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1.  Mathematical Modeling of Corticosteroid Pharmacogenomics in Rat Muscle following Acute and Chronic Methylprednisolone Dosing 
Molecular pharmaceutics  2008;5(2):328-339.
The pharmacogenomic effects of a corticosteroid (CS) were assessed in rat skeletal muscle using microarrays. Adrenalectomized (ADX) rats were treated with methylprednisolone (MPL) by either 50 mg/kg intravenous injection or 7-day 0.3 mg/kg/h infusion through subcutaneously implanted pumps. RNAs extracted from individual rat muscles were hybridized to Affymetrix Rat Genome Genechips. Data mining yielded 653 and 2316 CS-responsive probe sets following MPL bolus and infusion treatments. Of these, 196 genes were controlled by MPL under both dosing conditions. Cluster analysis revealed that 124 probe sets exhibited three typical expression dynamic profiles following acute dosing. Cluster A consisted of up-regulated probe sets which were grouped into five subclusters each exhibiting unique temporal patterns during the infusion. Cluster B comprised down-regulated probe sets which were divided into two subclusters with distinct dynamics during the infusion. Cluster C probe sets exhibited delayed down-regulation under both bolus and infusion conditions. Among those, 104 probe sets were further grouped into subclusters based on their profiles following chronic MPL dosing. Several mathematical models were proposed and adequately captured the temporal patterns for each subcluster. Multiple types of dosing regimens are needed to resolve common determinants of gene regulation as chronic exposure results in unexpected differences in gene expression compared to acute dosing. Pharmacokinetic/pharmacodynamic (PK/PD) modeling provides a quantitative tool for elucidating the complexities of CS pharmacogenomics in skeletal muscle.
doi:10.1021/mp700094s
PMCID: PMC4196382  PMID: 18271548
Microarray studies; pharmacokinetics; pharmacodynamics; mathematical models; computational biology
2.  Microarray analysis of the temporal response of skeletal muscle to methylprednisolone: comparative analysis of two dosing regimens 
Physiological genomics  2007;30(3):282-299.
The transcriptional response of skeletal muscle to chronic corticosteroid exposure was examined over 168 h and compared with the response profiles observed following a single dose of corticosteroid. Male adrenalectomized Wistar rats were given a constant-rate infusion of 0.3 mg•kg−1•h−1 methylprednisolone for up to 7 days via subcutaneously implanted minipumps. Four control and forty drug-treated animals were killed at ten different time points during infusion. Liver total RNAs were hybridized to 44 individual Affymetrix REA230A gene chips. Previously, we described a filtration approach for identifying genes of interest in microarray data sets developed from tissues of rats treated with methylprednisolone (MPL) following acute dosing. Here, a similar approach involving a series of three filters was applied sequentially to identify genes of interest. These filters were designed to eliminate probe sets that were not expressed in the tissue, not regulated by the drug, or did not meet defined quality control standards. Filtering eliminated 86% of probe sets, leaving a remainder of 2,316 for further consideration. In a previous study, 653 probe sets were identified as MPL regulated following administration of a single (acute) dose of the drug. Comparison of the two data sets yielded 196 genes identified as regulated by MPL in both dosing regimens. Because of receptor downregulation, it was predicted that genes regulated by receptor-glucocorticoid response element interactions would exhibit tolerance in chronic profiles. However, many genes did not exhibit steroid tolerance, indicating that present perspectives on the mechanism of glucocorticoid action cannot entirely explain all temporal profiles.
doi:10.1152/physiolgenomics.00242.2006
PMCID: PMC4186702  PMID: 17473217
glucocorticoids; corticosteroids; Affymetrix gene chips; gene expression; time series
3.  Application of Scaling Factors in Simultaneous Modeling of Microarray Data from Diverse Chips 
Pharmaceutical research  2007;24(4):643-649.
Purpose
Microarrays have been utilized in many biological, physiological and pharmacological studies as a high-throughput genomic technique. Several generations of Affymetrix GeneChip® microarrays are widely used in gene expression studies. However, differences in intensities of signals for different probe sets that represent the same gene on various types of Affymetrix chips make comparison of datasets complicated.
Materials and Methods
A power coefficient scaling factor was applied in the pharmacokinetic/ pharmacodynamic (PK/PD) modeling to account for differences in probe set sensitivities (i.e., signal intensities). Microarray data from muscle and liver following methylprednisolone 50 mg/kg i.v. bolus and 0.3 mg/kg/h infusion regimens were taken as an exemplar.
Results
The scaling factor applied to the pharmacodynamic output function was used to solve the problem of intensity differences between probe sets. This approach yielded consistent pharmacodynamic parameters for the applied models.
Conclusions
Modeling of pharmacodynamic/pharmacogenomic (PD/PG) data from diverse chips should be performed with caution due to differential probe set intensities. In such circumstances, a power scaling factor can be applied in the modeling.
doi:10.1007/s11095-006-9215-y
PMCID: PMC4181592  PMID: 17318415
bioinformatics; computational biology; pharmacodynamics; pharmacogenomics; pharmacokinetics
4.  Modeling Circadian Rhythms of Glucocorticoid Receptor and Glutamine Synthetase Expression in Rat Skeletal Muscle 
Pharmaceutical research  2006;23(4):670-679.
