Intranasal administration of PACAP: uptake by brain and regional brain targeting with cyclodextrins

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent neurotrophic and neuroprotectant that is transported across the blood-brain barrier in amounts sufficient to affect brain function. However, its short half-life in blood makes it difficult to administer peripherally. Here, we determined whether the radioactively labeled 38 amino acid form of PACAP can enter the brain after intranasal (i.n.) administration. Occipital cortex and striatum were the regions with the highest uptake, peaking at levels of about 2-4% of the injected dose per gram of brain region. Inclusion of unlabeled PACAP greatly increased retention of I-PACAP by brain probably because of inhibition of the brain-to-blood efflux transporter for PACAP located at the blood-brain barrier. Sufficient amounts of PACAP could be delivered to the brain to affect function as shown by improvement of memory in aged SAMP8 mice, a model of Alzheimer's disease. We found that each of three cyclodextrins when included in the i.n. injection produced a unique distribution pattern of I-PACAP among brain regions. As examples, β-cyclodextrin greatly increased uptake by the occipital cortex and hypothalamus, α-cyclodextrin increased uptake by the olfactory bulb and decreased uptake by the occipital cortex and striatum, and (2-hydropropyl)-β-cyclodextrin increased uptake by the thalamus and decreased uptake by the striatum. These results show that therapeutic amounts of PACAP can be delivered to the brain by intranasal administration and that cyclodextrins may be useful in the therapeutic targeting of peptides to specific brain regions.

Fig. 1

A-L. Appearance of I-PACAP in olfactory bulb (A: Olf), occipital cortex (B: OC), striatum (C: St), frontal cortex (D: FC), hypothalamus (E: Hyth), thalamus (F: Th), hippocampus (G: Hip), parietal cortex (H: PC), cerebellum (I: Cb), midbrain (J: Mb), pons-medulla (K: PM) and whole brain (L: WBr) at 5, 15, 30, 60, and 120 min after i.n. administration of I-PACAP with or without CD. The peak value without CD was at 60 min and with CD at 30 min in Olf (A), OC (B), and St (C). The peak value without CD was at 60 min and without CD at 120 min in FC (D) and Th (F). The peak value without CD was at 15 min and with CD at 120 min in Hyth (E). The peak value without CD was at 60 min and with CD at 60 min in Hip (G), PC (H), Cb (I), Mb (J), PM (K), and WBr (L).

Fig. 1

A-L. Appearance of I-PACAP in olfactory bulb (A: Olf), occipital cortex (B: OC), striatum (C: St), frontal cortex (D: FC), hypothalamus (E: Hyth), thalamus (F: Th), hippocampus (G: Hip), parietal cortex (H: PC), cerebellum (I: Cb), midbrain (J: Mb), pons-medulla (K: PM) and whole brain (L: WBr) at 5, 15, 30, 60, and 120 min after i.n. administration of I-PACAP with or without CD. The peak value without CD was at 60 min and with CD at 30 min in Olf (A), OC (B), and St (C). The peak value without CD was at 60 min and without CD at 120 min in FC (D) and Th (F). The peak value without CD was at 15 min and with CD at 120 min in Hyth (E). The peak value without CD was at 60 min and with CD at 60 min in Hip (G), PC (H), Cb (I), Mb (J), PM (K), and WBr (L).

Fig. 2

Appearance of I-PACAP with or without CD in serum after i.n. administration. The peak value with or without CD was at 60 min. I-PACAP without CD produced much lower levels in serum as shown by w-way ANOVA.

Fig. 3

Data in Figure 1 combined over time period of study and presented as single value per animal. Effect of CD on I-PACAP uptake by individual regions was compared by t-tests. CD produced a statistically significant (p<0.05) increase in values for occipital cortex (OC), frontal cortex (FC), hypothalamus (Hyth), thalamus (Th), hippocampus (Hip), parietal cortex (PC), cerebellum (Cb), midbrain (Mb), pons-medulla (PM), and whole brain (WBr).

Fig. 4

Acid precipitation (%) of I-PACAP without CD at 5, 30, 60, and 120 min after i.n. administration. Panel A shows the occipital cortex (OC), olfactory bulb (Olf), and serum and panel B shows the hypothalamus (Hyth), hippocampus (Hip), and pons-medulla (PM).

Fig. 5

%Inj/g of I-PACAP ± unlabeled PACAP in olfactory bulb (Olf), occipital cortex (OC), hypothalamus (Hyth), hippocampus (Hip), cerebellum (Cb), and pons-medulla (PM) at 30 min after i.n. administration. Unlabeled PACAP produced significant increases (* p < 0.05) in OC and Hyth and arithmetic increases in other brain regions (Panel A) and a significant increase in %Inj/ml in serum (Panel B) as assessed by t-test.

Figure 6

Intranasal PACAP dose of 0.01 microg improves memory in the SAMP8 mouse model of Alzheimer’s disease. *p<0.05 compared to vehicle or 0.1 microg dose, n = 9/group.

Figure 6

Intranasal PACAP dose of 0.01 microg improves memory in the SAMP8 mouse model of Alzheimer’s disease. *p<0.05 compared to vehicle or 0.1 microg dose, n = 9/group.

Fig. 7

A-L. %Inj/g of I-PACAP without CD, I-PACAP with α-CD, and I-PACAP with Hydro-β-CD in olfactory bulb (A: Olf), occipital cortex (B: OC), striatum (C: St), frontal cortex (D: FC), hypothalamus (E: Hyth), thalamus (F: Th), hippocampus (G: Hip), parietal cortex (H: PC), cerebellum (I: Cb), midbrain (J: Mb), pons-medulla (K: PM) and whole brain (L: WBr) at 60 min after i.n. administration. ANOVAs showed difference for Olf, OC, St, FC, Hyth, Th, Hip, PC, Cb, Mb, PM and WBr. Newman-Keuls Multiple Comparison Test showed Olf, OC, St, FC, Hyth, Th, Hip, PC, Cb, Mb, PM, and WBr in I-PACAP without CD, I-PACAP with α-CD, and IPACAP with Hydro-β-CD (* p < 0.05, ** p < 0.01, *** p < 0.001). n = 6 - 11 mice/groups.

Fig. 8

%Inj/ml of I-PACAP without CD, I-PACAP with α-CD, and I-PACAP with Hydro-β-CD in serum at 60 min after i.n. administration. No change occurred in serum levels as assessed by ANOVA.