Ibrutinib Efficacy, Safety, and Pharmacokinetics in Chinese Patients with Relapsed or Refractory Waldenström's Macroglobulinemia: A Multicenter, Single-Arm, Phase 4 Study.

INTRODUCTION: Waldenström's macroglobulinemia (WM) is a rare malignant B cell lymphoma which occurs in around 1-2% of all hematologic tumors. Ibrutinib was approved in China for WM on the basis of two global pivotal studies which enrolled no Chinese patients. The aim of this study was to determine the efficacy, safety, and pharmacokinetics of ibrutinib in Chinese patients with relapsed or refractory (r/r) WM. METHODS: This was an open-label, single-arm, multicenter phase 4 study conducted across five sites in China. Enrolled patients with clinicopathological confirmed WM received ibrutinib 420 mg once daily orally until disease progression or unacceptable toxicity. The primary endpoint was major response rate (MRR, partial response [PR], or better) according to the modified consensus criteria from the Sixth International Workshop on WM. RESULTS: Seventeen patients were enrolled; at data cutoff (March 19, 2022), MRR was 64.7% (90% confidence interval [CI] 42.0-83.4) and overall response rate was 100% (90% CI 83.8-100.0). One (5.9%) patient achieved very good PR, 10 (58.8%) achieved PR, and six (35.3%) achieved minor response. The median duration of response (PR or better) was 14.8 months (95% CI 10.8-not estimable [NE]). Median progression-free survival was 18.4 months (95% CI 12.9-NE). All patients experienced at least one treatment-emergent adverse event (TEAE) related to the study drug, and grade ≥ 3 TEAEs were reported in 13 (76.5%) patients. There were no TEAEs leading to dose reduction or death. The median model estimated maximum plasma concentration and area under the plasma concentration-time curve during 24 h after dosing at steady state were 40.5 ng/mL and 204 ng·h/mL, respectively. CONCLUSIONS: Ibrutinib demonstrated durable responses in Chinese patients with r/r WM. Treatment was well tolerated with no new safety signals compared with the pivotal global studies. Ibrutinib exposure was also comparable between Chinese and non-Chinese patients. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT04042376.

FaMYB5 Interacts with FaBBX24 to Regulate Anthocyanin and Proanthocyanidin Biosynthesis in Strawberry (Fragaria × ananassa).

MYB and BBX transcription factors play important roles in flavonoid biosynthesis. Here, we obtained transgenic woodland strawberry with stable overexpression of FaMYB5, demonstrating that FaMYB5 can increase anthocyanin and proanthocyanidin content in roots, stems and leaves of woodland strawberry. In addition, bimolecular fluorescence complementation assays and yeast two-hybridization demonstrated that the N-terminal (1-99aa) of FaBBX24 interacts with FaMYB5. Transient co-expression of FaBBX24 and FaMYB5 in cultivated strawberry 'Xiaobai' showed that co-expression strongly promoted the expression of F3'H, 4CL-2, TT12, AHA10 and ANR and then increased the content of anthocyanin and proanthocyanidin in strawberry fruits. We also determined that FaBBX24 is also a positive regulator of anthocyanin and proanthocyanidin biosynthesis in strawberry. The results reveal a novel mechanism by which the FaMYB5-FaBBX24 module collaboratively regulates anthocyanin and proanthocyanidin in strawberry fruit.

Regulation of flavonoids in strawberry fruits by FaMYB5/FaMYB10 dominated MYB-bHLH-WD40 ternary complexes.

Anthocyanins endowing strawberry fruit red color are regulated by the MYB-bHLH-WD40 complex. By analyzing the MYBs involved in the flavonoid biosynthesis in strawberry, we found that R2R3-FaMYB5 promoted the content of anthocyanin and proanthocyanidins in strawberry fruits. Yeast two-hybrid and BiFC assays confirmed that MBW complexes connected with flavonoid metabolism were FaMYB5/FaMYB10-FaEGL3 (bHLH)-FaLWD1/FaLWD1-like (WD40). Transient overexpression and qRT-PCR analysis revealed that disparate MBW models hold different patterns in the regulation of flavonoid biosynthesis in strawberry fruits. Compared with FaMYB10, FaMYB5 and its dominant complexes showed a more specific regulatory range on strawberry flavonoid biosynthetic pathway, while FaMYB10 was more extensive. In addition, the complexes involved in FaMYB5 facilitated PAs accumulation primarily through the LAR tributary while FaMYB10 mainly by the ANR branch. FaMYB9 and FaMYB11 tremendously elicited the accumulation of proanthocyanidins by up-regulating the expression levels of both LAR and ANR, and also affected anthocyanin metabolism by changing the ratio of Cy3G and Pg3G which were constituted as two major anthocyanin monomers in strawberries. Our study also illustrated that FaMYB5-FaEGL3-FaLWD1-like directly targeted the promoters of F3'H, LAR, and AHA10 thus committing to flavonoid accumulation. These results allow the specific members involved in the MBW complex to be deciphered and provide new insights into the regulatory mechanisms of anthocyanins and proanthocyanidins regulated by the MBW complex.

