Designing of an orally active complement C3a agonist peptide with anti-analgesic and anti-amnesic activity

Peptides 22 (2001) 25–32

Designing of an orally active complement C3a agonist peptide with anti-analgesic and anti-amnesic activity Yunden Jinsmaa, Yasuuki Takenaka, Masaaki Yoshikawa* Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan Received 26 June 2000; accepted 19 October 2000

Abstract Complement C3a is an anti-opioid peptide, having anti-analgesic and anti-amnesic effects after intracerebroventricular administration. However, the peptide is inactive after oral administration. Orally active C3a agonist peptide was designed based on the structure of oryzatensin, a C3a agonist peptide derived from rice albumin. Tyr-Pro-Leu-Pro-Arg, a pentapeptide at the carboxyl terminus of oryzatensin is the minimally essential structure for exerting C3a activity. Due to the affinity for ␮-opioid receptor, both oryzatensin and Tyr-Pro-LeuPro-Arg showed analgesia after intracerebroventricular administration in mice which was blocked by the opioid antagonist naloxone. Tyr-Pro-Leu-Pro-Arg lost opioid activity by substitution the amino terminus tyrosine with other hydrophobic residues. Among the newly designed peptides, Trp-Pro-Leu-Pro-Arg was found to possess the strongest C3a activity. The peptide antagonized morphine-induced analgesia at 300 mg/kg after oral administration and also improved scopolamine- and ischemia-induced amnesia in a step-through passive avoidance test. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Analgesia; Anti-opioid; Complement C3a; Learning

1. Introduction Many bioactive peptides are released from food proteins by enzymatic digestion. Some of them are resistant to proteases, thereby being effective even after oral administration. Such findings imply the possibility that we can secure more potent and orally effective peptides by substitution amino acids in these parent peptides. Complement C3a is a 77 amino acid peptide found in serum. It is cleaved from complement C3 protein during activation of the complement system. C3a mediates a number of biologic effects such as chemotaxis of mast cells and eosinophils, degranulation of mast cells and basophils, smooth muscle contraction, induction of lysosomal release from leukocytes, and increasing vascular permeability [18]. Receptors for C3a are expressed in the cells of myeloid lineage, such as neutrophils, macrophages, eosinophils, basophils and mast cells [8,19,37]. The mouse C3a receptor was recently cloned and found to be a member of the rhodopsin family of G-protein* Corresponding author. Tel.: ⫹81-774-38-3725; fax: ⫹81-774-383774. E-mail address: [email protected] (M. Yoshikawa).

coupled receptors [1,6,15,30,35]. It has been demonstrated that C3a receptors are abundantly expressed throughout the body and the brain [1]. In the central nervous system (CNS), the receptor expression was found in neurons, especially in cortical and hippocampal neurons, and in glial cells [7]. We isolated C3a agonist peptides from various protein digests. Besides exhibiting ileum-contracting and phagocytosis-stimulating activities, these peptides show antiopioid activity in a guinea-pig ileum (GPI) assay [33,34]. Recently, we found that complement C3a had anti-analgesic and anti-amnesic activities after intracerebroventricular (i.c.v.) administration in mice [17]. However, neither C3a nor C3a agonists show the central effects after oral administration, probably because of their molecular size and susceptibility to peptidases. It is quite interesting to design C3a agonist peptides, which exert anti-analgesic and anti-amnesic activities even after oral administration. In this study, we designed an orally active C3a agonist peptide based on the structural modification of oryzatensin, a C3a agonist peptide which was originally isolated as an ileum-contracting peptide from trypsin digest of rice albumin [32,33].

