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1.  External Validation of the Bilirubin–Atazanavir Nomogram for Assessment of Atazanavir Plasma Exposure in HIV-1-Infected Patients 
The AAPS journal  2012;15(2):308-315.
Atazanavir increases plasma bilirubin levels in a concentration-dependent manner. Due to less costly and readily available assays, bilirubin has been proposed as a marker of atazanavir exposure. In this work, a previously developed nomogram for detection of suboptimal atazanavir exposure is validated against external patient populations. The bilirubin nomogram was validated against 311 matching bilirubin and atazanavir samples from 166 HIV-1-infected Norwegian, French, and Italian patients on a ritonavir-boosted regimen. In addition, the nomogram was evaluated in 56 Italian patients on an unboosted regimen. The predictive properties of the nomogram were validated against observed atazanavir plasma concentrations. The use of the nomogram to detect non-adherence was also investigated by simulation. The bilirubin nomogram predicted suboptimal exposure in the patient populations on a ritonavir-boosted regimen with a negative predictive value of 97% (95% CI 95–100). The bilirubin nomogram and monitoring of atazanavir concentrations had similar predictive properties for detecting non-adherence based on simulations. Although both methods performed adequately during a period of non-adherence, they had lower predictive power to detect past non-adherence episodes. Using the bilirubin nomogram for detection of suboptimal atazanavir exposure in patients on a ritonavir-boosted regimen is a rapid and cost-effective alternative to routine measurements of the actual atazanavir exposure in plasma. Its application may be useful in clinical settings if atazanavir concentrations are not available.
doi:10.1208/s12248-012-9440-8
PMCID: PMC3618856  PMID: 23224752
atazanavir; bilirubin; nomogram
2.  Performance comparison of various maximum likelihood nonlinear mixed-effects estimation methods for dose-response models 
The AAPS Journal  2012;14(3):420-432.
Estimation methods for nonlinear mixed-effects modelling have considerably improved over the last decades. Nowadays several algorithms implemented in different softwares are used. The present study aimed at comparing their performance for dose-response models.
Eight scenarios were considered using a sigmoid Emax model, with varying sigmoidicity factors and residual error models. 100 simulated datasets for each scenario were generated. 100 individuals with observations at 4 doses constituted the rich design and at 2 doses for the sparse design. Nine parametric approaches for maximum likelihood estimation were studied: FOCE in NONMEM and R, LAPLACE in NONMEM and SAS, adaptive Gaussian quadrature (AGQ) in SAS, and SAEM in NONMEM and MONOLIX (both SAEM approaches with default and modified settings). All approaches started first from initial estimates set to the true values, and second using altered values. Results were examined through relative root mean squared error (RRMSE) of the estimates.
With true initial conditions, full completion rate was obtained with all approaches except FOCE in R. Runtimes were shortest with FOCE and LAPLACE, and longest with AGQ. Under the rich design with true initial conditions, all approaches performed well except FOCE in R. When starting from altered initial conditions, AGQ, and then FOCE in NONMEM, LAPLACE in SAS, and SAEM in NONMEM and MONOLIX with tuned settings, consistently displayed lower RRMSE than the other approaches.
For standard dose-response models analyzed through mixed-effects models, differences could be identified in the performance of estimation methods available in current software.
doi:10.1208/s12248-012-9349-2
PMCID: PMC3385815  PMID: 22528503
MAXIMUM LIKELIHOOD ESTIMATION; FOCE; LAPLACE; ADAPTIVE GAUSSIAN QUADRATURE; SAEM
3.  Performance Comparison of Various Maximum Likelihood Nonlinear Mixed-Effects Estimation Methods for Dose–Response Models 
The AAPS Journal  2012;14(3):420-432.
