Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling of Anti-Neoplastic Agents

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Introduction
Development of tumor resistance to chemotherapeutics is related to inherent tumor variations regarding sensitivity to chemotherapeutics and to sub-optimal dosing regimens, including variation in patient pharmacokinetics that result in suboptimal exposure of tumor cells to anti-neoplastic drugs [1,2]. The rate and extent of drug efficacy depends on the extent of drug exposure at the tumor site and the time above the effective concentration [3]. In vitro models that incorporate these pharmacokinetic and pharmacodynamic (PK/PD) principles to optimize therapeutic response may be considered the method of choice for optimizing dosing schedules before translating data from static assays to animals and clinical trials [4,5]. The hollow fiber bioreactor was recently used to evaluate pharmacokinetic/pharmacodynamic (PK/PD) effects of gemcitibine in lung and breast cancers and to model HIV treatments [4][5][6].

Hollow fiber bioreactor model
The hollow fiber bioreactor model ( Figure 1) is a dynamic system that provides a cell culture with constant flow of fresh medium through a 3D polysulfone cartridge (support for cell growth) with capillary sized passages (cells grow in the extracapillary space), at physiologically relevant flow rates. Automatic programmable pumps accurately regulate dosing of chemotherapeutics into the system.
The hollow fiber cartridge consists of two compartments, the porous capillaries through which medium flows and the extra-capillary space (ECS) that separates the cell culture from the flowing medium. Medium is constantly perfused through the fibers into the ECS, carrying nutrients and infused drugs to the cell culture. Due to the dynamic nature of the Fig. 1. Schematic of the bioreactor setup model, cells can be exposed to carefully chosen drug concentrations that simulate patient pharmacokinetics from published clinical data, simulating drug half-life and renal clearance [6]. This change in concentration over time cannot be achieved in static assays. To monitor cell growth and viable counts in undisturbed hollow fiber cultures, we used an assay based on measurement of fluorescence resulting from the reduction of non-fluorescent resazurin to its red fluorescent form (resorufin) by metabolically active cells [7]. The intensity of the fluorescence is proportional to the viable cell count [8]. Achievement of cell cycle arrest, as a surrogate marker of the therapeutic outcome, was measured by flow cytometry. We hypothesized that we could use the proposed dynamic model to accurately simulate (R)roscovitine (ROSC) pharmacodynamics, incorporating pharmacokinetic data. To achieve this, we used the MCF-7 human breast cancer cell line and compared the peak values from two proposed dosing regimens of ROSC to untreated controls. MCF-7 cells are derived from the pleural effusion of an estrogen receptor positive, human breast adenocarcinoma [9,10]. Cells were cultured in EMEM, 2mM L-glutamine, 0.1mM non-essential amino acids, 1mM sodium pyruvate, 0.01mg/mL bovine insulin and 10% fetal bovine serum; grown in 5% CO 2 at 37 o C and dispersed when confluent with of 0.25% trypsin/0.5mM EDTA. Hollow fiber cartridges were prepared according to manufacturers instructions. To prepare hollow fiber cultures, briefly, trypsinized cells were diluted to 1.67 x 10 6 /mL; 3mL were seeded into the ECS of each cartridge. Two cartridges were seeded for each experiment; one for drug treatment and one control. Cartridges were placed on a dual recirculating pump in the incubator at 37°C with 5% CO 2 to circulate fresh sterile medium to each cartridge simultaneously while establishing the cultures. www.intechopen.com

Pharmacokinetic simulation for (R)-roscovitine
After 24 hours, cartridges were removed from the recirculating pump, connected to a linear pump system (Masterflex) and a digital syringe pump was used to control the rate of drug infusion ( Figure 1). Mathematical pharmacokinetic drug profiles were simulated by adjusting the input of ROSC and medium in the system. Infusion of the drug at peak concentration was followed by simulation of its clearance rate over a 6 hour period. Controls were perfused with drug free medium under the same conditions. Each dosing schedule was performed at least in duplicate.

