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. Author manuscript; available in PMC: 2014 Aug 5.
Published in final edited form as: Biomed Chromatogr. 2013 Apr 5;27(8):1034–1040. doi: 10.1002/bmc.2901

Ultra-performance liquid chromatography tandem mass spectrometry method for the determination of AZ66, a sigma receptor ligand, in rat plasma and its application to in vivo pharmacokinetics

Seshulatha Jamalapuram a, Pradeep Kumar Vuppala a, Ahmed H Abdelazeem b,c, Christopher R McCurdy c, Bonnie A Avery a,*
PMCID: PMC4122523  NIHMSID: NIHMS608667  PMID: 23558564

Abstract

Methamphetamine abuse continues as a major problem in the USA owing to its powerful psychological addictive properties. AZ66, 3-[4-(4-cyclohexylpiperazine-1-yl)pentyl]-6-fluorobenzo[d]thiazole-2(3H)-one, an optimized sigma receptor ligand, is a promising therapeutic agent against methamphetamine. To study the in vivo pharmacokinetics of this novel sigma receptor ligand in rats, a sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed in rat plasma and validated. The developed method requires a small volume of plasma (100 μL) and a simple liquid–liquid extraction. The chromatographic separations were achieved in 3.3 min using an Acquity UPLC BEH Shield RP18 column. The mass spectrophotometric detection was carried out using a Waters Micromass Quattro MicroTM triple-quadrupole system. Multiple reaction monitoring was used for the quantitation with transitions m/z 406→m/z 181 for AZ66 and m/z 448→m/z 285 for aripiprazole. The method was validated over a concentration range of 1–3500 ng/mL and the lower limit of quantitation was determined to be 1 ng/mL. Validation of the assay demonstrated that the developed UPLC/MS/MS method was sensitive, accurate and selective for the determination of AZ66 in rat plasma. The present method has been successfully applied to an i.v. pharmacokinetic study in Sprague–Dawley rats.

Keywords: mass spectrometry, AZ66, pharmacokinetics, rats, method validation

Introduction

Methamphetamine (METH) is a psychomotor stimulant, neurotoxic, addictive and the second most illicit drug abused world wide (Kiyatkin and Sharma, 2009; Lai et al., 2009; Sharma and Kiyatkin, 2009). Methamphetamine can be synthesized readily from over-the-counter drugs. Current surveys estimate that around 15–16 million people have abused methamphetamine (Krasnova and Cadet, 2009). The short-term stimulant effects of METH include euphoria, hyperthermia, enhanced energy, increased physical activity and decreased appetite (Krasnova and Cadet, 2009; Kaushal and Matsumoto, 2011). Repeated METH use has been reported to result in addiction, psychosis, changes in brain structure and function involving terminals of dopamine and serotonin neurons, memory loss and neurodegeneration (Ricaurte et al., 1980; Sharma and Kiyatkin, 2009; Kousik et al., 2012). Multiple mechanisms that contribute to the METH-induced neurotoxicity include oxidative stress owing to formation of oxygen and nitrogen reactive species, abnormal dopamine and glutamate transmission, mitochondrial dysfunction, astroglial and microglial activation and brain hyperthermia (Brown et al., 2003; Thomas et al., 2004; Cadet et al., 2005; Sharma and Kiyatkin, 2009). However, the mechanisms underlying these neurodegenerative effects are not yet thoroughly understood. Methamphetamine enhances the synaptic levels of norepinephrine, dopamine and serotonin by reversing the transport that facilitates excess release of these monoamines and also by preventing their re-uptake (Rothman et al., 2001; Kousik et al., 2012). Various effects of METH have been reported to be related to its interaction with monoamine transporters. Nevertheless, METH also has an affinity for both sigma-1 and sigma-2 receptors and it interacts with sigma receptors at physiologically attainable concentrations, suggesting that they play a role in the effect of METH (Nguyen et al., 2005; Kaushal et al., 2011; Rodvelt et al., 2011; Seminerio et al., 2012b). Sigma receptors are unique proteins discovered by Martin et al. in 1976 (Martin et al., 1976; Zeng et al., 2007; Garces-Ramirez et al., 2011; Jamalapuram et al., 2012). Based on the specific pharmacological and functional characteristics, sigma receptors are divided into two subtypes, sigma-1 and sigma-2 (Hellewell et al., 1994; Crawford and Bowen, 2002; Kashiwagi et al., 2009). The sigma-1 receptor is a 25–29 kDa protein with 223 amino acids and was cloned in 1996 (Hanner et al., 1996; Monassier and Bousquet, 2002; Romieu et al., 2002; Daniels et al., 2006). It is localized in various organs, such as liver, heart and the gastrointestinal tract. The limbic system of brain is particularly rich in sigma-1 receptors (Crawford and Bowen, 2002; Monassier and Bousquet, 2002). The sigma-2 receptor is an 18–21 kDa protein that has not yet been cloned (Wheeler et al., 2000; Kashiwagi et al., 2009). Sigma receptors are distributed in systems that mediate the actions of METH such as the dopaminergic system in the brain. The activation of these receptors results in the synthesis and release of dopamine (Weiser et al., 1995; Matsumoto et al., 2008; Rodvelt et al., 2011). At this time, there is no pharmacotherapy approved by the US Food and Drug Administration (FDA) to treat the harmful effects of METH (Kaushal and Matsumoto, 2011; Graves and Napier, 2012).

