Long-term daily oral administration of intestinal permeation enhancers is safe and effective in mice

Abstract

Although protein drugs are powerful biologic therapeutics, they cannot be delivered orally because their large size and hydrophilicity limit their absorption across the intestinal epithelium. One potential solution is the incorporation of permeation enhancers into oral protein formulations; however, few have advanced clinically due to toxicity concerns surrounding chronic use. To better understand these concerns, we conducted a 30-day longitudinal study of daily oral permeation enhancer use in mice and resultant effects on intestinal health. Specifically, we investigated three permeation enhancers: sodium caprate (C₁₀), an industry standard, as well as 1-phenylpiperazine (PPZ) and sodium deoxycholate (SDC). Over 30 days of treatment, all mice gained weight, and none required removal from the study due to poor health. Furthermore, intestinal permeability did not increase following chronic use. We also quantified the gene expression of four tight junction proteins (claudin 2, claudin 3, ZO-1, and JAM-A). Significant differences in gene expression between untreated and permeation enhancer-treated mice were found, but these varied between treatment groups, with most differences resolving after a 1-week washout period. Immunofluorescence microscopy revealed no observable differences in protein localization or villus architecture between treated and untreated mice. Overall, PPZ and SDC performed comparably to C₁₀, one of the most clinically advanced enhancers, and results suggest that the chronic use of some permeation enhancers may be therapeutically viable from a safety standpoint.

Keywords: intestinal epithelium; oral drug delivery; permeation enhancer.

FIGURE 1

The permeation enhancers 1‐phenylpiperazine (PPZ), sodium deoxycholate (SDC), and sodium caprate (C₁₀) increased oral macromolecular absorption. FITC‐dextran 4 kDa (FD4) was co‐delivered to mice with either PBS (Control), PPZ, SDC, or C₁₀ by oral gavage. Chemical structures are shown in (a). (b) The concentration profiles varied between the enhancers, with C₁₀ and SDC producing rapid increases in blood concentration that dropped by hour 2 and PPZ producing a steady increase in blood concentration over 3 h. (c) All enhancers increased the area under the curve (AUC) of FD4 compared to control over 3 h. n = 6, error bars represent SEM, **p < 0.01, ****p < 0.0001 compared to untreated control by unpaired, two‐tailed Student's t‐test.

FIGURE 2

The permeation enhancers PPZ, SDC, and C₁₀ affected gene expression of tight junction proteins. Mice received a single dose of PBS (Control) or one of the three enhancers examined in this study. After 3 h, small intestine and colon tissue samples were collected, and mRNA expression was determined by qRT‐PCR for four tight junction proteins: pore‐forming claudin 2, barrier‐forming claudin 3, ZO‐1, and JAM‐A. (a) In the small intestine, PPZ and C₁₀ induced significant gene expression changes compared to the control. (b) In the colon, PPZ and SDC altered gene expression compared to the control. n = 6, error bars represent SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired, two‐tailed Student's t‐test.

FIGURE 3

Enhancers did not permanently increase intestinal permeability after 4 weeks of daily oral administration. Baseline untreated intestinal permeability was measured 1 week before treatment began (gray squares‐Pre). Mice were dosed with (a) PBS, (b) PPZ, (c) SDC, or (d) C₁₀ every day for 30 days, and the concentration of FD4 in the blood was measured five times throughout the 30‐day period and again after a 1‐week washout period (gray squares‐Post). Over the course of treatment, SDC and C₁₀ caused slight increases in permeability that were not significant, while the Control and PPZ groups had no difference between the permeability on Days 1 and 30. The observed increases in FD4 permeability for the SDC and C₁₀ groups were no longer present after the washout period. n = 6–12, error bars represent SEM. No statistical differences were found between any groups, with significance defined as p < 0.05 by unpaired, two‐tailed Student's t‐tests.

FIGURE 4

Chronic permeation enhancer exposure does not negatively affect health indicators. (a) Daily permeation enhancer treatment did not cause weight loss. Nonfasted mice were weighed daily during the study. Shown here is the weight gain between 1 week before the study began and treatment day 29. While all groups showed weight gain, weight gains were smaller in the PPZ group compared to the control group. n = 9–12, error bars represent SEM, *p < 0.05 by a two‐tailed, unpaired Student's t‐test. (b) Serum zonulin concentrations were not elevated by enhancer treatment. Serum was collected from mice on treatment day 30 (solid symbols) and after a 1‐week washout period (open symbols), and zonulin concentration was measured by ELISA. n = 4–6, error bars represent SEM. (c) Only SDC affected the quality of mouse stool. Once per week, mouse stool was collected and assessed for stool solidity, mucus in the stool, and blood in the stool (pos. hemoccult). Only SDC administration caused an increase in fecal score over time with the effect decreasing after a 1‐week washout period.

FIGURE 5

Chronic exposure to permeation enhancers affects gene expression of tight junction proteins. Small intestine and colon tissue samples were collected on treatment day 30 and after a 1‐week washout period, and mRNA expression was determined by qRT‐PCR for four tight junction proteins: pore‐forming claudin 2, barrier‐forming claudin 3, ZO‐1, and JAM‐A. (a) On treatment day 30, JAM‐A expression in the small intestine decreased for the SDC and C₁₀ groups. (b) None of the expression differences persisted after the washout period, but claudin 2 expression was lower for the SDC group compared to control. (c) In the colon, only PPZ treatment increased expression compared to control. (d) In the colon, after washout, PPZ treatment altered the expression of claudins 2 and 3, and C₁₀ elevated ZO‐1 expression. n = 6, error bars represent SEM, *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired, two‐tailed Student's t‐test.

FIGURE 6

Intestinal architecture and tight junction protein localization were not affected by 4 weeks of treatment with permeation enhancers. (a) Sections of small intestine from mice receiving PBS, PPZ, SDC, or C₁₀ were stained for nuclei (blue, Hoechst), the barrier‐forming claudin 3 (green, AF488), and the tight junction protein ZO‐1 (red, AF594). (b) Separate sections were stained for nuclei (blue, Hoechst) and F‐actin (red, AF594).