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 Complexed with CpG as Intratumoral Immunotherapy in Combination with Anti-PD-1.

CpG oligodeoxynucleotides are toll-like receptor 9 agonists capable of inducing potent pro-inflammatory immune responses. Although CpG oligodeoxynucleotides have shown promising antitumor effects, their systemic activity can trigger immune-related toxicity, limiting therapeutic application. We previously identified glatiramer acetate (GA), a cationic polypeptide approved for the treatment of relapsing-remitting multiple sclerosis, as an intratumoral delivery agent capable of complexing with CpG, thereby pinning it to the injection site and limiting systemic exposure. Here, we investigated whether the combination of CpG or GA-CpG polyplexes and intraperitoneal anti-PD-1 therapy would result in synergistic efficacy in AT84 and CT26 murine syngeneic models of head and neck and colon cancers, respectively. In both AT84 and CT26 tumor models, intratumoral CpG or GA-CpG treatment similarly suppressed tumor growth, but the efficacy was not amplified with anti-PD-1. Nevertheless, combination treatment increased cytotoxic T cell, helper T cell, and natural killer cell infiltration into AT84 tumors. Surprisingly, the combination of intratumoral GA and intraperitoneal anti-PD-1 treatment resulted in elevated systemic GM-CSF and IL-2 cytokine levels and demonstrated synergistic antitumor effects in the CT26 mouse tumor model. Moreover, tumors that responded most significantly to anti-PD-1 plus GA treatment showed increased markers of infiltration of CD4+ T cells and natural killer cells. Combinations of intratumoral GA or GA-CpG polyplexes with anti-PD-1 treatment warrant further investigation as combination cancer immunotherapy strategies.

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.

Intratumoral Cancer Chemotherapy with a Carrier-Based Immunogenic Cell-Death Eliciting Platinum (IV) Agent.

A carrier-based, immunogenic cell death (ICD)-eliciting platinum(IV) chemotherapeutic agent was synthesized via complexation between an axially derivatized Pt(IV)-tocopherol and hyaluronan (HA)-tocopherol nanocarrier. The resultant HA-Pt(IV) complex demonstrated antiproliferative activity and induced calreticulin translocation, an indicator of ICD, in murine and human head and neck cancer (HNC) cells. The intratumorally administered HA-Pt(IV) treatments were tolerable and efficacious in both immunocompetent and immunodeficient mice with HNC, partially because of the direct cytotoxicity. Superior efficacy and survival were observed in the immunocompetent group, suggesting a possible Pt(IV)-induced immunological response, which would only manifest in animals with an intact immune system. Subsequent imaging of tumor tissues demonstrated increased macrophage infiltration in the HA-Pt(IV)-treated tumors compared to the nontreated controls and the cisplatin-treated tumors, suggesting favorable inflammatory activation. RNA sequencing of HA-Pt(IV)-treated tumors indicated that carbohydrate and vitamin metabolisms were the most important Kyoto Encyclopedia of Genes and Genomes pathways, and molecular function, biological process, and cellular component were highly enriched gene ontology categories.

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.

Human intratumoral therapy: Linking drug properties and tumor transport of drugs in clinical trials.

Cancer therapies aim to kill tumor cells directly or engage the immune system to fight malignancy. Checkpoint inhibitors, oncolytic viruses, cell-based immunotherapies, cytokines, and adjuvants have been applied to prompt the immune system to recognize and attack cancer cells. However, systemic exposure of cancer therapies can induce unwanted adverse events. Intratumoral administration of potent therapies utilizes small amounts of drugs, in an effort to minimize systemic exposure and off-target toxicities. Here, we discuss the properties of the tumor microenvironment and transport considerations for intratumoral drug delivery. Specifically, we consider various tumor tissue factors and physicochemical factors that can affect tumor retention after intratumoral injection. We also review approved and clinical-stage intratumoral therapies and consider how the molecular and biophysical properties (e.g. size and charge) of these therapies influences intratumoral transport (e.g. tumor retention and cellular uptake). Finally, we offer a critical review and highlight several emerging approaches to promote tumor retention and limit systemic exposure of potent intratumoral therapies.

Formulation and preclinical evaluation of a toll-like receptor 7/8 agonist as an anti-tumoral immunomodulator.

The toll-like receptor 7 and 8 (TLR7/8) agonist Resiquimod (R848) has been recognized as a promising immunostimulator for the treatment of cutaneous cancers in multiple clinical trials. However, systemic administration of R848 often results in strong immune-related toxicities while having limited therapeutic effects to the tumor. In the present study, a prodrug-based nanocarrier delivery system was developed that exhibited high therapeutic efficiency. R848 was conjugated to α-tocopherol to constitute an R848-Toco prodrug, followed by formulating with a tocopherol-modified hyaluronic acid (HA-Toco) as a polymeric nano-suspension. In vitro evaluation showed that the delivery system prolonged the release kinetics while maintaining TLR agonist activities. When administered subcutaneously, the nano-suspension formed a depot at the injection site, inducing localized immune responses without systemic expansion. This formulation also suppressed tumor growth and recruited immune cells to the tumor in a murine model of head and neck cancer. In a preclinical canine study of spontaneous mast cell tumors, the treatment led to a 67% response rate (three partial remissions and one complete remission).