Sigma receptor antagonists are currently being investigated as potential agents for the treatment of METH abuse and toxicity. Targeting these receptors could become a promising therapeutic approach in treating addiction to METH (Kaushal et al., 2011; Seminerio et al., 2012b). In addition, the neurotoxic and stimulant effects of METH were shown to be attenuated in mouse by sigma receptor antagonists such as AC927 and CM156 (Kaushal et al., 2011, 2012). Even though several sigma receptors antagonists have been synthesized, a primary concern with these compounds is that they are not purely selective for sigma receptors (Xu et al., 2010). In addition to their affinity for sigma receptors, most of these compounds also interact with dopamine transporters, opioid receptors or N-methyl-D-aspartate receptors.

AZ66 {3-[4-(4-cyclohexylpiperazin-1-yl)pentyl]-6-fluorobenzo[d] thiazole-2(3H)-one; Fig. 1} is a synthetic piperazine derivative identified as a promising lead for the treatment of METH abuse, based on its toxicological and pharmacological data. Radioligand binding studies have shown that AZ66 has high nanomolar affinity for both subtypes of sigma receptors and is highly selective over a wide battery of CNS targets (Seminerio et al., 2012b). In addition, AZ66 exhibited an appreciably longer half-life in vitro compared with CM156 {3-[4-(4-cyclohexylpiperazin-1-yl)butyl]benzo[d]thiazole- 2(3H)-thione}, a precursor of AZ66 that exhibited an in vitro half-life of 4.62 min in rat liver microsomes. AZ66 was found to significantly attenuate convulsions in mice treated with a toxic dose of cocaine, demonstrating its anticocaine activity (Seminerio et al., 2012b). After intraperitoneal administeration to Swiss Webster mice, AZ66 was shown to attenuate METH-induced dopaminergic neurotoxicity and hyperthermia. AZ66 also attenuated the memory impairment caused by the repeated intake of METH in mouse (Seminerio et al., 2012a).

Figure 1.

Figure 1

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Chemical structure of AZ66 (a) and aripiprazole (b).

To aid discovery programs, accurate pharmacokinetics and metabolism data must be collected as early as possible. To completely describe the pharmacokinetic behavior of drug candidates in laboratory animals and in humans, an array of bioanalytical methods are required. In the present study, a sensitive and reproducible analytical method was developed and validated for quantification of AZ66 in rat plasma using UPLC/MS/MS. The sample preparation is simple and employs a single step liquid–liquid extraction. The assay was validated for selectivity, sensitivity, linearity, recovery, matrix effect, accuracy, precision and stability. The developed and validated method was successfully utilized to study the pharmacokinetic behavior of AZ66 in male Sprague–Dawley rats, following i.v. administration.

Experimental

Chemicals and reagents

AZ66 (>99% purity as determined by HPLC) was synthesized in the Department of Medicinal Chemistry, The University of Mississippi (Oxford, MS, USA) as previously reported (Seminerio et al., 2012a). Aripiprazole (99% purity), internal standard (IS), was purchased from Sigma-Aldrich (St Louis, MO, USA). Acetic acid and ammonium acetate were purchased from Sigma-Aldrich (St Louis, MO, USA). Solvents (HPLC grade) such as methanol, acetonitrile and water were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Rat plasma was obtained from Innovative Research (Peary Court Novi, MI, USA).

Calibration standards and quality control sample preparation

An accurately weighed amount (0.31 mg) of AZ66 was dissolved in water (310 μL) to obtain a stock solution at a concentration of 1.0mg/mL. A series of working standard solutions of AZ66 were prepared at concentrations from 10 to 35,000 ng/mL by diluting the stock solution with methanol. The IS stock solution (1mg/mL) was prepared by dissolving 0.24mg of aripiprazole in 240 μL methanol. The working standard solution (3 μg/mL) of the IS was prepared by diluting 3 μL of 1mg/mL solution with 997 μL of methanol. All stock solutions were stored at −20 °C. Calibration standards were freshly prepared by diluting blank rat plasma (100 μL) with the working standard solutions to obtain final concentrations of 1, 10, 50, 100, 500, 1000, 2000 and 3500 ng/mL. Quality control (QC) samples were prepared at three concentrations of 2, 400 and 3000 ng/mL by diluting rat plasma (100 μL) with the appropriate amounts of each working standard solution.

