Pharmacokinetics and metabolism of CNS-targeted natural products

Considerable progress has been made to increase the success rate of bringing new therapeutics to the market by implementation of drug metabolism and pharmacokinetics (DMPK) screening strategies in early drug discovery. DMPK screenings help to select leads with good oral bioavailability, low clearance, optimal half-life, and a desirable metabolic profile. In previous studies with natural products, the flavonoids kaempferol and quercetin, and the alkaloid piperine have been characterized in vivo as central nervous system (CNS) acting compounds.To gain a better understanding of anxiolytic effects reported for the flavonoids, PK studies after oral and intravenous administrations in rats were conducted. UHPLC-MS/MS methods for quantification of the compounds of interest in rat plasma were developed and validated according to the principles of regulatory guidelines for industry to support PK studies. The validated methods were successfully applied to determine the concentration levels of the analytes in rat plasma, and PK parameters were calculated with the aid of the industry standard software WinNonlin. The findings suggest that poor oral bioavailability and extensive first-pass metabolism limit plasma exposure of kaempferol. Based on the results, it is more likely that the anxiolytic effect reported for this flavonoid is rather attributed to its metabolites.The major colonic metabolites of kaempferol and quercetin are 4-hydroxyphenylacetic acid (4-HPAA), 3-hydroxyphenylacetic acid (3-HPAA), and 3,4-dihydroxyphenylacetic acid (DOPAC). Moreover, anxiolytic activity has been reported for 4-HPAA and DOPAC. Thus, we aimed to obtain PK profiles of the metabolites upon intravenous application. It has been found that the metabolites were rapidly eliminated with a half-life of 20-30 min, so that effective concentrations in the brain do not appear to be reached. During a screening of natural products for γ-aminobutyric acid type A (GABAA) receptor activity, piperine was characterized as a positive allosteric modulator targeting a benzodiazepine-independent binding site. Due to pharmacological promiscuity of piperine, its structure was systematically modified, and a library of piperine analogs was prepared. The most potent and efficacious analogs were identified from in vitro and in vivo studies. The information on metabolically labile sites of the selected analogs was needed to guide further lead optimization process. Thus, the objective of the second part of the PhD thesis was to investigate metabolism of the selected analogs. Metabolic stability of compounds was tested in the presence of pooled human liver microsomes. Intrinsic clearance was calculated using the substrate depletion approach. Metabolites were analyzed by UHPLC-Q-TOF-MS, and with the aid of metabolite identification software Mass-MetaSite. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. CYP450 reaction phenotyping studies were carried out with Silensomes™. Microsomal stability assays revealed piperine as the metabolically most stable compound, whereas its analogs demonstrated high metabolic liability. The principal routes of oxidative metabolism were found to be aliphatic hydroxylation, and N- and O-dealkylation. It appeared that piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Moreover, extensive binding to blood constituents was observed for all compounds. Our findings showed that analogs were rapidly metabolized and demonstrated strong binding to blood constituents due to increased lipophilicity. The next cycle of medicinal chemistry optimizations should, therefore, be focused on reducing lipophilicity to lower metabolic liability and extensive binding of analogs

4-Hydroxyphenylacetic Acid Prevents Acute APAP-Induced Liver Injury by Increasing Phase II and Antioxidant Enzymes in Mice

Acetaminophen (APAP) overdose is the principal cause of drug-induced acute liver failure. 4-hydroxyphenylacetic acid (4-HPA), a major microbiota-derived metabolite of polyphenols, is involved in the antioxidative action. This study seeks to investigate the ability of 4-HPA to protect against APAP-induced hepatotoxicity, as well as the putative mechanisms involved. Mice were treated with 4-HPA (6, 12, or 25 mg/kg) for 3 days, 1 h after the last administration of 4-HPA, a single dose of APAP was intraperitoneally infused for mice. APAP caused a remarkable increase of oxidative stress markers, peroxynitrite formation, and fewer activated phase II enzymes. 4-HPA increased Nrf2 translocation to the nucleus and enhanced the activity of phase II and antioxidant enzymes, and could thereby ameliorate APAP-induced liver injury. Studies reveal that 4-HPA, as an active area of bioactive dietary constituents, could protect the liver against APAP-induced injury, implying that 4-HPA could be a new promising strategy and natural hepatoprotective drug.This work was supported by National Key Research and Development Program of China (Grant No. 2017YFD0501400), SCO Regional collaborative innovation project (Grant No. 2016E03012), Xinjiang; the Key Construction Program of International Cooperation Base in S&T, Shaanxi Province (Grant No. 2015SD0018), Program for Science & Technology Innovation Talents in Universities of Henan Province (Grant No. 18HASTIT035], China strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P., by the Ministerio da Ciencia, Tecnologia e Ensino Superior (MCTES) and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), Portugal

