Investigation of the factors responsible for the low oral bioavailability of alizarin using a sensitive LC-MS/MS method: In vitro, in situ, and in vivo evaluations.

Alizarin (1,2-dihydroxyanthraquinone) is an anthraquinone reddish dye widely used for painting and textile dyeing. As the biological activity of alizarin has recently attracted increasing attention from researchers, its therapeutic potential as complementary and alternative medicine is of interest. However, no systematic research has been conducted on the biopharmaceutical and pharmacokinetic aspects of alizarin. Therefore, this study aimed to comprehensively investigate the oral absorption and intestinal/hepatic metabolism of alizarin using a simple and sensitive tandem mass spectrometry method developed and validated in-house. The present method for the bioanalysis of alizarin has merits, including a simple pretreatment procedure, small sample volume, and adequate sensitivity. Alizarin exhibited pH-dependent moderate lipophilicity and low solubility with limited intestinal luminal stability. Based on the in vivo pharmacokinetic data, the hepatic extraction ratio of alizarin was estimated to be 0.165-0.264, classified as a low level of hepatic extraction. In an in situ loop study, considerable fractions (28.2%-56.4%) of the alizarin dose were significantly absorbed in gut segments from the duodenum to ileum, suggesting that alizarin may be classified as the Biopharmaceutical Classification System class II. An in vitro metabolism study using rat and human hepatic S9 fractions revealed that glucuronidation and sulfation, but not NADPH-mediated phase I reactions and methylation, are significantly involved in the hepatic metabolism of alizarin. Taken together, it can be estimated that the fractions of oral alizarin dose unabsorbed from the gut lumen and eliminated by the gut and liver before reaching the systemic circulation are 43.6%-76.7%, 0.474%-36.3%, and 3.77%-5.31% of the dose, respectively, resulting in a low oral bioavailability of 16.8%. Therefore, the oral bioavailability of alizarin depends primarily on its chemical degradation in the gut lumen and secondarily on first-pass metabolism.

Effects of 1α,25-dihydroxyvitamin D3 on the pharmacokinetics and biodistribution of ergothioneine, an endogenous organic cation/carnitine transporter 1 substrate, in rats.

Purpose: This study aimed to investigate the effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression levels of organic cation/carnitine transporter 1 (OCTN1) as well as the pharmacokinetics and biodistribution of ergothioneine, an OCTN1 substrate, in rats. Methods: Rats pretreated with 1,25(OH)2D3 (2.56 nmol/kg/day) for four days were administered ergothioneine (2 mg/kg) intravenously. The expression levels of rat OCTN1 (rOCTN1) in organs were determined using real-time quantitative polymerase chain reaction. Ergothioneine levels in plasma, urine, and organs (with and without intravenous injection of exogenous ergothioneine) were determined using liquid chromatography-tandem mass spectrometry. Results: 1,25(OH)2D3 pretreatment resulted in a significant decrease in rOCTN1 mRNA expression levels in the kidney and brain, a significant increase in basal plasma levels of ergothioneine (from 48 h), and a significant decrease in the tissue-plasma partition coefficient (Kp) in all tissues (except the heart and lungs) and the basal urine levels of ergothioneine. After intravenous administration, the pharmacokinetic profiles of ergothioneine were consistent with the basal levels of endogenous ergothioneine, with an increase in AUC∞ by 85%, a significant decrease in total clearance by 49%, and a decrease in Vss by 32% in 1,25(OH)2D3-treated rats. The Kp value and urinary recovery of ergothioneine also decreased in the 1,25(OH)2D3-treated group. Conclusion: This study showed the effects of 1,25(OH)2D3 on the expression and function of rOCTN1 by investigating the interaction between 1,25(OH)2D3 and ergothioneine. Dose adjustment and possible changes in bioavailability should be considered before the co-administration of vitamin D or its active forms and OCTN1 substrates. Supplementary Information: The online version contains supplementary material available at 10.1007/s40005-022-00563-1.

The Role of Processed Aloe vera Gel in Intestinal Tight Junction: An In Vivo and In Vitro Study.