Sample preparation

A simple liquid–liquid extraction method was used for the extraction of AZ66 from rat plasma. Plasma samples (100 μL) were spiked with 10 μL of the IS and vortexed for 30 s. The samples were extracted with 800 μL of chloroform by vortex-mixing for 10min and the resultant mixture was then centrifuged at 10,000 g for 10min at 4 °C. The organic phase was transferred in to an eppendorf tube and evaporated to dryness in a vacuum oven at 25 °C. The residue was dissolved in 100 μL of methanol by vortex-mixing for 1 min. An aliquot of 10 μL of sample was used for the UPLC/MS/MS analysis.

UPLC/MS/MS conditions

An Acquity UPLC (Waters Corp., Milford, MA, USA) system coupled with a tandem mass spectrophotometer was used for the quantitative analysis of AZ66. Chromatographic separations were achieved on an Acquity UPLC BEH Shield RP 18 column (2.1 × 150mm). The mobile phase consisted of 10mM ammonium acetate containing 0.1% acetic acid and methanol (40:60, v/v) and pumped at a flow rate of 0.3mL/min. The sample injection volume was 10 μL. The total run time was 3.3min.

A Micromass Quattro micro™ system (Waters Corp., Manchester, UK) equipped with an electrospray ionization source (ESI) was used for the mass spectrophotometric detection. The ESI was operated in the positive ionization mode. The acquisitions of AZ66 and IS were performed using multiple reaction monitoring. The mass transitions chosen for the quantitation of the compounds were m/z 406→m/z 181 for AZ66 and m/z 448→m/z 285 for the IS. The optimized mass spectrophotometric parameters were: capillary, cone, extractor and RF lens voltages were set at 4.88 kV, 44 V, 3 V and 0.5 V, respectively. The source temperature and desolvation temperature were set at 100 and 250 °C, respectively. The desolvation and cone gas flows were set at 500 and 60 L/h, respectively. Argon was used as the collision gas at 3.5 × 10−3 Pirani. Collision energies were set at 30 and 21 eV for AZ66 and the IS, respectively.

Validation of assay

The analytical method was validated according to the FDA guidelines for bioanalytical method validation (US Food and Drug Administration, 2001).

Selectivity and sensitivity

The selectivity of the method was assessed by comparing the chromatograms of the blank non-pooled rat plasma from six different batches with the corresponding rat plasma samples spiked with AZ66 and the IS. The lower limit of quantification (LLOQ) was defined as the lowest concentration that resulted in a signal to noise (S/N) ratio of 10. The acceptable criterion for the LLOQ was relative standard deviation (RSD) and accuracy values less than ±20%. The LLOQ was evaluated by analyzing samples in six replicates on three consecutive days. The limit of detection (LOD) was defined as the concentration of analyte that yielded an S/N ratio of 3.

Linearity

Calibration standards were prepared by spiking blank rat plasma with standard working solutions of AZ66 to obtain standards at concentrations of 1, 10, 50, 100, 500, 1000, 2000 and 3500 ng/mL. An aliquot of 10 μL of the IS was also added to obtain a concentration of 3 μg/mL. The calibration curve was constructed by plotting the peak area ratios of analyte to the IS vs analyte concentrations.

Extraction recovery and matrix effect

The extraction recovery of AZ66 from rat plasma was determined at low, medium and high concentrations (2, 400 and 3000 ng/mL). The extraction recovery was calculated by comparing the peak area ratios of blank plasma samples spiked with analyte and IS before extracting the plasma, with peak area ratios of blank plasma samples to which analyte and IS were added after the extraction of blank plasma.

The matrix effect was assessed at three QC levels (2, 400 and 3000 ng/mL) by comparing the analyte/IS peak area ratios of extracted plasma samples with those of the corresponding AZ66 standard solutions prepared inmethanol. This study was conducted with blank rat plasma from six different lots.

Precision and accuracy

The intra-day and inter-day precision and accuracy of the method were assessed from the QC samples at concentrations 2, 400 and 3000 ng/mL. To determine intra-day precision and accuracy, six replicates of QC samples were analyzed at all the three concentration levels on the same day. To determine the inter-day precision and accuracy all the three QC samples were analyzed on three consecutive days. The precision was expressed as relative standard deviation (RSD) and the accuracy as percentage bias. The intra-and inter-day precision and accuracy should not be more than 15% of RSD and 15% of percentage bias, respectively.

Percentagebias=100×[(measured-actual)/actual]

Stability

Stability of a drug in plasma is a function of the chemical properties of the drug, the storage conditions, the matrix and the containers used for sample storage and processing. Stability studies should be conducted at different storage conditions that might be encountered during regular sample handling and processing.

