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Quantitative determination of the anticancer prodrug combretastatin A1 phosphate (OXi4503, CA1P), the active CA1 and its glucuronide metabolites in human urine and of CA1 in plasma by HPLC with mass spectrometric detection
29 May 2012,
10:25:41
Publication year:
2012
Source:Journal of Chromatography B, Volume 898
Michael R.L. Stratford, Lisa K. Folkes
Validated methods for the determination of CA1, the active agent derived from the prodrug CA1P, in human plasma and urine, and of CA1P and three glucuronides CA1G1, CA1G2 and CA1DG in human urine were developed using LC–MS. Plasma CA1 was extracted using solid phase extraction and validated over the range 5–1000nM. Urine samples were analysed without extraction, and the assays validated over the range 50–2000nM (CA1P), 25–2000nM (CA1), 50–40,000nM (CA1G1 and CA1G2) and 25–4000nM (CA1DG). The mean correlation coefficient (r 2) was ≥0.997 for all assays. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical methods (<15%). Mean recovery of CA1 from plasma was 101%, and 97% from urine. Mean urine recovery of CA1P was 98%, CA1G1 96%, CA1G2 93% and CA1DG 93%. The method was applied to plasma and urine samples from a recently completed clinical trial of the prodrug. Peak plasma concentrations of up to 470nM CA1 were seen. The majority of drug-related material measured in urine comprised of the two monoglucuronides; CA1 and the diglucuronide were about 10-fold lower. No CA1P was detectable in urine.
Source:Journal of Chromatography B, Volume 898
Michael R.L. Stratford, Lisa K. Folkes
Validated methods for the determination of CA1, the active agent derived from the prodrug CA1P, in human plasma and urine, and of CA1P and three glucuronides CA1G1, CA1G2 and CA1DG in human urine were developed using LC–MS. Plasma CA1 was extracted using solid phase extraction and validated over the range 5–1000nM. Urine samples were analysed without extraction, and the assays validated over the range 50–2000nM (CA1P), 25–2000nM (CA1), 50–40,000nM (CA1G1 and CA1G2) and 25–4000nM (CA1DG). The mean correlation coefficient (r 2) was ≥0.997 for all assays. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical methods (<15%). Mean recovery of CA1 from plasma was 101%, and 97% from urine. Mean urine recovery of CA1P was 98%, CA1G1 96%, CA1G2 93% and CA1DG 93%. The method was applied to plasma and urine samples from a recently completed clinical trial of the prodrug. Peak plasma concentrations of up to 470nM CA1 were seen. The majority of drug-related material measured in urine comprised of the two monoglucuronides; CA1 and the diglucuronide were about 10-fold lower. No CA1P was detectable in urine.
Graphical abstract
Graphical abstract Highlights
► Validated methods for the vascular targeted cancer drug combretastatin A1 in human plasma and urine by HPLC–MS. ► Validated methods for the phosphate pro-drug and three glucuronides found in human urine by HPLC–MS. ► Application to the analysis of patient samples following a Phase I clinical trial of combretastatin A1 phosphate.Novel membrane extraction procedure for the purification of hepatitis B surface antigen from Pichia pastoris
29 May 2012,
10:25:41
Publication year:
2012
Source:Journal of Chromatography B, Volume 898
Aravind Patil, Navin Khanna
The recombinant hepatitis B surface antigen (HBsAg) vaccine provides excellent protection against hepatitis B virus (HBV). However, high costs of its production prevents many underdeveloped and developing nations from implementing HBsAg vaccination. This in turn increases the risk of contracting HBV related diseases. Majority of the commercial HBV vaccines are derived from purified HBsAg expressed in recombinant yeasts. Most of the cost in production of the vaccine is incurred during the downstream processing. The costs associated with HBsAg purification can be decreased by optimizing the pre-chromatography steps and by reducing the impurity burden on chromatography operations. Here in this work we present a novel strategy for the enriched extraction of recombinant HBsAg from Pichia pastoris membranes. We have also developed a simple, easy to operate process for the purification of HBsAg VLPs from the membranes of P. pastoris. This novel strategy, while utilizing a single column chromatographic step in the purification scheme results in the highest recovery of HBsAg VLPs reported in the literature. The yield of HBsAg at the end of purification was nearly 5% (85μg/g of induced wet cell biomass). The HBsAg purified from this process has shown the presence of VLPs. The immunization of these VLPs in BALB/c mice with alhydrogel adjuvant has shown good titers of neutralizing antibodies.
