Polt’s undergraduate study of Metallurgy at Purdue as a National Merit Scholar was interrupted by service in the US Army, where he was trained to repair cryptographic equipment (COMSEC). Upon discharge after active duty, he studied Chemistry at IUPUI using his Vietnam Era GI Bill benefits. In addition to lecturing and laboratory teaching during 31 years at the University of Arizona, he has mentored a number of undergraduate, graduate (20 Ph.D.’s granted, 5 in progress) and post-doctoral students who have taken positions in academia, industry and the US government. Ph.D.s from the Polt Group have hailed from the US, Czech Republic*, China, India, Iran*, Ireland, Kenya*, South Korea, Germany, Mexico* and Sri Lanka. Four of these Ph.D. students (*) have gone on to become naturalized citizens. Recent undergraduates who worked in the Polt Lab have gone to prestigious graduate schools (e.g. Harvard U., MIT, Boston U., Univ. of Wisconsin, and Columbia U., as well as directly into industry (e.g. Pfizer, Astra-Zenica), or into government service (e.g. FDA, FBI). He has also mentored two daughters, one is a clinical audiologist and the other is a best-selling children’s science author. Pastimes include: Gardening, growing succulents, playing bridge, and training his various Neapolitan Mastiffs.
*(Naturalized US Citizens.)
The Big Idea– the Biousian Hypothesis Generally speaking, all endogenous peptide neurotransmitters found in the mammalian brain are highly amphipathic substances, and prefer a membrane environment to aqueous solution. This is logical since their target GPCRs are membrane-bound glycoproteins, and one can view “docking” of these ligands as a membrane event, and this concept (membrane compartment theory) has been thoroughly discussed by Robert Schwyzer.[i] The physical chemistry of amphipathic helices has been thoroughly explored by Jere Segrest.[ii] What we have found is that by judicious attachment of a carbohydrate or other water soluble moiety (phosphate, sulfonate), that we can extend the lifetimes of peptide “messages” in vivo, and also promote penetration of the BBB.[iii],[iv] (Figure 1)
Figure 1. BBB Penetration Is Independent of M.W. Over the past 20 years the Polt group has, in collaboration with a number of research groups, shown that glycosylation can produce brain-penetrant “peptide messages” with no loss of biological activity, and that this transport process is independent of molecular weight (at least up to ~3,500).
The endocytotic mechanism (transcytosis) of BBB penetration (c.f. Figure 2) by the glycopeptides is supported by several lines of research.[v] We postulate that the glycopeptides reversibly aggregate on a cell surface, thus promoting negative membrane curvature, which in turn promotes invagination and endocytosis. If the resulting vesicles drift across, or are transported across the endothelial cell, they can undergo exocytosis to deliver the glycopeptide to the luminal side of the endothelium. Since the glycopeptides reversibly associate with the membrane, the enkephalins can then dissociate from the luminal surface inside the brain. This mode of transport is superior to other modes since it is non-saturable, or only weakly saturable, does not require the presence of transporter enzymes at the endothelial cell surface, and does not expose the glycopeptide drugs to the cytosolic compartment of the cell where they would face a gauntlet of glycosidases, peptidases and oxidative enzymes. Several alternative modes of transport have been definitively ruled out by a previous study,[vi] and evidence strongly supports adsorptive endocytosis over fluid phase endocytosis.[vii] The opioid tetrapeptides TAPA, ADAB, and ADAMB have been independently postulated to penetrate the BBB by a charge-driven adsorptive endocytotic process.[viii]
Figure 2. Putative Mechanism for the BBB Transport Process.
a. Glycopeptides in serum. b. Adsorption. c. Aggregation. d. Invagination. e. Vesicle formation. f–g. Vesicular trafficking events. h. Membrane fusion. i–k. Exocytosis. l. Glycopeptides in cortex. This is a simplified analysis based on membrane biophysics, and neglects the presence of astrocytes, pericytes and microglia, which may play important roles in the overall transport process.
