Our initial attempts at improving the CNS bioavailability of opioid peptides came from collaborations between the Chemistry and Pharmacology Departments at the University of Arizona led by Victor Hruby [62
]. In 1983 this group successfully synthesized and characterized a series of cyclic penicillamine containing enkephalin analogues (e.g., DPDPE) that had higher affinity and selectivity for DOP [63
]. Incorporation of an unnatural amino acid (D-penicillamine) and the cyclic constraint into the peptide enhanced both its stability and DOP selectivity. One hypothesis was that by increasing the lipophilicity of an already quite lipophilic DPDPE, BBB penetration could be increased, which was confirmed in an in vitro
BBB model that used bovine brain microvessel endothelial cells [64
Concurrently, we tested an alternative hypothesis, which, in retrospect, was naive and incorrect, whereby attachment of a glucose molecule to the modified enkephalin peptide would make the overall ligand a substrate for the Glut-1 transporter [65
]. In an effort to enhance CNS bioavailability, we synthesized a series of enkephalin analogues (–)
. It was predicted that CNS delivery of the enkephalin molecule across the BBB would be increased, and that we would observe antinociceptive activity following systemic administration. While the Glut-1 hypothesis eventually proved to be incorrect, the enkephalin glycosides did penetrate the BBB very effectively and produced potent and long-lasting antinociception in mice after intravenous (iv.) or intraperitoneal (ip.) injection [66
Cyclic disulfides related to DPDPE†.
Linear glycopeptide amides as μ receptor/δ receptor opioid agonists.
highlights some of the initial enkephalin glycopeptides that were synthesized [67
]. Glycoside placement proved critical for affinity as determined by radioligand binding studies, and efficacy, as determined by GPI and MVD assays. Glycosylation sites close to the N-terminus resulted in reduced affinity for both DOP and MOP. Extension of the modified enkephalin peptide at the C-terminus allowed for glycosylation while preserving opioid receptor affinity, with some compounds retaining moderate DOP selectivity while others had approximately equal affinity for DOP and MOP. Two glycopeptides, β-glucoside 3
and 4 ()
were tested in mice for their ability to produce CNS-mediated antinociception after systemic administration, and were compared with the unglycosylated peptide control 1
. Both glycopeptides 3
produced dose- and time-related antinociception following ip. injection into mice, whereas the unglycosylated control peptides did not produce any measurable effects. The primary obstacles for better characterization of these glycopeptides were the somewhat tedious synthesis of the cyclic disulfides and the relatively low potency of the compounds (~30 mg/kg A50
Larger quantities of a linear enkephalin glycoside based on Roques's so-called ‘delta-enkephalin’ or DTLET (YtGFLT, Tyr-d
-Thr-Gly-Phe-Leu-Thr) were produced. The parent unglycosylated peptides, 9
, retained high affinity for both DOP and MOP, relatively weak binding to KOP, and displayed a slight preference (~tenfold) for functional activity in the mouse MVD assay versus GPI assay [68
]. Both compounds were extremely potent (<0.1 nmol A50
values) following intra cerebroventricular (i.c.v.) administration but required very large doses iv. to produce any antinociception in the mouse 55°C tail-flick. The addition of a glucoside to a serine in the sixth position of the peptide resulted in retention of modest selectivity for DOP over MOP in functional MVD/ GPI tissue assays and in receptor-binding studies [69
]. Both the parent peptide (9)
and glycosylated analogue (12)
were extremely potent in the mouse tail-flick assay following intra cerebroventricular injection. However, glycopeptide 12
was significantly more potent following systemic routes of administration (iv., ip. and sc.). In situ
BBB studies in rats also indicated, despite the increase in MW and increased water solubility, that the glycopeptide penetrated the BBB more effectively than its unglycosylated peptide counterpart 9
]. When compared with morphine, glycopeptide 12
resulted in lower levels of physical dependence as indicated by naloxone-precipitated withdrawal.
In an effort to further explore the structure-activity of glycosylation [71
], a number of glycopeptides were synthesized to determine if the type of monosaccharide altered the transport characteristics and systemic potency of the lead peptide pharmacophore; if di- or tri-saccharides provided any additional benefit to pharmacokinetic and pharmacodynamic properties; and if bis- or tris-monosaccharides were viable alternative strategies for improving BBB transport and systemic potency [72
]. In addition, several other modifications were made to explore the geometry of the attachment point (D vs L amino acid) of the glycoside and to see if the more sterically hindered threonine attachment differed from serine in its effects on activity. It should be noted that we stayed with the linear enkephalin parent peptide as it had roughly equal affinity for DOP and MOP receptors. This was important in assessing potential effects of glycosylation on preferential biasing for DOP or MOP.
