Structure-based design of glycosylated oxytocin analogues with improved selectivity and antinociceptive activity

doi:10.21203/rs.3.rs-1995802/v1

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Structure-based design of glycosylated oxytocin analogues with improved selectivity and antinociceptive activity

https://doi.org/10.21203/rs.3.rs-1995802/v1

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Pain, both acute and chronic, is often treated with opioids despite severe negative side effects, such as physical dependence, respiratory depression and overdose. In the United States the misuse of opioid analgesics has given rise to the opioid crisis or opioid epidemic. As the frequency of overdoses increases, the need for alternative, non-addictive analgesics has become increasingly urgent. Oxytocin, a pituitary hormone, has shown robust evidence for analgesia and shows promise for treatment and prevention of opioid use disorder. Despite decades of research, clinical implementation is hindered by the poor pharmacokinetic profile of the native hormone oxytocin, which is cyclized by a labile disulfide bond. We addressed this by replacing the disulfide bond with a more stable lactam; additionally, we have glycosylated the cyclic peptides to yield brain penetrant oxytocin analogues. These analogues show exquisite selectivity for the oxytocin receptor and potent in vivo antinociception in mice following peripheral administration, suggesting further study toward clinical applications for pain treatment.

Opioid analgesics are at the center of the current opioid crisis. However, they are legitimately prescribed for otherwise intractable cases of pain. In the United States, pain is the leading cause of distress and comorbidity due to its negative affect.¹ Opioid analgesics are the most effective and frequently prescribed treatments for moderate to severe pain.^2,3 They effectively relieve pain but also have serious side effects, including physical dependency and fatal overdose. Even when medically appropriate, prolonged opioid use can cause physical dependency, tolerance, withdrawal symptoms when discontinued, which can lead to opioid-induced hyperalgesia.⁴ The prevalence of prescription opioid misuse across the United States has increased more than threefold over the past two decades, leading to a public health crisis.³ Prescription opioids are now among the most misused drugs in the US.⁵

There are currently no opioid medications without the risk of misuse, abuse, and addiction,¹ and opioids are responsible for 80% of healthy lives lost from disability and premature death from substance use disorders (SUD).⁵ At least 50% of these deaths involve fentanyl, a m opioid receptor agonist (MOR) that is 50-100 times more potent than morphine whether alone or added to illicit drugs.⁵ Fentanyl has become an increasingly significant contributor to the opioid epidemic and is directly linked to opioid-induced respiratory depression (OIRD), the principal cause of death in most overdoses.⁶ Opioid overdose deaths are responsible for over 75% of the drug overdose deaths reported in 2021 through April,^7,8 indicating that the COVID-19 pandemic exacerbated these problems.

Oxytocin (OT) has been investigated for treating pain for three decades in both animal and human studies.^9-12 Most of these studies studies showed antinociceptive effects¹² following a variety of OT administration modes, including systemic, peripheral, intrathecal, intracerebroventicular, intranasal, i.v., or inhaled vapor administration of OT; fewer studies showed no significant antinociception.¹³ OT has also been evaluated for analgesia in humans,^9,12 with studies showing mixed results, at least partly due to design inconsistencies.^14-17

Unfortunately, OT remains a poor drug candidate.^10,18 Debate over its effective BBB penetration has persisted, although recent evidence indicates success.⁶ Yet its half-life in blood circulation is estimated at 5 minutes in humans and rats, and about 20 minutes in rat cerebrospinal fluid (CSF).¹⁰ OT is not completely selective for the oxytocin receptor (OTR), with similar binding affinities for V1a vasopressin receptor (V1a) and the Transient Receptor Potential Vanilloid type-1 (TRVP1),¹⁰ and moderate binding affinities for other arginine vasopressin receptor subtypes (V1b and V2).¹⁸ Until recently, there was little known about the active conformation of OTR until its crystal structure was determined by cryo-electron microscopy (cryo-EM),¹⁹ and within the past year, two crystal structures were published of OT bound to OTR via cryo-EM, giving insight into the OTR:OT activation mechanism.^20,21

