The CYP3A inducer dexamethasone affects the pharmacokinetics of sunitinib by accelerating its metabolism in rats.

Sunitinib, a novel anti-tumor small molecule targeting VEGFR, is prescribed for advanced RCC and GISTs. Sunitinib is primarily metabolized by the CYP3A enzyme. It is well-known that dexamethasone serves as a potent inducer of this enzyme system. Nonetheless, the effect of dexamethasone on sunitinib metabolism remains unclear. This study examined the effect of dexamethasone on the pharmacokinetics of sunitinib and its metabolite N-desethyl sunitinib in rats. The plasma levels of both compounds were measured using UHPLC-MS/MS. Pharmacokinetic parameters and metabolite ratio values were calculated. Compare to control group, the low-dose dexamethasone group and high-dose dexamethasone group decreased the AUC(0-t) values of sunitinib by 47 % and 45 %, respectively. Meanwhile, the AUC(0-t) values of N-desethyl sunitinib were increased by 2.2-fold and 2.4-fold in low-dose dexamethasone group and high-dose dexamethasone group, respectively. The CL values for sunitinib were both approximately 45 % higher in the two dexamethasone groups. Remarkably, metabolite ratio values increased over 5-fold in both low-dose dexamethasone group and high-dose dexamethasone group, indicating a significant enhancement of sunitinib metabolism by dexamethasone. Moreover, the total levels of sunitinib and its metabolite are also significantly increased. The impact of interactions on sunitinib metabolism, as observed with CYP3A inducers such as dexamethasone, is a crucial consideration for clinical practice. To optimize the dosage and prevent adverse drug events, therapeutic drug monitoring can be employed to avoid the toxicity from such interactions.

Interpreting mono- and poly-SCRA intoxications from an activity-based point of view: JWH-018 equivalents in serum as a comparative measure.

Synthetic cannabinoid receptor agonists (SCRAs) are a class of synthetic drugs that mimic and greatly surpass the effect of recreational cannabis. Acute SCRA intoxications are in general difficult to assess due to the large number of compounds involved, differing widely in both chemical structure and pharmacological properties. The rapid pace of emergence of unknown SCRAs hampers on one hand the timely availability of methods for identification and quantification to confirm and estimate the extent of the SCRA intoxication. On the other hand, lack of knowledge about the harm potential of emerging SCRAs hampers adequate interpretation of serum concentrations in intoxication cases. In the present study, a novel comparative measure for SCRA intoxications was evaluated, focusing on the cannabinoid activity (versus serum concentrations), which can be measured in serum extracts with an untargeted bioassay assessing ex vivo CB1 activity. Application of this principle to a series of SCRA intoxication cases (n = 48) allowed for the determination of activity equivalents, practically entailing a conversion from different SCRA serum concentrations to a JWH-018 equivalent. This allowed for the interpretation of both mono- (n = 34) and poly-SCRA (n = 14) intoxications, based on the intrinsic potential of the present serum levels to exert cannabinoid activity (cf. pharmacological/toxicological properties). A non-distinctive toxidrome was confirmed, showing no relation to CB1 activity. The JWH-018 equivalent was partly related to the poison severity score (PSS) and causality of the clinical intoxication elicited by the SCRA. Altogether, this equivalent concept allows to comparatively and timely interpret (poly-)SCRA intoxications based on CB1 activity.

Development and clinical implementation of an LC-HRMS method for ivacaftor, lumacaftor, tezacaftor and elexacaftor in human plasma and breast milk.

The four cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, ivacaftor, lumacaftor, tezacaftor, and elexacaftor, have revolutionised the treatment of CF by direct action on the protein target behind the disease's development. The aim was to develop and validate a quantification method for these CFTR modulators in plasma and breast milk to better understand inter-patient variability in pharmacokinetics and treatment outcome, including the risk of adverse drug reactions. The ability to monitor CFTR modulators in breast milk enables the estimation of the exposure of breastfed infant, with a potential concern for CFTR modulator-induced liver injury. The analysis was performed on a Thermo Vanquish Flex Binary UHPLC system coupled to a high-resolution mass spectrometer (HRMS), Thermo Q Exactive. The analytes were detected using positive electrospray ionisation in full scan mode. After sample preparation by protein precipitation, the supernatant was injected onto the LC system and the analytes were separated using a Zorbax SB-C18 Rapid Res HPLC column (3.5 µm, 4.6 × 75 mm). This is the first published method for CFTR modulators in breast milk. The validated quantification range for ivacaftor is 0.0050-10 µg/mL with a coefficient of variation < 6% and a mean accuracy of 97-106%; for lumacaftor, tezacaftor, and elexacaftor, the validated quantification range is 0.050-100 µg/mL with a coefficient of variation < 8% and a mean accuracy 93-106%. A simple and sensitive quantification method for CFTR modulators has been developed and used for routine analysis of human plasma and breast milk samples since 2022.