A novel R2R3-MYB transcription factor FaMYB5 positively regulates anthocyanin and proanthocyanidin biosynthesis in cultivated strawberries (Fragaria × ananassa).

Flavonoids have a major contribution to the fruit quality in cultivated strawberries and are regulated by MYB, bHLH and WD40 transcriptional factors. We reported here the identification of the FaMYB5, an R2R3-MYB transcription factor, which positively regulated the accumulation of anthocyanins and proanthocyanidins through the trans-activation of the F3'H and LAR. The strawberry FaEGL3 and FaLWD1/FaLWD1-like interact with the R2R3-FaMYB5 to form an MYB-bHLH-WD40 complex (MBW), enhancing the regulatory efficiency. The R2R3-FaMYB5 was constitutively expressed in various tissues and in fruits of different developmental stages, which was strikingly contrasting to the fruit-specific expression patterns of FaMYB10. Meanwhile, R2R3-FaMYB5 failed to promote a stable accumulation of anthocyanin glycosides in the mature fruits of the myb10 mutant, mainly due to the suppressed expression of TT19. The R2R3-FaMYB5 was regulated by an antisense long noncoding RNA lncRNA-myb5. Additionally, the R2R3-FaMYB5 protein could interact with FaBT2 and was degraded through the ubiquitin/26 S proteasome pathway. Transcriptome and metabolome data showed that R2R3-FaMYB5 enhanced the gene expression and the metabolite accumulation involved in the flavonoid, phenylpropanoid and lignin biosynthesis pathways. Collectively, we conclude that the FaMYB5 is an R2R3-MYB activator involved in the composition of MBW, which positively regulates the biosynthesis of anthocyanin and proanthocyanidin. These findings provided new insights into the molecular mechanisms that regulate flavonoids in strawberry fruits.

Genome-Wide Identification of Strawberry C2H2-ZFP C1-2i Subclass and the Potential Function of FaZAT10 in Abiotic Stress.

C2H2-type zinc finger proteins (C2H2-ZFPs) play a key role in various plant biological processes and responses to environmental stresses. In Arabidopsisthaliana, C2H2-ZFP members with two zinc finger domains have been well-characterized in response to abiotic stresses. To date, the functions of these genes in strawberries are still uncharacterized. Here, 126 C2H2-ZFPs in cultivated strawberry were firstly identified using the recently sequenced Fragaria × ananassa genome. Among these C2H2-ZFPs, 46 members containing two zinc finger domains in cultivated strawberry were further identified as the C1-2i subclass. These genes were unevenly distributed on 21 chromosomes and classified into five groups according to the phylogenetic relationship, with similar physicochemical properties and motif compositions in the same group. Analyses of conserved domains and gene structures indicated the evolutionary conservation of the C1-2i subclass. A Ka/Ks analysis indicated that the C1-2i members were subjected to purifying selection during evolution. Furthermore, FaZAT10, a typical C2H2-ZFP, was isolated. FaZAT10 was expressed the highest in roots, and it was induced by drought, salt, low-temperature, ABA, and MeJA treatments. It was localized in the nucleus and showed no transactivation activity in yeast cells. Overall, these results provide useful information for enriching the analysis of the ZFPs gene family in strawberry, and they provide support for revealing the mechanism of FaZAT10 in the regulatory network of abiotic stress.

Importance of FaWRKY71 in Strawberry (Fragaria × ananassa) Fruit Ripening.