Enkephalins and “Short” Opioid Glycopeptides We began our studies in 1990 with the synthesis of glycosylated enkephalins for the treatment of pain. Replacement of the -CH2CH2OH moiety of DAMGO with glycosylated serine amide led to a series of highly µ-selective compounds, which showed fentanyl-levels of antinociceptive activity in mouse tail-flick studies.[ix] While these compounds were all quite potent in terms of analgesia, they showed classical opiate side effects typically associated with the µ-opioid receptor (MOR).
The mixed d/m-agonist Lactomorphin showed greatly reduced locomotor side-effects (hyperlocomotion), relative to morphine,[x],[xi] and it was this effect of the delta-agonist opioids[xii] that led us to exploit the use of “endorphin-like” address regions,[xiii],[xiv] (c.f. Figure 3) and to explore the effects of PACAP/VIP on Parkinsonism (vide infra). Presynaptic d-opioid receptors play an important role in the regulation of the excitability of spinally projecting LC neurons and the descending NE inhibitory system. Prolonged morphine treatment (m-agonism) does not lead to change in the expression of m-opioid receptors (MOR), but there is transcriptional regulation of other receptors such as DA, NMDA, GABAA and a2A adrenoceptor.[xv] Opiate-induced changes in the NA produce changes in locomotion, and these changes have been used to predict addictive potential (abuse liability),[xvi] and other side effects[xvii] of individual opiate drugs. A unique aspect of our approach is that the mixed d/m agonists not only bind to m-receptors in these areas (VTA, NA & LC), but also produces a d/m balance that might be similar to the balance achieved by endogenous endorphins. Almost all opioids used in the clinic are m-agonists (e.g. morphine, Oxycontin®) or m-partial agonists (e.g. nalbuphine, buprenorphine). In order to produce d-opioid agonism we abandoned the cyclic “Hruby” peptides (c.f. DPDPE) that are constrained by disulfide bonds, and used Roque’s acyclic sequence (YtGFL~) for further development.
While morphine and similar alkaloid-based pharmaceuticals all produce a cascade of metabolites, each of which have their own pharmacological effects, glycopeptides degrade to naturally-occurring amino acid and carbohydrate metabolites devoid of further pharmacological effects.
Figure 3. Summary of Known SAR for Opioid GPCRs and Peptide “Ligands.” 50 years after their discovery, a large number of amino acid substitutions are known for the opioid peptides, which function both as neuromodulators for pain and reward circuits, as well as hormones. Although these substitutions have been largely successful in promoting affinity for GPCRs and selectivity between MOR, DOR and KOR opioid subtypes, stability and BBB penetration by the native peptides remains problematic.
Relevant Mesolimbic Structures. As shown for the rat, dopamine (DA) systems from the ventral tegmental area (VTA) include projections from cell bodies of the VTA to the nucleus accumbens (NA), amygdala, and prefrontal cortex; glutamatergic (GLU) projections from the prefrontal cortex to the NA and the VTA; and projections from the g-aminobutyric acid (GABA) neurons of the NA to the prefrontal cortex. Opioid interneurons modulate the GABA-inhibitory action on the VTA and influence the firing of norepinephrine (NE) neurons in the locus ceruleus (LC). Descending NE pathways emanate from the LC to the dorsal horn of the spinal column, and constitute the descending pain pathways.
Over 200 grams of Lactomorphin was synthesized cGMP (ONR-funded study), with the notion that it would provide morphine-like analgesic effects, but would not induce hypotension in the hypovolemic patient (wounded soldier), nor have strong sedative effects. Although we were not able to test this hypothesis in the hypovolemic pig due to a change in emphasis of the Combat Casualty Care (the program was transferred from the ONR to USArmy, who chose Intranasal Ketamine as their off-the-shelf solution to the problem). Nevertheless, Lactomorphin (MMP-2200) remains to be a drug candidate of clinical interest,[xviii] and we are presently formulating Lactomorphin[xix] and other glycopeptides for intranasal delivery in collaboration with the Mansour Group,[xx] and others.