Simultaneous determination of icotinib, osimertinib, aumolertinib, and anlotinib in human plasma for therapeutic drug monitoring by UPLC-MS/MS.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as icotinib, osimertinib, and aumolertinib have emerged as promising treatment options for EGFR mutated Non-small cell lung cancer (NSCLC) patients. Additionally, anlotinib, an anti-angiogenic agent targeting VEGFR, FGFR, and PDGFR, has been used in combination with EGFR-TKIs in NSCLC cases. A method utilizing ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated for quantifying icotinib, osimertinib, aumolertinib and anlotinib simultaneously in clinical TDM. The chromatographic separation was performed using a Kinetex C18 column (100 mm × 2.1 mm) and an elution gradient of ammonium acetate in water acidified with 0.1 % formic acid and in acetonitrile. The assay was validated over a linear range of 4-2000 ng/mL for icotinib, 2-1000 ng/mL for osimertinib, 1-500 ng/mL for aumolertinib, and 0.8-400 ng/mL for anlotinib, following the guidelines on bioanalytical methods by FDA. The quantification method exhibited satisfactory performance in terms of selectivity, accuracy (from 91.3 % to 107 %), precision (intra- and inter-day coeffficients of variation ranged from 0.944 % to 7.48 %), linearity, recovery (from 86.0 % to 91.9 %), matrix effect (IS-normalized matrix factors were from 96.7 % to 102 %), and stability. Overall, the method proved to be sensitive, reliable, and straightforward, enabling successful simultaneous determination of blood concentrations of icotinib, osimertinib, aumolertinib, and anlotinib in patients. The validity of the method has been confirmed across various instruments.

Does a postmortem redistribution affect the concentrations of the 7 azaindole-derived synthetic cannabinoid 5F-MDMB-P7AICA in tissues and body fluids following pulmonary administration to pigs?

Many fatal intoxications have been reported in connection with the consumption of newer, highly potent synthetic cannabinoids. Yet, a possible postmortem redistribution (PMR) might complicate reliable interpretation of analytical results. Thus, it is necessary to investigate the PMR-potential of new synthetic cannabinoids. The pig model has already proven to be suitable for this purpose. Hence, the aim of this study was to study the PMR of the synthetic cannabinoid 5F-MDMB-P7AICA and its main metabolite 5F-MDMB-P7AICA-dimethylbutanoic acid (DBA). 5F-MDMB-P7AICA (200 µg/kg body weight) was administered by inhalation to anesthetized and ventilated pigs. At the end of the experiment, the animals were euthanized and stored at room temperature for 3 days. Tissue and body fluid samples were taken daily. Specimens were analyzed after solid phase extraction using a standard addition method and LC-MS/MS, blood was quantified after protein precipitation using a validated method. In perimortem samples, 5F-MDMB-P7AICA was found mainly in adipose tissue, bile fluid, and duodenum contents. Small amounts of 5F-MDMB-P7AICA were found in blood, muscle, brain, liver, and lung. High concentrations of DBA were found primarily in bile fluid, duodenum contents, urine, and kidney/perirenal fat tissue. In the remaining tissues, rather low amounts could be found. In comparison to older synthetic cannabinoids, PMR of 5F-MDMB-P7AICA was less pronounced. Concentrations in blood also appear to remain relatively stable at a low level postmortem. Muscle, kidney, fat, and duodenum content are suitable alternative matrices for the detection of 5F-MDMB-P7AICA and DBA, if blood specimens are not available. In conclusion, concentrations of 5F-MDMB-P7AICA and its main metabolite DBA are not relevantly affected by PMR.

A rapid LC-MS/MS method for the simultaneous quantification of ivacaftor, lumacaftor, elexacaftor, tezacaftor, hexyl-methyl ivacaftor and ivacaftor carboxylate in human plasma.