Estimation methods for nonlinear mixed-effects modelling have considerably improved over the last decades. Nowadays, several algorithms implemented in different software are used. The present study aimed at comparing their performance for dose–response models. Eight scenarios were considered using a sigmoid Emax model, with varying sigmoidicity and residual error models. One hundred simulated datasets for each scenario were generated. One hundred individuals with observations at four doses constituted the rich design and at two doses, the sparse design. Nine parametric approaches for maximum likelihood estimation were studied: first-order conditional estimation (FOCE) in NONMEM and R, LAPLACE in NONMEM and SAS, adaptive Gaussian quadrature (AGQ) in SAS, and stochastic approximation expectation maximization (SAEM) in NONMEM and MONOLIX (both SAEM approaches with default and modified settings). All approaches started first from initial estimates set to the true values and second, using altered values. Results were examined through relative root mean squared error (RRMSE) of the estimates. With true initial conditions, full completion rate was obtained with all approaches except FOCE in R. Runtimes were shortest with FOCE and LAPLACE and longest with AGQ. Under the rich design, all approaches performed well except FOCE in R. When starting from altered initial conditions, AGQ, and then FOCE in NONMEM, LAPLACE in SAS, and SAEM in NONMEM and MONOLIX with tuned settings, consistently displayed lower RRMSE than the other approaches. For standard dose–response models analyzed through mixed-effects models, differences were identified in the performance of estimation methods available in current software, giving material to modellers to identify suitable approaches based on an accuracy-versus-runtime trade-off.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9349-2) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-012-9349-2
PMCID: PMC3385815  PMID: 22528503
adaptive Gaussian quadrature; FOCE; LAPLACE; maximum likelihood estimation; SAEM
4.  External Validation of the Bilirubin–Atazanavir Nomogram for Assessment of Atazanavir Plasma Exposure in HIV-1-Infected Patients 
The AAPS Journal  2012;15(2):308-315.
Atazanavir increases plasma bilirubin levels in a concentration-dependent manner. Due to less costly and readily available assays, bilirubin has been proposed as a marker of atazanavir exposure. In this work, a previously developed nomogram for detection of suboptimal atazanavir exposure is validated against external patient populations. The bilirubin nomogram was validated against 311 matching bilirubin and atazanavir samples from 166 HIV-1-infected Norwegian, French, and Italian patients on a ritonavir-boosted regimen. In addition, the nomogram was evaluated in 56 Italian patients on an unboosted regimen. The predictive properties of the nomogram were validated against observed atazanavir plasma concentrations. The use of the nomogram to detect non-adherence was also investigated by simulation. The bilirubin nomogram predicted suboptimal exposure in the patient populations on a ritonavir-boosted regimen with a negative predictive value of 97% (95% CI 95–100). The bilirubin nomogram and monitoring of atazanavir concentrations had similar predictive properties for detecting non-adherence based on simulations. Although both methods performed adequately during a period of non-adherence, they had lower predictive power to detect past non-adherence episodes. Using the bilirubin nomogram for detection of suboptimal atazanavir exposure in patients on a ritonavir-boosted regimen is a rapid and cost-effective alternative to routine measurements of the actual atazanavir exposure in plasma. Its application may be useful in clinical settings if atazanavir concentrations are not available.
doi:10.1208/s12248-012-9440-8
PMCID: PMC3618856  PMID: 23224752
atazanavir; bilirubin; nomogram
5.  Assessment of a Spectrophotometric Assay for Monoacylglycerol Lipase Activity 
The AAPS journal  2010;12(2):197-201.
doi:10.1208/s12248-010-9180-6
PMCID: PMC2844520  PMID: 20186507
monoacylglycerol lipase; spectrophotometric assay
6.  Assessment of a Spectrophotometric Assay for Monoacylglycerol Lipase Activity 
The AAPS Journal  2010;12(2):197-201.
doi:10.1208/s12248-010-9180-6
PMCID: PMC2844520  PMID: 20186507
monoacylglycerol lipase; spectrophotometric assay
7.  Micro-Flow Imaging: Flow Microscopy Applied to Sub-visible Particulate Analysis in Protein Formulations 
The AAPS Journal  2010;12(3):455-464.
ABSTRACT
The need to monitor, measure, and control sub-visible proteinaceous particulates in biopharmaceutical formulations has been emphasized in recent publications and commentaries. Some of these particulates can be highly transparent, fragile, and unstable. In addition, for much of the size range of concern, no practical measurement method with adequate sensitivity and repeatability has been available. A complication in measuring protein particulates in many formulations is the simultaneous presence of other particle types such as silicone micro-droplets, air bubbles, and extrinsic contaminants. The need has therefore been identified for new analytical methods which can accurately measure and characterize sub-visible particulates in formulations. Micro-flow imaging has been shown to provide high sensitivity in detecting and imaging transparent protein particles and a unique capability to independently analyze such populations even when other particle types are present.
doi:10.1208/s12248-010-9205-1
PMCID: PMC2895433  PMID: 20517661
light obscuration; micro-flow imaging; particle sizing; protein aggregation; protein formulation
8.  Micro-Flow Imaging: Flow Microscopy Applied to Sub-visible Particulate Analysis in Protein Formulations 
The AAPS Journal  2010;12(3):455-464.
