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One-month subchronic toxicity study of cell-penetrating peptides for insulin nasal delivery in rats
European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 736–743
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Research paper
One-month subchronic toxicity study of cell-penetrating peptides for insulin nasal delivery in rats El-Sayed Khafagy a,b, Noriyasu Kamei a, Ebbe Juel Bech Nielsen a,c, Reiji Nishio d, Mariko Takeda-Morishita a,⇑ a
Laboratory of Drug Delivery Systems, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt Novo Nordisk A/S, Måløv, Denmark d New Frontiers Research Laboratories, Toray Industries, Inc., Kamakura, Japan b c
a r t i c l e
i n f o
Article history: Received 20 June 2013 Accepted in revised form 13 September 2013 Available online 21 September 2013 Keywords: Cell-penetrating peptide Penetratin Toxicity Insulin Nasal delivery Long-term administration
a b s t r a c t Recently, cell-penetrating peptides (CPPs) based vehicles have been developed for the delivery of different payloads in animals. Our studies have shown that nasal absorption of insulin and other therapeutic peptides and proteins can be improved significantly by co-administration of the CPP penetratin. Successful development of suitable CPP-based delivery systems, however, will depend not only on the efficiency of CPPs to transport therapeutic agents across the biological barriers of the nasal cavity, but also on the risk of adverse effects such as toxicity and undesired immunogenicity, especially in chronic therapy. In this study, we investigated the bioavailability (BA) of insulin and the adverse effects on the nasal mucosa in rats following a long-term dosing regimen of L-penetratin and the novel penetratin analogue ‘‘PenetraMax.’’ Following nasal delivery, a significantly higher BA for insulin (almost 100% relative to subcutaneous (s.c.) injections) was observed for PenetraMax in comparison with the parent penetratin peptide after chronic administrations in rats. Importantly, there was negligible biomarker leakage in nasal lavage fluid and the integrity of the nasal epithelium remained unaffected when PenetraMax was used in long-term multiple administrations. In addition, no significant difference in the release of inflammatory and immunogenicity mediators in plasma was observed after nasal administration of PenetraMax with or without insulin solution. In conclusion, PenetraMax, a novel CPP candidate, can open a new avenue in clinical trials for noninvasive nasal insulin delivery. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Cell-penetrating peptides (CPPs) have proven their ability for intracellular delivery of various macromolecular therapies such as nucleic acids, proteins and peptides, as well as drug delivery carriers [1–5]. CPPs comprise a growing family of peptides that have opened a new avenue for noninvasive systemic delivery of a vast collection of biomolecules otherwise restricted in crossing the plasma membrane [6,7]. CPPs are short polybasic and/or amphipathic peptides (<40 amino acids) with a net positive charge derived from signal peptides, viral peptides or other sources and they have been used successfully to translocate covalently or non-covalently bound cargo across cell membranes [8–10]. Previously, we have shown that CPPs are capable of transcellular delivery of peptides and proteins across the intestinal and nasal ⇑ Corresponding author. Laboratory of Drug Delivery Systems, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe, Hyogo 650-8586, Japan. Tel.: +81 78 974 4816; fax: +81 78 974 4820. E-mail address: [email protected] (M. Takeda-Morishita). 0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.09.014
epithelial membranes [11–16]. Recently, systemic delivery of proteins and peptides via the nasal route has received increased attention because of the great potential for noninvasive biodrug delivery. Sufficient bioavailability, however, has to date been a challenge to achieve [17–19]. We have previously demonstrated that penetratin, a peptide derived from the Drosophila Antennapedia Homeoprotein, to be amongst the most successful CPPs to overcome the permeability problems of peptides and proteins in the nasal cavity. Although the relative bioavailability of insulin can reach up to 50% after co-administration with penetratin, a relatively high dose of penetratin is required for this enhancement of nasal insulin absorption [13]. To address the efficiency of penetration, a screening study of various penetratin analogues suggested that the chain length, hydrophobicity, amphipathicity and basicity of the CPP contribute to their absorption-enhancing efficiency [20]. Penetratin structure modifications introduced in the ‘‘shuffle (R, K fix) 2’’ sequence were successful in overcoming the permeability barrier for peptides and proteins in the nasal cavity. These findings were further supported in an in silico study based on molecular orbital analysis with Self-Organizing Maps
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(SOM) classification in which the structural and physiochemical factors mentioned above were associated with the intestinal insulin absorption-enhancing effect of CPPs. As predicted by SOM clustering, a novel penetratin analogue (Sample 6) had significantly greater capacity to interact with insulin and to enhance intestinal insulin absorption than that of the original penetratin sequence [21] (Now this optimised peptide Sample 6 was named as ‘‘PenetraMax.’’) Insufficient information, however, regarding long-term CPP toxicity restricts their application in vivo and preclinical development in general as a successful delivery system will depend equally on the efficiency of CPPs to transport therapeutic agents through the biological barriers as well as the risk of associated side effects such as toxicity and undesired immunogenicity [22,23]. Hence, it is of great interest to develop a novel highly effective nasal absorption promoter with negligible toxic effect on the nasal mucosa and low systemic toxicity, especially for chronic therapy. In this study, we explored the potential of a novel CPP, PenetraMax, for nasal delivery of insulin in comparison with the parent penetratin peptide at varying concentrations in a one-month twice daily administration study in rats. In a clinical setting, absorption enhancers like CPPs would be used chronically and as such, their safety must be guaranteed for chronic administration. Thus, following the same dosing scheme as mentioned above, we also examined systemic toxicity by quantitatively assessing nasal biomarker release (lactate dehydrogenase (LDH), interleukin-1a (IL-1a), and tumour necrosis factor-a (TNF-a)) and systemic release of IL-1a and TNF-a. In addition, local effects on the nasal mucosa after 7 and 30 days repeated daily administrations were investigated by histopathological examination of the nasal epithelium.
2. Materials and methods 2.1. Materials Recombinant human insulin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). L-penetratin and its analogue PenetraMax listed in Table 1 were synthesised by Sigma–Genosys, Life Science Division of Sigma–Aldrich Japan Co. (Hokkaido, Japan). All other chemicals were of analytical grade and are commercially available.
2.2. Preparation of the mixed insulin/CPP solutions To prepare the insulin solution, specific amounts of recombinant human insulin were dissolved in 50 lL of 0.1 M HCl. The insulin-HCl solution was diluted to 1.4 mL with phosphate-buffered saline (PBS, pH 6.0) containing 0.001% methylcellulose, which prevents the adsorption of the insulin to the tube surface, and normalised with 50 lL of 0.1 M NaOH. Specific amounts of L-penetratin or PenetraMax were dissolved in PBS (pH 6.0) containing 0.001% methylcellulose. Aliquot of insulin solution was added to L-penetratin or PenetraMax solution, mixed gently, and adjusted to a specific concentration. Each insulin/CPP solution was clear after mixing.
Table 1 Molecular weight and amino acid sequences of L-penetratin and PenetraMax. Sequence
Mw
L-Penetratin
RQIKIWFQNRRMKWKK
2246.7
PenetraMax
KWFKIQMQIRRWKNKR
2246.7
F, phenylalanine; I, isoleucine; K, lysine; M, methionine; N, asparagine; Q, glutamine; R, arginine; W, tryptophan.