To determine the stability of AZ66, QC samples (six replicates) at concentrations of 10, 400 and 3000 ng/mL were exposed to various storage conditions. Freeze–thaw stability was investigated for three successive freeze–thaw cycles at −20 °C. For long-term stability, the samples were stored at −20 °C for 30 days. Short-term temperature stability was assessed at room temperature for 12 h. Post-operative stability was also evaluated by analyzing the processed samples stored in the auto-sampler at 25 °C for 24 h. The stability of the stock solutions of analyte and IS was tested daily for a period of 1 week at −20 °C. The concentrations of the stored samples for all the stability studies were compared with the freshly prepared standards.

Application to an intravenous pharmacokinetic study in rats

To assess the applicability of the developed method, the intravenous pharmacokinetic profile of AZ66 in rats was determined. The rat pharmacokinetic protocol was approved by the Institutional Animal Care and Use Committee of the University of Mississippi (OLAW Assurance number is A3356-01). Male Sprague–Dawley rats (n = 6) weighing 260–280 g were obtained from Harlan Laboratories (Indianapolis, IN, USA). The rats were fasted overnight before the administration of the compound and then had free access to water after dosing. The rats were also allowed free access to food beginning 4 h after dosing. A single i.v. dose of AZ66 was administered at a dose of 5mg/kg through the jugular-vein cannula. Blood samples were collected via the jugular-vein cannula just before dosing and subsequently at times of 2, 15, 30, 60, 120, 240, 480 min, and 12, 24, 30, 36 and 42 h after dosing. To clear the heparinized saline from the cannula, a blood volume of 0.05mL was withdrawn and a clean syringe was used to collect 0.15mL of blood sample. After each blood sampling, the cannula was flushed with 0.2 mL of a heparin-saline (10 IU/mL) solution. Blood samples were transferred into heparinized microcentrifuge tubes and centrifuged immediately at 10,000 g for 10 min at 4 °C. The plasma was separated from the blood cells, transferred to clean Eppendorf tube and stored at −20 °C until analyzed. The pharmacokinetic study samples were processed as described above. Pharmacokinetic parameters were determined by analyzing the plasma concentration-time data by non-compartmental analysis using WinNonlin 5.2 (Pharsight, Mountain View, CA, USA).

Results and discussion

Method development

AZ66 exhibited a higher mass spectrophotometric response using the ESI positive mode compared with the negative mode. The most intense analyte signal appeared at m/z of 406. Several fragment ions were observed in the product ion spectra, but the most abundant product ions were noted at m/z of 181 and 285 for AZ66 and the IS, respectively. The mass spectrophotometric parameters such as collision energy, source temperature, capillary voltage and gas flow were optimized to enhance this signal intensity. Figure 2 shows the product ion scans of [M + H]+ of AZ66 and the IS. Aripiprazole was chosen as the internal standard owing to its similar chromatographic and mass spectrophotometric characteristics to AZ66 and negligible endogenous interferences at m/z 448 (Vuppala et al., 2011).

Figure 2.

Figure 2

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Product ion mass spectra of AZ66 (a) and aripiprazole (b).

The chromatographic conditions were optimized through several trials using various columns and mobile phases to achieve good chromatographic behavior and better ionization of AZ66 and the IS. Columns such as the Acquity UPLC C18 (2.1 × 50mm), C8 (2.1 × 50mm) and Atlantis dC18 (2.1 × 50mm) were examined. These columns resulted in broader peaks with 0.4–0.5 min width. In order to achieve sharper peaks we tried the Acquity UPLC HILIC (2.1 × 50mm) and BEH Shield RP 18 (2.1 × 150mm) columns. The unique bonding chemistry (the embedded carbamate group in the bonded phase ligand) of the BEH Shield RP18 column enhances the peak shape for basic compounds. It resulted in the best performance with a peak width of 7–9 s. Ammonium acetate, formic acid and acetic acid were used alone or in combination at various concentrations in aqueous phase for the baseline separation of the analyte and the IS and to achieve higher ionization efficiency and the sensitivity. Ultimately, a 0.1% acetic acid solution with 10mM ammonium acetate in water and methanol (40:60, v/v) was chosen as the mobile phase. This mobile phase resulted in good peak shapes and high sensitivity (LOD 0.5 ng/mL). The retention times of AZ66 and the IS were 1.3 and 1.0 min, respectively, resulting in a total run time of 3.3 min.