Synthetic modifications to increase the peripheral bioavailability of OT or its active fragments are well studied, leading researchers to the discovery of carbetocin in 1974.²² Frič et al. modified OT to yield a long-acting uterotonic octapeptide analogue, simultaneously addressing aminopeptidase cleavage and disulfide bond breakage. The peripheral half-life of carbetocin increased from 5 minutes (OT)¹⁰ to 85-100 minutes.²³ Despite increased metabolic stability, carbetocin has about 10-fold lower affinity for OTR than OT has, and there have been no studies into its CNS effects.²³ To date, many synthetic strategies have been employed to improve OT’s pharmaceutical profile.^10,24,25

Design Strategies for Oxytocin Analogues. OT, a neurohormone conserved across species for 700 million years,^11,26 plays a major role in many fundamental physiological processes in both the central and peripheral nervous system.^11,18,26,27 Its primary role in behavior is “to facilitate stability in changing environments, rooted in its evolutionary origins as a system to promote survival.”²⁸ This biological significance led to sustained interest in its pharmaceutical application to a range of disorders,²⁷ including pain. Its extensive conservation suggests that at least some of its native structure is advantageous. We made minimal structural changes to address the pharmacological limitations of oxytocin: lack of receptor specificity and poor bioavailability. We aimed to optimize its druggability and elicit antinociception in mouse models.

Specifically, we modified the native amino acid sequence and glycosylated the C-terminus of the peptide chain. Replacing the labile disulfide bond with a more robust amide bond produced highly potent glycopeptides, which are exquisitely selective for the OT receptor, brain penetrant, and antinociceptive after intravenous administration in mice. The amide bond was also used to vary the size of the resultant macrocyclic ring. Our synthetic modifications drew on established design strategies in peptide drug design, such as macrocyclization and glycosylation. Macrocyclization is used for increasing stability towards proteolysis^29,30 and providing an entropic advantage in receptor binding (as compared to more flexible linear peptides).³¹ Glycosylation slows the metabolism of peptide drugs, increasing their bioavailability, and promotes BBB penetration.^32–38 It is hypothesized that glycosylation increases receptor selectivity because of “membrane hopping” due to “biousian behavior,” and that the carbohydrate acts as a shield to protect the peptide from enzymatic cleavage.^32,33,38,39

Receptor Potencies in CHO Cells. All novel oxytocin derivatives were tested for their potency and selectivity at the Oxytocin Receptor (OTR) and closely related Arginine-Vasopressin 1a/1b Receptors (AVP1a/b). They were evaluated using an In Cell Western assay for ERK MAPK activation in CHO cells expressing human OTR/AVP. We found that oxytocin (OT) and vasopressin (VP) control peptides activated the cells with high potency, as expected (Table 1). The 8 OT derivatives showed a range of receptor potencies at the OTR, from the very high (8) to low (1–2) with ligands performing in between. Several compounds improved on the potency of OT, demonstrating that our modifications can enhance both pharmacodynamic and pharmacokinetic performance (3, 5, 6, and 8). Notably, all derivatives showed no activity at the AVP1a/1b receptors, suggesting that they may be selective in vivo and avoid blood pressure-related side effects caused by AVP activation (Table 1).