0196-9781/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 0 ) 0 0 3 5 2 - 1

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2. Methods 2.1. Chemicals and reagents Trp-Pro-Leu-Pro-Arg, Tyr-Pro-Leu-Pro-Arg, Ile-ProLeu-Pro-Arg, Met-Pro-Leu-Pro-Arg, Phe-Pro-Leu-Pro-Arg, Leu-Pro-Leu-Pro-Arg, Ala-Pro-Leu-Pro-Arg, Val-Pro-LeuPro-Arg and oryzatensin were synthesized by t-Boc method. Human complement C3a was from Calbiochem-Novabiochem Co. (San Diego, CA.). Scopolamine was from Sigma Chemical Co. (ST. Louis, MO.). Morphine was from Takeda Chemical Industries Ltd. (Osaka, Japan). [3H]DAMGO and [125I]C3a were from Amersham (Little Chalfont, Buckinghamshire, England). RBL-2H3 cells transfected with C3a receptor cDNA was kindly provided by Dr. Robert S. Ames, Smith-Kline Beecham Pharmaceuticals, King of Prussia, PA. 2.2. Animals Male Hartley guinea pigs (250 –350 g) and male ddy mice (4 weeks old, 22–25 g) were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). Animals were housed in standard plastic cages in a temperature-controlled room (23°C) on a daily 12 h light: 12 h dark cycle with food and water freely available. Pharmacological tests and care of the animals were in accordance with standard ethical guidelines (NIH, 1985). 2.3. Radioreceptor assay C3a receptor assay was performed using RBL-2H3 cells transfected with C3a receptor cDNA essentially as described [35] with some modification. Transfected cells (1.0 ⫻ 106) were incubated in HBSS buffer, containing 0.5% BSA with 20 pM [125I]C3a (2000 Ci/mmol) in the presence or absence of unlabeled 20 nM C3a for 20 min at 37°C. Cocktail of inhibitors (30 ␮M bestatin, 2 ␮M lleucyl-l-leucine, 0.3 ␮M thiorphan, 10 ␮M captopril) was added to the assay mixture. Unbound radioactivity was removed by filtration through glass microfiber filters GF/C (Whatman Inter. Ltd., England) which had been soaked in 0.1% polyethylenimine for one hour to reduce nonspecific binding. The filter was washed 2 times with 3 ml of the cold buffer. Radioactivity remaining on the filter was determined by ␥-counter. The IC50 value is the concentration of peptides to inhibit the binding of labeled ligand by 50%. ␮-Opioid receptor assay was performed by measuring the binding of [3H]DAMGO to rat brain membrane. Rat brain was homogenized in 10 volumes (v/w) of 50 mM Tris-HCl buffer (pH 7.4) at 4°C with Brinkmann Polytron PT-10. The homogenate was centrifuged at 1,500 g for 5 min. The sediment was resuspended in the buffer and centrifuged again to remove the crude of nuclear fraction. The supernatant fluids were combined and centrifuged for 30 min at 50,000 g. The pellet was washed in 50 mM Tris-HCl buffer to obtain crude synaptosomal membrane. Finally the

pellet was resuspended in 10 volumes (v/w) of Tris-HCl buffer and stored at -80°C. Binding assay for ␮-opioid receptor was done in the presence of 1 nM [3H]DAMGO (47.8 Ci/mmol) and rat brain membrane according to the method of Pert and Snyder [27]. Cocktail of inhibitors as described above was added to the mixture. Unbound radioactivity was removed by filtration through glass microfiber filters GF/B (Whatman Inter. Ltd., England). The filter was suspended in 5 ml of PCS (Amersham-Searle; Phase Combining System) and radioactivity was counted by liquid scintillation counter. 2.4. GPI assay Ileum-contracting activity was measured in the longitudinal muscle strips of GPI as described previously [13]. Briefly, the strips were suspended in magnus chamber in Krebs-Ringer solution (120 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 11 mM glucose) at 37°C and bubbled with a mixture of 95% O2: 5% CO2. The ileal contraction induced by peptides was measured using an isometric transducer (TB-612T, Nihon Koden). The assay was accomplished in the presence of 30 ␮M bestatin. C3a agonist peptides cause a biphasic contraction, rapid and slow one’s; the former is blocked by pyrilamine, and the latter by tetrodotoxin and atropine [33]. The EC50 value was defined as the concentration of the peptides to induce a half-maximal contraction. To measure EC50 value for slow contraction of individual peptides precisely, 1 ␮M pyrilamine was added to the incubation mixture. Opioid activity of peptides was measured by inhibition of electrically stimulated (12–13 V, 0.5 ms, 0.1 Hz) contraction of longitudinal muscle strips of GPI. The ileum having no response to C3a agonist was chosen for the assay. IC50 value was defined as the concentration of peptides to inhibit contraction by 50%. 2.5. Analgesic and anti-analgesic test Analgesic effect of peptides was measured every 5 min after peptide administration by tail pinch test. Naloxone was injected 15 min before peptide administration. Anti-analgesic effect of C3a was measured on morphineinduced analgesia in mice by tail pinch test. Peptide solution in PBS was given by i.c.v. (5 min) or perorally (p.o.) (30 min) before morphine administration and the anti-analgesic effect was measured every 15 min for 1 h after intraperitoneal (i.p.) injection of morphine (5 mg/kg). 2.6. Passive avoidance test Amnesia was induced by i.p. administration of 0,1 mg/kg of scopolamine 30 min before training (scopolamine amnesia) or by stopping the blood flow of bilateral common carotid arteries for 10 min 3 days before training to induce