ABSTRACT
The need to monitor, measure, and control sub-visible proteinaceous particulates in biopharmaceutical formulations has been emphasized in recent publications and commentaries. Some of these particulates can be highly transparent, fragile, and unstable. In addition, for much of the size range of concern, no practical measurement method with adequate sensitivity and repeatability has been available. A complication in measuring protein particulates in many formulations is the simultaneous presence of other particle types such as silicone micro-droplets, air bubbles, and extrinsic contaminants. The need has therefore been identified for new analytical methods which can accurately measure and characterize sub-visible particulates in formulations. Micro-flow imaging has been shown to provide high sensitivity in detecting and imaging transparent protein particles and a unique capability to independently analyze such populations even when other particle types are present.
doi:10.1208/s12248-010-9205-1
PMCID: PMC2895433  PMID: 20517661
light obscuration; micro-flow imaging; particle sizing; protein aggregation; protein formulation
9.  Preservation of the Immunogenicity of Dry-powder Influenza H5N1 Whole Inactivated Virus Vaccine at Elevated Storage Temperatures 
The AAPS Journal  2010;12(2):215-222.
Stockpiling of pre-pandemic influenza vaccines guarantees immediate vaccine availability to counteract an emerging pandemic. Generally, influenza vaccines need to be stored and handled refrigerated to prevent thermal degradation of the antigenic component. Requirement of a cold-chain, however, complicates stockpiling and the logistics of vaccine distribution. We, therefore, investigated the effect of elevated storage temperatures on the immunogenicity of a pre-pandemic influenza A H5N1 whole inactivated virus vaccine. Either suspended in liquid or kept as a freeze-dried powder, vaccines could be stored for 1 year at ambient temperature (20°C) with minimal loss of immunogenicity in mice. Elevation of the storage temperature to 40°C, however, resulted in a significant loss of immunogenic potency within 3 months if vaccines were stored in liquid suspension. In sharp contrast, freeze-dried powder formulations were stable at 40°C for at least 3 months. The presence of inulin or trehalose sugar excipients during freeze-drying of the vaccine proved to be critical to maintain its immunogenic potency during storage, and to preserve the characteristic Th1-type response to whole inactivated virus vaccine. These results indicate that whole inactivated virus vaccines may be stored and handled at room temperature in moderate climate zones for over a year with minimal decline and, if converted to dry-powder, even in hot climate zones for at least 3 months. The increased stability of dry-powder vaccine at 40°C may also point to an extended shelf-life when stored at 4°C. Use of the more stable dry-powder formulation could simplify stockpiling and thereby facilitating successful pandemic intervention.
doi:10.1208/s12248-010-9179-z
PMCID: PMC2844510  PMID: 20195930
freeze-drying; inulin; pandemic influenza; vaccine stockpiling; whole inactivated influenza vaccine (H5N1)
10.  Preservation of the Immunogenicity of Dry-powder Influenza H5N1 Whole Inactivated Virus Vaccine at Elevated Storage Temperatures 
The AAPS Journal  2010;12(2):215-222.
Stockpiling of pre-pandemic influenza vaccines guarantees immediate vaccine availability to counteract an emerging pandemic. Generally, influenza vaccines need to be stored and handled refrigerated to prevent thermal degradation of the antigenic component. Requirement of a cold-chain, however, complicates stockpiling and the logistics of vaccine distribution. We, therefore, investigated the effect of elevated storage temperatures on the immunogenicity of a pre-pandemic influenza A H5N1 whole inactivated virus vaccine. Either suspended in liquid or kept as a freeze-dried powder, vaccines could be stored for 1 year at ambient temperature (20°C) with minimal loss of immunogenicity in mice. Elevation of the storage temperature to 40°C, however, resulted in a significant loss of immunogenic potency within 3 months if vaccines were stored in liquid suspension. In sharp contrast, freeze-dried powder formulations were stable at 40°C for at least 3 months. The presence of inulin or trehalose sugar excipients during freeze-drying of the vaccine proved to be critical to maintain its immunogenic potency during storage, and to preserve the characteristic Th1-type response to whole inactivated virus vaccine. These results indicate that whole inactivated virus vaccines may be stored and handled at room temperature in moderate climate zones for over a year with minimal decline and, if converted to dry-powder, even in hot climate zones for at least 3 months. The increased stability of dry-powder vaccine at 40°C may also point to an extended shelf-life when stored at 4°C. Use of the more stable dry-powder formulation could simplify stockpiling and thereby facilitating successful pandemic intervention.