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2.3. Nasal absorption study This research was performed at Kobe Gakuin University and complied with the regulations of the Committee on Ethics in the Care and Use of Laboratory Animals. Male Sprague Dawley rats weighing 180–220 g were purchased from Japan SLC, Inc. (Shizouka, Japan). The animals were housed in temperature (23 ± 1 °C) and relative humidity (55 ± 5%) controlled rooms with free access to water and food during acclimatisation. Animals were fasted for 24 h before the experiments; however, they were allowed to drink water ad libitum. Under an intraperitoneal (i.p.) injection of sodium pentobarbital (12.5 mg/kg; Somnopentyl; Kyoritsu Seiyaku Corp., Tokyo, Japan), insulin or insulin/CPP mixture was applied intranasally twice a day for 1, 7 or 30 days. Rats were restrained in a supine position during administration at an angle of 15° and total 10 lL of drug (5 lL/each nostril) was instilled at 1 cm depth in each nostril by micropipette with a narrow tip fitted. The procedure was performed gently and slowly and lasted 1–2 min allowing the rats to receive all of the prepared sample. Insulin doses were 1 IU/kg body weight and CPP concentrations were 0.5 and 2 mM. Blood sampling to determine plasma insulin concentrations was performed on all groups after the first dose on days 1, 7 and 30 depending on the group. Following their anesthetization with an i.p. injection of sodium pentobarbital (50 mg/kg), the rats were restrained in a supine position on a thermostatically controlled board at 37 °C. Additional i.p. injections of sodium pentobarbital (12.5 mg/kg) were given every 1 h to maintain anaesthesia during the experiment for collection of blood samples. A 0.25 mL blood aliquot was taken from the jugular vein using 1 mL tuberculin heparinized syringes at 5, 10, 15, 30, 60, 120 and 180 min after dosing. Plasma was separated by centrifugation at 12,000 rpm (13,400g) for 1 min and stored at 80 °C until analysis. The plasma insulin concentration was determined using a human insulin ELISA kit (Mercodia AB, Uppsala, Sweden) and the absorbance at kmax 450 nm was detected by using a microplate reader (POWERSCAN HT, DS Pharma Biomedical Co. Ltd., Osaka, Japan). 2.4. Pharmacokinetic analysis The bioavailability of nasally administered insulin was calculated relative to a s.c. injection (1 IU/kg). Briefly, an insulin solution was prepared by dissolving an appropriate amount of insulin in PBS. The peak plasma concentration (Cmax) and time to reach Cmax (Tmax) were directly determined from plasma insulin concentration–time curves. The total area under the insulin concentration curve (AUC) for 0–3 h was estimated from the sum of successive trapezoids between each data point. The relative bioavailability (BA) of insulin was calculated relative to the s.c. injection as follows:
BA ð%Þ ¼ ð½AUC=doseÞ 100=ð½AUCs:c:=doses:c: Þ 2.5. Biomarker assay Inflammatory blood biomarkers (IL-1a and TNF-a) for insulin solution (control), L-penetratin with and without insulin and PenetraMax with and without insulin were analysed on the first day of nasal administration for each group and at the end of each respective dosing regimen (after 1, 7 and 30 days of nasal administrations). Nasal lavage fluid was collected for analysis of LDH, IL-1a and TNF-a release by the nasal mucosal cells at the end of days 1, 7 and 30 respectively. Briefly, after blood sampling rats were killed with an i.p. overdose of pentobarbital. The nasal cavity was perfused with 10 mL PBS warmed to 37 °C at a flow rate 2 mL/
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min using an infusion pump (KD Scientific Inc., Holliston, MA, USA. LDH activity in the nasal lavage fluid was determined using the CytoTox-96 assay kit (Promega Corp., Madison, WI, USA) as per manufacturer’s instructions. The positive control group received 5% (w/v) sodium taurodeoxycholate in same manner as other treatment groups. Results were determined at kmax 490 nm. Rat IL-1a was measured using a Quantikine IL-1a/IL-1F1 immunoassay kit (R&D Systems, Inc., Minneapolis, MN, USA) as per manufacturer’s instructions. Results were determined against a manufacturer supplied rat IL-1a standard at kmax 450 nm. Rat TNF-a was measured using a rat TNF-a immunoassay kit (R&D Systems, Inc.) as per manufacturer’s instructions. Results were determined against a manufacturer supplied rat TNF-a standard at kmax 450 nm. All measurements of the absorbance were conducted by using a POWERSCAN HT (DS Pharma Biomedical Co. Ltd.). 2.6. Histopathological examination After 1, 7 and 30 days of nasal administrations of the test solutions and after collection of biomarkers in the nasal lavage fluid as described above, 10 mL of 20% formalin neutral buffer solution (Mildform 20 N; Wako Pure Chemical Industries Ltd., Osaka, Japan) was perfused through the nasal cavity at a flow rate of 2 mL/min. The nasal cavity samples of all rats were preserved and fixed in Mildform 20 N. After fixation, samples were decalcified by an 8% Formic acid + 10% ion-exchange resin solution. For histopathological evaluation, samples are taken from the upper incisor teeth to the incisive papilla, according to the specific palatal landmarks (Level II), and then embedded in paraffin wax. Three lm-thick sections of Level II were cut and stained with haematoxylin and eosin (Kanto Chemical Co., Inc., Tokyo, Japan), and were examined by a light microscope (NIKON ECLIPSE E600; Nikon Corp., Tokyo, Japan). 2.7. Statistical analysis Data are presented as mean ± SEM. The significance of differences in mean values was evaluated using Student’s unpaired t test. For multiple comparisons, a one-way layout analysis of variance (ANOVA) with Dunnett’s test was applied. Differences were considered significant when p was less than 0.05. 3. Results and discussion
insulin absorption [21]. Thus building on our previous data for PenetraMax, we explored this novel CPP as an absorption enhancer for nasally delivered insulin in this one-month twice daily administration study. Time profiles for plasma insulin concentration after co-administration of insulin and L-penetratin or PenetraMax in different concentrations (0.5 and 2 mM) are shown in Fig. 1. Increasing concentrations of PenetraMax (0.5 or 2 mM) enhanced the permeability of the nasal mucosa to insulin more markedly than L-penetratin and insulin alone. The pharmacokinetic parameters derived from the insulin concentration–time profiles following nasal administration of insulin with L-penetratin and PenetraMax are shown in Table 2. Treatment with 0.5 mM PenetraMax resulted in a relative increase in insulin BA by 1.9-times compared to L-penetratin and treatment with 2 mM PenetraMax resulted in a 2.3-times increase in the nasal insulin BA compared with L-penetratin. Thus, PenetraMax greatly increased the Cmax, AUC, and BA of insulin compared to L-penetratin which can be attributed to a dramatically increased absorption. The superior effect of PenetraMax on nasal insulin absorption may be attributed to the change in the positions of the Trp molecules (Table 1) [20,27]. This allows a high degree of conformational flexibility of the interacting moieties, thus stabilizing the conformation of the peptide on the water–lipid interface and facilitating the insertion of PenetraMax into the lipid bilayer [28]. Our recent study on the enhancement effect of by penetratin analogues by SOM clustering analysis showed the relationship between the essential physical and electrical parameters, as casual factors, and the increase in the insulin AUC, as response variable. The higher capacity of PenetraMax to interact with insulin than the original penetratin might be involved in its enhanced stimulatory effect on insulin absorption, especially in our approach using physical mixtures of the drug and CPPs rather than their conjugates [15,21,29]. To verify L-penetratin and PenetraMax for nasal insulin absorption for long-term administration, we examined their effects on nasal insulin absorption for 7 days and compared the PenetraMax enhancing effect with that of the original L-penetratin using insulin solution as a control. Insulin concentration–time profiles following intranasal administration for 7 consecutive days show that PenetraMax was the most effective promoter of insulin absorption compared with L-penetratin as shown in Fig. 2 and Table 3. On the first day of administration, as shown in Table 3, co-administration of 0.5 or 2 mM L-penetratin increased nasal
3.1. Effect of L-penetratin and PenetraMax on nasal insulin absorption Our recent work has shown that absorption of insulin and other medicinal peptides and proteins from the nasal cavity can be significantly improved by the co-administration of functional CPPs [13,14]. Amongst the CPPs examined in our previous studies, penetratin derived from the Drosophila Antennapedia Homeoprotein [24–26] enhanced the nasal absorption of insulin most effectively [13]. The dose dependent relationship of L-penetratin and insulin bioavailability has been investigated and we found a BA of insulin reached up to 50% when insulin was co-administered with 2 mM L-penetratin [13]. However, the penetratin concentration was relatively high considering cost and possible adverse effects after multiple administrations. Therefore, to identify CPPs that were more effective, we compared the enhancing effect of L-penetratin and 20 of its analogues on nasal insulin absorption. Surprisingly, the ‘‘shuffle (R, K fix) 2’’ analogue greatly increased insulin absorption reaching a relative bioavailability value 1.85-times that of original penetratin [20]. Moreover, an in silico analysis of penetratin analogues based on molecular orbital analysis with SOM classification, predicted a novel CPP, PenetraMax, as the sequence with the strongest enhancing effect on intestinal
Fig. 1. Plasma insulin concentrations vs. time profiles following single nasal administration of insulin (1 IU/kg) with L-penetratin or PenetraMax (0.5 and 2 mM). Each data represents the mean ± SEM (n = 4).
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Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
1.9 ± 0.3 88.2 ± 10.3b 138.6 ± 15.6b 215.2 ± 9.6b 544.3 ± 18.8b
7.5 ± 1.4 12.5 ± 1.4b 13.7 ± 1.2b 10.0 ± 0.0 13.7 ± 1.2b
1.7 ± 0.2 79.3 ± 32.6a 111.0 ± 18.6b 152.0 ± 13.9b 254.2 ± 29.9b
0.7 ± 0.1 31.0 ± 12.8a 43.4 ± 7.3b 59.7 ± 5.4b 99.5 ± 11.7b
Each value represents the mean ± SEM (n = 4). Cmax, maximum concentration; Tmax, time to reach Cmax; AUC, area under the curve; BA, relative bioavailability compared with s.c. a p < 0.05. b p < 0.01, significant difference compared with the corresponding ‘‘insulin solution.’’
insulin absorption. 0.5 and 2 mM of PenetraMax enhanced the BA of nasal insulin more strongly than L-penetratin reaching 2.8-times and 2.0-times in the first of administration. On day 7, 0.5 and 2 mM L-penetratin similarly increased nasal insulin absorption. In comparison, 0.5 and 2 mM PenetraMax increased insulin AUC, and the BA of nasal insulin was increased by 2.5- and 1.8-times to that of L-penetratin. We also examined the effects of PenetraMax on the nasal absorption of insulin for 30 consecutive days (Fig. 3 and Table 4). The time profiles of plasma insulin concentrations after co-administration of insulin and L-penetratin or PenetraMax were consistent with our results for 7 consecutive days of nasal administration. On the first day of administration, the absorption-enhancing effect of L-penetratin markedly enhanced the nasal absorption of insulin. PenetraMax enhanced the absorption of insulin stronger than original penetratin. Co-administration of 0.5 or 2 mM PenetraMax
significantly increased the insulin BA by 2.3- and 2.3-times compared with L-penetratin. In line with the results after 7 days of administration, PenetraMax had the most significantly increased absorption of insulin after 30 days of consecutive administrations. Co-administration with 0.5 or 2 mM PenetraMax clearly enhanced the nasal insulin absorption more than original penetratin. At 0.5 and 2 mM, PenetraMax, when co-administered with insulin, increased the BA of nasally delivered insulin compared to L-penetratin to 2.1-times and 2.2-times on the last day of the dosing regimen. These pharmacokinetic results demonstrate that both L-penetratin and PenetraMax enhance nasal insulin absorption significantly even after long-term multiple daily administrations. Our study, however, revealed a marked increase in nasal insulin absorption when co-administering with PenetraMax instead of the parent penetratin peptide. 3.2. Effect of repeated nasal CPP administration on mucosal toxicity and systemic release of biomarkers
Fig. 2. Plasma insulin concentrations vs. time profiles following multiple nasal administrations of insulin (1 IU/kg) with L-penetratin or PenetraMax (0.5 and 2 mM) for 7 days. Panels (a) and (b) show the results following the first instillation of the twice a day nasal administrations on the first day and 7th day, respectively. Each data point represents the mean ± SEM (n = 4).
