INO-4995 therapeutic efficacy is enhanced with repeat dosing in cystic fibrosis knockout mice and human epithelia

Abstract

Progressive lung damage in cystic fibrosis (CF) has been linked to inadequate airway mucosal hydration. We previously demonstrated that an inositol tetrakisphosphate analog, 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl)ester (INO-4995), regulates airway secretory and absorptive processes, affecting mucosal hydration by prolonged (24 h) inhibition of Na(+) and fluid absorption in CF human nasal epithelia (CFHNE). The objectives of this study were to further assess clinical potential of INO-4995 in CF through ascertaining in vivo activity in mice with CF, determining the effects of repeated administration on potency and determining cytoplasmic half-life. Uptake and metabolism of [(3)H]INO-4995 was monitored with HPLC to calculate intracellular half-life. INO-4995 was administered in vitro repeatedly over 4 to 8 days to CFHNE. Fluid absorption was assessed by blue dextran exclusion, and basal short-circuit current was measured in Ussing chambers. INO-4995 (1-100 microg/kg) was dosed intranasally either as a single dose or once per day over 4 days to gut-corrected CF mice. [(3)H]INO-4995 was rapidly taken up by epithelial cultures and converted to the active drug, which had a half-life of 40 hours. Repeated daily application of INO-4995 to CFHNE lowered the effective concentration for inhibition of fluid absorption and amiloride-sensitive short-circuit current in cultured CFHNE, and reduced nasal potential difference to nearly control levels in gut-corrected CF mice. Ca(2+)-activated Cl(-) channel activity was also boosted in cultures. Mouse nasal levels fell from abnormal levels to within 2 muA of normal with repeated exposure to 0.8 microg/kg over 4 days. These data support further development of INO-4995 for the treatment of CF.

Figure 1.

Pathway depicting 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl)ester (INO-4995) and de-esterification products, 1-O-octyl-2-O-butyryl-myo inositol 3,4,5,6-tetrakisphosphate (INO-4913), and 1-O-octyl-2-O-butyryl-myo inositol 3,4,5,6-tetrakisphosphate (INO-4949). INO-4995 entry into cells is facilitated by the propionoxymethylester groups (PMs), which conceal the charges on the phosphate groups. Once inside the cell, endogenous carboxyesterases cleave the protecting PMs, resulting in an inositol tetrakisphosphate analog, INO-4913 (2). Further hydrolysis of the 2-butyrate yields another inositol polyphosphate analog, INO-4949 (3). Although it is not known whether the 1-octyl group would be cleaved by plasmologenases, which would yield the endogenous inositol polyphosphate, Ins(3,4,5,6)P₄, both INO-4913 and INO-4949 are subject to dephosphorylation by way of phosphatases, yielding, successively, InsP₃, InsP₂, InsP₁ analogs, and 1-octyl-inositol. In the current study, only products labeled with the [³H]octyl group would be detectable by radio-HPLC.

Figure 2.

HPLC of de-esterified products of [³H]INO-4995 extracted at various time points from T₈₄ cells. (A) Effect of duration of exposure to [³H]INO-4995 on levels of InsP₄ analogs in T₈₄ cells. Squares, total intracellular counts; circles, InsP₄ analogs. (B) Comparison of levels of tritiated inositol polyphosphate derivatives during pulse-chase analysis of [³H]INO-4995 metabolism in T₈₄ cells. Squares, InsP₁ analogs; circles, InsP₂ analogs; triangles, InsP₃ analogs; inverted triangles, InsP₄ analogs.

Figure 3.

Repeat dosing effects on fluid absorption. The effect of repeated exposure to INO-4995 (2 h/day for 8 d) on fluid absorption was measured over 18 hours with blue dextran, as previously described (4). A portion of the control data has been described elsewhere (10 and 50 μM), and are reproduced here for comparison only (4). Comparison of effect of single (open bars) versus multiple doses (closed bars, 4 d) of INO-4995 on fluid absorption expressed as mean percent control ± SEM. *P = 0.0015, Student's unpaired t test comparing 1 μM single dose versus 1 μM multiple dose (n = 6).

Figure 4.

The effect of repeated exposure to INO-4995 on basal short-circuit current (I_sc) in cystic fibrosis (CF) human nasal epithelia (CFHNE). Comparison of amiloride-inhibitable I_sc in single versus multiple dose (4 d basolateral or 8 d repeated apical exposure, the latter denoted with ^) measured 20 minutes after mounting in Ussing chamber. Data (I_sc, % control) are expressed as means (±SEM). Control values were calculated from averaging basal I_sc from coincubated monolayers 20 minutes after mounting in Ussing chambers. *Multiple dose exposure to 1 μM was statistically different from a single dose exposure by Student's unpaired t test (1 μM, P = 0.0032; 1 μM^, P = 0.0006); **similarly, 2.5 μM multiple exposure was statistically different from single exposure to 2 μM (P = 0.0001).

Figure 5.

Response to ATP is enhanced after a 4-day exposure to 2.5 μM INO-4995. Comparison of the peak and duration of ATP-stimulated I_sc in the presence of amiloride after repeated 4-day exposure of CFHNE cells to 2.5 μM INO-4995 versus controls treated with vehicle. (A) Traces represent the average of I_sc over time from nine individual chambers, three separate experiments in triplicate (mean ± SEM; n = 9). The data were normalized to baseline just before the point of addition of 100 μM ATP. Closed circles, control; open circles, 2.5 μM INO-4995. (B) Peak ΔI_sc from the experiment depicted in A showing the difference between CFHNE subjected to repeated exposure to INO-4995 versus vehicle (P < 0.0002, Student's two-tailed, unpaired t test).

Figure 6.

Effect of a single dosing of INO-4995 on nasal potential difference (NPD) in gut-corrected fatty acid–binding protein/CF transmembrane regulator knockout mice measured 24 hours after exposure compared with values in the same mice 3 days before dosing. INO-4995 was dripped into mouse nasal passages. NPD measurements were taken 3 days before drug exposure (−3 d), and 24 hours after drug delivery (+1 d). Comparison of NPD 3–5 days before addition of INO-4995 or 24 hours after addition of 20 μl of INO-4995 at either 0.001 mg/ml (0.8 μg/kg; n = 4) or 0.01 mg/ml (8 μg/kg; n = 3). The effect at 24 hours after addition of 0.01 mg/ml was statistically significant compared with the PD in the same mice 3 days before dosing (Student's paired t test, P < 0.05).

Figure 7.

Effect of repeat dosing with INO-4995 (20 μl of 0.001 mg/ml or 0.8 μg/kg was delivered to mouse nasal passages once per day for 4 d). On the fifth day, NPD was measured. (A) Scatter graph depicting control (diamonds) and treated (squares) values in individual mice. The control represents NPD for the same mice 3–5 days before the administration of INO-4995. (B) Bar graph showing difference in NPD between mice before and after 4-day treatment with INO-4995. Dotted line shows average wild-type NPD. Data are means (±SEM) (n = 16). Significance was evaluated with a Student's paired t test (P < 0.005). The values for the group treated with 20 μl of 0.001 mg/ml INO-4995 for 4 days was also significantly different from the values for the group treated with the same dose once and displayed in Figure 5 (Student's unpaired t test, P = 0.013).