Metabolism of a Selective Serotonin and Norepinephrine Reuptake Inhibitor Duloxetine in Liver Microsomes and Mice.

Duloxetine (DLX) is a dual serotonin and norepinephrine reuptake inhibitor, widely used for the treatment of major depressive disorder. Although DLX has shown good efficacy and safety, serious adverse effects (e.g., liver injury) have been reported. The mechanisms associated with DLX-induced toxicity remain elusive. Drug metabolism plays critical roles in drug safety and efficacy. However, the metabolic profile of DLX in mice is not available, although mice serve as commonly used animal models for mechanistic studies of drug-induced adverse effects. Our study revealed 39 DLX metabolites in human/mouse liver microsomes and mice. Of note, 13 metabolites are novel, including five N-acetyl cysteine adducts and one reduced glutathione (GSH) adduct associated with DLX. Additionally, the species differences of certain metabolites were observed between human and mouse liver microsomes. CYP1A2 and CYP2D6 are primary enzymes responsible for the formation of DLX metabolites in liver microsomes, including DLX-GSH adducts. In summary, a total of 39 DLX metabolites were identified, and species differences were noticed in vitro. The roles of CYP450s in DLX metabolite formation were also verified using human recombinant cytochrome P450 (P450) enzymes and corresponding chemical inhibitors. Further studies are warranted to address the exact role of DLX metabolism in its adverse effects in vitro (e.g., human primary hepatocytes) and in vivo (e.g., Cyp1a2-null mice). SIGNIFICANCE STATEMENT: This current study systematically investigated Duloxetine (DLX) metabolism and bioactivation in liver microsomes and mice. This study provided a global view of DLX metabolism and bioactivation in liver microsomes and mice, which are very valuable to further elucidate the mechanistic study of DLX-related adverse effects and drug-drug interaction from metabolic aspects.

Bioaccumulation of therapeutic drugs by human gut bacteria.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

Brain Targeting of Duloxetine HCL via Intranasal Delivery of Loaded Cubosomal Gel: In vitro Characterization, ex vivo Permeation, and in vivo Biodistribution Studies.

PURPOSE: Duloxetine (DLX) is dual serotonin and norepinephrine reuptake inhibitor suffering from limited bioavailability (≈ 40%) due to extensive hepatic metabolism. This work aims to formulate and evaluate DLX intranasal thermoreversible cubosomal gels to enhance its bioavailability and ensure efficient brain targeting. MATERIALS AND METHODS: Cubo-gels were prepared by 33 central composite design with three independent factors, lipid ratio (glycerol monooleate: glycerol tripalmitate), Pluronic F127%, and Pluronic F68%. The prepared formulations were evaluated for their particle size (PS), gelling temperature (GT), entrapment efficiency (EE%), and in vitro release. The cubo-gel with the highest desirability (0.88) was chosen as the optimized formulation. DLX cubo-gel was evaluated using differential scanning calorimetry, Fourier-transform infrared spectroscopy, X-ray powder diffraction, and transmission electron microscopy. Cytotoxicity study, ex vivo permeation study and in vivo bio-distribution study were conducted to evaluate the safety and efficacy of brain targeting. RESULTS: The optimum cubo-gel was composed of 3.76 lipid ratio, 20% w/v PF127, and 5% w/v PF68. It had PS of 265.13 ± 9.85 nm, GT of 32 ± 0.05°C, EE% of 98.13 ± 0.50%, and showed controlled release behavior where 33% DLX was released within 6 hrs. The plain in situ cubo-gel had a significantly higher IC50 compared to DLX solution and DLX-loaded in situ cubo-gel. The ex vivo permeation study showed 1.27 enhancement in the drug permeation from DLX in situ cubo-gel. According to the in vivo bio-distribution study in plasma and brain, the intranasal DLX in situ cubo-gel showed a 1.96 fold improvement in brain bioavailability compared to the intranasal solution. Its BTE% and DTP% were 137.77 and 10.5, respectively, indicating efficient brain targeting after intranasal administration. CONCLUSION: Accordingly, intranasal DLX in situ cubo-gel can be considered as an innovative nano-carrier delivery system for bioavailability enhancement and efficient brain targeting of DLX to maximize its effect.

Application of an Inter-Species Extrapolation Method for the Prediction of Drug Interactions between Propolis and Duloxetine in Humans.

Duloxetine (DLX) is a potent drug investigated for the treatment of depression and urinary incontinence. DLX is extensively metabolized in the liver by two P450 isozymes, CYP2D6 and CYP1A2. Propolis (PPL) is one of the popular functional foods known to have effects on activities of CYPs, including CYP1A2. Due to the high probability of using DLX and PPL simultaneously, the present study was designed to investigate the potent effect of PPL on pharmacokinetics (PKs) of DLX after co-administration in humans. A PK study was first conducted in 18 rats (n = 6/group), in which the plasma concentration of DLX and its major metabolite 4-hydroxy duloxetine (4-HD) with or without administration of PPL was recorded. Population PKs and potential effects of PPL were then analyzed using NONMEM software. Lastly, these results were extrapolated from rats to humans using the allometric scaling and the liver blood flow method. PPL (15,000 mg/day) exerts a statistically significant increase in DLX exposures at steady state, with a 20.2% and 24.6% increase in DLX C m a x , s s and the same 28.0% increase in DLX A U C s s when DLX (40 or 60 mg) was administered once or twice daily, respectively. In conclusion, safety issues are required to be attended to when individuals simultaneously use DLX and PPL at high doses, and the possibility of interactions between DLX and PPL might be noted.