Another objective of this long-term multiple daily administration study was to obtain information on local and systemic toxicity in rats treated with L-penetratin and PenetraMax in optimal absorption-enhancing concentrations. The treatments investigated were nasal installations of total 10 lL twice daily for 1, 7 and 30 days of either an insulin solution (control), L-penetratin with and without insulin, and PenetraMax with and without insulin. In order to quantify the effect on the nasal mucosa we evaluated LDH and biomarker release into the nasal cavity after 180 min and after 7 and 30 days of twice daily nasal administrations. LDH (a cytosolic enzyme) reflects mucosal damage in the form of rupture of epithelial cell membranes releasing cytosolic contents [30]. Fig. 4 shows data on LDH in the absence (insulin solution) or in the presence of L-penetratin and PenetraMax 180 min after dose administration. Groups receiving insulin with L-penetratin show a slight upward trend in the LDH release with increasing CPP dose compared to animals receiving the CPPs only. The overall results of LDH leakage in the CPP groups, however, were not significantly different from insulin alone whereas sodium taurodeoxycholate that was used as positive control significantly increased LDH leakage compared with L-penetratin or PenetraMax. Additionally, long-term administration and exposure to CPPs might be immunogenic which is crucial to avoid in delivery systems meant for chronic treatment [22,23,31]. Therapeutic proteins and peptides used in clinical settings are generally considered nontoxic, but an adaptive immune response in the form of antibodies may develop during the treatment. In order to determine the degree of immune system activation, we investigated the release of an inflammatory mediator (IL-1a) from the nasal mucosa that signals the onset of an inflammatory response [32,33]. In addition, TNF-a, a cytokine that has been implicated in immune responses to foreign therapeutic proteins – responses resembling that seen
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Table 3 Pharmacokinetic parameters following multiple nasal administrations of insulin (1 IU/kg) with various concentrations of L-penetratin or PenetraMax for 7 days. Day 1
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
Day 7
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
1.6 ± 0.3 90.7 ± 20.4a 216.4 ± 34.7a 354.8 ± 47.5a 549.6 ± 28.6a
12.5 ± 1.4 13.7 ± 1.2 12.5 ± 1.4 10.0 ± 0.0 10.0 ± 0.0
1.8 ± 0.4 63.1 ± 8.8a 137.1 ± 10.7a 175.8 ± 15.6a 272.3 ± 12.7a
0.7 ± 0.2 24.7 ± 3.5a 53.6 ± 4.2a 68.9 ± 6.1a 106.6 ± 5.0a
2.3 ± 0.1 111.0 ± 8.0a 244.8 ± 15.4a 306.7 ± 10.9a 554.0 ± 15.5a
11.2 ± 1.2 10.0 ± 0.0 11.2 ± 1.2 10.0 ± 0.0 10.0 ± 0.0
2.8 ± 1.0 66.9 ± 2.2a 150.0 ± 11.3a 167.7 ± 9.1a 266.4 ± 12.6a
1.1 ± 0.4 26.2 ± 0.9a 58.7 ± 4.4a 65.6 ± 3.6a 104.3 ± 4.9a
Each value represents the mean ± SEM (n = 4). Cmax, maximum concentration; Tmax, time to reach Cmax; AUC, area under the curve; BA, relative bioavailability compared with s.c. a p < 0.01, significant difference compared with the corresponding ‘‘insulin solution.’’
Fig. 4. LDH release in nasal lavage fluid after single or multiple nasal administrations of L-penetratin or PenetraMax with or without insulin. LDH leakage is expressed as a percentage of the positive control (5 w/v% sodium taurodeoxycholate). Each column represents the mean ± SEM (n = 3–4). p < 0.01, significant difference compared with ‘‘insulin (1 IU/kg)’’ at Day 1.
Fig. 3. Plasma insulin concentrations vs. time profiles following multiple nasal administrations of insulin (1 IU/kg) with L-penetratin or PenetraMax (0.5 and 2 mM) for 30 days. Panels (a) and (b) show the results following the first instillation of twice a day nasal administrations on the first day and 30th day, respectively. Each data point represents the mean ± SEM (n = 4).
against pathogens or vaccines involving T-cells, B-cells and the innate immune system [34], was also selected for analysis. After 1, 7 and 30 days of administrations of 0.5 and 2 mM L-penetratin or PenetraMax with and without insulin, IL-1a and TNF-a secretion on the mucosal side of the nasal membrane in nasal lavage fluid did not differ from that found in animals given insulin alone (Table 5). In order to confirm the systemic toxicity of the CPPs administered nasally, ELISA analysis of plasma samples detected no evidence of enhanced IL-1a or TNF-a cytokine production after 1, 7 or 30 days of treatment with L-penetratin and PenetraMax (0.5 and 2 mM) as shown in Table 6. Our data were compared to a previous study [30] which also evaluated biomarkers for mucosal damage in the nasal cavity as a way to assess the irritancy potential of nasal formulations. The biomarker release values obtained after CPP/insulin treatment were in line with saline treated control animals in the abovementioned study. Since saline treated controls reflect the baseline levels normally found in the nasal lining fluid,
Table 4 Pharmacokinetic parameters following multiple nasal administrations of insulin (1 IU/kg) with various concentrations of L-penetratin or PenetraMax for 30 days. Day 1
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
Day 30
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
2.1 ± 0.3 80.6 ± 5.2b 153.0 ± 12.2b 204.1 ± 24.3b 544.3 ± 18.8b
6.2 ± 1.2 13.7 ± 1.2b 15.0 ± 0.0b 11.2 ± 1.2a 13.7 ± 1.2b
1.9 ± 0.2 81.4 ± 34.1a 118.9 ± 12.7b 186.7 ± 41.9b 272.9 ± 13.9b
0.8 ± 0.1 31.9 ± 13.3a 46.5 ± 5.0b 73.1 ± 16.4b 106.8 ± 5.5b
2.1 ± 0.9 137.2 ± 8.3b 223.4 ± 5.7b 413.3 ± 26.0b 598.4 ± 98.3b
16.2 ± 4.7 10.0 ± 0.0 11.6 ± 1.6 11.2 ± 1.2 11.2 ± 1.2
2.7 ± 1.1 86.1 ± 16.4a 124.6 ± 13.7b 179.5 ± 22.5b 268.3 ± 33.7b
1.1 ± 0.5 33.7 ± 6.4a 48.8 ± 5.4b 70.3 ± 8.8b 105.0 ± 13.2b
Each value represents the mean ± SEM (n = 3–4). Cmax, maximum concentration; Tmax, time to reach Cmax; AUC, area under the curve; BA, relative bioavailability compared with s.c. a p < 0.05. b p < 0.01, significant difference compared with the corresponding ‘‘insulin solution.’’
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E.-S. Khafagy et al. / European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 736–743 Table 5 IL-1a and TNF-a release in nasal lavage following administration of L-penetratin or PenetraMax with or without insulin (1 IU/kg, ‘‘control’’) into rat nasal cavities. IL-1a (ng/mL)
0.5 mM 2.0 mM 0.5 mM 2.0 mM
L-penetratin L-penetratin
PenetraMax PenetraMax
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
TNF-a (ng/mL)
Day 1
Day 7
Day 30
Day 1
Day 7
Day 30
0.020 ± 0.000 0.021 ± 0.001 0.021 ± 0.001 0.021 ± 0.001
0.018 ± 0.000 0.018 ± 0.000 0.022 ± 0.001 0.020 ± 0.001
0.019 ± 0.001 0.019 ± 0.000 0.019 ± 0.000 0.020 ± 0.001
0.077 ± 0.001 0.076 ± 0.000 0.073 ± 0.001 0.074 ± 0.001
0.076 ± 0.001 0.077 ± 0.001 0.076 ± 0.001 0.075 ± 0.001
0.074 ± 0.000 0.074 ± 0.000 0.076 ± 0.000 0.076 ± 0.000
0.020 ± 0.001 0.020 ± 0.000 0.022 ± 0.002 0.021 ± 0.001 0.019 ± 0.001
0.018 ± 0.000 0.018 ± 0.000 0.020 ± 0.001 0.021 ± 0.001 0.021 ± 0.001
0.023 ± 0.004 0.026 ± 0.001 0.023 ± 0.002 0.021 ± 0.001 0.022 ± 0.001
0.076 ± 0.001 0.076 ± 0.001 0.076 ± 0.001 0.074 ± 0.001 0.074 ± 0.001
0.077 ± 0.001 0.075 ± 0.000 0.075 ± 0.000 0.074 ± 0.000 0.074 ± 0.000
0.075 ± 0.001 0.074 ± 0.000 0.075 ± 0.000 0.076 ± 0.000 0.077 ± 0.000
Each value represents the mean ± SEM (n = 3–4).
