Therapeutic Potential of Orally Administered Rubiscolin-6

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1		Yusuke Karasawa	--	1922	2023-06-21 12:56:43	\|
2	format	Jason Zhu	Meta information modification	1922	2023-06-25 04:23:12	\|

This entry is adapted from the peer-reviewed paper 10.3390/ijms24129959

Rubiscolins are naturally occurring opioid peptides derived from the enzymatic digestion of the ribulose bisphosphate carboxylase/oxygenase protein in spinach leaves. They are classified into two subtypes based on amino acid sequence, namely rubiscolin-5 and rubiscolin-6. In vitro studies have determined rubiscolins as G protein-biased delta-opioid receptor agonists, and in vivo studies have demonstrated that they exert several beneficial effects via the central nervous system. The most unique and attractive advantage of rubiscolin-6 over other oligopeptides is its oral availability. Therefore, it can be considered a promising candidate for the development of a novel and safe drug.

spinach rubiscolin peptide opioid

1. Introduction

Proteins play crucial roles in the human body, serving as enzymes, hormones, neurotransmitters, and bone and muscle components. Several proteins or peptides exert strong bioactivities even when they are present in small amounts; therefore, they may be used as therapeutic agents. Currently, advancements in biotechnology, biologics, and proteins, such as antibodies and hormones, have been developed and exploited in clinical practice ^[1]. From the perspective of drug development, the specificity of biologics to target sites is beneficial because it makes them safer to use than traditional non-specific drugs consisting of small molecules. However, there are a few disadvantages of biologics, such as in vivo instability, low permeability into the intestinal tract, and expensive manufacturing and quality control processes.

In recent years, naturally occurring or chemically synthesized peptides have been considered for novel drug development, including opioid peptides ^[2]^[3]. The approaches for developing a peptide drug are based on the expectation that peptides will be more effective, considering their higher specificity and lower toxicity than small molecules, which are broadly distributed in the body. Moreover, peptides have the advantages of both small molecule and biological drugs, including reduced running cost and increased specificity to the target site ^[4]. Nevertheless, the method of administration of peptide drugs is basically restricted to injection, similar to that of biologics. Generally, orally ingested proteins or oligopeptides are thought to be rapidly disassembled by digestive or proteolytic enzymes, and owing to their high polarity and molecular weights, they rarely penetrate the gastrointestinal mucosa while maintaining their original structure ^[5]. Therefore, peptide drugs become inactive when orally administered. Given that injection is not preferred by patients because of pain at the injection site, treatment with peptide drug injection could possibly result in treatment termination because of poor adherence. To address these issues, several approaches to enhance permeability, chemically modify the molecular structure, and exploit novel drug delivery systems have been explored ^[6]. However, the development of a peptide drug that is active after oral administration has not been achieved.

Rubiscolins, which are composed of penta- or hexapeptide (Tyr-Pro-Leu-Asp-Leu and Tyr-Pro-Leu-Asp-Leu-Phe), are spinach-derived naturally occurring oligopeptides produced by pepsin digestion of _D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the major enzyme involved in carbon dioxide fixation and photorespiration ^[7]. The pharmacological properties of rubiscolins as delta-opioid receptor (DOR) agonists have been elucidated ^[7]^[8]^[9]^[10]^[11]^[12]^[13]^[14]. In general, the DOR is responsible for regulating a wide range of physiological functions and is associated with disease symptoms, including persistent pain, emotional disorders, and neurological disorders ^[15]^[16]. To date, none of the DOR agonists have been approved for clinical use by regulatory bodies, unlike mu-opioid receptor (MOR) agonists, which have been widely utilized for a long time for relieving pain related to various pathologies ^[17]. However, MOR agonists are occasionally associated with severe adverse effects, such as sedation, respiratory depression, and tolerance. Therefore, novel and safe options are urgently needed to replace existing opioids.

There are two subtypes of rubiscolins, rubiscolin-6, which evokes the DOR agonistic effect equivalent to that evoked by SNC-80, an existing specific DOR agonist, and rubiscolin-5, which has been demonstrated to be relatively weaker than both SNC-80 and rubiscolin-6 ^[18]. The robust efficacy of rubiscolin-6 has been investigated in several in vivo studies using animal models mimicking a variety of disease states ^[7]^[8]^[11]^[12]^[13]^[14]. In addition, rubiscolins have an attractive property of oral availability. Furthermore, rubiscolin-6 is a G protein-biased specific DOR full agonist that is not activated in the β-arrestin-mediated pathway upon binding to any type of ORs, indicating that it could be safer than available opioids. Additionally, the limited effects on endogenous ligands or opioid analgesics activating MOR or kappa-OR (KOR) could enable its use with an MOR agonist when the total amount of MOR agonist used has to be reduced. Considering the adverse effects of available opioids and the circumstances of the “opioid crisis” ^[19], it is noteworthy that rubiscolin-6 could be a potentially novel and safer drug than MOR agonists as it evokes pain-relieving effects and various central effects via DOR ^[18].