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Fig. 1. Homology among C3a and Oryzatensin. Identical residues are shown in boxes. Non-identical but similar residues are shown in dotted box.

ischemia on mouse (ischemic amnesia). The anti-amnesic effect of peptides was tested by a step-through passive avoidance experiment as described previously [17]. The peptide was given just after the training by i.c.v. administration and its effect on scopolamine- or ischemia-induced amnesia was measured 24 h after the training. For scopolamine-induced amnesia group statistical analysis was performed against the non-treated group. In the ischemia-induced amnesia, statistical analysis was performed against sham-operated group, in which mice were operated just as in the other experimental groups, without pinching the cervical artery. 2.7. Statistical analysis Statistical analysis for analgesic and anti-analgesic effects were performed by Bonferroni test. For passive avoidance experiment data were expressed as the median and interquartile ranges and statistically analyzed by means of a Mann-Whitney test.

3. Results Oryzatensin (Gly-Tyr-Pro-Met-Tyr-Pro-Leu-Pro-Arg) is a C3a agonist peptide isolated from trypsin digest of rice albumin [32,33]. The peptide has homology to the complement C3a (Fig. 1) and shows contracting activity of longitudinal muscle strips of GPI. Tyr-Pro-Leu-Pro-Arg, a carboxyl terminal pentapeptide of oryzatensin is the shortest peptide having C3a activity [33]. It has been reported that Leu1 in Leu-Gly-Leu-Ala-Arg (C3a (73–77)) can be substituted by an aromatic or large aliphatic amino acids with little or no loss in ileum contraction [36]. Ala4 in Leu-GlyLeu-Ala-Arg has been reported to be specific for C3a activity [36]. Previously, we also found that the contracting activity of Tyr-Gly-Leu-Ala-Arg was diminished by substitution of Gly2 or Ala4 with proline [33]. Interestingly, the peptide substituted at both Gly2 and Ala4 by proline (TyrPro-Leu-Pro-Arg) showed ileum contracting activity [33]. Furthermore, 2 proline residues in Tyr-Pro-Leu-Pro-Arg might make the peptide resistant for digestion in gastrointestinal tract. Such a favorable feature of Tyr-Pro-Leu-ProArg prompted us to investigate whether Tyr-Pro-Leu-ProArg could show anti-analgesic and anti-amnesic activities after oral administration.

