![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Laboratory of Molecular Pharmacology, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
(Received June 18, 2006; accepted December 12, 2006)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResults and DiscussionReferences |
|---|
; Wang et al., 1999). However, in vivo administration of the inactive D-enantiomer (D-NNA) produced the same biological effects as its L-enantiomer (L-NNA), such as increasing blood pressure that was blocked by administration of L-arginine but not D-arginine (Wang et al., 1991, 1993), leading to a notion that D-NNA was converted into L-NNA in vivo (Wang et al., 1993). Wang et al. (1999) from another laboratory showed that D-NNA was indeed unidirectionally converted into L-NNA in vivo by using chiral high-performance liquid chromatography. Furthermore, the kidney was confirmed to be the major organ, accounting for approximately 80% of chiral inversion of D-NNA in vivo (Xin et al., 2005).
It was hypothesized that D-NNA unidirectionally converts to L-NNA by a two-step pathway involving the oxidation of D-NNA to NG-nitro-5-gunidino-2-oxopentanoic acid by D-amino acid oxidase (DAAO), followed by amination to L-NNA by an unidentified transaminase (Wang et al., 1999; Xin et al., 2006). Several lines of evidence support the above hypothesis: 1) DAAO incubation with D-NNA in vitro reduced D-NNA content (Wang et al., 1999); 2) the chiral inversion rate of D-NNA was parallel with the DAAO activity in tissue homogenates (Xin et al., 2005); and 3) injection of the inhibitor of DAAO benzoate completely blocked the pressor response to naive D-NNA in conscious rats (Xin et al., 2005). However, further studies, such as testing DAAO inhibitors on chiral inversion of D-NNA in vitro and in vivo, are needed to confirm the above hypothesis.
It is known that chiral drugs may exhibit stereoselective pharmacokinetics. Stereoselective disposition of NNA was observed in anesthetized rats in which D-NNA had a faster clearance than that of L-NNA (Wang et al., 1999). Chiral inversion is one type of drug metabolism that contributes to drug clearance. However, it is not yet known whether chiral inversion of D-NNA accounts for pharmacokinetic stereoselectivity of NNA.
This study aimed to test whether the DAAO inhibitor sodium benzoate blocks the chiral inversion of D-NNA in rat kidney homogenates and conscious rats. This study also intended to confirm pharmacokinetic stereospecificity of NNA in conscious rats, as well as deduce whether chiral inversion of D-NNA contributes to this stereospecificity. Our results showed that benzoate completely blocked chiral inversion of D-NNA in vitro and in vivo, suggesting that D-NNA conversion is via the metabolism of DAAO. Moreover, NNA exhibited its pharmacokinetics in a stereospecific manner in which the D-enantiomer cleared much faster (5-fold higher) as a result of its chiral inversion because administration of sodium benzoate mostly reduced the clearance of D-NNA.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
Animal Preparation. Male Sprague-Dawley rats (350–400 g) from Fudan University Medical Animal Center (Shanghai, China) were anesthetized with sodium pentobarbital (65 mg/kg, i.p.). A polyethylene cannula (PE50, Becton Dickinson Co., Franklin Lakes, NJ) was inserted into the left femoral vein for the collection of blood samples. The vascular cannula were tunneled s.c. and exteriorized at the back of the neck. The rats were given at least a 24-h recovery from anesthesia and surgery before use.
Preparation of Tissue Homogenates. Rats were sacrificed, and the kidneys were removed and washed with Tris-HCl buffer (4°C, 0.1 M, pH 8.2). Tissue samples (1 g each) were homogenized in Tris-HCl buffer (3 ml, pH 8.2) at 4°C with a homogenizer (Fluko Equipment Co., Shanghai, China). The homogenates were centrifuged at 1500 rpm for 10 min, and the supernatants were used for the measurements of the DAAO activity and D-NNA conversion.
Determination of DAAO Activity. The activity of DAAO was determined according to the "keto acid method" (D'Aniello et al., 1993; Sarower et al., 2003). The supernatants (200 µl) of the homogenates were incubated with 0.1 M D-alanine (200 µl, dissolved in the above Tris-HCl buffer) plus additional sodium benzoate solution (100 µl, concentration range 0.00025–500 mg/ml) for 30 min in a shaking bath (700 rpm) at 37°C. The control samples were treated similarly, except the benzoate solution (100 µl) was replaced by Tris-HCl buffer (100 µl). Afterward, trichloroacetic acid (200 µl, 25%) was added to the incubated mixture, mixed again, and centrifuged at 12,000 rpm for 10 min. The supernatant (400 µl) was mixed with 2,4-dinitrophenylhydrazine (400 µl of 1 mM in 1 M HCl) and incubated at 37°C for 10 min. NaOH (800 µl of 1.5 M) was subsequently added and mixed. The mixture was kept at room temperature for 10 min, and the absorbance was read at 445 nm against a blank sample consisting of the same mixture without D-alanine. The activity of DAAO in the homogenates was quantified against the standard curve of pyruvic acid (from 0.35 nM to 2.80 nM, r2 > 0.99).
