utilizing a cocktail approach. Graphical Abstract Workflow overwiew of cocktail CYP-phenotyping study.

Inhibitory effects and oxidation of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin via human CYP2A6 and its mouse and pig orthologous enzymes.

1. Information about the metabolism of compounds is essential in drug discovery and development, risk assessment of chemicals and further development of predictive methods. 2. In vitro and in silico methods were applied to evaluate the metabolic and inhibitory properties of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin with human CYP2A6, mouse CYP2A5 and pig CYP2A19. 3. 6-Methylcoumarin was oxidized to fluorescent 7-hydroxy-6-methylcoumarin by CYP2A6 (Km: 0.64-0.91 µM; Vmax: 0.81-0.89 min(-1)) and by CYP2A5 and CYP2A19. The reaction was almost completely inhibited at 10 µM 7-methylcoumarin in liver microsomes of human and mouse, but in pig only 40% inhibition was obtained with the anti-CYP2A5 antibody or with methoxsalen and pilocarpine. 7-Methylcoumarin was a mechanism-based inhibitor for CYP2A6, but not for the mouse and pig enzymes. 7-Formylcoumarin was a mechanism-based inhibitor for CYP2As of all species. 4. Docking and molecular dynamics simulations of 6-methylcoumarin and 7-methylcoumarin in the active sites of CYP2A6 and CYP2A5 demonstrated a favorable orientation of the 7-position of 6-methylcoumarin towards the heme moiety. Several orientations of 7-methylcoumarin were possible in CYP2A6 and CYP2A5. 5. These results indicate that the active site of CYP2A6 has unique interaction properties for ligands and differs in this respect from CYP2A5 and CYP2A19.

[Drug therapy and the most common drugs for childhood psychiatric disorders]. / Lasten psykiatristen häiriöiden lääkehoito ja tavallisimmat lääkkeet.

Psychotropic drugs are more commonly prescribed for children, although scientific evidence about psychotrophic medication and long-term effects thereof in children is scarce. The drugs are often used off-label. ADHD drugs, antipsychotics and antidepressants and melatonin are the most commonly used drugs. ADHD medication possesses the most established status. Antipsychotic drugs are utilized for the treatment of psychoses, bipolar disorder, and conduct disorder symptoms in particular. Antidepressants are utilized for the treatment of childhood depression and anxiety disorders, melatonin for the treatment of children's sleep problems. Drug therapy should always be carried out as part of other psychiatric therapy.

Time-Dependent Inhibition of CYP2C19 by Isoquinoline Alkaloids: In Vitro and In Silico Analysis.

The cytochrome P450 2C19 (CYP2C19) enzyme plays an important role in the metabolism of many commonly used drugs. Relatively little is known about CYP2C19 inhibitors, including compounds of natural origin, which could inhibit CYP2C19, potentially causing clinically relevant metabolism-based drug interactions. We evaluated a series (N = 49) of structurally related plant isoquinoline alkaloids for their abilities to interact with CYP2C19 enzyme using in vitro and in silico methods. We examined several common active alkaloids found in herbal products such as apomorphine, berberine, noscapine, and papaverine, as well as the previously identified mechanism-based inactivators bulbocapnine, canadine, and protopine. The IC50 values of the alkaloids ranged from 0.11 to 210 µM, and 42 of the alkaloids were confirmed to be time-dependent inhibitors of CYP2C19. Molecular docking and three-dimensional quantitative structure-activity relationship analysis revealed key interactions of the potent inhibitors with the enzyme active site. We constructed a comparative molecular field analysis model that was able to predict the inhibitory potency of a series of independent test molecules. This study revealed that many of these isoquinoline alkaloids do have the potential to cause clinically relevant drug interactions. These results highlight the need for studying more profoundly the potential interactions between drugs and herbal products. When further refined, in silico methods can be useful in the high-throughput prediction of P450 inhibitory potential of pharmaceutical compounds.

Ultrafast protein structure-based virtual screening with Panther.

