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NEUROPHARMACOLOGY

Utilization of Combined Chemical Modifications to Enhance the Blood-Brain Barrier Permeability and Pharmacological Activity of Endomorphin-1

Department of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, Lanzhou, People's Republic of China; (H.-M.L., X.-F.L., J.-L.Y., C.-L.W., Y.Y., R.W.); and Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou, People's Republic of China (R.W.)

Received for publication April 18, 2006
Accepted June 26, 2006.

	Abstract

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The endogenous µ-opioid receptor agonist, endomorphin (EM)-1, cannot be delivered into the central nervous system (CNS) in sufficient quantity to elicit analgesia when given systemically because it is severely restricted by the blood-brain barrier (BBB). To improve the physicochemical characteristics of EM-1 and subsequently achieve greater BBB permeation, we synthesized a series of EM-1 analogs by combining successful chemical modifications, including N-terminal cationization, C-terminal chloro-halogenation, and unnatural amino acid (D-Ala, Sar, and D-Pro-Gly) substitutions in position 2. Presently, their binding and bioassay activity, lipophilicity, stability, and antinociceptive activity were determined and compared. Guanidino-addition and chloro-halogenation attenuated the µ-receptor affinity to some extent, but they demonstrated differences in the influence on stability. It appeared that guanidino-addition contributed to brain stability enhancement for the greater part, whereas chloro-halogenation together with amino acid substitutions in position 2 was of more importance for the stability enhancement in serum than in brain. Determination of the octanol/buffer coefficient revealed that chloro-halogenation did compromise the decreased lipophilicity caused by guanidino-addition, and introduction of D-Ala as well as D-Pro-Gly, but not Sar, in place of L-Pro², also increased the overall lipophilicity to some extent. Among the peptides tested, intracerebroventricular injection of guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 showed the strongest analgesia, being 3 times more potent than the parent peptide. We also found that in comparison with EM-1, the four D-Ala-containing tetrapeptides and the chloro-halogenated D-Pro-Gly-containing pentapeptide elicited significant and prolonged central-mediated analgesia upon subcutaneous administration, indicating that more peptides reached the CNS, eliciting greater analgesic effect.

The recently discovered endogenous opioid peptides, endomorphin (EM)-1 and EM-2, are two potent and highly selective µ-opioid receptor agonists (Zadina et al., 1997). They are thought to inhibit pain without some of the undesirable side effects of morphine. Particularly, the rewarding effect of EM-1 can be separated from analgesia (Wilson et al., 2000), and it is less prone to induce respiratory depression and cardiovascular effects at effective antinociceptive doses (Czapla et al., 2000). However, EM-1 still suffers from serious limitations including short duration of action, lack of oral activity, relative inability to cross the BBB into the CNS, and poor metabolic stability (Tömböly et al., 2002). Therefore, it is essential to enhance the CNS entry of EM-1 and its resistance to enzymatic degradation if it is to be considered for therapeutic use.

To date, numerous strategies have been developed for enhancing peptide delivery to the CNS (Temsamani et al., 2000). With various modifications that were successful, it may be a good idea to combine them. For this purpose, herein we have taken several approaches that mainly fall into three groups. The first group is cationization of EM-1 by guanidino-addition on the Tyr¹. There is accumulating evidence that cationization can improve pharmacological parameters of peptides such as membrane permeability (Kumagai et al., 1987) and proteolytic stability. Hau et al. (2002) showed in a study that cationization of EM-2 by guanidino-addition resulted in a significant increase in metabolic stability, BBB permeability, and analgesic profile. Almost at the same time, Ogawa et al. (2002) found that N-terminal amidination of several dermorphin tetrapeptide analogs led to compounds with strong and long-lasting activity after oral administration. The second group is chloro-halogenation at the para-position of Phe⁴. As one might expect, cationic group addition would possibly decrease the lipophilicity of peptides (Hau et al., 2002), whereas previous studies have shown that addition of halogens to enkephalin analogs may enhance overall lipophilicity of the compound resulting in greater BBB permeability (Weber et al., 1991, 1993; Abbruscato et al., 1996; Gentry et al., 1999). Thus, to ensure the necessary lipophilicity for passive transport of the cationized EM-1 analogs through the BBB, structural modification was also made at the C terminus. The third group is replacement of L-Pro in position 2 with unnatural amino acids (D-Ala and Sar) or dipeptide fragment (D-Pro-Gly). This is mainly for stability enhancement purpose. It was found that incorporation of D-amino acid in position 2 of the peptide sequence significantly increased their stability (Dooley et al., 1994). Recent studies indicated that [Sar²] EM-2 showed enhanced stability in vitro (Janecka et al., 2006). In early work, it was recognized that replacement of L-Pro with D-Pro in EM-2 significantly attenuated the µ affinity (Okada et al., 2000) but produced more potent and longer-lasting antinociception after ventricular administration (Shane et al., 1999). Here, we also introduced Gly as a spacer residue in position 3 with the hope of ensuring a -turn conformation of the peptide. Moreover, analogs without C-terminal modification were also synthesized to clarify whether chloro-halogenation would affect the stability of peptides besides lipophilicity.