Table 1

In Vitro Molecular Pharmacology of Novel Oxytocin Derivatives. Bold amino acids indicate cyclization site. Dap = L-1,3-diaminopropionic acid, Aib = α-amino-isobutyric acid. NA = no activity. Potency values at the 3 receptors reported as the mean ± SEM of N = 3 independent experiments.
	Structure	MW / RT_HPLC	OTR EC₅₀ (nM)	AVP1a EC₅₀ (nM)	AVP1b EC₅₀ (nM)
OT	^C[Cys-Tyr-Ile-Gln-Asn-Cys]-Pro-Leu-Gly-NH₂	1007.19 / —	2.3 ± 1.7	–	–
VP	^C[Cys-Tyr-Phe-Gln-Asn-Cys]-Pro-Arg-Gly-NH₂	1008.2	–	32 ± 23	11 ± 8
1	^C[Asp-Tyr-Ile-Gln-Asn-Dap]-Pro-Leu-Gly-NH₂	986.61 / 9.82	3800 ± 3100	NA	NA
2	^C[Glu-Tyr-Ile-Gln-Asn-Dap]-Pro-Leu-Gly-NH₂	1178.71 / 9.29	7000 ± 3000	NA	NA
3	^C[Asp-Tyr-Ile-Gln-Asn-Dap]-Pro-Leu-Ser(Lact)-NH₂	1340.76 / 8.42	0.54 ± 0.48	NA	NA
4	^C[Asp-Tyr-Ile-Gln-Asn-Dap]-Pro-Leu-Gly-Ser(β-Lact)-NH₂	1398.69 / 9.50	160 ± 160	NA	NA
5	^C[Asp-Tyr-Ile-Gln-Asn-Dap]-Pro-Aib-Ser(β-Glc)-NH₂	1150.64 /8.84	0.28 ± 0.17	NA	NA
6	^C[Glu-Tyr-Ile-Gln-Asn-Dap]-Pro-Leu-Ser(β-Glc)-NH₂	1197.72 / 9.66	0.73 ± 0.49	NA	NA
7	^C[Glu-Tyr-Ile-Gln-Asn-Dap]-Pro-Aib-Ser(β-Glc)-NH₂	1164.67 / 8.50	220 ± 110	NA	NA
8	^C[Glu-Tyr-Ile-Gln-Asn-Dap]-Pro-Aib-Gly-Ser(β-Glc)-NH₂	1221.67 / 8.74	0.06 ± 0.01	NA	NA

Based on their activity in cells, we next selected oxytocin derivatives to evaluate their antinociceptive ability in vivo. We chose 1 as a comparison since it is not glycosylated and only contains the stabilized amide bond, and selected 8 as the most potent glycoside (38-fold more potent than OT). All drugs were delivered by the intravenous route to mice. When we tested antinociceptive activity by the tail flick test, we found robust and efficacious pain relief for both 1 and 8, with 8 slightly higher than 1. Vehicle and oxytocin control produced no effect, while morphine produced robust antinociception, validating the assay. These results strongly suggest that our stabilized oxytocin derivatives can penetrate the brain to produce pain relief.

Conformational Studies of Oxytocin Analogues by NMR. Following the cell-based efficacy data from in vitro assays, we selected compound 8 for extensive NMR characterization. Importantly, researchers in the past have found the 20-membered ring of OT to be essential for its binding and activity.^40,41 To our knowledge, only one other 21-membered analogue has produced OTR agonism, and this alkene-containing derivative required much higher dosage than our analogue does to elicit effects.⁴² The results from analogue 8, suggest the 20-membered ring motif is not essential, since 8 has greater specificity for and activity at OTR despite its 21-membered ring. We obtained NMR spectra in solution corresponding to data in the prior aqueous NMR studies of oxytocin.^43–45 High-field (¹H and ¹³C 800 MHz and 200 MHz, respectively) 1D and 2D (¹H-¹³C-HSQC, ¹H-¹³C-HMBC, ¹H-¹H-COSY, 1H-1H NOESY, 1H-1H TOCSY, and ¹H-¹⁵N HSQC) NMR studies were used to unambiguously confirm the structure of the compound and obtain sequence specific resonance assignments. Water suppression was achieved with excitation sculpting in the pulse sequence. Sequential NOEs (nuclear Overhauser effects) between the HG/HG’ of Glu¹ and HG in Dap⁶ confirm the cyclization between those amino acids (Fig. 2). Sequential HN-HA NOEs were not significantly different in strength as compared to the sequential HN-HN NOEs. Chemical shifts for Ha, Ca, and backbone carbonyl carbons were compared to Wishart’s Chemical-Shift Index (CSI) to predict secondary structure^46,47 Variable temperature ¹H NMR (VT NMR) data collected at five temperatures was used to quantify chemical shift changes of the amide protons (temperature coefficient, TC, DdNH/DT), which are used to predict the presence of intramolecular hydrogen bonding (IMHB).^48,49 TC calculations showed evidence of intramolecular hydrogen bonding between residues in the cyclic moiety, although that evidence is weakened by the CSI and NOESY analysis. Preliminary TC data in ionic (SDS) micelles provides much greater evidence for these interactions, although the extent of the contribution from the charge has yet to be determined (manuscript in preparation). Additional spectra, assignments, TC calculations, and CSI analysis can be found in the supplemental information.

