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Book Subtitle : In Vitro Methods. Editors : Gary W. Series Title : Methods in Pharmacology and Toxicology. Publisher : Humana Totowa, NJ. Hardcover ISBN : Softcover ISBN : Series ISSN : Edition Number : 2. Topics : Pharmacology. Skip to main content. Search SpringerLink Search. Editors: view affiliations Gary W. Provides updated variations on assays as well as numerous new protocols for drug discovery optimization Features readily reproducible methods and protocols that are lab-tested and successful Includes expert tips and key implementation advice.

Buying options eBook EUR Softcover Book EUR Hardcover Book EUR Learn about institutional subscriptions. Table of contents 34 protocols Search within book Search. Page 1 Navigate to page number of 2. Front Matter Pages i-xvii. Caldwell, Becki Hasting, John A. Masucci, Zhengyin Yan Pages Mark Kao Pages Induction is the increase in intestinal and hepatic enzyme activity as a result of increased mrna transcription leading to protein levels higher than normal physiologic values Amacher, When this happens, there is a corresponding increase in the rate of drug metabolism affecting both the oral bioavailability and the systemic disposition Argikar et al.

In the formulation and dosage design of oral medications, allowance is often made for pre-systemic metabolism in order to achieve predictable systemic bioavailability. A disruption in this balance can result in significant changes in blood concentrations of the drugs. As shown in subsequent sections, some herbal products have been shown to be capable of inducing CYP.

Concomitant administration of enzyme-inducing herbal products and Apart from enzyme induction, herbal products can also inhibit enzyme activities. The inhibition of CYP and other metabolic enzymes is usually competitive with instantaneous and inhibitor concentration-dependent effects Zhang and Wong, Most inhibitors are also substrates of CYP Zhou, This phenomenon alters pharmacokinetic profiles of xenobiotics significantly.

As a result of the suppression of the anticipated pre-systemic intestinal and hepatic metabolism, unusually high plasma levels of xenobiotics are observed. Toxic manifestation could be the ultimate effect of this observation. An equally clinically important consequence of enzyme inhibition is drug accumulation due to subdued hepatic clearance. These effects will be of particular concern in drugs with narrow therapeutic window or steep dose-response profiles.

Studies from case reports indicate that, due to its inducing effects on CYP3A4, it significantly reduces the plasma levels of CYP3A4 substrates including cyclosporine, simvastatin, indinavir, warfarin, amitriptyline, tacrolimus, oxycodone and nevirapine Johne et al.

It has been reported that the alteration in the blood serum concentration of cyclosporine due to SJW has led to organ rejection in patients Ernst, ; Murakami et al. Reports of breakthrough bleeding and unplanned pregnancies due to interaction between SJW and oral contraceptives have been documented Hu et al. The group of drugs with a high potential for clinically significant pharmacokinetic drug interaction with SJW is the antidepressants as SJW itself is consumed by patients with depression.

Its concomitant use with SSRIs like sertraline and paroxetine has been reported to result in symptoms of central serotonergic syndrome Barbenel et al. The risk of therapeutic failure is thus high due to induction of CYP3A4- dependent metabolism activities resulting in poor oral bioavailability.

It also inhibits the production of SN, an active metabolite of irinotecan, in cancer patients Caraci et al. Other CYP and P-gp substrates whose pharmacokinetic profile have been altered by SJW include anticoagulants like phenprocoumon and warfarin; antihistamines like fexofenadine; antiretroviral drugs including protease inhibitors and reverse transcriptase inhibitors; hypoglycemic agents such as tolbutamide; immunosuppressants like cyclosporine, tacrolimus and mycophenolic acid; anticonvulsants such as carbamazepine; anticancer like irinotecan; bronchodilators like theophylline; antitussives like dextromethorphan; cardiovascular drugs like statins, digoxin and dihydropyridine calcium channel blockers; oral contraceptives; opiates like methadone and loperamide; and benzodiazepines including alprazolam and midazolam Greeson et al.

Compared with in vitro inductive concentrations, in vivo extrapolation suggests a high possibility of in vivo pharmacokinetic drug interaction Agrosi et al. Dresser and co-workers demonstrated that SJW is capable of inducing CYP3A4 in healthy subjects through the observation of increased clearance of midazolam. Thus animal and human studies further confirm SJW as containing both inhibitory and inducing constituents on various CYP isozymes.

These effects depend on dosage and duration of administration, and are species- and tissue-specific. While the individual phytochemical constituents of SJW have elicited varying effects on the metabolic activity of the CYP isozymes, whole extracts and major constituents especially hyperforin have been reported to induce the metabolic activities of CYP1A2, 2C9, 2C19, 2D6 and 3A4 as shown in in vitro studies and in vivo studies Lee et al.

Ginkgo biloba have been reported to induce CYP 2Cdependent omeprazole metabolism in healthy human subjects Yin et al. Its effects on the pharmacokinetics of warfarin have been reported in animal models Taki et al. A related CYP inhibitor is rotenone. By interfering with the electron transfer of the heme iron, rotenone, a naturally occurring phytochemical found in several plants such as the jicama vine plant is known to inhibit CYP activity Sanderson et al. Resveratrol, a natural polymer, and tryptophan, an amino acid have been documented as potent CYP inhibitors Rannug et al.

Some herbal medications and their phytochemical constituents capable of interacting with CYP are presented in Table 3. While cytochrome Pmediated HDI have been extensively investigated in various studies, the effects of herbal extracts on phase II enzymes have not been adequately studied. However, there is sufficient evidence to suggest the potential of phase II enzymes to induce clinically significant HDI.

