In normal cardiac myocytes, the action potential duration (APD) is several hundred milliseconds. However, experimental studies showed that under certain conditions, APD could be excessively long (or ultralong), up to several seconds. Unlike the normal APD, the ultralong APD increases sensitively with pacing cycle length even when the pacing rate is very slow, exhibiting a sensitive slow rate-dependence. In addition, these long action potentials may or may not exhibit early afterdepolarizations (EADs). Although these phenomena are well known, the underlying mechanisms and ionic determinants remain incompletely understood. In this study, computer simulations were performed with a simplified action potential model. Modifications to the L-type calcium current (ICa,L) kinetics and the activation time constant of the delayed rectifier K current were used to investigate their effects on APD. We show that: 1) the ultralong APD and its sensitive slow rate-dependence are determined by the steady-state window and pedestal ICa,L currents and the activation speed and the recovery of the delayed rectifier K current; 2) whether an ultralong action potential exhibits EADs or not depends on the kinetics of ICa,L; 3) increasing inward currents elevates the plateau voltage, which in general prolongs APD, however, this can also shorten APD when the APD is already ultralong under certain conditions; and 4) APD alternans occurs at slow pacing rates due to the sensitive slow rate-dependence and the ionic determinants are different from the ones causing APD alternans at fast heart rates.

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e13013 Background: Hot flashes or vasomotor symptoms (VMS) are a common side effect of hormone deprivation (HD) therapy. Up to 80 % of cancer patients treated with tamoxifen (antiestrogen treatment) or leuprolide (androgen deprivation) have VMS. In some cases, patients discontinue HD therapy due to VMS severity and lower quality of life; therefore, reducing VMS is critical for patient compliance. NK3 receptor (NK3R) antagonists have previously been shown to reduce VMS in postmenopausal women. ACER-801 is a candidate NK3R antagonist drug intended to alleviate VMS severity when used with HD therapy. We used Quantitative Systems Pharmacology (QSP) modeling to predict the likely efficacy of ACER-801 in patients on HD therapy and evaluate the potential of hepatic drug-drug interactions between ACER-801 and tamoxifen or leuprolide. Methods: The model is composed of KNDy neurons in the arcuate nucleus with NKB, dynorphin, and estradiol effects on KNDy neurons, HPG axis, sex hormones, and neuroendocrine feedback. ACER-801, tamoxifen, and leuprolide PK, PD, and hepatic metabolism are included in the model. The model was developed, qualified, and tested using literature data. A tamoxifen-treated postmenopausal female virtual patient (VP) and leuprolide-treated male VP were created based on typical patients included in clinical trials. VMS severity and frequency were estimated based on the level of NKB binding to NK3R. Results: In the male VP, simulated leuprolide administration induced hypertrophy of KNDy neurons and VMS over six months. Coadministration of ACER-801 with leuprolide reduced VMS frequency and severity to near 0 in short-term (5-week) simulations. Simulations predict ACER-801 will not alter leuprolide metabolism nor increase plasma testosterone concentrations. Tamoxifen treatment increased VMS by 15% in simulations with the postmenopausal VP. Coadministration of ACER-801 with tamoxifen in this VP reduced VMS by 75% compared to tamoxifen monotherapy. ACER-801 had minimal effects on plasma estradiol concentrations in the postmenopausal VP. Drug-drug interactions between ACER-801 and tamoxifen were dependent on the simulated bioavailability of ACER-801. Using current estimates of ACER-801 bioavailability in the model, the hepatic concentration of ACER-801 had limited effects on tamoxifen metabolism, which are not expected to necessitate dose adjustments. Conclusions: Using a QSP neurobiology model as a research tool enabled us to evaluate the efficacy of the NK3R antagonist, ACER-801, to treat HD therapy-induced VMS. Simulations show ACER-801 may be highly efficacious for the treatment of induced-VMS. The research provided estimates of DDI with ACER-801 and tamoxifen and what clinical experiments would be needed to confirm those estimates.

