Analytical characterization and comparison of the blood–brain barrier permeability of eight opioid peptides
Introduction
Opioid-based pain treatment mainly relies on interaction of opioid analgesics with opioid receptors [48]. Three major opioid receptor types were demonstrated by Martin et al. [68]: μ-, δ- and κ-receptors (MOR, DOR and KOR, respectively). These receptor types are further divided in the following subtypes: μ1, μ2, μ3; δ1, δ2; κ1α, κ1β, κ2 and κ3, which are distributed throughout the nervous system, i.e. centrally and peripherally (see Table 1). Additionally, the opioid receptor-like (ORL) receptor was discovered, which is very similar to MOR, DOR and KOR, although it is not able to bind opioid ligands [72].
Among these receptor classes, the MOR is the main target of analgesics for pain treatment and shows the greatest clinical importance [70], [88]. Morphine, the classic opioid drug, interacts mainly with the MOR and is still often used in the treatment of severe pain. However, its clinical efficiency is limited due to a number of well-known side-effects, such as respiratory depression [93], [96]. Therefore, therapeutic peptides, exhibiting these undesirable side-effects to a lesser extent, were synthesized [21], [47], [49], [57], [65], [66], [84], [91], [101], [103]. As pain management occurs mainly within the central nervous system (CNS), the opioid peptides should be able to cross the blood–brain barrier (BBB) intact [42], [44]. The BBB separates blood from brain, preventing molecular transmembrane transport due to the presence of tight junctions. Thus, the entry of compounds into the brain is strictly regulated [1], [56]. Peptides are generally not transported through this barrier by passive transmembrane diffusion due to their limited lipophilicity. However, some peptides do cross the BBB by transmembrane diffusion as shown in numerous publications [7], [9], [36], [99]. Moreover, their BBB permeability can be enhanced by modification, e.g. by lipidization, glycosylation, halogenation or pegylation [15], [34], [46], [64], [71], [100]. Additionally, several specific influx transport systems are present at the BBB, transporting compounds by a receptor-, absorptive- or carrier-mediated transfer mechanism [15], [25], [33]. In contrast, efflux transporters are described at the BBB as well, so that peptides transported into the brain are pumped out again [12], [61], [90]. Opiate peptides are effluxed by peptide transport system-1 (PTS-1) [6], [10], [11].
The BBB transport properties of some opioid peptides have already been reported [2], [4], [5], [17], [23], [26], [29], [32], [40], [41], [53], [54], [55], [67], [74], [76], [79], [87]. However, inconsistent methods were used, influx or efflux aspects are not reported and some opioid peptides remain unstudied (see Supporting information). Moreover, only fragmentary data are available due to the use of different techniques; even within a certain method large variations are observed for the same peptide [53], [54]. Thus, the overall view is lacking.
Furthermore, peptide metabolism is an integral part of the BBB transport analysis since metabolites are less likely to exert a similar pharmacological effect. It was described that dermorphin produces inactive fragments, while the bioactivity of glycosylated dermorphin increased owing to the more active fragments [74]. This can be explained by the fact that the N-terminal tetrapeptide is the minimal fragment for analgesic activity [18]. The production of active fragments was also described for dynorphin A (1–8). This peptide interacts with the κ opioid receptor and is susceptible to several peptidases, leading to the following predominant fragments: [Leu5]-enkephalin and [Leu5]-enkephalin-Arg6. These two fragments have low affinity for the κ opioid receptor, but binding to μ and δ sites was observed [13]. Thus, the bioactivity of fragments, resulting from cleaving, should be investigated in metabolism studies [14], [20], [24], [37], [58], [63], [73], [80]. Although most of the opioid peptides undergo rapid enzymatic degradation by brain peptidases (such as aminopeptidases, carboxypeptidases and enkephalinases) [19], [27], [30], [35], [45], [50], [60], [62], [81], [85], EM-1 (endomorphin-1) and EM-2 (endomorphin-2) are assumed to be relatively more stable than other endogenous opioid peptides due to the presence of proline [94]. Appropriate modifications may create synthetic opioid peptide analogs with increased resistance to enzyme degradation [31], [65], [86], [88], [97], [98], [101]. Possible chemical modifications include the incorporation of d-amino acids [51], insertion of unnatural amino acids or cyclization [3]. However, metabolic data is not available for all peptides and sometimes different animal species are used (sheep, rat, and mouse), thus these data cannot be comparatively used due to possible species differences (see Supporting information). Moreover, metabolic stability was mostly investigated using non-standardized different tissue homogenates [28], [41], [65].