WRKY transcription factors play a nonnegligible role in plant growth and development, but little is known about the involvement of WRKY transcription factors in the regulation of fruit ripening. In this study, FaWRKY71 was identified to be closely related to fruit maturation in octoploid strawberry. FaWRKY71 protein localized in the nucleus and responded to cold, salt, low phosphate, ABA, and light quality in strawberry seedlings. The temporal and spatial pattern expression analysis indicated that FaWRKY71 was expressed in all the detected tissues, especially in the full red fruits. In addition, FaWRKY71 gave rise to the accumulation of anthocyanin content by promoting the expression of structural genes FaF3'H, FaLAR, FaANR, and transport factors FaTT19 and FaTT12 in the flavonoid pathway, and softening the texture of strawberry via up-regulating the abundance of FaPG19 and FaPG21. Furthermore, FaWRKY71 was a positive regulator that mediated resistance against reactive oxygen species by enhancing the enzyme activities of SOD, POD, and CAT, reducing the amount of MDA. Altogether, this study provides new and comprehensive insight into the regulatory mechanisms facilitating fruit ripening in strawberry.

Pharmacokinetics and disposition of anlotinib, an oral tyrosine kinase inhibitor, in experimental animal species.

Anlotinib is a new oral tyrosine kinase inhibitor; this study was designed to characterize its pharmacokinetics and disposition. Anlotinib was evaluated in rats, tumor-bearing mice, and dogs and also assessed in vitro to characterize its pharmacokinetics and disposition and drug interaction potential. Samples were analyzed by liquid chromatography/mass spectrometry. Anlotinib, having good membrane permeability, was rapidly absorbed with oral bioavailability of 28%-58% in rats and 41%-77% in dogs. Terminal half-life of anlotinib in dogs (22.8±11.0 h) was longer than that in rats (5.1±1.6 h). This difference appeared to be mainly associated with an interspecies difference in total plasma clearance (rats, 5.35±1.31 L·h-1·kg-1; dogs, 0.40±0.06 L·h-1/kg-1). Cytochrome P450-mediated metabolism was probably the major elimination pathway. Human CYP3A had the greatest metabolic capability with other human P450s playing minor roles. Anlotinib exhibited large apparent volumes of distribution in rats (27.6±3.1 L/kg) and dogs (6.6±2.5 L/kg) and was highly bound in rat (97%), dog (96%), and human plasma (93%). In human plasma, anlotinib was predominantly bound to albumin and lipoproteins, rather than to α1-acid glycoprotein or γ-globulins. Concentrations of anlotinib in various tissue homogenates of rat and in those of tumor-bearing mouse were significantly higher than the associated plasma concentrations. Anlotinib exhibited limited in vitro potency to inhibit many human P450s, UDP-glucuronosyltransferases, and transporters, except for CYP3A4 and CYP2C9 (in vitro half maximum inhibitory concentrations, <1 µmol/L). Based on early reported human pharmacokinetics, drug interaction indices were 0.16 for CYP3A4 and 0.02 for CYP2C9, suggesting that anlotinib had a low propensity to precipitate drug interactions on these enzymes. Anlotinib exhibits many pharmacokinetic characteristics similar to other tyrosine kinase inhibitors, except for terminal half-life, interactions with drug metabolizing enzymes and transporters, and plasma protein binding.

Pharmacokinetics-Based Identification of Potential Therapeutic Phthalides from XueBiJing, a Chinese Herbal Injection Used in Sepsis Management.

XueBiJing, an injectable five-herb preparation, has been incorporated into routine sepsis care in China. Phthalides, originating from XueBiJing's component herbs Ligusticum chuanxiong rhizomes and Angelica sinensis roots, are believed to contribute to its therapeutic effects due to their presence in the preparation and antisepsis-related properties. This investigation aimed to identify potential therapeutic phthalides that are bioavailable to act on XueBiJing's therapeutic targets and that could serve as pharmacokinetic markers to supplement classic biomarkers for sepsis care. Among 10 phthalides detected in XueBiJing, senkyunolides I and G were the major circulating phthalides in human subjects, but their different pharmacokinetics might influence their contribution to XueBiJing's therapeutic action. Senkyunolide I exhibited a large distribution volume (1.32 l/kg) and was moderately bound in plasma (54% unbound), whereas senkyunolide G exhibited a small distribution volume (0.10 l/kg) and was extensively bound in plasma (3% unbound). Clearance of senkyunolide I from the systemic circulation was governed by UGT2B15-mediated hepatic glucuronidation; the resulting electrophilic glucuronides were conjugated with glutathione in the liver. Senkyunolide G was selectively bound to albumin (99%) in human plasma. To our knowledge, the human pharmacokinetic data of XueBiJing's phthalides are reported here for the first time. Based on this investigation and such investigations of the other component herbs, follow-up pharmacodynamic assessments of bioavailable herbal compounds are planned to elucidate XueBiJing's chemical basis responsible for its therapeutic action. Senkyunolides I and G, having the preceding disposition characteristics that could be detectably altered by septic pathophysiology, could serve as pharmacokinetic markers for sepsis care.

