Design of a Tumor Binding GMCSF as Intratumoral Immunotherapy of Solid Tumors.

Next-generation cancer immunotherapies may utilize immunostimulants to selectively activate the host immune system against tumor cells. Checkpoint inhibitors (CPIs) like anti-PD1/PDL-1 that inhibit immunosuppression have shown unprecedented success but are only effective in the 20-30% of patients that possess an already "hot" (immunogenic) tumor. In this regard, intratumoral (IT) injection of immunostimulants is a promising approach since they can work synergistically with CPIs to overcome the resistance to immunotherapies by inducing immune stimulation in the tumor. One such immunostimulant is granulocyte macrophage-colony-stimulating factor (GMCSF) that functions by recruiting and activating antigen-presenting cells (dendritic cells) in the tumor, thereby initiating anti-tumor immune responses. However, key problems with GMCSF are lack of efficacy and the risk of systemic toxicity caused by the leakage of GMCSF from the tumor tissue. We have designed tumor-retentive versions of GMCSF that are safe yet potent immunostimulants for the local treatment of solid tumors. The engineered GMCSFs (eGMCSF) were synthesized by recombinantly fusing tumor-ECM (extracellular matrix) binding peptides to GMCSF. The eGMCSFs exhibited enhanced tumor binding and potent immunological activity in vitro and in vivo. Upon IT administration, the tumor-retentive eGMCSFs persisted in the tumor, thereby alleviating systemic toxicity, and elicited localized immune activation to effectively turn an unresponsive immunologically "cold" tumor "hot".

Glatiramer acetate enhances tumor retention and innate activation of immunostimulants.

Cancer immunotherapy aims to stimulate immune cells to recognize and attack tumor tissue. The immunostimulatory polyanions polyI:C and CpG induce potent pro-inflammatory immune responses as TLR3 and TLR9 agonists, respectively. Clinical trials of TLR agonists, however, have been fraught with immune-related adverse events, even when injecting intratumorally in an effort to minimize systemic exposure. We identified Glatiramer Acetate (GA), a positively-charged polypeptide approved for multiple sclerosis, as a delivery agent capable of complexing with polyI:C or CpG and reducing the mobility of these actives. Small nanoparticles termed polyplexes form when mixing positively-charged GA and negatively-charged immunostimulant (polyI:C or CpG). The ratio of GA to immunostimulant directly affected the potency of TLR activation and the mobility of these actives in simulated tumor tissue. Polyplexes of GA and CpG were injected intratumorally in a tumor model of head and neck cancer (HNC) and significantly mitigated tumor growth as compared to the vehicle controls. Intratumoral injections of CpG showed the slowest tumor growth but exhibited dramatically higher systemic proinflammatory cytokine levels compared to polyplexes of GA with CpG. Sequencing of RNA from resected tumors revealed a similar pattern of upregulated proinflammatory cytokines for CpG and polyplexes, a finding supported by histological tumor staining showing similar infiltration of immune cells induced by these treatments. Intratumoral administration of polyplexes of GA with immunostimulant represents a translational approach to enhance local immune responses while mitigating systemic immune-related adverse events.

Constructing a Biomaterial to Simulate Extracellular Drug Transport in Solid Tumors.

Designing an in vitro model of the tumor extracellular microenvironment to screen intratumoral drugs is an active challenge. As recent clinical successes of human intratumoral therapies stimulate research on intratumoral delivery, a need for a 3D tumor model to screen intratumoral therapies arises. When injecting the drug formulation directly into the tumor, the biophysics affecting intratumoral retention must be considered; especially for biologic therapies, which may be dominated by extracellular transport mechanisms. Fibrotic regions in solid tumors are typically rich in collagen I fibers. Using shear rheology, head and neck tumors with higher collagen density show a higher stiffness. Similarly, the stiffness of the hyaluronic acid (HA) hydrogel models is increased by adding collagen fibers to model the bulk biomechanical properties of solid tumors. HA hydrogels are then used as intratumoral injection site simulators to model in vitro the retention of glatiramer acetate (GA) and polyethylene glycol (PEG) administered intratumorally. Both compounds are also injected in murine tumors and retention is studied ex vivo for comparison. Retention of GA in the hydrogels is significantly longer than PEG, analogous to the solid tumors, suggesting the utility of HA hydrogels with collagen I fibers for screening extracellular drug transport after intratumoral administration.

Formation of platinum (II) as a six member ring for sustained polymeric delivery.

A new pH-activated polymer chelate of cisplatin was synthesized using a scalable and green aqueous technique. Synthesis of the chelate was based on formation of a 6-member ring of platinum(II) with acetyl-homo-Lysine (Ac-homo-Lys), which was accomplished under completely aqueous conditions using a traceless photocleavable protection chemistry. Synthesis preceded by, first, amidation of a photocaged homo-Ac-Lys with hyaluronic acid (HA) in water using a p-hydroxyphenacyl (pHP) group as the photoremovable protecting group, followed by reaction of cisplatin (diaqua form) in water to form the reversible chelate. Platinum drug release was pH rate controlled, with more rapid release (t1/2 20 h) at acidic pH similar to the tumor microenvironment yet slower release (t1/2 35 h) at normal physiological pH.

