Safety and Pharmacokinetics of Intranasally Administered Heparin

Abstract

Purpose: Intranasally administered unfractionated heparin (UFH) and other sulfated polysaccharides are potential prophylactics for COVID-19. The purpose of this research was to measure the safety and pharmacokinetics of clearance of intranasally administered UFH solution from the nasal cavity.

Methods: Double-blinded daily intranasal dosing in C57Bl6 mice with four doses (60 ng to 60 μg) of UFH was carried out for fourteen consecutive days, with both blood coagulation measurements and subject adverse event monitoring. The pharmacokinetics of fluorescent-labeled UFH clearance from the nasal cavity were measured in mice by in vivo imaging. Intranasal UFH at 2000 U/day solution with nasal spray device was tested for safety in a small number of healthy human subjects.

Results: UFH showed no evidence of toxicity in mice at any dose measured. No significant changes were observed in activated partial thromboplastin time (aPTT), platelet count, or frequency of minor irritant events over vehicle-only control. Human subjects showed no significant changes in aPTT time, international normalized ratio (INR), or platelet count over baseline measurements. No serious adverse events were observed. In vivo imaging in a mouse model showed a single phase clearance of UFH from the nasal cavity. After 12 h, 3.2% of the administered UFH remained in the nasal cavity, decaying to background levels by 48 h.

Conclusions: UFH showed no toxic effects for extended daily intranasal dosing in mice as well as humans. The clearance kinetics of intranasal heparin solution from the nasal cavity indicates potentially protective levels for up to 12 h after dosing.

Keywords: Covid-19; SARS-CoV-2; heparin; intranasal delivery.

Fig. 1

Sample results from 14 day toxicology trial of intranasal heparin in mice. Images of representative A. vehicle control and B. 60 μg/day mouse nose after two weeks. C. Change in body weight from pre-dose to after the 14th day dose

Fig. 2

Dose dependence of frequency of minor observations reported for 14-day heparin trial in mice. No dose dependence in the frequency of minor observations (nasal discharge, dried nasal residue, mild nasal discoloration, etc.) was observed, and no difference between heparin-dosed and vehicle-only control animals were observed

Fig. 3

Blood platelet count after 14 days of daily administration of 60 μg of intranasal heparin in mice. No significant difference in platelet count was observed (p = 0.766)

Fig. 4

aPTT assay of mouse plasma after 14-day heparin trial. No significant differences in aPTT clotting time was observed between heparin at any dose versus vehicle only. Significant differences were observed between vehicle only and the vehicle plasma with exogenous heparin added directly at concentrations of 1 U/mL or 0.5 U/mL (p < 0.0001)

Fig. 5

Buried food retrieval anosmia assay of naïve, vehicle-only, and 60 μg/day heparin in mice. No significant differences were observed between groups (p = 0.2387)

Fig. 6

In vivo imaging of 1.67 μg of nasally administered CF680R-heparin in mice. (A) Analytical size exclusion chromatography verifies that purification has removed nearly all free dye, resulting in all IR fluorescence signal coming from CF680R-heparin. (B) Clearance from the nasal cavity follows a one-phase exponential decay (black line; 95% CI shown as red dotted line) to reach background IR levels (normalized to zero); K = 0.01762, Y₀ = 1.070, half life =39.34 min. Signal is normalized to the measured radiant efficiency at 5 min. (C) In vivo imaging shows accumulation and residence in the nasal cavity, with clear evidence of clearance through the gut

Fig. 7

Blood results from intranasal heparin human subjects. Clinically normal range shaded in green. (A) aPTT results taken during screening, acute and chronic phases, and wash-out. Each line represents a subject. (B) INR results taken during screening, after chronic phase, and after wash-out. (C) Platelet count taken during screening, after chronic phase, and after wash-out. Each line represents a subject