2. Orally Administered Rubiscolins

Several effects of orally administered rubiscolins observed in animal studies are shown in Table 1.

Table 1. Summary of evidence of the effects of orally administered rubiscolins.

	Effect	ID	Animal	Effective Dose	Findings
1	Antinociceptive	Yang et al. ^[7]	Male ddY mice	Rubiscolin-5: 300 mg/kg, rubiscolin-6: 100 mg/kg	Both peptides exhibited an antinociceptive effect and were blocked by a selective δ opioid antagonist, naltrindole.
2	Memory-enhancing	Yang et al. ^[8]	Male ddY mice	Rubiscolin-6: 100 mg/kg	Rubiscolin-6 showed memory-enhancing effects; it was blocked by naltrindole. Notably, rubiscolin-5 failed to elicit this effect, even at 300 mg/kg.
3	Anxiolytic	Hirata et al. ^[11]	Male ddY mice	Rubiscolin-6: 100 mg/kg	Rubiscolin-6 exerted an anxiolytic effect and was blocked by naltrindole. The effect was mediated by σ1 and dopamine D1 receptors.
4	Orexigenic	Kaneko et al. ^[12]	Male ddY or C57BL/6 mice	Rubiscolin-6: 0.3 mg/kg	Rubiscolin-6 stimulated food intake and was blocked by naltrindole. The effect was mediated by cyclooxygenase-2 and lipocalin-type PGDS.
4	Orexigenic	Miyazaki et al. ^[14]	Male C57BL/6N mice	Rubiscolin-6: 1 mg/kg	Rubiscolin-6 stimulated food intake even in aged mice with ghrelin resistance and was blocked by naltrindole.
5	Anorexigenic	Kaneko et al. ^[13]	Male ddY or C57BL/6 mice	Rubiscolin-6: 0.3 mg/kg	Rubiscolin-6 suppressed food intake in mice on a high-fat diet and was blocked by naltrindole and HS024.

2.1. Antinociceptive Effect

Rubiscolins were initially expected to exhibit analgesic activity, similar to other opioid compounds. A previous study revealed that orally administered rubiscolins have antinociceptive effects ^[7]. While rubiscolin-6 exhibited the effect at a dose of 100 mg/kg, at least 300 mg/kg rubiscolin-5 was needed to observe the same effect, indicating that rubiscolin-6 was three times more potent than rubiscolin-5. Given that the effect was blocked by intracerebroventricularly (i.c.v.) injected naltrindole, a specific DOR antagonist, the effect was mediated via the DOR pathway in the central nervous system (CNS). To date, rubiscolins are the only naturally occurring oligopeptides that have been shown to exert analgesic effects after oral administration.

2.2. Memory-Enhancing Effect

Research using a step-through type passive avoidance test has demonstrated that rubiscolin-6 at a dose of 100 mg/kg enhances memory consolidation ^[8]. In contrast, rubiscolin-5 showed no significant effect by not only per os (p.o.) administration but also i.c.v. administration. The effect of rubiscolin-6 was blocked by i.c.v. naltrindole, suggesting that the effect involved the central DOR pathway.

2.3. Anxiolytic Effect

Rubiscolin-6 at a dose of 100 mg/kg showed anxiolytic activity in an elevated plus-maze test ^[11]. The activity was blocked by i.c.v. naltrindole and a dopamine D1 antagonist, SCH23390, but not by a dopamine D2 antagonist, raclopride. The effect was also blocked by the sigma 1 receptor antagonists BMY14802 and BD1047. These results suggest that the anxiolytic effect of rubiscolin-6 is mediated by the dopamine D1 and sigma1 receptors downstream of DOR.