The initial Roques-based linear peptides tested had either an l-Ser or l-Thr added to the sixth position of the peptide. The geometry of the glucoside attachment did not impact functional potency/efficacy in the in vitro or in vivo assays (l-Ser vs d-Ser or l-Thr vs d-Thr). For the monosaccharides, the β-xylose was approximately two-times more potent than β-glucose or α-mannose following iv. administration. The three disaccharides (β-lactose, β-maltose and β-melibiose) were all more potent than the best monosaccharide tested, with the β-melibioside being the most potent of the three.
Based on these results, we synthesized additional glycopeptides that incorporated a trisaccharide (β-maltotriose) to see if additional size/ bulk of the carbohydrate moiety would lead to further increases in iv. potency. The experimen iv. potency. The experimental data indicated a modest fall off in binding affinity and potency in the in vitro and in vivo functional assays. We also extended the hexa-peptide to include one to two additional Ser or Thr attachment points with β-glucose (bis- and tris-monosaccharides) to more fully explore the structure–activity relationship. In all cases, the additional glycosyl bulk reduced potency following i.c.v. administration, and the one compound tested iv. was significantly less potent than the original glycopeptide (β-glucoside and l-serine attachment).
Additional studies confirmed aspects of the in vivo
]. Larger carbohydrates reduced octanol:saline partitioning (logD value), indicating greater water solubility (parent peptide < monosaccharide < disaccharide < trisccharide). Serum and brain stability of the glycopeptides also increased with these substitutions. In an in situ
model of BBB transport the disaccharide proved to be the most readily transported with the trisaccharide having a reduced RBr
value (though still superior to the unglycosylated control). We extended the in vivo
work by adding additional pain assays to assess efficacy. The disaccharide 17
produced potent antinociception in the formic acid, acetic acid and carrageenan assays following systemic administration (all of these pain assays have an inflammatory component).
Based on this modest library of glycopeptides, we chose the β-lactoside (17) as the lead molecule to pursue more advanced in vivo characterization. While not the most potent of the disaccha-rides, the compound was much easier and less costly to synthesize compared with melibiose. Glycopeptide 17 also had some additional desirable characteristics, including being highly water soluble (>50 mg/ml). Based on these findings, we advanced 17 into a more complete characterization of its antinociceptive efficacy and side-effect profile.
As expected, 17 produced full efficacy in the GTPγS assay with a modest selectivity for DOP over MOP. This profile was similar to what was observed in the functional MVD and GPI tissue assays. We confirmed this profile in vivo by pre-treating mice with various opioid antagonists. The general antagonist naloxone completely blocked the actions of 17 in the 55°C tail-flick assay in mice. In contrast, the peripherally selective antagonist naloxone methiodide did not alter the agonist actions of the compound. Subtype selective DOP (naltrindole) and MOP (β-FNA) antagonists each partially blocked the antinociceptive actions of 17 and, when combined, they completely eliminated the agonist actions. The KOP-selective antagonist nor-BNI was without effect.
As a lead molecule, 17
was also tested in several rat models of pain to determine how broad an antinociceptive spectrum the compound might have. The first assay used was a post-surgical incision model of the hind paw [73
]. Morphine and 17
both produced dose-related reversal of the tactile allodynia associated with the injury [D Giuvelis et al., Unpublished Data
]. On a μmol/kg basis, 17
was almost equal to morphine in terms of potency. Similar results were seen in a subchronic inflammatory pain model induced by complete Freund's adjuvant, although, in this case, 17
had greater potency than morphine, possibly due to enhanced DOP signaling under inflammatory conditions. Finally, 17
was compared with gabapentin in a rat spinal nerve ligation model of neuropathic pain. Glycopeptide 17
produced potent reversal of the tactile allodynia and thermal hyperalgesia post-ligation, whereas gabapentin only reversed tactile allodynia at the doses examined [D Giuvelis et al., Unpublished Data
]. Collectively, the data indicate that a mixed DOP/MOP agonist has a broad spectrum of antinociceptive effects in acute and chronic pain models, including ones that have inflammatory and/or neuropathic pain components.
