(T1330-04-21) Repurposing Paclitaxel via Vector-Guided Brain Delivery for Alzheimer Disease Treatment

Purpose: Alzheimer’s disease (AD) is an age-associated progressive neurodegenerative disorder affecting millions of people worldwide along the combined presence of two lesions in the brain: the extracellular amyloid-beta plaques and the intraneuronal neurofibrillary tangles (NFTs) caused by tau protein [1]. Tau protein, an intracellular microtubule-associated protein (MAPT) crucial for stabilizing axonal microtubules and facilitating intracellular transport, has emerged as a pivotal factor in AD’s pathology. Its abnormal hyperphosphorylation and subsequent aggregation into twisted paired helical NFTs deposited within the neurons are closely linked with the cognitive deficits observed in AD [2]. Despite initial enthusiasm, anti-tau therapies have been hindered by the formidable blood-brain barrier (BBB) [2,3]. Our research introduces a novel approach utilizing paclitaxel (PTX), a well-known microtubule-stabilizing agent, formulated at low dosages to mitigate tau-related neuronal damage without inducing the toxic effects commonly associated with higher doses [4,5]. To address the challenges of BBB penetration and enhance the therapeutic efficacy of PTX, we hypothesize that P-PTX-AP2, an innovative conjugate combining PTX with a HPMA (N-(2-hydroxypropyl)methacrylamide) copolymer and Angiopep-2 (AP2), could significantly enhance its delivery across the BBB, increasing its therapeutic potential against tauopathies. Leveraging AP2 to harness LRP1 (low density lipoprotein receptor related protein 1) receptor on endothelial cells for transcytosis [6], alongside the advantageous pharmacokinetic and pharmacodynamic profiles of HPMA polymer. The targeted delivery of PTX can effectively stabilize microtubules, mitigate tau-related neuronal damage, and reverse neurofibrillary tangles formation.

Methods: AP2 (TFFYGGSRGKRNNFKTEEY) was prepared by solid phase peptide synthesis. P-PTX-AP2 was synthesized in two steps: first a copolymer of HPMA, APMA (N-(3-aminopropyl)methacrylamide), and a polymerizable derivative of PTX (MA-GFLG-PTX, MA is methacryloyl) was synthesized by RAFT copolymerization; in the second step the amino groups at copolymer side chain termini were converted to maleimide groups using SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) followed by attachment of cysteine modified AP2 (Cys-AP2). The unreacted maleimide groups were capped with 2-mercaptoethanol. To evaluate the safety profile of P-PTX-AP2 compared to free PTX neurotoxicity assays were carried out on human neuroblastoma SH-SY5Y cells, human hCMEC/D3 endothelial cells, and mouse bEnd.3 endothelial cells. Additionally, the effectiveness of AP2 in mediating BBB transcytosis was quantified using both in vitro Transwell BBB models (Cy5-labeled conjugates) and in vivo mouse models (Cy5.5-labeled conjugates). Transendothelial Electrical Resistance (TEER) value was employed to confirm the integrity of in vitro BBB models. Radial Water Tread (RWT) maze analyzed spatial learning and memory in aged 3xTg-AD mice and AD pathophysiology and phenotypic manifestations in 3xTg-AD mouse models was assessed via immunohistochemical staining of tau protein, amyloid-beta plaques, microglia and astrocytes.