Taking into account these aspects (MOR selectivity, BBB transport and metabolism), the following eight opioid peptides were studied: EM-1, EM-2, DAMGO ([d-Ala2, MePhe4, Gly5-ol]-enkephalin), dermorphin, TAPP ([d-Ala2]-endomorphin-2), TAPS (Tyr-d-Arg-Phe-Sar), CTAP (d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2) and CTOP (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2) (see Table 2). These eight peptides were selected because they show a high affinity and selectivity for the MOR and may thus be applied in pain treatment (agonists) or in treatment of addiction (antagonists). Besides, some aspects of BBB transport and metabolism of these peptides is missing in literature. Moreover, TAPP was chosen because its BBB transport properties were not investigated before.
The first two opioid peptides, EM-1 and EM-2, are endogenous agonists, serving as lead peptides for the development of new analogs [102]. TAPP was developed as an analog of EM-2 by replacing the second amino acid residue [22]. Dermorphin is a biological peptide derived from frog skin. Due to derivatization of the N-terminal tetrapeptide fragment of dermorphin, which is the minimum sequence for opioid activity, the analog TAPS was synthesized [82]. Modification of the enkephalin sequence produced DAMGO [43]. In addition to these linear modified peptides, cyclic peptides were developed. These include the two antagonist opioid peptides, CTAP and CTOP, both structurally similar to somatostatin [38], [78]. Selectivity for the MOR was proven for all these peptides as given by their Ki (δ/μ) binding ratio, being 5447 for EM-2 [75], 3131 for EM-1 [75], 2530 for CTAP [16], 1724 for dermorphin [69] and only 559 for DAMGO showing also binding to KOR [102]. Thus, DAMGO is able to bind the three opioid receptor types, while TAPP, TAPS and CTOP only interact with the MOR type.
The BBB transport properties and metabolic stability of these opioid peptides were investigated in mouse. This structured investigation and comparison will provide more insight in opioid peptide characteristics.
Section snippets
Animals
Male Institute for Cancer Research, Caesarean Derived-1 (ICR-CD-1) mice (Harlan Laboratories, Venray, The Netherlands), weighing 25–30 g, were used according to the Ethical Committee principles of laboratory animal welfare as approved by our institute (Ghent University, Faculty of Veterinary Medicine, 2009-052). Mouse plasma was obtained from Harlan Laboratories (Venray, The Netherlands).
Peptides
The eight opioid peptides (EM-1, EM-2, CTAP, CTOP, TAPP, TAPS, DAMGO, and dermorphin) were synthesized by
Blood-to-brain transport measurement
In order to determine whether the peptides are able to cross the blood–brain barrier, multiple time regression analysis was performed.
Table 3 lists the calculated Kin and Vi values, which could be divided into three classes depending on the values obtained:
- (1)
very high influx into the brain for the peptides: dermorphin (Kin = 2.18 μl/(g × min));
- (2)
high influx constant rates: EM-1, EM-2 and TAPP (Kin = 1.06–1.14 μl/(g × min));
- (3)
very low to no influx: DAMGO, TAPS, CTOP and CTAP (Kin = −0.025 to +0.40 μl/(g × min)).
The
Discussion
The results obtained in the influx experiment (MTR) indicate that four opioid peptides were able to cross the BBB significantly: dermorphin, EM-1, EM-2 and TAPP. Within this set of peptides, dermorphin represented the highest influx rate. TAPP is the only synthetic peptide of the selected test set that is transported into the brain at a high influx rate. Dermorphin and TAPP demonstrate the same influx profile, reaching a plateau within ten minutes. The peptides entering the brain slowly (DAMGO,
Conclusion
This study examined the ability of eight opioid peptides (EM-1, EM-2, CTAP, CTOP, DAMGO, dermorphin, TAPP, TAPS) to cross the BBB and induce their CNS effects. Multiple time regression analysis revealed high apparent influx rates for four peptides (dermorphin, EM-1, EM-2 and TAPP), with the highest influx rate observed for dermorphin. Only little influx was shown for one MOR antagonist peptide (CTOP) and two MOR agonists (DAMGO and TAPS). CTAP, the other MOR antagonist opioid peptide tested,
Acknowledgments
This research is funded by a PhD grant (no. 71402) of “Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen)”. We are grateful to Olga Jedlickova and Nadia Lemeire for their technical assistance.
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