Synthetic Studies of Neoclerodane Diterpenes from Salvia divinorum: Identification of a Potent and Centrally Acting µ Opioid Analgesic with Reduced Abuse Liability.

Opioids are widely used to treat millions suffering from pain, but their analgesic utility is limited due to associated side effects. Herein we report the development and evaluation of a chemical probe exhibiting analgesia and reduced opioid-induced side effects. This compound, kurkinorin (5), is a potent and selective µ-opioid receptor (MOR) agonist (EC50 = 1.2 nM, >8000 µ/κ selectivity). 5 is a biased activator of MOR-induced G-protein signaling over ß-arrestin-2 recruitment. Metadynamics simulations of 5's binding to a MOR crystal structure suggest energetically preferred binding modes that differ from crystallographic ligands. In vivo studies with 5 demonstrate centrally mediated antinociception, significantly reduced rewarding effects, tolerance, and sedation. We propose that this novel MOR agonist may represent a valuable tool in distinguishing the pathways involved in MOR-induced analgesia from its side effects.

Synthesis and κ-opioid receptor activity of furan-substituted salvinorin A analogues.

The neoclerodane diterpene salvinorin A, found in the leaves of Salvia divinorum, is a potent κ-opioid receptor agonist, making it an attractive scaffold for development into a treatment for substance abuse. Although several successful semisynthetic studies have been performed to elucidate structure-activity relationships, the lack of analogues with substitutions to the furan ring of salvinorin A has prevented a thorough understanding of its role in binding to the κ-opioid receptor. Herein we report the synthesis of several salvinorin A derivatives with modified furan rings. Evaluation of these compounds in a functional assay indicated that sterically less demanding substitutions are preferred, suggesting the furan ring is bound in a congested portion of the binding pocket. The most potent of the analogues successfully reduced drug-seeking behavior in an animal model of drug-relapse without producing the sedation observed with other κ-opioid agonists.

Studies toward the Development of Antiproliferative Neoclerodanes from Salvinorin A.

The success rate for central nervous system (CNS) drug candidates in the clinic is relatively low compared to the industry average across other therapeutic areas. Penetration through the blood-brain barrier (BBB) to reach the therapeutic target is a major obstacle in development. The rapid CNS penetration of salvinorin A has suggested that the neoclerodane nucleus offers an excellent scaffold for developing antiproliferative compounds that enter the CNS. The Liebeskind-Srogl reaction was used as the main carbon-carbon bond-forming step toward the synthesis of quinone-containing salvinorin A analogues. Quinone-containing salvinorin A analogues were shown to have antiproliferative activity against the MCF7 breast cancer cell line, but show no significant activity at the κ-opioid receptors. In an in vitro model of BBB penetration, quinone-containing salvinorin A analogues were shown to passively diffuse across the cell monolayer. The analogues, however, are substrates of P-glycoprotein, and thus further modification of the molecules is needed to reduce the affinity for the efflux transporter.

Functional selectivity of 6'-guanidinonaltrindole (6'-GNTI) at κ-opioid receptors in striatal neurons.

There is considerable evidence to suggest that drug actions at the κ-opioid receptor (KOR) may represent a means to control pain perception and modulate reward thresholds. As a G protein-coupled receptor (GPCR), the activation of KOR promotes Gαi/o protein coupling and the recruitment of ß-arrestins. It has become increasingly evident that GPCRs can transduce signals that originate independently via G protein pathways and ß-arrestin pathways; the ligand-dependent bifurcation of such signaling is referred to as "functional selectivity" or "signaling bias." Recently, a KOR agonist, 6'-guanidinonaltrindole (6'-GNTI), was shown to display bias toward the activation of G protein-mediated signaling over ß-arrestin2 recruitment. Therefore, we investigated whether such ligand bias was preserved in striatal neurons. Although the reference KOR agonist U69,593 induces the phosphorylation of ERK1/2 and Akt, 6'-GNTI only activates the Akt pathway in striatal neurons. Using pharmacological tools and ß-arrestin2 knock-out mice, we show that KOR-mediated ERK1/2 phosphorylation in striatal neurons requires ß-arrestin2, whereas Akt activation depends upon G protein signaling. These findings reveal a point of KOR signal bifurcation that can be observed in an endogenous neuronal setting and may prove to be an important indicator when developing biased agonists at the KOR.

Differential signaling properties at the kappa opioid receptor of 12-epi-salvinorin A and its analogues.

The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alter the course of mood, anxiety and addictive disorders or reduce response to stress. In a search for highly potent and selective KOPR partial agonists as pharmacological tools, we have modified 12-epi-salvinorin A, a compound which we have previously observed to be a KOPR partial agonist. Five analogues of 12-epi-salvinorin A were synthesized and their effects on G protein activation as well as ß-arrestin2 recruitment were evaluated. Only 12-epi-salvinorin A (1) partially activated signaling through G proteins, yet acted as a full agonist in the ß-arrestin 2 DiscoveRx assay. Other salvinorin analogues tested in these functional assays were full agonists in both assays of KOPR activation. By comparison, the non-selective opioid ligand nalbuphine, known to be a partial agonist for G-protein activation, was also a partial agonist for the ß-arrestin mediated signaling pathway activated through KOPR.