Cystic fibrosis is one of the most common genetic diseases among caucasian population. This disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding for the CFTR protein. Lumacaftor, elexacaftor, tezacaftor, and ivacaftor were currently used as the treatment to Cystic fibrosis. In this study, we describe a new method for the simultaneous quantification of four molecules: lumacaftor, elexacaftor, tezacaftor, and ivacaftor, alongside two metabolites of ivacaftor, specifically hexyl-methyl ivacaftor and ivacaftor carboxylate by liquid chromatography-tandem mass spectrometry. This method holds significant utility for therapeutic drug monitoring and the optimization of treatments related to CFTR modulators. Molecules were extracted from 100 µL of plasma by a simple method of protein precipitation using acetonitrile. Following extraction, chromatographic separation was carried out by reverse chromatography on a C18 analytical column, using a gradient elution of water (0.05 % formic acid, V/V) and acetonitrile (0.05 % formic acid, V/V). The run time was 7 minutes at a flow rate of 0.5 mL/min. After separation, molecules were detected by electrospray ionization on a Xevo TQD triple-quadrupole-mass-spectrometer (Waters®, Milford, USA). The calibration range were: 0.053-20.000 mg/L for elexacaftor, tezacaftor and lumacaftor, 0.075-14.000 mg/L for ivacaftor, and 0.024-6.500 mg/L for hexyl-methyl ivacaftor and ivacaftor carboxylate. The proposed method underwent throughout validation demonstrating satisfactory precision (inter- and intra-day coefficients of variation less than 14.3 %) and a good accuracy (inter- and intra-day bias ranging between -13.7 % and 14.7 %) for all the analytes. The presented method for the simultaneous quantification of CFTR modulators and their metabolites in human plasma has undergone rigorous validation process yielding good results including strong precision and accuracy for all analytes. This method has been effectively used in routine analytical analysis and clinical investigations within our laboratory.

Characterization and optimization of magnetic molecularly imprinted nanofibers for determination of sunitinib in human serum and capsule samples.

This article reports a spectrofluorometric method for the determination of sunitinib (STB) drug based on molecularly imprinted nanofibers fabricated by the electrospinning method and modified by magnetic nanoparticles as sorbent. The characterization of magnetic molecularly imprinted nanofibers (MMINs) was carried out using X-ray diffraction (XRD), scanning electron microscope (SEM), and transmission electron microscopy (TEM), which confirmed the successful synthesis of MMINs with well-distributed magnetite nanoparticles. Drug adsorption and desorption were optimized and important parameters such as sample pH, nanofiber mass, adsorption and desorption time, eluent solvent and sample volume were analyzed. The results demonstrated that the MMINs act as a selective sorbent for STB and can be readily collected through an external magnetic field. Methanol was used as the best eluent solvent for STB desorption from MNIN. A linear correlation was observed between the STB concentrations and fluorescence intensities in the range of 0.01-15.0 mg L-1. The detection limit for this method was 0.002 mg L-1. The relative standard deviation (RSD) of 2.6 % for 1.0 mg L-1 and 1.1 % for 10 mg L-1 of STB (n = 3) were obtained, which indicates that the developed method is precise in determining STB. Human serum and capsule analysis show the applicability of the proposed sensor for real samples.

A versatile ratiometric fluorescence nanoprobe for the determination of clonazepam in patients' plasma samples.

Despite the necessity of the study of therapeutic drug monitoring of clonazepam (CLZ), there are only a few fast detection methods available for determining CLZ in biological media. This study aims to develop a cost-effective and ratiometric probe for the quantification of CLZ in plasma samples. Fluorescent polydopamine nanoparticles were produced through a self-polymerization process at a pH of 8.5. Rhodamine B molecules were employed as a fluorescent reference material, emitting stable fluorescence in the visible range. The fabricated probe exhibited a specific detection capability for CLZ. The fluorescence emission of the probe was enhanced in two concentration ranges: from 50 ng/mL to 1.0 µg/mL and from 1.0 to 15.0 µg/mL with a lower limit of quantification of 50 ng/mL, indicating the sensitivity of the probe for detecting CLZ plasma levels. The accuracy of the probe is favorable which could be recommended for CLZ monitoring in the biological media. Furthermore, this probe is highly specific towards CLZ in the presence of various interfering agents which is mainly caused by its ratiometric nature. The developed platform showed high reliability in quantifying CLZ concentrations in patients' plasma samples. Hence, the fabricated probe could be recommended as a reliable method for the routine detection of CLZ in clinical settings.

