(M0930-05-27) In Vivo Preclinical Pharmacokinetics of Nortriptyline and Its Application to Develop a Physiologically Based Pharmacokinetic Model

Purpose: Tricyclic antidepressants (TCAs) are well-established drugs with favorable safety profiles, making them strong candidates for drug repurposing.¹ TCAs provide neuropathic pain relief and exhibit anti-tumoral effects across various cancers, including lymphoma, breast cancer, small cell carcinoma, and brain cancers.² Nortriptyline, an active metabolite of amitriptyline, is known to be efficacious in pineoblastoma; but its use in medulloblastoma, a leading cause of cancer-related mortality in children-has not been studied. This study aims to repurpose nortriptyline for the treatment of medulloblastoma by characterizing its in vivo pharmacokinetic profile and applying it to the development of a physiologically based pharmacokinetic (PBPK) model to predict therapeutic doses.

Methods: A safe dose for pharmacokinetic study was determined by identifying the maximum tolerated dose (MTD). The MTD was identified in healthy male and female NSG mice by the standard up-down method. A starting dose of 5 mg/kg was administered and increased 1.8-fold (every 48 hours) until toxicity. The in vivo pharmacokinetics and biodistribution profile of nortriptyline was assessed in healthy NSG mice following a single intraperitoneal (IP) dose of 15 mg/kg. Plasma and tissue concentrations were quantified using a validated and robust liquid chromatography–tandem mass spectrometry method, with protein precipitation as the extraction technique. Non-compartmental analysis (NCA) was performed on plasma concentration data using Phoenix WinNonlin. NCA parameters with standardized units, along with published physicochemical properties, were used to develop a PBPK model of nortriptyline in SimCYP software (V23). A permeability-limited model was used with a first-order input into venous blood to simulate IP dosing. Biodistribution was modeled using observed tissue partition coefficients (K_p) for brain, heart, lungs, liver, kidney, and spleen (30.7, 24.7, 146.9, 37.5, 112.5, and 45.1, respectively), and a Kp scalar of 0.18 to simulate in vivo the volume of distribution (V_d/F). The elimination phase was defined using a combination of hepatic (CYP-mediated metabolism) and renal clearance. The developed PBPK model was verified against the in vivo plasma concentration-time profile, and model verification was considered successful if the fold difference between observed and predicted parameters was less than 2.

Results: The in vivo plasma concentration–time profile indicated rapid absorption, reaching a mean maximum plasma concentration (C_max) of 818.3 ± 85.7 ng/mL in 0.15 ± 0.09 h (t_max) with the area under the curve (AUC₀-_t) of 2294.3 ± 316.8 ng.h/mL following an IP injection of 15 mg/kg (Figure 1a). Nortriptyline exhibited a short elimination half-life (t_1/2 2.3 h), high V_d /F (20.8 ± 2.6 L/kg) and systemic clearance (CL/F 6.3 ± 0.6 L/h/kg). The biodistribution study revealed that the brain concentration peaked at approximately 14 μg/g at 2 h, resulting in a high brain-to-plasma ratio (K_{p, brain} 30.7) (Figure 1b). Our findings suggest that an IP dose of 15 mg/kg produces brain concentrations above the IC₅₀ value (1844 ng/L in HD-MB03 cells) for more than 8 h, underscoring the potential of repurposing nortriptyline for brain tumors. Additionally, high lipophilicity of nortriptyline led to its extensive distribution into other tissues as well (Figure 1b). A MTD of 38.6 mg/kg was identified with insignificant weight loss, normal hematology, and normal tissue histology (data not shown). This highlights the potential to increase the dose/dosing frequency of nortriptyline to maintain therapeutic drug levels in the brain. The developed PBPK model accurately captured the absorption, distribution, and elimination phases (Figure 2). The predicted vs. observed systemic PK parameters aligned closely, C_max (829.8 vs. 818.3 ng/mL), T_max (0.19 vs. 0.15 h), AUC₀-_t (2078.62 vs. 2294.3 ng·h/mL), V_d/F (18.1 vs. 20.8 L/kg), t_1/2 (2.8 vs. 2.3 h), and the CL/F (3.0 vs. 2.8 mL/min), respectively. The predicted vs. observed brain concentrations at 2 h (11843.3 vs. 13796.4 ng/g), and AUC_0-t (63816.5 vs. 78633.9 ng.h/g), respectively, also overlapped (Table 1). The fold-difference between the systemic and brain predicted and observed pharmacokinetic parameters was within 2-fold (0.81-1.28), successfully validating the developed PBPK model (Table 1). The verified PBPK model of nortriptyline will be used to predict doses and dosing intervals for identifying an effective therapeutic regimen for the treatment of medulloblastoma.

Conclusion: This study characterized nortriptyline pharmacokinetics and biodistribution in NSG mice, achieving brain levels above the therapeutic threshold after a single dose. The PBPK model, developed and validated using the preclinical pharmacokinetic profile, effectively predicted systemic and brain pharmacokinetic parameters within an acceptable 2-fold difference. These findings demonstrate the utility of the PBPK model for dose optimization and provide a strong foundation for repurposing nortriptyline in the treatment of medulloblastoma. Future work will focus on leveraging this model to design therapeutic regimens and evaluate the efficacy of nortriptyline in preclinical studies.

References: (1) Asensi-Cantó, A.; López-Abellán, M. D.; Castillo-Guardiola, V.; Hurtado, A. M.; Martínez-Penella, M.; Luengo-Gil, G.; Conesa-Zamora, P. Antitumoral Effects of Tricyclic Antidepressants: Beyond Neuropathic Pain Treatment. Cancers 2022, 14 (13), 3248.
(2) Petrosyan, E.; Fares, J.; Cordero, A.; Rashidi, A.; Arrieta, V. A.; Kanojia, D.; Lesniak, M. S. Repurposing autophagy regulators in brain tumors. Int J Cancer 2022, 151 (2), 167-180. DOI: 10.1002/ijc.33965 From NLM.

Acknowledgements:

Funding: This research was supported in part by the State of Nebraska through the Pediatric Cancer Research Group, part of the Child Health Research Institute (Dr. Daryl J. Murry and Dr. Siddharth Mahapatra)


Figure 1: Preclinical (a) plasma concentration time profile (mean ± SD, n=5) and (b) biodistribution (mean ± SD, n=5) of nortriptyline following a single intraperitoneal dose of 15 mg/kg


Figure 2: Observed vs predicted systemic concentrations of nortriptyline using an optimized physiologically based pharmacokinetic model (PBPK)


Table 1: Fold difference between observed and predicted preclinical systemic and brain pharmacokinetic parameters using the optimized PBPK model of nortriptyline