In a study carried out in rat models by Sheweita and co-workers , extracts of hypoglycemic herbs, Cymbopogon proximus, Zygophylum coccineum and Lipinus albus reduced the activity of GST and GSH. Curcumin, from Curcuma longa, an herbal antioxidant with anti-inflammatory and antitumor properties increased the activity of GST and quinone reductase in the ddy mice liver Iqbal et al. Kampo, a traditional Japanese medicine made of a mixture of several medicinal herbs has shown inhibitory effects on some phase II enzymes.

This result agrees with Katoh et al. Apart from the well-known effects of Ginkgo biloba on CYP enzymes as illustrated earlier section , its extracts have demonstrated potent inhibition of mycophenolic acid glucuronidation investigated in human liver and intestinal microsomes Mohamed and Frye, In a study to investigate the influence of 18 herbal remedies on the activity of human recombinant sulfotransferase 1A3 employing dopamine and ritodrine as substrates, extracts of grape seed, milk thistle, gymnema, SJW, ginkgo leaf, banaba, rafuma and peanut seed coat showed potent inhibition with IC 50 values lower than putative gastrointestinal concentrations Nagai et al.

While the clinical significance of these findings is yet to be determined, it is noteworthy that phase II metabolic enzymes may play significant roles in HDIs Inhibition and induction of transport and efflux proteins The ATP-binding cassette ABC family of drug transporters plays significant roles in the absorption, distribution and elimination of drugs. It is constitutively expressed in a number of body tissues and concentrated on the apical epithelial surfaces of the bile canaliculi of the liver, the proximal tubules of the kidneys, the pancreatic ductal cells, the columnar mucosal cells of the small intestine, colon and the adrenal glands Marzolini et al.

It is actively involved in drug absorption, distribution and elimination from the intestines the liver, kidneys and the brain. Thus, the modulation of P-gp or competitive affinity as substrates for its binding sites by co-administered herbs presents a potential for alteration in the pharmacokinetic profile of the drug. Pharmacokinetic interaction occurs when herbal drugs inhibit or decrease the normal activity level of drug transporters through a competitive or non-competitive mechanism.

Interactions can also occur through the induction of transport proteins via the increase of the mrna of the relevant protein. Studies have identified a number of clinically important P-gp inhibitors including phytochemicals flavonoids, furanocoumarins, reserpine, quinidine, yohimbine, vincristine, vinblastine among others Krishna and Mayer, ; Zhou et al.

Borrel and co-workers reported that mobile ionophores such as valinomycin, nonactin, nigericin, monencin, calcimycin, and lasalocid inhibit the efflux of anthracycline by P-gp whereas channel-forming ionophores such as gramicidin do not Larsen et al. A number of herbal products which interact with CYP also have similar effects on transport proteins Table 3. The transport proteins are actively involved in the pharmacokinetics of The influence of some herbs on transport proteins with respect to anti-cancer drugs is presented in Table 4.

Clinically relevant interactions between herbal medicine and chemotherapeutic agents are detailed in a recent review by Yap and co-workers Changes in the gastrointestinal ph and other biochemical factors Complexation and chelation, leading to the formation of insoluble complexes and competition at the sites of absorption especially with site-specific formulations can greatly affect the absorption of medicines.

Anthranoid-containing plants cassia Cassia senna , Cascara Rhamnus purshiana , rhubarb Rheum officinale and soluble fibres including guar gum and psyllium can decrease drug absorption by decreasing GI transit time. They are known to increase GIT motility. During concomitant use with prescribed medication, significant alteration in the absorption of the latter has been reported due to decreased GI transit time Fugh-Berman, This significantly increased intestinal transit due to the alteration in the intestinal water and salt absorption and the subsequent fluid accumulation.

In a study conducted by Munday and Munday , a garlic-derived compound was shown to increase the tissue activities of quinone reductase and glutathione transferase in the gastrointestinal tract of the rat. In view of their roles in metabolism, both enzymes are considered chemoprotective especially from chemical carcinogens. In addition to CYP and P-gp mediated mechanisms, ginseng may cause pharmacokinetic HDI through its inhibitory effects on gastric secretion Suzuki et al.

The potential of rhein and danthron to increase the absorption of furosemide, a poorly water-soluble drug, has been demonstrated through in vitro studies Laitinen et al. In a study carried out in mice, a Chinese herbal plant, Polygonum paleaceum, showed the potential to depress the motility of the gastrointestinal tract, inhibit defecation reflex and delay gastric emptying Zhang, A similar study demonstrated the inhibitory effects of two Chinese traditional herbal prescriptions, Fructus aurantii immaturus and Radix paeoniae alba on gastrointestinal movement Fang et al.

The absorption of drugs such as phenoxymethylpenicillin, metformin, glibenclamide and lovastatin may be reduced by high-fiber herbal products through the sequestration of bile acids Colalto, Mochiki and co-workers reported the ability of kampo, a traditional Japanese medicine, to stimulate elevated intestinal blood flow, and to induce increased secretion of gastrointestinal hormones including motilin, vasoactive intestinal peptide, and calcitonin gene-related peptide.

Similarly, another traditional Japanese medicine has been shown to increase the intestinal secretion of ghrelin, a hunger-related hormone, leading to delayed gastric emptying Tokita et al. Also, Qi and co-workers demonstrated the capability of Da-Cheng-Qi-Tang, a traditional Chinese herbal formula, to increase plasma These effects have the potential of reducing the intestinal transit time of concurrently administered drug, with the risk of reduced absorption Alteration in renal elimination This involves herbal products capable of interacting with renal function, leading to altered renal elimination of drugs.

