(T1030-05-27) Dendrimer Conjugated nSMase2 Inhibitor (D-DPTIP) as a Novel Treatment for Alzheimer’s Disease

Tuesday, October 18, 2022

10:30 AM – 11:30 AM ET

Presenting Author(s)

Arindom Pal, PhD (he/him/his)

Post Doctoral Fellow
Johns Hopkins Drug Discovery
Baltimore, Maryland, United States

Main Author(s)

Arindom Pal, PhD (he/him/his)

Post Doctoral Fellow
Johns Hopkins Drug Discovery
Baltimore, Maryland, United States

Purpose: Approximately 1 in 9 Americans age 65 and older are suffering from Alzheimer’s disease (AD) and this number is expected to rise as high as 16 million by 2050¹. Current treatments only provide symptomatic improvement². Moreover, a lack of therapeutics in the development pipeline and their attrition rate suggest disease-modifying treatments may not be available in the foreseeable future³. Recently a novel AD therapeutic target has emerged from the Extracellular Vesicle (EV) field. EVs are small vesicular carriers containing proteins, nucleic acids, and bioactive lipids that are shed from cells in response to various stimuli. Under disease conditions, EVs can carry pathological cargo and play an active role in disease progression^4-11. There is cumulative evidence from animal models and human studies supporting roles for EVs in AD^12-15. The brain enzyme neutral sphingomyelinase2 (nSMase2) is a critical regulator of EV biogenesis¹⁶. Pharmacological inhibition and genetic deletion of nSMase2 has been shown to reduce EV secretion, Aβ plaque formation and tau propagation, and improve cognition in mouse models of AD^12-15. Thus, nSMase2 inhibition represents a promising new therapeutic approach for AD that would focus on inhibiting disease progression^12-14. Our laboratory recently discovered 2,6-dimethoxy-4-(5-phenyl-4-(thiophen-2-yl)-1H-imidazol-2-yl) phenol (DPTIP), which is a highly potent and selective inhibitor of nSMase2 (IC₅₀ = 30 nM)¹⁷. However, DPTIP exhibits poor pharmacokinetic properties with low oral bioavailability ( < 5 F%), poor brain penetration (AUC_brain/ AUC_plasma < 0.2) and rapid clearance (t_1/2<0.5h). In this study, we sought to improve DPTIP’s pharmacokinetic properties and brain delivery by conjugating the drug to dendrimer nanoparticles.

Methods: We synthesized dendrimer-DPTIP (D-DPTIP) conjugates with generation-4 (G4) and generation-6 (G6) hydroxyl PAMAM dendrimers as shown in scheme-1. The number of drug molecules attached to the conjugate was calculated using 1H NMR comparing the integration of dendrimer amidic protons to ester methylene protons and triazole ring protons. We evaluated the G4 and G6 D-DPTIP for pH-dependent chemical stability at pH 1.2 (simulated gastric fluid), 4.5, 6.8 (simulated intestinal fluid), and 7.4. In addition, we also evaluated stability in mouse plasma, mouse brain homogenate, and in the presence of purified carboxylesterase (CES) enzyme. Next, we conducted in vivo time-dependent pharmacokinetic evaluation in PS19 mice¹⁸ (a transgenic mouse model of AD overexpressing human P301S mutant tau) with a head-to-head comparison to DPTIP, G4, and G6 D-DPTIP conjugates (100 mg/kg DPTIP equivalent, PO). We also evaluated the inhibition of IL-1β-induced EV release by G4 and G6 D-DPTIP (100 mg/kg DPTIP equivalent, PO) using a GFAP–EGFP transgenic mouse model of brain injury.

Results: Both G4 and G6 D-DPTIP demonstrated excellent chemical stability, with less than 5% and 10% DPTIP release from G6 and G4 D-DPTIP, respectively, over 60 min under all pH conditions tested. However, G4 D-DPTIP showed instability in mouse plasma, brain homogenate, and purified pig liver CES, releasing ~100%, ~60%, ~95% of DPTIP at 60 min, respectively. Whereas, G6 D-DPTIP showed ~100%, and ~93% release of DPTIP over 60 min in plasma and purified CES, respectively, but an ~8% DPTIP release in the brain. When evaluated in vivo in PS19 mice, G4 D-DPTIP showed a 3-fold increase (AUC_brain3064 pmol.h/g) and G6 D-DPTIP showed ~2.5-fold (AUC_brain2606 pmol.h/g) increase in brain exposures compared to DPTIP (AUC_brain990 pmol.h/g). Both G4 and G6 D-DPTIP showed ~4-fold lower plasma exposures compared to DPTIP, thereby demonstrating greatly improved brain-to-plasma ratios, with ~12 and 10.5-fold increase, respectively. G4 and G6 conjugates also showed a remarkably improved apparent half-life in the brain (~50h and 38h respectively) compared to DPTIP (half-life ~30 min). These improvements in the brain exposures were also reflected by the significant inhibition of microglial nSMase2 activity 24h and 48h post-dosing in the brain samples from the PS19 mice. When tested in an IL-1β induced GFAP–EGFP transgenic mouse model of brain injury, both G4 and G6 (100 mg/kg DPTIP equivalent dose) D-DPTIP showed inhibition of EV release (total and brain-derived) up to 48h, whereas DPTIP did not show any inhibitory activity. This was further confirmed by the significant inhibition of nSMase2 activity in the brain striatal samples from the IL-1β induced mice.

Conclusion: We have demonstrated a significant enhancement in the pharmacokinetic properties and brain penetration of DPTIP using dendrimer-conjugation. Future efforts are focused on the G4 DP-DPTIP since this conjugate provided overall higher brain exposure and apparent half-life compared to the G6 conjugate.

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Acknowledgments: This work is funded by an NIH grant (1R01AG063831).

Scheme-1: Synthesis protocol of G4 and G6 D-DPTIP conjugates. (A) Conjugation of PEG linker using the corresponding PEG-azide acid; (B) Coupling of DPTIP-PEG-azide with G4 or G6 dendrimer with alkyne modified terminal using click (CuAAC) chemistry yielding G4 and G6 D-DPTIP.

Figure-1: Pharmacokinetic evaluation. DPTIP (A), G4 D-DPTIP (B) and G6 D-DPTIP were dosed orally in PS19 mice 100 mg/kg DPTIP equivalent dose; (D) Inhibition of microglial nSMase2 activity in the brain samples of the treated mice from the same study; (E) Pharmacokinetic parameters of DPTIP, G4 and G6 D-DPTIP.

Figure-2: Inhibition of EVs release. (A) Study design; Quantification of total EVs (B) and GFP labeled EVs (C) and measurement of nSMase2 activity in brain striata following treatment with Vehicle, DPTIP, G4 or G6 D-DPTIP orally.