Fig. 2. Analgesic activity of Tyr-Pro-Leu-Pro-Arg (YPLPR) in mice. Peptide was given at doses of 30 and 60 nmole/mouse intracerebroventricularly (A) or at a dose of 300 mg/kg orally (B). Analgesic effect was measured by tail pinch test. Physiological saline was given to control mice. Naloxone was injected subcutaneously (s.c.) 15 min before peptide administration. Statistical analysis was done by Bonferroni test. Each point represents the mean ⫾ s.e.m. ** P ⬍ 0.01 vs control, * P ⬍ 0.05 vs control (n ⫽ 6)

3.1. Central effect of Tyr-Pro-Leu-Pro-Arg Effect of Tyr-Pro-Leu-Pro-Arg on pain perception was measured after i.c.v. and p.o. administration in mouse. Unexpectedly, as shown in Fig. 2A, Tyr-Pro-Leu-Pro-Arg showed analgesic instead of anti-analgesic effect after i.c.v. administration, which was blocked by the opioid antagonist naloxone. The analgesic effect was also detected after oral administration at 300 mg/kg and lasted for 50 min after the administration (Fig. 2B). In fact, Tyr-Pro-Leu-Pro-Arg and its original peptide oryzatensin also showed affinity to the ␮-opioid receptor and inhibited electrically stimulated contraction of longitudinal muscle strips of GPI (Table 1). Opioid activities of Tyr-Pro-Leu-Pro-Arg are explained by the presence of tyrosine residue at the amino terminus which is essential for binding to the opioid receptor. Thus, Tyr-Pro-Leu-Pro-Arg has affinities for both C3a and ␮-opioid receptors.

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Table 1 Opioid activity of Oryzatensin and YPLPR IC50 (␮M)

Peptide

Oryzatensin GYPMYPLPR YPLPR

GPI assay

␮-receptor assay

32 ⬎300

39 488

Opioid activity of peptides was measured by inhibition of electrically stimulated contraction of longitudinal muscle strip in GPI assay. ␮-Opioid receptor assay was performed by measuring the binding of [3H]DAMGO to rat brain membrane.

Fig. 3. Ileum-contracting activity of Tyr-Pro-Leu-Pro-Arg (YPLPR) and Trp-Pro-Leu-Pro-Arg (WPLPR). Both peptides were added to ileal strip at final concentration 3 ␮M. The ileal strip was rested for 15 min after washing (w) until the next application.

3.2. Designing of novel C3a agonist peptides To eliminate opioid activity, tyrosine residue at the amino terminus of Tyr-Pro-Leu-Pro-Arg was replaced by various hydrophobic amino acid residues such as alanine, methionine, leucine, isoleucine, valine, phenylalanine and tryptophan. As expected, opioid activity disappeared by substitution of tyrosine (Table 2). As shown in Fig. 3, the ileum-contracting activity of Trp-Pro-Leu-Pro-Arg was stronger than Tyr-Pro-Leu-ProArg in GPI assay. At higher concentrations C3a agonist peptides cause biphasic contractions, rapid and slow ones; the former mediated by release of histamine and the latter by prostaglandin E2 and acethylcholine [33]. As depicted in Table 2, Trp-Pro-Leu-Pro-Arg, Ile-ProLeu-Pro-Arg and Leu-Pro-Leu-Pro-Arg showed stronger ileum-contracting activity with IC50 for slow contraction of 1.7 ␮M, 3.0 ␮M and 4.6 ␮M, respectively, than Tyr-ProLeu-Pro-Arg of which IC50 was 6.8 ␮M. Similar result was observed in binding assay with C3a receptor (Table 2). The IC50 values of Trp-Pro-Leu-Pro-Arg, Ile-Pro-Leu-Pro-Arg and Leu-Pro-Leu-Pro-Arg were 70 ␮M, 100 ␮M and 150 ␮M, respectively, while that of Tyr-Pro-Leu-Pro-Arg was 240 ␮M. Because of the absence of suitable C3a antagonist, crosstachyphylaxis with C3a was used to demonstrate that a peptide acts through the same C3a receptor [33]. Second

addition of the same C3a agonist peptide into the ileum without washout did not induce ileum contraction (tachyphylaxis). Neither rapid nor slow contractions of C3a (70 – 77) were observed after second addition of the peptide (Fig. 4). Trp-Pro-Leu-Pro-Arg also showed the same effect. Addition of the other C3a receptor agonist into the ileum also induce no contractions, because contractions are mediated

Table 2 Ileum-contracting activity and receptor affinity of YPLPR related peptides Peptide

