Methodology and effects of repeated intranasal delivery of DNSP-11 in a rat model of Parkinson's disease

Abstract

Background: To circumvent the challenges associated with delivering large compounds directly to the brain for the treatment of Parkinson's disease (PD), non-invasive procedures utilizing smaller molecules with protective and/or restorative actions on dopaminergic neurons are needed.

New method: We developed a methodology for evaluating the effects of a synthetic neuroactive peptide, DNSP-11, on the nigrostriatal system using repeated intranasal delivery in both normal and a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of PD.

Results: Normal rats repeatedly administered varying doses of DNSP-11 intranasally for 3 weeks exhibited a significant increase in dopamine (DA) turnover in both the striatum and substantia nigra (SN) at 300μg, suggestive of a stimulative effect of the dopaminergic system. Additionally, a protective effect was observed following repeated intranasal administration in 6-OHDA lesioned rats, as suggested by: a significant decrease in d-amphetamine-induced rotation at 2 weeks; a decrease in DA turnover in the lesioned striatum; and an increased sparing of tyrosine hydroxylase (TH) positive (+) neurons in a specific sub-region of the lesioned substantia nigra pars compacta (SNpc). Finally, tracer studies showed (125)I-DNSP-11 distributed diffusely throughout the brain, including the striatum and SN, as quickly as 30min after a single intranasal dose.

Comparison with existing methods: The results of bilateral intranasal administration of DNSP-11 are compared to our unilateral single infusion studies to the brain in rats.

Conclusions: These studies support that DNSP-11 can be delivered intranasally and maintain its neuroactive properties in both normal rats and in a unilateral 6-OHDA rat model of PD.

Keywords: 6-Hydroxydopamine; Intranasal administration; Neurochemistry; Neuroprotection; Nigrostriatal pathway; Peptide.

Figure 1. Illustration of intranasal administration of DNSP-11 in F344 rats

For all in vivo intranasal studies rats were anesthetized with isoflurane (A) and administered either vehicle or DNSP-11 while in a supine position. (B) For intranasal dosing a micropipetter containing 12.5 μl of either vehicle or DNSP-11 in an equivalent volume of vehicle was administered.

Figure 2. Experimental timeline for all intranasal studies

Three major studies designs were used to determine the efficacy of repeated intranasally administered DNSP-11’s effects on the nigrostriatal system in both normal and unilateral 6-OHDA- lesioned rats using isoflurane anesthesia. (A) To determine the efficacy of our intranasal delivery method and adequate dosing, normal rats were subjected to repeated intranasal administration of various doses of DNSP-11, 5 days a week for 3 weeks (21 days). (B) Utilizing the determined dosage from experiment A, the efficacy of intranasally administered DNSP-11’s effects on the nigrostriatal system were examined in a unilateral 6-OHDA (right hemisphere) lesion model of PD where rats were treated with either vehicle or DNSP-11 daily, 1 week (7 days) prior to unilateral 6-OHDA infusion and 5 weeks (35 ± 3 days) post-surgery. Prior to the start of the study baseline d-amphetamine rotation data were determined and at both 2 and 4 weeks post unilateral 6-OHDA infusion to assess lesion severity. (A–B) For experiments A and B, researchers were blinded to the treatments throughout the entirety of the studies. (C) Tracer studies examined ¹²⁵I-labeled DNSP-11’s distribution at 30, 60, and 360 minutes after the start of the first intranasal dose in blood, CSF and the CNS.

Figure 3. HPLC-EC studies of DA turnover [(DOPAC+HVA)/DA] following intranasal administration of DNSP-11 or vehicle. of the nigrostriatal system in normal rats

Normal rats were repeatedly administered either vehicle (open circles), 100 (red squares), 300 (green diamonds) or 1000 (blue triangles) μg of DNSP-11 intranasally 5 days a week for 3 weeks. (A) At 300 μg a significant increase (p = 0.0230) in basal DA turnover was observed compared to vehicle in the striatum (A). (F_(3,23)=4.520, p=0.0124) (B) In addition, a significant increase in DA turnover was observed in the SN at both 300 (p = 0.0129) and 1000 μg (p = 0.0217) compared to vehicle (F_(3,23)=9.818, p=0.0002). All data were analyzed using a one-way ANOVA with Bonferroni’s post-hoc test *p<0.05. All data are shown as mean ± SEM, n=9 vehicle and n=6 DNSP-11 treated rats.