Source:Journal of Chromatography B, Volume 898
Aravind Patil, Navin Khanna
The recombinant hepatitis B surface antigen (HBsAg) vaccine provides excellent protection against hepatitis B virus (HBV). However, high costs of its production prevents many underdeveloped and developing nations from implementing HBsAg vaccination. This in turn increases the risk of contracting HBV related diseases. Majority of the commercial HBV vaccines are derived from purified HBsAg expressed in recombinant yeasts. Most of the cost in production of the vaccine is incurred during the downstream processing. The costs associated with HBsAg purification can be decreased by optimizing the pre-chromatography steps and by reducing the impurity burden on chromatography operations. Here in this work we present a novel strategy for the enriched extraction of recombinant HBsAg from Pichia pastoris membranes. We have also developed a simple, easy to operate process for the purification of HBsAg VLPs from the membranes of P. pastoris. This novel strategy, while utilizing a single column chromatographic step in the purification scheme results in the highest recovery of HBsAg VLPs reported in the literature. The yield of HBsAg at the end of purification was nearly 5% (85μg/g of induced wet cell biomass). The HBsAg purified from this process has shown the presence of VLPs. The immunization of these VLPs in BALB/c mice with alhydrogel adjuvant has shown good titers of neutralizing antibodies.
Highlights
► HBsAg protein, when over expressed in P. patoris gets associated with membranes. ► The membrane association of HBsAg can be disrupted by 2% Tween-20. ► A centrifugation step followed by lysis ensures removal of supernatant impurities. ► Phenyl-600M Toyopearl resin can purify HBsAg to homogeneity. ► Membrane purified HBsAg elicits protective antibody responses in mice.A synthetic Protein G adsorbent based on the multi-component Ugi reaction for the purification of mammalian immunoglobulins
29 May 2012,
10:25:41
Publication year:
2012
Source:Journal of Chromatography B, Volume 898
Jianing Qian, Graziella El Khoury, Hamzah Issa, Khaled Al-Qaoud, Penelope Shihab, Christopher R. Lowe
Numerous efforts have been devoted to develop synthetic affinity ligands mimicking natural immunoglobulin-binding proteins, such as Proteins A and L, in order to overcome intrinsic drawbacks involving their high cost and acidic pH elution. However, few reports have focused on a Protein G mimic. This work describes the use of the solid phase multi-component Ugi reaction to generate a low cost, rationally designed, affinity ligand to mimic Protein G for the purification of mammalian immunoglobulins, including the heavy-chain only camelid IgGs, with effective elution at neutral pH. An aldehyde-functionalised Sepharose™ resin constituted one component (aldehyde) of the four-component Ugi reaction, whilst the other three components (a primary or secondary amine, a carboxylic acid and an isonitrile) were varied to generate a tri-substituted Ugi scaffold, with a wide range of functionality, suitable for mimicking peptides for immunoglobulin purification. Ligand A2C11I1 was designed to mimic Asn35 and Trp43 of Protein G (PDB: 1FCC) and in silico docking into the Fc domain showed a key binding interface closely resembling native Protein G. This candidate ligand demonstrated affinity towards IgGs derived from human, cow, goat, mouse, sheep, pig, rabbit and rat serum, chicken IgY and recombinant camelid Fc domain, out of which cow and sheep IgG demonstrated 100% binding under the conditions selected. Preparative chromatography of IgG from human serum under a standardised buffer regime eluted IgG of ∼65% purity, compared to ∼62% with Protein G. This adsorbent achieved highest elution of IgG at neutral pH (0.1M sodium phosphate pH 7.0, 30%, v/v, ethylene glycol), an advantage for purifying antibodies sensitive to extremes of pH. The ligand demonstrated a static binding capacity of 24.6mgIgGml−1 resin and a dissociation constant (K d) of 4.78×10−6 M. The solid phase Ugi scaffold provides a strategy to develop pseudo-biospecific ligands to purify immunoglobulins and other potentially high-value biotherapeutic proteins.