YPLPR WPLPR IPLPR LPLPR MPLPR FPLPR VPLPR APLPR

Ileum-contracting activity of slow contraction in GPI assay EC50 (␮M)

IC50 (␮M) in receptor assay C3a

␮-opioid

6.8 1.7 3.0 4.6 8.6 18.0 17.0 24.0

240 70 100 150 300 ⬎1000 ⬎1000 ⬎1000

488 – – – – – – –

C3a receptor assay was performed using RBL-2H3 cells transfected with C3a receptor cDNA

Fig. 4. Cross-tachyphylaxis between C3a (70 –77) and Trp-Pro-Leu-ProArg (WPLPR). Applications of C3a (70 –77) and Trp-Pro-Leu-Pro-Arg at final concentration of 10 ␮M are shown by arrows (B). Ileum-contracting activity of Trp-Pro-Leu-Pro-Arg is shown in (A).

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Fig. 5. Antagonistic effect of Trp-Pro-Leu-Pro-Arg (WPLPR) on morphine induced antinociception in mice. Peptide was given 5 min before morphine (5 mg/kg s.c.) administration by i.c.v. (A, B) or 30 min before morphine by p.o. (C, D). Analgesic effect was measured by tail pinch test. Physiological saline was given to control mice. Statistical analysis was done by Bonferroni test. Each point represents the mean ⫾ s.e.m. *** P ⬍ 0.001 vs control, * P ⬍ 0.05 vs control (n ⫽ 6). AUC means area under curve.

by the same receptor (cross-tachyphylaxis). Trp-Pro-LeuPro-Arg showed cross-tachyphylaxis in the contraction with C3a (70 –77) (Fig. 4), suggesting that the contractions of Trp-Pro-Leu-Pro-Arg and C3a (70 –77) are mediated by the same C3a receptor. All of these findings strongly indicate that these peptides are specific C3a receptor agonists and Trp-Pro-Leu-Pro-Arg is the strongest C3a agonist designed on the basis of the structure of Tyr-Pro-Leu-Pro-Arg.

observed after oral administration of the peptide at a dose of 300 mg/kg. Less potent peptide Leu-Pro-Leu-Pro-Arg also exhibited anti-analgesic effect at 100 nmole/mouse after i.c.v. injection, however this effect was not significantly observed orally (data not shown). Thus, Trp-ProLeu-Pro-Arg, a new orally active C3a agonist peptide based on the structure of oryzatensin derived from rice albumin. 3.4. Anti-amnesic effect of Trp-Pro-Leu-Pro-Arg

3.3. Anti-analgesic activity of Trp-Pro-Leu-Pro-Arg The strongest C3a activity of Trp-Pro-Leu-Pro-Arg stimulated our investigation to test anti-analgesic effect. TrpPro-Leu-Pro-Arg inhibited morphine-induced analgesia after i.c.v. administration at a dose of 30 nmole/mouse (Fig. 5A). As shown in the Fig. 5B, similar effect was also

As described previously, complement C3a improves scopolamine- and ischemia-induced amnesia after i.c.v. administration in mouse [17]. Step-through type passive avoidance test was performed in mice in order to elucidate whether Trp-Pro-Leu-Pro-Arg shows the same effects. As illustrated in the Fig. 6, scopolamine induced amnesia at a dose

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Fig. 6. Effect of Trp-Pro-Leu-Pro-Arg (WPLPR) on scopolamine (Scop)induced amnesia. Learning experiment was done by passive avoidance test in the step-through cage. Peptide was given after the training to scopolamine-treated mice by i.c.v. (A) or by p.o. (B). Data are expressed as the median ⫾ quarter point by Mann-Whitney test. * P ⬍ 0.05 vs control, # P ⬍ 0.05 vs scop. group, ## P ⬍ 0.01 vs scop. group (n ⫽ 9)

of 0.1 mg/kg (i.p.) whereas Trp-Pro-Leu-Pro-Arg improved the amnesia after i.c.v. and p.o. administration (Fig. 6A, B). On the other hand, C3a was reported to improve ischemia-induced amnesia after i.c.v. administration. This type of amnesia was improved to the control level by administration of Trp-Pro-Leu-Pro-Arg at 300 mg/kg (p.o.) (Fig. 7).

