Photochemical Internalization Enhanced Vaccination Is Safe, and Gives Promising Cellular Immune Responses to an HPV Peptide-Based Vaccine in a Phase I Clinical Study in Healthy Volunteers.

Background and Aims: Photochemical internalization (PCI) is a technology for inducing release of endocytosed antigens into the cell cytosol via a light-induced process. Preclinical experiments have shown that PCI improves MHC class I antigen presentation, resulting in strongly enhanced CD8+ T-cell responses to polypeptide antigens. In PCI vaccination a mixture of the photosensitizing compound fimaporfin, vaccine antigens, and an adjuvant is administered intradermally followed by illumination of the vaccination site. This work describes an open label, phase I study in healthy volunteers, to assess the safety, tolerability, and immune response to PCI vaccination in combination with the adjuvant poly-ICLC (Hiltonol) (ClinicalTrials.gov Identifier: NCT02947854). Methods: The primary objective of the study was to assess the safety and local tolerance of PCI mediated vaccination, and to identify a safe fimaporfin dose for later clinical studies. A secondary objective was to analyze the immunological responses to the vaccination. Each subject received 3 doses of HPV16 E7 peptide antigens and two doses of Keyhole Limpet Hemocyanin (KLH) protein. A control group received Hiltonol and vaccine antigens only, whereas the PCI groups in addition received fimaporfin + light. Local and systemic adverse effects were assessed by standard criteria, and cellular and humoral immune responses were analyzed by ELISpot, flow cytometry, and ELISA assays. Results: 96 healthy volunteers were vaccinated with fimaporfin doses of 0.75-50 µg. Doses below 17.5 µg were safe and tolerable, higher doses exhibited local tolerability issues in some study subjects, mainly erythema, and pain during illumination. There were few, and only mild and expected systemic adverse events. The employment of PCI increased the number of subjects exhibiting a T-cell response to the HPV peptide vaccine about 10-fold over what was achieved with the antigen/Hiltonol combination without PCI. Moreover, the use of PCI seemed to result in a more consistent and multifunctional CD8+ T-cell response. An enhancement of the humoral immune response to KLH vaccination was also observed. Conclusions: Using PCI in combination with Hiltonol for intradermal vaccination is safe at fimaporfin doses below 17.5 µg, and gives encouraging immune responses to peptide and protein based vaccination.

Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination.

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs) in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses.

Endosome Targeting meso-Tetraphenylchlorin-Chitosan Nanoconjugates for Photochemical Internalization.

Four amphiphilic covalently linked meso-tetraphenylchlorin-chitosan nanoconjugates were synthesized and evaluated for use in photochemical internalization (PCI) in vitro and in vivo. The synthetic protocol for the preparation of two different hydrophobic chlorin photosensitizers, 5-(4-aminophenyl)-10,15,20-triphenylchlorin and 5-(4-carboxyphenyl)-10,15,20-triphenylchlorin, was optimized. These monofunctional photosensitizers were covalently attached to carrier chitosan via silyl-protected 3,6-di-O-tert-butyldimethylsilyl-chitosan (Di-TBDMS-chitosan) with 0.10 degree of substitution per glucosamine (DS). Hydrophilic moieties such as trimethylamine and/or 1-methylpiperazine were incorporated with 0.9 DS to give fully water-soluble conjugates after removal of the TBDMS groups. A dynamic light scattering (DLS) study confirmed the formation of nanoparticles with a 140-200 nm diameter. These nanoconjugates could be activated at 650 nm (red region) light, with a fluorescence quantum yield (ΦF) of 0.43-0.45, and are thus suitable candidates for use in PCI. These nanoconjugates were taken up and localized in the endocytic vesicles of HCT116/LUC human colon carcinoma cells, and upon illumination they substantially enhanced plasmid DNA transfection. The nanoconjugates were also evaluated in preliminary in vivo experiments in tumor-bearing mice, showing that the nanoconjugates could induce a strong photodynamic therapy (PDT) and also PCI effects in treatment with bleomycin.

Cytosolic Delivery of Liposomal Vaccines by Means of the Concomitant Photosensitization of Phagosomes.