Table 6 IL-1a and TNF-a release in plasma following administration of L-penetratin or PenetraMax with or without insulin (1 IU/kg, ‘‘control’’) into rat nasal cavities. IL-1a (ng/mL)
0.5 mM 2.0 mM 0.5 mM 2.0 mM
L-penetratin L-penetratin
PenetraMax PenetraMax
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
TNF-a (ng/mL)
Day 1
Day 7
Day 1
Day 30
Day 1
Day 7
Day 1
Day 30
0.020 ± 0.000 0.020 ± 0.000 0.019 ± 0.000 0.019 ± 0.000
0.019 ± 0.000 0.018 ± 0.000 0.019 ± 0.000 0.018 ± 0.000
0.030 ± 0.008 0.026 ± 0.003 0.028 ± 0.002 0.023 ± 0.000
0.019 ± 0.000 0.020 ± 0.000 0.019 ± 0.000 0.020 ± 0.001
0.081 ± 0.001 0.081 ± 0.001 0.080 ± 0.002 0.080 ± 0.001
0.079 ± 0.000 0.077 ± 0.001 0.078 ± 0.000 0.078 ± 0.001
0.076 ± 0.000 0.077 ± 0.001 0.076 ± 0.001 0.076 ± 0.002
0.072 ± 0.001 0.074 ± 0.001 0.072 ± 0.000 0.075 ± 0.004
0.031 ± 0.001 0.020 ± 0.000 0.027 ± 0.002 0.021 ± 0.001 0.019 ± 0.000
0.029 ± 0.009 0.021 ± 0.001 0.028 ± 0.007 0.021 ± 0.001 0.019 ± 0.005
0.025 ± 0.002 0.028 ± 0.004 0.029 ± 0.003 0.022 ± 0.001 0.025 ± 0.002
0.020 ± 0.000 0.024 ± 0.003 0.023 ± 0.002 0.020 ± 0.000 0.020 ± 0.001
0.081 ± 0.000 0.079 ± 0.001 0.080 ± 0.001 0.081 ± 0.001 0.079 ± 0.001
0.080 ± 0.002 0.080 ± 0.002 0.079 ± 0.002 0.079 ± 0.001 0.081 ± 0.002
0.073 ± 0.000 0.073 ± 0.000 0.074 ± 0.001 0.079 ± 0.005 0.074 ± 0.001
0.074 ± 0.002 0.075 ± 0.002 0.072 ± 0.001 0.076 ± 0.002 0.075 ± 0.001
Each value represents the mean ± SEM (n = 3–4).
we therefore conclude that the irritancy and immunogenic potential of our formulation in a preclinical subchronic setting is negligible and that L-penetratin and PenetraMax induce no systemic toxicity after 1, 7 and 30 days of consecutive nasal administrations compared to insulin alone.
These results are in line with data obtained from in vitro and in vivo studies of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs) peptides (which were obtained by chemically modifying the TP10 peptide sequence), and also with TAT and stearyl-(RxR)4 peptides, showing no influence on the host
Fig. 5. Photomicrographs of the nasal respiratory mucosal membranes of vertical sections through the anterior rat nasal cavity following nasal administration of L-penetratin or PenetraMax (2.0 mM) with or without insulin for 30 consecutive days. Sodium taurodeoxycholate (5 w/v%) was used as positive control. The bars indicate 50 lm. Tissues were decalcified and stained with haematoxylin and eosin after fixation in 20% formalin neutral buffer solution. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 7 Histopathological findings of rat nasal cavity treated with L-penetratin or PenetraMax with or without insulin (insulin 1 IU/kg) for 30 consecutive days, or treated with 5% w/v sodium taurodeoxycholate for 3 h. Histopathological findings
0.5 mM 2.0 mM 0.5 mM 2.0 mM
L-penetratin L-penetratin
PenetraMax PenetraMax
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax 5 w/v% Sodium taurodeoxycholate
Respiratory epithelium necrosis
Olfactory epithelium necrosis
Dorsal meatus
Upper septum
Nasoturbinate
Maxillo turbinate
+++
+++
+++
++
++
+++
Abbreviations: , no change; ++, moderate change; +++, marked change. Each score represents triplicate experiments.
system. None of the peptides used for i.v. injections for nucleic acid delivery induced an increase in the cytokine levels in blood serum or caused pathological deviations in liver, kidney, lung, and spleen of the treated animals [35,36] suggesting that certain CPPs can be considered as non-toxic and non-immunogenic tools in drug delivery. 3.3. Histopathological irritation scores in the nasal membrane following 30 days of CPP administration The purpose of this study was to obtain histological information on disruptive reactions in the nasal mucosa of rats treated with L-penetratin and PenetraMax after long-term repeated administrations. The photomicrographs in Fig. 5 show no apparent histological damage of the mucosal membranes caused by Lpenetratin and PenetraMax (2 mM) with or without insulin in comparison to insulin treatment after 30 days. In contrast, severe damage of the nasal mucosal membrane was made by 3 h treatment with sodium taurodeoxycholate used as a positive control. The nasal membrane histopathological findings and irritation scores are summarised in Table 7. The histopathological signs used to assess the nasal respiratory mucosal membranes in vertical sections through the anterior rat nasal cavity following nasal administration of CPPs (0.5 mM and 2 mM) with or without coadministration of insulin for 180 min for 30 consecutive days show no observable change in membrane integrity in the different regions of nasal respiratory tissues such as respiratory and olfactory epithelium, dorsal meatus, upper nasal septum, nasoturbinate, and maxillo turbinate. These data show that administration of L-penetratin and PenetraMax safely enhanced insulin delivery without altering the integrity of the nasal membrane. In contrast, positive control taurodeoxycholate showed significant irritation scores of the nasal epithelium membrane, septum and turbinate (Table 7). Our results indicate that tryptophan, lysine and arginine backbone spacing can affect the CPP uptake efficiency while maintaining the low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view [37]. Finally, our study showed no cytotoxicity in the nasal mucosal membrane. The histopathological scoring confirmed the negligible LDH and biomarkers leakage picturing a healthy and undamaged nasal membrane after 30 days of multiple administrations. As a result, PenetraMax can be considered a non-toxic insulin absorption enhancer for nasal delivery showing significant BA.
4. Conclusions The study presented here was conducted to determine the effect of sequence modifications of L-penetratin on the nasal absorption-enhancing capacities. The novel CPP, PenetraMax, allows for optimal systemic delivery of insulin from the nasal cavity resulting in high BA for single and multiple long-term administrations. The BA reached almost 100% relatively to s.c. insulin administration without detectable epithelial membrane damage. In conclusion, we found that L-penetratin and PenetraMax are not toxic either systemically or locally at two selected concentrations in this subchronic rat study. The present results open the way to for rational engineering of CPPs as delivery tools and hold great promise for noninvasive delivery of biotherapeutic peptides without risk of local toxicity and inflammation/immunogenicity – the latter being crucial for peptidic based pharmaceuticals in preparation for clinical trials. Acknowledgments This study was supported in part by JSPS KAKENHI Grant Number 23590056 and the Research Programme for the Development of Innovative Technology of the Japan Science and Technology Agency. This work was also supported in part by The Nagai Foundation Tokyo. The authors are grateful to the undergraduate students of the Laboratory of Drug Delivery Systems in Kobe Gakuin University for their technical assistance. References [1] S.R. Schwarze, A. Ho, A. Vocero-Akbani, S.F. Dowdy, In vivo protein transduction: delivery of a biologically active protein into the mouse, Science 285 (1999) 1569–1572. [2] B. Gupta, T.S. Levchenko, V.P. Torchilin, Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides, Adv. Drug Deliv. Rev. 57 (2005) 637–651. [3] C. Foerg, H.P. Merkle, On the biomedical promise of cell penetrating peptides: limits versus prospects, J. Pharm. Sci. 97 (2008) 144–162. [4] El-S. Khafagy, M. Morishita, Oral biodrug delivery using cell-penetrating peptide, Adv. Drug Deliv. Rev. 64 (2012) 531–539. [5] A.T. Jones, E.J. Sayers, Cell entry of cell penetrating peptides: tales of tails wagging dogs, J. Control. Release 161 (2012) 582–591. [6] C. Foged, H.M. Nielsen, Cell-penetrating peptides for drug delivery across membrane barriers, Expert Opin. Drug Deliv. 5 (2008) 105–117. [7] L.N. Patel, J.L. Zaro, W.C. Shen, Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives, Pharm. Res. 24 (2007) 1977–1992. [8] J. Hawiger, Noninvasive intracellular delivery of functional peptides and proteins, Curr. Opin. Chem. Biol. 3 (1999) 89–94.