Method validation

Selectivity and sensitivity

Typical chromatograms obtained from the LC/MS/MS analysis of blank rat plasma, blank rat plasma spiked with AZ66 and the IS, and a rat plasma sample obtained 1 h after the intravenous administration of AZ66 (5 mg/kg) are represented in Fig. 3. No obvious interferences from endogenous components were observed at the retention times of AZ66 and the IS. The LLOQ for AZ66 in plasma was found to be 1 ng/mL with precision (RSD) below 20% (2.2) and accuracy (percentage bias) within ±20% (2.1). A corresponding chromatogram is shown in Fig. 3(b). The LOD was defined at an S/N ratio of 3:1 (Jamalapuram et al., 2012) and was found to be 0.5 ng/mL.

Figure 3.

Figure 3

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Multiple reaction monitoring chromatograms of AZ66 and aripiprazole (IS) in rat plasma: (A) a blank plasma; (B) spiked plasma with AZ66 at 1 ng/mL and IS and 3 μg/mL; (C) rat plasma sample obtained at 1.0 h after i.v. administration at a dose of 5 mg/kg.

Linearity of calibration curve

The calibration curves for AZ66 were linear over the concentration range of 1–3500 ng/mL (r2>0.99). The representative regression equation for the calibration curve was:

y=0.0511x+0.1682

where y represents the peak area ratios of AZ66 to the IS and x represents concentrations of analyte in plasma. The RSD of the slope was 0.00018 and the RSD of the intercept was 0.0031. Table 1 represents the linearity of calibration curves for AZ66.

Table 1.

Linearity of calibration curves for AZ66 in rat plasma

Intra-day RSD (%) Inter-day RSD (%)
y- Intercept 0.0031 0.0034
Slope 0.0002 0.0002
r2 0.01 0.01
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RSD, Relative standard deviation.

Precision and accuracy of the assay

The results of the intraand inter-day precision and accuracy are presented in Table 2. The intra-day precisions for the three QC levels of AZ66 were 2.0, 0.1 and 0.1% (RSD) and the inter-day precisions were 3.0, 0.4 and 0.1% (RSD). The intra-day accuracies for the three QC levels were 1.7, 0.4 and 0.3% (percentage bias) and the inter-day accuracies were 6.3, 0.7 and 0.3% (percentage bias) for the all three QC concentrations. The data indicates that the precision and accuracy were within the acceptable limits specified by the FDA where the precision (RSD) determined should not exceed 15% and accuracy (percentage bias) should be within ±15% of the actual value. These results suggest that the reported method is consistent and reproducible for the quantitation of AZ66 in rat plasma.

Table 2.

Inter- and intra-day precision and accuracy of the assay in rat plasma

Spiked concentration (ng/mL) Recovery
Intra-day precision and accuracy
Inter-day precision and accuracy
Bias (%) Mean ± SD (ng/mL) RSD (%) Bias (%) Mean ± SD (ng/mL) RSD (%) Bias (%)
1 - 1.1 ± 0.01 2.2 2.1 1.0 ± 0.01 2.4 4.6
2 0.8 2.0 ± 0.02 2.0 1.7 2.1 ± 0.03 3.0 6.3
400 0.6 401.4 ± 0.5 0.1 0.4 402.8 ± 1.6 0.4 0.7
3000 1.1 3008 ± 3.7 0.1 0.3 3009 ± 4.3 0.1 0.3
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The precision is expressed as RSD and the accuracy as percentage bias.

Extraction recovery and matrix effect

According to FDA guidelines, the extraction recovery of analyte and IS needs to be invariable and reproducible and need not be 100%. The extraction recoveries of AZ66 were 97.7 ± 3.4, 98.2 ± 2.9 and 98.6 ± 3.7% for QC samples at the concentrations of 2, 400 and 3000 ng/mL, respectively. The percentage bias values for the recovery at three QC levels were 0.8, 0.6 and 1.1%. These results suggested that the recovery of AZ66 was precise and reproducible at all the three concentrations. The extraction recovery of the IS was consistent and found to be 67.5 ± 3.8%.

The matrix effect of AZ66 was evaluated by analyzing QC samples at three concentration levels of 2, 400 and 3000 ng/mL. The mean matrix effect values ranged from 96.5 to 101.4%. The matrix effect of the IS was 95.4%. This indicated that the ion suppression from plasma matrix for AZ66 and IS was negligible.

Stability

To investigate the stability of AZ66 in rat plasma, QC samples at concentrations of 10, 400 and 3000 ng/mL were stored under various conditions that may be encountered during routine sample handling, processing and analysis. Table 3 summarizes the results of the freeze–thaw, short-term, long-term and postoperative stabilities of AZ66 in rat plasma. The results suggested that AZ66 was found to be stable after three freeze–thaw cycles, at room temperature for 12 h (25 °C) and at −20 °C for 30 days. The compound was also stable in reconstituted samples stored in the auto-sampler at 25°C for 24 h. AZ66 and the IS stock solutions were stable for 12 h maintained at room temperature and for a week stored at −20°C. No loss of compound was observed at all the storage condition examined, suggesting that the plasma samples can be stored at −20 °C for a period of 30 days.