Molecular docking is by far the most common method used in protein structure-based virtual screening. This paper presents Panther, a novel ultrafast multipurpose docking tool. In Panther, a simple shape-electrostatic model of the ligand-binding area of the protein is created by utilizing the protein crystal structure. The features of the possible ligands are then compared to the model by using a similarity search algorithm. On average, one ligand can be processed in a few minutes by using classical docking methods, whereas using Panther processing takes <1 s. The presented Panther protocol can be used in several applications, such as speeding up the early phases of drug discovery projects, reducing the number of failures in the clinical phase of the drug development process, and estimating the environmental toxicity of chemicals. Panther-code is available in our web pages (http://www.jyu.fi/panther) free of charge after registration.

Rational design of novel CYP2A6 inhibitors.

Inhibition of CYP2A6-mediated nicotine metabolism can reduce cigarette smoking. We sought potent and selective CYP2A6 inhibitors to be used as leads for drugs useful in smoking reduction therapy, by evaluating CYP2A6 inhibitory effect of novel formyl, alkyl amine or carbonitrile substituted aromatic core structures. The most potent CYP2A6 inhibitors were thienopyridine-2-carbaldehyde, benzothienophene-3-ylmethanamine, benzofuran-5-carbaldehyde and indole-5-carbaldehyde, with IC50 values below 0.5 µM for coumarin 7-hydroxylation. Nicotine oxidation was effectively inhibited in vitro by two alkyl amine compounds and benzofuran-5-carbonitrile. Some of these molecules could serve as potential lead molecules when designing CYP2A6 inhibitory drugs for smoking reduction therapy.

Prodrugs--from serendipity to rational design.

The prodrug concept has been used to improve undesirable properties of drugs since the late 19th century, although it was only at the end of the 1950s that the actual term prodrug was introduced for the first time. Prodrugs are inactive, bioreversible derivatives of active drug molecules that must undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug, which can then elicit its desired pharmacological effect in the body. In most cases, prodrugs are simple chemical derivatives that are only one or two chemical or enzymatic steps away from the active parent drug. However, some prodrugs lack an obvious carrier or promoiety but instead result from a molecular modification of the prodrug itself, which generates a new active compound. Numerous prodrugs designed to overcome formulation, delivery, and toxicity barriers to drug utilization have reached the market. In fact, approximately 20% of all small molecular drugs approved during the period 2000 to 2008 were prodrugs. Although the development of a prodrug can be very challenging, the prodrug approach represents a feasible way to improve the erratic properties of investigational drugs or drugs already on the market. This review introduces in depth the rationale behind the use of the prodrug approach from past to present, and also considers the possible problems that can arise from inadequate activation of prodrugs.

Design, synthesis, and evaluation of novel cyclic phosphates of 5-aminosalicylic acid as cytochrome p450-activated prodrugs.

Four novel cyclic phosphates of the anti-inflammatory agent 5-aminosalicylic acid (5-ASA) were designed and synthesized as cytochrome P450 (CYP)-activated prodrugs. These prodrugs can be used for targeting into gut wall, since these types of cyclic phosphates are known to be activated mainly by CYP3A forms, which are expressed not only in the liver but also in the small intestine and to a lesser extent in the colon. The present study shows that aromatic ring activating substituents, like chlorine, are definitely needed to obtain the desired enzymatic cleavage of the cyclic phosphate prodrugs of 5-ASA. However, the position of the activating substituent has also a strong impact on the chemical stability, and therefore, an appropriate balance between the rates of prodrug bioactivation and chemical stability needs to be taken into consideration in future studies on cyclic phosphate prodrugs of 5-ASA.

Pinoresinol stimulates keratinocyte proliferation and downregulates TNF-α secretion in peripheral blood mononuclear cells: An experimental in vitro study.