PK/PD simulation for the bioreactor
A one compartment model, oral absorption and first order elimination was constructed using STELLA software (isee systems inc., Lebanon, NH). This model simulated pharmacokinetic profiles of peak and trough plasma levels after 800 mg twice daily (BID) and three times daily (TID) dosage regimens using pharmacokinetic parameters from a previously published clinical trial [11]. STELLA simulation provided a profile range of several C max from which to mimic the C ss (steady state concentration) in the bioreactor. To accomplish this infusion, peak and trough concentrations of the C ss range were used to determine the final dose, which represented the population pharmacokinetics and was infused to the central reservoir. Simulated BID peak and trough concentrations were 3167 ng/mL and 808 ng/mL, respectively. Simulated TID peak and trough concentrations were 3893 ng/mL and 1923 ng/mL, respectively. Flow rates for the pumps were determined using the following equation for oral administration: where C l is clearance of drug and is equal to the rate set on the Masterflex pumps, k e is the elimination rate constant for ROSC, V d is central reservoir volume, C 1 is C max from the simulation during the steady state, C 2 is C min from the simulation during the steady state, and t (time) is the experiment duration. The rate of the digital pumps simulated the patient drug clearance.

LC/MS
To confirm the achievement of peak drug concentrations, samples were collected from the ECS of the hollow fiber cartridge and analyzed by LC/MS. were analyzed using Shimadzu's data analysis program. Peak integrations were compared to a standard curve to obtain the concentrations of ROSC.

Resazurin assay
Fluorescent conversion of resazurin is directly related to the number of metabolically active cells in culture and serves as a surrogate probe for viable cell count. Medium containing 5% v/v resazurin was added to the hollow fiber system and circulated for 30 minutes for homogenous distribution of resazurin containing medium. System valves were closed and the cartridge incubated 2 hours; 200 µl samples were drawn from medium in the ECS. Fluorescence was measured on a microplate reader (Biotek, Winooski, VT). Resazurin was removed by pumping fresh medium through the system with linear pumps, collecting the resazurin medium as waste. Assays were performed before and after treatment for assessing baseline fluorescence and the effect of drug on cell proliferation. Resazurin has been reported to be cytotoxic at high concentrations and exposure beyond 24 hours [12]. Since cell lines respond differently to resazurin, [13], we conducted a static assay to establish that 5% (v/v) resazurin could be used over at least a 24 hour period without toxicity to the MCF-7 cell line. Cells incubated with resazurin, and untreated controls, were harvested, stained with trypan blue and counted at the conclusion of the experiment. This confirmed that growth and viability were unaffected by resazurin at 5% v/v (unpublished data).

Flow cytometry
MCF-7 cells were harvested from the hollow fiber cartridge by trypsinization and centrifuged at 300x g for 5 minutes. Supernatant was removed and the pellet resuspended in 0.5ml of PBS-T with 20 µg/mL of RNAse and incubated at 37°C for 30 min. 10μg/ml propidium iodide was added to the cell suspension and incubated at 37°C for 30 min. The stained nuclei (200,000 per sample) were analyzed with a Becton Dickinson FACSCalibur (San Jose, California) flow cytometer using medium flow mode. Cell cycle distribution was analyzed with Modfit LT program (Verity, Maine).

