Transport of Cryptotanshinone, a Major Active Triterpenoid in Salvia Miltiorrhiza Bunge Widely Used in the Treatment of Stroke and Alzheimer’s Disease, Across the Blood-Brain Barrier
Abstract: Cryptotanshinone (CTS), a major constituent from the roots of Salvia miltiorrhiza (Danshen), is widely used in the treatment of coronary heart disease, stroke and less commonly Alzheimer’s disease. Our recent study indicates that CTS is a substrate for P- glycoprotein (PgP/MDR1/ABCB1). This study has investigated the nature of the brain distribution of CTS across the brain-blood barrier (BBB) using several in vitro and in vivo rodent models. A polarized transport of CTS was found in rat primary microvascular endothelial cell (RBMVEC) monolayers, with facilitated efflux from the abluminal side to luminal side. Addition of a PgP (e.g. verapamil and quinidine) or multi-drug resistance protein 1/2 (MRP1/2) inhibitor (e.g. probenecid and MK-571) in both luminal and abluminal sides at- tenuated the polarized transport. In a bilateral in situ brain perfusion model, the uptake of CTS into the cerebrum increased from 0.52 ± 0.1% at 1 min to 11.13 ± 2.36 ml/100 g tissue at 30 min and was significantly greater than that of sucrose. Co-perfusion of a PgP/MDR1 (e.g. verapamil) or MRP1/2 inhibitor (e.g. probenecid) significantly increased the brain distribution of CTS by 35.1163.6%. The brain levels of CTS were only about 21% of those in plasma, and were significantly increased when coadministered with verapamil or probenecid in rats. The brain levels of CTS in rats subjected to middle cerebral artery occlusion and rats treated with quinolinic acid (a neurotoxin) were about 2- to 2.5-fold higher than the control rats. Moreover, the brain levels in mdr1a(-/-) and mrp1(-/-) mice were 10.9- and 1.5-fold higher than those in the wild-type mice, respectively. Taken collectively, these findings indicate that PgP and Mrp1 limit the brain penetration of CTS in rodents, suggesting a possible role of PgP and MRP1 in limiting the brain penetration of CTS in patients and causing drug resistance to Danshen therapy and interactions with conventional drugs that are substrates of PgP and MRP1. Further stud- ies are needed to explore the role of other drug transporters in restricting the brain penetration of CTS and the clinical relevance.
Key Words: Cryptotanshinone; blood-brain barrier; P-glycoprotein; multidrug resistance associated protein; middle cerebral artery occlusion; Alzheimer’s disease.
INTRODUCTION
The worldwide market for therapies for central nervous system (CNS) diseases was worth more than 50 billion dollars in 2004 and is set to grow substantially in the years ahead [1]. This is due to the facts that: a) the incidence of common neuron-degenerating CNS diseases (e.g., Alzheimer’s disease, stroke, and Parkinson’s disease) increases exponentially in the elderly; b) the number of the elderly in the world over 65 years is increasing dramatically because of a marked rise in fertility since the 1950s and the increase in people’s life-span due to significantly improved healthcare systems. Alz- heimer’s disease is a late-onset mental illness that is characterized by the loss of memory and an impairment of multiple cognitive functions. It is estimated that by the year 2050, 50% of people worldwide (approximately 370 million) who are 85 years of age or older will be afflicted with Alzheimer’s disease [1]. Stroke is a life- threatening disease characterized by rapidly developing clinical signs of focal or global disturbance of cerebral function due to cerebral ischemia [2]. Early detection, prevention, and therapeutic interventions are urgently needed for these devastating CNS disor- ders. A number of neuron-protective drugs have been used in the treatment of stroke and neurodegenerative diseases, but most of them do not exhibit satisfactory clinical outcomes due to drug resis- tance, limited brain penetration and other patient- and drug- associated factors [1, 3]. It has been reported that the blood-brain barrier (BBB) blocks delivery of more than 98% of CNS-acting drugs [4]. The BBB is formed by the tight junctions that connect the brain endothelial cells, thus restricting the entry of compounds from the circulating blood to the brain via paracellular and transcellular routes [5]. Paracellular diffusion does not occur to any great extent at the BBB, due to the presence of tight junctions. The endothelial cells that line microvessels of the brain express a multitude of ATP- binding cassette transporters such as P-glycoprotein (PgP/MDR1/ ABCB1) and multi-drug resistance proteins (MRP1/ABCC1 & MRP2/ABCC2) 1 & 2 that decrease the entry of substrate drugs [6].
Recently, much of the research interest of the neuropharma- cologists has focused on the antioxidant and neuro-protective prop- erties of natural products and application in the treatment of neuro- degenerative CNS diseases such as stroke and Alzheimer’s disease [7]. Among these natural products, the dried root of Salvia Miltior- rhiza Bunge (Danshen) is widely used in the treatment of coronary heart disease, stroke and Alzheimer’s disease [8, 9]. Danshen contains more than one dozen constituents, including water-insoluble diterpene quinones and water soluble phenolic acid derivatives [10- 15]. The former category of compounds include tanshinones I, tan- shinone IIA (TSA), isotanshinones, dihydrotanshinone, and crypto- tanshinone (CTS, (Fig. 1)) and the latter group includes Danshensu (salvianic acid A), protocatechuic acid, rosmarinic acid, and salvi- anolic acid A, B and C. CTS has been purified and formulated and is given orally for protracted regimens in the treatment of inflam- mation, stroke, ischemic heart disease and Alzheimer’s disease, due to its anti-inflammatory, anti-oxidative and neuro-protective effects [8, 9]. Several clinical studies in China have showed that Danshen extracts appear to exhibit neuro-protective activities in patients with ischemic stroke, with few or no adverse effects [9, 16]. However, at this time it is unlikely to draw any evidence-based conclusions on the clinical efficacy of Danshen and CTS in the treatment of ischemic diseases because all clinical trials of Danshen extracts conducted in China are of poor quality with problematic designs. Our previous study has shown that the oral bioavailability of CTS in rats was very low (2.1%) and CTS is a substrate for PgP with a Km of 0.9 M [17]. It is also a substrate for multidrug resistance associated protein 1 (MRP1) (data unpublished, Zhou et al.). In this study, we hypothesize that the brain penetration of CTS is restricted by the BBB due to the contribution of PgP and MRP1 expressed in the brain capillary endothelial cells. To test this hypothesis, we have investigated the nature of brain transport of CTS using the following in vitro and in vivo models: a) primary rat brain mi- crovascular endothelial cells (RBMVECs); b) in situ rat brain per- fusion model; c) healthy rats treated with CTS alone or in combina- tion with a PgP or MRP1/2 inhibitor; d) rats subject to middle cere- bral artery occlusion (MCAO) and rats treated with the neurotoxin, quinolinic acid; and e) mdr1a and mrp1 gene deficient mice.
MATERIALS AND METHODS
Chemicals and Reagents
CTS and tanshinone IIA (used as internal standard, IS) ex- tracted from the root of S. miltiorrhiza Bunge were purchased from the National Institute for the Control of Pharmaceutical and Bio- logical Products (Beijing, China). The two compounds have a pu- rity >99.0%, as determined by high performance liquid chromatog- raphy (HPLC) with ultraviolet detection. Fetal bovine serum, 0.05% trypsin-ethylenediaminetetraacetic acid, penicillin-streptomycin, sterilized Hank’s balanced salt solution (HBSS) at pH 7.4 contain- ing 25 mM N-[2-hydroxyethyl] piperazine-N9-[4-butanesulfonic acid] and 25 mM glucose, and non-essential amino acids were all obtained from Invitrogen (Carlsbad, CA). Trypan blue, DMSO, quinolinic acid, verapamil hydrochloride, nifedipine, probenecid, none in Primary Rat Brain Microvascular Endothelial Cells.
The cytotoxic effect of CTS on RBMVECs was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay as described previously [19-21]. The uptake and efflux of CTS in RBMVECs were examined with regard to substrate concentration and incubation time in confluent cell cul- tures grown on 60-mm plastic culture dishes as described previ- ously [17]. For uptake and efflux assays, cells were exposed to CTS at 0100 µM over 120 min at 37oC. The cells were harvested and the cell pellet was suspended in 200 µl of extraction solution (ace- tonitrile:methanol, 1:1, v/v, with 0.01N HCl) with the addition of 10 µl 1.0 mg/ml tanshinone IIA (used as an internal standard). Sub- sequently, the mixture was sonicated, vortexed and centrifuged. The supernatants were dried under nitrogen gas and reconstituted with 100 µl of mobile phase. An aliquot (10-20 µl) was injected into the liquid chromatography mass spectrometry (LC-MS) for CTS con- centration determination. Control uptake and efflux assays and transport assays in RBMVEC monolayers were performed with 0.3 µCi/well of the extracellular markers [3H]-sucrose and [3H]- propranolol (GE Healthcare, Buckinghamshire, UK). Transport experiments of CTS in the RBMVEC monolayers were performed when the cells had reached integrated confluence 6 to 10 days after seeding as described previously [17]. The effects of pH, Na+, glu- cose, and temperature on CTS transport across the monolayers were also investigated by varying the incubation conditions. The uptake of daunomycin, efflux of [3H]-vinblastine and transepithelial trans- port of [3H]-digoxin in the monolayers were also included as con- trol incubations. The radioactivity in culture medium and cellular lysates was determined by an LC-6000 liquid scintillation counter (Beckman Instruments, Fullerton, CA). Preliminary experiments showed that daunomycin uptake was at equilibrium after 6090 min of incubation with RBMVECs. Therefore, cells were incubated for 30 min with 1.0 µM daunomycin (0.3 µCi/well [3H]-daunomycin and unlabelled daunomycin with or without verapamil at 100 µM. The percentage of CTS transported across the monolayers was plot- ted as a function of time, the slope of which is related to the first order rate constant (K = slope/100) for the steady-state flux and effective permeability (Peff) as follows: MK-571 (L-660,711), was a gift from Dr Ford Hutchinson (Merck Frosst Canada, Inc., Kirkland, Québec, Canada). Mouse mono- clonal antibodies to MDR1 (C219) or MRP1 were purchased from Abcam Co. (Cambridge, UK). All other chemicals and reagents were of analytical or HPLC grade as appropriate.
Where V and Area are the volume of the donor chamber and effective cellular surface area of the insert, respectively. Moreover, the effects of various compounds, including sodium azide, 2, 4- dinitrophenol, verapamil, nifedipine, quinidine, MK-571, pro- benecid, and celecoxib, on CTS uptake and efflux in RBMEVECs and transport in the monolayers were investigated in at least three independent experiments. All inhibitors except sodium azide and 2, 4-dinitrophenol are known drug transporter inhibitors [22]. Despite a lack of selectivity, these transporter inhibitors are commonly used in inhibition studies as the data can provide initial supporting evi- dence for the involvement of one or more drug transporters. These inhibitors at indicated concentrations showed little cytotoxicity (<5%) to the cells tested when incubated for 4 hr. DMSO was used to dissolve all inhibitors, with a final concentration of 0.2% (v/v). For the uptake inhibition assays, all inhibitors were pre-incubated with cells for 2 hr and co-incubated for a further 30 min in the pres- ence of CTS at 0.1 or 1.0 M [23, 24]. For the efflux inhibition assay, CTS was loaded at 0.1 or 1.0 M and incubated for 120 min to achieve maximum drug uptake. Thereafter, after five washes with 4oC HBSS of the cells to eliminate extracellular drug and stop any transporter-mediated drug flux, cells were incubated in culture medium for 30 min at 37oC with the inhibitor.
Bilateral in Situ Brain Perfusion in Rats
Adult male Sprague-Dawley rat brain perfusion was performed using the method reported previously [25]. CTS was introduced via a side arm into the perfusion medium at 0.5 ml/min and achieved a final concentration of 0.5 M in the perfusion medium. [3H]- sucrose was used as a brain intravascular volume marker. At prede- termined time points, the rat was decapitated, and the individual cerebral hemispheres and cerebellum were weighed, rinsed and homogenized. In a separate series of single-time point (10 min) experiments, the CNS uptake of CTS at 0.5 µM was examined in the presence of verapamil, nifedipine, or quinidine, MK-571, or probenecid. In vivo BBB permeability (Kin, µl/min/g of brain) was calculated using the single-time point analysis as described previ- ously [26, 27] after correcting for the remaining intravascular CTS, estimated from the apparent brain uptake of [3H]-sucrose.
Brain and Tissue Distribution of Cryptotanshinone in mdr1a(-/-) and mrp1(-/-) Mice
Male FVB (2035 g), mdr1a gene-deficient and mrp1(-/-) mice (2530 g, 812 week of age) were purchased from Taconic Farms, Inc. (Germantown, NY). The mice were treated with oral CTS at 5 mg/kg by gavage (n = 5 per time points). At pre-determined time points, the mice were sacrificed by neck
dislocation. Blood, brain, heart, liver, lung, spleen and kidney were immediately collected, rinsed with saline, and weighed.
Liquid Chromatography Mass Spectrometry (LC-MS) Analysis of Cryptotanshinone
The concentrations of CTS in rat plasma and tissues, transport medium in cellular monolayers, and cellular lysates were deter- mined by an LC-MS system equipped with an Agilent 1100 LC connected to an Applied Biosystems Q-Trap 4000 mass spectrome- ter through an electrospray ionization source under optimal condi- tions as described previously [17]. The monitor ion and collision energy were m/z 297 → 251 and −32 eV for CTS and m/z 295 → 249 and −32 eV for IS (tanshinone IIA). The lower limit of Where T is the length of perfusion in minutes, and Rbrain is the amount of CTS in the brain over that in the perfusate.
Brain and Tissue Distribution of Cryptotanshinone in Healthy Rats with or Without Combined PgP or Mrp1/2 Inhibitor
Healthy male Sprague Dawley rats (200-250 g) were random- ized to receive different types of treatment: CTS at 5 mg/kg by gavage plus water; CTS at 5 mg/kg by p.o. in combination with oral verapamil at 25 or 100 mg/kg; CTS at 5 mg/kg p.o. in combination with quinidine at 10 or 50 mg/kg by p.o.; and CTS at 5 mg/kg in combination with probenecid at 50 or 200 mg/kg by p.o.. The in- hibitor was administered 2 hr before CTS dosing. At pre- determined time points (15 min, 30 min, 1, 2, 4, 6, 8, 12 and 24 hr), the rats were sacrificed under ether anesthesia, and blood and other tissues were immediately collected for analysis. To examine the specificity of the effect of verapamil and quinidine, the brain uptake and Ri values of the paracellular transport marker [3H]-mannitol at 5 and 15 min after i.v. administration (1 µCi/rat) were also deter- mined according to the method described above.
Microdialysis and Brain Distribution of Cryptotanshinone in fitted using the Prism 3.0 program (Graphpad Software Co., San Diego, CA) as described previously [32, 33]. The Student’s t-test was conducted for the between-group comparisons with a signifi- cance level of P 0.05.
RESULTS
Cytotoxicity and Metabolism of Cryptotanshinone and Expres- sion of PgP and Mrp1 in Primary Rat Brain Microvascular Endothelial Cells CTS at 0.1100 M did not show significant cytotoxicity (<8.5%) to RBMVEC cells when incubated for up to 72 hr as de- termined by the MTT assay. No detectable metabolites (e.g. TSA) were observed when CTS at 0.1100 M was incubated with RBMVECs when incubated for 248 hr as determined by HPLC and LC-MS analysis.
Western blot analysis using the PgP monoclonal antibody C219 and antibody against MRP1 detected a marked single band at ap- proximately 170 180 kDa and a weak band at about 190 kDa in RBMVEC cells (Fig. 2), indicating that this cell culture model ex- hibits important features of the BBB.
The effects of a 2-hr pre-incubation and a 30-min co-incubation of various compounds on the uptake of CTS by RBMVECs are shown in (Fig. 4). When sodium azide, 2,4-dinitrophenol, verapa- mil, nifedipine, or quinidine was pre-incubated for 2 hr with RBMVEC cells, the uptake of CTS at 0.1 M was significantly (P < 0.05 or P < 0.01) increased by 65.1 8.9%, 77.9 11.2%, 121.1 19.3%, 139.7 21.4% and 169.5 27.2 %, respectively (Fig. 4). MK-571 (100 µM) and probenecid (200 M) also significantly increased CTS uptake in the cells (P < 0.05). These compounds also significantly decreased CTS efflux in the cells (P < 0.05). However, celecoxib (100 M) did not significantly alter the uptake or efflux of CTS in RBMVECs. Similar results were found when the concentration of CTS was increased to 1.0 M. These findings demonstrated that the uptake of CTS by RBMVEC was ATP- dependent, and pre-incubation with PgP or Mrp1/2 inhibitors such as verapamil and MK-571, but not a MRP4 inhibitor (celecoxib), significantly altered the uptake and efflux of CTS in RBMVECs. These data also provided further evidence indicating that CTS is a substrate for PgP and Mrp1/2, but not for Mrp4.
Transport of Cryptotanshinone in Rat Brain Microvascular Endothelial Monolayers
In our in vitro rat BBB model, the Peff values across the mono- layer from the luminal (AP) to abluminal (BL) direction for sucrose and propranolol were 2.17 ± 0.23 10-6 cm/sec and 3.52 0.41 10-6 cm/sec, respectively, indicating the integrity of RBMVEC monolayers [34]. Moreover, the initial integrity of RBMVEC mon- olayers before each experiment was monitored and confirmed by measuring the effective transepithelial electrical resistance values; the mean value was 650.3 ± 55.1 •cm2. After incubation of CTS at 0.1–100 M loaded at the AP or BL side, the sample was col- lected from the receiving side for LC-MS analysis. No detectable metabolites (e.g. tanshinone IIA) were observed when CTS was loaded at all concentrations over 60 min. The time course and concentration effect of CTS flux from the AP to BL or BL to AP have been examined and the results are shown in (Fig. 5). After a luminal or abluminal loading, CTS appeared on the receiving side by 5 min.
The flux rate of CTS from the AP to BL or BL to AP was largely proportional to its concentrations over 0.1100 M and was linear up to 60 min of incubation time. The transport rate of CTS across RBMVEC monolayers from the BL to AP side was significantly (about 3- to 5-fold) higher than that from the AP to BL. The Peff of CTS from the BL to AP side (0.998.93 10-6 cm/sec) was about 3- to 5-fold higher than that from the AP to BL side (0.182.36 10-6 cm/sec) with a marked decrease in Peff values for both direc- tional flux at increasing CTS concentration (Fig. 5). The Rnet values ranged from 3.0 to 4.9. These results demonstrated a polarization in the RBMVEC transport toward CTS and predominantly efflux from the abluminal side to luminal side (i.e. from brain to systemic circu- lation). The BL to AP efflux rate of CTS increased with increasing concentrations over 0.1100 M but appeared saturable when its concentration was 25 M as indicated by a non-proportional in- crease in the efflux. Consistently, there was a significant decrease in Peff values for the BL to AP flux at CTS concentrations 25 M (P < 0.01). Reducing the apical pH to 6.5-7.0 caused a significantly (P < 0.05) increased flux by 25.436.2% at 0.1 M CTS and 18.939.4% at 1.0 M CTS from the AP to BL or BL to AP compared to the values at pH 7.4. A maximum Peff was observed at pH 7.4 for both AP-BL and BL-AP transports at 0.1 and 1.0 M CTS concentrations. Lowering the pH may reduce the ionization of CTS and thus increase its flux. In contrast, the substitution of sodium salts in the transport medium with potassium salts had insignificant (P > 0.05) effect on the flux of CTS for either AP to BL or BL to AP. In addition, reducing the incubation temperature from 37oC to 4oC significantly (P < 0.01) decreased the Peff of CTS at 0.1 and 1.0 M from the AP to BL or BL to AP side, with a 47.665.2% reduc- tion. Moreover, the absence of glucose in the transport medium slightly increased the AP to BL flux of CTS at either 0.1 or 1.0 M.
In contrast, depletion of glucose significantly (P < 0.05) decreased the BL to AP flux of CTS at 0.1 and 1.0 M by 36.543.8%.Addition of transport buffer to both sides with sodium azide, 2, 4-dinitrophenol, verapamil, nifedipine, quinidine, probenecid, or MK-571 significantly increased the AB-BL flux of CTS at 0.1 M by 60.2 8.9% to 106.6 15.7% (P < 0.05 or 0.01) (Fig. 5). In contrast, these compounds caused a significant (P < 0.05 or 0.01) decrease in the BL to AP flux of CTS at 0.1 M by 51.2 4.6% to 98.7 10.8%. Similar results were observed when the concentra- tion of CTS was increased to 1.0 M in the presence of the above inhibitors with an increase of Peff from AP-BL direction by 55.4 7.2% to 95.6 10.3% and an decrease in Peff from BL-AP direction by 43.2 4.2% to 100.7 12.4%. However, celecoxib did not sig- nificantly alter the AP-BL or BL-AP Peff values (P > 0.05), suggest- ing that MRP4 plays a minor or negligible role in the brain trans- port of CTS.