Injectable Chemotherapy Downstaged Oral Squamous Cell Carcinoma from Nonresectable to Resectable in a Rescue Dog: Diagnosis, Treatment, and Outcome.

This case report documents the diagnosis, treatment, and outcome of a nonresectable oral squamous cell carcinoma in a dog with initial poor prognosis. An approximately 4-year-old female Staffordshire Bull Terrier presented with a large mass on the front of lower jaw which was diagnosed as oral papillary squamous cell carcinoma by histopathology. CT scans revealed invasion of the cancer to the frenulum of the tongue. The mass was inoperable due to location, expansiveness, and metastatic lymph nodes. The dog received 4 treatments of intralesional hyaluronan-platinum conjugates (HylaPlat™, HylaPharm LLC, Lawrence, Kansas) at 3-week intervals. Clinical chemistry and complete blood count were performed one week after each treatment and results were within normal limits. Complications included bleeding due to tumor tissue sloughing, as well as a single seizure due to unknown causes. Upon completion of chemotherapy, CT showed that the mass had regressed and was no longer invading the lingual frenulum, and multiple lymph nodes were free of metastasis. The mass thus became resectable and the dog successfully underwent rostral bilateral mandibulectomy. Over one year after chemotherapy and surgery, the cancer remains in complete remission.

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.

Phase I-II clinical trial of hyaluronan-cisplatin nanoconjugate in dogs with naturally occurring malignant tumors.

OBJECTIVE To conduct a phase I-II clinical trial of hyaluronan-cisplatin nanoconjugate (HA-Pt) in dogs with naturally occurring malignant tumors. ANIMALS 18 healthy rats, 9 healthy mice, and 16 dogs with cancer. PROCEDURES HA-Pt was prepared and tested by inductively coupled plasma mass spectrometry; DNA-platinum adduct formation and antiproliferation effects of cisplatin and HA-Pt were compared in vitro. Effects of cisplatin (IV) and HA-Pt (SC) in rodents were tested by clinicopathologic assays. In the clinical trial, dogs with cancer received 1 to 4 injections of HA-Pt (10 to 30 mg/m(2), intratumoral or peritumoral, q 3 wk). Blood samples were collected for pharmacokinetic analysis; CBC, serum BUN and creatinine concentration measurement, and urinalysis were conducted before and 1 week after each treatment. Some dogs underwent hepatic enzyme testing. Tumors were measured before the first treatment and 3 weeks after each treatment to assess response. RESULTS No adverse drug effects were detected in pretrial assessments in rodents. Seven of 16 dogs completed the study; 3 had complete tumor responses, 3 had stable disease, and 1 had progressive disease. Three of 7 dogs with oral and nasal squamous cell carcinoma (SCC) that completed the study had complete responses. Myelosuppression and cardiotoxicosis were identified in 6 and 2 dogs, respectively; none had nephrotoxicosis. Four of 5 dogs with hepatic enzymes assessed had increased ALT activities, attributed to diaquated cisplatin products in the HA-Pt. Pharmacokinetic data fit a 3-compartment model. CONCLUSIONS AND CLINICAL RELEVANCE HA-Pt treatment resulted in positive tumor responses in some dogs, primarily those with SCC. The adverse effect rate was high. IMPACT FOR HUMAN MEDICINE Oral SCC in dogs has characteristics similar to human head and neck SCC; these results could be useful in developing human treatments.

Hyaluronan-Lysine Cisplatin Drug Carrier for Treatment of Localized Cancers: Pharmacokinetics, Tolerability, and Efficacy in Rodents and Canines.

The purpose of this study was to develop a safe and efficacious drug delivery platform for sustained release of cisplatin after locoregional administration. We successfully synthesized hyaluronan-cisplatin nanoconjugates (HA-Lys-Pt) using an N-Ac-lysine linker, which formed a thermodynamically stable five-membered ring with the platinum. The conjugate was characterized for release kinetics, in vitro anti-proliferative activity, degradability, impurity content, formation of Pt-DNA adducts, pharmacokinetics, tolerability in rodents and canines, and for efficacy in rodents. The 75 kD HA-Lys-Pt (75HA-Lys-Pt) sustained release of platinum with a 69 h half-life in phosphate buffered saline without substantial burst release. Compared to intravenous cisplatin, subcutaneously injected 75HA-Lys-Pt formed 3.2-fold more Pt-DNA adducts in rat peripheral blood mononuclear cells compared to intravenous cisplatin over 96 h. Subcutaneous 75HA-Lys-Pt was tolerable in rats at 40 mg/kg (4 × LD50 of conventional cisplatin) and resulted in 62.5% partial response and 37.5% stable disease in murine xenografts of head and neck squamous cell cancer (20 mg/kg/wk × 3 weeks). 75HA-Lys-Pt demonstrated extended tmax and improved area-under-the-curve compared to cisplatin in rats and canines. Canine safety was demonstrated by liver enzyme and electrolyte levels, complete blood count, and urinalysis.

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.