Molecular Modeling Studies. Published studies based on molecular modeling, photoaffinity labeling, and site-directed mutagenesis indicate that the oxytocin macrocycle Cys¹-Tyr²-Ile³-Gln⁴-Asn⁵-Cys⁶ binds into the upper third of the oxytocin receptor pocket at the transmembrane helices TM3, TM4, and TM6. The linear tail Pro-Leu-Gly-amide is bound closer to the surface and interacts with TM2 and TM3.^50,51 Oxytocin-like biological activity (e.g. selectivity for OT vs AVP) is marked by the presence of two conserved intramolecular hydrogen bonds in the ligand backbone. This bonding system produces a Type I β-turn within the macrocyclic ring (see Supplemental Info for details).^52,53 In addition, the activity of oxytocin is also associated with more compact structure as indicated with radius of gyration (Rg) centered at 5Å and below.⁵³ In our studies all analogues were modeled using MOE^®54 by building the structures of the compounds guided by either crystal structure of deamino-oxytocin,⁵² oxytocin,²⁰ or by MOE^® based energy of each amino acid sequence. Each structure was subjected to charge correction, energy minimization and conformational search using the LowModeMD method available in MOE^®. Analysis of each ensemble for the presence of β-turn conformers, Rg and dE (i.e. strain energy of the conformation relative to the lowest energy conformation) across all the conformers showed that they all retained an accessible Type I β-turn. Descriptors of solubility, lipophilicity and total polar surface area were calculated for all analogues under evaluations. See supplementary methods for details of the calculations.

Stability Assessments. The stability of these compounds was assessed in vitro in artificial cerebrospinal fluid (aCSF) to verify the utility of our structural modifications (see Supplemental Information for data). Substitution of the disulfide cyclization with the amide linker shows a significant increase in tested compounds as compared to oxytocin. Interestingly, unlike most (glyco)peptides assessed in our lab, the half-lives of the glycosylated analogues was not statistically different than their un-glycosylated “partners,” which indicates the amide cyclization substitution is a greater contributing factor to stability in vitro. The candidates were also studied in vivo to assess their serum stability and BBB penetration. The in vivo results are also different than most comparisons of glycosylated/un-glycosylated peptides in our lab. In the case of the studied OT analogues, the glycosylated peptide was slightly less stable in vivo, degrading initially to the unglycosylated peptide. However, in every case the glycosylated “partner” showed greater CSF concentration than the corresponding un-glycosylated peptide, but this improvement was not always statistically significant. BBB penetration was probed by measuring CSF concentration using microdialysis and MS². The tested OT compounds showed a peak in concentration around 10-20 minutes, and the CSF levels were sufficiently high to suggest BBB penetration, consistent with the in vivo antinociception studies.

Discussion and Outlook. OT remains a poor drug candidate, however, 700 million years of evolutionary conservation across species and almost 100 years of clinical applications in humans support the merit of much of its structure. Following this logic and guidance from Sir Francis Bacon who taught, “Nature to be commanded must be obeyed,”⁵⁵ we made minimal synthetic modifications. OT is known for its short in vivo half-life, primarily due to its unstable disulfide bond. Replacement of the labile S–S bond with the more robust lactam (HN–C=O) increased the in vitro serum stability from 1.4 ± 0.6 min to 25.8 ± 0.9 min, and glycosylation increased the in vitro stability to 34.5 ± 0.9 min. The increased BBB penetration observed with the OT glycosides seems largely due to the increased stability of the lactam rings. (See SI for details.)