2.4. Orexigenic and Anorexigenic Effects

A modulating effect of rubiscolin-6 on food intake has been reported ^[12]^[13]^[14]. First, rubiscolin-6 at doses of 0.3–1.0 mg/kg stimulated food intake, but this effect was blocked by i.c.v. naltrindole ^[12]. The orexigenic effect was also inhibited by intraperitoneally administered celecoxib, a cyclooxygenase (COX)-2 inhibitor, but not by intraperitoneally administered SC-560, a COX-1 inhibitor, suggesting that the effect was mediated by COX-2. In addition, leptomeningeal lipocalin-type prostaglandin D synthase (L-PGDS) in addition to the DP1 and Y1 receptors was involved in the pathway. Another study showed that rubiscolin-6 at a dose of 1 mg/kg stimulated food intake in both young and aged mice (2–27 months old), although the aged mice were resistant to ghrelin, an oligopeptide known to stimulate food intake in animals and humans via hypothalamic neuropeptide Y-mediated signaling ^[14]. Conversely, the anorexigenic effect of rubiscolin-6 at a dose of 0.3–1.0 mg/kg was observed in mice on a high-fat diet ^[13]. This effect was blocked by i.c.v. administered naltrindole as well as by i.c.v. administered HS024, an antagonist for the melanocortin 4 receptor. Taken together with the results of additional tests using wild-type and L-PGDS knock-out mice, the effect of rubiscolin-6 is mediated via pathways downstream of the central DOR.

3. In Vivo Oligopeptide Transportation

Evidence for the pharmacokinetics of rubiscolins remains scarce, despite their attractive property of oral availability. Generally, orally administered opioid peptides are inactive owing to rapid degradation into dipeptides or tripeptides by endogenous peptidases in the process of digestion before absorption. However, it is likely that some amount of rubiscolin-6 escapes the system. Stefanucci et al., investigated the in vitro intestinal stability and bioavailability of rubiscolin-6 using single layers of CaCo2 cells as a model of absorption in the small intestine ^[20]. They reported that approximately 10% of rubiscolin-6 was transepithelially transported. The mechanism by which rubiscolin-6 crosses the blood–brain barrier (BBB) is also unknown. Hence, two pathways should be elucidated to understand the pharmacokinetics of rubiscolin-6, which can activate DOR via oral administration: first, the mechanism by which rubiscolin-6 is absorbed while maintaining its original structure; second, the mechanism by which rubiscolin-6 can penetrate the BBB to evoke central effects.

Regarding the first mechanism, several previous studies have focused on transporters related to the absorption of oligopeptides in the intestinal tract, especially sodium-coupled oligopeptide transporters (SOPTs), which are of two subtypes, namely SOPT-1 and SOPT-2; these SOPTs are involved in oligopeptide transportation ^[21]. They are distinct from traditional peptide transporters known as peptide transporter-1 (PEPT-1) or PEPT-2 ^[22], which only transport dipeptides and tripeptides. SOPT-1 and SOPT-2 are expressed in the small intestinal epithelium, as well as in retinal pigment epithelial cells and neurons ^[21]^[23]^[24]. In the intestinal cells, they transport endogenous, exogenous, and synthetic oligopeptides, which consist of five or more amino acid residues, including Tat47–57, a fragment of the Tat protein encoded by human immunodeficiency virus-1, and DADLE ([_D-Ala2, _D-Leu5]-enkephalin), a selective DOR agonist ^[21]. Among the two subtypes of SOPT, SOPT-2 is considered to be involved in transporting an opioid peptide as a substrate.

Regarding the second mechanism, rubiscolin-6 has been considered to enter the CNS via BBB, although the mechanism of opioid peptide drug delivery to the CNS remains unclear ^[25]. Four types of peptide transporters, namely peptide transport system (PTS)-1, PTS-2, PTS-3, and PTS-4, play a role in transporting oligopeptides in brain microvascular endothelial cells. Among them, PTS-1 has been reported to be involved in the penetration of opioid peptides ^[26]^[27]. However, the detailed mechanism has not yet been elucidated. In a study on the intravenous injection method, endogenous opioid peptides, such as Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH₂), Met-enkephalin (Tyr-Gly-Gly-Phe-Met), Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu), and β-casomorphin (Tyr-Pro-Phe-Pro-Gly-Pro-Ile), were transported by PTS-1, whereas β-endorphin (31 amino acid residues), kyotorphin (Tyr-Arg), dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser), and morphiceptin (Tyr-Pro-Phe-Pro) were not. PTS-1 demonstrated the ability to transport opioid oligopeptides with five or more amino acid residues, but it did not transport dipeptide kyotorphin and dermorphin, which have a seven amino acid sequence, similar to that of β-casomorphin. Accordingly, it may not be easy to determine the transportation of peptides by considering only the number of amino acid residues. Although the detailed mechanism remains unknown, factors, such as size, structure, polarity, charge, and the protein binding rate of a peptide are considered to be related to the selectivity for transportation by a transport system ^[28].

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Yusuke Karasawa

Kanako Miyano

Masahiro Yamaguchi

Miki Nonaka

Keisuke Yamaguchi

Masako Iseki

Izumi Kawagoe

Yasuhito Uezono

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Update Date: 25 Jun 2023

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