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1.  Modeling effects of dexamethasone on disease progression of bone mineral density in collagen-induced arthritic rats 
A mechanism-based model was developed to characterize the crosstalk between proinflammatory cytokines, bone remodeling biomarkers, and bone mineral density (BMD) in collagen-induced arthritic (CIA) rats. Male Lewis rats were divided into five groups: healthy control, CIA control, CIA receiving single 0.225 mg kg−1 subcutaneous (SC) dexamethasone (DEX), CIA receiving single 2.25 mg kg−1 SC DEX, and CIA receiving chronic 0.225 mg kg−1 SC DEX. The CIA rats underwent collagen induction at day 0 and DEX was injected at day 21 post-induction. Disease activity was monitored throughout the study and rats were sacrificed at different time points for blood and paw collection. Protein concentrations of interleukin (IL)-1β, IL-6, receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), and tartrate-resistant acid phosphatase 5b (TRACP-5b) in paws were measured by enzyme-linked immunosorbent assays (ELISA). Disease progression and DEX pharmacodynamic profiles of IL-1β, IL-6, RANKL, and OPG were fitted simultaneously and parameters were sequentially applied to fit the TRACP-5b and BMD data. The model was built according to the mechanisms reported in the literature and modeling was performed using ADAPT 5 software with naïve pooling. Time profiles of IL-1β and IL-6 protein concentrations correlated with their mRNAs. The RANKL and OPG profiles matched previous findings in CIA rats. DEX inhibited the expressions of IL-1β, IL-6, and RANKL, but did not alter OPG. TRACP-5b was also inhibited by DEX. Model predictions suggested that anti-IL-1β therapy and anti-RANKL therapy would result in similar efficacy for prevention of bone loss among the cytokine antagonists.
PMCID: PMC4618640  PMID: 26516581
Biomarkers; bone modeling; dexamethasone; pharmacodynamics; systems model
2.  Therapeutic Protein Drug–Drug Interactions: Navigating the Knowledge Gaps–Highlights from the 2012 AAPS NBC Roundtable and IQ Consortium/FDA Workshop 
The AAPS Journal  2013;15(4):933-940.
The investigation of therapeutic protein drug–drug interactions has proven to be challenging. In May 2012, a roundtable was held at the American Association of Pharmaceutical Scientists National Biotechnology Conference to discuss the challenges of preclinical assessment and in vitro to in vivo extrapolation of these interactions. Several weeks later, a 2-day workshop co-sponsored by the U.S. Food and Drug Administration and the International Consortium for Innovation and Quality in Pharmaceutical Development was held to facilitate better understanding of the current science, investigative approaches and knowledge gaps in this field. Both meetings focused primarily on drug interactions involving therapeutic proteins that are pro-inflammatory cytokines or cytokine modulators. In this meeting synopsis, we provide highlights from both meetings and summarize observations and recommendations that were developed to reflect the current state of the art thinking, including a four-step risk assessment that could be used to determine the need (or not) for a dedicated clinical pharmacokinetic interaction study.
PMCID: PMC3787234  PMID: 23794076
cytochrome P450s; drug–drug interactions; pro-inflammatory cytokines; small molecule; therapeutic protein
3.  Quantitative Dynamic Models of Arthritis Progression in the Rat 
Pharmaceutical research  2008;26(1):196-203.
This comparison employs mathematical disease progression models to identify a rat model of arthritis with the least inter-animal variability and features lending to better study designs.
Arthritis was induced with either collagen (CIA) or mycobacterium (AIA) in either Lewis or Dark Agouti (DA) rats. Disease progression was monitored by paw edema and body weight. Models with production, loss, and feedback components were constructed and population analysis using NONMEM software was employed to identify inter-animal variability in the various disease progression parameters.
Onset time was the only parameter different within all four groups (DA–AIA 11.5 days, DA–CIA 16.5 days, Lewis–AIA 11.9 days, Lewis–CIA 13.9 days). The loss-of-edema rate constant was 20% slower in DA (0.362 h−1) than Lewis (0.466 h−1) rats. Most models exhibited peak paw edema 20 days post-induction. Edema in CIA returned to 150% of the initial value after the disease peaked. DA rats displayed more severe overall responses.
No statistical differences between groups were observed for inter-animal variation in disease onset, progression and severity parameters. Onset time varies and should be noted in the design of future studies. DA rats may offer a more dynamic range of edema response than Lewis rats.