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Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
One-month subchronic toxicity study of cell-penetrating peptides for insulin nasal delivery in rats El-Sayed Khafagy a,b, Noriyasu Kamei a, Ebbe Juel Bech Nielsen a,c, Reiji Nishio d, Mariko Takeda-Morishita a,⇑ a
Laboratory of Drug Delivery Systems, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt Novo Nordisk A/S, Måløv, Denmark d New Frontiers Research Laboratories, Toray Industries, Inc., Kamakura, Japan b c
a r t i c l e
i n f o
Article history: Received 20 June 2013 Accepted in revised form 13 September 2013 Available online 21 September 2013 Keywords: Cell-penetrating peptide Penetratin Toxicity Insulin Nasal delivery Long-term administration
a b s t r a c t Recently, cell-penetrating peptides (CPPs) based vehicles have been developed for the delivery of different payloads in animals. Our studies have shown that nasal absorption of insulin and other therapeutic peptides and proteins can be improved significantly by co-administration of the CPP penetratin. Successful development of suitable CPP-based delivery systems, however, will depend not only on the efficiency of CPPs to transport therapeutic agents across the biological barriers of the nasal cavity, but also on the risk of adverse effects such as toxicity and undesired immunogenicity, especially in chronic therapy. In this study, we investigated the bioavailability (BA) of insulin and the adverse effects on the nasal mucosa in rats following a long-term dosing regimen of L-penetratin and the novel penetratin analogue ‘‘PenetraMax.’’ Following nasal delivery, a significantly higher BA for insulin (almost 100% relative to subcutaneous (s.c.) injections) was observed for PenetraMax in comparison with the parent penetratin peptide after chronic administrations in rats. Importantly, there was negligible biomarker leakage in nasal lavage fluid and the integrity of the nasal epithelium remained unaffected when PenetraMax was used in long-term multiple administrations. In addition, no significant difference in the release of inflammatory and immunogenicity mediators in plasma was observed after nasal administration of PenetraMax with or without insulin solution. In conclusion, PenetraMax, a novel CPP candidate, can open a new avenue in clinical trials for noninvasive nasal insulin delivery. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Cell-penetrating peptides (CPPs) have proven their ability for intracellular delivery of various macromolecular therapies such as nucleic acids, proteins and peptides, as well as drug delivery carriers [1–5]. CPPs comprise a growing family of peptides that have opened a new avenue for noninvasive systemic delivery of a vast collection of biomolecules otherwise restricted in crossing the plasma membrane [6,7]. CPPs are short polybasic and/or amphipathic peptides (<40 amino acids) with a net positive charge derived from signal peptides, viral peptides or other sources and they have been used successfully to translocate covalently or non-covalently bound cargo across cell membranes [8–10]. Previously, we have shown that CPPs are capable of transcellular delivery of peptides and proteins across the intestinal and nasal ⇑ Corresponding author. Laboratory of Drug Delivery Systems, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe, Hyogo 650-8586, Japan. Tel.: +81 78 974 4816; fax: +81 78 974 4820. E-mail address: [email protected] (M. Takeda-Morishita). 0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.09.014
epithelial membranes [11–16]. Recently, systemic delivery of proteins and peptides via the nasal route has received increased attention because of the great potential for noninvasive biodrug delivery. Sufficient bioavailability, however, has to date been a challenge to achieve [17–19]. We have previously demonstrated that penetratin, a peptide derived from the Drosophila Antennapedia Homeoprotein, to be amongst the most successful CPPs to overcome the permeability problems of peptides and proteins in the nasal cavity. Although the relative bioavailability of insulin can reach up to 50% after co-administration with penetratin, a relatively high dose of penetratin is required for this enhancement of nasal insulin absorption [13]. To address the efficiency of penetration, a screening study of various penetratin analogues suggested that the chain length, hydrophobicity, amphipathicity and basicity of the CPP contribute to their absorption-enhancing efficiency [20]. Penetratin structure modifications introduced in the ‘‘shuffle (R, K fix) 2’’ sequence were successful in overcoming the permeability barrier for peptides and proteins in the nasal cavity. These findings were further supported in an in silico study based on molecular orbital analysis with Self-Organizing Maps
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(SOM) classification in which the structural and physiochemical factors mentioned above were associated with the intestinal insulin absorption-enhancing effect of CPPs. As predicted by SOM clustering, a novel penetratin analogue (Sample 6) had significantly greater capacity to interact with insulin and to enhance intestinal insulin absorption than that of the original penetratin sequence [21] (Now this optimised peptide Sample 6 was named as ‘‘PenetraMax.’’) Insufficient information, however, regarding long-term CPP toxicity restricts their application in vivo and preclinical development in general as a successful delivery system will depend equally on the efficiency of CPPs to transport therapeutic agents through the biological barriers as well as the risk of associated side effects such as toxicity and undesired immunogenicity [22,23]. Hence, it is of great interest to develop a novel highly effective nasal absorption promoter with negligible toxic effect on the nasal mucosa and low systemic toxicity, especially for chronic therapy. In this study, we explored the potential of a novel CPP, PenetraMax, for nasal delivery of insulin in comparison with the parent penetratin peptide at varying concentrations in a one-month twice daily administration study in rats. In a clinical setting, absorption enhancers like CPPs would be used chronically and as such, their safety must be guaranteed for chronic administration. Thus, following the same dosing scheme as mentioned above, we also examined systemic toxicity by quantitatively assessing nasal biomarker release (lactate dehydrogenase (LDH), interleukin-1a (IL-1a), and tumour necrosis factor-a (TNF-a)) and systemic release of IL-1a and TNF-a. In addition, local effects on the nasal mucosa after 7 and 30 days repeated daily administrations were investigated by histopathological examination of the nasal epithelium.
2. Materials and methods 2.1. Materials Recombinant human insulin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). L-penetratin and its analogue PenetraMax listed in Table 1 were synthesised by Sigma–Genosys, Life Science Division of Sigma–Aldrich Japan Co. (Hokkaido, Japan). All other chemicals were of analytical grade and are commercially available.
2.2. Preparation of the mixed insulin/CPP solutions To prepare the insulin solution, specific amounts of recombinant human insulin were dissolved in 50 lL of 0.1 M HCl. The insulin-HCl solution was diluted to 1.4 mL with phosphate-buffered saline (PBS, pH 6.0) containing 0.001% methylcellulose, which prevents the adsorption of the insulin to the tube surface, and normalised with 50 lL of 0.1 M NaOH. Specific amounts of L-penetratin or PenetraMax were dissolved in PBS (pH 6.0) containing 0.001% methylcellulose. Aliquot of insulin solution was added to L-penetratin or PenetraMax solution, mixed gently, and adjusted to a specific concentration. Each insulin/CPP solution was clear after mixing.
Table 1 Molecular weight and amino acid sequences of L-penetratin and PenetraMax. Sequence
Mw
L-Penetratin
RQIKIWFQNRRMKWKK
2246.7
PenetraMax
KWFKIQMQIRRWKNKR
2246.7
F, phenylalanine; I, isoleucine; K, lysine; M, methionine; N, asparagine; Q, glutamine; R, arginine; W, tryptophan.