Purpose
The circadian rhythm of endogenous corticosterone (CS) may produce fluctuations of downstream gene expression in normal rats. This study examined changes in glucocorticoid receptor (GR) and glutamine synthetase (GS) expression in rat skeletal muscle in relation to plasma CS over a 24-h period.
Methods
Fifty-four normal male Wistar rats were sacrificed at 18 time points (n = 3) over 24 h. Plasma CS concentrations and gastrocnemius muscle GR and GS mRNA and GS activity were measured.
Results
The circadian rhythm of plasma CS was captured by a two-harmonic function. The expression of GR and GS mRNA and GS activity follow a circadian rhythm in normal rat skeletal muscle. GR mRNA reaches a trough at 4 h after the peak of plasma CS and it fluctuates between 0.55 and 0.9 fmol g tissue−1. GS mRNA and activity reach peaks at 6 and 12 h after the endogenous CS peak. GS mRNA oscillates between 3 and 6 fmol g tissue−1, whereas GS activity fluctuates between 17 and 23 µmol min−1 g protein−1. Mechanistic receptor/gene-mediated pharmacodynamic models were applied to describe the temporal patterns of GR mRNA, GS mRNA, and GS activity within the circadian cycle.
Conclusions
The integrated models were able to capture the circadian expression patterns of plasma CS, and GR and GS in normal rat skeletal muscle showing a dependence of tissue gene expression on plasma CS.
doi:10.1007/s11095-005-9608-3
PMCID: PMC4178542  PMID: 16673181
biological rhythm; mathematical model; pharmacodynamics; pharmacokinetics
5.  Pharmacokinetic/Pharmacodynamic Modeling of Corticosterone Suppression and Lymphocytopenia by Methylprednisolone in Rats 
Journal of pharmaceutical sciences  2008;97(7):2820-2832.
Adrenal suppression and lymphocytopenia are commonly monitored pharmacological responses during systemic exposure to exogenously administered corticosteroids. The pharmacodynamics of plasma corticosterone (CS) and blood lymphocytes were investigated in 60 normal rats which received either 50 mg/kg methylprednisolone (MPL) or vehicle intramuscularly. Blood samples were collected between 0.5 and 96 h following treatment. Plasma CS displayed a transient suppression with re-establishment of a normal circadian rhythm 24 h following drug treatment. An indirect response model with suppression of production well captured plasma CS profiles. An early stress-induced rise in CS was also factored into the model. Blood lymphocyte numbers exhibited a sharp decline and then returned to a new circadian rhythm which was half of the original baseline level. An integrated pharmacodynamic (PD) model with inhibition of lymphocyte trafficking from tissue to blood by both MPL and CS and induction of cell apoptosis by MPL reasonably captured this lymphocytopenia. Rats and humans differ in lymphocyte responses with humans showing full recovery of baselines. Modeling provides a valuable tool in quantitative assessment of dual, complex drug responses.
doi:10.1002/jps.21167
PMCID: PMC3726057  PMID: 17828751
pharmacokinetics; pharmacodynamics; hormones; mathematical model; pharmacokinetic/pharmacodynamic models; corticosteroid; lymphocyte; cell trafficking; indirect response model; circadian rhythm
6.  Charge variants in IgG1 
mAbs  2010;2(6):613-624.
Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7–9.1 and was composed of about 20% acidic variants, 12% basic variants and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity or the PK properties in rats.
doi:10.4161/mabs.2.6.13333
PMCID: PMC3011216  PMID: 20818176
mAb IgG1; charge heterogeneity; isoelectric point; neonatal Fc receptor (FcRn); pharmacokinetics; potency
7.  Pharmacodynamic/Pharmacogenomic Modeling of Insulin Resistance Genes in Rat Muscle After Methylprednisolone Treatment: Exploring Regulatory Signaling Cascades 
Corticosteroids (CS) effects on insulin resistance related genes in rat skeletal muscle were studied. In our acute study, adrenalectomized (ADX) rats were given single doses of 50 mg/kg methylprednisolone (MPL) intravenously. In our chronic study, ADX rats were implanted with Alzet mini-pumps giving zero-order release rates of 0.3 mg/kg/h MPL and sacrificed at various times up to 7 days. Total RNA was extracted from gastrocnemius muscles and hybridized to Affymetrix GeneChips. Data mining and literature searches identified 6 insulin resistance related genes which exhibited complex regulatory pathways. Insulin receptor substrate-1 (IRS-1), uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), fatty acid translocase (FAT) and glycerol-3-phosphate acyltransferase (GPAT) dynamic profiles were modeled with mutual effects by calculated nuclear drug-receptor complex (DR(N)) and transcription factors. The oscillatory feature of endothelin-1 (ET-1) expression was depicted by a negative feedback loop. These integrated models provide testable quantitative hypotheses for these regulatory cascades.
PMCID: PMC2733097  PMID: 19787081
corticosteroid; glucocorticoid; microarrays; mathematical modeling; insulin resistance

Results 1-7 (7)