In-vitro and in situ assessment of the efflux of five antidepressants by breast cancer resistance protein.

OBJECTIVES: Antidepressants need to penetrate the blood-brain barrier (BBB) to exert their functions in the central nervous system. Breast cancer resistance protein (BCRP), an efflux transporter abundantly expressed in the BBB, prevents the accumulation of many drugs in the brain. This study aimed to identify whether five commonly used antidepressants (sertraline, duloxetine, fluoxetine, amitriptyline and mirtazapine) are BCRP substrates. METHODS: A combination of bidirectional transport and intracellular accumulation experiments was conducted on BCRP-overexpressing MDCKII and wild-type (WT) cells, and in situ brain perfusion was conducted in rats. KEY FINDINGS: The bidirectional transport study revealed that the net efflux ratio (NER) of sertraline reached 2.08 but decreased to 1.06 when co-incubated with Ko143, a selective BCRP inhibitor. Conversely, the other four antidepressants did not appear to be BCRP substrates, due to their low NER values (<1.5). The accumulation of sertraline in MDCKII-BCRP cells was significantly lower than that in MDCKII-WT cells. The presence of Ko143 significantly increased the sertraline accumulation in MDCKII-BCRP cells but not in MDCKII-WT cells. Brain perfusion showed that the permeability of 1 and 5 µm sertraline was significantly higher in the presence of Ko143. CONCLUSIONS: Taken together, BCRP is involved in sertraline efflux.

Differences in Duloxetine Dosing Strategies in Smoking and Nonsmoking Patients: Therapeutic Drug Monitoring Uncovers the Impact on Drug Metabolism.

BACKGROUND: For certain psychotropic drugs, such as clozapine or olanzapine, the influence of smoking on drug metabolism is well studied. Tobacco smoke increases the metabolism of drugs that are substrates for cytochrome P450 (CYP) 1A2 due to CYP induction. The antidepressant duloxetine, acting as a serotonin-norepinephrine reuptake inhibitor, is mainly metabolized via CYP1A2. To date, little is known about the influence of smoking on serum duloxetine concentrations. METHODS: A therapeutic drug monitoring database consisting of plasma concentrations of duloxetine collected from January 2013 to June 2017 was analyzed. A group of nonsmoking patients undergoing treatment with duloxetine (n = 89) was compared to a group of active smokers also receiving duloxetine (n = 36). Serum concentrations of duloxetine and dose-adjusted serum concentrations were compared using non-parametric tests. RESULTS: Groups did not differ concerning sex (P = .063), but the group of active smokers was younger (P < .001) and received higher daily doses of duloxetine (P = .001). Smokers showed significantly lower median serum duloxetine concentrations (38.4% lower, P = .002) and 53.6% lower dose-adjusted serum concentrations (0.325 [ng/mL]/[mg/d] in smokers vs 0.7 [ng/mL]/[mg/d] in nonsmokers, P < .001). CONCLUSIONS: Despite higher daily doses, smokers had considerably lower serum duloxetine concentrations. The induction of CYP1A2 by tobacco smoke is a clinically relevant factor for drugs that are substrates for CYP1A2. Clinicians should actively assess smoking status, inform patients about the effect of smoking on duloxetine metabolism, and anticipate higher serum concentrations in the case of smoking cessation. Therapeutic drug monitoring ensures treatment efficacy by enabling the personalizing of treatment, as smokers need higher duloxetine doses to target serum concentrations within the therapeutic reference range.

Formulation and optimization of duloxetine hydrochloride buccal films: in vitro and in vivo evaluation.

Duloxetine hydrochloride (DH) is a serotonin-norepinephrine reuptake inhibitor (SSNRI) indicated for the treatment of depression. Duloxetine suffers from reduced oral bioavailability (≈50%) due to hepatic metabolism. This study aims to develop DH buccoadhesive films to improve its bioavailability. DH buccoadhesive films were prepared adopting the solvent casting method using hydroxypropyl methylcellulose (HPMC) and polyvinyl alcohol (PVA). The prepared films were evaluated for weight uniformity, drug content, surface pH, swelling index, mucoadhesion strength and drug release percentages. Accelerated stability and bioavailability studies in healthy human volunteers were also performed for the selected films. Results of the evaluation tests showed that the optimum physicochemical characters were obtained by the films prepared with 2% HPMC using 10% propylene glycol (F2 films). Accelerated stability studies revealed that DH showed proved stability throughout the experiment time. DH bioavailability from F2 films was determined and compared with that of the marketed oral capsules (Cymbalta® 30 mg). The pharmacokinetic results showed that Cmax for F2 was higher than the market product. In addition, ANOVA analysis showed that a Tmax of F2 film was significantly lower, while, the AUC0-72 of F2 was significantly higher than that of Cymbalta capsules. The percentage relative bioavailability of DH from F2 was found to be 296.39%. Therefore, the prepared buccal films offer an alternative route for the administration of DH with the possibility of improving its bioavailability.

Chemoenzymatic synthesis of (S)-duloxetine using carbonyl reductase from Rhodosporidium toruloides.

A chemoenzymatic strategy was developed for (S)-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli, RtSCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both RtSCR9 and glucose dehydrogenase, (S)-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000mM) in a space-time yield per gram of biomass (DCW) of 22.9mmolL(-1)h(-1)gDCW(-1) at a 200-g scale. The subsequent synthetic steps from RtSCR9-catalyzed (S)-3a were further performed, affording (S)-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee.