• Archives of Pharmacal Research
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• 제29권4호
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• pp.310-317
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• 2006

We investigated the effects of trinitrobenzene sulfonic acid (TNBS), an amino-group reagent, on the human ether-a-go-go-related gene (HERG) $K^+$ channels expressed in Xenopus oocytes. TNBS neutralizes the positively charged amino-groups of peptide N-terminal and lysine residues. External application of TNBS at 10 mM for 5 min irreversibly shifted the curves for currents at the end of the pulse and tail currents of HERG to a more negative potential and decreased the maximal amplitude of the $I_{tail}$ curve $(I_{tail,max})$. TNBS had little effect on either the activated current-voltage relationship or the reversal potential of HERG current, indicating that TNBS did not change ion selectivity properties. TNBS shifted the time constant curves of both activation and deactivation of the HERG current to a more hyperpolarized potential; TNBS's effect was greater on channel opening than channel closing. External $H^+$ is known to inhibit HERG current by shifting $V_{1/2}$ to the right and decreasing $I_{tail,max}$. TNBS enhanced the blockade of external $H^+$ by exaggerating the effect of $H^+$ on $I_{tail,max}$, not on $V_{1/2}$. Our data provide evidence for the presence of essential amino-groups that are associated with the normal functioning of the HERG channel and evidence that these groups modify the blocking effect of external $H^+$ on the current.

• Archives of Pharmacal Research
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• 제29권4호
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• pp.318-322
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• 2006

Many quaternary ammonium salts are incompletely absorbed after their oral administration and may also be actively secreted into the intestine. However, the underlying mechanism(s) that control the transport of these cations across the intestinal epithelium is not well understood. In this study, the mechanism of absorption of quaternary ammonium salts was investigated using Caco-2 cell monolayers, a human colon carcinoma cell line. Tributylmethylammonium (TBuMA) was used as a model quaternary ammonium salts. When TBuMA was administrated at a dose of 13.3 imole/kg via iv and oral routes, the AUC values were $783.7{\pm}43.6\;and\;249.1{\pm}28.0{\mu}mole\;min/L$ for iv and oral administration, indicating a lower oral bioavailability of TBuMA $(35.6\%)$. The apparent permeability across Caco-2 monolayers from the basal to the apical side was 1.3 times (p<0.05) greater than that from the apical to the basal side, indicating a net secretion of TBuMA in the intestine. This secretion appeared to be responsible for the low oral bioavailability of the compound, probably mediated by p-gp (p-glycoprotein) located in the apical membrane. In addition, the uptake of TBuMA by the apical membrane showed a $Na^+$ dependency. Thus, TBuMA appears to absorbed via a $Na^+$ dependent carrier and is then secreted via p-gp related carriers.

• Archives of Pharmacal Research
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• 제29권4호
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• pp.323-327
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• 2006

The objective of this study was to examine the pharmacokinetics of organic cations in intrahepatic cholestatic rats. A pretreatment with $17{\alpha}$-ethynylestradiol was used to induce intrahepatic cholestasis, and tributylmethylammonium (TBuMA) was used as a representative model organic cation. When $[^3H]$TBuMA was intravenously administered, the AUC value for TBuMA was significantly increased by $79\%$ in cholestasis, and its total systemic clearance was consequently decreased by $46\%$. In addition, the in vivo hepatic uptake clearance of TBuMA from the plasma to the liver was decreased by $50\%$ in cholestasis. The concentration of bile salts in plasma was increased by 2.1 fold in cholestatic rats. Since TBuMA forms ion-pair complexes with anionic components such as bile salts, the decreased hepatic uptake of TBuMA in cholestasis may be due to a change in endogenous components, e.g., bile salts in the plasma. In isolated normal hepatocytes, the uptake clearance for TBuMA in the presence of cholestatic plasma was decreased by $20\%$ compared with normal plasma. Therefore, we conclude that the inhibition of the hepatic uptake process by the cholestasis may be in part due to the increased formation of ion-pair complexes of TBuMA with bile salts in the plasma.

• Archives of Pharmacal Research
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• 제29권4호
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• pp.339-342
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• 2006

A simple high performance liquid chromatographic (HPLC) method was developed for the determination of tolperisone in human plasma. Tolperisone and internal standard (chlorphenesin) were isolated from 1 mL of plasma using 8 mL of dichlormethane. The organic phase was collected and evaporated under nitrogen gas. The residue was then reconstituted with 300 mL aliquot of mobile phase and a 100 mL aliquot was injected onto the $C_{18}$ reverse-phased column. The mobile phase, $45\%$ methanol containing $1\%$ glacial acetic acid and $0.05\%$ 1-hexanesulfonic acid was run at a flow rate of 1 mL/min. The column effluent was monitored using UV detector at 260 nm. The retention times for tolperisone and the internal standard were approximately 7.1 and 8.4 min, respectively. The standard curve was linear with minimal intra-day and inter-day variability. The quantification limit of tolperisone in human plasma was 10 ng/ mL. The proposed method has been applied to the determination of pharmacokinetic profile of tolperisone in Koreans. The T max of tolperisone in Koreans $(0.94{\pm}0.42\;h)$ was not significantly differ from that reported in Europeans (0.5-1 h), but the mean half-life in Koreans $(1.14{\pm}0.27\;h)$ was shorter than that in Europeans $(2.56{\pm}0.2\;h)$. The proposed HPLC method is simple, accurate, reproducible and suitable for pharmacokinetic study of tolperisone.

• Archives of Pharmacal Research
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• 제29권4호
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• pp.282-286
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• 2006

Ochnaflavone is a medicinal herbal product isolated from Lonicera japonica that inhibits cyclooxygenase-2 (COX-2) dependent phases of prostaglandin $D_2(PGD_2)$ generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with $IC_{50}$ values of $0.6{\mu}M$. Western blotting probed with specific anti-COX-2 antibodies showed that the decrease in quantity of the $PGD_2$ product was accompanied by a decrease in the COX-2 protein level. In addition, this compound consistently inhibited the production of leukotriene $C_4 (LTC_4)$ in a dose dependent manner, with an $IC_{50}$ value of $6.56 {\mu}M$. These results demonstrate that ochnaflavone has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity. Furthermore, this compound strongly inhibited degranulation reaction in a dose dependent manner, with an $IC_{50}$ value of $3.01{\mu}M$. Therefore, this compound might provide a basis for novel anti-inflammatory drugs.

• Archives of Pharmacal Research
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• 제29권4호
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• pp.293-297
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• 2006

The fruits of Evodia rutaecarpa Benth (Rutaceae) has long been used for inflammatory disorders and some anti-inflammatory actions of its constituents such as dehydroevodiamine, evodiamine and rutaecarpine were previously reported. Since the pharmacological data is not sufficient to clearly establish the scientific rationale of anti-inflammatory medicinal use of this plant material and the search for its active principles is limited so far, three major constituents (evodiamine, rutaecarpine, goshuyuamide II) were evaluated for their anti-inflammatory cellular action mechanisms in the present study. From the results, evodiamine and rutaecarpine were found to strongly inhibit prostaglandin $E_2$ synthesis from lipopolysaccharide-treated RAW 264.7 cells at $1-10{\mu}M$. Evodiamine inhibited cyclooxygenase-2 induction and NF-kB activation, while rutaecarpine did not. On the other hand, goshuyuamide II inhibited 5-lipoxygenase from RBL-1 cells $(IC_{50}=6.6{\mu}M)$, resulting in the reduced synthesis of leukotrienes. However, these three compounds were not inhibitory against inducible nitric oxide synthase-mediated nitric oxide production from RAW cells up to $50{\mu}M$. These pharmacological properties may provide the additional scientific rationale for anti-inflammatory use of the fruits of E. rutaecarpa.

• Archives of Pharmacal Research
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• 제29권4호
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• pp.302-309
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• 2006

Lupus-like syndrome is characterized by multiple organ injuries including lungs and kidneys. Endotoxin induces a transiently intent systemic inflammatory response and indirectly transient acute lung injury in normal condition. However, whether endotoxin may trigger the persistent development of lung injury in chronic, inflammatory lupus-like syndrome compared with normal condition remains unclear. We examined the pulmonary vascular permeability and production of proinflammatory cytokines, such as TNF-${\alpha}$, IL-6, IL-10 and IFN-${\gamma}$, which play prominent roles in the pathogenesis of lupus-like tissue injury, 6 hand 72 h after i.p. lipopolysaccharide (LPS; endotoxin) injection in pristane-primed chronic inflammation ICR mice characterized by a lupus-like syndrome. These results demonstrated that levels of serum IL-6, IL-10 and IFN-${\gamma}$ and bronchoalveolar lavage (BAL) IL-6 and IFN-${\gamma}$ were remarkably increased 6 h in LPS-exposed pristane-primed mice compared with pristane-primed controls, while pulmonary vascular permeability and levels of serum and BAL TNF-${\alpha}$ were not. And levels of BAL TNF-${\alpha}$, IL-6 and IL-10 were significantly enhanced 72 h in LPS-exposed pristane-primed mice compared with pristane-primed controls. Also, LPS significantly induced the increased in vitro production of TNF-${\alpha}$, IL-6 and IL-10 by lung cells obtained from LPS-exposed pristane-primed mice compared with LPS-exposed normal mice. Our findings indicate that LPS may trigger persistent progression of lung injury through late overproduction of BAL TNF-${\alpha}$, IL-6, and IL-10 in lupuslike chronic inflammation syndrome compared with normal condition.