Tissue Homogenate Incubation with D-NNA. The effects of kidney homogenates (n = 4) on the conversion of D-NNA into L-NNA were determined. The incubation systems of D-NNA and kidney homogenates for conversion were treated similarly to that for DAAO activity detection, except that in the D-alanine solution (200 µl of 0.1 M), the substrate of DAAO was replaced by D-NNA solution (200 µl of 50 mM, dissolved in the same Tris-HCl buffer). In some experiments, the homogenates were first denatured through heating (10 min at 100°C). The incubation supernatants were used for ex vivo determination of D-NNA and L-NNA by capillary electrochromatography (CEC).
Measurement of D-NNA and L-NNA in Biological Samples. D-NNA and L-NNA were measured by CEC in the chiral ligand exchange mode (Xin et al., 2005). Plasma samples (100 µl) or incubation samples (100 µl) of D-NNA or L-NNA were deproteinated through mixing for 20 min with methanol/acetone (1000 µl; v/v = 1:1). After centrifugation at 10,000 rpm for 20 min, the organic layer was dried at –20°C, and the residue was dissolved in acetate buffer (pH 5.0, plasma in 100 µl and incubation samples in 5 ml). After mixing for 1 min, a 10-nl aliquot of the sample was injected into the CEC system (Unimicro Technologies, Inc., Pleasanton, CA). Cupric acetate (1 mM) and aspartame (2 mM) were dissolved in the methanol/acetate buffer (pH 5.0, v/v = 1:20) to allow the formation of diastereomeric pairs with D-NNA or L-NNA. The diastereomeric pairs were separated through a reverse-phase C18 column (75 µm x 20 cm; Unimicro Tech. Ltd, Shanghai, China) at a detection wavelength of 280 nm. The retention times for L-NNA and D-NNA were 17 and 19 min, respectively. The samples were quantified against standard curves of L-NNA and D-NNA that ranged from 0.025 to 0.75 mM (r2 > 0.98). The between-day (n = 5) precision values measured by the time and the area under curve were 3.5 and 3.9% for L-NNA and D-NNA, respectively. The within-day precision of the quality control samples was 2% for both L-NNA and D-NNA.
Pharmacokinetic Studies. Conscious rats were divided into four groups (n = 5 in each group). Two groups of rats were injected with D-NNA (32 mg/kg) or L-NNA (16 mg/kg), respectively. The other two groups of rats were pretreated with injection of sodium benzoate (400 mg/kg), a selective inhibitor of DAAO that effectively blocks DAAO activity at this dose (Moses et al., 1996; Xin et al., 2005, 2006). Twenty minutes later, the benzoate-pretreated group was injected with D-NNA (32 mg/kg) or L-NNA (16 mg/kg), respectively. All the drugs were injected as an i.v. bolus via the indwelling cannula. The cannula was flushed with saline solution (600 µl, 0.9%) to ensure no contamination during blood sampling. Blood samples (200 µl) were obtained at 5, 20, 40, 60, 90, 120, 180, 240, 300, and 420 min after administration. Blood volume was replaced by injection of an equal volume of saline solution (0.9% NaCl) after each sampling. Plasma samples obtained were stored at –20°C for later analysis by CEC.
|
):
 |
The area under the curve (AUC) was calculated by the trapezoidal rule. Data Analyses. IC50 values for benzoate to block DAAO and D-NNA conversion were determined by using a curve fitting program, DoseResp of Origin 7.5 (Northampton, MA). All the results were expressed as mean ± S.E.M. and analyzed by the analysis of variance followed by Duncan's multiple range test. Differences with values of P < 0.05 were considered statistically significant.
|
Results and Discussion |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
; Xin et al., 2005, 2006). However, further experiments such as those using DAAO inhibitors are needed to confirm the above hypothesis. Sodium benzoate was selected as a DAAO inhibitor for this study to examine whether DAAO inhibitors block chiral inversion of D-NNA in vitro and in vivo.
Our previous study indicated that D-NNA but not L-NNA was the substrate of DAAO, and the Kcat/Km of DAAO to D-NNA was approximately 10% of that to D-alanine, the optimum substrate for kidney DAAO (Xin et al., 2006). In kidney homogenates, DAAO activity was measured by using the "keto acid method," in which 0.1 M D-alanine was used (D'Aniello et al., 1993; Sarower et al., 2003). Sodium benzoate inhibited DAAO activity in a sigmoid concentration-response manner (Fig. 1), with the IC50 value of 6.7 mM (95% confidence interval, 5.72–7.68 mM). Our results, together with a previous report that the Kd for benzoate to inhibit DAAO was approximately 0.2 mM (Pilone, 2000), suggest that benzoate is a weak inhibitor of DAAO. Moreover, the chiral inversion of D-NNA in this study was observed at 50 mM D-NNA, in contrast to the measurement of DAAO activity in which 0.1 M D-alanine was used. Benzoate also blocked the chiral inversion of D-NNA in kidney homogenates, with the concentration-response curve correlating very well with that of the inhibition of DAAO activity (Fig. 1). The IC50 value for benzoate to block D-NNA disposition was 2.1 mM (95% confidence interval, 1.31–2.88 mM), which was close to the inhibition of the DAAO activity. These results suggest benzoate blocks D-NNA conversion via the inhibition of the DAAO activity in kidney homogenates.