In the present study, in an effort to make EM-1 overcome the problems of enzymatic degradation and inability to cross the BBB into the brain, thus being able to produce analgesia after systemic administration, we have synthesized a series of EM-1 analogs by combined chemical modifications, and determined their opioid receptor affinity and selectivity as well as lipophilicity and stability. We further characterized their antinociceptive activities by a tail-flick test after i.c.v. and s.c. administrations. These analogs could help identify the best possible drug candidate for clinical nociceptive pain management.

	Materials and Methods

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Chemicals. Radioligands [³H]H-Tyr-D-Ala-Gly-MePhe-Gly-ol and [³H]H-Tyr-c[D-Pen-Gly-Phe-D-Pen]-OH were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Commercial N-Boc/Cbz-protected amino acids were obtained from GL Biochem (Shanghai) Ltd. (China). 1H-pyrazole-1-carboxamidine hydrochloride was purchased from Aldrich Chemical Co. (Milwaukee, WI). Naloxone hydrochloride and naloxone methiodide were purchased from Sigma-Aldrich (St. Louis, MO).

Peptide Synthesis. EM-1 and its analogs (Table 1) were obtained by combining the step-by-step elongation of the peptide chain with segment-coupling peptide synthesis strategy. Synthesis of all of the peptides was conducted by conventional solution methods with N-Boc, N-Cbz, and O-Bzl protection and N,N'-dicyclohexylcarbodiimide/N-hydroxysuccinimide coupling. The first step in the synthesis of all of the chloro-halogenated analogs involved the preparation of the amide of Boc-p-Cl-Phe-OH that was easily achieved by N,N'-dicyclohexylcarbodiimide/N-hydroxysuccinimide coupling with ammonia. After N-terminal dipeptides were obtained, guanylating modification was performed by removal of the Boc protection group of Boc-Tyr-(Bzl)-Xaa-OH (Xaa = D-Ala, Sar, L-Pro, D-Pro) followed by treatment with a mixture of 1-(bis-benzyloxycarbonylguanyl) pyrazole and diisopropylethylamine in dimethylformamide. Then, the N-terminal guanylated dipeptide and C-terminal fragment were coupled to give the protected tetra- or pentapeptides. The progress of the coupling reactions was monitored by thin-layer chromatography (TLC) and electrospray ionization-mass spectrometry (MS). Deprotection of Boc was performed using hydrochloric acid in ethyl acetate or trifluoroacetic acid cleavage, and for deprotection of the Cbz and O-Bzl group, catalytic hydrogenation over Pd/C was employed. It is important to note that strict catalytic hydrogenation time is required in case of the deprotection of chlorine at the same time. All of the final products were purified by column chromatography on silica gel.

View this table: [in this window] [in a new window]   TABLE 1 Sequences of EM-1 and its analogs; the structure of a guanidino group is shown below

Animals. For guinea pig ileum (GPI) assay, guinea-pigs (National Institute of the Biological Products, Gansu, People's Republic of China) of either sex weighing 300 to 350 g were used. Male Kunming mice (30-35 g; Lanzhou Pharmaceutical Factory, Lanzhou, People's Republic of China) were used for mouse vas deferens (MVD) assay and stability studies, whereas for the analgesia studies, male Kunming mice weighing 18 to 22 g were employed. Rats were obtained from the Animal Center of Medical College of Lanzhou University. They were housed in a temperature-controlled environment (22 ± 2°C) under standard 12-h light/dark conditions and received food and water ad libitum. All animals were cared for and experiments were carried out in accordance with the principles and guidelines of the American Council on Animal Care. All protocols were approved by the Ethics Committee of Lanzhou Medical College.

