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Formulation and Delivery - Biomolecular

(T1230-10-54) Validation of the Potential of Oligoarginine-Linked Hyaluronic Acid as an Absorption Enhancer Through Animal Studies from Rodents to Primates

Tuesday, October 22, 2024
12:30 PM – 1:30 PM MT
Purpose: Protein drugs with poor membrane permeability are generally developed as injections that are administered by physicians. Injections provide reliable treatments; however, the physician management reduces quality of life, especially for outpatients. It is desirable to develop dosage forms of protein drugs that are self-managed by patients, such as the use of mucosal pathways. There are limited numbers of those commercial products, such as intranasal dosage forms of oxytocin, desmopressin, buserelin, nafarelin, calcitonin, and glucagon, and oral dosage forms of desmopressin and semaglutide, and intrapulmonary dosage forms of insulin. However, since membrane permeability of protein drugs reduces as their molecular weights increase, none of current technologies including sodium salcaprozate (SNAC) formulated in semaglutide tablets as an absorption enhancer enable to improve mucosal absorption of protein drugs whose molecular weights exceed approximately 6.0 kDa. There is an unmet need for absorption enhancers that improve mucosal absorption of protein drugs with large molecular weights. Absorption enhancers are chemically categorized into anionic middle-chain fatty acids and cationic chemical substances. Cell-penetrating peptides, which are positively charged oligopeptides that comprise mainly basic amino acids, such as arginine and lysine, are a typical one of the latter enhancer. Cell-penetrating peptides that interact electrostatically with negatively charged proteoglycans on the cell membrane are taken up into cells via endocytic pathways, such as macropinocytosis. We have been investigating the potential use of macromolecules where oligoarginines were chemically grafted as a novel absorption enhancer. We have developed a couple of macromolecules: biocompatible/nondegradable one using poly(N-vinylacetamide-co-acrylic acid) as a platform to which oligoarginines are anchored and biodegradable one using endogenous hyaluronic acid as the platform. A series of our mouse and rat studies (1–6) demonstrated that these macromolecules possessed universal absorption-enhancing abilities independent to molecular weights of protein drugs including antibodies under intranasal administration and that hyaluronic acid modified with L-tetraarginine via a diglycine spacer (G2R4-HA) was the most suitable candidate as an absorption enhancer whose ability and safety were well-balanced. Mechanism studies using gene-knocked down cells indicated that syndecan-4-induced macropinocytosis played a crucial role on acceleration of uptake of protein drugs into cells. The present study validated the potential of G2R4-HA as an absorption enhancer through the animal scale up.

Methods: Diglycine-L-tetraarginine was conjugated to the free form of hyaluronic acid via amide bonds to yield G2R4-HA (Mw: 199 kDa; the weight % of L-tetraarginine in G2R4-HA: 34%). Octreotide, bacitracin, ranibizumab, and trastuzumab were selected as a model protein drug. Drug solutions with or without G2R4-HA were intranasally administered to either mice or monkeys under anesthesia, and blood samples were collected at predetermined time intervals. Drug concentrations in the plasma were measured by LC/MS/MS or ELISA. Separately, western blot analysis was performed to estimate expression levels of four endocytosis-associated proteins (clathrin, caveolin-1, syndecan-4, and dynamin-1) on the nasal mucosa collected from mice and monkeys. Alveolar epithelial type-1 and type-2 cells, which are responsible for gas-exchange and secretion, respectively, in the lung, were primary cultured from the lung of rats. The cell studies using tetraglycine-L-octaarginine-linked hyaluronic acid (G4R8-HA), whose absorption-enhancing ability is identical to G2R4-HA, and FITC-IgG were performed under inhibitor (dyngo-4a)-present conditions with the aim of estimating the contribution of dynamin endocytosis.

Results: As shown in Fig. 1, G2R4-HA significantly enhanced intranasal absorption of octreotide and bacitracin with molecular weights on the order of 1 kDa. Similar enhancement was observed for trastuzumab, although pharmacokinetic analysis of trastuzumab with a long half-life was incomplete because a tiny body of the mouse restricted the number of blood sampling. Monkey experiments also demonstrated that G2R4-HA possessed the absorption-enhancing ability, irrespective of molecular weights of protein drugs. Ranibizumab with a relatively short half-life which was substituted to trastuzumab was not absorbed intranasally at all when administered alone. Its absorption clearly elevated when coadministered with G2R4-HA. As shown in Fig. 2, there was no significant difference in expression levels of endocytosis-associated proteins other than dynamin-1 between mice and monkeys. IgG uptake was not inhibited by dyngo-4a, indicating that dynamin was not involved in cellular uptake of protein drugs in the presence of G2R4-HA and G4R8-HA. The present animal studies and a series of our past studies supported that our oligoarginine-linked hyaluronic acid would enhance the nasal absorption mainly via syndecan-4-mediated macropinocytosis, irrespective of animal species.