PMCID: PMC3725549  PMID: 18758921
arthritis; disease; model; progression; rat
4.  Pharmacokinetics of Dexamethasone in a Rat Model of Rheumatoid Arthritis 
Dexamethasone (DEX) is often given for the treatment of rheumatoid arthritis and clinical dosing regimens of DEX have often been based empirically. This study tests whether the inflammation processes in a rat model of rheumatoid arthritis alters the clearance and volume of distribution of DEX when compared with healthy controls. Groups of healthy and arthritic male Lewis rats received either a low (0.225 mg/kg) or high (2.25 mg/kg) intramuscular dose of DEX. Arthritis was induced by intradermal injection of type II porcine collagen in incomplete Freund's adjuvant emulsion at the base of the tail. DEX was dosed in the arthritic animals 22 days post arthritis induction. Plasma DEX concentrations were determined by HPLC. Plasma concentration versus time data were analysed by non-compartmental analysis and pharmacokinetic model fitting using the population pharmacokinetic software NONMEM V. A linear bi-exponential pharmacokinetic model with extravascular input described the data for both healthy and arthritic animals. Clearance was the only parameter determined statistically different between both groups (healthy=1.05 l/h/kg, arthritic=1.19 l/h/kg). The steady-state volume of distribution for both groups was 4.85 l/kg. The slight difference in clearance was visibly undetectable and unlikely to produce meaningful changes in DEX disposition in arthritic rats.
PMCID: PMC3712282  PMID: 18613033
dexamethasone; pharmacokinetics; arthritis; collagen
5.  Down-regulation of heat shock protein 70 improves arsenic trioxide and 17-DMAG effects on constitutive signal transducer and activator of transcription 3 activity 
Signal transducer and activator of transcription 3 (STAT3) has been shown to be constitutively active in approximately 50% of patients with acute myeloid leukemia and is associated with worse outcome. Arsenic trioxide (ATO) synergizes with the heat shock protein (HSP) 90 inhibitor, 17-DMAG, to down-regulate STAT3 activity. However, both agents up-regulate HSP70, an anti-apoptotic protein. We therefore examined whether down-regulating HSP70 with short interference (si) RNA will affect ATO and 17-DMAG effects on constitutive STAT3 activity.
Experimental design
A semi-mechanistic pharmacodynamic model was used to characterize concentration–effect relationships of ATO and 17-DMAG effects on constitutive STAT3 activity and HSP70 expression with or without siRNA against HSP70 in a cell line model.
Treatment with siRNA for HSP70 resulted in a stronger degree of synergism on down-regulation of STAT3 activity by ATO and 17-DMAG. However, treatment with siRNA for HSP70 resulted in less synergism on up-regulation of HSP70 by the two drugs.
Down-regulation of HSP70 improves ATO and 17-DMAG effects on constitutive STAT3 activity. These results further provide a basis for studying the combined role of ATO with a HSP90 inhibitor such as 17-DMAG in AML with constitutive STAT3 activity.
PMCID: PMC3152797  PMID: 20035426
Pharmacodynamic modeling; Heat shock protein 70; Heat shock protein 90; Arsenic trioxide; Signal transducer and activator of transcription 3
6.  Targeting 11q23 positive acute leukemia cells with high molecular weight-melanoma associated antigen-specific monoclonal antibodies 
Acute leukemia with 11q23 aberrations is associated with a poor outcome with therapy. The lack of efficacy of conventional therapy has stimulated interest in developing novel strategies. Recent studies have shown that 11q23-positive acute leukemia cells express the high molecular weight-melanoma associated antigen (HMW-MAA). This tumor antigen represents a useful target to control growth of human melanoma tumors in patients and in severe combined immunodeficient (SCID) mice, utilizing antibody-based immunotherapy. This effect appears to be mediated by inhibition of the HMW-MAA function such as triggering of the focal adhesion kinase/proline-rich tyrosine kinase 2 (Pyk2) pathways. Therefore, in this study we tested whether HMW-MAA-specific monoclonal antibodies (mAb) could inhibit growth of 11q23-positive leukemia cells in SCID mice.
HMW-MAA-specific mAb were tested for their ability to inhibit the in vitro proliferation of an 11q23-positive acute myeloid leukemia (AML) cell line and blasts from four patients with 11q23 aberrations and their in vivo growth in subcutaneous and disseminated xenograft models.
The HMW-MAA-specific mAb did not affect in vitro proliferation although they down-regulated phosphorylated (P) Pyk2 expression. Furthermore, the mAb enhanced the in vitro anti-proliferative effect of cytarabine. In vivo the mAb inhibited the growth of leukemic cells in a dose-dependent fashion. However, the difference did not reach statistical significance. No effect was detected on P-Pyk2 expression. Furthermore, HMW-MAA-specific mAb in combination with cytarabine did not improve tumor inhibition. Lastly, the combination of two mAb which recognize distinct HMW-MAA determinants had no detectable effect on survival in a disseminated xenograft model.
HMW-MAA-specific mAb down-regulated P-Pyk2 expression and enhanced the anti-proliferative effect of cytarabine in vitro, but had no detectable effect on survival or growth of leukemia cells in vivo. Whether the HMW-MAA-specific mAb can be used as carriers of toxins or chemotherapeutic agents against 11q23-acute leukemia remains to be determined.