Radioligand Binding Assay. Membranes were prepared from Wistar rat brain (without cerebellum) according to the literature method (Simon et al., 1986). All binding experiments were performed in 50 mM Tris-HCl buffer, pH 7.4, at a final volume of 0.5 ml containing 300 to 500 µg/ml protein (protein concentration was determined by the method of Bradford, 1976). In competition experiments, the following conditions were used for incubations: [³H]H-Tyr-D-Ala-Gly-MePhe-Gly-ol (0.5 nM), 1 h; and [³H]H-Tyr-c[D-Pen-Gly-Phe-D-Pen]-OH (1 nM), 3 h. Incubations were started by the addition of membrane suspension in a rotating incubator at 25°C and terminated by rapid vacuum filtration through GF/C filters using cell harvester. The filters were washed thrice with 6 ml of ice-cold buffer and then dried for 1 h at 80°C. The radioactivity was measured by a Wallac Microbeta 1450 Trilux scintillation counter (GE Healthcare) after 12 h of incubation in the scintillation cocktail. The extent of nonspecific binding was determined in the presence of 10 µM naloxone. All experiments were carried out in duplicate assays and repeated at least three times. K_i values were calculated according to the equation of Cheng and Prusoff (1973).

In Vitro Bioactivity Assays. In vitro opioid activities of peptides were tested in the GPI and MVD bioassays as reported elsewhere (Shook et al., 1987). The GPI tissue and MVD tissue were mounted in a 10-ml bath that contained aerated (95% O₂, 5% CO₂) Krebs-Henseleit solution at 37 and 36°C, respectively. Twitch contractions were evoked by rectangular pulses with the following parameters: 0.1-Hz, 50-V, 0.5-ms pulse width for GPI assay; pairs (100-ms pulse distance) of rectangular impulses (1-ms pulse width, 9 V/cm, i.e., supramaximal intensity) were repeated by 10 s for MVD assays. Dose-response curves were constructed, and IC₅₀ values (concentration causing a 50% reduction of the electrically induced twitches) were calculated graphically. The values are arithmetic means of five to eight measurements. To measure whether µ-opioid receptor-mediated antagonism occurred in the MVD, naltrindole (a selective -receptor antagonist, 200 nM) was added to the tissue preparation, and after 5-min incubation, the test compound was added at the IC₅₀ dose value, and the percentage recovery (reversal rate) of electrically evoked contraction was then calculated.

Octanol/Buffer Distribution. Partition coefficients for peptides were expressed as the ratio of peptide found in the octanol phase to that found in the aqueous phase. Equal volumes of octanol and 0.05 M HEPES buffer in 0.1 M NaCl, pH 7.4, were mixed and allowed to equilibrate for 12 h. The layers were then separated and stored at 4°C. At testing, 50 µg of peptide was added to 500 µl of the HEPES buffer and mixed with 500 µl of octanol by vortexing for 2 min. The octanol/buffer solution was centrifuged in a Beckman microfuge (Beckman Coulter, Fullerton, CA) for 1 min at 4000 rpm. After separation into aqueous and octanol phases, peptide content of the aqueous phase was quantified by RP-HPLC. A portion of the octanol phase was lyophilized and reconstituted in methanol before RP-HPLC analysis. All octanol/buffer distribution studies were performed in triplicate. The octanol/buffer distribution coefficient (D) was calculated as the ratio of octanol layer to the buffer layer.

Metabolic Stability. Enzymatic degradation studies of EM-1 and its analogs were carried out using mouse brain homogenate and mouse serum. To obtain 100% mouse serum, adult Kunming male mice (30-35 g) were anesthetized (1 g/kg urethane intraperitoneally), and blood was collected from the carotid with a heparinized syringe. The blood was kept at 4°C overnight and then centrifuged for 20 min at 20,000g (4°C). The supernatant was separated and stored at -80°C. The 15% mouse brain homogenate was prepared as described previously (Gillespie et al., 1992). Protein content of the suspension was confirmed by the method of Bradford (1976). A final protein concentration of 2.3 mg/ml in 50 mM Tris buffer, pH 7.4, was used for all incubations.

The stability of peptides was determined by RP-HPLC analysis. Approximately 10 µl of a 10 mM peptide stock solution was added to 190 µl of the matrix. To resuspended 15% brain homogenate or serum were added peptides to a final concentration of 500 µM. Incubations were carried out at 37°C for 0, 60, 120, 180, 240, and 300 min in triplicate. For peptide that was found to have a half-life less than 60 min, additional incubations were performed by using time intervals of 5 and 15 min. Aliquots of 20 µl were withdrawn from the incubation mixtures, and enzyme activity was terminated by precipitating proteins with 90 µl of glacial acetonitrile, vortexing, and maintaining the sample on ice for a couple of minutes. Samples were then diluted with 90 µl of 0.5% acetic acid to prevent further enzymatic breakdown and centrifuged at 13,000g for 15 min. The supernatants were collected for analysis by RP-HPLC as described below. The rate constants of degradation (k) were obtained by least square linear regression analysis of logarithmic peptides peak areas [ln (A_t/A₀)] versus time courses, using a minimum of five points. Degradation half-lives (t_1/2) were calculated from the rate constants as ln2/k.