737
2.3. Nasal absorption study This research was performed at Kobe Gakuin University and complied with the regulations of the Committee on Ethics in the Care and Use of Laboratory Animals. Male Sprague Dawley rats weighing 180–220 g were purchased from Japan SLC, Inc. (Shizouka, Japan). The animals were housed in temperature (23 ± 1 °C) and relative humidity (55 ± 5%) controlled rooms with free access to water and food during acclimatisation. Animals were fasted for 24 h before the experiments; however, they were allowed to drink water ad libitum. Under an intraperitoneal (i.p.) injection of sodium pentobarbital (12.5 mg/kg; Somnopentyl; Kyoritsu Seiyaku Corp., Tokyo, Japan), insulin or insulin/CPP mixture was applied intranasally twice a day for 1, 7 or 30 days. Rats were restrained in a supine position during administration at an angle of 15° and total 10 lL of drug (5 lL/each nostril) was instilled at 1 cm depth in each nostril by micropipette with a narrow tip fitted. The procedure was performed gently and slowly and lasted 1–2 min allowing the rats to receive all of the prepared sample. Insulin doses were 1 IU/kg body weight and CPP concentrations were 0.5 and 2 mM. Blood sampling to determine plasma insulin concentrations was performed on all groups after the first dose on days 1, 7 and 30 depending on the group. Following their anesthetization with an i.p. injection of sodium pentobarbital (50 mg/kg), the rats were restrained in a supine position on a thermostatically controlled board at 37 °C. Additional i.p. injections of sodium pentobarbital (12.5 mg/kg) were given every 1 h to maintain anaesthesia during the experiment for collection of blood samples. A 0.25 mL blood aliquot was taken from the jugular vein using 1 mL tuberculin heparinized syringes at 5, 10, 15, 30, 60, 120 and 180 min after dosing. Plasma was separated by centrifugation at 12,000 rpm (13,400g) for 1 min and stored at 80 °C until analysis. The plasma insulin concentration was determined using a human insulin ELISA kit (Mercodia AB, Uppsala, Sweden) and the absorbance at kmax 450 nm was detected by using a microplate reader (POWERSCAN HT, DS Pharma Biomedical Co. Ltd., Osaka, Japan). 2.4. Pharmacokinetic analysis The bioavailability of nasally administered insulin was calculated relative to a s.c. injection (1 IU/kg). Briefly, an insulin solution was prepared by dissolving an appropriate amount of insulin in PBS. The peak plasma concentration (Cmax) and time to reach Cmax (Tmax) were directly determined from plasma insulin concentration–time curves. The total area under the insulin concentration curve (AUC) for 0–3 h was estimated from the sum of successive trapezoids between each data point. The relative bioavailability (BA) of insulin was calculated relative to the s.c. injection as follows:
BA ð%Þ ¼ ð½AUC=doseÞ 100=ð½AUCs:c:=doses:c: Þ 2.5. Biomarker assay Inflammatory blood biomarkers (IL-1a and TNF-a) for insulin solution (control), L-penetratin with and without insulin and PenetraMax with and without insulin were analysed on the first day of nasal administration for each group and at the end of each respective dosing regimen (after 1, 7 and 30 days of nasal administrations). Nasal lavage fluid was collected for analysis of LDH, IL-1a and TNF-a release by the nasal mucosal cells at the end of days 1, 7 and 30 respectively. Briefly, after blood sampling rats were killed with an i.p. overdose of pentobarbital. The nasal cavity was perfused with 10 mL PBS warmed to 37 °C at a flow rate 2 mL/
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min using an infusion pump (KD Scientific Inc., Holliston, MA, USA. LDH activity in the nasal lavage fluid was determined using the CytoTox-96 assay kit (Promega Corp., Madison, WI, USA) as per manufacturer’s instructions. The positive control group received 5% (w/v) sodium taurodeoxycholate in same manner as other treatment groups. Results were determined at kmax 490 nm. Rat IL-1a was measured using a Quantikine IL-1a/IL-1F1 immunoassay kit (R&D Systems, Inc., Minneapolis, MN, USA) as per manufacturer’s instructions. Results were determined against a manufacturer supplied rat IL-1a standard at kmax 450 nm. Rat TNF-a was measured using a rat TNF-a immunoassay kit (R&D Systems, Inc.) as per manufacturer’s instructions. Results were determined against a manufacturer supplied rat TNF-a standard at kmax 450 nm. All measurements of the absorbance were conducted by using a POWERSCAN HT (DS Pharma Biomedical Co. Ltd.). 2.6. Histopathological examination After 1, 7 and 30 days of nasal administrations of the test solutions and after collection of biomarkers in the nasal lavage fluid as described above, 10 mL of 20% formalin neutral buffer solution (Mildform 20 N; Wako Pure Chemical Industries Ltd., Osaka, Japan) was perfused through the nasal cavity at a flow rate of 2 mL/min. The nasal cavity samples of all rats were preserved and fixed in Mildform 20 N. After fixation, samples were decalcified by an 8% Formic acid + 10% ion-exchange resin solution. For histopathological evaluation, samples are taken from the upper incisor teeth to the incisive papilla, according to the specific palatal landmarks (Level II), and then embedded in paraffin wax. Three lm-thick sections of Level II were cut and stained with haematoxylin and eosin (Kanto Chemical Co., Inc., Tokyo, Japan), and were examined by a light microscope (NIKON ECLIPSE E600; Nikon Corp., Tokyo, Japan). 2.7. Statistical analysis Data are presented as mean ± SEM. The significance of differences in mean values was evaluated using Student’s unpaired t test. For multiple comparisons, a one-way layout analysis of variance (ANOVA) with Dunnett’s test was applied. Differences were considered significant when p was less than 0.05. 3. Results and discussion
insulin absorption [21]. Thus building on our previous data for PenetraMax, we explored this novel CPP as an absorption enhancer for nasally delivered insulin in this one-month twice daily administration study. Time profiles for plasma insulin concentration after co-administration of insulin and L-penetratin or PenetraMax in different concentrations (0.5 and 2 mM) are shown in Fig. 1. Increasing concentrations of PenetraMax (0.5 or 2 mM) enhanced the permeability of the nasal mucosa to insulin more markedly than L-penetratin and insulin alone. The pharmacokinetic parameters derived from the insulin concentration–time profiles following nasal administration of insulin with L-penetratin and PenetraMax are shown in Table 2. Treatment with 0.5 mM PenetraMax resulted in a relative increase in insulin BA by 1.9-times compared to L-penetratin and treatment with 2 mM PenetraMax resulted in a 2.3-times increase in the nasal insulin BA compared with L-penetratin. Thus, PenetraMax greatly increased the Cmax, AUC, and BA of insulin compared to L-penetratin which can be attributed to a dramatically increased absorption. The superior effect of PenetraMax on nasal insulin absorption may be attributed to the change in the positions of the Trp molecules (Table 1) [20,27]. This allows a high degree of conformational flexibility of the interacting moieties, thus stabilizing the conformation of the peptide on the water–lipid interface and facilitating the insertion of PenetraMax into the lipid bilayer [28]. Our recent study on the enhancement effect of by penetratin analogues by SOM clustering analysis showed the relationship between the essential physical and electrical parameters, as casual factors, and the increase in the insulin AUC, as response variable. The higher capacity of PenetraMax to interact with insulin than the original penetratin might be involved in its enhanced stimulatory effect on insulin absorption, especially in our approach using physical mixtures of the drug and CPPs rather than their conjugates [15,21,29]. To verify L-penetratin and PenetraMax for nasal insulin absorption for long-term administration, we examined their effects on nasal insulin absorption for 7 days and compared the PenetraMax enhancing effect with that of the original L-penetratin using insulin solution as a control. Insulin concentration–time profiles following intranasal administration for 7 consecutive days show that PenetraMax was the most effective promoter of insulin absorption compared with L-penetratin as shown in Fig. 2 and Table 3. On the first day of administration, as shown in Table 3, co-administration of 0.5 or 2 mM L-penetratin increased nasal
3.1. Effect of L-penetratin and PenetraMax on nasal insulin absorption Our recent work has shown that absorption of insulin and other medicinal peptides and proteins from the nasal cavity can be significantly improved by the co-administration of functional CPPs [13,14]. Amongst the CPPs examined in our previous studies, penetratin derived from the Drosophila Antennapedia Homeoprotein [24–26] enhanced the nasal absorption of insulin most effectively [13]. The dose dependent relationship of L-penetratin and insulin bioavailability has been investigated and we found a BA of insulin reached up to 50% when insulin was co-administered with 2 mM L-penetratin [13]. However, the penetratin concentration was relatively high considering cost and possible adverse effects after multiple administrations. Therefore, to identify CPPs that were more effective, we compared the enhancing effect of L-penetratin and 20 of its analogues on nasal insulin absorption. Surprisingly, the ‘‘shuffle (R, K fix) 2’’ analogue greatly increased insulin absorption reaching a relative bioavailability value 1.85-times that of original penetratin [20]. Moreover, an in silico analysis of penetratin analogues based on molecular orbital analysis with SOM classification, predicted a novel CPP, PenetraMax, as the sequence with the strongest enhancing effect on intestinal
Fig. 1. Plasma insulin concentrations vs. time profiles following single nasal administration of insulin (1 IU/kg) with L-penetratin or PenetraMax (0.5 and 2 mM). Each data represents the mean ± SEM (n = 4).
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Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
1.9 ± 0.3 88.2 ± 10.3b 138.6 ± 15.6b 215.2 ± 9.6b 544.3 ± 18.8b
7.5 ± 1.4 12.5 ± 1.4b 13.7 ± 1.2b 10.0 ± 0.0 13.7 ± 1.2b
1.7 ± 0.2 79.3 ± 32.6a 111.0 ± 18.6b 152.0 ± 13.9b 254.2 ± 29.9b
0.7 ± 0.1 31.0 ± 12.8a 43.4 ± 7.3b 59.7 ± 5.4b 99.5 ± 11.7b
Each value represents the mean ± SEM (n = 4). Cmax, maximum concentration; Tmax, time to reach Cmax; AUC, area under the curve; BA, relative bioavailability compared with s.c. a p < 0.05. b p < 0.01, significant difference compared with the corresponding ‘‘insulin solution.’’
insulin absorption. 0.5 and 2 mM of PenetraMax enhanced the BA of nasal insulin more strongly than L-penetratin reaching 2.8-times and 2.0-times in the first of administration. On day 7, 0.5 and 2 mM L-penetratin similarly increased nasal insulin absorption. In comparison, 0.5 and 2 mM PenetraMax increased insulin AUC, and the BA of nasal insulin was increased by 2.5- and 1.8-times to that of L-penetratin. We also examined the effects of PenetraMax on the nasal absorption of insulin for 30 consecutive days (Fig. 3 and Table 4). The time profiles of plasma insulin concentrations after co-administration of insulin and L-penetratin or PenetraMax were consistent with our results for 7 consecutive days of nasal administration. On the first day of administration, the absorption-enhancing effect of L-penetratin markedly enhanced the nasal absorption of insulin. PenetraMax enhanced the absorption of insulin stronger than original penetratin. Co-administration of 0.5 or 2 mM PenetraMax
significantly increased the insulin BA by 2.3- and 2.3-times compared with L-penetratin. In line with the results after 7 days of administration, PenetraMax had the most significantly increased absorption of insulin after 30 days of consecutive administrations. Co-administration with 0.5 or 2 mM PenetraMax clearly enhanced the nasal insulin absorption more than original penetratin. At 0.5 and 2 mM, PenetraMax, when co-administered with insulin, increased the BA of nasally delivered insulin compared to L-penetratin to 2.1-times and 2.2-times on the last day of the dosing regimen. These pharmacokinetic results demonstrate that both L-penetratin and PenetraMax enhance nasal insulin absorption significantly even after long-term multiple daily administrations. Our study, however, revealed a marked increase in nasal insulin absorption when co-administering with PenetraMax instead of the parent penetratin peptide. 3.2. Effect of repeated nasal CPP administration on mucosal toxicity and systemic release of biomarkers
Fig. 2. Plasma insulin concentrations vs. time profiles following multiple nasal administrations of insulin (1 IU/kg) with L-penetratin or PenetraMax (0.5 and 2 mM) for 7 days. Panels (a) and (b) show the results following the first instillation of the twice a day nasal administrations on the first day and 7th day, respectively. Each data point represents the mean ± SEM (n = 4).