Such interaction can result from the inhibition of tubular secretion, tubular reabsorption or interference with glomerular filtration Isnardd et al. In addition to this group of herbal products are those products consumed as diuretics. The mechanism of herbal diuresis is complex and non-uniform. Certain herbs increase the glomerular filtration rate but do not stimulate electrolyte secretion while some others act as direct tubular irritants Crosby et al.

Some herbs capable of interacting with renal function and drug elimination are presented in Table 5. The resulting accumulation of cortisol in the kidney stimulates the aldosterone receptors in cells of the cortical leading to increased BP, sodium retention and hypokalaemia.

This may potentiate the action of drugs such as digoxin 4 1 Martinez et al. This can precipitate pharmacodynamic toxicity or antagonistic effects Table 6. Like most other herbs, SJW contains complex mixture of phytochemicals including phenylpropanes, naphthodanthrones, acylphloglucinols, flavonoids, flavanol glycosides and biflavones. Hyperforin is known to inhibit the reuptake of neurotransmitters dopamine, serotonin, and noradrenalin and is believed to be the main bioactive moiety responsible for the antidepressant activity of SJW.

Conclusion Concomitant use of herbs and conventional drugs may present with untoward events. Available evidence indicates various mechanisms through which this can occur. By interacting with conventional medication, herbal remedies may precipitate manifestations of toxicity or in the other extreme, therapeutic failure. A good knowledge of the potential of commonly consumed herbal medicines to interact with prescription medicines, irrespective of the nature of evidence available, will equip health professionals in their practice.

Apart from those demonstrated in significant numbers in human subjects, not all reported HDIs are clinically significant. As such, more clinically relevant research in this area is necessary. The enzymes contained in the cytochrome P superfamily present predominantly in the liver have been identified as the single most important catalyst of drug metabolism and have formed the bedrock of most matured technologies for in vitro drug biotransformation studies. With the development of a number of liver-based technologies, in vitro metabolism has gained significant popularity in the past three decades.

This has come in response to several demanding factors including the waning relevance of data from animal studies; the high cost and stringent regulatory and ethical requirements, as well as safety issues involved with studies using human subjects; and the need for high throughput due to the wide range of chemical entities for routine tests. In vitro liver-based metabolic technologies have found ready application in generating the desired information on the substrate and inhibitor specificity of most metabolic enzymes.

The technologies available vary from whole liver to subcellular fractions. These include isolated fresh liver; liver slices; primary, cultured and cryopreserved hepatocytes; microsomes; cytosolic fractions; and purified or heterologously expressed drug-metabolizing enzymes.

The choice of a particular method depends on the metabolic questions to be answered. A common advantage to all the in vitro technologies is the reduced complexity of the study system. In this review, the general principles of in vitro enzyme kinetics are discussed. The review also discusses the major features, advantages and disadvantages of the various liver-based in vitro methodologies for drug metabolism study. Liver-based in vitro technologies for drug biotransformation studies - a review.

Curr Drug Metab Feb;13 2 :. Introduction Drug biotransformation study is an integral part of preclinical screening for new drug candidates Pelkonen and Raunio, This assessment usually involves both in vitro and in vivo models in animal species where the main pharmacokinetic, pharmacodynamic and toxicological profiles are investigated Singh Biotransformation involves the physiologic conversion of nonpolar, lipophilic and pharmacologically active drugs into polar, hydrophilic, inactive or nontoxic molecules.

In certain instances as with prodrugs like tamoxifen, enalapril, levodopa among others, products of biotransformation can be bioactive, and further routes of disposition are responsible for their clearance. The liver is responsible for the biotransformation of the majority of drugs. Other organs including the kidneys, skin, lungs and intestine contribute significantly to the biotransformation of xenobiotics Krishna and Klotz, ; De Kanter et al.

The biotransformation pathway of a drug could be mediated by any or combination of phase I hydrolysis, oxidation and reduction , phase II conjugations and phase III further modification, efflux transport, excretion reactions Xu et al. Phase I biotransformation is mediated predominantly by the cytochrome P CYP enzyme superfamily present in the liver, intestines, kidney, lung and other organs.

This superfamily of hemoproteins is expressed throughout the phylogenic spectrum and is responsible for catalytic metabolism of a wide range of endogenous and foreign chemicals. Initially thought to be hepatic, CYP has been found to be significantly expressed in other body organs including the intestines, spleen and the kidneys Ingelman-Sundberg, Phase II enzymes including uridine diphosphoglucuronosyl transferase UGT , N-acetyl transferase NAT , glutathione S-transferase GST and sulfotransferase ST mediate the attachment of polar and ionizable groups to phase I metabolites aiding their elimination while drug transporters phase III exert their influence on the absorption, distribution and elimination of drugs Petzinger and Geyer, The use of human subjects for comprehensive clinical pharmacology studies seems to be ideal.

However, the risks involved, and the stringent regulatory demands make this practically challenging with attendant high cost and long time duration. Results from animal studies pose challenges regarding their clinical significance. Current guidelines for drug development encourage the use of in vitro systems for qualitative and supportive assessments. In general, in vivo studies are deemed necessary only after positive in vitro results. The European Centre for the Validation of Alternative Methods ECVAM promotes the scientific and regulatory acceptance of alternative methods which reduce, refine or replace the use of laboratory animals Eric et al.