RP-HPLC Analysis. Samples from octanol/buffer distribution and stability studies were analyzed by RP-HPLC on a Water Delta Park C₁₈ column (3.9 x 150 mm; Waters, Milford, MA) and with the absorbance monitored at 280 nm. The solvents for analytical HPLC were as follows: A, 0.1% trifluoroacetic acid in water; and B, 0.1% trifluoroacetic acid in acetonitrile. The column was eluted at a flow rate of 0.6 ml/min with a linear gradient of A:B = 80:20 to A:B = 20:80 for 12 min and A:B = 20:80 to A:B = 80:20 for 3 min.

Assessment of Antinociception. Peptides were administered i.c.v. in a volume of 4 µl of sterile physical saline under ether anesthesia as described previously (Haley and McCormick, 1957). The anesthetic did not affect antinociceptive measurements up to 60 min later (data not shown). For s.c. injections, the compounds were dissolved in saline and injected in 30 mg/kg body weight doses. For the study involving the opioid antagonist, animals were pretreated with naloxone both centrally (10 nmol/mouse i.c.v.) and peripherally (2 mg/kg s.c.) before s.c. challenge with peptides (30 mg/kg s.c.). We also evaluated the antagonist effect of naloxone methiodide (10 mg/kg s.c.), which does not readily cross the BBB.

Antinociception was assessed using the 50°C warm water tail-flick test. Nociception was evoked by immersing the mouse's tail in hot water (50 ± 0.2°C) and measuring the latency to withdrawal. Before treatment, each mouse was tested, and the latency to tail-flick was recorded [control latency (CL)]. Mice not responding within 5 s were excluded from further testing; the tail-flick responses were measured at different times after i.c.v. or s.c. injection of drugs. The latency to tail-flick was defined as the test latency; a cut-off of 10 s was adopted; 0.9% saline was used as control. The antinociceptive response was expressed as percentage of maximal possible effect (%MPE), calculated by the following equation: %MPE = 100 x (test latency - CL)/(10 - CL). The ED₅₀ values and their 95% confidence limits were determined by using the graded dose-response procedure.

Statistical Analysis. The data are expressed as the mean with S.E.M. Comparisons of data were made with a one-way analysis variance followed by the Student's t test (comparisons between two groups) and Dunnett's test (for comparison of multiple groups with one saline control group). A level of P < 0.05 was considered significant.

	Results

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Peptide Synthesis. All of the purified peptides were characterized by electrospray ionization-MS, reverse-phase high-performance liquid chromatography (RP-HPLC), and TLC (Table 2). Retention factors were determined on silica gel 60 F₂₅₄-precoated glass plates. The solvent system was ethyl acetate/methanol/ammonia water (6:2:1). Purities were determined to be 95 to 99% by analytical RP-HPLC (data not shown).

Radioligand Binding and in Vitro Bioactivity Assays. The affinity and selectivity of EM-1 and all its analogs were evaluated by radioligand binding assay using rat brain membranes and by bioassays using GPI and MVD preparations. Their binding affinities for µ- and -opioid receptors are summarized in Table 3. In the radioligand binding assay, substitution by a single D-Ala residue gave [D-Ala²]EM-1 with a 1.9-fold increased µ-affinity and a 4.2-fold increased -affinity compared with EM-1, but it still retained the highest µ selectivity among all of the synthesized analogs. Its chloro-halogenated correlate, [D-Ala², p-Cl-Phe⁴]EM-1, showed a µ-opioid receptor affinity approximately 2-fold lower than EM-1 and a 3-fold higher -opioid receptor affinity. Guanidino-addition decreased the binding affinity to the µ-opioid receptor. Guanidino-[D-Ala², p-Cl-Phe⁴]EM-1, and guanidino-[D-Ala²]EM-1 displayed moderate µ-opioid receptor affinities, which were approximately 3.9- and 3-fold lower than that of EM-1, respectively. Guanidino-[Sar²]EM-1 possessed a µ-opioid receptor affinity characteristic close to that of guanidino-[D-Ala²]EM-1, whereas its halogenated form, guanidino-[Sar², p-Cl-Phe⁴]EM-1, exhibited a 21-fold lower µ-affinity than the parent. For guanidino-[p-Cl-Phe⁴]EM-1 and guanidino-EM-1 in which no substitution was made in position 2, comparable weak µ-receptor affinities were observed. Substitution of L-Pro² with D-Pro-Gly led to the only two pentapeptides (guanidino-[D-Pro², Gly³, p-Cl-Phe⁵]EM-1 and guanidino-[D-Pro², Gly³]EM-1) that both showed significant decrease in µ-receptor affinity but slight increase in -receptor affinity, thus resulting in a remarkable decrease in µ-receptor selectivity. In addition, all of the analogs displayed low -affinities, with K_i higher than 1000 nM.