Another objective of this long-term multiple daily administration study was to obtain information on local and systemic toxicity in rats treated with L-penetratin and PenetraMax in optimal absorption-enhancing concentrations. The treatments investigated were nasal installations of total 10 lL twice daily for 1, 7 and 30 days of either an insulin solution (control), L-penetratin with and without insulin, and PenetraMax with and without insulin. In order to quantify the effect on the nasal mucosa we evaluated LDH and biomarker release into the nasal cavity after 180 min and after 7 and 30 days of twice daily nasal administrations. LDH (a cytosolic enzyme) reflects mucosal damage in the form of rupture of epithelial cell membranes releasing cytosolic contents [30]. Fig. 4 shows data on LDH in the absence (insulin solution) or in the presence of L-penetratin and PenetraMax 180 min after dose administration. Groups receiving insulin with L-penetratin show a slight upward trend in the LDH release with increasing CPP dose compared to animals receiving the CPPs only. The overall results of LDH leakage in the CPP groups, however, were not significantly different from insulin alone whereas sodium taurodeoxycholate that was used as positive control significantly increased LDH leakage compared with L-penetratin or PenetraMax. Additionally, long-term administration and exposure to CPPs might be immunogenic which is crucial to avoid in delivery systems meant for chronic treatment [22,23,31]. Therapeutic proteins and peptides used in clinical settings are generally considered nontoxic, but an adaptive immune response in the form of antibodies may develop during the treatment. In order to determine the degree of immune system activation, we investigated the release of an inflammatory mediator (IL-1a) from the nasal mucosa that signals the onset of an inflammatory response [32,33]. In addition, TNF-a, a cytokine that has been implicated in immune responses to foreign therapeutic proteins – responses resembling that seen
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Table 3 Pharmacokinetic parameters following multiple nasal administrations of insulin (1 IU/kg) with various concentrations of L-penetratin or PenetraMax for 7 days. Day 1
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
Day 7
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
1.6 ± 0.3 90.7 ± 20.4a 216.4 ± 34.7a 354.8 ± 47.5a 549.6 ± 28.6a
12.5 ± 1.4 13.7 ± 1.2 12.5 ± 1.4 10.0 ± 0.0 10.0 ± 0.0
1.8 ± 0.4 63.1 ± 8.8a 137.1 ± 10.7a 175.8 ± 15.6a 272.3 ± 12.7a
0.7 ± 0.2 24.7 ± 3.5a 53.6 ± 4.2a 68.9 ± 6.1a 106.6 ± 5.0a
2.3 ± 0.1 111.0 ± 8.0a 244.8 ± 15.4a 306.7 ± 10.9a 554.0 ± 15.5a
11.2 ± 1.2 10.0 ± 0.0 11.2 ± 1.2 10.0 ± 0.0 10.0 ± 0.0
2.8 ± 1.0 66.9 ± 2.2a 150.0 ± 11.3a 167.7 ± 9.1a 266.4 ± 12.6a
1.1 ± 0.4 26.2 ± 0.9a 58.7 ± 4.4a 65.6 ± 3.6a 104.3 ± 4.9a
Each value represents the mean ± SEM (n = 4). Cmax, maximum concentration; Tmax, time to reach Cmax; AUC, area under the curve; BA, relative bioavailability compared with s.c. a p < 0.01, significant difference compared with the corresponding ‘‘insulin solution.’’
Fig. 4. LDH release in nasal lavage fluid after single or multiple nasal administrations of L-penetratin or PenetraMax with or without insulin. LDH leakage is expressed as a percentage of the positive control (5 w/v% sodium taurodeoxycholate). Each column represents the mean ± SEM (n = 3–4). p < 0.01, significant difference compared with ‘‘insulin (1 IU/kg)’’ at Day 1.
Fig. 3. Plasma insulin concentrations vs. time profiles following multiple nasal administrations of insulin (1 IU/kg) with L-penetratin or PenetraMax (0.5 and 2 mM) for 30 days. Panels (a) and (b) show the results following the first instillation of twice a day nasal administrations on the first day and 30th day, respectively. Each data point represents the mean ± SEM (n = 4).
against pathogens or vaccines involving T-cells, B-cells and the innate immune system [34], was also selected for analysis. After 1, 7 and 30 days of administrations of 0.5 and 2 mM L-penetratin or PenetraMax with and without insulin, IL-1a and TNF-a secretion on the mucosal side of the nasal membrane in nasal lavage fluid did not differ from that found in animals given insulin alone (Table 5). In order to confirm the systemic toxicity of the CPPs administered nasally, ELISA analysis of plasma samples detected no evidence of enhanced IL-1a or TNF-a cytokine production after 1, 7 or 30 days of treatment with L-penetratin and PenetraMax (0.5 and 2 mM) as shown in Table 6. Our data were compared to a previous study [30] which also evaluated biomarkers for mucosal damage in the nasal cavity as a way to assess the irritancy potential of nasal formulations. The biomarker release values obtained after CPP/insulin treatment were in line with saline treated control animals in the abovementioned study. Since saline treated controls reflect the baseline levels normally found in the nasal lining fluid,
Table 4 Pharmacokinetic parameters following multiple nasal administrations of insulin (1 IU/kg) with various concentrations of L-penetratin or PenetraMax for 30 days. Day 1
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
Day 30
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
Cmax (lU/m1)
Tmax (min)
AUC (lU h/m1)
BA (%)
2.1 ± 0.3 80.6 ± 5.2b 153.0 ± 12.2b 204.1 ± 24.3b 544.3 ± 18.8b
6.2 ± 1.2 13.7 ± 1.2b 15.0 ± 0.0b 11.2 ± 1.2a 13.7 ± 1.2b
1.9 ± 0.2 81.4 ± 34.1a 118.9 ± 12.7b 186.7 ± 41.9b 272.9 ± 13.9b
0.8 ± 0.1 31.9 ± 13.3a 46.5 ± 5.0b 73.1 ± 16.4b 106.8 ± 5.5b
2.1 ± 0.9 137.2 ± 8.3b 223.4 ± 5.7b 413.3 ± 26.0b 598.4 ± 98.3b
16.2 ± 4.7 10.0 ± 0.0 11.6 ± 1.6 11.2 ± 1.2 11.2 ± 1.2
2.7 ± 1.1 86.1 ± 16.4a 124.6 ± 13.7b 179.5 ± 22.5b 268.3 ± 33.7b
1.1 ± 0.5 33.7 ± 6.4a 48.8 ± 5.4b 70.3 ± 8.8b 105.0 ± 13.2b
Each value represents the mean ± SEM (n = 3–4). Cmax, maximum concentration; Tmax, time to reach Cmax; AUC, area under the curve; BA, relative bioavailability compared with s.c. a p < 0.05. b p < 0.01, significant difference compared with the corresponding ‘‘insulin solution.’’
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E.-S. Khafagy et al. / European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 736–743 Table 5 IL-1a and TNF-a release in nasal lavage following administration of L-penetratin or PenetraMax with or without insulin (1 IU/kg, ‘‘control’’) into rat nasal cavities. IL-1a (ng/mL)
0.5 mM 2.0 mM 0.5 mM 2.0 mM
L-penetratin L-penetratin
PenetraMax PenetraMax
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
TNF-a (ng/mL)
Day 1
Day 7
Day 30
Day 1
Day 7
Day 30
0.020 ± 0.000 0.021 ± 0.001 0.021 ± 0.001 0.021 ± 0.001
0.018 ± 0.000 0.018 ± 0.000 0.022 ± 0.001 0.020 ± 0.001
0.019 ± 0.001 0.019 ± 0.000 0.019 ± 0.000 0.020 ± 0.001
0.077 ± 0.001 0.076 ± 0.000 0.073 ± 0.001 0.074 ± 0.001
0.076 ± 0.001 0.077 ± 0.001 0.076 ± 0.001 0.075 ± 0.001
0.074 ± 0.000 0.074 ± 0.000 0.076 ± 0.000 0.076 ± 0.000
0.020 ± 0.001 0.020 ± 0.000 0.022 ± 0.002 0.021 ± 0.001 0.019 ± 0.001
0.018 ± 0.000 0.018 ± 0.000 0.020 ± 0.001 0.021 ± 0.001 0.021 ± 0.001
0.023 ± 0.004 0.026 ± 0.001 0.023 ± 0.002 0.021 ± 0.001 0.022 ± 0.001
0.076 ± 0.001 0.076 ± 0.001 0.076 ± 0.001 0.074 ± 0.001 0.074 ± 0.001
0.077 ± 0.001 0.075 ± 0.000 0.075 ± 0.000 0.074 ± 0.000 0.074 ± 0.000
0.075 ± 0.001 0.074 ± 0.000 0.075 ± 0.000 0.076 ± 0.000 0.077 ± 0.000
Each value represents the mean ± SEM (n = 3–4).