|
), as well as results from a previous study that showed benzoate led to the inhibition of DAAO activity (Williams and Lock, 2005). As seen in Fig. 2B, benzoate completely blocked D-NNA conversion in vivo, in agreement with our previous study that showed benzoate abolished the pressor response to naive D-NNA (Xin et al., 2005). In contrast to control rats, in which nearly no D-NNA was detected in plasma samples at 3 h after administration, plasma D-NNA was kept at 
10 µg/ml at 3 h and was still detectable at 7 h after D-NNA dosing in benzoate-pretreated rats. As a result of the blockade of chiral inversion of D-NNA, the mean clearance of D-NNA was significantly reduced (P < 0.05) by nearly 50% from 0.98 ± 0.07 l/h/kg to 0.49 ± 0.12 l/h/kg on coadministration (Table 1). Table 1 also shows that benzoate significantly increased t1/2 of D-NNA and approximately doubled the AUC of D-NNA. The effectiveness of benzoate to block chiral inversion of D-NNA and partially reduce clearance of D-NNA is in agreement with the previous report that pretreatment of sodium benzoate (1000 mg/kg, i.p.) led to a 40% reduction of D-ethionine disposition in 4 h in rats (Brada and Bulba, 1980). Similar results have also been shown in the study of D-methionine metabolism, in which i.p. treatment with sodium benzoate (0.9 g/kg) reflected a gradual clearance of D-methionine from the blood and a decrease of hepatic metabolism to D-methionine (London and Gabel, 1988).
|
|
). Unlike in D-NNA injection, in which L-NNA was immediately detected, the D-enantiomer of L-NNA was not detected in plasma samples during the period observed after L-NNA administration, confirming that chiral inversion of D-NNA was unidirectional (Wang et al., 1999). On the other hand, benzoate pretreatment, as shown in Fig. 3B, did not significantly affect pharmacokinetic parameters of L-NNA (Table 1), excluding the possibility that the effect of benzoate on D-NNA chiral inversion and clearance was nonspecific. Moreover, in the presence of benzoate for complete blockade of chiral inversion, the difference in pharmacokinetic parameters between D-NNA and L-NNA became significantly smaller (only approximately 2-fold) (Table 1). The results suggest that chiral inversion mostly contributes to clearance stereoselectivity of NNA.
According to the Pang and Kwan formula (1993), the calculated inversion rate of D-NNA to L-NNA was 36.7 ± 0.6%. The chiral inversion ratio of D-NNA to L-NNA calculated from this study was in agreement with the potency ratio (40%) of L-NNA/D-NNA to increase blood pressure in conscious rats (Wang et al., 1991), as well as the chiral inversion of D-NNA in anesthetized rats (Wang et al., 1999).
It is well known that most chiral drugs exhibit stereospecificity in their pharmacokinetics. Enantioselectivity of pharmacokinetics may be influenced by enzymatic metabolism, carrier transport, protein binding, distribution, and elimination of different stereoisomers (Burke and Henderson, 2002). In this study, we suggest that chiral inversion is an additional factor that contributes to pharmacokinetic stereospecificity, particularly in clearance. It was recently reported that many chiral pharmaceuticals undergo chiral inversion in vivo, such as certain D-amino acids (Lehmann et al., 1983; Hasegawa et al., 2000), 2-arylpropionic acid analogs (Drummond et al., 1990), the new quinoxaline topoisomerase poison 2-[4-(7-chloro-2-quinoxaliny)oxy] phenoxy propionic acid (Zheng et al., 2002), and thalidomide (Eriksson et al., 1998). Therefore, chiral inversion may be an important contributing factor for pharmacokinetic stereospecificity of these chiral drugs.
In summary, D-NNA and L-NNA exhibited biphasic disposition in conscious rats with different pharmacokinetic parameters. Unidirectional chiral inversion of D-NNA but not L-NNA was found in animals. In addition to its inhibition of D-NNA conversion in kidney homogenates, DAAO inhibitor sodium benzoate completely blocked chiral inversion of D-NNA and partially reduced clearance of D-NNA. The results suggest DAAO plays an essential role in chiral inversion of D-NNA, and chiral inversion contributes mostly to the pharmacokinetic stereospecificity of NNA.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: NNA, NG-nitro-arginine; D-NNA, NG-nitro-D-arginine; L-NNA, NG-nitro-L-arginine; DAAO, D-amino acid oxidase; CEC, capillary electrochromatography; AUC, area under the curve; CL, clearance.
Address correspondence to: Yong-Xiang Wang, Laboratory of Systems Pharmacology, School of Pharmacy, Shanghai Jiao Tong University, Biology Building No. 6 (Room 102), 800 Dongchuan Road, Shanghai 200240, China. E-mail: yxwang{at}sjtu.edu.cn
|
References |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) and the chiral inversion of D-NNA (
) in rat kidney homogenates (n = 4 samples in each point). All the readings are mean ± S.E.M.
L-NNA; 