Hong-mei Liu

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

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.

Structure-activity study of endomorphin-2 analogs with C-terminal modifications by NMR spectroscopy and molecular modeling.

Endomorphin-2 (EM-2) is a putative endogenous mu-opioid receptor ligand. To get insight into the important role of C-terminal amide group of EM-2, we investigated herein a series of EM-2 analogs by substitution of the C-terminal amide group with -NHNH(2), -NHCH(3), -N(CH(3))(2), -OCH(3), -OCH(2)CH(3), -OC(CH(3))(3), and -CH(2)-OH. Their binding affinity and bioactivity were determined and compared. Despite similar (analogs 1, 4, and 7) or decreased (analogs 2, 3,5, and 6) mu affinity in binding assays, all analogs showed low guinea pig ileum (GPI) and mouse vas deferens (MVD) potencies compared to their parent peptide. Interestingly, as for analogs 2 and 3 (a single and double N-methylation of C-terminal amide), the potency order with the K(i) (mu) values was 2>3; for the C-terminal esterified analogs 4-6, the potency order with the K(i) (mu) values was 4>5>6. Thus, we concluded that the steric hindrance of C-terminus might play an important role in opioid receptor affinity. We further investigated the conformational properties of these analogs by 1D and 2D (1)H NMR spectroscopy and molecular modeling. Evaluating the ratios of cis- and trans-isomers, aromatic interactions, dihedral angles, and stereoscopic views of the most convergent conformers, we found that modifications at the C-terminal amide group of EM-2 affected these analog conformations markedly, therefore changed the opioid receptor affinity and in vitro bioactivity.

Cardiovascular effects of endomorphins in alloxan-induced diabetic rats.

Endomorphins, the endogenous, potent and selective mu-opioid receptor agonists, have been shown to decrease systemic arterial pressure (SAP) in rats. In the present study, responses to endomorphins were investigated in systemic vascular bed of alloxan-induced diabetic rats and in non-diabetic rats. Diabetes was induced by alloxan (220 mg/kg, i.p.) in male Wistar rats. At 4-5 weeks after the onset of diabetes, intravenous injections of endomorphins (1-30 nmol/kg) led to an increase of SAP and heart rate (HR) consistently and dosed-dependently. SAP increased 7.68+/-3.73, 11.19+/-4.55, 21.19+/-2.94 and 27.48+/-6.21% from the baseline at the 1, 3, 10 and 30 nmol/kg dose, respectively, of endomorphin 1 (n=4; p<0.05), and similar changes were observed in response to endomorphin 2. The hypertension could be antagonized markedly by i.p. 2 mg/kg of naloxone. On the other hand, bilateral vagotomy would attenuate the effects of hypertension and diminished the changes of HR in response to endomorphins. With diabetic rats, 6-10 weeks after the induction of diabetes, intravenous injections of endomorphins produced non-dose-related various changes in SAP, such as a single decrease, or a single increase, or biphasic changes characterized by an initial decrease followed by a secondary increase, or no change at all. These results suggest that diabetes may lead to the dysfunction of the cardiovascular system in response to endomorphins. Furthermore, the diabetic rats of 4-5 weeks after alloxan-treatment, the increase in SAP and HR caused by i.v. endomorphins might be explained by a changed effect of vagus and by a naloxone-sensitive mechanism.

Structure-activity study on the spatial arrangement of the third aromatic ring of endomorphins 1 and 2 using an atypical constrained C terminus.

The discovery of endomorphins (EMs) has opened the possibility of searching for new analgesics. However, the design of peptide analgesics has proven to be very difficult as a result of their conformational flexibility and a lack of clarity in structure–activity relationships (SAR). In EMs, the amino acid side chains exhibit considerable conformational flexibility, especially in the third aromatic ring, which is free to adopt a bioactive conformation. To resolve these problems, a series of C terminus EM analogues, [Xaa4‐R]EMs, modified through the substitution of Phe4 with nonaromatic residues and termination with benzyl groups, were designed to generate conformational constrains of the third aromatic ring by amide bond and torsion angles (ϕ4 and ψ4) of Xaa4. Introduction of (S)‐α‐methyl or (S)/(R)‐α‐carboxamide on the methylene unit of the benzyl group was designed to produce an atypical topographical constraint (ϕ5) of the third aromatic ring rotation. Interestingly, some EM derivatives, with elimination of the C‐terminal carboxamide group and significant changes in the address sequence (Phe4‐NH2), still exhibited higher μ‐opioid receptor (MOR) affinity than unmodified EMs. In contrast, some analogues with incorrectly constrained C termini displayed very low affinity and pharmacological activities. Thus, our results indicate that these EM analogues, with atypical constrained C termini, provide model compounds with potent MOR agonism. They also give evidence that the proper spatial orientation and conformational restriction of the third aromatic ring are crucial for the interaction of EMs with MOR.