АППРОКСИМАЦИЯ ЛУЗИНА ФУНКЦИЙ ИЗ КЛАССОВ СОБОЛЕВА НА УЛЬТРАМЕТРИЧЕСКИХ ПРОСТРАНСТВАХ С УСЛОВИЕМ УДВОЕНИЯ

In this article, we consider an analog of the Luzin theorem on the correction for Sobolev-type spaces on ultrametric spaces with a doubling condition. The correcting function belongs to the Hölder class and approximates a given function in the metrics of the initial space. Dimensions of exceptional sets are evaluated in terms of capacities and Hausdorff volumes. This result was previously obtained for the special case of the p-adic vector space.В работе рассматривается аналог теоремы Лузина об исправлении для пространств соболевского типа на ультраметрических пространствах с условием удвоения. Исправляющая функция принадлежит классу Гельдера и приближает заданную функцию в метрике исходного пространства. Размеры исключительных множеств оцениваются в терминах емкостей и вместимостей Хаусдорфа. Этот результат был ранее получен для частного случая пространства p-адических векторов

Anadromy and residency in steelhead and rainbow trout (Oncorhynchus mykiss): a review of the processes and patterns

Oncorhynchus mykiss form partially migratory populations with anadromous fish that undergo marine migrations and residents that complete their life cycle in fresh water. Many populations’ anadromous components are threatened or endangered, prompting interest in understanding ecological and evolutionary processes underlying anadromy and residency. In this paper, we synthesize information to better understand genetic and environmental influences on O. mykiss life histories, identify critical knowledge gaps, and suggest next steps. Anadromy and residency appear to reflect interactions among genetics, individual condition, and environmental influences. First, an increasing body of literature suggests that anadromous and resident individuals differ in the expression of genes related to growth, smoltification, and metabolism. Second, the literature supports the conditional strategy theory, where individuals adopt a life history pattern based on their conditional status relative to genetic thresholds along with ultimate effects of size and age at maturation and iteroparity. However, except for a generally positive association between residency and high lipid content plus a large attainable size in fresh water, the effects of body size and growth are inconsistent. Thus, individuals can exhibit plasticity in variable environments. Finally, patterns in anadromy and residency among and within populations suggested a wide range of possible environmental influences at different life stages, from freshwater temperature to marine survival. Although we document a number of interesting correlations, direct tests of mechanisms are scarce and little data exist on the extent of residency and anadromy. Consequently, we identified as many data gaps as conclusions, leaving ample room for future research

Silymarin secretion and its elicitation by methyl jasmonate in cell cultures of Silybum marianum is mediated by phospholipase D-phosphatidic acid

The flavonolignan silymarin is released to the extracellular medium of Silybum marianum cultures and its production can be stimulated by the elicitor methyljasmonate (MeJA). The sequence of the signalling processes leading to this response is unknown at present. It is reported in this work that MeJA increased the activity of the enzyme phospholipase D (PLD). Treatment with mastoparan (Mst), a PLD activity stimulator, also enhanced PLD and caused a substantial increase in silymarin production. The application of the product of PLD activity, phosphatidic acid (PA) promoted silymarin accumulation. Altering PLD activity by introducing in cultures n-butanol (nBuOH), which inhibits PA production by PLD, prevented silymarin elicitation by MeJA or Mst and also impeded its release in non-elicited cultures. Treatment with iso-, sec- or tert- butanol had no effect on silymarin production. The exogenous addition of PA reversed the inhibitory action of nBuOH, both in control and MeJA-treated cultures. These results suggest that the enzyme PLD and its product PA mediate silymarin secretion to the medium of S. marianum cultures

RARE DISEASES OF THE BLOOD SYSTEM: A CLINICAL CASE OF VIRUS-ASSOCIATED BONE MARROW APLASIA IN A PATIENT WITH A HEREDITARY MEMBRANOPATHY (REVIEW)