Table 3.

Stability of AZ66 in rat plasma at various storage conditions

Storage conditions Concentration (ng/mL)
RSD (%) Bias (%)
Spiked Measured
Three freeze–thaw cycles 10 10.2 ± 0.7 6.9 −3.1
400 396.1 ± 12.7 3.2 −0.6
3000 2990 ± 42.9 1.4 0.2
Long-term for 30 days (−20 °C) 10 10.2 ± 0.4 3.9 0.3
400 402.4 ± 7.1 1.8 −0.4
3000 3028 ± 55.0 1.8 −0.6
Short-term for 12 h (25 °C) 10 10.2 ± 0.2 2.4 1.3
400 401.4 ± 3.5 0.9 −0.5
3000 3015 ± 30.6 0.9 1.0
Auto-sampler for 24 h (25 °C) 10 10.1 ± 0.2 2.0 1.7
400 403.2 ± 18.6 4.6 0.8
3000 3222 ± 79.4 2.5 4.1
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Application to an i.v. pharmacokinetic study in rats

The developed method was used to determine the plasma concentration of AZ66 in male Sprague–Dawley rats following i.v. (5 mg/kg) administration of the compound. Figure 4 shows the mean plasma concentration–time profiles of AZ66 after i.v. administration. The pharmacokinetic parameters are summarized in Table 4. After i.v. administration the peak plasma concentration (Cmax) was found to be 1.2 ± 0.2 μg/mL. AZ66 concentrations in plasma declined with a half-life of 7.9 ± 0.3 h after i.v. dosing. The volume of distribution was found to be 30.1 ± 3.6 L/kg. The half-life of the compound may be owing to the high volume of distribution and low clearance (2.6 ± 0.4).

Figure 4.

Figure 4

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Mean plasma concentration of AZ66 after i.v. administration at a dose of 5 mg/kg.

Table 4.

Noncompartmental pharmacokinetic parameters in rats after i.v. administration of 5 mg/kg of AZ66 (n=6)

Parameter Mean ± SD
t1/2 (h) 7.9 ± 0.3
Cmax (μg/mL) 1.2 ± 0.2
Vd (L/kg) 30.1 ± 3.6
CL (L/h/kg) 2.6 ± 0.4
MRT (h) 7.6 ± 0.7
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t1/2, Elimination half-life; Cmax, peak plasma concentration; Vd, volume of distribution; CL, total clearance; MRT, mean residence time; SD, standard deviation.

Conclusions

A sensitive and reproducible UPLC/MS/MS method for the determination of AZ66 in rat plasma has been developed and validated. The developed method uses simple liquid–liquid extraction for sample pretreatment. This method demonstrated a short chromatographic run time, and excellent sensitivity, linearity, precision and accuracy. The calibration curve was linear over the range of 1–3500 ng/mL with an LLOQ of 1 ng/mL. Inter- and intra-day assay variations were found to be <15%. AZ66 and the IS were successfully separated from endogenous interferences. The UPLC/MS/MS assay was applied to a pharmacokinetic study of AZ66 after i.v. administration in rats.

Acknowledgments

This work was supported in part by grants from the National Institute on Drug Abuse (DA023205, C.R.M.) and the National Center for Research Resources (5P20RR021929).