• Archives of Pharmacal Research
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• 제26권9호
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• pp.739-746
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• 2003

Ultraviolet B (UVB) is known to induce apoptosis in human melanocytes. Here we show the cytoprotective effect of sphingosine-1-phosphate (S1P) against UVB-induced apoptosis. We also show that UVB-induced apoptosis of melanocytes is mediated by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, and that S1P prevents apoptosis by inhibiting this apoptotic pathway. We further investigated three major mitogen-activated protein (MAP) kinases after UVB irradiation. UVB gradually activated c-Jun N-terminal kinase (JNK) and p38 MAP kinase, while extracellular signal-regulated protein kinase (ERK) was inactivated transiently. Blocking of the p38 MAP kinase pathway using SB203580 promoted cell survival and inhibited the activation of caspase-3 and PARP cleavage. These results suggest that p38 MAP kinase activation may play an important role in the UVB-induced apoptosis of human melanocytes. To explain this cytoprotective effect, we next examined whether S1P could inhibit UVB-induced JNK and p38 MAP kinase activation. However, S1P was not found to have any influence on UVB-induced JNK or p38 MAP kinase activation. In contrast, S1P clearly stimulated the phosphorylation of ERK, and the specific inhibition of the ERK pathway using PD98059 abolished the cytoprotective effect of S1P. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that S1P may show its cytoprotective effect through ERK activation in human melanocytes.

• Archives of Pharmacal Research
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• 제26권9호
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• pp.768-772
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• 2003

Tween 80 (Polysorbate 80) is a hydrophilic nonionic surfactant commonly used as an ingredient in dosing vehicles for pre-clinical in vivo studies (e.g., pharmacokinetic studies, etc.). Tween 80 increased apical to basolateral permeability of digoxin in Caco-2 cells suggesting that Tween 80 is an in vitro inhibitor of P-gp. The overall objective of the present study was to investigate whether an inhibition of P-gp by Tween 80 can potentially influence in vivo absorption of P-gp substrates by evaluating the effect of Tween 80 on the disposition of digoxin (a model P-gp substrate with minimum metabolism) after oral administration in rats. Rats were dosed orally with digoxin (0.2 mg/kg) formulated in ethanol (40％, v/v) and saline mixture with and without Tween 80 (1 or 10％, v/v). Digoxin oral AUC increased 30 and 61％ when dosed in 1 ％ and 10％ Tween 80, respectively, compared to control (P＜0.05). To further examine whether the increase in digoxin AUC after oral administration of Tween 80 is due, in part, to a systemic inhibition of digoxin excretion in addition to an inhibition of P-gp in the GI tract, a separate group of rats received digoxin intravenously (0.2 mg/kg) and Tween 80 (10％ v/v) orally. No significant changes in digoxin IV AUC was noted when Tween 80 was administered orally. In conclusion, Tween 80 significantly increased digoxin AUC and Cmax after oral administration, and the increased AUC is likely to be due to an inhibition of P-gp in the gut (i.e., improved absorption). Therefore, Tween 80 is likely to improve systemic exposure of P-gp substrates after oral administration. Comparing AUC after oral administration with and without Tween 80 may be a viable strategy in evaluating whether oral absorption of P-gp substrates is potentially limited by P-gp in the gut.

• Archives of Pharmacal Research
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• 제29권7호
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• pp.566-569
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• 2006

This study examined whether or not acute stress is linked to increases in the neurosteroid levels, which is a well-known neurotransmitters associated with stress stimuli. The ginsenoside, Rb1, was tested in order to better understand its potential effects on altering the neurosteroid levels and ultimately attenuating stress. The optimal stressed condition was checked by measuring the 5a-dihydroprogesterone (DHP) and allopregnanolone (THP) levels in the brain after immobilization stress at various times. Based on this result, an acute stress model was set up to give 30 min of immobilization stress. The DHP and THP brain levels of the stressed mice were then investigated after administering Rb1 orally (10 mg/kg). These results were compared with the neurosteroid level in the stressed mice not given Rb1. Saline was administered orally to the nonstressed mice to check the placebo effect. Acute immobilization stress induced an increase in the THP and DHP concentration in the frontal cortex and cerebellum. When Rb1 was administered orally prior to immobilization stress, the THP level in the frontal cortex and cerebellum was significantly lower than that in the stressed animals not given Rb1. On the other hand, the DHP level was lower in the cerebellum only. This suggests that the metabolism of the brain neurosteroids is linked to psychological stress, and Rb1 attenuates the stressinduced increase in neurosteroids.