Conclusion: The set of data supported that hyaluronic acid modified with oligoarginines would locally perform on the mucosal surface and enhance membrane permeation of drugs under its colocalization, irrespective of animal species difference.

References: 1. Kohei Miyata, Masami Ukawa, Kohta Mohri, Kozue Fujii, Mako Yamada, Sohei Tanishita, Satoshi Higashitarumi, Seiya Ishizaki, Hironori Kumagai, Kyohei Ochiai, Ken-ichiro Hiwatari, Kazufumi Tsubaki, Koichi Shigeno, Etsuo Tobita, Hideo Kobayashi, Shinji Sakuma, Biocompatible polymers modified with D-octaarginine as an absorption enhancer for nasal peptide delivery. Bioconjug. Chem., 29 (2018) 1748-1755.
2. Masami Ukawa, Sohei Tanishita, Haruya Yagi, Yuki Yoshida, Takumi Tomono, Koichi Shigeno, Etsuo Tobita, Tomofumi Uto, Masanori Baba, Shinji Sakuma, Biodegradable hyaluronic acid modified with tetraglycine-L-octaarginine as a safe adjuvant for mucosal vaccination, Mol. Pharmaceutics., 16 (2019) 1105-1118.
3. Takumi Tomono, Haruya Yagi, Masami Ukawa, Seiya Ishizaki, Takahiro Miwa, Mao Nonomura, Ryoji Igi, Hironori Kumagai, Kohei Miyata, Etsuo Tobita, Hideo Kobayashi, Shinji Sakuma, Nasal absorption enhancement of protein drugs independent to their chemical properties in the presence of hyaluronic acid modified with tetraglycine-L-octaarginine, Eur. J. Pharm. Biopharm., 154 (2020) 186-194.
4. Takumi Tomono, Haruya Yagi, Ryoji Igi, Akihiro Tabaru, Koichi Fujimoto, Kaho Enomoto, Masami Ukawa, Kohei Miyata, Koichi Shigeno, Shinji Sakuma, Mucosal absorption of antibody drugs enhanced by cell-penetrating peptides anchored to a platform of polysaccharides, Int. J. Pharm., 647 (2023) 123499.
5. Takumi Tomono, Haruya Yagi, Saki Kanemoto, Masami Ukawa, Kohei Miyata, Koichi Shigeno, Shinji Sakuma, Acquisition of absorption-enhancing abilities of cationic oligopeptides with short chain arginine residues through conjugation to hyaluronic acid, Int. J. Pharm., 616 (2022) 121519.
6. Haruya Yagi, Takumi Tomono, Yuma Handa, Natsuki Saito, Masami Ukawa, Kohei Miyata, Koichi Shigeno, Shinji Sakuma,Performance of cell-penetrating peptides anchored to polysaccharide platforms applied via various mucosal routes as an absorption enhancer, Mol. Pharmaceutics., 20 (2023) 303-313.

Acknowledgements: We thank Axcelead Drug Discovery Partners, Inc. (Kanagawa, Japan) where monkey experiments were performed under contract. Experimental protocols for mice were reviewed and approved by the Ethical Review Committee of Setsunan University. Experimental protocols for monkeys were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Shonan Health Innovation Park (Kanagawa, Japan), which possesses test facilities certified by The Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. The authors declare no competing financial interest.

Figure 1. Plasma concentration-time profiles of octreotide, bacitracin, ranibizumab, and trastuzumab administered intranasally with (●) or without (○) G2R4-HA in mice (A, mean ± S.D., n = 3) and monkeys (B, mean of 2 experiments, individual data at the top right).

Figure 2. (A) Expression levels of endocytosis-associated proteins on the nasal mucosa in mice (opened bars) and monkeys (left-hashed bars) (mean ± S.D., n = 4). (B) Cellular uptake of FITC-IgG in rat alveolar epithelial type-1 and type-2 cells in the presence (with or without dyngo-4a (an inhibitor of dynamin endocytosis) or absence of G4R8-HA (mean ± S.D., n = 4).