doi:10.1208/s12248-010-9179-z
PMCID: PMC2844510  PMID: 20195930
freeze-drying; inulin; pandemic influenza; vaccine stockpiling; whole inactivated influenza vaccine (H5N1)
11.  Glial–Neuronal Interactions—Implications for Plasticity and Drug Addiction 
The AAPS Journal  2009;11(1):123-132.
Among neuroscientists, astrocytes have for long played Cinderella to their neuron stepsisters. While the importance of glia in regulating brain activity was predicted by Ramon y Cajal more than a century ago (Garcia-Marin et al., Trends. Neurosci. 30:479–787, 2007), these cells, until recently, have been thought to play mainly a passive part in synaptic signaling. Results obtained over the last decade have begun to suggest otherwise. Experiments carried out in a number of labs have shown that glial cells, especially astrocytes, directly participate in synaptic signaling and potentially regulate synaptic plasticity and network excitability. The presence of signaling pathways on astrocytes that are analogous to those at presynaptic terminals suggests a role for these cells in network plasticity. Findings that the same signaling pathways can be activated by receptors for drugs of abuse present on astrocytes suggest a role for these cells in the addictive process. In this review, we summarize current understanding of astrocytic role in synaptic signaling and suggest that a complete understanding of the process of addiction requires a better understanding of the functional role of these cells.
doi:10.1208/s12248-009-9085-4
PMCID: PMC2664886  PMID: 19238557
astrocyte; calcium; gliotransmission; nicotinic; synapse; tripartite synapse
12.  Intranasal Delivery of Influenza Subunit Vaccine Formulated with GEM Particles as an Adjuvant 
The AAPS Journal  2010;12(2):109-116.
Nasal administration of influenza vaccine has the potential to facilitate influenza control and prevention. However, when administered intranasally (i.n.), commercially available inactivated vaccines only generate systemic and mucosal immune responses if strong adjuvants are used, which are often associated with safety problems. We describe the successful use of a safe adjuvant Gram-positive enhancer matrix (GEM) particles derived from the food-grade bacterium Lactococcus lactis for i.n. vaccination with subunit influenza vaccine in mice. It is shown that simple admixing of the vaccine with the GEM particles results in a strongly enhanced immune response. Already after one booster, the i.n. delivered GEM subunit vaccine resulted in hemagglutination inhibition titers in serum at a level equal to the conventional intramuscular (i.m.) route. Moreover, i.n. immunization with GEM subunit vaccine elicited superior mucosal and Th1 skewed immune responses compared to those induced by i.m. and i.n. administered subunit vaccine alone. In conclusion, GEM particles act as a potent adjuvant for i.n. influenza immunization.
doi:10.1208/s12248-009-9168-2
PMCID: PMC2844513  PMID: 20058113
influenza vaccine; intranasal vaccine; Lactococcus lactis GEM particles
13.  Intranasal Delivery of Influenza Subunit Vaccine Formulated with GEM Particles as an Adjuvant 
The AAPS Journal  2010;12(2):109-116.
Nasal administration of influenza vaccine has the potential to facilitate influenza control and prevention. However, when administered intranasally (i.n.), commercially available inactivated vaccines only generate systemic and mucosal immune responses if strong adjuvants are used, which are often associated with safety problems. We describe the successful use of a safe adjuvant Gram-positive enhancer matrix (GEM) particles derived from the food-grade bacterium Lactococcus lactis for i.n. vaccination with subunit influenza vaccine in mice. It is shown that simple admixing of the vaccine with the GEM particles results in a strongly enhanced immune response. Already after one booster, the i.n. delivered GEM subunit vaccine resulted in hemagglutination inhibition titers in serum at a level equal to the conventional intramuscular (i.m.) route. Moreover, i.n. immunization with GEM subunit vaccine elicited superior mucosal and Th1 skewed immune responses compared to those induced by i.m. and i.n. administered subunit vaccine alone. In conclusion, GEM particles act as a potent adjuvant for i.n. influenza immunization.
doi:10.1208/s12248-009-9168-2
PMCID: PMC2844513  PMID: 20058113
influenza vaccine; intranasal vaccine; Lactococcus lactis GEM particles
14.  Physical Approaches for Nucleic Acid Delivery to Liver 
The AAPS Journal  2008;10(4):589-595.
