Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally

Abstract

To determine if the dose of peptide administered or the plasma level was more important, doses of 0.15, 0.45, 4.5, or 45 mg/kg/day of the peptide D-4F were administered orally or subcutaneously (SQ) to apoliptotein (apo)E null mice. Plasma levels of peptide were ∼1,000-fold higher when administered SQ compared with orally. Regardless of the route of administration, doses of 4.5 and 45 mg/kg significantly reduced plasma serum amyloid A (SAA) levels and the HDL inflammatory index (P < 0.0001); doses of 0.15 or 0.45 mg/kg did not. A dose of 45 mg/kg/day administered to apoE null mice on a Western diet reduced aortic atherosclerosis by ∼50% (P < 0.0009) whether administered orally or SQ and also significantly reduced plasma levels of SAA (P < 0.002) and lysophosphatidic acid (P < 0.0009). Remarkably, for each dose administered, the concentration and amount of peptide in the feces was similar regardless of whether the peptide was administered orally or SQ. We conclude: i) the dose of 4F administered and not the plasma level achieved determines efficacy; ii) the intestine may be a major site of action for the peptide regardless of the route of administration.

Fig. 1.

Plasma levels of D-4F after subcutaneous (SQ) injection. Female apoE null mice 6–7 months of age (n = 48) were placed on a Western diet for 2 weeks and then fasted overnight in individual cages. In the morning, water was removed for 3 h and each mouse was administered 900 μg of D-4F (45 mg/kg) by SQ injection on the back. The mice were returned to their cages after injection and allowed to drink up to 3 ml of water. The mice were bled at the time points shown on the X-axis and the plasma concentration of intact D-4F peptide was determined by LC-ESI-MS/MS as described in Materials and Methods. The data shown are mean ± SD for three mice that were used for each time point.

Fig. 2.

Plasma levels of D-4F after administration of the peptide in the drinking water. Female apoE null mice 6–7 months of age (n = 24) were placed on a Western diet for 2 weeks and then fasted overnight in individual cages. In the morning, water was removed for 3 h after which drinking water containing 300 μg/ml of D-4F was provided and the mice were allowed to drink 3 ml of water over a period of 3 h and the mice were not injected with D-4F. The mice were bled at the time points shown on the X-axis. The data shown are mean ± SD for three mice that were used for each time point.

Fig. 3.

Administering D-4F by SQ injection in apoE null mice produced plasma levels 100- to 1,000-fold higher compared with the same dose administered orally in the drinking water, but the reduction in SAA levels was similar. Female apoE null mice 6–7 months of age (n = 12 per group) were fed a Western diet and were administered D-4F in the drinking water (B) to provide each mouse a daily dose of peptide of 3, 9, 90, or 900 μg per day of D-4F (Oral) or the mice received drinking water without peptide but received the same dose of D-4F by daily SQ injection on the back (A). In the morning of the 8th day of treatment after an overnight fast, water was removed for 3 h and then replaced with water that did or did not contain D-4F at the concentration each mouse had been receiving. The mice that received water without D-4F were injected SQ on the back with the dose of D-4F that they had been receiving and 2 h later were bled. The mice that received drinking water with D-4F were allowed to drink for 3 h and were then bled. The plasma from four mice in each group was pooled and plasma levels of D-4F were determined as described in Materials and Methods. The data shown are mean ± SD and are representative of two of two separate experiments. C: The plasma of the mice described in panels A and B was analyzed for SAA levels as described in Materials and Methods. The data shown are mean ± SD and are representative of two of two separate experiments.

Fig. 4.

The route of administration does not determine SAA levels but the dose administered does. Female apoE null mice (n = 16 per group) 6–7 months of age were fed a Western diet and were administered drinking water with or without D-4F at the following doses: 3, 9, 90, or 900 μg per day per mouse. Some of the mice received scrambled D-4F (Sc-D-4F) in the drinking water instead of D-4F. The doses of Sc-D-4F provided to these mice were 90 or 900 μg per mouse per day of Sc-D-4F. Mice that did not receive D-4F in their drinking water received D-4F SQ at doses of 3, 9, 90, or 900 μg per mouse per day. Some of the mice received Sc-D-4F SQ instead of D-4F at doses of 90 or 900 μg per mouse per day of Sc-D-4F. After 6 weeks, in the morning after an overnight fast, water was removed for 3 h and then replaced with water that did or did not contain D-4F at the concentration each mouse had been receiving. The mice that received water without D-4F were injected SQ on the back with the dose of D-4F that they had been receiving and 2 h later were bled. The mice that received drinking water with D-4F were allowed to drink for 3 h and were then bled. Plasma SAA levels were determined as described in Materials and Methods. The symbols represent individual values for each mouse that received D-4F; the longer horizontal line represents the mean and the shorter horizontal lines define 1 SD above and below the mean.

Fig. 5.

Plasma levels of peptide did not predict improvement in the HDL-inflammatory index (HII). Eight groups of female apo E null mice 7–8 months of age (n = 23 per group) were fed a Western diet and treated daily with 3, 9, 90, or 900 μg of D-4F administered in the drinking water daily (Oral) or SQ daily for 2 weeks at which time they were bled. The plasma levels of D-4F after SQ administration (A) and after oral administration (B) was determined as described in Materials and Methods. The data shown are the mean ± SD. For determination of HII, plasma from three to four mice was pooled to yield six pools of plasma for each treatment group and HDL from each pool was isolated by FPLC and the HII was determined as described in Materials and Methods (C). In C, the symbols represent the values for each of the six pools for each treatment group; the longer horizontal line represents the mean and the shorter horizontal lines define 1 SD above and below the mean.

Fig. 6.

Administering D-4F SQ daily or administering the same dose incorporated into the diet reduced aortic atherosclerosis to the same degree. A: Female apoE null mice 8–9 months of age were fed a Western diet. The mice received 900 μg per mouse per day of D-4F or Sc-D-4F SQ daily or received the same daily dose orally by incorporating the peptide in the diet. After 8 weeks on the Western diet, mice from each group were euthanized daily; by the 9th week, all mice had been euthanized. The percent of aorta containing lesions was determined by en face analysis as described in Materials and Methods. The symbols represent individual values for each mouse; the longer horizontal line represents the mean and the shorter horizontal lines define 1 SD above and below the mean. In the evening 2 days before euthanasia, food was removed from the mice described in A and the mice were fasted overnight. B: The next morning (1 day prior to euthanasia), the mice receiving peptide SQ were injected with D-4F or Sc-D-4F at a dose of 900 μg per mouse. Two hours later, these mice were bled and D-4F levels were determined by LC-ESI-MS/MS in 10 randomly chosen plasma samples. C: For the mice receiving peptides in the diet, on the morning prior to euthanasia, after the overnight fast, the mice were given frozen blocks of diet prepared as described in Materials and Methods, each containing 900 μg of D-4F or Sc-D-4F. The mice were allowed to eat all of the diet presented to them over a period of 3–4 h at which time they were bled and D-4F levels were determined by LC-ESI-MS/MS in 10 randomly chosen plasma samples. The symbols represent individual values for each mouse; the longer horizontal line represents the mean and the shorter horizontal lines define 1 SD above and below the mean. D: On the day of euthanasia, after an overnight fast, a terminal bleed was performed in the mice described in A and SAA levels were determined as described in Materials and Methods. The data shown are mean ± SD. E: Lysophosphatidic acid (18:1) (LPA) levels were determined as described in Materials and Methods on the plasma taken from the mice described in D. The symbols represent individual values for each mouse; the longer horizontal line represents the mean and the shorter horizontal lines define 1 SD above and below the mean.