One of the most significant pharmacodynamic shortcomings of OT is its lack of receptor selectivity. It is well known that promiscuous binding can lead to unwanted and potentially dangerous side effects.^56,57 Since OT binds to a wide range of receptors, including those involved in physiological regulatory mechanisms we have no interest in modulating (e.g. V1a receptor binding has been linked to bradycardia and body temperature changes after peripheral OT administration)⁴¹, we aim to design analogues that are selective for OTR. Additional support for this aim comes from Brackley et al., who found that V1aR cross-activation from high doses of their OT agonist compounds compromised OIRD reversal in their study of OTR agonism for fentanyl-induced OIRD.⁶

NMR data of 8 in aqueous solution do not indicate a preference for any specific secondary structure. Increasing the intensity of the spectra produces signals that indicate conformational isomers, which is expected for peptides in solution and especially for those containing proline residues.⁵⁸ Glycopeptide 8 showed changes in TC (ΔδNH/ΔT) that indicate the potential for some structural preference, but this evidence is challenged by the CSI analysis for both proton and carbon spectra (see Supplemental Information). Conformational data of OT in aqueous solution has been published, and the receptor-bound conformation of OT•OTR was recently determined via cryo-EM.¹⁹ The crystal structure of OT bound to OTR has been recently determined via cryo-EM.^20,21 Further NMR studies in micelles (both charged and neutral) will be used and compared with previous literature examining OT in nonpolar solvents (e.g., CDCl₃, DMSO)⁴¹ and in charged micelles.⁵⁹

Several of our OT analogues showed increased potency as compared to OT, demonstrating that our modifications can improve both pharmacodynamic and pharmacokinetic performance. All of the compounds produced no activity at the AVP1a/1b receptors, suggesting that they may be selective in vivo and avoid side effects caused by AVP activation. Antinociception was evaluated using the 52°C tail flick test in analogue 1 and its glycosylated “partner” 8, which showed robust, efficacious pain relief. Intravenous delivery in mice required BBB penetration, and the antinociceptive effects suggest that penetration was successful. Serum stability studies in vivo support this belief from increased dialysate levels 10-20 minutes after administration. These results strongly suggest that our stabilized oxytocin derivatives can penetrate the brain to produce antinociceptive effects. The half-lives of several glycopeptides were measured both in vitro and in vivo and showed no statistically significant differences between the glycosylated and un-glycosylated peptides of the same sequence, indicating that the amide replacement of the labile disulfide bond is much more significant in stabilizing the compounds than the glycosylation state.

An important consideration of novel pain therapeutics is avoiding the negative side effects of opioids, including reinforcing behavior. OT has been investigated as a potential OUD treatment in animals. Exogenous OT administration has been shown to mitigate classical addiction metrics, including self-administration, withdrawal and tolerance.^14-17,60 More recently, clinical trials have lacked consistency and yielded mixed results,¹⁴ though strong evidence indicates that intranasal administration of OT leads to increased compliance with therapeutic interventions, addressing a well-established barrier to effective OUD/SUD treatment. OT was found to be a safe, effective attenuation for nonopioid respiratory depression in patients at increased risk for OIRD.^6,61 Cardiorespiratory depression caused by fentanyl in rats was dose-dependently reversed by OTR activation, both with OT and with a nonpeptide partial agonist in a recent study by Brackley et al.⁶

Preliminary data will guide our future work generating additional efficacious analogues by consideration of conformational analysis, cell-based potencies, and extent of BBB penetration to guide our design efforts. This data suggests that the 20-membered ring in OT is not required for its activity at OTR, contributing to the growing insights into binding and conformation, and will inform our future drug design efforts. We will modify the structure of 8 and then study its receptor binding affinity, antinociceptive activity, and serum stability. NMR studies (both solution and micellar conditions) will be used to provide additional insight into in silico modeling efforts. Ultimately, in vivo studies will assess the addictive potential of these compounds to confirm that their administration yields no reward that might lead to overuse and/or misuse analogous to that which is associated with opioids.