Pharmacokinetics and disposition of monoterpene glycosides derived from Paeonia lactiflora roots (Chishao) after intravenous dosing of antiseptic XueBiJing injection in human subjects and rats.

AIM: Monoterpene glycosides derived from Paeonia lactiflora roots (Chishao) are believed to be pharmacologically important for the antiseptic herbal injection XueBiJing. This study was designed to characterize the pharmacokinetics and disposition of monoterpene glycosides. METHODS: Systemic exposure to Chishao monoterpene glycosides was assessed in human subjects receiving an intravenous infusion and multiple infusions of XueBiJing injection, followed by assessment of the pharmacokinetics of the major circulating compounds. Supportive rat studies were also performed. Membrane permeability and plasma-protein binding were assessed in vitro. RESULTS: A total of 18 monoterpene glycosides were detected in XueBiJing injection (content levels, 0.001-2.47 mmol/L), and paeoniflorin accounted for 85.5% of the total dose of monoterpene glycosides detected. In human subjects, unchanged paeoniflorin exhibited considerable levels of systemic exposure with elimination half-lives of 1.2-1.3 h; no significant metabolite was detected. Oxypaeoniflorin and albiflorin exhibited low exposure levels, and the remaining minor monoterpene glycosides were negligible or undetected. Glomerular-filtration-based renal excretion was the major elimination pathway of paeoniflorin, which was poorly bound to plasma protein. In rats, the systemic exposure level of paeoniflorin increased proportionally as the dose was increased. Rat lung, heart, and liver exposure levels of paeoniflorin were lower than the plasma level, with the exception of the kidney level, which was 4.3-fold greater than the plasma level; brain penetration was limited by the poor membrane permeability. CONCLUSION: Due to its significant systemic exposure and appropriate pharmacokinetic profile, as well as previously reported antiseptic properties, paeoniflorin is a promising XueBiJing constituent of therapeutic importance.

Systemic and cerebral exposure to and pharmacokinetics of flavonols and terpene lactones after dosing standardized Ginkgo biloba leaf extracts to rats via different routes of administration.

BACKGROUND AND PURPOSE: Flavonols and terpene lactones are putatively responsible for the properties of Ginkgo biloba leaf extracts that relate to prevention and treatment of cardiovascular disease and cerebral insufficiency. Here, we characterized rat systemic and cerebral exposure to these ginkgo compounds after dosing, as well as the compounds' pharmacokinetics. EXPERIMENTAL APPROACH: Rats received single or multiple doses of ShuXueNing injection (prepared from GBE50 for intravenous administration) or GBE50 (a standardized extract of G. biloba leaves for oral administration). Brain delivery of the ginkgo compounds was assessed with microdialysis. Various rat samples were analysed using liquid chromatography/mass spectrometry. KEY RESULTS: Slow terminal elimination features of the flavonols counterbalanced the influence of poor oral bioavailability on their systemic exposure levels, which also resulted in significant accumulation of the compounds in plasma during the subchronic treatment with ShuXueNing injection and GBE50. Unlike the flavonols, the terpene lactones had poor enterohepatic circulation due to their rapid renal excretion and unknown metabolism. The flavonol glycosides occurred as major forms in plasma after dosing with ShuXueNing injection, while the flavonol aglycone conjugates were predominant in plasma after dosing with GBE50. Cerebral exposure was negligible for the flavonols and low for the terpene lactones. CONCLUSION AND IMPLICATIONS: Unlike the significant systemic exposure levels, the levels of cerebral exposure to the flavonols and terpene lactones are low. The elimination kinetic differences between the two classes of ginkgo compounds influence their relative systemic exposure levels. The information gained is relevant to linking ginkgo administration to the medicinal effects.