Table 6 IL-1a and TNF-a release in plasma following administration of L-penetratin or PenetraMax with or without insulin (1 IU/kg, ‘‘control’’) into rat nasal cavities. IL-1a (ng/mL)
0.5 mM 2.0 mM 0.5 mM 2.0 mM
L-penetratin L-penetratin
PenetraMax PenetraMax
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax
TNF-a (ng/mL)
Day 1
Day 7
Day 1
Day 30
Day 1
Day 7
Day 1
Day 30
0.020 ± 0.000 0.020 ± 0.000 0.019 ± 0.000 0.019 ± 0.000
0.019 ± 0.000 0.018 ± 0.000 0.019 ± 0.000 0.018 ± 0.000
0.030 ± 0.008 0.026 ± 0.003 0.028 ± 0.002 0.023 ± 0.000
0.019 ± 0.000 0.020 ± 0.000 0.019 ± 0.000 0.020 ± 0.001
0.081 ± 0.001 0.081 ± 0.001 0.080 ± 0.002 0.080 ± 0.001
0.079 ± 0.000 0.077 ± 0.001 0.078 ± 0.000 0.078 ± 0.001
0.076 ± 0.000 0.077 ± 0.001 0.076 ± 0.001 0.076 ± 0.002
0.072 ± 0.001 0.074 ± 0.001 0.072 ± 0.000 0.075 ± 0.004
0.031 ± 0.001 0.020 ± 0.000 0.027 ± 0.002 0.021 ± 0.001 0.019 ± 0.000
0.029 ± 0.009 0.021 ± 0.001 0.028 ± 0.007 0.021 ± 0.001 0.019 ± 0.005
0.025 ± 0.002 0.028 ± 0.004 0.029 ± 0.003 0.022 ± 0.001 0.025 ± 0.002
0.020 ± 0.000 0.024 ± 0.003 0.023 ± 0.002 0.020 ± 0.000 0.020 ± 0.001
0.081 ± 0.000 0.079 ± 0.001 0.080 ± 0.001 0.081 ± 0.001 0.079 ± 0.001
0.080 ± 0.002 0.080 ± 0.002 0.079 ± 0.002 0.079 ± 0.001 0.081 ± 0.002
0.073 ± 0.000 0.073 ± 0.000 0.074 ± 0.001 0.079 ± 0.005 0.074 ± 0.001
0.074 ± 0.002 0.075 ± 0.002 0.072 ± 0.001 0.076 ± 0.002 0.075 ± 0.001
Each value represents the mean ± SEM (n = 3–4).
we therefore conclude that the irritancy and immunogenic potential of our formulation in a preclinical subchronic setting is negligible and that L-penetratin and PenetraMax induce no systemic toxicity after 1, 7 and 30 days of consecutive nasal administrations compared to insulin alone.
These results are in line with data obtained from in vitro and in vivo studies of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs) peptides (which were obtained by chemically modifying the TP10 peptide sequence), and also with TAT and stearyl-(RxR)4 peptides, showing no influence on the host
Fig. 5. Photomicrographs of the nasal respiratory mucosal membranes of vertical sections through the anterior rat nasal cavity following nasal administration of L-penetratin or PenetraMax (2.0 mM) with or without insulin for 30 consecutive days. Sodium taurodeoxycholate (5 w/v%) was used as positive control. The bars indicate 50 lm. Tissues were decalcified and stained with haematoxylin and eosin after fixation in 20% formalin neutral buffer solution. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 7 Histopathological findings of rat nasal cavity treated with L-penetratin or PenetraMax with or without insulin (insulin 1 IU/kg) for 30 consecutive days, or treated with 5% w/v sodium taurodeoxycholate for 3 h. Histopathological findings
0.5 mM 2.0 mM 0.5 mM 2.0 mM
L-penetratin L-penetratin
PenetraMax PenetraMax
Insulin solution (1 IU/kg) +0.5 mM L-penetratin +2.0 mM L-penetratin +0.5 mM PenetraMax +2.0 mM PenetraMax 5 w/v% Sodium taurodeoxycholate
Respiratory epithelium necrosis
Olfactory epithelium necrosis
Dorsal meatus
Upper septum
Nasoturbinate
Maxillo turbinate
+++
+++
+++
++
++
+++
Abbreviations: , no change; ++, moderate change; +++, marked change. Each score represents triplicate experiments.
system. None of the peptides used for i.v. injections for nucleic acid delivery induced an increase in the cytokine levels in blood serum or caused pathological deviations in liver, kidney, lung, and spleen of the treated animals [35,36] suggesting that certain CPPs can be considered as non-toxic and non-immunogenic tools in drug delivery. 3.3. Histopathological irritation scores in the nasal membrane following 30 days of CPP administration The purpose of this study was to obtain histological information on disruptive reactions in the nasal mucosa of rats treated with L-penetratin and PenetraMax after long-term repeated administrations. The photomicrographs in Fig. 5 show no apparent histological damage of the mucosal membranes caused by Lpenetratin and PenetraMax (2 mM) with or without insulin in comparison to insulin treatment after 30 days. In contrast, severe damage of the nasal mucosal membrane was made by 3 h treatment with sodium taurodeoxycholate used as a positive control. The nasal membrane histopathological findings and irritation scores are summarised in Table 7. The histopathological signs used to assess the nasal respiratory mucosal membranes in vertical sections through the anterior rat nasal cavity following nasal administration of CPPs (0.5 mM and 2 mM) with or without coadministration of insulin for 180 min for 30 consecutive days show no observable change in membrane integrity in the different regions of nasal respiratory tissues such as respiratory and olfactory epithelium, dorsal meatus, upper nasal septum, nasoturbinate, and maxillo turbinate. These data show that administration of L-penetratin and PenetraMax safely enhanced insulin delivery without altering the integrity of the nasal membrane. In contrast, positive control taurodeoxycholate showed significant irritation scores of the nasal epithelium membrane, septum and turbinate (Table 7). Our results indicate that tryptophan, lysine and arginine backbone spacing can affect the CPP uptake efficiency while maintaining the low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view [37]. Finally, our study showed no cytotoxicity in the nasal mucosal membrane. The histopathological scoring confirmed the negligible LDH and biomarkers leakage picturing a healthy and undamaged nasal membrane after 30 days of multiple administrations. As a result, PenetraMax can be considered a non-toxic insulin absorption enhancer for nasal delivery showing significant BA.
4. Conclusions The study presented here was conducted to determine the effect of sequence modifications of L-penetratin on the nasal absorption-enhancing capacities. The novel CPP, PenetraMax, allows for optimal systemic delivery of insulin from the nasal cavity resulting in high BA for single and multiple long-term administrations. The BA reached almost 100% relatively to s.c. insulin administration without detectable epithelial membrane damage. In conclusion, we found that L-penetratin and PenetraMax are not toxic either systemically or locally at two selected concentrations in this subchronic rat study. The present results open the way to for rational engineering of CPPs as delivery tools and hold great promise for noninvasive delivery of biotherapeutic peptides without risk of local toxicity and inflammation/immunogenicity – the latter being crucial for peptidic based pharmaceuticals in preparation for clinical trials. Acknowledgments This study was supported in part by JSPS KAKENHI Grant Number 23590056 and the Research Programme for the Development of Innovative Technology of the Japan Science and Technology Agency. This work was also supported in part by The Nagai Foundation Tokyo. The authors are grateful to the undergraduate students of the Laboratory of Drug Delivery Systems in Kobe Gakuin University for their technical assistance. References [1] S.R. Schwarze, A. Ho, A. Vocero-Akbani, S.F. Dowdy, In vivo protein transduction: delivery of a biologically active protein into the mouse, Science 285 (1999) 1569–1572. [2] B. Gupta, T.S. Levchenko, V.P. Torchilin, Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides, Adv. Drug Deliv. Rev. 57 (2005) 637–651. [3] C. Foerg, H.P. Merkle, On the biomedical promise of cell penetrating peptides: limits versus prospects, J. Pharm. Sci. 97 (2008) 144–162. [4] El-S. Khafagy, M. Morishita, Oral biodrug delivery using cell-penetrating peptide, Adv. Drug Deliv. Rev. 64 (2012) 531–539. [5] A.T. Jones, E.J. Sayers, Cell entry of cell penetrating peptides: tales of tails wagging dogs, J. Control. Release 161 (2012) 582–591. [6] C. Foged, H.M. Nielsen, Cell-penetrating peptides for drug delivery across membrane barriers, Expert Opin. Drug Deliv. 5 (2008) 105–117. [7] L.N. Patel, J.L. Zaro, W.C. Shen, Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives, Pharm. Res. 24 (2007) 1977–1992. [8] J. Hawiger, Noninvasive intracellular delivery of functional peptides and proteins, Curr. Opin. Chem. Biol. 3 (1999) 89–94.
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