4. Discussion In this study, we designed a novel C3a agonist peptide Trp-Pro-Leu-Pro-Arg through a modification of the struc-

Fig. 7. Effect of Trp-Pro-Leu-Pro-Arg (WPLPR) on ischemia-induced amnesia. Step-through type passive avoidance test was used for learning experiment. Peptide was given after the training to ischemia-operated mice by i.c.v. (A) or by p.o. (B) administration. Statistical analysis was performed by Mann-Whitney test. Data are expressed as the median ⫾ quarter point. *P ⬎ 0.05 vs sham, # P ⬍ 0.05 vs ischemia

ture of oryzatensin derived from rice albumin. We found that oryzatensin and its carboxyl terminus pentapeptide TyrPro-Leu-Pro-Arg had affinity for both C3a and opioid receptors. Besides fitting to the essential structure for complement C3a activity, Tyr-Pro-Leu-Pro-Arg contains TyrPro-X at the aminoterminus, which found in many opioid peptides. In ␤-casomorphin, hemorphin and endomorphins, X are aromatic residues [3,4,40]. However, X are aliphatic residues in gluten exorphins A and C, Tyr-MIF-1, and neocasomorphin [13,14,16,39]. Thus, it is quite reasonable that Tyr-Pro-Leu-Pro-Arg showed opioid activity. Tyrosine

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residue at the aminoterminus is the essential for opioid activity. Tyr-Pro-Leu-Pro-Arg lost opioid activity by substitution of amino terminus tyrosine with hydrophobic amino acid residues as expected. Among the newly designed peptides, Trp-Pro-Leu-Pro-Arg was evidenced to have the strongest affinity for C3a receptor. Thus, binding ability or biologic activities of peptides can be alterated by the substitution of amino acids or removing unwanted residues. Many endogenous bioactive peptides show central effects such as modulation of pain perception [24,29,40], learning [9,20], locomotion [10,11,26] and regulation of food intake [25,28,31] after central administration, although a few of them are effective only after peripheral injection. Of interest to note is that none of them exert central effect after oral administration. In sharp contrast, some of bioactive peptides, derived from food proteins, having effects on blood pressure [12,22] and opioid [38] systems are known to be effective after oral administration. However, C3a agonist peptides isolated from food protein digests are not active when orally given. Trp-Pro-Leu-Pro-Arg, a newly designed C3a agonist showed central effects such as antianalgesia and anti-amnesia after oral administration. TrpPro-Leu-Pro-Arg was demonstrated to be resistant to gastrointestinal proteases in vitro (data not shown). However, the dose to induce such effects is still high. Therefore, further modification of this peptide will need to be addressed in the hope of getting more derivatives showing central effects at lower doses. By using site-directed mutagenesis technique of genes, it is possible to introduce sequence of such novel bioactive peptides comprising all L-amino acids into food proteins. There are many reports on introduction of exogenous genes into plant such as potato [2], tomato [23], banana [5], tobacco [21] etc. to produce crops conductive to exerting regulatory function for animals, whereas there still remains other problem whether the wanted peptides are precisely released from ingested food proteins in vivo, depending on a cleavage site by gastrointestinal proteases. Nevertheless, it is expected that Trp-Pro-Leu-Pro-Arg might be released in vivo by action of pepsin and trypsin. By introducing this sequence into food proteins such as rice albumin and soybean proteins through site-directed mutagenesis technique of genes, we will be able to produce new crops which have beneficial effects. In conclusion, Trp-Pro-Leu-Pro-Arg is a newly designed C3a agonist peptide, which shows central effects such as anti-analgesia and anti-amnesia after oral administration.

Acknowledgments We thank Prof. Robert S. Ames, the Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA, USA for his kind providing cells transfected with human C3a receptor.

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