Results: Conjugate synthesis and characterization are shown in Figure 1. The polymer precursor (P-PTX) exhibited a molecular weight of 62.5 kDa, with a narrow polydispersity index. Two distinct batches of P-PTX-AP2 were synthesized, containing either 6 or 13 AP2 moieties per polymer chain (Figure 1B). The expression of LRP-1 in bEnd.3 and hCMEC/D3 cell lines was confirmed (Figure 2A), and AP2 binding to the cell surface on the LRP-1 was assessed via confocal microscopy (Figure 2B) and flow cytometry (Figure 2C). An in vitro transcytosis model utilized the hCMEC.D3 cell line was developed and TEER confirmed cell layer integrity. On day 5, both batches of P-PTX-AP2 were introduced into the apical compartment, and the basolateral to apical concentration ratio (B/A ratio) was determined (Figure 2D). The 6 AP2/chain conjugate demonstrated superior in vitro transcytosis compared to the 13 AP2/chain conjugate. The data show that P-PTX-AP2 has significantly lower neurotoxicity than free PTX, with IC50 values indicating improved safety profiles in bEnd.3, SH-SY5Y, and hCMEC/D3 cell lines. Specifically, IC50 values for P-PTX-AP2 were 88μM, 148 nM, and 4 μM respectively, while those for the generic PTX were 34 μM, 33 nM, and 153 nM (Figure 2E). In vivo experiments in aged 3xTg-AD mice (0.6 mg PTX/kg, administered intravenously in 4 doses, 2 weeks apart) significantly ameliorated cognitive deficits as demonstrated by the RWT (Figure 3A). Histological analysis showed a marked reduction in phospho-tau positive cells in the brains of treated mice, underscoring the efficacy of P-PTX-AP2 in targeting and mitigating tau pathology (Figure 3B). Furthermore, AP2's role in enhancing brain delivery was corroborated through a biodistribution in vivo study using fluorescent imaging , showing higher intensity of conjugates in the brains of mice administered P-AP2-Cy5.5 compared to P-Cy5.5 (Figure 3C).

Conclusion: Our study presents a promising strategy for AD therapy using a novel P-PTX-AP2 conjugate. By leveraging the unique properties of AP2 to mediate BBB transcytosis, we have demonstrated that it is possible to enhance the delivery of PTX to the brain, thereby directly addressing tau-related pathologies. This innovative approach demonstrated promising outcomes in preclinical AD model. It offers a potential therapeutic advancement for AD but also sets a precedent for the development of other CNS-targeted therapies. Further studies are warranted to optimize this delivery system and evaluate its long-term efficacy and safety in clinical settings.

References: [1] M.A. DeTure et al., Mol. Neurodegener. 14, 32 (2019).
[2] E.E. Congdon et al., Nat. Rev. Neeurol. 14, 399 (2018).
[3] E.A. Horne et al., Neurooncol. Adv. 3, 165 (2021).
[4] O.A. Shemesh et al., Neurobiol. Dis. 43, 163 (2011).
[5] D.J. Cross et al., J. Alzheimer Dis. 83, 379 (2021).
[6] M. Demeule et al., J. Neurochem. 106, 1534 (2008).

Acknowledgements: The research was supported by a grant from Alzheimer’s Association (to JY and DC).


Figure 1. (A) Synthesis of P-PTX-AP2. (B) The MW of polymer precursor is 62.5 kDa with a narrow polydispersity. Two different batches of P-PTX-AP2 were obtained, containing 6 or 13 AP2 per polymer chain.


Figure 2. (A) Expression of LRP-1 on bEnd.3 and hCMEC/D3 cells. (B) Colocalization of Cy3-AP2 and anti-LRP-1 antibody. (C) Binding affinity of AP2 peptide to hCMEC/D3. (D) Transwell in vitro BBB model; TEER value of the cell model; transcytosis ratio of P-PTX-AP2 and P-PTX. (E) Cytotoxicity of PTX and P-PTX-AP2 toward different cell lines.


Figure 3. (A) Testing spatial learning and memory with Radial Water Tread following P-PTX-AP2 treatment of aged 3xTg-AD mice (n=12). Saline (n=11) and wild type (WT) mice (n=12) were used as controls. Day 1-4 (learning and acquisition phase), Day 5 (short term), and Day12 (long term memory). (B) Immunohistochemical staining of Tau protein and microglia in untreated or P-PTX-AP2 treated mice. (C) Transport of Cy5.5 labeled P-AP2 across the BBB following intravenous administration to mice. HPMA copolymer without AP2 (PHPMA) was used as control.