Source:Journal of Chromatography B, Volume 898
Jianing Qian, Graziella El Khoury, Hamzah Issa, Khaled Al-Qaoud, Penelope Shihab, Christopher R. Lowe
Numerous efforts have been devoted to develop synthetic affinity ligands mimicking natural immunoglobulin-binding proteins, such as Proteins A and L, in order to overcome intrinsic drawbacks involving their high cost and acidic pH elution. However, few reports have focused on a Protein G mimic. This work describes the use of the solid phase multi-component Ugi reaction to generate a low cost, rationally designed, affinity ligand to mimic Protein G for the purification of mammalian immunoglobulins, including the heavy-chain only camelid IgGs, with effective elution at neutral pH. An aldehyde-functionalised Sepharose™ resin constituted one component (aldehyde) of the four-component Ugi reaction, whilst the other three components (a primary or secondary amine, a carboxylic acid and an isonitrile) were varied to generate a tri-substituted Ugi scaffold, with a wide range of functionality, suitable for mimicking peptides for immunoglobulin purification. Ligand A2C11I1 was designed to mimic Asn35 and Trp43 of Protein G (PDB: 1FCC) and in silico docking into the Fc domain showed a key binding interface closely resembling native Protein G. This candidate ligand demonstrated affinity towards IgGs derived from human, cow, goat, mouse, sheep, pig, rabbit and rat serum, chicken IgY and recombinant camelid Fc domain, out of which cow and sheep IgG demonstrated 100% binding under the conditions selected. Preparative chromatography of IgG from human serum under a standardised buffer regime eluted IgG of ∼65% purity, compared to ∼62% with Protein G. This adsorbent achieved highest elution of IgG at neutral pH (0.1M sodium phosphate pH 7.0, 30%, v/v, ethylene glycol), an advantage for purifying antibodies sensitive to extremes of pH. The ligand demonstrated a static binding capacity of 24.6mgIgGml−1 resin and a dissociation constant (K d) of 4.78×10−6 M. The solid phase Ugi scaffold provides a strategy to develop pseudo-biospecific ligands to purify immunoglobulins and other potentially high-value biotherapeutic proteins.
Highlights
► De novo design and synthesis by the Ugi multicomponent reaction of a ligand mimicking Protein G binding to immunoglobulins (IgGs). ► Purification of IgGs from various mammalian species. ► Effective elution at neutral pH conditions. ► Comparison of the Ugi adsorbent with Protein A and Protein G adsorbents. ► Purification of camelid IgGs including heavy-chain only antibodies from camel milk.Computational design and synthesis of molecular imprinted polymers for selective extraction of allopurinol from human plasma
29 May 2012,
10:25:41
Publication year:
2012
Source:Journal of Chromatography B, Volume 898
Mehrdad Tabandeh, Soheila Ghassamipour, Heydar Aqababa, Meisam Tabatabaei, Meisam Hasheminejad
The present study was focused on the rational development of polymers for selective extraction of allopurinol (ALP) from human plasma. Therefore, a computational modeling approach was combined with the molecular imprinting technology to obtain the polymers. The computational approach was used in order to screen the functional monomers as well as the polymerization solvents for rational design of molecular imprinted polymers (MIPs). It was based on the comparison of the binding energy (ΔE) of the formed complexes between the template molecule and different functional monomers. In the design, the effect of the polymerization solvent was also included using the polarizable continuum model. The theoretical calculation results showed that among virtual solvents tested, acrylamide (AAM) gave the largest ΔE while acrylonitrile (ACN) gave the smallest ΔE in acetone. Therefore, the MIP prepared using AAM as functional monomer in acetone was desired. To examine the validity of this approach, three MIPs were synthesized with different functional monomers i.e. AAM, acrylic acid (AA), and ACN, and then evaluated using Langmuir–Freundlich (LF) isotherm. The results obtained from this experiment confirmed the computational results that the MIP prepared by AAM was the most appropriate adsorbent. Subsequently, the MIP was used to develop a molecular imprinted solid-phase extraction (MISPE) procedure. Finally, the MISPE procedure followed by HPLC was developed for selective extraction and determination of allopurinol in human plasma. For the proposed MISPE method, the linearity between peak area and concentration was found in the range of 0.100–25.000μM with a linear regression coefficient (R 2) of 0.995. The limit of detection (LOD) and quantification (LOQ) in plasma were 0.028 and 0.093μM, respectively. The results of this study indicated the possibility of using computer aided design for rational selection of functional monomers and solvents for preparation of the MIPs capable of extracting allopurinol from human plasma.