C-terminal amide to alcohol conversion changes the cardiovascular effects of endomorphins in anesthetized rats

Endomorphin1-ol (Tyr-Pro-Trp-Phe-ol, EM1-ol) and endomorphin2-ol (Tyr-Pro-Phe-Phe-ol, EM2-ol), with C-terminal alcohol (-ol) containing, have been shown to exhibit higher affinity and lower intrinsic efficacy in vitro than endomorphins. In the present study, in order to investigate the alterations of systemic hemodynamic effects induced by C-terminal amide to alcohol conversion, responses to intravenous (i.v.) or intracerebroventricular (i.c.v.) injection of EM1-ol, EM2-ol and their parents were compared in the system arterial pressure (SAP) and heart rate (HR) of anesthetized rats. Both EM1-ol and EM2-ol induced dose-related decrease in SAP and HR when injected in doses of 3-100 nmol/kg, i.v. In terms of relative vasodepressor activity, it is interesting to note that EM2-ol was more potent than endomorphin2 [the dose of 25% decrease in SAP (DD25) = 6.01+/-3.19 and 13.99+/-1.56 nmol/kg, i.v., respectively] at a time when responses to EM1-ol were less potent than endomorphin1. Moreover, decreases in SAP in response to EM1-ol and EM2-ol were reduced by naloxone, atropine sulfate, L-NAME and bilateral vagotomy. It indicated that the vasodepressor responses were possibly mediated by a naloxone-sensitive, nitric oxide release, vagus-activated mechanism. It is noteworthy that i.c.v. injections of -ol derivatives produced dose-related decreases in SAP and HR, which were significantly less potent than endomorphins and were attenuated by naloxone and atropine sulfate. In summary, the results of the present study indicated that the C-terminal amide to alcohol conversion produced different effects on the vasodepressor activity of endomorphin1 and endomorphin2 and endowed EM2-ol distinctive hypotension characters in peripheral (i.v.) and central (i.c.v.) tissues. Moreover, these results provided indirect evidence that amidated C-terminus might play an important role in the regulation of the cardiovascular system.

Type 1 Diabetes attenuates the modulatory effects of endomorphins on mouse colonic motility

Our previous studies have shown that endomorphins (EMs), endogenous ligands for mu-opioid receptor, display a significant potentiation effect on mouse colonic motility. In the present study, to assess whether diabetes alters these modulatory effects of EMs on colonic motility, we investigated the effects of EMs in type 1 diabetic mouse colon in vitro. At 4 weeks after the onset of diabetes, carbachol-induced contractions in the longitudinal muscle of distal colon were significantly reduced compared to those of non-diabetic mice. Furthermore, the contractile effects induced by EMs in the longitudinal muscle of distal colon and in the circular muscle of proximal colon were also significantly reduced by type 1 diabetes. It is noteworthy that EMs-induced longitudinal muscle contractions were not significantly affected by atropine, Nomega-nitro-l-arginine methylester (l-NAME), phentolamine, propranolol, hexamethonium, methysergide and naltrindole. On the other hand, tetrodotoxin, indomethacin, naloxone, beta-funaltrexamine, naloxonazine and nor-binaltorphimine completely abolished these effects. These mechanisms responsible for EMs-induced modulatory effects in type 1 diabetes were in good agreement with those of non-diabetes, indicating similar mechanisms in both diabetes and non-diabetes. At 8 weeks after the onset of diabetes, both carbachol- and EMs-induced longitudinal muscle contractions were similar to those of short-time (4 weeks) diabetic mice. In summary, all the results indicated that type 1 diabetes significantly attenuated the modulatory effects of EMs on the mouse colonic motility, but the mechanisms responsible for these effects were not significantly altered.

In vitro and in vivo characterization of opioid activities of endomorphins analogs with novel constrained C-terminus: evidence for the important role of proper spatial disposition of the third aromatic ring.

In the present study, the C-terminus of endomorphin (Tyr(1)-Pro(2)-Trp/Phe(3)-Phe(4)-NH(2), EMs) analogs [Xaa(4)-R]EMs, modified by substitution of a non-aromatic residue for Phe(4) and ending up with -NH-benzyl, were designed to generate an atypical conformationally constrained peptide set. We investigated the effects of these analogs on the opioid receptors affinity, guinea pig ileum (GPI) and mouse vas deferens (MVD) activity, system arterial pressure (SAP), heart rate (HR), antinociception and colonic motility. Analogs 5 ([D-V(4)-Bzl]EM1) and 10 ([D-V(4)-Bzl]EM2), which exhibit appropriate spatial orientations of the third aromatic ring, were about 3-4 times more potent than their parents both in vivo and in vitro. However, a drastic loss of activity was found in analogs 2 ([A(4)-Bzl]EM1) and 7 ([A(4)-Bzl]EM2), which possess improper spatial orientations of the third aromatic ring. Interestingly, analog 7 or 3 ([G(4)-Bzl]EM1), when injected intravenously (i.v.), produced significantly different changes in SAP from their parents. Surprisingly, analog 4 displayed relatively higher vasodepressor activity but significantly less potent colonic contractile activity than analog 5. This may be elicited by the differences in the spatial disposition of the third aromatic ring, which were verified by molecular modeling. Our results indicate that the proper spatial disposition of the third aromatic ring plays an important role in the regulation of pharmacological activities of EMs.