Abbreviations used

ESI

electrospray ionization

METH

methamphetamine

References

  1. Brown PL, Wise RA, Kiyatkin EA. Brain hyperthermia is induced by methamphetamine and exacerbated by social interaction. Journal of Neuroscience. 2003;23:3924–3929. doi: 10.1523/JNEUROSCI.23-09-03924.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cadet JL, Jayanthi S, Deng X. Methamphetamine-induced neuronal apoptosis involves the activation of multiple death pathways. Neurotoxicity Research. 2005;8:199–206. doi: 10.1007/BF03033973. [DOI] [PubMed] [Google Scholar]
  3. Crawford KW, Bowen WD. Sigma-2 receptor agonists activate a novel apoptotic pathway and potentiate antineoplastic drugs in breast tumor cell lines. Cancer Research. 2002;62:313–322. [PubMed] [Google Scholar]
  4. Daniels A, Ayala E, Chen W, Coop A, Matsumoto RR. N-[2-(m-methoxyphenyl) ethyl]-N-ethyl-2-(1-pyrrolidinyl)ethylamine (UMB 116) is a novel antagonist for cocaine-induced effects. European Journal of Pharmacology. 2006;542:61–68. doi: 10.1016/j.ejphar.2006.03.062. [DOI] [PubMed] [Google Scholar]
  5. Garces-Ramirez L, Green JL, Hiranita T, Kopajtic TA, Mereu M, Thomas AM, Mesangeau C, Narayanan S, McCurdy CR, Katz JL, Tanda G. Sigma receptor agonists: receptor binding and effects on mesolimbic dopamine neurotransmission assessed by microdialysis. Biological Psychiatry. 2011;69:208–217. doi: 10.1016/j.biopsych.2010.07.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Graves SM, Napier TC. SB 206553, a putative 5-HT2C inverse agonist, attenuates methamphetamine-seeking in rats. BMC Neuroscience. 2012;13:1–10. doi: 10.1186/1471-2202-13-65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hanner M, Moebius FF, Flandorfer A, Knaus HG, Striessnig J, Kempner E, Glossmann H. Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proceedings of the National Academy of Sciences of the United States of America. 1996;93:8072–8077. doi: 10.1073/pnas.93.15.8072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hellewell SB, Bruce A, Feinstein G, Orringer J, Williams W, Bowen WD. Rat liver and kidney contain high densities of sigma 1 and sigma 2 receptors: characterization by ligand binding and photoaffinity labeling. European Journal of Pharmacology. 1994;268:9–18. doi: 10.1016/0922-4106(94)90115-5. [DOI] [PubMed] [Google Scholar]
  9. Jamalapuram S, Vuppala PK, Mesangeau C, McCurdy CR, Avery BA. Determination of a highly selective mixed-affinity sigma receptor ligand, in rat plasma by ultra performance liquid chromatography mass spectrometry and its application to a pharmacokinetic study. Journal of Chromatography B. 2012;891–892:1–6. doi: 10.1016/j.jchromb.2011.12.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kashiwagi H, McDunn JE, Simon PO, Jr, Goedegebuure PS, Vangveravong S, Chang K, Hotchkiss RS, Mach RH, Hawkins WG. Sigma-2 receptor ligands potentiate conventional chemotherapies and improve survival in models of pancreatic adenocarcinoma. Journal of Translational Medicine. 2009;7:1–8. doi: 10.1186/1479-5876-7-24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaushal N, Matsumoto RR. Role of sigma receptors inmethamphetamine-induced neurotoxicity. Current Neuropharmacology. 2011;9:54–57. doi: 10.2174/157015911795016930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaushal N, Seminerio MJ, Shaikh J, Medina MA, Mesangeau C, Wilson LL, McCurdy CR, Matsumoto RR. CM156, a high affinity sigma ligand, attenuates the stimulant and neurotoxic effects of methamphetamine in mice. Neuropharmacology. 2011;61:992–1000. doi: 10.1016/j.neuropharm.2011.06.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaushal N, Elliott M, Robson MJ, Iyer AK, Rojanasakul Y, Coop A, Matsumoto RR. AC927, a sigma receptor ligand, blocks methamphetamine- induced release of dopamine and generation of reactive oxygen species in NG108-15 cells. Molecular Pharmacology. 2012;81:299–308. doi: 10.1124/mol.111.074120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kiyatkin EA, Sharma HS. Acute methamphetamine intoxication: brain hyperthermia, blood–brain barrier, brain edema, and morphological cell abnormalities. International Review of Neurobiology. 2009;88:65–100. doi: 10.1016/S0074-7742(09)88004-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kousik SM, Napier TC, Carvey PM. The effects of psychostimulant drugs on blood brain barrier function and neuroinflammation. Frontiers in Pharmacology. 2012;3:121. doi: 10.3389/fphar.2012.00121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Krasnova IN, Cadet JL. Methamphetamine toxicity and messengers of death. Brain Research Reviews. 2009;60:379–407. doi: 10.1016/j.brainresrev.2009.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lai H, Zeng H, Zhang C, Wang L, Tso MO, Lai S. Toxic effect of methamphetamine on the retina of CD1 mice. Current Eye Research. 2009;34:785–790. doi: 10.1080/02713680903079641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Martin WR, Eades CE, Thompson JA, Huppler RE. The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. Journal of Pharmacology and Experimental Therapeutics. 1976;197:517–532. [PubMed] [Google Scholar]
