PBPK with FcRn and Apoptosis Dynamics

Physiologically-Based Pharmacokinetic (PBPK) Model
FcRn and Apoptosis Dynamics

· Physiologically-Based Pharmacokinetic (PBPK) Model

o Tumor, Bone, Heart

§ Expansion of Baxter model (pharmacodynamics)

· Apoptosis

§ Two-pore transcapillary flux model

· Two-pore model under non-isogravimetric conditions

· Solute transport across a permeable barrier in the presence of volume flow (I)

· Solute transport across a permeable barrier in the presence of volume flow (II)

· Summary of the two-pore model (*.doc)

o Skin and Muscle

§ Expansion of Baxter model (pharmacodynamics)

· Apoptosis

§ Two-pore transcapillary flux model

· Two-pore model under non-isogravimetric conditions

· Solute transport across a permeable barrier in the presence of volume flow (I)

· Solute transport across a permeable barrier in the presence of volume flow (II)

· Summary of the two-pore model (*.doc)

§ novel FcRn sub-model

· Mathematical Model of FcRn sub-model

· FcRn expression in various organs

o Kidney

§ Expansion of Baxter model (pharmacodynamics)

· Apoptosis

§ Two-pore transcapillary flux model

· Two-pore model under non-isogravimetric conditions

· Solute transport across a permeable barrier in the presence of volume flow (I)

· Solute transport across a permeable barrier in the presence of volume flow (II)

· Summary of the two-pore model (*.doc)

o Liver

§ Expansion of Baxter model (pharmacodynamics)

· Apoptosis

§ Two-pore transcapillary flux model

· Two-pore model under non-isogravimetric conditions

· Solute transport across a permeable barrier in the presence of volume flow (I)

· Solute transport across a permeable barrier in the presence of volume flow (II)

· Summary of the two-pore model (*.doc)

o Lung

§ Expansion of Baxter model (pharmacodynamics)

· Apoptosis

§ Two-pore transcapillary flux model

· Two-pore model under non-isogravimetric conditions

· Solute transport across a permeable barrier in the presence of volume flow (I)

· Solute transport across a permeable barrier in the presence of volume flow (II)

· Summary of the two-pore model (*.doc)

o Plasma (central pool)

o GI and Spleen

§ Expansion of Baxter model (pharmacodynamics)

· Apoptosis

§ Two-pore transcapillary flux model

· Two-pore model under non-isogravimetric conditions

· Solute transport across a permeable barrier in the presence of volume flow (I)

· Solute transport across a permeable barrier in the presence of volume flow (II)

· Summary of the two-pore model (*.doc)

o Augmentations

§ FcRn Submodel

§ Variable Tumor Mass Submodel

o Parameters & Parameter Estimation

§ Parameters used in PBPK model

· Parameter units and definitions

§ Adjustable Parameters

§ SAAM II Simulation and Parameter Estimation Conditions

§ Results of SAAM II parameter estimation

o Statistics

§ Objective function values

§ Covariance Matrix

§ Correlation Matrix

o Simulations

§ Blood, Tumor, Lungs, Kidneys, Liver, Spleen, GI Tract, Bone, Carcass

o Predict F(ab’)₂ Biodistribution

§ Using modified intact mAb PBPK model

§ Set k_on for interaction between antibody Fc portion and FcRn to zero

§ Fit tumor growth model to data from F(ab’)₂ biodistribution experiment

§ Change antibody specific parameters as listed in Baxter et al.

§ Predicted curve for anti-CEA F(ab’)2 fragment & biodistribution data: Blood, Tumor, Lungs, Kidneys, Liver, Spleen, GI Tract, Bone Carcass

o SAAM II

§ intact mAb

§ F(ab’)₂

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· FcRn and Apoptosis Dynamics (image key)

o FcgR

§ FcgR expressing cells amplify CD20-mediated apoptosis in Ramos B cells

§ Fc common g chain (component of activating FcRs) knock-out mice exhibit decreased susceptibility to mAb / FcRg meditated anti-tumor action

§ FcgRIIB (inhibitory FcR) knock-out mice exhibit increased susceptibility to mAb / FcRg meditated anti-tumor action

§ In vitro and in vivo properties of the D255A mutant antibody

o Ca2+

§ Intracellular calcium mobilization initiated by anti-CD20 mAb + GAM

§ Ca²⁺ chelators inhibit apoptosis induced by hypercrosslinking CD20

§ Calcium chelators inhibit CD20-mediated apoptosis in Ramos B cells

§ PP2 inhibits CD20 mediated calcium influx (Ca²+ influx is downstream of Lck/Fyn/Hck phosphorylation)