The liver is a key organ for numerous metabolic pathways and involves many inherited diseases that, although being different in their pathology, are often caused by lack or overproduction of a critical gene product in the diseased cells. In principle, a straightforward method to fix such problem is to introduce into these cells with a gene-coding sequence to provide the missing gene product or with the nucleic acid sequence to inhibit production of the excessive gene product. Practically, however, success of nucleic acid-based pharmaceutics is dependent on the availability of a method capable of delivering nucleic acid sequence in the form of DNA or RNA to liver cells. In this review, we will summarize the progress toward the development of physical methods for nucleic acid delivery to the liver. Emphasis is placed on the mechanism of action, pros, and cons of each method developed so far. We hope the information provided will encourage new endeavor to improve the current methodologies or develop new strategies that will lead to safe and effective delivery of nucleic acids to the liver.
doi:10.1208/s12248-008-9067-y
PMCID: PMC2628207  PMID: 19083101
gene delivery; liver; nonviral vectors; physical method; transfection
15.  Physical Approaches for Nucleic Acid Delivery to Liver 
The AAPS journal  2008;10(4):589-595.
Liver is a key organ for numerous metabolic pathways and involves many inherited diseases that, although being different in their pathology, are often caused by lack or overproduction of a critical gene product in the diseased cells. In principle, a straightforward method to fix such problem is to introduce into these cells with a gene-coding sequence to provide the missing gene product, or with the nucleic acid sequence to inhibit production of the excessive gene product. Practically, however, success of nucleic acid-based pharmaceutics is dependent on availability of a method capable of delivering nucleic acid sequence in the form of DNA or RNA to liver cells. In this review, we will summarize the progress toward development of physical methods for nucleic acid delivery to liver. Emphasis is placed on the mechanism of action, pros and cons of each method developed so far. We hope the information provided will encourage new endeavor to improve the current methodologies or develop new strategies that will lead to safe and effective delivery of nucleic acids to liver.
doi:10.1208/s12248-008-9067-y
PMCID: PMC2628207  PMID: 19083101
Gene delivery; non-viral vectors; physical method; liver; transfection
16.  A Bayesian Approach for Quantifying Trace Amounts of Antibody Aggregates by Sedimentation Velocity Analytical Ultracentrifugation 
The AAPS Journal  2008;10(3):481-493.
Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become an important tool for the characterization of the purity of protein therapeutics. The work presented here addresses a need for methods orthogonal to size-exclusion chromatography for ensuring the reliable quantitation of immunogenic oligomers, for example, in antibody preparations. Currently the most commonly used approach for SV-AUC analysis is the diffusion-deconvoluted sedimentation coefficient distribution c(s) method, previously developed by us as a general purpose technique and implemented in the software SEDFIT. In both practical and theoretical studies, different groups have reported a sensitivity of c(s) for trace oligomeric fractions well below the 1% level. In the present work we present a variant of c(s) designed for the purpose of trace detection, with customized Bayesian regularization. The original c(s) method relies on maximum entropy regularization providing the most parsimonious distribution consistent with the data. In the present paper, we use computer simulations of an antibody system as example to demonstrate that the standard maximum entropy regularization, due to its design, leads to a theoretical lower limit for the detection of oligomeric traces and a consistent underestimate of the trace populations by ∼0.1% (dependent on the level of regularization). This can be overcome with a recently developed Bayesian extension of c(s) (Brown et al., Biomacromolecules, 8:2011–2024, 2007), utilizing the known regions of sedimentation coefficients for the monomer and oligomers of interest as prior expectation for the peak positions in the distribution. We show that this leads to more clearly identifiable and consistent peaks and lower theoretical limits of quantization by approximately an order of magnitude for some experimental conditions. Implications for the experimental design of SV-AUC and practical detection limits are discussed.
doi:10.1208/s12248-008-9058-z
PMCID: PMC2696691  PMID: 18814037
analytical ultracentrifugation; Bayesian analysis; hydrodynamic separation; sedimentation velocity; size-distribution; trace aggregates
17.  Pharmacogenetic and Metabolic Differences Between Dog Breeds: Their Impact on Canine Medicine and the Use of the Dog as a Preclinical Animal Model 
The AAPS journal  2008;10(1):110-119.