Results
Pharmacokinetic simulation of ROSC was used to predict the C max of BID and TID dosing regimens. The predicted bolus dose values for the BID and TID were 3167ng/mL (8.9µM) and 3893ng/mL (10.9µM), respectively. From this data, we modeled a one dose peak profile that we could assess in the in vitro hollow fiber model. Actual concentrations of ROSC achieved in the model reached a C max of 2953 ng/mL (8.3µM) for the BID schedule, an accuracy of 93.2% compared to the predicted value. Actual concentration for the TID schedule was 3307ng/mL (9.3µM) as compared to the mathematical profile C max of 3893ng/mL, an accuracy of 85.0%. This level of accuracy for infused drug is consistent with the reports of Kirstein, et al. in their PK/PD studies in the hollow fiber model [5,6]. To monitor cell proliferation and viability before treatment, without disturbing established cultures, we used the resazurin assay on all cartridges. There was no significant difference in cell proliferation between cartridges before treatment (p=0.86), indicating consistency in seeding the hollow-fiber cartridges. In post-treatment resazurin assays, ROSC treated cartridges had decreased cell proliferation compared to control cartridges. The TID (9.3µM) simulated dose had a 15-20% decrease in growth that was not evident with the BID (8.3 µM) www.intechopen.com simulated dose (Figure 2). To determine the effect of ROSC on cell cycle, cells were collected by trypsinization from the hollow-fiber cartridge at 24 hours post-treatment and analyzed by flow cytometry. An increase was seen in the proportion of cells in the G1 phase in both the BID and TID treatments compared to the control. Correspondingly, there was a statistically significant decrease of the proportion of S phase cells in both the TID (p=0.004) and BID (p=0.003) doses compared to the control and a statistically significant increase in G2 for BID simulation (Table 1).

Discussion
Uncontrolled proliferation is a defining characteristic of tumor cells. This is known to occur through mutation in genes that produce regulatory proteins which modulate cell cycle, such as the Cyclin Dependent Kinases (Cdks) [14][15][16]. Cdks are highly conserved serine/threonine kinases whose activation depends on formation of a heterodimeric complex with cyclins [17][18][19]. Thus, Cdks are a promising therapeutic target for cancer treatment [20]. ROSC is a novel Cdk inhibitor that induces cell cycle arrest and apoptosis through multiple intracellular targets in a variety of tumor cell lines. When tested in static in vitro assays, IC 50 values of about 14µM are reported with 24 hours of drug exposure [21][22][23][24]. However, dosing schedules of 2500 mg/day over 5 days in 3 week cycles or the reduced dose of 1600mg/day BID for 10 days were recommended based on early human trials [25] and a concomitant Phase one trial that indicated a dose limiting toxicity of 800 mg BID for 7 days. With this dosing schedule, peak plasma levels were achieved in 1-4 hours with elimination by 2-5 hours. The highest plasma concentration achieved clinically was 10µM within a treatment period. There was no tumor response to this dose, although the disease was stabilized over several courses of treatment [11]. To investigate the effect of administration of ROSC as a monotherapy by altering the dosing schedule, we employed a dynamic hollow fiber model in this study. We established our model using STELLA to simulate pharmacokinetic profiles of ROSC derived from clinical data. These profiles were simulated in the in vitro hollow fiber model for assessing the effectiveness of two dosing regimens against MCF-7 cells by using flow cytometry to measure cell cycle distribution under each condition. The results revealed increasing numbers of MCF-7 cells accumulating in G1 phase after simulating the BID and TID dosing peaks with ROSC, and indicating a significant difference between TID and the control. Also, BID dosing simulation had a significant increase in G2 arrest (p<0.05). This data is consistent with previous studies which indicated arrest of MCF-7 cell proliferation at the G1/S transition and G2/M transition with accumulation in G2. However, Wesierska-Gadek, et al. revealed ROSC activity only at the G2/M transition and accumulation of arrested MCF-7 cells only in the G2 phase. The differences may be attributed to differences in their experimental design, as they exposed MCF-7 cells in a static system to 10µM ROSC for 24 hours [26]. In this study, we tested concentrations of ROSC based on human clinical trial values of a maximum value of 10µM over 1-4 hours, with ROSC clearance within 5 hours, which were below the levels tested in static systems [11]. In this case, dose limiting toxicity of the drug precludes achievement of effective plasma concentrations in humans, which is consistent with the clinical trials. Consequently, ROSC is now being tested in combination therapies [27]. In conclusion, this study demonstrated the application of the dynamic hollow fiber model with controlled pharmacokinetic profiles to simulate clinically relevant dosing schedules of ROSC for treatment of breast tumor cells and the use of a resazurin assay for monitoring cell viability of hollow fiber cell cultures. With this methodology, cells can be recovered for further analysis. The applied hollow fiber model in this study can be used for PK/PD assessment of different anti-neoplastic drugs (single and in combination), with different exposure times against many tumor cell lines.