Such models which range from whole cell systems intact perfused liver, human hepatocytes cultures, hepatic and transfected cell lines to enzyme preparations liver microsomes and isolated enzymes cultures are now increasingly applied for quantitative and qualitative assessment in preclinical drug development, post-approval routine checks, identification of metabolic determinant factors and prediction of drug-drug, herb-drug and food-drug interactions. Apart from helping researchers and drug discovery scientists extract important information on chemical and metabolic characteristics from early screening in the drug discovery phase, results from in vitro investigations are relevant determinants for clinical trials of new drugs.

Results have been utilized for physiology-based pharmacokinetic modeling Baneyx et al. In addition, they help to predict drug-drug interactions at metabolic levels. One of the most important features of in vitro drug biotransformation research is how to make reliable extrapolations relevant to clinical practice Wrighton et al.

This has been achieved with a number of the liver-based technologies. Each in vitro model presents with its own peculiar advantages and limitations. The choice of method however is determined mainly by the goal of evaluation. Factors to be considered in the choice of an in vitro model include in vivo resemblance, ethical considerations, cost and availability.

The aim of the current review is to provide an update on the various in vitro liver-based models currently utilized in drug biotransformation studies. The major features of each in vitro technology are highlighted Principles of in vitro metabolism Depending on the throughput demand, biotransformation assays in most in vitro models can be conducted manually or robotically.

Some of the in vitro models require exogenous supply of cofactors for activity. These include primary hepatocytes, cell lines, liver slices and The choice of incubation conditions is believed to have significant influence on the outcome of in vitro metabolism Venkatakrishnan et al.

The in vivo physicochemical environment can be mimicked by incubating at ph values similar to the in vivo conditions. For insoluble test compounds and substrates, care should be taken in the choice of lipophilic solvents. Studies have demonstrated that beyond certain concentrations, organic solvents such as ethanol, dimethyl sulfoxide, isopropanol, methanol, acetonitrile, dimethyl formamide, acetone and polyethylene glycol can greatly interfere with the metabolic activities of in vitro enzymes Tang et al.

Thus solvent effects are often part of the optimization process for in vitro metabolism. The metabolic activities of in vitro models are assessed through the rate of metabolite formation or substrate depletion Jones and Houston, Most single-substrate enzymatic reactions follow the Michaelis-Menten principle. The initial rate of enzymatic reactions is usually linearly proportional to the substrate concentration.

The initial reaction rate, V 0 depends on the position of the substrate-binding equilibrium and the rate of metabolite formation Kou et al. Beyond the substrate concentration where reaction rate is maximal V max , there is loss of linearity between the rate of metabolite formation and substrate concentration [S]. The substrate concentration corresponding to half the V max is referred to as the Michaelis-Menten constant K m Hsu et al.

The Michaelis-Menten equation as expressed in Equation 3. The rate of product formation depends on both the enzyme and substrate concentrations reaching its peak at equilibrium where the saturation of the enzyme binding site yields the highest rate of product formation. A typical Michaelis-Menten curve is therefore non-linear and various attempts have been made to modify it in a way that enzyme kinetic parameters can be estimated at a linear glance.

One of the most common linearized modifications to the Michaelis-Menten curve is the Hanes-Woolf Plot. It is based on the rearrangement of Michaelis-Menten equation as depicted in Equation 3. Equation 3. The Hanes-Woolf Plot has been used for rapid and easy determination of major kinetic parameters such as the K m and the V max. When 2 or more CYP isoforms with distinct affinities are involved in catalyzing a given metabolic pathway, a biphasic model is used as expressed in equation 3. Softwares are now available for easier and more accurate determination of enzyme kinetic parameters.

IC 50 and K i values are then determined. It is calculated as shown in equation 3. Non-linear regression equations can also be generated by profiling the inhibitor concentration against observed inhibition. Such equation can be used to determine the IC K i is a measure of the affinity of the compound to the enzyme determined graphically. The values of IC 50 and K i are determined by taking the concentration of marker substrates around their corresponding K m values and about fold of their corresponding K m values respectively.

Enzyme inhibition can also be expressed as percentage of control Wang et al. The use of in vitro technologies in drug biotransformation studies has thus evolved with standard procedures. However, only animal liver in small scale has been employed. The model usually involves the perfusion of isolated liver with Krebs-Henseleit buffer as perfusate Lafranconi and Huxtable ; Jia and Liu, The three-dimensional cytoarchitecture, the presence of non-hepatic cells and transporters make this model the closest in similarity to the in vivo environment.

The presence of functional bile canaliculi also makes this model suitable for bile collection and analysis Groneberg et al. However this model is difficult to handle, has very short viable period of three hours due to the poor perfusion of the cells by oxygen and nutrients, poor reproducibility and low throughput. In addition, human liver is difficult to source Brandon et al. First developed in s by Otto Heinrich Warburg, its use and reliability have further been improved by the development of high-precision tissue slicers De Kanter et al.

Unlike in perfused liver, the preparation of liver slices does not require harmful proteases Seglen, This model offer intact cellular interaction, normal spatial arrangement and the possibility of morphological studies. Like the perfused liver, it has functional drug metabolizing enzymes, transporters and bile canaliculi. It has reasonably higher throughput than perfused liver. In addition, viable cell enrichment is not possible with liver slices, the liver slices are not inducible by CYP inducers, biles collection for analysis may not be possible as in perfused liver and the presence of necrotic cells may affect drug transport studies Olinga et al.

In addition to their application in drug biotransformation and toxicity studies, they are important tools for studying drug efficacy, hepatic proliferation and as supportive tools in other bioartificial studies Wang et al. Primary hepatocytes have strong resemblance of in vivo situation with metabolic results closely correlated to in vivo yields.