Source:Journal of Chromatography B, Volume 898
Mehrdad Tabandeh, Soheila Ghassamipour, Heydar Aqababa, Meisam Tabatabaei, Meisam Hasheminejad
The present study was focused on the rational development of polymers for selective extraction of allopurinol (ALP) from human plasma. Therefore, a computational modeling approach was combined with the molecular imprinting technology to obtain the polymers. The computational approach was used in order to screen the functional monomers as well as the polymerization solvents for rational design of molecular imprinted polymers (MIPs). It was based on the comparison of the binding energy (ΔE) of the formed complexes between the template molecule and different functional monomers. In the design, the effect of the polymerization solvent was also included using the polarizable continuum model. The theoretical calculation results showed that among virtual solvents tested, acrylamide (AAM) gave the largest ΔE while acrylonitrile (ACN) gave the smallest ΔE in acetone. Therefore, the MIP prepared using AAM as functional monomer in acetone was desired. To examine the validity of this approach, three MIPs were synthesized with different functional monomers i.e. AAM, acrylic acid (AA), and ACN, and then evaluated using Langmuir–Freundlich (LF) isotherm. The results obtained from this experiment confirmed the computational results that the MIP prepared by AAM was the most appropriate adsorbent. Subsequently, the MIP was used to develop a molecular imprinted solid-phase extraction (MISPE) procedure. Finally, the MISPE procedure followed by HPLC was developed for selective extraction and determination of allopurinol in human plasma. For the proposed MISPE method, the linearity between peak area and concentration was found in the range of 0.100–25.000μM with a linear regression coefficient (R 2) of 0.995. The limit of detection (LOD) and quantification (LOQ) in plasma were 0.028 and 0.093μM, respectively. The results of this study indicated the possibility of using computer aided design for rational selection of functional monomers and solvents for preparation of the MIPs capable of extracting allopurinol from human plasma.
Highlights
► Rational development of polymers for selective extraction of allopurinol from human plasma. ► Computational modeling combined with molecular imprinting technology to obtain polymers. ► Production of MIPs capable of extracting allopurinol from human plasma.Development and utilization of a combined LC–UV and LC–MS/MS method for the simultaneous analysis of tegafur and 5-fluorouracil in human plasma to support a phase I clinical study of oral UFT®/leucovorin
29 May 2012,
10:25:41
Publication year:
2012
Source:Journal of Chromatography B, Volume 898
Cody J. Peer, Terence J. McManus, Herbert I. Hurwitz, William P. Petros
Tegafur is a 5-fluorouracil (5-FU) prodrug widely used outside the United States to treat colorectal cancer as well as cancers of the head and neck. The resulting plasma concentrations of tegafur are much higher than those of 5-FU; thus, analytical methods are needed that are sensitive enough to detect low plasma concentrations of 5-FU and robust enough to simultaneously analyze tegafur. Previous LC–MS/MS methods have either failed to demonstrate the ability to simultaneously measure low 5-FU and high tegafur plasma levels, or failed to be applicable in clinical studies. Our goal was to develop a method capable of measuring low concentrations of 5-FU (8–200ng/ml) and high concentrations of tegafur (800–20,000ng/ml) in human plasma and to subsequently evaluate the utility of the method in patient samples collected during a phase I clinical study where oral doses of either 200mg or 300mg UFT®/LV (uracil and tegafur in a 4:1 molar ratio plus leucovorin) were administered. A combined LC–MS/MS and LC–UV method was developed utilizing negative ion atmospheric pressure ionization (API). The method provides an accuracy and precision of <10% and <6%, respectively, for both analytes. Material recoveries from the liquid–liquid extraction technique were 97–110% and 86–91% for tegafur and 5-FU, respectively. Utilization of this method to determine tegafur and 5-FU plasma concentrations followed by noncompartmental pharmacokinetic analyses successfully estimated pharmacokinetic parameters (C MAX, t MAX and AUC0–10h) in the clinical study patients. Overall, this method is ideal for the simultaneous bioanalysis of low levels of 5-FU and relatively higher levels of its prodrug, tegafur, in human plasma for clinical pharmacokinetic analysis.