Despite decades of efforts, chronic pain treatment remains challenging to researchers and clinicians alike.⁶² Growing insight into pain perception mechanisms are aiding the search for effective treatments. However, prescription opioids remain the primary approach to pain treatment, and their use is irrefutably dangerous. It is imperative for researchers to discover alternative non-opioid analgesics to address the persistent problem of pain without exacerbating the opioid epidemic.

Synthesis of Oxytocin Analogues. Glycosylated amino acid residues (Fmoc-Ser[O-β-D-Glc(OAc)₄]-OH, Fmoc-Ser[O-β-D-Glc(OAc)₃-β-1,4-D-Gal(OAc)₄]-OH) were prepared using published procedures.⁶³ Compounds 1—8 were synthesized using standard procedures for solid phase synthesis^64,65 starting from Rink resin. The synthesis was performed using a Prelude® automated peptide synthesizer from Gyros-PTI. Glycosylated amino acids were manually loaded and coupled with Cl-HOBt and DIC in NMP. The resin was protected with Ac₂O and DIEA in DCM. Fmoc removal was accomplished using DBU/piperidine (2% of each) in DMF. The subsequent Fmoc-protected amino acids were automatically coupled with HBTU and NMP using the same protocol to achieve the desired sequence. Cyclization was achieved with the side-chain protected residues Fmoc-Asp(All) or Fmoc-Glut(All) and Fmoc Dap(Alloc) at the cyclization site.⁶⁶ After the full peptide was synthetized on the resin, the All and Alloc protecting groups were removed with Pd(PPh₃)₄ and PhSiH₃ in DCM and washed with DMF. The lactam ring was formed PyClock and DIEA in NMP.⁶⁷ The resin was washed with 0.5% sodium DEDTC in DMF. Acetyl groups were removed from the sugar moieties with hydrazine hydrate in NMP (50% H₂NNH₂•H₂O/NMP). The solution was drained and more 50% H₂NNH₂•H₂O/NMP was again added and gently mixed. This solution was drained, the resin was washed with DMF, and then dried in vacuo. An acidic cleavage cocktail (TFA/DCM/H₂O/Et₃SiH/anisole,9:1:0.2:0.3:0.5) was used to simultaneously remove the amino acid sidechain protecting groups and cleave the cyclic glycopeptide from the resin by gentle tumbling. The solution was collected from the resin and the volume reduced under argon before the crude glycopeptide was precipitated from solution by addition of cold Et₂O. This material was lyophilized prior to purification by reverse phase high performance liquid chromatography (RP-HPLC) using a semi-preparative (250X22 mm) C-18 column with a CH₃CN-H₂O gradient containing 0.1% TFA. Purity of the products (typically 5—7.0 mg) was confirmed using analytical RP-HPLC (250X4.0 mm) and MS/MS with Collision Induced Decomposition (CID). See supplementary methods for details of the syntheses.

NMR Methods. NMR data was collected on an inverse-probe 800 MHz Bruker Avance at 298 K. All samples were 600 uL. Samples for ¹H NMR, ¹³C NMR, COSY, HSQC, and HMBC spectra were prepared in buffered D₂O (50 mM NaHPO₄, 0.45% NaCl, 0.05% NaN₃, pH 6.5). Samples for NOESY, ROESY, and TOCSY spectra were prepared in buffered 90:10 H₂O:D₂O (50 mM NaOAc, 0.45% NaCl, 0.05% NaN₃, pH 5.2). All NMR data was referenced to (3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid sodium salt, TSP-d₄ and processed using MNova 14.2. NMR samples were roughly 3 mM.

Cell Lines and In Vitro Functional Assay. CHO-K1 cells from ATCC were used as the parental line for molecular pharmacology studies. Human 3X-HA N-tagged OTR, AVP1a, and AVP1b expression constructs with neomycin resistance cassettes were obtained from Genecopoeia. The cells were electroporated with 10 µg of each DNA, and receptor-expressing populations were selected with 500 µg/mL of G418. Receptor expression was validated by immunocytochemistry. Cells were maintained with 1:1 DMEM/F12 with 10% heat-inactivated FBS and 1X pencillin/streptomycin supplement, all from Gibco/ThermoFisher, and stable cell lines were further maintained with 500 µg/mL G418. Cells were grown at 37°C, 5% CO₂, in a humidified incubator.