There is limited information describing species related pharmacogenetic differences in animals. Despite the lack of genetic information in veterinary medicine, breed specific responses to endogenous and exogenous substances have been reported across many species. This finding underscores the importance of obtaining insight into the genotypic and phenotypic variation present across breeds. This article provides a summary of the literature pertaining to canine breed differences in physiology, drug response, drug pharmacokinetics, and metabolic idiosyncrasies. The existing knowledge of pedigrees and the known phenotypes and genotypes of dogs provides important information for determining mode of inheritance, penetration, and other major characteristics of heritable traits. Understanding these breed differences will improve canine population predictions (for canine drug products) and may be of value when extrapolating toxicology data from dogs to humans.
doi:10.1208/s12248-008-9011-1
PMCID: PMC2747081  PMID: 18446511
bioavailability; breed-related differences; canine pharmacodynamics; canine pharmacogenetics; canine pharmacokinetics; drug response; population diversity
18.  Glial–Neuronal Interactions—Implications for Plasticity and Drug Addiction 
The AAPS journal  2009;11(1):123-132.
Among neuroscientists, astrocytes have for long played Cinderella to their neuron stepsisters. While the importance of glia in regulating brain activity was predicted by Ramon y Cajal more than a century ago (Garcia-Marin et al., Trends. Neurosci. 30:479–787, 2007), these cells, until recently, have been thought to play mainly a passive part in synaptic signaling. Results obtained over the last decade have begun to suggest otherwise. Experiments carried out in a number of labs have shown that glial cells, especially astrocytes, directly participate in synaptic signaling and potentially regulate synaptic plasticity and network excitability. The presence of signaling pathways on astrocytes that are analogous to those at presynaptic terminals suggests a role for these cells in network plasticity. Findings that the same signaling pathways can be activated by receptors for drugs of abuse present on astrocytes suggest a role for these cells in the addictive process. In this review, we summarize current understanding of astrocytic role in synaptic signaling and suggest that a complete understanding of the process of addiction requires a better understanding of the functional role of these cells.
doi:10.1208/s12248-009-9085-4
PMCID: PMC2664886  PMID: 19238557
astrocyte; calcium; gliotransmission; nicotinic; synapse; tripartite synapse
19.  A Bayesian Approach for Quantifying Trace Amounts of Antibody Aggregates by Sedimentation Velocity Analytical Ultracentrifugation 
The AAPS journal  2008;10(3):481-493.
Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become an important tool for the characterization of the purity of protein therapeutics. The work presented here addresses a need for methods orthogonal to size-exclusion chromatography for ensuring the reliable quantitation of immunogenic oligomers, for example, in antibody preparations. Currently the most commonly used approach for SV-AUC analysis is the diffusion-deconvoluted sedimentation coefficient distribution c(s) method, previously developed by us as a general purpose technique and implemented in the software SEDFIT. In both practical and theoretical studies, different groups have reported a sensitivity of c(s) for trace oligomeric fractions well below the 1% level. In the present work we present a variant of c(s) designed for the purpose of trace detection, with customized Bayesian regularization. The original c(s) method relies on maximum entropy regularization providing the most parsimonious distribution consistent with the data. In the present paper, we use computer simulations of an antibody system as example to demonstrate that the standard maximum entropy regularization, due to its design, leads to a theoretical lower limit for the detection of oligomeric traces and a consistent underestimate of the trace populations by ∼0.1% (dependent on the level of regularization). This can be overcome with a recently developed Bayesian extension of c(s) (Biomacromolecules (2007), 8, 2011-2024), utilizing the known regions of sedimentation coefficients for the monomer and oligomers of interest as prior expectation for the peak positions in the distribution. We show that this leads to more clearly identifiable and consistent peaks and lower theoretical limits of quantization by approximately an order of magnitude for some experimental conditions. Implications for the experimental design of SV-AUC and practical detection limits are discussed.
doi:10.1208/s12248-008-9058-z
PMCID: PMC2696691  PMID: 18814037
sedimentation velocity; analytical ultracentrifugation; trace aggregates; hydrodynamic separation; size-distribution; Bayesian analysis
20.  Modulation of Microglial Pro-inflammatory and Neurotoxic Activity for the Treatment of Parkinson’s Disease 
The AAPS journal  2006;8(3):E606-E621.