Leaky gut is a condition of increased paracellular permeability of the intestine due to compromised tight junction barriers. In recent years, this affliction has drawn the attention of scientists from different fields, as a myriad of studies prosecuted it to be the silent culprit of various immune diseases. Due to various controversies surrounding its culpability in the clinic, approaches to leaky gut are restricted in maintaining a healthy lifestyle, avoiding irritating factors, and practicing alternative medicine, including the consumption of supplements. In the current study, we investigate the tight junction-modulating effects of processed Aloe vera gel (PAG), comprising 5-400-kD polysaccharides as the main components. Our results show that oral treatment of 143 mg/kg PAG daily for 10 days improves the age-related leaky gut condition in old mice, by reducing their individual urinal lactulose/mannitol (L/M) ratio. In concordance with in vivo experiments, PAG treatment at dose 400 µg/mL accelerated the polarization process of Caco-2 monolayers. The underlying mechanism was attributed to enhancement in the expression of intestinal tight junction-associated scaffold protein zonula occludens (ZO)-1 at the translation level. This was induced by activation of the MAPK/ERK signaling pathway, which inhibits the translation repressor 4E-BP1. In conclusion, we propose that consuming PAG as a complementary food has the potential to benefit high-risk patients.

Investigation of the Factors Responsible for the Poor Oral Bioavailability of Acacetin in Rats: Physicochemical and Biopharmaceutical Aspects.

Acacetin, an important ingredient of acacia honey and a component of several medicinal plants, exhibits therapeutic effects such as antioxidative, anticancer, anti-inflammatory, and anti-plasmodial activities. However, to date, studies reporting a systematic investigation of the in vivo fate of orally administered acacetin are limited. Moreover, the in vitro physicochemical and biopharmaceutical properties of acacetin in the gastrointestinal (GI) tract and their pharmacokinetic impacts remain unclear. Therefore, in this study, we aimed to systematically investigate the oral absorption and disposition of acacetin using relevant rat models. Acacetin exhibited poor solubility (≤119 ng/mL) and relatively low stability (27.5-62.0% remaining after 24 h) in pH 7 phosphate buffer and simulated GI fluids. A major portion (97.1%) of the initially injected acacetin dose remained unabsorbed in the jejunal segments, and the oral bioavailability of acacetin was very low at 2.34%. The systemic metabolism of acacetin occurred ubiquitously in various tissues (particularly in the liver, where it occurred most extensively), resulting in very high total plasma clearance of 199 ± 36 mL/min/kg. Collectively, the poor oral bioavailability of acacetin could be attributed mainly to its poor solubility and low GI luminal stability.

Preparation of an oil suspension containing ondansetron hydrochloride as a sustained release parenteral formulation.

Ondansetron hydrochloride (ODS) is a selective 5-hydroxytryptamine type 3 antagonist for nausea and emesis prevention in neoplastic patients. To reduce dosing frequency and side effects and improve patient compliance, a sustained release parenteral formulation of ODS was developed. Microparticles of methylcellulose (MC) and ODS were prepared using the spray-drying method and suspended in oils to form oil suspensions. The formulations were evaluated for residual moisture, drug content, size distribution, DSC, XRD, FTIR, SEM, drug release, and pharmacokinetic studies. The effects of polymers and oils on the drug release were evaluated. MC showed the most prominent sustained release effect among various polymers examined with the optimum MC/ODS ratio of 2:1 (w/w). The particle size of the produced microparticles was in the mean diameter of approximately 3 µm. Physicochemical characterization suggested that ODS existed in an amorphous matrix within the microparticles and interacted with MC via hydrogen bonds. Corn oil was selected as the appropriate oil for suspension due to the sustained release of ODS and the appropriate viscosity. The optimized sustained release formulation of ODS was the corn oil suspension of spray-dried microparticles containing MC and ODS (2:1, w/w). It showed an in vitro drug sustained release up to 120 h, while the oil suspension of ODS without any polymer released the drug within 2 h. Following subcutaneous administration in rats, the optimized formulation could prolong the drug release until 72 h with the enhanced bioavailability in comparison with the ODS solution. The oil suspension of spray-dried microparticles might be an efficient approach for prolongation of the drug effect in the management of nausea and emesis. Graphical abstract.

Therapeutic Evaluation of Synthetic Peucedanocoumarin III in an Animal Model of Parkinson's Disease.