The concept of producing BBB penetrating glycopeptides from endogenous peptide neurotransmitters such as the opioids enkephalins, endorphins and dynorphins, as well as non-opioids related to angiotensin, PACAP and VIP will be extremely important going forward, particularly when their delivery can be optimized by incorporation into chylomicron-like particles. Ideally, these would be Solid Lipid Nanoparticles (SLNs) composed of glycolipids that self-assemble to form micelles. Unlike SDS micelles, the headgroups here are not ionic, so that the micelles are robust over a large pH range, and increasing the ionic strength of the matrix increases the stability of the glycolipid micelles, whereas SDS micelles are destabilized by divalent ions such as Mg++, Ca++ and Mn++.
Our work with the Hay Group led to the design of PNA5 as an anti-inflammatory treatment following brain insults. This drug was derived from the hormone Angiotensin1—7, a metabolite of the well know blood-pressure hormone, Angiotensin II. We now seek to exploit this drug in the treatment of COVID-19 infection, as PNA5 binds to ACE2, the viral receptor for SARS-CoV-2. The peptidase ACE2 actually produces the peptide hormone Ang-(1-7), which plays important protective roles in reducing inflammation and symptoms associated with acute respiratory distress syndrome, which is recognized now as a significant predictor of mortality from COVID-19.
Fig 4. Glycosylation of Angiotensin1–7 Produces Membrane-Hopping Drug for Treatment of Vascular Dementia. PNA-5 has also shown efficacy in treating both pain and cognitive impairment associated with traumatic brain injury. Research and development of the technology is being supported by the National Institutes of Health and others. ProNeurogen, Inc., developer of the technology, exclusively licenses the technology from the University of Arizona.
[i] Robert Schwyzer 100 YEARS LOCK-AND-KEY CONCEPT - ARE PEPTIDE KEYS SHAPED AND GUIDED TO THEIR RECEPTORS BY THE TARGET-CELL MEMBRANE. Biopolymers 37, 5—16 (1997).
[ii] Segrest, J.P.; De Loof, H.; Dohlman, J.G.; Brouillette, C.G.; Anantharamaiah, G.M. Amphipathic helix motif: Classes and properties. Proteins 8, 103—117 (1990).
[iii] Apostol, C.A.; Hay, M.; Polt, R. Glycopeptide drugs: A pharmacological dimension between “Small Molecules” and “Biologics.” Peptides 131, 170369 (2020). https://doi.org/10.1016/j.peptides.2020.170369
[iv] Mabrouk O, Falk T, Sherman SJ, Kennedy RT, Polt R. CNS penetration of the opioid glycopeptide MMP-2200: A microdialysis study. Neurosci. Letters 531, 99—103 (2012).
[v] Egleton, R.D.; Mitchell, S.A.; Huber, J.D.; Palian, M.M.; Polt, R.; Davis, T.P. Improved Blood-Brain Barrier Penetration and Enhanced Analgesia of an Opioid Peptide by Glycosylation. J. Pharm. Exp. Ther. 299, 967—972 (2001).
[vi] Williams, S.A.; Abbruscato, T.J.; Szabó, L.; Polt, R.; Hruby, V.; Davis, T.P. The Effect of Glycosylation on the Uptake of an Enkephalin Analogue into the Central Nervous System. In: Biology and Physiology of the Blood-Brain Barrier. Couraud & Scherman, Eds., Plenum Press, New York, 69—77 (1996).
[vii] Palian, M.M.; Boguslavsky, V.I.; O’Brien, D.F.; Polt, R. Glycopeptide-Membrane Interactions: Glycosyl Enkephalin Analogs Adopt Turn Conformations by NMR and CD in Amphipathic Media. J. Am. Chem. Soc. 125, 5823—5831 (2003).
[viii] a) Deguchi, Y.; Naito, Y.; Ohtsuki, S.; Yusaku Miyakawa, Y.; Morimoto, K.; Hosoya, K.; Sakurada, S.; Terasaki, T. Blood-Brain Barrier Permeability of Novel [D-Arg2]Dermorphin (1-4) Analogs: Transport Property Is Related to the Slow Onset of Antinociceptive Activity in the Central Nervous System. J. Pharm. Exp. Therap. 310, 177—184 (2004). b) Ogawa, T.; Miyamae, T.; Murayama, K.; Okuyama, K.; Okayama, T.; Hagiwara, M.; Sakurada, S.; Morikawa, T. Synthesis and structure-activity relationships of an orally available and long-acting analgesic peptide, Na-amidino-Tyr-DArg-Phe-MebAla-OH (ADAMB). J. Med. Chem. 45, 5081–5089 (2002).