We next examined the in vitro biological activities of the peptides by assessing their ability to inhibit electrically stimulated contractions of GPI and MVD preparations. The former tissues contain predominantly µ receptors, but also receptors, whereas the latter include predominantly receptors but contain µ and receptors, too. As shown in Table 3, in the GPI assay, [D-Ala², p-Cl-Phe⁴]EM-1 and [D-Ala²]EM-1 (IC₅₀ = 4.99 and 15.5 nM, respectively) were found to be more potent or similar to EM-1 (IC₅₀ = 11.4 nM), respectively. In agreement with predictions from the binding data, guanidino-[Sar², p-Cl-Phe⁴]EM-1, guanidino-[p-Cl-Phe⁴]EM-1, and guanidino-EM-1 showed low GPI potencies, whereas the only two pentapeptides were almost devoid of activity. It should be noted that guanidino-[Sar²]EM-1, with a K_i (µ) being only 3-fold lower than that of EM-1 in the radioligand binding assay, showed a 15-fold lower GPI potency. In the MVD assay, guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 and guanidino-[D-Ala²]EM-1 exhibited unexpectedly higher potencies than that based on the -opioid receptor affinity. To confirm receptor preferences, the MVD activity of EM-1 and all of the analogs was examined using a specific -opioid receptor antagonist, naltrindole. We found that the MVD activity of analogs as well as that of EM-1 was only partly inhibited by naltrindole, with the percentage recovery of electrically stimulated contractions being around 18 to 49%, suggesting that the inhibitory effects of these analogs were exerted mainly or partly on µ-opioid receptors that coexisted in the MVD tissues.

Octanol/Buffer Distribution. The relative lipophilicity of peptides as determined by octanol/buffer distribution is shown in Table 4. Comparisons of lipophilicity between EM-1 and guanidino-EM-1 revealed that N-terminal cationization alone drastically decreased the lipophilicity by approximately 19-fold, whereas for analogs containing D-Ala in the second position, the decrease was less significant. Both guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 and guanidino-[D-Ala²]EM-1 had only a 9-fold lower affinity for the octanol phase than their corresponding noncationized peptides ([D-Ala², p-Cl-Phe⁴]EM-1 and [D-Ala²]EM-1). All chloro-halogenated analogs were found to be more lipophilic than those related nonhalogenated analogs, and the increase of D values was in the range of 6- to 10-fold. Specially, [D-Ala², p-Cl-Phe⁴]EM-1, without N-terminal guanidino-addition, was the most lipophilic of all of the peptides tested, with a D value being approximately 10-fold greater than that of the parent peptide. Furthermore, guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 and guanidino-[D-Pro², Gly³, p-Cl-Phe⁵]EM-1 (D = 13.5 and 11.2, respectively) had almost the same lipophilicity compared with EM-1(D = 12.5). Comparison of the D values for guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 with that of [D-Ala²]EM-1 showed that guanidination accompanying halogenation only resulted in a slight decrease of lipophilicity. A similar trend was also observed for guanidino-[p-Cl-Phe⁴]EM-1 versus EM-1.