The motor and nonmotor symptoms of Parkinson's disease (PD) correlate with the formation and propagation of aberrant α-synuclein aggregation. This protein accumulation is a pathological hallmark of the disease. Our group recently showed that peucedanocoumarin III (PCIII) possesses the ability to disaggregate ß sheet aggregate structures, including α-synuclein fibrils. This finding suggests that PCIII could be a therapeutic lead compound in PD treatment. However, the translational value of PCIII and its safety information have never been explored in relevant animal models of PD. Therefore, we first designed and validated a sequence of chemical reactions for the large scale organic synthesis of pure PCIII in a racemic mixture. The synthetic PCIII racemate facilitated clearance of repeated ß sheet aggregate (ß23), and prevented ß23-induced cell toxicity to a similar extent to that of purified PCIII. Given these properties, the synthetic PCIII's neuroprotective function was assessed in 6-hydroxydopamine (6-OHDA)-induced PD mouse models. The PCIII treatment (1 mg/kg/day) in a 6-OHDA-induced PD mouse model markedly suppressed Lewy-like inclusions and prevented dopaminergic neuron loss. To evaluate the safety profiles of PCIII, high dose PCIII (10 mg/kg/day) was administered intraperitoneally to two-month-old mice. Following 7 days of PCIII treatment, PCIII distributed to various tissues, with substantial penetration into brains. The mice that were treated with high dose PCIII had no structural abnormalities in the major organs or neuroinflammation. In addition, high dose PCIII (10 mg/kg/day) in mice had no adverse impact on motor function. These findings suggest that PCIII has a relatively high therapeutic index. Given the favorable safety features of PCIII and neuroprotective function in the PD mouse model, it may become a promising disease-modifying therapy in PD to regulate pathogenic α-synuclein aggregation.

Determination of isoorientin levels in rat plasma after oral administration of Vaccinum bracteatum Thunb. methanol extract by high-performance liquid chromatography-tandem mass spectrometry.

A simple, sensitive and rapid liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination of plasma isoorientin levels in rats. After simple protein precipitation using methanol, chromatographic analysis was performed using a Synergi 4µ polar-RP 80A column (150 × 2.0 mm, 4µm) under isocratic conditions and a mobile phase consisting of 0.1% formic acid in water and methanol (80:20, v/v) at a flow rate of 0.2 mL/min. In positive electrospray ionization mode, the protonated precursor and product ion transitions of isoorientin (m/z 449.0 → 299.1) and of puerarin (the internal standard; m/z 417.1 → 297.1) were acquired by multiple reaction monitoring. Calibration curves obtained for plasma showed good linearity over the concentration range 1-1000 ng/mL. The lower limit of quantification was 1 ng/mL. Intra- and inter-day precisions were within 8.8% relative standard deviation. Accuracies ranged from 92.1 and 109.7%. The isoorientin stability in rat plasma under typical handling/storage conditions also found to be acceptable. The developed method was applied successfully to a pharmacokinetic study of isoorientin orally administered as the methanol extract of Vaccinium bracteatum Thunb. or administered as pure isoorientin.

Vitamin D Receptor-Mediated Upregulation of CYP3A4 and MDR1 by Quercetin in Caco-2 cells.

To examine whether quercetin interacts with vitamin D receptor, we investigated the effects of quercetin on vitamin D receptor activity in human intestinal Caco-2 cells. The effects of quercetin on the expression of the vitamin D receptor target genes, vitamin D3 24-hydroxylase, cytochrome P450 3A4, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were measured using quantitative polymerase chain reaction. The vitamin D receptor siRNA was used to assess the involvement of the vitamin D receptor. Vitamin D receptor activation using a vitamin D responsive element-mediated cytochrome P450 3A4 reporter gene assay was investigated in Caco-2 cells transfected with human vitamin D receptor. We also studied the magnitude of the vitamin D receptor activation and/or synergism between 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] and quercetin-like flavonoids. Slight but significant increases in the mRNA expression of cytochrome P450 3A4, vitamin D3 24-hydroxylase, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were observed after 3 days of continual quercetin treatment. The silencing effect of vitamin D receptor by vitamin D receptor siRNA in Caco-2 cells significantly attenuated the induction of the vitamin D receptor target genes. Moreover, quercetin significantly enhanced cytochrome P450 3A4 reporter activity in Caco-2 cells in a dose-dependent manner, and the expression of exogenous vitamin D receptor further stimulated the vitamin D receptor activity. Quercetin-like flavonoids such as kaempferol stimulated the vitamin D receptor activity in a manner similar to that seen with quercetin. Taken together, the data indicates that quercetin upregulates cytochrome P450 3A4 and multidrug resistance protein 1 expression in Caco-2 cells likely via a vitamin D receptor-dependent pathway.