Receptor activation was measured using an In Cell Western assay for ERK MAPK activation, performed precisely as in our previous work. ^68,69 All drugs, vehicle control, and oxytocin/vasopressin controls were incubated with the cells for 10 minutes before processing as reported. Each assay was performed three separate times, and the resulting data reported as the mean ± SEM of these 3 independent replicates. In each independent assay, 4 replicate wells per data point were used, which was averaged to produce one value for that replicate. The plates were read using an Azure Sapphire laser imager. The data was fit with 3 variable concentration/response curves using GraphPad Prism 9.3, which calculated the potency (EC₅₀) for each replicate.

Tail Flick Antinociceptive Studies in Mice. Male CD-1 mice between 5–8 weeks of age were used for all studies (Charles River). The mice were housed in a 12:12 hour light cycle, temperature and humidity controlled room in the University of Arizona AAALAC-accredited vivarium with standard chow and water available ad libitum. All studies were approved by the UA IACUC, and performed in accordance with the standards of the NIH Guide for the Care and Use of Laboratory Animals and the guidelines of the International Association for the Study of Pain.

Animals were randomly assigned in cage blocks to treatment group and brought up to the experiment room at least 1 hour prior to start for acclimation. The tail flick study (52°C, 10 sec cutoff) was performed as in our previous work. Baseline measurements were taken, then drug or Vehicle control injected at 10 mg/kg by the intravenous route. A measurement time course of 2 hours was followed for all animals. The experimenter was blinded to treatment group by the delivery of coded drug vials by a lab member.

Determination of in vitro Stability of Peptides in Rat Serum and Quantification of the Glycopeptides in Rat CSF Following iv Administration. Oxytocin is known for its short in vivo half-life due to its unstable disulfide bond. Replacement of the labile S–S bond with the more robust lactam (HN–C = O) increased the in vitro serum stability from 1.4 ± 0.6 min to 25.8 ± 0.9 min, and glycosylation increased the in vitro stability to 34.5 ± 0.9 min. The increased BBB penetration observed with the OT glycosides seems largely due to the increased stability of the lactam rings. (See SI for details.)

Acknowledgements: Support for these studies was provided by the Flynn Foundation and by Tech Launch Arizona as part of its Asset Development Program. The University of Arizona Comprehensive Pain and Addiction Center also provided infrastructure support for cell and mouse studies.

Author Contributions: HJG collected and analyzed NMR data and drafted the manuscript, PT created the cell lines, measured in vitro pharmacology, and performed mouse tail-flick data, LZ synthesized and purified peptides and glycopeptides, SS synthesized and purified glycopeptides, CL collected MS data and performed stability studies, FAH performed in silico structural calculations, VKK modified pulse sequences for NMR data acquisition and aided with data analysis, MLH directed microdialysis and MS² studies, JMS directed cell and mouse studies, RP designed drug candidates and provided overall project direction. All authors had editorial input into the manuscript.

Competing Interests: JMS, MH, and RP are co-founders of Teleport Pharmaceuticals, LLC, a company for the development and commercialization of glycosylated peptides for neurodegenerative disease and other neurological conditions. JMS is an equity holder in Botanical Results, LLC, a local cannabidiol company; no company products or interests were tested here. The authors have no other relevant conflicts of interest to declare.

Supporting Information: See attached document.

Data Availability: Data is collected in the Supporting Information.

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Yes there is potential Competing Interest. JMS, MH, and RP are co-founders of Teleport Pharmaceuticals, LLC, a company for the development and commercialization of glycosylated peptides for neurodegenerative disease and other neurological conditions. JMS is an equity holder in Botanical Results, LLC, a local cannabidiol company; no company products or interests were tested here. The authors have no other relevant conflicts of interest to declare.

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Structure-based design of glycosylated oxytocin analogues with improved selectivity and antinociceptive activity

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