Inhalation delivery of proteins from ethanol suspensions

Abstract

To circumvent inherent problems associated with pulmonary administration of aqueous-solution and dry-powder protein drugs, inhalation delivery of proteins from their suspensions in absolute ethanol was explored both in vitro and in vivo. Protein suspensions in ethanol of up to 9% (wt/vol) were readily aerosolized with a commercial compressor nebulizer. Experiments with enzymic proteins revealed that nebulization caused no detectable loss of catalytic activity; furthermore, enzyme suspensions in anhydrous ethanol retained their full catalytic activity for at least 3 weeks at room temperature. With the use of Zn(2+)-insulin, conditions were elaborated that produced submicron protein particles in ethanol suspensions. The latter (insulin/EtOH) afforded respirable-size aerosol particles after nebulization. A 40-min exposure of laboratory rats to 10 mg/ml insulin/EtOH aerosols resulted in a 2-fold drop in the blood glucose level and a marked rise in the serum insulin level. The bioavailability based on estimated deposited lung dose of insulin delivered by inhalation of ethanol suspension aerosols was 33% (relative to an equivalent s.c. injection), i.e., comparable to those observed in rats after inhalation administration of dry powder and aqueous solutions of insulin. Inhalation of ethanol in a relevant amount/time frame resulted in no detectable acute toxic effects on rat lungs or airways, as reflected by the absence of statistically significant inflammatory or allergic responses, damage to the alveolar/capillary barrier, and lysed and/or damaged cells.

Figure 1

Rat blood glucose concentration (A) and serum insulin concentration (B) as a function of time after the animals inhaled for 40 min the aerosols created by nebulizing a 10 mg/ml suspension of Zn²⁺-insulin in absolute ethanol. Curve a refers to the exposed animals (2–11 rats); curve b corresponds to the control animals that breathed room air instead of insulin/EtOH aerosols (5–7 rats). The insulin dose inhaled by a rat was calculated to be ≈100 μg. For other experimental conditions, see Materials and Methods.

Figure 2

The rat serum insulin concentration as a function of time after a s.c. injection of 10 μl of a 10 mg/ml suspension of Zn²⁺-insulin in absolute ethanol diluted with 340 μl of PBS (curve a) or a 40-min inhalation administration of aerosols of the same suspension (curve b). For the s.c. injections, four or five rats were used. For other experimental conditions, see the legend to Fig. 1 and Materials and Methods.