Background and Aims: Natural coniferous resins are used in traditional medicine for the treatment of skin wounds. Coniferous wood resins ("callus" resin) are a mixture of abietic (resin) acids, lignans such as pinoresinol, and p-coumaric acid. The wound-healing properties of resins are thought to be related to their antimicrobial properties, but also to their effects on cell proliferation and inflammation. The purpose of this study was to identify and investigate the effects of novel aqueous dispersions of resin and its main components in the proliferation of human primary keratinocytes in vitro and in the expression of proinflammatory cytokines in human peripheral blood mononuclear cells. Methods: The proliferation studies were performed under low and high calcium conditions with or without added growth stimulators at the time points of 2 and 6 days using AlamarBlue Cell Viability Reagent. The cytokine release assay was carried out by incubating the cells with the test articles for 18 h, after which the levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, and IL-8 were measured in the supernatant by enzyme-linked immunosorbent assay. Results: Resin and the purified lignan PINO, but not p-coumaric acid or abietic acid (industrial tall oil rosin), enhanced the proliferation of human keratinocytes in vitro and inhibited the expression of TNF-α, and to a lesser extent the expression of IL-1ß in peripheral blood mononuclear cells. Conclusions: In this study, novel aqueous dispersions of spruce resin were used to investigate the effects of main resin components on keratinocyte proliferation and on the expression of key proinflammatory cytokines known to be associated with chronic wounds. The observations suggest that lignans, such as PINO, but not resin acids, are the components of resins that mediate the proliferative and TNF-α-suppressing effects. Lignans including PINO were identified as novel potential compounds in the treatment of chronic skin ulcers.

Metabolism of bilirubin by human cytochrome P450 2A6.

The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic "Bilirubin Oxidase" (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14-22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K(i) of 2.23 µM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human "Bilirubin Oxidase" where bilirubin is potentially a substrate and a regulator of the enzyme.

CYP2A6: genetics, structure, regulation, and function.

The human CYP2A gene subfamily consists of three members, CYP2A6, CYP2A7, and CYP2A13. The CYP2A6 gene is highly polymorphic with approximately 40 annotated allelic variants. Individuals homozygous for some of these alleles have a total lack of CYP2A6 activity. The CYP2A6 protein is most abundant in liver and is expressed, although at much lower levels, in some other tissues, especially nasal mucosa. CYP2A6 differs from other human liver CYP forms in that it participates in the metabolism of very few currently used drugs. The two most relevant substrates for CYP2A6 are coumarin and nicotine. Coumarin is the marker substance for determining CYP2A6 activity both in vitro and in vivo. Approximately 80% of a nicotine dose is eliminated by CYP2A6, and there is a clear link between CYP2A6 genotypes, smoking behavior, and lung cancer risk.

A Comprehensive Evaluation of Sdox, a Promising H2S-Releasing Doxorubicin for the Treatment of Chemoresistant Tumors.

Sdox is a hydrogen sulfide (H2S)-releasing doxorubicin effective in P-glycoprotein-overexpressing/doxorubicin-resistant tumor models and not cytotoxic, as the parental drug, in H9c2 cardiomyocytes. The aim of this study was the assessment of Sdox drug-like features and its absorption, distribution, metabolism, and excretion (ADME)/toxicity properties, by a multi- and transdisciplinary in silico, in vitro, and in vivo approach. Doxorubicin was used as the reference compound. The in silico profiling suggested that Sdox possesses higher lipophilicity and lower solubility compared to doxorubicin, and the off-targets prediction revealed relevant differences between Dox and Sdox towards several cancer targets, suggesting different toxicological profiles. In vitro data showed that Sdox is a substrate with lower affinity for P-glycoprotein, less hepatotoxic, and causes less oxidative damage than doxorubicin. Both anthracyclines inhibited CYP3A4, but not hERG currents. Unlike doxorubicin, the percentage of zebrafish live embryos at 72 hpf was not affected by Sdox treatment. In conclusion, these findings demonstrate that Sdox displays a more favorable drug-like ADME/toxicity profile than doxorubicin, different selectivity towards cancer targets, along with a greater preclinical efficacy in resistant tumors. Therefore, Sdox represents a prototype of innovative anthracyclines, worthy of further investigations in clinical settings.