PMCID: PMC2741306  PMID: 18677475
Acute leukemia; 11q23; HMW-MAA; Immunotherapy
7.  Modeling Corticosteroid Effects in a Rat Model of Rheumatoid Arthritis I: Mechanistic Disease Progression Model for the Time Course of Collagen-Induced Arthritis in Lewis Rats 
A mechanism-based model was developed to describe the time course of arthritis progression in the rat. Arthritis was induced in male Lewis rats with type II porcine collagen into the base of the tail. Disease progression was monitored by paw swelling, bone mineral density (BMD), body weights, plasma corticosterone (CST) concentrations, and TNF-α, IL-1β, IL-6, and glucocorticoid receptor (GR) mRNA expression in paw tissue. Bone mineral density was determined by PIXImus II dual energy x-ray densitometry. Plasma CST was assayed by HPLC. Cytokine and GR mRNA were determined by quantitative real-time polymerase chain reaction. Disease progression models were constructed from transduction and indirect response models and applied using S-ADAPT software. A delay in the onset of increased paw TNF-α and IL-6 mRNA concentrations was successfully characterized by simple transduction. This rise was closely followed by an up-regulation of GR mRNA and CST concentrations. Paw swelling and body weight responses peaked approximately 21 days post induction while bone mineral density changes were greatest at 23 days post induction. After peak response the time course in IL-1β, IL-6 mRNA, and paw edema slowly declined towards a disease steady-state. Model parameters indicate TNF-α and IL-1β mRNA most significantly induce paw edema while IL-6 mRNA exerted the most influence on BMD. The model for bone mineral density captures rates of turnover of cancellous and cortical bone and the fraction of each in the different regions analyzed. This small systems model integrates and quantitates multiple factors contributing to arthritis in rats.
PMCID: PMC2574807  PMID: 18448865
8.  Modeling Corticosteroid Effects in a Rat Model of Rheumatoid Arthritis II: Mechanistic Pharmacodynamic Model for Dexamethasone Effects in Lewis Rats with Collagen-Induced Arthritis 
A mechanism-based model for pharmacodynamic effects of dexamethasone (DEX) was incorporated into our model for arthritis disease progression in the rat to aid in identification of the primary factors responsible for edema and bone loss. Collagen-induced arthritis (CIA) was produced in male Lewis rats following injection of type II porcine collagen. DEX was given subcutaneously in single doses of 0.225 or 2.25 mg/kg or 7-day multiple doses of 0.045 or 0.225 mg/kg at 21 days post disease induction. Effects on disease progression were measured by paw swelling, bone mineral density (BMD), body weights, plasma corticosterone (CST), and TNF-α, IL-1β, IL-6, and GR mRNA expression in paw tissue. Lumbar and femur BMD was determined by PIXImus-II dual energy x-ray absorptiometry. Plasma CST was assayed by HPLC. Cytokine and GR mRNA were assayed by quantitative real-time PCR. Indirect response models, drug-interaction models, transduction processes, and the 5th-generation model of corticosteroid dynamics were integrated and applied using S-ADAPT software to describe how dexamethasone binding to GR can regulate diverse processes. Cytokine mRNA, GR mRNA, plasma CST, and paw edema were suppressed following DEX administration. TNF-α mRNA expression and BMD appeared to increase immediately after dosing but were ultimately reduced. Model parameters indicated that IL-6 and IL-1β were most sensitive to inhibition by DEX. TNF-α appeared to primarily influence edema while IL-6 contributed the most to bone loss. Lower doses of corticosteroids may be sufficient to suppress the cytokines most relevant to bone erosion.
PMCID: PMC2574741  PMID: 18448864
9.  Synergism between Arsenic Trioxide and Heat Shock Protein 90 Inhibitors on Signal Transducer and Activator of Transcription Protein 3 Activity - Pharmacodynamic Drug-Drug Interaction Modeling 
Constitutive signal transducer and activator of transcription (STAT) 3 activity, observed in approximately 50% of acute myeloid leukemia (AML) cases and associated with adverse treatment outcome, is down-regulated by arsenic trioxide (ATO). Heat shock protein (HSP) 90 is a molecular chaperone involved in signal transduction pathways. We hypothesized that HSP90 inhibitors will potentiate ATO effect on constitutive STAT3 activity and cell killing. One concern was that the effect of ATO and HSP90 inhibitors will result in up-regulation of HSP70, a protein known to inhibit apoptosis.
Experimental Design
We have used a semi-mechanistic pharmacodynamic model to characterize concentration-effect relationships of ATO and HSP90 inhibitors on constitutive STAT3 activity, HSP70 expression and cell death in a cell line model.
Pharmacodynamic interaction of ATO and three HSP90 inhibitors showed synergistic interactions in inhibiting constitutive STAT3 activity and inducing cell death, in spite of a concurrent synergistic up-regulation of HSP70.
These preliminary results provide a basis for studying the combined role of ATO with HSP90 inhibitors in AML with constitutive STAT3 activity.
PMCID: PMC2715964  PMID: 17404111

Results 1-9 (9)