They reflect the heterogeneity of CYP-expression in human liver and are thus popularly employed for in vitro drug biotransformation studies Hewitt et al. Different methods have been developed to obtain hepatocytes for drug biotransformation Guguen- Guillou et al.

An important feature of primary hepatocytes is the preservation of drug metabolizing enzymes at in vivo levels. Further advantage is the relative ease to use and reasonably higher throughput Wang et al. However, lack of cell polarity, cell-cell and cell-matrix contacts limits the in vivo resemblance of isolated hepatocytes in drug biotransformation research. Isolated hepatocytes also have limited viability hrs Bi et al. This offers the advantage of longer viability compared to primary hepatocytes Guillouzo et al.

Cultured hepatocytes gradually lose viability and liver-specific functions including decreased CYP expression with time Luttringer et al. Another major challenge with the use of hepatocyte is the dependence of its availability on the handiness of fresh liver tissue. This makes it impossible to perform studies on demand, or to repeat studies with hepatocytes from same donor. To solve these challenges, cryopreservation procedures have been developed for hepatocytes, retaining the activity of most phase I and phase II enzymes Zaleski et al.

This has made the commercial availability of human hepatocytes possible and accessibility independent of fresh liver tissue availability. The hepatocytes preserved in this form are a potential tool for chronic toxicity and drug-drug interaction studies Hewitt et al. Hepatocyte cultures may require special supplements in media and, depending on the technology used to preserve the tissue function, cultured hepatocytes may maintain differentiated function and possess a functional bile canaliculi LeCluyse et al.

One of the major challenges with the use of human hepatocytes is the considerable inter-individual variation carried over from the source of the liver. This can be overcome by using mixtures of heptocytes from multiple donors Hewitt et al.

Human hepatocytes do not reflect completely the in vivo environment as non-hepatic cells like Kupffer cells which may be necessary for cofactor supply are absent. Although hepatocytes often give better in vitro in vivo correlations than cell lines and subcellular fractions, perfused cell lines and liver slices are considered better in this regard Boess et al.

Their cellular characteristics are de-differentiated and they lack complete expression of all families of metabolic enzymes Hoekstra and Chamuleau, ; Strick-Marchand and Weiss, They can be obtained as isolated cells from primary tumors of the liver parenchyma seen after chronic hepatitis or cirrhosis Knasmuller et al.

Each of these cell lines The most widely used and best characterized liver cell line is the hepatoma cell line Hep G2 Brandon et al. The metabolic activity of liver cell lines is generally low compared to freshly isolated human hepatocytes Khetani and Bhatia, Liver cell lines require culture medium, the composition of which also greatly influences the metabolic activity Xu et al.

The main advantages of liver cell lines compared to other in vitro models are their ease to culture and their possession of relatively stable enzyme concentration Khalil et al. On the other hand, the absence or low expression of most phase I and phase II enzymes is a major disadvantage.

Metabolites are not easily detected and the activities of individual CYP and other enzymes are difficult to investigate. These factors limit the application of liver cell lines in drug biotransformation studies Mersch-Sundermann et al. They contain both microsomal and cytosolic fractions expressing a wide range of metabolic enzymes CYP, flavinmonooxygenases, carboxylesterases, epoxide hydrolase, UDP-glucuronosyl transferases, sulfotransferases, methyltransferases, acetyltransferases, glutathione S-transferases, among others.

They have been widely employed for metabolic, toxicity and mutagenicity studies mainly in combination with the Ames test Hakura et al. S9 fractions offer a more complete representation of the metabolic profile compared to microsomes and cytosol, as they contain both phase I and phase II activity. The major disadvantage is the lower enzyme activity in the S9 fraction compared to microsomes or cytosol which may leave some metabolites unnoticed Hakura et al.

It expresses the soluble phase II enzymes like N-acetyl transferase, glutathione S-transferase, sulfotransferase, carboxylesterases, soluble epoxide hydrolases, diamine oxidases, xanthine oxidases and alcohol dehydrogenase. Human liver cytosolic fractions are used often in drug biotransformation research for enzyme-specific studies Maser et al. Human liver cytosol expressing the specific NAT isozymes in cytosol without the other soluble phase II enzymes is commercially available.

The most important advantage offered by this model is the presence of only few cystolic enzymes at concentrations higher than those in the liver S9 fraction. This limited enzyme expression also account for the main disadvantage, as other enzymes especially the UGT, which is located on the endoplasmic reticulum, are absent and cannot be investigated via this model Li et al.

Microsomes from the liver and small intestines are employed to investigate the potential for pre-systemic metabolism following oral drug administration while those from small intestine, kidney are often utilized to evaluate extrahepatic metabolism Bu, Liver microsomes consist of vesicles of the hepatocyte endoplasmic reticulum prepared by differential centrifugation, and can be collected from liver preparations including fresh human liver, liver slices, liver cell lines and primary hepatocytes Asha and Vidyavathi, They are used for the evaluation of phase I enzymes because of the rich expression of CYP, flavin monoxygenases, carboxyl esterases, epoxides, hydrolase and UDP glucuronyl transferases Newton et al.

The CYP retain their activity for many years in microsomes at low The reaction is initiated by the addition of NRS, incubation at 30 C in a shaking water bath or shaking incubator. The metabolic reaction is terminated by the addition of a stop solution comprising ice-cold acetonitrile, trichloroacetic acid, ice-cold carbonate buffer, sodium hydroxide or hydrochloric acid, in order to precipitate the microsomal protein.