  19. Matsumoto RR, Shaikh J, Wilson LL, Vedam S, Coop A. Attenuation of methamphetamine-induced effects through the antagonism of sigma (sigma) receptors: evidence from in vivo and in vitro studies. European Neuropsychopharmacology. 2008;18:871–881. doi: 10.1016/j.euroneuro.2008.07.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Monassier L, Bousquet P. Sigma receptors: from discovery to highlights of their implications in the cardiovascular system. Fundamental and Clinical Pharmacology. 2002;16:1–8. doi: 10.1046/j.1472-8206.2002.00063.x. [DOI] [PubMed] [Google Scholar]
  21. Nguyen EC, McCracken KA, Liu Y, Pouw B, Matsumoto RR. Involvement of sigma (sigma) receptors in the acute actions of methamphetamine: receptor binding and behavioral studies. Neuropharmacology. 2005;49:638–645. doi: 10.1016/j.neuropharm.2005.04.016. [DOI] [PubMed] [Google Scholar]
  22. Ricaurte GA, Schuster CR, Seiden LS. Long-term effects of repeated methylamphetamine administration on dopamine and serotonin neurons in the rat brain: a regional study. Brain Research. 1980;193:153–163. doi: 10.1016/0006-8993(80)90952-x. [DOI] [PubMed] [Google Scholar]
  23. Rodvelt KR, Oelrichs CE, Blount LR, Fan KH, Lever SZ, Lever JR, Miller DK. The sigma receptor agonist SA4503 both attenuates and enhances the effects of methamphetamine. Drug and Alcohol Dependence. 2011;116:203–210. doi: 10.1016/j.drugalcdep.2010.12.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Romieu P, Phan VL, Martin-Fardon R, Maurice T. Involvement of the sigma (1) receptor in cocaine-induced conditioned place preference: possible dependence on dopamine uptake blockade. Neuropsychopharmacology. 2002;26:444–455. doi: 10.1016/S0893-133X(01)00391-8. [DOI] [PubMed] [Google Scholar]
  25. Rothman RB, Baumann MH, Dersch CM, Romero DV, Rice KC, Carroll FI, Partilla JS. Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse. 2001;39:32–41. doi: 10.1002/1098-2396(20010101)39:1<32::AID-SYN5>3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  26. Seminerio MJ, Robson MJ, Abdelazeem AH, Mesangeau C, Jamalapuram S, Avery BA, McCurdy CR, Matsumoto RR. Synthesis and pharmacological characterization of a novel sigma receptor ligand with improved metabolic stability and antagonistic effects against methamphetamine. The AAPS Journal. 2012a;14:43–51. doi: 10.1208/s12248-011-9311-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Seminerio MJ, Hansen R, Kaushal N, Zhang HT, McCurdy CR, Matsumoto RR. The evaluation of AZ66, an optimized sigma receptor antagonist, against methamphetamine-induced dopaminergic neurotoxicity and memory impairment in mice. The International Journal of Neuropsychopharmacology. 2012b;29:1–12. doi: 10.1017/S1461145712000831. [DOI] [PubMed] [Google Scholar]
  28. Sharma HS, Kiyatkin EA. Rapid morphological brain abnormalities during acute methamphetamine intoxication in the rat: an experimental study using light and electron microscopy. Journal of Chemical Neuroanatomy. 2009;37:18–32. doi: 10.1016/j.jchemneu.2008.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas DM, Dowgiert J, Geddes TJ, Francescutti-Verbeem D, Liu X, Kuhn DM. Microglial activation is a pharmacologically specific marker for the neurotoxic amphetamines. Neuroscience Letters. 2004;367:349–354. doi: 10.1016/j.neulet.2004.06.065. [DOI] [PubMed] [Google Scholar]
  30. US Food and Drug Administration. FDA Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research; Rockville, MD: 2001. [Google Scholar]
  31. Vuppala P, Boddu S, Furr E, McCurdy CR, Avery BA. Simple, sensitive, high-throughput method for the quantification of mitragynine in rat plasma using UPLC-MS and its application to an intravenous pharmacokinetic study. Chromatographia. 2011;74:703–710. [Google Scholar]
  32. Weiser SD, Patrick SL, Mascarella SW, Downing-Park J, Bai X, Carroll FI, Walker JM, Patrick RL. Stimulation of rat striatal tyrosine hydroxylase activity following intranigral administration of sigma receptor ligands. European Journal of Pharmacology. 1995;275:1–7. doi: 10.1016/0014-2999(94)00718-m. [DOI] [PubMed] [Google Scholar]
  33. Wheeler KT, Wang LM, Wallen CA, Childers SR, Cline JM, Keng PC, Mach RH. Sigma-2 receptors as a biomarker of proliferation in solid tumours. British Journal of Cancer. 2000;82:1223–1232. doi: 10.1054/bjoc.1999.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Xu YT, Kaushal N, Shaikh J, Wilson LL, Mesangeau C, McCurdy CR, Matsumoto RR. A novel substituted piperazine, CM156, attenuates the stimulant and toxic effects of cocaine in mice. Journal of Pharmacology and Experimental Therapeutics. 2010;333:491–500. doi: 10.1124/jpet.109.161398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zeng C, Vangveravong S, Xu J, Chang KC, Hotchkiss RS, Wheeler KT, Shen D, Zhuang ZP, Kung HF, Mach RH. Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy. Cancer Research. 2007;67:6708–6716. doi: 10.1158/0008-5472.CAN-06-3803. [DOI] [PubMed] [Google Scholar]

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