Metabolic Stability. The metabolic stability of EM-1 and its analogs was assessed in mouse brain homogenate and serum. Table 4 summarizes the half-lives determined for all of the test peptides in twice-washed 15% mouse brain membrane homogenate and 100% mouse serum. Within the brain homogenate, EM-1 disappeared rapidly, with half-life being 21.2 min. The replacement of L-Pro² in EM-1 with D-Ala² resulted in a significant enhancement of the stability, but the half-lives of [D-Ala², p-Cl-Phe⁴]EM-1 and [D-Ala²]EM-1 were much shorter compared with those cationized analogs (guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 and guanidino-[D-Ala²]EM-1). All of the cationic analogs were extremely stable in the brain homogenate, with half-lives exceeding 180 min, indicating that cationization did significantly increase their brain stability. In the serum, EM-1 also demonstrated a short half-life of 9.4 min. However, the four D-Ala-containing tetrapeptides, either with or without guanidino-addition, had the longest metabolic half-lives. They were only slightly degraded after incubating in the mouse serum for 300 min, being still present in around 90% of the initial amount. Although other analogs also showed a significant increase in half-lives, they were far less stable than the D-Ala-containing analogs. It should be noted that besides guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 and guanidino-[D-Ala²]EM-1, the two pentapeptides, guanidino-[D-Pro², Gly³, p-Cl-Phe⁵]EM-1 and guanidino-[D-Pro², Gly³]EM-1, were also highly stable in both biological media. Their stability in brain was increased by greater than 20-fold, whereas serum stability was increased by greater than 12-fold. Interestingly, we found that the half-lives of those halogenated analogs were significantly (P < 0.05) shorter than those of the nonhalogenated forms in the brain homogenate, whereas in the serum, the former were found to be more stable than the latter.

Antinociception. Antinociceptive activities of EM-1 and its analogs were studied in the tail-flick test in mice after i.c.v. and s.c. administration. The antinociception was initially measured at a fixed dose of 20 nmol/kg following i.c.v. administration, after which analogs with high potencies were selected for determination of ED₅₀ values. The ED₅₀ value for EM-1 after i.c.v. administration is 11.7 nmol/kg, which is in agreement with that of Sanchez-Blanquez et al. (1999). From all of the peptides tested, only [D-Ala², p-Cl-Phe⁴]EM-1, guanidino-[D-Ala², p-Cl-Phe⁴]EM-1, guanidino-[D-Ala²]EM-1, and guanidino-[p-Cl-Phe⁴]EM-1 showed almost equipotent or slightly better analgesia relative to the parent peptide. Intracerebroventricular administration of EM-1 and these four analogs at doses from 0.67 to 20 nmol/kg produced a dose- and time-dependent inhibition of the tail-flick response (Fig. 1, A-E). Judging from the ED₅₀ values (Table 4), guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 was the most potent analog in terms of potency of the analgesic response, with an ED₅₀ value 3 times lower than that of EM-1. Meanwhile, it was also the only analog that shifted its maximal analgesic effect from 5 to 10 min, whereas the antinociceptive effects of EM-1 (20 nmol/kg) and the other three analogs reached their peaks at 5 min. Furthermore, all of the four analogs showed an increased duration of action compared with the parent peptide (30-45 min in contrast to 20 min with EM-1).

View larger version (29K): [in this window] [in a new window]   Fig. 1. Time course of the antinociceptive effect of i.c.v. EM-1 (A) and its analogs [D-Ala2, p-Cl-Phe4]EM-1 (B), guanidino-[D-Ala2, p-Cl-Phe4]EM-1 (C), guanidino-[D-Ala2]EM-1 (D), and guanidino-[p-Cl-Phe4]EM-1 (E) in the mouse tail-flick test. Groups of mice were administered an i.c.v. injection of different doses of EM-1 or its analogs. The doses used are shown in the figure. The tail-flick responses were measured at 5, 10, 15, 20, 25, 30, and 45 min after the injection. Each value represents the mean with S.E.M. for 10 mice. Control mice treated with saline only did not show any significant change of nociceptive threshold, and these data are not shown.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Time course of the antinociceptive effect of s.c. [D-Ala2, p-Cl-Phe4]EM-1 and [D-Ala2]EM-1 (A), guanidino-[D-Ala2, p-Cl-Phe4]EM-1 and guanidino-[D-Ala2]EM-1 (B), and guanidino-[D-Pro2, Gly3, p-Cl-Phe5]EM-1 and EM-1 (C) at a dose of 30 mg/kg in the mouse tail-flick test. The tail-flick responses were measured at 5, 10, 15, 20, 25, 30, 35, 40, 45, 60, and 90 min after the injection. Each value represents the mean with S.E.M. for six to eight mice. Control mice treated with saline only did not show any significant change of nociceptive threshold, and these data are not shown.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Antagonism of antinociception elicited by 30 mg/kg s.c. [D-Ala2, p-Cl-Phe4]EM-1 (analog 1), [D-Ala2]EM-1 (analog 2), guanidino-[D-Ala2, p-Cl-Phe4]EM-1 (analog 3), guanidino-[D-Ala2]EM-1 (analog 4), and guanidino-[D-Pro2, Gly3, p-Cl-Phe5]EM-1 (analog 9) in the tail-flick assay by naloxone (10 nmol/mouse i.c.v., administered 5 min before drugs; 2 mg/kg s.c., administered 10 min before drugs) and by naloxone methiodide (10 mg/kg s.c., administered 10 min before drugs). Each value represents the mean with S.E.M. for five to seven mice. Vertical line, S.E.M. of the mean. *, P < 0.05, compared with the drug-injected control.