The reaction mixture is then centrifuged and analyzed Asha and Vidyavathi, Exogenous cofactors are required for in vitro microsomal activity, the choice of which depends on the desired enzyme activity. Alternatively, NADPH may be added directly to the incubation mixture to supply the required energy for metabolism.

HLM is employed in the drug discovery process for metabolite identification, comparison of metabolism by different species, prediction of in vivo clearance, reaction phenotyping and metabolic pathway identification Bourrie et al. The activity of HLMs can vary substantially between individuals.

This problem however can be successfully solved by the application of microsomes pooled from a large bank of individual liver tissues while individual microsomes are used to study inter-individual variability in drug metabolism. Microsomes from single-sex sources are also available and are employed in the evaluation of gender-based discrepancies in drug metabolism The utilization of HLM offers major advantages including ease of application, affordability, well established procedure and the possibility of its use in studying inter-individual and population-based metabolic variation.

It provides qualitative estimations of in vivo drug metabolism and is convenient tools for high throughput screening of compounds, lead optimization studies during drug development and drug interaction studies. The use of HLM is however limited in its quantitative clinical extrapolative relevance.

Thus, results obtained from HLM cannot be used for quantitative estimations of in vivo human biotransformation Brandon et al. This is because the microsomal fraction is enriched with CYPs and UGTs and there is no competition with other enzymes. This leads to relatively higher biotransformation rates in microsomes compared to the human in vivo situation, but also compared to primary hepatocytes and liver slices.

This will necessarily lead to a reduction in the expected metabolic competition with limited number of metabolites. Secondly, the fraction of drug bound to plasma versus microsomal protein is an important determinant of extent of drug metabolism in vivo. The impact of this factor is difficult to assess in vitro. They are enzyme-induced cells, often used in cytotoxic studies whose preparation involves the production of individual enzymes in the endoplasmic reticulum of an eukaryote host cell including yeast and mammalian cells Donato et al.

Stable expression of human CYPs in a human cell line was first successfully performed in by Crespi and co-workers. Cell lines may be transfected at high efficiency using protoplast fusion, centrifugation of lysozyme-treated bacteria bearing the desired vector with the parent cells in the presence of polyethylene glycol protoplast fusion Crespi et al. The main advantages of transgenic cell lines are the ease of culturing, high expression of CYP and UGT enzymes and the possibility of single enzyme study as in supersomes.

However, they are more expensive than most other enzyme-based technologies. They also do not completely reflect the in vivo situation Ferro and Doyle, Non-mammalian cells containing expressed CYP and UGT enzymes CYP expression for this model includes bacterial expression in Escherichia coli and yeast cells.

This model is the final and most focused enzyme only system in which the most popular is the baculovirus-based model allowing functional expression of recombinant proteins in insect cells Guengerich et al. Insect cells lack endogenous CYP and uridine diphosphoglucuronosyl transferase activity. Microsomes from insect cells therefore This has proven to be a useful tool in in vitro biotransformation studies Miller et al.

This model of baculovirus mediated microsomes of insect cells is sometimes referred to as baculosomes or supersomes Gentest. A control experiment - incubation with nontransfected supersomes, must always be conducted. After the determination of the metabolic pathway using the HLM, the use of cdna-expressed CYPs can be an excellent way to confirm results Kurkela et al. Although studies with supersomes may lack quantitative in vivo relevance, they address very particular questions with respect to the role of specific CYP isozymes and their roles in the metabolic pathways of xenobiotics.

Supersomes can be used to study not only isozyme-specific drug biotransformation but also drug-drug interactions Wang et al. However, in UGT supersomes, the UGT active site is shielded behind a hydrophobic barrier resulting in latency of glucuronidations. This disadvantage can be overcome by using a pore-forming agent like alamethicin Brandon et al. The various technologies are compared in Table 9. These techniques have made major contributions towards the development of various therapeutic agents and are still the preferable tools for future studies.

It has been pointed out that the methods vary with their ease of use and the conclusions that can be inferred from them. They also present with unique advantages and disadvantages. The choice of any of the methods will depend on the research question to be answered. However, in certain instances, a combination of different in vitro tools may be required for a reliable assessment.

This is because of its ease to use, commercial availability, long-time viability, ethical and technical simplicity and the ability for high throughput. HLM is the most often used in similar studies. Positive results from HLM can be confirmed through the use of hepatocytes or in vivo human studies. By standard practice, if there is no enzyme inhibition in HLM in The disadvantage of the use of HLM as mentioned earlier, is poor quantitative extrapolation to human.

In addition, cryopreserved hepatocytes were utilized to establish CYP3A4 inhibition by Lessertia frutescens. This is because hepatocytes represent a closer system to human liver than HLM, but require more technical and complex procedures in the sourcing and usage.

The activities of the selected herbs in this study on drug transporters were investigated employing cell lines stably expressing the specific transporters. Summary While consumers may purchase herbal products over-the-counter, many patients consult traditional health practitioners THPs who prescribe and dispense to them, various herbal products in accordance with their presenting complaints. This part of the study was conducted to identify medicinal herbs utilized by traditional health practitioners to manage various conditions.

It involved the collection of samples and the administration of semistructured interview. In all, 15 plant species belonging to 14 families were collected and identified scientifically. The parts used, methods of preparation and administration, together with specific indications were documented.

An overview of published studies on the identified was provided. The absence of information on HDI potential of the medicinal herbs was identified. Introduction Traditional medicine especially the use of medicinal herbs is popular among South Africans where THPs are the first point of call for health services in most rural areas.