Source:Journal of Chromatography B, Volume 898
Cody J. Peer, Terence J. McManus, Herbert I. Hurwitz, William P. Petros
Tegafur is a 5-fluorouracil (5-FU) prodrug widely used outside the United States to treat colorectal cancer as well as cancers of the head and neck. The resulting plasma concentrations of tegafur are much higher than those of 5-FU; thus, analytical methods are needed that are sensitive enough to detect low plasma concentrations of 5-FU and robust enough to simultaneously analyze tegafur. Previous LC–MS/MS methods have either failed to demonstrate the ability to simultaneously measure low 5-FU and high tegafur plasma levels, or failed to be applicable in clinical studies. Our goal was to develop a method capable of measuring low concentrations of 5-FU (8–200ng/ml) and high concentrations of tegafur (800–20,000ng/ml) in human plasma and to subsequently evaluate the utility of the method in patient samples collected during a phase I clinical study where oral doses of either 200mg or 300mg UFT®/LV (uracil and tegafur in a 4:1 molar ratio plus leucovorin) were administered. A combined LC–MS/MS and LC–UV method was developed utilizing negative ion atmospheric pressure ionization (API). The method provides an accuracy and precision of <10% and <6%, respectively, for both analytes. Material recoveries from the liquid–liquid extraction technique were 97–110% and 86–91% for tegafur and 5-FU, respectively. Utilization of this method to determine tegafur and 5-FU plasma concentrations followed by noncompartmental pharmacokinetic analyses successfully estimated pharmacokinetic parameters (C MAX, t MAX and AUC0–10h) in the clinical study patients. Overall, this method is ideal for the simultaneous bioanalysis of low levels of 5-FU and relatively higher levels of its prodrug, tegafur, in human plasma for clinical pharmacokinetic analysis.
Highlights
► Published analytic methods for tegafur are suboptimal for pharmacokinetic studies. ► An analytic method was developed for the anticancer drug tegafur and its metabolite. ► Accuracy, precision and recovery of the analytes from plasma were acceptable. ► The method was used to successfully determine human pharmacokinetic parameters.Simultaneous determination of pesticides, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and phthalate esters in human adipose tissue by gas chromatography–tandem mass spectrometry
29 May 2012,
10:25:41
Publication year:
2012
Source:Journal of Chromatography B, Volume 898
Na Wang, Deyang Kong, Zhengjun Shan, Lili Shi, Daoji Cai, Yanzhong Cao, Yongming Liu, Guofang Pang
This paper describes a method for the simultaneous determination of 284 environmental contaminants, including 57 pesticides, 15 polycyclic aromatic hydrocarbons (PAHs), 209 polychlorinated biphenyls (PCBs) and 3 phthalate esters (PAEs), in adipose tissue samples. For the first time, a gas chromatography–tandem mass spectrometry (GC–MS/MS) method following a homogenised extraction using acetonitrile and purification by gel permeation chromatography (GPC) was used. Various performance characteristics, such as the limit of detection (LOD), limit of quantification (LOQ), linear range, recovery and precision, were determined for each analyte. The LOD for most analytes was below 0.01mg/kg. The recoveries and relative standard deviations (RSDs) were determined by spiking untreated samples with the analytes at the LOQ, 2×LOQ and 4×LOQ levels. The average recovery for most pesticides was between 70% and 120% and the precision values, expressed as RSD, were all below 20.4% (n =6). This method may provide an efficient tool for evaluating the extent of exposure to organic contaminants using human adipose tissue.