The aim of study was to research and present literature data regarding the investigations of some combined types of anemia in pregnancy and to describe the clinical case of pregnancy associated with primary parvovirus B19 infection and newly diagnosed hereditary erythrocyte membrane disorder. The contemporary information about the etiology, the prevalence, structure and some features of pathogenesis of parvovirus infection based on literature data was given. The huge prevalence of this infection (according to data of different sources the serum level of parvovirus B19 specific antibodies may be detect in 90 % of population), its threat for fetus and risk of severe complications in patients with immunodeficiency, as well as possibility of development of total depression of hematopoiesis in patients with hereditary erythrocyte membrane disorders, such as Minkowsky-Chauffard disease, determine the relevance and significance of the discussed topic. Furthermore, this clinical case, which illustrates the features of primary parvovirus B19 infection in patient with hereditary hemolytic anemia and the difficulties of differential diagnosis, demonstrates the importance of parvovirus infection markers detection, which is not available in the most TORCH-panel

Single dose pharmacokinetics of intravenous 3,4-dihydroxyphenylacetic acid and 3-hydroxyphenylacetic acid in rats

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (3-HPAA) are intestinal metabolites of the dietary flavonoid quercetin. DOPAC reportedly showed anxiolytic activity after i.p. administration in rats. The fate of these metabolites after consumption, and the pharmacological properties of 3-HPAA in the body are largely unknown. The aim of the current study was to characterize pharmacokinetic properties of DOPAC and 3-HPAA after intravenous bolus application in rats. UHPLC-MS/MS methods for quantification of DOPAC and 3-HPAA levels in lithium heparin Sprague Dawley rat plasma were developed and validated according to international regulatory guidelines. Non-compartmental and compartmental analyses were performed. Pharmacokinetic profiles of DOPAC and 3-HPAA followed a two-compartment body model, with a fast distribution into peripheral tissues (half-lives of 3.27–5.26 min) and rapid elimination from the body (half-lives of 18.4–33.3 min)

Pharmacokinetics and metabolism of dietary kaempferol and its metabolite 4-hydroxy-phenylacetic acid in rats

Scope Kaempferol is a major flavonoid in the human diet and in medicinal plants. The compound exerts anxiolytic activity when administered orally in mice, while no behavioural changes were observed upon intraperitoneal administration, or upon oral administration in gut sterilized animals. 4-Hydroxyphenylacetic acid (4-HPAA), which possesses anxiolytic effects when administered intraperitoneally, is a major intestinal metabolite of kaempferol. Pharmacokinetic properties of the compounds are currently not clear. Methods and results UHPLC-MS/MS methods were validated to support pharmacokinetic studies of kaempferol and 4-HPAA in rats. Non-compartmental and compartmental analyses were performed. After intravenous administration, kaempferol followed a one-compartment model, with a rapid clearance (4.40–6.44 l/h/kg) and an extremely short half-life of 2.93–3.79 min. After oral gavage it was not possible to obtain full plasma concentration–time profiles of kaempferol. Pharmacokinetics of 4-HPAA was characterized by a two-compartment model, consisting of a quick distribution phase (half-life 3.04–6.20 min) followed by a fast elimination phase (half-life 19.3–21.1 min). Conclusion Plasma exposure of kaempferol is limited by poor oral bioavailability and extensive metabolism. Both compounds are rapidly eliminated, so that effective concentrations at the site of action do not appear to be reached. At present, it is not clear how the anxiolytic-like effects reported for the compounds can be explained

Validation of UHPLC-MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to in vitro BBB and intestinal drug permeability studies

Sedative and anxiolytic-like properties of flavonoids such as kaempferol and quercetin, and of some of their intestinal metabolites, have been demonstrated in pharmacological studies. However, routes of administration were shown to be critical for observing in vivo activity. Therefore, the ability to cross intestinal and blood-brain barriers was assessed in cell-based models for kaempferol (KMF), and for the major intestinal metabolite of KMF, 4-hydroxyphenylacetic acid (4-HPAA). Intestinal transport studies were performed with Caco-2 cells, and blood-brain barrier transport studies with an immortalized monoculture human model and a primary triple-co-culture rat model. UHPLC–MS/MS methods for KMF and 4-HPAA in Ringer-HEPES buffer and in Hank’s balanced salt solution were validated according to industry guidelines. For all methods, calibration curves were fitted by least-squares quadratic regression with 1/X2 as weighing factor, and mean coefficients of determination (R2) were >0.99. Data obtained with all barrier models showed high intestinal and blood-brain barrier permeation of KMF, and no permeability of 4-HPAA, when compared to barrier integrity markers