Pluronic based β-cyclodextrin polyrotaxanes for treatment of Niemann-Pick Type C disease

Abstract

Niemann-Pick Type C disease (NPC) is a rare metabolic disorder characterized by disruption of normal cholesterol trafficking within the cells of the body. There are no FDA approved treatments available for NPC patients. Recently, the cycloheptaglucoside 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) has shown efficacy as a potential NPC therapeutic by extending lifetime in NPC mice, delaying neurodegeneration, and decreasing visceral and neurological cholesterol burden. Although promising, systemic HP-β-CD treatment is limited by a pharmacokinetic profile characterized by rapid loss through renal filtration. To address these shortcomings, we sought to design a family of HP-β-CD pro-drug delivery vehicles, known as polyrotaxanes (PR), capable of increasing the efficacy of a given injected dose by improving both pharmacokinetic profile and bioavailability of the HP-β-CD agent. PR can effectively diminish the cholesterol pool within the liver, spleen, and kidney at molar concentrations 10-to-100-fold lower than monomeric HP-β-CD. In addition to this proof-of-concept, use of PR scaffolds with differing physiochemical properties reveal structure-activity relationships in which PR characteristics, including hydrophobicity, threading efficiency and surface charge, were found to both decisively and subtly effect therapeutic efficacy. PR scaffolds exhibit absorption, pharmacokinetics, and biodistribution patterns that are significantly altered from monomeric HP-β-CD. In all, PR scaffolds hold great promise as potential treatments for visceral disease in NPC patients.

Figure 1. Molecular Structures of PR Components.

The overall PR molecular structure (a) consists of cyclodextrin-derivative macrocycles non-covalently threaded onto tri-block copolymer cores. CD macrocycles (b) will consist of either HP-β-CD (X) alone or a mixture of HP-β-CD and SBE-β-CD (Y). Tri-block copolymer cores (c) consist of a central PPG block and outer PEG blocks of varying lengths. Terminal cholesterol substituents serve to block the CD macrocycles from dethreading from the polymer core. L81: m = 6.25, n = 43; F127: m = 200, n = 65.

Figure 2. PR are capable of reducing cholesterol accumulation in the visceral organs of NPC^nih mice.

Unesterified cholesterol extracted from the visceral organs was quantified by LC/MS/MS. Cholesterol concentrations in the (a) liver, (b) kidney, (c) spleen, and (d) lung are shown. For WT control, n = 7. For all others, n = 6. Statistical analyses are relative to untreated NPC control. *p = 0.05, **p = 0.01, ***p = 0.005, ****p = 0.001.

Figure 3. Cholesterol concentrations in the central nervous system of NPC^nih mice are unaffected by PR treatment.

Unesterified cholesterol concentrations in the (a) brain and the (b) cerebellum specifically were determined by LC/MS/MS after extraction. For WT control n = 7. For all others n = 6. Statistical analyses are relative to untreated NPC control. ****p = 0.001.

Figure 4. Incubation with L81-HP PR and L81-HP/SBE PR affect the substrate turnover of Cytochrome P450 isoforms.

Isoforms studied included (a) 3A4, (b) 2C9, and (c) 2D6. Studies characterized the cleavage of fluorogenic CYP substrates in the presence of PR compounds or known inhibitors ketoconazole (3A4), sulphaphenazole (2C9), and quinidine (2D6). The intensity of fluorescence indicates the extent of CYP activity perturbation. Experiments were done in triplicate and expressed as a percent of uninhibited control. *p = 0.05, **p = 0.01, ***p = 0.005, ****p = 0.001.

Figure 5. Magnetic resonance images of Balb/c mice after IP administration of Gd³⁺-labeled L81-HP/SBE PR and Gd³⁺-labeled HP-β-CD.

Both materials were injected into the IP cavity at 0.03 mmol Gd³⁺/kg. Changes in contrast were evident over 6 h of imaging in the IP space (top), kidney (middle) and liver (bottom). Red arrows indicate the IP space and bladder contrast. Yellow arrows indicate contrast in kidneys. Light blue arrows indicate liver. Images were acquired using a 7T Bruker scanner.

Figure 6. L81-HP/SBE PR pharmacokinetics and biodistribution after intraperitoneal injection.

Gd³⁺:DOTA labeled L81-HP/SBE was administered to WT mice by IP injection. Samples were collected at the specified times before HNO₃ digestion and analysis for Gd³⁺ content by ICPMS. (a) PR biodistribution at 24 h revealed predominantly liver deposition (n = 4). (b) PR pharmacokinetics showed low blood concentrations over time (n = 4; except at 24 h where n = 7 and 48 h where n = 3). Gd³⁺ concentrations measured in the (c) blood, liver and lymph nodes and recovered from the (d) IP cavity at 24 and 48 h (n = 4). (e) Gd³⁺-containing material that remained adhered to the parietal peritoneum at 48 h (n = 4). ***p = 0.005, ****p = 0.001.