Correlation between trough concentration and AUC for elexacaftor, tezacaftor and ivacaftor.

Therapeutic drug monitoring (TDM) of elexacaftor, tezacaftor, ivacaftor (ETI) could be a useful tool to increase efficacy and decrease the risk of adverse effects in people with Cystic Fibrosis (pwCF). It is however unclear whether drug exposure should be monitored by assessment of trough (Cmin) levels or determination of the area under the curve (AUC). Hence, in this study the correlation between measured Cmin concentration and AUC was evaluated. Serial plasma samples, including Cmin, were drawn after administration of ETI in order to calculate the AUC and assess the correlation between the two parameters. A linear correlation between Cmin and AUC0-24h was found, with Pearson's r correlation coefficients of 0.963, 0.908 and 0.860 for elexacaftor, tezacaftor and ivacaftor, respectively. Exposure of ETI may be monitored by assessment of Cmin levels.

GC-MS/MS analysis of synthetic cannabinoids 5F-MDMB-PICA and 5F-CUMYL-PICA in forensic cases.

Aim: Validate a method to quantify 1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide (5F-CUMYL-PICA) and methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate (5F-MDMB-PICA) in blood samples using GC-MS/MS. Materials & methods: A solid-phase extraction (SPE) method has been developed to quantify 5F-MDMB-PICA and 5F-CUMYL-PICA in authentic human blood samples. Results & conclusion: The limit of detection (LOD) was 0.1 and 0.11 ng/ml for 5F-CUMYL-PICA and 5F-MDMB-PICA, respectively, while the limit of quantification (LOQ) was 0.50 ng/ml for both two compounds. Recovery was 91.40, 82.54 and 85.10% for SPE, supported liquid extraction (SLE) and ISOLUTE C18; matrix effects 15, 24 and 22.5% for SPE, SLE and ISOLUTE C18; accuracy was 2.4-5.5 and 3.9-7.3% for SPE, SLE and ISOLUTE C18, while precision was 4.6-7.7 and 6.4-8.3% for SPE, SLE and ISOLUTE C18, respectively. The concentrations of 5F-CUMYL-PICA and 5F-MDMB-PICA in the authentic human blood samples were 2.18 and 3.07 ng/ml, respectively. The validated method was successfully used in supporting the quantification of analytes in blood.

[Box: see text].

The gut metabolite indole-3 propionate promotes nerve regeneration and repair.

The regenerative potential of mammalian peripheral nervous system neurons after injury is critically limited by their slow axonal regenerative rate1. Regenerative ability is influenced by both injury-dependent and injury-independent mechanisms2. Among the latter, environmental factors such as exercise and environmental enrichment have been shown to affect signalling pathways that promote axonal regeneration3. Several of these pathways, including modifications in gene transcription and protein synthesis, mitochondrial metabolism and the release of neurotrophins, can be activated by intermittent fasting (IF)4,5. However, whether IF influences the axonal regenerative ability remains to be investigated. Here we show that IF promotes axonal regeneration after sciatic nerve crush in mice through an unexpected mechanism that relies on the gram-positive gut microbiome and an increase in the gut bacteria-derived metabolite indole-3-propionic acid (IPA) in the serum. IPA production by Clostridium sporogenes is required for efficient axonal regeneration, and delivery of IPA after sciatic injury significantly enhances axonal regeneration, accelerating the recovery of sensory function. Mechanistically, RNA sequencing analysis from sciatic dorsal root ganglia suggested a role for neutrophil chemotaxis in the IPA-dependent regenerative phenotype, which was confirmed by inhibition of neutrophil chemotaxis. Our results demonstrate the ability of a microbiome-derived metabolite, such as IPA, to facilitate regeneration and functional recovery of sensory axons through an immune-mediated mechanism.

A Simple LC-MS/MS Method for the Quantification of PDA-66 in Human Plasma.