Parkinson’s disease (PD) is a debilitating movement disorder resulted from a progressive degeneration of the nigrostriatal dopaminergic pathway and depletion of neurotransmitter dopamine in the striatum. Molecular cloning studies have identified nearly a dozen genes or loci that are associated with small clusters of mostly early onset and genetic forms of PD. The etiology of the vast majority of PD cases remains unknown and the precise molecular and biochemical processes governing the selective and progressive degeneration of the nigrostriatal dopaminergic pathway is poorly understood. Current drug therapies for PD are symptomatic and appear to bear little impact on the progressive neurodegenerative process. Studies of post mortem PD brains and various cellular and animal models of PD in the last two decades strongly suggest that the generation of pro-inflammatory and neurotoxic factors by the resident brain immune cells, microglia, plays a prominent role in mediating the progressive neurodegenerative process. This review will discuss literature supporting the possibility of modulating the activity of microglia as a neuroprotective strategy for the treatment of PD.
doi:10.1208/aapsj080369
PMCID: PMC2668934  PMID: 17025278
Dopamine neuron; Parkinson’s disease; Movement disorder; Microglia; Neuroprotection; Free radical
21.  Pharmacogenomic Responses of Rat Liver to Methylprednisolone: An Approach to Mining a Rich Microarray Time Series 
The AAPS journal  2005;7(1):E156-E194.
A data set was generated to examine global changes in gene expression in rat liver over time in response to a single bolus dose of methylprednisolone. Four control animals and 43 drug-treated animals were humanely killed at 16 different time points following drug administration. Total RNA preparations from the livers of these animals were hybridized to 47 individual Affymetrix RU34A gene chips, generating data for 8799 different probe sets for each chip. Data mining techniques that are applicable to gene array time series data sets in order to identify drug-regulated changes in gene expression were applied to this data set. A series of 4 sequentially applied filters were developed that were designed to eliminate probe sets that were not expressed in the tissue, were not regulated by the drug treatment, or did not meet defined quality control standards. These filters eliminated 7287 probe sets of the 8799 total (82%) from further consideration. Application of judiciously chosen filters is an effective tool for data mining of time series data sets. The remaining data can then be further analyzed by clustering and mathematical modeling techniques.
doi:10.1208/aapsj070117
PMCID: PMC2607485  PMID: 16146338
Data mining; gene arrays; glucocorticoids; mathematical modeling; pharmacogenomics
23.  Overview of the Proton-coupled MCT (SLC16A) Family of Transporters 
The AAPS journal  2008;10(2):311-321.
The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs). Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been functionally characterized. Despite their sequence homology, only MCT1-4 have been demonstrated to be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1-4 vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates, MCTs are involved in the transport of pharmaceutical agents, including γ-hydroxybuytrate (GHB), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide. MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such they have the potential to impact a number of processes contributing to the disposition of xenobiotic substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant administration of GHB and l-lactate to rats results in an approximately two-fold increase in GHB renal clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective strategy for increasing renal and total GHB elimination in overdose situations. Further studies are required to more clearly define the role of MCTs on drug disposition and the potential for MCT-mediated detoxification strategies in GHB overdose.
doi:10.1208/s12248-008-9035-6
PMCID: PMC2574616  PMID: 18523892
butyrate; gamma-hydroxybutyrate; lactate; monocarboxylate transporters; SLC16A
24.  O-Phospho-L-Serine, Multi-functional Excipient for B Domain Deleted Recombinant Factor VIII 
The AAPS journal  2007;9(2):E251-E259.
Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain, A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, ∼15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients’ plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid binding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free-vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDrFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.
doi:10.1208/aapsj0902028
PMCID: PMC2573386  PMID: 17907766
B domain deleted recombinant factor VIII; O-phospho-L-serine; protein formulation; excipient; physical stability; immunogenicity; inhibitor development
25.  O-phospho-L-serine, multi-functional excipient for B domain deleted recombinant factor VIII 
The AAPS Journal  2007;9(2):E251-E259.
Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, ∼15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients' plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid bindinding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free-vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDRFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.
doi:10.1208/aapsj0902028
PMCID: PMC2573386  PMID: 17907766
B domain deleted recombinant factor VIII; O-phospho-L-serine; protein formulation; excipient; physical stability; immunogenicity; inhibitor development

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