	Discussion

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EM-1 and EM-2 are two highly selective µ-opioid receptor agonists. However, with relatively rapid degradation and limited delivery to the CNS, it is unlikely that these neuropeptides could be used for the clinical treatment of pain. In regard to stability, a promising solution is to develop stable analogs. The unique approaches undertaken here relied upon utilizing N-terminal cationization by guanylating and unnatural amino acid substitutions in position 2. In addition to the necessity for enzymic stability, it is widely accepted that peptides must cross the BBB to reach the CNS in an amount sufficient to act on appropriate receptors to exert the desired pharmacological effects (Gentry et al., 1999; Habgood et al., 2000). Recently, EM-1 and EM-2 have been demonstrated to have a saturable efflux system at the BBB (Kastin et al., 2001; Somogyvari-Vigh et al., 2004), which removes the peptides from the CNS, thus limiting brain uptake. Therefore, peptides need to find alternative transcellular pathways to increase their entry into the CNS for their potent biological activity, for example, by passive diffusion. Considering that halogenation has been recently applied to improve the entry of therapeutic peptides into the brain, herein we incorporated a chlorine at the Phe⁴ residue of EM-1. Moreover, since the strategy of cationization of peptides was employed, we cannot rule out the possibility that other transport mechanisms might be involved, for instance, absorptive-mediated endocytosis (Hau et al., 2002). These various modifications by themselves or in combination would be expected to improve the stability and BBB permeability of these analogs, thereby resulting in enhanced entry into the CNS and elicit analgesia when given peripherally.

It was reported that N-terminal amidination of dermorphin tetrapeptides decreased their opioid receptor affinity (Marastoni et al., 1987). However, previous studies suggested that opioid peptides carrying a net positive charge would accumulate in the vicinity of the µ-opioid receptor and, therefore, would show µ-opioid receptor preference (Schiller et al., 1989). The study of the structure-activity relationship of EM-2 conducted by our research group showed that a neutral or weakly basic group at the C-terminal of EM-2 might result in an increased binding selectivity (Gao et al., 2005). Here, the present receptor binding results showed that guanidino-addition decreased the binding affinity to the µ-opioid receptor and subsequently decreased its µ-opioid receptor selectivity. In addition, C-terminal chloro-halogenation produced peptides that all displayed a decrease in both µ- and -receptor affinity relative to related analogs, an observation previously reported for similar analogs (Toth et al., 1990). It thus appeared that this subtle structural modification of the EM-1 derivatives in C terminus affected the receptor binding that may be related to a steric interference or the local hydrophobicity of the C-terminal substituents. Furthermore, it should be noted that there was a lack of direct relationship between the receptor binding affinity and biological activity. These properties may be explained in part by the fact that µ-receptor subtypes in the brain and peripheral tissues were different with regard to their structural requirements for ligand binding.

Passage of opioids through the BBB to gain access to the CNS by passive diffusion appeared to correlate with their lipid solubility. The more lipophilic a peptide is, the more likely it will interact with the cell membrane that, in fact, is the first step for the transcellular pathway (Habgood et al., 2000). Determination of the octanol/buffer coefficients of peptides suggested that the C-terminal halogenation indeed decreased the negative impact on lipophilicity caused by N-terminal cationization, consistent with our initial design concept. Meanwhile, by comparing the D values of all analogs, it was concluded that the introduction of D-Ala as well as D-Pro-Gly, but not Sar, in place of L-Pro², also contributed to the enhancement of the overall lipophilicity, which may be due to the differences in hydrophobicity of each amino acid.

On the other hand, entry into the brain is a complex phenomenon that depends on multiple factors. To improve bioavailability, the effects of lipophilicity on membrane permeability have to be balanced with first pass metabolism, for the peptidases within the blood and brain can rapidly degrade most peptides, including naturally occurring neuropeptides (Brownless and Williams, 1993; Witt et al., 2001). Therefore, the stability appears to be a deciding factor for the potency of peptide drugs after systemic administration. The present results revealed that although N-terminal guanidino-addition significantly increased the metabolic half-lives of peptides in both biological media (Table 5), the increase in serum stability seemed to mainly arise from the replacement of L-Pro² by unnatural amino acids in position 2. Interestingly, in the face of the influence of chloro-halogenation on the stability of peptides, it appeared that it had a disparate effect on the brain and serum stability. Overall, this provided evidence that C-terminal chloro-halogenation not only increased the lipophilicity but also protected this part of peptide from peptidases in the serum.