The treatment of cancer is one of the most important pharmacotherapeutic challenges. To this end, chemotherapy has for some time been complemented by targeted therapies against specific structures. PDA-66, a structural analogue of the inhibitor of serine-threonine kinase glycogen synthase kinase 3ß SB216763, has shown preclinical antitumour effects in various cell lines, with the key pathways of its anticancer activity being cell cycle modulation, DNA replication and p53 signalling. For the monitoring of anticancer drug treatment in the context of therapeutic drug monitoring, the determination of plasma concentrations is essential, for which an LC-MS/MS method is particularly suitable. In the present study, a sensitive LC-MS/MS method for the quantification of the potential anticancer drug PDA-66 in human plasma with a lower limit of quantification of 2.5 nM is presented. The method was successfully validated and tested for the determination of PDA-66 in mouse plasma and sera.

Pharmacokinetic comparison of four arbidol hydrochloride preparations in beagle dogs.

This study aimed to compare the pharmacokinetic properties of four preparations (dispersible tablets, ordinary tablets, capsules and granules) of arbidol hydrochloride, a broad-spectrum antiviral drug, in beagle dogs. Briefly, a single dose of 100 mg of the four preparations of arbidol hydrochloride was orally administered to dogs; blood was then collected from the veins of the foreleg at different times after administration to prepare plasma samples. The plasma concentration of arbidol hydrochloride was measured using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that when orally administered with dispersible tablets, ordinary tablets, capsules and granules suspended with water, there were no significant differences in the pharmacokinetic parameters (including peak time, peak concentration, elimination half-life, area under the curve (AUC0-t ), and mean retention time) of arbidol hydrochloride. However, in the case of the dispersible tablets, the pharmacokinetics of arbidol hydrochloride was significantly affected by the mode of administration. Compared with direct feeding, peak time [0.50 (0.13, 0.50) vs. 1.00 (0.50, 2.00)] was significantly shortened (P = 0.033) and the AUC0-48 h (8726.5 ± 2509.3 vs. 3650.8 ± 1536.9 ng h/ml) was significantly increased (P = 0.012) when the dispersible tablets were orally administered as water dispersion. In conclusion, the pharmacokinetics of four preparations of arbidol hydrochloride were not significant different in beagle dogs. However, compared with direct feeding, the absorption of arbidol hydrochloride was faster and the bioavailability was better when the dispersible tablets were orally administered as water dispersion.

Indole-3-Propionic Acid, a Gut-Derived Tryptophan Metabolite, Associates with Hepatic Fibrosis.

BACKGROUND AND AIMS: Gut microbiota-derived metabolites play a vital role in maintenance of human health and progression of disorders, including obesity and type 2 diabetes (T2D). Indole-3-propionic acid (IPA), a gut-derived tryptophan metabolite, has been recently shown to be lower in individuals with obesity and T2D. IPA's beneficial effect on liver health has been also explored in rodent and cell models. In this study, we investigated the association of IPA with human liver histology and transcriptomics, and the potential of IPA to reduce hepatic stellate cell activation in vitro. METHODS: A total of 233 subjects (72% women; age 48.3 ± 9.3 years; BMI 43.1 ± 5.4 kg/m2) undergoing bariatric surgery with detailed liver histology were included. Circulating IPA levels were measured using LC-MS and liver transcriptomics with total RNA-sequencing. LX-2 cells were used to study hepatoprotective effect of IPA in cells activated by TGF-ß1. RESULTS: Circulating IPA levels were found to be lower in individuals with liver fibrosis compared to those without fibrosis (p = 0.039 for all participants; p = 0.013 for 153 individuals without T2D). Accordingly, levels of circulating IPA associated with expression of 278 liver transcripts (p < 0.01) that were enriched for the genes regulating hepatic stellate cells (HSCs) activation and hepatic fibrosis signaling. Our results suggest that IPA may have hepatoprotective potential because it is able to reduce cell adhesion, cell migration and mRNA gene expression of classical markers of HSCs activation in LX-2 cells (all p < 0.05). CONCLUSION: The association of circulating IPA with liver fibrosis and the ability of IPA to reduce activation of LX-2 cells suggests that IPA may have a therapeutic potential. Further molecular studies are needed to investigate the mechanisms how IPA can ameliorate hepatic fibrosis.

Disrupting biological sensors of force promotes tissue regeneration in large organisms.

Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1.

Impact of plasma 5-hydroxyindoleacetic acid, a serotonin metabolite, on clinical outcome in septic shock, and its effect on vascular permeability.