The inyanga is a traditional doctor or herbalist, typically male, who often uses medicinal herbs to treat various diseases. The faith healer integrates religious ritual and traditional practices Freeman and Motsei, The reason for the persistent popularity of traditional healers across South Africa is rooted in traditional and cultural acceptance, relative affordability and accessibility. There is therefore, an increasing acceptance of the need to incorporate THP into the mainstream health care Mills et al.

This advocacy has grown despite the absence of sufficient data on the safety and efficacy of herbal medicines. This becomes very important because of the many documented cases of herbal toxicity, herb-drug interaction and therapy failure consequent upon concomitant use of herbs and prescription medicines. The long history of the use of medicinal herbs has impacted on the socio-cultural identities of indigenous Africans. There are research efforts globally to document the ethno-medical data of medicinal plants used in various communities.

This becomes necessary because most THPs, who are seen as the custodian of the herbal knowledge, seldom keep records of their practice. Medical claims for the medicinal herbs are not well proven scientifically Aim and Objectives The aim of this chapter was to identify and select medicinal plants for HDI studies. Mfuleni is a predominantly Black township about 40 kilometres from Cape Town, South Africa with a population of about 10, according to census.

The trado-cultural practices including traditional medicine are thus a reflection of the traditions of the Eastern Cape. Delft is a much bigger township about 34 kilometres North-East of Cape Town with a mixed population of Black and Coloured residents estimated in the year to be 92, The sourcing of the medicinal herbs involved three stages. Samples of herbal materials used for various chronic ailments were obtained from them.

The herbal samples in their natural habitat were also obtained in Goso, near Ngcobo, in Eastern Cape Province about kilometres away , and identified by their local IsiXhosa names with the assistance of the THPs. The collected plants were identified with the help of botanical experts in the Compton Herbarium, Cape Town and Voucher specimens were prepared and housed at the Division of Pharmacology, University of Stellenbosch. Further, a literature search was conducted through the databases of Pubmed, Embase and Google Scholar to published studies on the identified plants.

The scientific and local names of the respective plants were used as search terms. The content of the questionnaire was translated from English to IsiXhosa, the first language of the THPs, with the help of a fluent speaker of both languages. The THPs, having made to understand their roles in the research, consented by appending their signatures in the presence of witnesses. The major The intellectual property of the THPs was protected by the strict adherence to the national guidelines governing the interactions with THPs and other indigenous knowledge custodians.

This included the notification of the department of Environmental Affairs of the Republic of South Africa of the intention to perform this research, and the filling of the Bioprospecting and Discovery Notification Form. These were done in collaboration with the Legal and Patent units of Stellenbosch University Material transfer agreement In addition to the administered semi-structured questionnaires, a Material transfer Agreement MTA was mutually agreed upon, prepared and signed by both the researchers and the THPs.

This was witnessed by the Legal and Patent units of Stellenbosch University. The content of the MTA ensured that the plant materials were the property of the THPs who had voluntarily made them available as a service to the research community. It also restricted the recipients to use the materials for research purposes only in which case, the materials are not transferable to any other person. The THPs allows the researchers to publish any findings therefrom and to acknowledge them.

The identified plants belong to 14 different families. Various disorders managed with these herbal remedies include gastrointestinal, respiratory, skin and infectious diseases. The identities and use of the plants are summarized in Table 10 and sample plant pictures are provided in Appendix G. Both THPs agreed on the information provided on the supplied medicinal herbs. Apiaceae Iqwili Rhizome Chewed or boiled, allowed to cool, and the infusion taken Respiratory problems - flu, coughs and chest complaints.

Bowiea volubilis Harv. Ex Hook. Chenopodiaceae Fat hen E , imbikicane embomvu Emex australis Polygonaceae Devil s thorn E , inkunzane Hypoxis hemerocallidea Hypoxidaceae African Potato E Afrika patat A , Inkomfe Z , ilabatheka Leaves Leaves Corms Crush in hot water to make infusion Boil in water to make decoction Boil bulb, eat whole or take aqueous decoction Anorexia, dysentery, diarrhoea, cough Constipation, stomach cramps, appetite stimulation Immune booster, skin rash Kedrostis africana L.

Cucurbitaceae utuvana Leaves Crush and boil in water for Skin rash oral use and topical wash Pachycarpus concolor E. Bulb Crush, boil in water to make infusion Araceae Inyibiba Leaves Warm leaves as plasters, infusion for oral use Uses infections, inflammation, skin rash STIs, genital sores, warts, haemorrhoid Diarhoea, dysentery Rheumatism, stomach and liver problems, diarrhoea, cancer, immune boosting Cold infusion to douche children; hot infusion for coughs, fever; as purgative Dressing for wounds, boils, sores and minor burns.

Oral use to aid wound healing Published studies on the selected plants A large number of studies have been conducted on the identified plants investigating their potential for therapeutic effects. Hypoxoside, sterols and sterolins are important components of Hypoxis hemerocallidea that have been identified and thought to be responsible for pharmacological benefits Albrecht et al.

A geophyte with bright yellow flowers giving it its common name yellow stars, H. Its major phytochemicals, hypoxoside and rooperol have been investigated for cancer therapy Smit et al. Various other studies have suggested more therapeutic effects derivable from the corm African potato Table Its decoction is employed in the treatment of open wounds, fever, chicken pox, gastrointestinal cramps, rheumatism, heartburn, haemorrhoids, diabetes, inflammation and eye infections among indigenous South Africans Van Wyk and Albrecht, It has been investigated for many of the anecdotal claims.