Comparative effects of 1α-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on transporters and enzymes in fxr(+/+) and fxr(-/-) mice.

Previous studies have shown that 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] treatment in mice resulted in induction of intestinal and renal Cyp24a1 and Trpv6 expression, increased hepatic Cyp7a1 expression and activity, as well as higher renal Mdr1/P-gp expression. The present study compared the equimolar efficacies of 1α-hydroxyvitamin D3 [1α(OH)D3 ] (6 nmol/kg i.p. q2d × 4), a lipophilic precursor with a longer plasma half-life that is converted to 1,25(OH)2 D3 , and 1,25(OH)2 D3 on vitamin D receptor (VDR) target genes. To clarify whether changes in VDR genes was due to VDR and not secondary, farnesoid X receptor (FXR)-directed effects, namely, lower Cyp7a1 expression in rat liver due to increased bile acid absorption, wildtype [fxr(+/+)] and FXR knockout [fxr(-/-)] mice were used to distinguish between VDR and FXR effects. With the exception that hepatic Sult2a1 mRNA was increased equally well by 1α(OH)D3 and 1,25(OH)2 D3 , 1α(OH)D3 treatment led to higher increases in hepatic Cyp7a1, renal Cyp24a1, VDR, Mdr1 and Mrp4, and intestinal Cyp24a1 and Trpv6 mRNA expression in both fxr(+/+) and fxr(-/-) mice compared to 1,25(OH)2 D3 treatment. A similar induction in protein expression and microsomal activity of hepatic Cyp7a1 and renal P-gp and Mrp4 protein expression was noted for both compounds. A higher intestinal induction of Trpv6 was observed, resulting in greater hypercalcemic effect following 1α(OH)D3 treatment. The higher activity of 1α(OH)D3 was explained by its rapid conversion to 1,25(OH)2 D3 in tissue sites, furnishing higher plasma and tissue 1,25(OH)2 D3 levels compared to following 1,25(OH)2 D3 -treatment. In conclusion, 1α(OH)D3 exerts a greater effect on VDR gene induction than equimolar doses of 1,25(OH)2 D3 in mice.

Effect of adjuvant on pharmacokinetics, organ distribution and humoral immunity of hepatitis B surface antigen after intramuscular injection to rats.

Two types of radioiodinated plasma driven antigens, heat-inactivated (¹²5I-h-HBsAg) and formalin-inactivated HBsAg (¹²5I-f-HBsAg) were investigated for the effect of immunoadjuvant, aluminium phosphate (AP) on pharmacokinetics, organ distribution and humoral immunity of differently inactivated hepatitis B surface antigens (HBsAg) in rats. As a result, most of h-HBsAg (90%) was retained and slowly eliminated from the injection site. The h-HBsAg was highly localized in regional lymph node (RLN), but resulted in low humoral immune response. On the other hand, f-HBsAg was less localized in the injection site and RLN, but mainly distributed into serum and liver (62.9 and 22.4%, respectively). However, both h-HBsAg and f-HBsAg slowly disappeared from the injection site with AP, resulting in the increased area under the amount-time curve (AUQ) of h-HBsAg and f-HBsAg in the injection site. Exposures of h-HBsAg and f-HBsAg in serum were increased (1.4 and 2.8 times increase in AUC, respectively) with AP. The RLN uptake of both antigens were dramatically increased (25 and 3.1 times increase in AUC, respectively) with higher humoral immune response. The antibody titres were also increased with AP. In conclusion, pharmacokinetics, organ distribution and humoral immunity of h-HBsAg were highly dependent on the inactivation method of antigen and the presence of immunoadjuvant such as AP.