Septic shock is characterized by dysregulated vascular permeability. We hypothesized that the vascular permeability of endothelial cells (ECs) would be regulated by serotonin via serotonin-Rho-associated kinase (ROCK) signaling. We aimed to determine the impact of 5-hydroxyindoleacetic acid (5-HIAA) on septic shock as a novel biomarker. Plasma 5-HIAA levels and disease severity indices were obtained from 47 patients with sepsis. The association between 5-HIAA levels and severity indices was analyzed. Permeability upon serotonin stimulation was determined using human pulmonary microvascular ECs. 5-HIAA were significantly higher in septic shock patients than in patients without shock or healthy controls (p = 0.004). These elevated levels were correlated with severity indexes (SOFA score [p < 0.001], APACHE II [p < 0.001], and PaO2:FiO2 [p = 0.02]), and longitudinally associated with worse clinical outcomes (mechanical ventilation duration [p = 0.009] and ICU duration [p = 0.01]). In the experiment, serotonin increased the permeability of ECs, which was inhibited by the ROCK inhibitor (p < 0.001). Serotonin increases vascular permeability of ECs via ROCK signaling. This suggests a novel mechanism by which serotonin disrupts endothelial barriers via ROCK signaling and causes the pathogenesis of septic shock with a vascular leak. Serotonin serves as a novel biomarker of vascular permeability.

A novel method for the quantification of anlotinib in human plasma using two-dimensional liquid chromatography.

A simple, efficient, and stable detection method of two-dimensional liquid chromatography (2D-LC) was established and validated to determine anlotinib in the human plasma. The 2D-LC system comprises a first-dimensional column (LC1), an intermediate transfer column, and a second-dimensional column (LC2). With simple protein precipitation treatment, the samples were processed directly for detection. The analysis cycle time was completed within 9.50 min. For the anlotinib concentrations, the calibration curve was linear over the 5.00-320.00 ng/mL range. The intra-day and inter-day precision ranges were 0.77-6.22% and 1.92-4.26%, respectively, for anlotinib concentrations. The recoveries were in the range of 97.85-102.50%. A total of 135 plasma samples from 94 patients were analyzed by our method. The plasma concentrations of patients were in the range of 5.17-106.38 ng/mL, in which the female had a higher plasma concentration (6.44-106.38 ng/mL). The simultaneous application of dexamethasone can increase the anlotinib concentration in the plasma. In our clinical application, we found that the factors that affect the plasma concentration include the time and dose of the medication, gender, and drug interactions. The method appears to be sensitive, precise, selective, and suitable for determining the concentration of anlotinib in the plasma sample.

Comparison of the tissue distribution and metabolism of AN1284, a potent anti-inflammatory agent, after subcutaneous and oral administration in mice.

This study is to compare the tissue distribution and metabolism of AN1284 after subcutaneous and oral administration at doses causing maximal reductions in IL-6 in plasma and tissues of mice. Anti-inflammatory activity of AN1284 and its metabolites was detected in lipopolysaccharide (LPS) activated RAW 264.7 macrophages. Mice were given AN1284 by injection or gavage, 15 min before LPS. IL-6 protein levels were measured after 4 h. Using a liquid chromatography/mass spectrometry method we developed, we showed that AN1284 is rapidly metabolized to the indole (AN1422), a 7-OH derivative (AN1280) and its glucuronide. AN1422 has weaker anti-inflammatory activity than AN1284 in LPS-activated macrophages and in mice. AN1284 (0.5 mg/kg) caused maximal reductions in IL-6 in the plasma, brain, and liver when injected subcutaneously and after gavage only in the liver. Similar reductions in the plasma and brain required a dose of 2.5 mg/kg, which resulted in 5.5-fold higher hepatic levels than after injection of 0.5 mg/kg, but 7, 11, and 19-fold lower ones in the plasma, brain, and kidneys, respectively. Hepatic concentrations produced by AN1284 were 2.5 mg/kg/day given by subcutaneously implanted mini-pumps that were only 12% of the peak levels seen after acute injection of 0.5 mg/kg. Similar hepatic concentrations were obtained by (1 mg/kg/day), administered in the drinking fluid. These were sufficient to decrease hepatocellular damage and liver triglycerides in previous experiments in diabetic mice. AN1284 can be given orally by a method of continuous release to treat chronic liver disease, and its preferential concentration in the liver should limit any adverse effects.

3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.