One of the initial screens for side effect was to inject increasing doses of 17 or morphine and collect locomotor data in an automated open field assay. Morphine and other MOP agonists stimulate forward locomotion in imprinting control region mice. This effect becomes pronounced at near maximal and supramaximal antinociceptive doses. The mixed MOP/DOP agonist 17 produced an initial and transient decrease in forward locomotion that was replaced by a very mild stimulation of activity at later time points. We further investigated the initial inhibition of locomotor activity by pretreating mice with naloxone methiodide or nor-BNI. Both pretreatments attenuated the effects of 17 on locomotion and completely eliminated both effects when the two opioid antagonists were administered simultaneously. This indicated that stimulation of peripheral opioid receptors can produce a transient decrease in exploratory locomotor behavior and there may be a modest κ-agonist effect of the compound in the CNS that contributes to reduced stimulation of locomotor activity but does not contribute to the antinociceptive effects in the 55°C tail-flick assay.
Interestingly, one of the other gross observable differences between 17 and morphine is a lack of Straub tail and muscular rigidity with 17. We quantified this effect in dose–response curves versus antinociception with both morphine and the mixed agonist 17. The potential of morphine to produce both effects overlapped, whereas it took much higher doses of 17 to produce the muscular rigidity and Straub tail compared with its antinociceptive effects.
Based on the mixed DOP/MOP profile of 17, we were interested in evaluating the tolerance and physical dependence liability of the compound relative to morphine. For tolerance studies, we used a common paradigm involving twice-daily injections of the approximate A90 doses of the agonist (or vehicle) for 3 days. On the morning of day 4, full dose–response curves were constructed for each compound in the agonist- and vehicle-treated animals. Repeated doses of morphine resulted in an approximately 13-fold rightward shift in the A50 value indicating substantial development of antinociceptive tolerance. Equivalent doses of 17 (in terms of analgesia) resulted in a significantly reduced rightward shift (<fivefold). Morphine and glycopeptide 17 have similar durations of action and AUC values, making the comparisons more straightforward.
For assessment of physical dependence liability, we used both an acute (single high-dose administration of agonist) and chronic (twice-daily injections for 3 days) dependence protocol. In both cases, injection of the general opioid antagonist naloxone was used to precipitate withdrawal and several indices of withdrawal were recorded (vertical jumps and paw tremors, for example). The level of physical dependence/severity of withdrawal was consistently lower with the 17
exposure compared with equivalent exposures of morphine [74
]. The working hypothesis for explaining these results is that the antinociceptive effects of the mixed DOP/MOP compound synergize at the cellular or network level, whereas the processes that drive tolerance and/or physical dependence are additive or subadditive. A predominantly MOP-selective agonist, on the other hand, requires significant occupation of the MOP receptors at sites both responsible for antinociception and tolerance/dependence. Other explanations are possible, including the formation of heterodimers with the glycopeptide (17)
versus the small molecule (morphine) that lead to activation of different signaling pathways.
To further characterize the side-effect profiles of the glycopeptide, two commonly used assays for assessing MOP effects were used (gastrointestinal transit and respiratory depression). We had predicted that the DOP/MOP profile would have a reduced effect on these parameters compared with equivalent doses of morphine. This was not the case. Glycopeptide 17 also inhibited upper-gastrointestinal transit and suppressed respiratory response to elevations in CO2 on the minute ventilation parameter. The former may have been due to the apparent higher concentrations of glycopeptide 17 in the peripheral circulation compared with CNS, thus, overwhelming the MOP populations in the enteric nervous system. These values were estimated from the i.c.v. versus iv. potency ratios to produce antinociception for 17 versus morphine (more formal pharmacokinetic measures are currently being conducted). The respiratory depression observations indicate that a slight preference for DOP over MOP is not sufficient to differentiate from a MOP selective agonist.
Additional studies were conducted with 17
with respect to its abuse liability. As mentioned previously, the level of locomotor stimulation with 17
was markedly reduced compared with morphine. The stimulation of forward locomotion is generally interpreted as an activation of mesolimbic dopamine systems and an indicator of abuse liability. Our group has also conducted preliminary studies using conditioned place preference and iv. drug self administration in rodents. In the conditioned place preference studies, morphine produced a significant place preference whereas antinociceptive equivalent doses of 17
did not (similar to vehicle). In rat self-administration studies 17
maintained significantly lower numbers of infusions than the more MOP selective agonists morphine and fentanyl. In addition, the cumulative latency to delivery of the first three infusions of 17
(at the peak of the dose–effect curve) was significantly longer than morphine and fentanyl [Stevenson et al. Manuscript in Preparation
]. These experiments suggest that the reinforcing effects of 17
are less than the prototypical MOP agonists morphine and fentanyl. The reinforcing effects of 17
were also evaluated in rhesus monkeys [75
]. Under the conditions examined 17
did not support self-administration in rhesus across a series of doses/concentrations, although the results are more difficult to interpret due to species differences with respect to pharmacokinetics.