Studies have suggested some possible anti-retroviral Harnett Tulbaghia violacea is traditionally used for the treatment of respiratory disorders including tuberculosis, asthma; gastrointestinal ailments, oesophageal cancer, fungal infection, fever and colds Van den Heever et al. It contains a C S lyase, cysteine and tetrathiaoctane derivatives Kubeca et al. It inhibits angiotensin converting enzyme ACE supporting its use in hypertension Duncan et al. Its extracts inhibited the growth of cancer cells and induced apoptosis in an in vitro study Bungu et al.

Other studies have suggested its anthelmintic McGaw et al. Spirostachys africana is widely distributed in the vegetations of Southern Africa. A number of phytochemicals including stachenone, diosphenol and diterpenoid derivatives have been isolated and identified as active constituents Duri et al.

The latex is traditionally used in South Africa and Zimbabwe as purgative, to stimulate emesis, and manage infective diarrhoea and dysentery Gelfand et al. It is used in South Africa, especially among the IsiXhosa-speaking people for the topical treatment of infantile cradle cap known locally as ishimca Beach et al. In vitro studies have shown antimicrobial activity of isolated compounds against Escherichia coli, Salmonella typhi, Shigella dysentery, Staphylococcus aureus and Vibrio cholera Mathabe et al.

A clinical study of the antimalarial preparation from S. Bowiea volubilis is known variously as climbing onion, ugibisisila or iguleni isizulu , umgaqana IsiXhosa , gibizisila IsiSwati and Knolklimop Afrikaans. Some studies have claimed that its extracts demonstrated moderate in vitro antibacterial and antifungal activity Buwa et al. Kee and co-workers reported that its extracts inhibited inhibition of thrombin-induced clotting in an in vitro study.

Chemopodium album is a popular edible herb known locally as Imbikilicane IsiZulu. It is consumed nutritionally and for gastrointestinal complaints. In vitro antioxidant and antiinflammatory activity of its extracts has been reported Lindsey et al. Alepidea amatymbica is one of the popular medicinal plants used for the treatment of abdominal disorders, constipation, respiratory tract infections and cold.

Mulaudzi and coworkers reported that its extracts demonstrated in vitro antibacterial and antiinflammatory activity. The leaves and bark of Acacia karroo contain proanthocyanidin and flavonols which possess antioxidant analgesic and membrane-stabilizing properties Malan and Swartz, ; Adedapo et al. Emex australis is a common weed in South Africa used traditionally for gastrointestinal disorders.

It possesses anti-inflammatory properties Akula and Odhav, It is used in the treatment of oral ulcers Hebbar et al. A study in animals has reported glucose-lowering activity in extracts from its leaves Selvamani et al. Pentanisia prunelloides is used traditionally among the Zulus to facilitate labour.

It has been shown to exhibit direct smooth muscle activity on rat uterus and ileum preparations Kaido et al. Its extract was reported to inhibit the activitity of cyclooxygenasein an in vitro study; demonstrate antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae; and inhibit viral replication of influenza A virus Yff et al.

Thus strong popularity of traditional health practices in Eastern Cape is carried through to these Black communities. Besides these three, most of the other plants identified in this study are often administered for other conditions. In traditional health practice, precise instruction for medicinal herb use is therefore important. Phytochemical studies usually show significant variation in the chemical constituents of the different plant parts.

This may explain the anecdotal belief in different indications for different parts of the same plants. Infusions and decoctions are the most common herbal preparations used by the THPs. Infusions are made usually from leaves in hot water, left or shaken for about 30 minutes to few hours while decoction involves boiling usually harder plant parts like roots, bark and stem.

All preparations are taken orally. In addition, decoction from Kedrostis africana is applied as topical wash for skin rash while extracts of Ranunculus multifidus are used as enemas. The involvement of multiple dosage routes for the same dosage formulation is a demonstration of the flexibility and sophistication of traditional medicine.

All the traditional indications for the identified plants have well-established conventional drug treatment. However, there are insufficient data from relevant clinical studies to verify the effectiveness of the herbal preparations. The literature search has demonstrated the extent of studies conducted on these plants. Apart from Kedrostis Africana, the search for which yielded no relevant study to the therapeutic claims, the identified plants in this study have been studied to some extent.

Such studies are limited to in vitro and limited animal studies. Safety data on the use of the medicinal herbs are still not available. Most importantly, the effect of the identified medicinal herbs on metabolic enzymes and drug transporters is unknown.

In the event of concomitant use conventional standard drug therapy of the respective indications, pharmacokinetic herb-drug interaction is a source of clinical concern see Chapter 2. This has been the basis for the selection of these medicinal herbs for HDI assessment in this study. All three are transcriptionally controlled by the aryl hydrocarbon receptor-aryl hydrocarbon receptor nuclear translocator AHR-ARNT and inducible by polycyclic aromatic hydrocarbons PAH and smoking Schmidt and Bradfield, ; Zevin and Benowitz, The expression of CYP1A1 in humans is generally low, with extrahepatic expressions in the lungs, lymphocytes, mammary gland, and placenta Raunio et al.

There is very little involvement of CYP1A1 in drug metabolism. It is however significant in its roles as activators of procarcinogens, thus playing important role in chemically-induced carcinogenesis Ji et al. Like CYP1A1, the expression of CYP1B1 is mainly extrahepatic mainly in kidney, prostate, mammary gland, and ovary with little involvement in the metabolism of xenobiotics Tang et al.

It is the primary enzyme responsible for the phase I metabolism of a number of drugs including tacrine, theophylline, and clozapine.

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