Role of P-glycoprotein in the intestinal absorption of tanshinone IIA, a major active ingredient in the root of Salvia miltiorrhiza Bunge

The extracts from the roots of Salvia miltiorrhiza Bunge (Danshen) are widely and traditionally used in the treatment of angina pectoris, acute myocardial infarct, hyperlipidemia and stroke in China and other Asian countries. In this study, we have investigated the role of P-glycoprotein (P-gp) in the intestinal absorption of tanshinone IIA (TSA), a major active constituent of Danshen, using several in vitro and in vivo models. The oral bioavailability of TSA was about 2.9

Role of P-glycoprotein in limiting the brain penetration of glabridin, an active isoflavan from the root of Glycyrrhiza glabra.

Purpose. Glabridin is a major active constituent of Glycyrrhiza glabra which is commonly used in the treatment of cardiovascular and central nervous system (CNS) diseases. Recently, we have found that glabridin is a substrate of P-glycoprotein (PgP/MDR1). This study aimed to investigate the role of PgP in glabridin penetration across the blood–brain barrier (BBB) using several in vitro and in vivo models.
Materials and Methods. Cultured primary rat brain microvascular endothelial cells (RBMVECs) were used in the uptake, efflux and transcellular transport studies. A rat bilateral in situ brain perfusion model was used to investigate the brain distribution of glabridin. The brain and tissue distribution of glabridin in rats with or without coadministered verapamil or quinidine were examined with correction for the tissue residual blood. In addition, the brain distribution of glabridin in mdr1a(-/-) mice was compared with the wild-type mice. Glabridin in various biological matrices was determined by a validated liquid chromatography mass spectrometric method.
Results. The uptake and efflux of glabridin in cultured RBMVECs were ATP-dependent and significantly altered in the presence of a PgP or multi-drug resistance protein (Mrp1/2) inhibitor (e.g. verapamil or MK-571). A polarized transport of glabridin was found in RBMVEC monolayers with
facilitated efflux from the abluminal (BL) to luminal (AP) side. Addition of a PgP or Mrp1/2 inhibitor in both luminal and abluminal sides attenuated the polarized transport across RBMVECs. In a bilateral in situ brain perfusion model, the uptake of glabridin into the cerebrum increased from 0.42 T 0.09% at 1 min to 9.27 T 1.69% (ml/100 g tissue) at 30 min and was significantly greater than that for sucrose. Coperfusion of a PgP or Mrp1/2 inhibitor significantly increased the brain distribution of glabridin by 33.6j142.9%. The rat brain levels of glabridin were only about 27% of plasma levels when corrected by tissue residual blood and it was increased to up to 44% when verapamil or quinidine was coadministered. The area under the brain concentration-time curve (AUC) of glabridin in mdr1a(-/-) mice was 6.0-fold higher than the wild-type mice.
Conclusions. These findings indicate that PgP limits the brain penetration of glabridin through the BBB and PgP may cause drug resistance to glabridin (licorice) therapy for CNS diseases and potential drugglabridin interactions. However, further studies are needed to explore the role of other drug transporters (e.g. Mrp1-4) in restricting the brain penetration of glabridin.

Role of the ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C_max) and area under the plasma concentration-time curve (AUC) with increasing dosage. The time to C_max (T_max) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P_app) based on TSB disappearance from the lumen (P_lumen) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (P_blood). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P_app values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC_0-24hr in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.

Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue

Background: ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks.
Results: In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H). High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals.
Conclusions: In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the potentially differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin.

Profiling of secondary metabolites in blue lupin inoculated with Phytophthora cinnamomi following phosphite treatment

In order to discover phytochemicals that are potentially bioactive against Phytophthora cinnamomi, (a soil-borne plant pathogen) a metabolite profiling protocol for investigation of metabolic changes in Lupinus angustifolius L. plant roots in response to pathogen challenge has been established. Analysis of the metabolic profiles from healthy and P. cinnamomi-inoculated root tissue with high resolution mass spectrometry and nuclear magnetic resonance spectroscopy confirmed that although susceptible, L. angustifolius upregulated a defence associated genistein and 2′-hydroxygenistein-based isoflavonoid and a soyasapogenol saponin at 12h post inoculation which increased in concentration at 72h post inoculation. In contrast to the typical susceptible interaction, the application of a phosphorous-based treatment to L. angustifolius foliage 48h before P. cinnamomi challenge negated the ability of the pathogen to colonise the root tissue and cause disease. Importantly, although the root profiles of water-treated and phosphite-treated plants post pathogen inoculation contained the same secondary metabolites, concentration variations were observed. Accumulation of secondary metabolites within the P. cinnamomi-inoculated plants confirms that pathogen ingress of the root interstitially occurs in phosphite-treated plants, confirming a direct mode of action against the pathogen upon breaching the root cells.

On the importance of the first observation in GLS detrending in unit root testing

First-differencing is generally taken to imply the loss of one observation, the first, or at least that the effect of ignoring this observation is asymptotically negligible. However, this is not always true, as in the case of generalized least squares (GLS) detrending. In order to illustrate this, the current article considers as an example the use of GLS detrended data when testing for a unit root. The results show that the treatment of the first observation is absolutely crucial for test performance, and that ignorance causes test break-down.