§ Effect of calcium chelators

o Rituxan

§ Rituxan is a Chimeric Anti-CD20 Monoclonal Antibody

§ Apoptotic effects of CD20 mAbs

§ Rituxan alone induces apoptosis while 1F5 and 2H7 require GAM mediated hyper-crosslinking (Ramos B cells analyzed by flow cytometry after 18-22 hours)

o Topology of CD20

o Fyn, Lck, Lyn

§ Fyn, Lck, and Lyn are constitutively associated with CD20

§ Inhibition of Lck and Fyn inhibits apoptosis (p < 0.05)

§ PP2 (Src family kinase inhibitor) inhibits CD20-mediated apoptosis in Ramos B cells

§ PP2 inhibits CD20-mediated caspase-3 activation

o PLCg2

§ Tyrosine phosphorylation of PLC-g1 and PLC-g2 is induced by CD20 stimulation

§ CD20 mediated PLCg2 phosphorylation

o B-cell receptor

§ Correlation between anti-CD20 and BCR-mediated apoptosis

§ Similar changes of c-myc and Berg-36 RNA expression level after CD20 and BCR cross-linking

§ CD20 and the BCR transiently colocalize on intact Ramos cells

§ BCR/CD20 dissociation requires BCR stimulation

o Bcl-2

o Bax

o Mcl-1 and XIAP

o Cytochrome c

o p38 MAPK

§ Rituximab-induced apoptosis and p38 MAP-kinase activity

§ p38 inhibitor decreases % apoptosis; MEK inhibitor has no effect on CD20 mediated apoptosis

o Jun / Fos

o AP-1

o Caspase-9, -3, PARP

§ Change in caspase and apoptosis proteins in vivo after treatment with rituximab

§ CD20XL induces cytochrome-c release, caspase-9 and –3 activation, and PARP cleavage

§ Effects of CD20 triggering on PARP, SP1, and Caspase-3

o SP1

o Mathematical Model [Ca²⁺]_cyt Oscillations

§ VisSim: Ferl-DeYoung-Marhl calcium model

o Evidence for a second, caspase independent pathway

§ Caspase inhibition does not completely block CD20 mediated apoptosis

§ Overexpression of Bcl-2 does not inhibit CD20 mediated apoptosis

§ Overexpression of Bcl-2 prevents cytochrome c release and dissipation of mitochondrial transmembrane potential (Δψ_m), but does not completely inhibit CD20-mediated apoptosis

o Protein Domain Interactions

§ Schematic of IP3 generation

§ Schematic of Lyn activation

· Evidence for the involvement of kinases other than Fyn and Lck

· Previously unidentified CD20 associated p75/80

1. CD20 association with 75/80- and 50-60-kDa proteins tyrosine phosphorylated in vivo

2. Association of CD20 with p75/80, p56^lck, and p59^fyn in CD20-transfected Molt-4 T cells

3. Deletions involving the cytoplasmic regions of CD20 do not eliminate associations with p75/80 or PTK

§ Schematic of regulation of cytochrome-c flux through mitochondrial membrane

§ Schematic of caspase-9 activation

§ Schematic of caspase-3 activation

§ Schematic of SP1 cleavage

§ Schematic of PARP cleavage

o Flux / Activation / Formation (arrows)

§ Equations for IP3 generation

· Simulation Equations

1. Parameters

1. Ad hoc parameter sensitivity analysis: n

2. Ad hoc parameter sensitivity analysis: “k_IP3”

2. VisSIM: Calcium 2002

· Michaelis - Menten kinetics

1. Derivation of Michaelis-Menten Equation (I)

2. Derivation of Michaelis-Menten Equation (II)

3. Derivation of Michaelis-Menten Equation: Example 1

4. Derivation of Michaelis-Menten Equation: Example 2

§ Equations for Ca²⁺ flux through cell membrane

§ Equations for FcgR induced Ca²⁺ flux through cell membrane

§ Ca2+ flux equations for mitochondria

· Marhl et al. model

1. Parameters

2. VisSIM: Marhl complex calcium oscillations

§ Ca2+ flux equations for ER

· Complete Marhl et al. Model

· Marhl et al. model

1. Parameters

2. VisSIM: Marhl complex calcium oscillations

· Complete De Young - Keizer Model

1. Parameters

2. IP3 Receptor Kinetics

3. VisSIM: De Young calcium model

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