Permeability of Cryptotanshinone in Bilateral Perfused Rat Brain
Entry of CTS into the CNS was examined by means of the in situ brain perfusion technique in rats and compared with results for the plasma space marker molecule sucrose (Fig. 6). The uptake of CTS into the cerebrum increased from 0.52 ± 0.1% at 1 min to 11.13 ± 2.36% (ml/100 g tissue) at 30 min and was significantly greater than that measured for sucrose at all measured time points (P < 0.01). The values for cerebellum were 2.44 0.44% at 1 min and 7.77 1.64% at 30 min. Lower values of CTS were observed in CSF, which were 1.33 0.27% at 1 min and 4.81 1.11% at 30 min. The rank order of CTS distribution in rat CNS was cerebrum > cerebellum > CSF. The permeability coefficient (Kin) of CTS in cerebrum, cerebellum and CSF significantly decreased when the substrate concentration was 10 M (Fig. 2), suggesting the in- volvement of a saturable mechanism for CTS uptake by the CNS. The CTS-specific Rbrain values at 0.1, 0.25, 0.5, 1, 2.5, 10, 25, 50 and 100 µM substrate concentrations were 8.96, 7.20, 5.07, 4.06, 3.68, 3.28, 2.81, 2.57, and 2.26 ml/min/g of cerebrum tissue after subtraction of the value of [3H]-sucrose, respectively, with a sig- nificant concentration-dependent decrease. Lower values were ob- served for cerebellum and CSF but significant concentration- dependent decrease remained. In addition, verapamil, nifedipine, quinidine, MK-571, and probenecid all significantly increased the Rcerebrum, Rcerebellum and RCSF values by 35.1 to 163.6% (Fig. 6) (P < 0.05 or 0.01).
Brain Levels of Cryptotanshinone in Rats Subjected to MCAO or Treated with Quinolinic Acid
Fig. 8 shows representative free plasma/brain concentrations- time profiles over 600 min in normal healthy rats and rats subject to MCAO for 2 hr with treatment of CTS at 5 or 25 mg/kg (i.p). In normal rats, the free plasma/brain concentration-time profiles of CTS resembled those when the total concentrations were measured, with mean Ri values of 0.21. The AUCs for free CTS concentrations were about 1.01.5% of those of total concentration, namely, the protein binding of CTS was about 1.01.5%. The AUC0-480min and Cmax in both rat brain and plasma were increased less proportionally when the dose of CTS was increased from 5 to 25 mg/kg.
DISCUSSION
Though Danshen as well as its major active constituent CTS are widely used in the treatment of stroke and Alzheimer’s disease in China and other countries, little is known about the nature of its brain distribution. This prompted us to investigate its CNS distribu- tion using several in vitro and in vivo models. Firstly, we used the RBMVEC cell line to characterize the transport of CTS across the BBB by examining its efflux, uptake and transport across cell membranes and cellular monolayers. The kinetic data and inhibition studies indicate that both active and passive transport are likely involved in the uptake and efflux of CTS across cells. The uptake and efflux of CTS across the cellular monolayers were pH-, energy-, and temperature-dependent, but not sodium-dependent. In particu- lar, PgP and MRP1 are highly likely to be involved in the influx and efflux of CTS in the cells, as indicated by the significant en- hanced accumulation and decreased efflux of the substrate in the presence of PgP or MRP1 inhibitors such as nifedipine, quinidine, or probenecid (Fig. 3). The permeability coefficients for the luminal to abluminal transport of CTS in RBMVEC monolayers were about 3- to 5-fold lower than those in the BL-AP direction (Fig. 5).
Secondly, we examined the permeability of CTS using the bi- lateral in situ brain perfusion model in rats. These results indicate that the BBB can restrict the entry of CTS into the CNS, probably due to the contribution of PgP and MRP1 to a lesser extent as indi- cated by the inhibition studies (Fig. 6). Notably, the brain uptake of CTS after considering vascular space was significantly greater than uptake into CSF (Fig. 6). Although it is appreciated that in in vivo studies the separation of uptake across either the BBB or the blood- CSF barrier is not possible, it would appear unlikely that the blood- CSF route would produce the CTS levels observed in the brain.