Background and Aims: Replacement FIX therapy (rIX) is an effective treatment for hemophilia B even with undetectable levels in the blood 1. However, the mechanistic reason for hemostasis with low plasma levels is not well understood. There is growing evidence that FIX interactions with one or multiple binding partners (BP), may play a significant role in the exposure and hemostatic efficacy of rIX 2,3. The aim of this study is to explore this hypothesis by comparing the plasma PK, tissue biodistribution, and in vivo endpoints of different rIX variants using a mouse QSP model. Method:  in vitro and in vivo FIX-KO mice studies and mathematical models were used to build a QSP model consisting of 8 tissue compartments , with each tissue divided into vascular, endothelial and interstitial spaces 4,5. The model simulates endogenous mouse IgG (mIgG), mouse serum albumin (MSA), and rIX dynamics including key clearance and distribution mechanisms. Competition for the endothelial FcRn receptor between Fc, albumin, mIgG, and MSA is explicitly modeled 6,7,8. The model was calibrated using mouse studies of radiolabeled rIX-Fc (Alprolix®), rIX-WT (BeneFIX®), and rIX-FP (Idelvion®). Tail-clip experiments following administration of rIX-Fc, rIX-WT, and rIX-FP were used to correlate the predicted exposures with the observed effects on bleeding time and total blood loss. Results: Preliminary simulations proved that having at least one BP best explains the rapid distribution of rIX-Fc and rIX-WT into the tissues, and the long plasma T 1/2 of rIX-Fc and rIX-FP. Visual predictive checks of the full PBPK model showed good agreement with the PK in the tissues. The best fit was achieved using a specific arrangement of four distinct binding partners: Shared BP (SBP) between all compounds (e.g. N-terminal binder) located within the vasculature with estimated K D of 470/600/4100 nM, for rIX-WT/rIX-FP/rIX-Fc, respectively. BP binding specific to rIX-WT (e.g. C-terminal binder) located in the interstitium of the tissue (varying densities) with estimated K D of 23 nM BP binding only for rIX-FP (e.g. albumin binder) located in both; the vasculature and interstitium of the tissue with estimated K D 20/0.05 μM (vascular/interstitial) BP binding only for rIX-Fc (e.g. Fc binder) located in the interstitium of tissue (varying densities) with estimated K D 3 μM The high degree of extravasation of rIX-Fc (and rIX-WT to a lesser degree) results in rapid distribution and sequestration in the tissues. The limited extravasation of rIX-FP and its high affinity to the SBP, results in increased recovery and a greater pool of bound rIX available in the tissue vasculature. Additionally, strong inverse correlation between the bound rIX in the vasculature and bleeding time/total blood loss suggests that the vascular pool plays a more significant role in FIX pharmacology, as compared to the pool in the extravascular space. Conclusion: The mouse QSP model demonstrated that the plasma and tissue biodistribution of rIX-Fc, rIX-FP, and rIX-WT cannot be explained without a BP, and that it is plausible to assume that different binding partners, both intra- and extravascular, for different rFIX variants exist. The correlation between the levels of bound rIX and the coagulation endpoints suggests that the vascular bound rIX may be the pharmacologically active pool or reservoir for haemostasis. The extravasation and sequestration of rIX-WT and rIX-Fc into the tissues may explain the decreased vascular exposure, and hence, the reduced efficacy (increased bleeding time/total blood loss) at later time points. Although the exact identity of the BP's remains to be further elucidated, the model estimates of their affinity, density and location provide guidance for further experimental investigations. Expansion of the QSP model with additional data and coagulation kinetics will further our understanding of the role of BPs in rIX pharmacology. References 1Srivastava A et al (2013) Haemophilia 19(1), e1-47 2Feng D et al (2013) JTH, Vol. 11 (12), 2176-2178 3Cheung WF et al (1996) PNAS USA, 93(20), 11068-11073 4Li L et al (2014) AAPS Journal 16(5), 1097-1109 5Shah DK & Betts AM (2012) J Pharmacokinet Pharmacodyn 39(1), 67-86 6Chia J et al (2018) J Biol Chem 293(17), 6363-6373 7Andersen JT et al (2010) J Biol Chem 285(7), 4826-4836 8Andersen JT et al (2013) J Biol Chem 288(33), 24277-24285 Disclosures Pestel: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Rezvani-Sharif: CSL Behring Ltd: Current Employment, Current equity holder in publicly-traded company. Muir: CSL Behring Ltd: Current Employment, Current holder of stock options in a privately-held company. Krupa: CSL Behring LLC: Current Employment, Current equity holder in publicly-traded company. Brechmann: CSL Behring Innovation GmbH, Ended employment in the past 24 months: Bayer Ag (Bayer Pharmaceuticals),: Current Employment, Ended employment in the past 24 months, Patents & Royalties: Bayer. Verhagen: CSL Behring Ltd: Current Employment, Current equity holder in publicly-traded company. Dower: CSL Behring Ltd: Current Employment, Current equity holder in publicly-traded company. Herzog: CSL Behring GmbH: Current Employment, Current equity holder in publicly-traded company.