Functional selectivity at the µ-opioid receptor: implications for understanding opioid analgesia and tolerance.

Opioids are the most effective analgesic drugs for the management of moderate or severe pain, yet their clinical use is often limited because of the onset of adverse side effects. Drugs in this class produce most of their physiological effects through activation of the µ opioid receptor; however, an increasing number of studies demonstrate that different opioids, while presumably acting at this single receptor, can activate distinct downstream responses, a phenomenon termed functional selectivity. Functional selectivity of receptor-mediated events can manifest as a function of the drug used, the cellular or neuronal environment examined, or the signaling or behavioral measure recorded. This review summarizes both in vitro and in vivo work demonstrating functional selectivity at the µ opioid receptor in terms of G protein coupling, receptor phosphorylation, interactions with ß-arrestins, receptor desensitization, internalization and signaling, and details on how these differences may relate to the progression of analgesic tolerance after their extended use.

Agonist-directed interactions with specific beta-arrestins determine mu-opioid receptor trafficking, ubiquitination, and dephosphorylation.

Morphine and other opiates mediate their effects through activation of the µ-opioid receptor (MOR), and regulation of the MOR has been shown to critically affect receptor responsiveness. Activation of the MOR results in receptor phosphorylation, ß-arrestin recruitment, and internalization. This classical regulatory process can differ, depending on the ligand occupying the receptor. There are two forms of ß-arrestin, ß-arrestin1 and ß-arrestin2 (also known as arrestin2 and arrestin3, respectively); however, most studies have focused on the consequences of recruiting ß-arrestin2 specifically. In this study, we examine the different contributions of ß-arrestin1- and ß-arrestin2-mediated regulation of the MOR by comparing MOR agonists in cells that lack expression of individual or both ß-arrestins. Here we show that morphine only recruits ß-arrestin2, whereas the MOR-selective enkephalin [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), recruits either ß-arrestin. We show that ß-arrestins are required for receptor internalization and that only ß-arrestin2 can rescue morphine-induced MOR internalization, whereas either ß-arrestin can rescue DAMGO-induced MOR internalization. DAMGO activation of the receptor promotes MOR ubiquitination over time. Interestingly, ß-arrestin1 proves to be critical for MOR ubiquitination as modification does not occur in the absence of ß-arrestin1 nor when morphine occupies the receptor. Moreover, the selective interactions between the MOR and ß-arrestin1 facilitate receptor dephosphorylation, which may play a role in the resensitization of the MOR and thereby contribute to overall development of opioid tolerance.

Synthesis of conolidine, a potent non-opioid analgesic for tonic and persistent pain.

Management of chronic pain continues to represent an area of great unmet biomedical need. Although opioid analgesics are typically embraced as the mainstay of pharmaceutical interventions in this area, they suffer from substantial liabilities that include addiction and tolerance, as well as depression of breathing, nausea and chronic constipation. Because of their suboptimal therapeutic profile, the search for non-opioid analgesics to replace these well-established therapeutics is an important pursuit. Conolidine is a rare C5-nor stemmadenine natural product recently isolated from the stem bark of Tabernaemontana divaricata (a tropical flowering plant used in traditional Chinese, Ayurvedic and Thai medicine). Although structurally related alkaloids have been described as opioid analgesics, no therapeutically relevant properties of conolidine have previously been reported. Here, we describe the first de novo synthetic pathway to this exceptionally rare C5-nor stemmadenine natural product, the first asymmetric synthesis of any member of this natural product class, and the discovery that (±)-, (+)- and (-)-conolidine are potent and efficacious non-opioid analgesics in an in vivo model of tonic and persistent pain.

Herkinorin analogues with differential beta-arrestin-2 interactions.

Salvinorin A is a psychoactive natural product that has been found to be a potent and selective kappa opioid receptor agonist in vitro and in vivo. The activity of salvinorin A is unusual compared to other opioids such as morphine in that it mediates potent kappa opioid receptor signaling yet leads to less receptor downregulation than observed with other kappa agonists. Our initial chemical modifications of salvinorin A have yielded one analogue, herkinorin ( 1c), with high affinity at the microOR. We recently reported that 1c does not promote the recruitment of beta-arrestin-2 to the microOR or receptor internalization. Here we describe three new derivatives of 1c ( 3c, 3f, and 3i) with similar properties and one, benzamide 7b, that promotes recruitment of beta-arrestin-2 to the microOR and receptor internalization. When the important role micro opioid receptor regulation plays in determining physiological responsiveness to opioid narcotics is considered, micro opioids derived from salvinorin A may offer a unique template for the development of functionally selective mu opioid receptor-ligands with the ability to produce analgesia while limiting adverse side effects.