[ix] Lowery JJ, Yeomans L, Keyari CM, Davis P, Porreca F, Knapp BI, Bidlack JM, Bilsky EJ, Polt R. Glycosylation improves the central effects of DAMGO. Chem. Biol. Drug Design 69, 41—47 (2007).
[x] Yue, X; Falk, T; Zuniga, LA; Szabò, L; Porreca, F; Polt, R; Sherman, SJ Effects of the novel glycopeptide opioid agonist MMP-2200 in preclinical models of Parkinson's disease. Brain Res. 1413, 72—83 (2011).
[xi] Flores, A.J.; Bartlett, M.J.; Root, B.K.; Parent, K.L.; Heien, M.L.; Porreca, F.; Polt, R.; Sherman, S.J.; Falk, T. The combination of the opioid glycopeptide MMP-2200 and a NMDA receptor antagonist reduced L-DOPA-induced dyskinesia and MMP-2200 by itself reduced dopamine receptor 2-like agonist-induced dyskinesia. Neuropsychopharmacology 141, 260—271 (2018).
[xii] Stevenson, G.W.; Giuvelis, D.; Cormier, J.; Cone, K.; Atherton, P.; Krivitsky, R.; Warner, E.; Laurent, B.; Dutra J.; Bidlack, J.M.; Szabò, L.; Polt, R.; Bilsky, E.J. Behavioral pharmacology of the mixed-action delta-selective opioid receptor agonist BBI-11008: studies on acute, inflammatory and neuropathic pain, respiration, and drug self-administration. Psychopharmacology 237, 1195—1208 (2020) https://doi.org/10.1007/s00213-019-05449-z
[xiii] Li Y, St. Louis L, Knapp BI, Muthu D, Anglin B, Giuvelis D, Bidlack JM, Bilsky EJ, Polt R. Amphipathic Helices Influence CNS Antinociceptive Activity of Glycopeptides Related to β-Endorphin. J. Med. Chem. 57, 2237—2246 (2014). https://doi.org.10.1021/jm400879w
[xiv] Lefever M, Li Y, Anglin B, Muthu, D, Lowery, JJ, Giuvelis D, Knapp BI, Bidlack, JM, Bilsky EJ, Polt R. Structural Requirements for CNS Active Opioid Glycopeptides. J. Med. Chem. 58, 5728—5741 (2015).
[xv] Ammon-Treiber, S.; Hollt, V. Morphine-induced changes of gene expression in the brain. Addiction Biology 10, 81—89 (2005).
[xvi] Cunningham, S.T.; Finn, M.; Kelley, A.E. Sensitization of the locomotor response to psychostimulants after repeated opiate exposure: Role of the nucleus accumbens. Neuropsychopharmacology 16, 147—155 (1997).
[xvii] Erdtmann-Vourliotis, M.; Mayer, P.; Linke, R.; Riechert, U.; Hollt, V. Long-lasting sensitization towards morphine in motoric and limbic areas as determined by c-fos expression in rat brain. Mol. Brain Res. 72, 1—16 (1999).
[xviii] Burgess, G.E.; Polt, R.; Rice, K.C.; Jutkiewicz, E.M. Characterization of Delta Opioid Receptor Activity of MMP2200, a Mixed Efficacy Mu and Delta Opioid Receptor Agonist. FASEB Journal 18 April 2020 https://doi.org/10.1096/fasebj.2020.34.s1.05714
[xix] Inventors: Polt, R.; Heien, M.L.; Pemberton, J.E. Glycopeptide drug delivery system for delivering a glycopeptide drug and for treating a neurodegenerative disease, pain, depression and behavioral disorder, comprises a glycopeptide drug integrated into a lipid portion of the micelle. Patent Number: WO2018031782-A1, Patent Assignee: Arizona Board of Regents, Publication Date 15 Feb 2018
[xx] Hickey, A. J.; Mansour, H. M., Inhalation Aerosols: Physical and Biological Basis for Therapy. . CRC Press/Taylor & Francis: London, United Kingdom, p 418— (2019).