Figure 4. d-amphetamine-induced rotation at 2 & 4 weeks post unilateral 6-OHDA lesion in rats after repeated intranasal administration of DNSP-11 or vehicle

d-amphetamine (2.5 mg/kg i.p.) rotation was assessed prior to 6-OHDA lesion and again at 2 and 4 weeks post-surgery and data are presented as the number of peek clockwise turns per hour. A significant decrease in rotation was observed at 2 weeks (*p = 0.0210) but not at 4 weeks (p = 0.1176) compared to vehicle treated rats. Data were analyzed using a two-way ANOVA with repeated measures (F_(1,18)=0.0210, p=0.0283) with Bonferroni’s post hoc test. Animals taken out to 6 weeks (data not shown) observed a 21% reduction in d-amphetamine-induced rotations in DNSP-11 (1069 ± 47; n = 3) compared to vehicle (840 ± 127; n = 2) treated animals. When animals taken out to 6 weeks were added back into the analysis, d-amphetamine-induced rotation became significant at both 2 (p = 0.0180) and 4 (p = 0.0315) weeks post lesion (data not shown) in DNSP-11 (n = 13) compared to vehicle (n = 12) treated rats. Data presented as mean ± SEM, n = 10 vehicle, n = 10 DNSP-11.

Figure 5. HPLC-EC studies of DA turnover (DOPAC + HVA)/DA in unilateral (right) 6-OHDA lesioned rats after repeated intranasal administration of DNSP-11 or vehicle

DNSP-11 treatment significantly decreased DA turnover in the ipsilateral (right/lesioned) striatum from 0.884 ± 0.074 to 0.538 ± 0.062 ng/g at 5 weeks in 6-OHDA lesioned rats (green diamonds) compared to vehicle (open circles). Data were analyzed using a one-way ANOVA (F_(3,24) = 24.62, p < 0.0001) with Bonferroni’s post hoc test **p < 0.01. Data presented as mean ± SEM, n = 8 vehicle, n = 6 DNSP-11.

Figure 6. Representative sections of the SNpc used for TH+ neurons and fiber area of the SNpr at three levels

rostral: − 5.24 mm; (A – D) middle: − 5.60 mm; caudal: − 5.96 mm of the SNpc and SNpr used for cell counting and fiber area. Scale bars = 200 μm in A – B and 100 μm in C–D.

Figure 7. The effects of repeated intranasal administration of DNSP-11 on TH+ dopaminergic neurons in the SNpc and fiber area of the SNpr in 6-OHDA unilaterally lesioned rats

(A–C) The total number of TH+ neurons were counted from three sections of the lesioned SNpc after repeated intranasal administration of either vehicle or DNSP-11: Rostral (t₍₁₁₎ = 0.2714, p = 0.7911), (A–D) Middle (t₍₁₁₎ = 2.703, p = 0.0205), and Caudal (t₍₁₁₎ = 1.223, p = 0.2468). There was no significant difference found between DNSP-11 treated rats or vehicle in the rostral or caudal regions of the SNpc (mean ± SEM, vehicle: 23 ± 6; DNSP-11: 25 ± 5) and (mean ± SEM, vehicle: 18 ± 1; DNSP-11: 26 ± 7). (B) There was a significant increase observed in the average number of TH+ positive neurons in DNSP-11 treated rats compared to vehicle (*p < 0.05) in the middle region of the lesioned SNpc (mean ± SEM, vehicle: 13 ± 2; DNSP-11: 29 ± 5). The data were analyzed by a two-tailed unpaired t test. Data are shown as mean ± SEM (n = 7 vehicle, n = 6 DNSP-11).

Figure 8. Tracking of a one-time ¹²⁵I-labeled DNSP-11 dose 60 minutes after intranasal administration

Normal F344 rats were given a one-time intranasal dose of ¹²⁵I-labeled DNSP-11, at 60 minutes blood (500 μl) and cerebrospinal fluid (100–120 μl) were collected from each rat and processed by gamma counting (n=3) and autoradiography (n=1). (A) Representative sagittal brain section (0.5 mm thick), exposed for 21 days on a GE phosphor screen. A qualitative increase in radioactive signal was found in the olfactory bulbs 60 minutes post intranasal administration and diffusely throughout the brain. (B) Data are shown as the normalized DNSP-11 concentrations (ng or μl/mg of wet sample weight) as analyzed by gamma counting at 60 minutes. (C) Blood and CSF; the visible increase in radioactive signal found in the olfactory bulbs at 60 minutes is consistent with autoradiography. Data are presented as the normalized DNSP-11 concentrations (ng or μl/mg). Rats treated with vehicle only were found to have CPM lower then background levels < 50 (data not pictured). * Denotes the olfactory bulb of a representative sagittal section of the midbrain after autoradiography analysis.