We assessed the antinociceptive activities of peptides in the tail-flick test after i.c.v. and s.c. injection. The comparatively high analgesic potency of [D-Ala², p-Cl-Phe⁴]EM-1 after i.c.v. and s.c. administration was well related with its close µ-receptor binding characteristic to the parent. Besides, it is possible that its extraordinarily high lipophilicity and increased stability in brain and serum also contributed to this increase of in vivo activity because these factors may lead to an improved distribution of peptide within the brain. However, although increased lipophilicity may facilitate drug uptake into the CNS, it also enhances efflux processes. Herein, the relatively rapid decline in analgesic effect of [D-Ala², p-Cl-Phe⁴]EM-1 after i.c.v. and s.c. administration could possibly be due to a quickly transport out of the CNS. As in the case of [D-Ala²]EM-1, although superior to EM-1 in stability and lipophilicity, it showed a significantly decreased analgesia after i.c.v. injection. Considering the high µ-opioid affinity and selectivity of [D-Ala²]EM-1, in case that its dose-response curve for the central analgesic effect showed a bell-shaped pattern, we then evaluated its analgesic activity at a lower dose (6.7 nmol/kg); still, it elicited a low analgesia (%MPE = 33.3). More interestingly, as shown in Fig. 2A, s.c. administration of [D-Ala²]EM-1 exhibited a biphasic effect, but the exact mechanism for these effects is not yet clear.

It is noteworthy that guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 and guanidino-[D-Ala²]EM-1 had only moderate µ-receptor affinity and potency and yet surprisingly high potency in the MVD assay; nonetheless, both of them elicited profound and prolonged analgesia after i.c.v. and s.c. administration. Likewise, guanidino-[D-Pro², Gly³, p-Cl-Phe⁵]EM-1 produced moderate (i.c.v.) (Table 5) and unexpected remarkable (s.c.) analgesia despite its much lower binding property and in vitro activity. Taken together, apparently these results could be interpreted to be in agreement with the increase in metabolic stability. Maybe the reduced metabolism led to higher plasma concentrations of these analogs after s.c. administration, thereby resulting in greater diffusion across the BBB.

The antinociceptive activities after peripheral administration of these analogs could be significantly reversed by naloxone (i.c.v. and s.c.) but not by naloxone methiodide (s.c.), which does not readily cross the BBB, indicating that the antinociception of these EM-1 analogs is mediated through a central mechanism. Since centrally mediated analgesia following peripheral administration was observed, the results reported here suggested that more peptides were available for the pharmacological activity that may be due to an enhanced BBB permeability and the improved stability provided by the physicochemical properties designed into these analogs.

In summary, the present study focused on the potential design of new analogs of EM-1 with an attempt to enhance its BBB permeability and pharmacological activity. Our results showed that modifications with combination of N-terminal guanidino-addition and C-terminal chloro-halogenation, together with D-Ala substitution in position 2 in EM-1, gave rise to guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 with improved physicochemical properties and remarkable activity in vivo. Particularly, the four D-Ala-containing tetrapeptides as well as the chloro-halogenated D-Pro-Gly-containing pentapeptide produced potent and prolonged analgesia upon s.c. administration through a central mechanism. Overall, the present study demonstrated that the modifications used in this research are successful for enhanced EM-1 delivery to the brain and may serve a role in the development of novel analogs of EM-1 with increased therapeutic potential.

	Footnotes

 
This work was supported by the National Natural Science Foundation of China (Grants 20525206, 20372028, and 20472026), by the Specialized Research Fund for the Doctoral Program in Higher Education Institutions, and by the Chang Jiang Program of the Ministry of Education of China.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.106484.

ABBREVIATIONS: CNS, central nervous system; BBB, blood-brain barrier; EM, endomorphin; TLC, thin-layer chromatography; MS, mass spectrometry; GPI, guinea pig ileum; MVD, mouse vas deferens; RP-HPLC, reversed-phase high-performance liquid chromatography; CL, control latency; %MPE, percentage of maximal possible effect; s.c., subcutaneous; i.c.v., intracerebroventricular

Address correspondence to: Dr. Rui Wang, Department of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, People's Republic of China. E-mail: wangrui{at}lzu.edu.cn

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