Fourthly, the CNS access of CTS in stroked rats was investi- gated, as there is evidence that the function of the BBB can be im- paired by a number of pathological and environmental factors in- cluding stroke, inflammation and Alzheimer’s disease [32, 33]. Compared to the brain distribution data in normal rats, the stroked and quinolinic acid-treated rats had approximately 2.5 and 2-fold higher brain levels of CTS (Fig. 7) & (Fig. 8), suggesting that stroke and quinolinic acid-induced brain injury disrupted the BBB function and thus increased the permeability of CTS across the BBB in rats. There are some reports where stroked rodents had 3- to 10-fold higher brain penetration of drugs acting on the CNS com- pared to normal animals [6]. In stroke, oxidative stress plays a criti- cal role and participates in the complex interplay between excito- toxicity, apoptosis and inflammation in ischemia. All these factors may contribute to increased permeability of the BBB in stroke. In addition, there is evidence that the BBB function is
disrupted in Alzheimer’s disease.
Our study demonstrated that drug combination and disease status (e.g. stroke) can alter the brain distribution of CTS. Since drug entry into the CNS depends on various parameters, the effects of other factors (e.g genetic factors, age, other ABC transporters and inflammation) on the brain distribution of CTS should be ex- amined. Knowledge about newly developed CNS-active drugs, such as CTS, is therefore of importance since these factors alter the ac- cess to the brain of CTS and subsequently its pharmacodynamics.
Finally, the importance of PgP and MRP1 on CTS transport across the BBB was confirmed in vivo in mdr1a or mrp1 gene defi- cient mice. CTS brain accumulation was clearly much higher in mdr1a(-/-) mice and mrp1(-/-) mice to a lesser extent than that in wild-type mice, while the distribution in other organs the difference was insignificant when corrected by plasma concentrations (i.e. by comparing the Ri values) (Fig. 11). The absence of active PgP or mrp1 in mdr1a or mrp1 knockout mice allows unrestricted access of PgP/MRP1 substrates to the brain, yielding significantly in- creased CNS concentrations often exceeding those observed in wild-type mice by orders of magnitude. Similar results have been found in previous studies [9], which demonstrated that in the same mdr1a knockout mouse models, the distribution of PgP substrates was strongly affected by PgP, in particular with respect to the brain.
Results from the present study suggest that PgP and MRP1 to a lesser extent play an important role in limiting the brain penetration of CTS across the BBB, but the contribution of other transporters such as MDR3 and MRP5-9 cannot be excluded. It would be inter- esting to investigate the contributions of these relevant P- glycoproteins and other MRPs in the transport of CTS. Moreover, PgP- and MRP1-mediated drug-drug interactions have been re- ported, drug interaction studies of CTS with PgP and MRP1 sub- strates/inhibitors should be considered in future preclinical and clinical trials.
Based on the findings from this study, we hypothesize that re- sistance to drug treatment of CNS diseases is highly possible with herbal medicines, such as Danshen, although more evidence is needed in future studies. To our knowledge, there are no studies documenting this important issue in the literature. In fact, drug resistance is a common phenomenon in the treatment of a number of diseases, including cancer, bacterial and viral infection, epilepsy, diabetes, hypertension, and stroke. It is, however, generally consid- ered that drug resistance to herbal medicines does not occur be- cause: a) the herbs are from natural sources; b) they are mixtures of multiple components at low levels; c) many of them are adaptogens which can interact with the body without altering the basic biochemical pathways and thus the body will not cause resistance to them; and d) many herbs have been found to overcome drug resis- tance when used in combination with anticancer drugs, antibiotics, anti-diabetic agents, and anticonvulsants in animal studies. The present study indicates that, like many CNS drugs, CTS is subject to a low brain penetration due to difficulty crossing through the BBB, which would provide an explanation for the therapeutic fail- ure in a majority of patients with stroke or Alzheimer’s treated with Danshen [4, 36].
In conclusion, we found that the entry to the brain of CTS is restricted both in vivo and in vitro, but disease status (e.g. stroke) and co-treatment of PgP or MRP1 inhibitors increase its brain ac- cess. PgP and MRP1 to a lesser extent in the BBB play an impor- tant role in limiting the brain entry of CTS in rodents, suggesting a possible role of PgP and MRP1 in limiting the brain penetration of CTS in patients and causing drug resistance to Danshen therapy and interactions with conventional drugs that are substrates of PgP and MRP1. Further research addressing the CNS entry of CTS in pa- tients and identifying proper methods to MK571 overcome its restricted brain access is warranted.