Source:Journal of Chromatography B, Volume 898
Na Wang, Deyang Kong, Zhengjun Shan, Lili Shi, Daoji Cai, Yanzhong Cao, Yongming Liu, Guofang Pang
This paper describes a method for the simultaneous determination of 284 environmental contaminants, including 57 pesticides, 15 polycyclic aromatic hydrocarbons (PAHs), 209 polychlorinated biphenyls (PCBs) and 3 phthalate esters (PAEs), in adipose tissue samples. For the first time, a gas chromatography–tandem mass spectrometry (GC–MS/MS) method following a homogenised extraction using acetonitrile and purification by gel permeation chromatography (GPC) was used. Various performance characteristics, such as the limit of detection (LOD), limit of quantification (LOQ), linear range, recovery and precision, were determined for each analyte. The LOD for most analytes was below 0.01mg/kg. The recoveries and relative standard deviations (RSDs) were determined by spiking untreated samples with the analytes at the LOQ, 2×LOQ and 4×LOQ levels. The average recovery for most pesticides was between 70% and 120% and the precision values, expressed as RSD, were all below 20.4% (n =6). This method may provide an efficient tool for evaluating the extent of exposure to organic contaminants using human adipose tissue.
Highlights
► We established a GC–MS/MS method for determination of 284 analytes in adipose sample. ► We conducted systematic method optimisation, including extraction and cleanup. ► Method validation showed performance characteristics for each analyte were satisfied. ► The method has been successfully applied for human adipose tissue samples. ► It was an efficient tool for evaluating human exposure extent of contaminants.Determination of reboxetine in rat brain microdialysates and plasma samples using liquid chromatography coupled to fluorescence detection
29 May 2012,
10:25:41
Publication year:
2012
Source:Journal of Chromatography B, Volume 898
Naser Shraim, Ralph Clinckers, Sophie Sarre, Yvette Michotte, Ann Van Eeckhaut
A liquid chromatographic method with fluorescence detection was developed and validated for the quantification of the antidepressant reboxetine (RBX), a selective noradrenalin reuptake inhibitor, in rat brain microdialysates. After modification of the method in terms of sample preparation and sensitivity, it was also validated for the quantification of RBX in rat plasma samples. To enable fluorescence detection, a pre-column derivatization step with 9-fluorenylmethyl chloroformate was included. Separations were performed on a reversed phase C18 column using gradient elution. The retention time for RBX was found to be 8.8min. The assay of RBX in brain microdialysis samples showed a linear relationship in the calibration curve from 2 to 200ng/mL, with a correlation coefficient ≥0.999. The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 0.6 and 2.0ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.5% and 11.7% with an average recovery of 101.2±8.2% (mean±SD, n =40). For the analysis of plasma samples, the calibration curve was linear between 20 and 700ng/mL with a correlation coefficient ≥0.999. LOD and LLOQ were 6 and 20ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.7% and 11.5% with an average recovery of 98.5±7.3% (mean±SD, n =40). We demonstrated the applicability of the method to determine the concentration–time profiles of RBX in brain and plasma following systemic administration.
Source:Journal of Chromatography B, Volume 898
Naser Shraim, Ralph Clinckers, Sophie Sarre, Yvette Michotte, Ann Van Eeckhaut
A liquid chromatographic method with fluorescence detection was developed and validated for the quantification of the antidepressant reboxetine (RBX), a selective noradrenalin reuptake inhibitor, in rat brain microdialysates. After modification of the method in terms of sample preparation and sensitivity, it was also validated for the quantification of RBX in rat plasma samples. To enable fluorescence detection, a pre-column derivatization step with 9-fluorenylmethyl chloroformate was included. Separations were performed on a reversed phase C18 column using gradient elution. The retention time for RBX was found to be 8.8min. The assay of RBX in brain microdialysis samples showed a linear relationship in the calibration curve from 2 to 200ng/mL, with a correlation coefficient ≥0.999. The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 0.6 and 2.0ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.5% and 11.7% with an average recovery of 101.2±8.2% (mean±SD, n =40). For the analysis of plasma samples, the calibration curve was linear between 20 and 700ng/mL with a correlation coefficient ≥0.999. LOD and LLOQ were 6 and 20ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.7% and 11.5% with an average recovery of 98.5±7.3% (mean±SD, n =40). We demonstrated the applicability of the method to determine the concentration–time profiles of RBX in brain and plasma following systemic administration.
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