One of the greatest pharmaceutical challenges in vaccinology is the delivery of antigens to the cytosol of antigen-presenting cells (APCs) in order to allow for the stimulation of major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell responses, which may act on intracellular infections or cancer. Recently, we described a novel method for cytotoxic T-lymphocyte (CTL) vaccination by combining antigens with a photosensitizer and light for cytosolic antigen delivery. The goal of the current project was to test this immunization method with particle-based formulations. Liposomes were prepared from dipalmitoylphosphatidylcholine and cholesterol, and the antigen ovalbumin (OVA) or the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) was separately encapsulated. C57BL/6 mice were immunized intradermally with OVA liposomes or a combination of OVA and TPCS2a liposomes, and light was applied the next day for activation of the photosensitizer resulting in cytosolic release of antigen from phagosomes. Immune responses were tested both after a prime only regime and after a prime-boost scheme with a repeat immunization 2 weeks post priming. Antigen-specific CD8(+) T-cell responses and antibody responses were analyzed ex vivo by flow cytometry and ELISA methods. The physicochemical stability of liposomes upon storage and light exposure was analyzed in vitro. Immunization with both TPCS2a- and OVA-containing liposomes greatly improved CD8(+) T-cell responses as compared to immunization without TPCS2a and as measured by proliferation in vivo and cytokine secretion ex vivo. In contrast, OVA-specific antibody responses (IgG1 and IgG2c) were reduced after immunization with TPCS2a-containing liposomes. The liposomal formulation protected the photosensitizer from light-induced inactivation during storage. In conclusion, the photosensitizer TPCS2a was successfully formulated in liposomes and enabled a shift from MHC class II to MHC class I antigen processing and presentation for stimulation of strong CD8(+) T-cell responses. Therefore, photosensitive particulate vaccines may have the potential to add to current vaccine practice a new method of vaccination that, as opposed to current vaccines, can stimulate strong CD8(+) T-cell responses.

Photosensitizer and Light Pave the Way for Cytosolic Targeting and Generation of Cytosolic CD8 T Cells Using PLGA Vaccine Particles.

The generation of CTLs is crucial in the immunological fight against cancer and many infectious diseases. To achieve this, vaccine Ags need to be targeted to the cytosol of dendritic cells, which can activate CD8 T cells via MHC class I (MHCI). Therefore, such targeting has become one of the major objectives of vaccine research. In this study, we aimed to bypass the unwanted and default MHC class II Ag presentation and trigger MHCI presentation by using a photosensitizer that, upon light activation, would facilitate cytosolic targeting of codelivered Ag. Poly(lactide-co-glycolide) microparticles ∼1 µm size were loaded with OVA and the photosensitizer tetraphenyl chlorine disulphonate (TPCS2a) and administered intradermally in mice, which were illuminated 1 d later for activation of the photosensitizer. Immunization in the presence of TPCS2a significantly increased activation of CD8 T cells compared with immunization without TPCS2a and as measured by CD8 T cell proliferation, production of proinflammatory IFN-γ, TNF-α, and IL-2, and prevention of tumor growth. Cytotoxicity was demonstrated by granzyme B production in vitro and by in vivo killing of CFSE-labeled targets. CD4-dependent Ab responses were abrogated in mice immunized with TPCS2a-containing particles, suggesting that photosensitization facilitated a shift from default MHC class II toward MHCI Ag presentation. Hence, vaccine particles with Ag and photosensitizers proved an effective vehicle or adjuvant for stimulation of CTLs, and they may find potential application in therapeutic cancer vaccination and in prophylactic and therapeutic vaccination against intracellular infections.

Photosensitisation facilitates cross-priming of adjuvant-free protein vaccines and stimulation of tumour-suppressing CD8 T cells.

Cancer vaccines aim to induce CD8 T cells infiltrating the tumour. For protein-based vaccines, the main biological barrier to overcome is the default MHC class-II-pathway, with activation of CD4 T cells rather than CD8 T cells. The latter requires antigens to access the cytosol and MHC class I antigen presentation. We applied photosensitiser and light to trigger disruption of antigen-containing endosomes and thereby MHC class I cross-presentation of a model cancer vaccine. This "photochemical internalisation" resulted in activation, proliferation, and IFN-γ production of cytotoxic CD8 T cells, which suppressed tumour growth by infiltrating CD8 T cells and caspase-3-dependent apoptosis. The process was independent of MHC class II, MyD88, and TLR4 signalling, but dependent on trypsin- and caspase-like proteasome activity and partly also on chloroquine. This novel method of vaccination may find applications in cancer immunotherapy where the activation of CD8 T cells is important.

Intradermal photosensitisation facilitates stimulation of MHC class-I restricted CD8 T-cell responses of co-administered antigen.

The protection or treatment of several immunological disorders is dependent on the antigen-specific and cytotoxic CD8 T cells. However, vaccines aimed at stimulating CD8 T-cell responses are typically ineffective because vaccine antigens are primarily processed by the MHC class-II and not the MHC class-I pathway of antigen presentation: the latter requires cytosolic delivery of antigen. In order to facilitate targeting of antigen to cytosol, the antigen was combined with the photosensitiser TPCS2a (disulfonated tetraphenyl chlorin) and administered intradermally to mice. The photosensitiser was activated by illumination of the injection site. This photochemical internalization (PCI) strongly increased the stimulation of CD8 T-cell responses as measured by antigen-specific proliferation and secretion of pro-inflammatory cytokines. Fluorescence microscopy showed that delivery to cytosol was TPCS2a dependent and occurred by light-induced disruption of TPCS2a- and antigen-containing endosomes. PCI-based vaccination prevented growth of malignant B16 cells as compared with vaccination without PCI. In conclusion, PCI represents a potent tool for delivery of antigens to cytosol for stimulation of cytotoxic CD8 T-cell responses. This study demonstrated a first proof-of-principle for PCI-mediated immunisation with potential application in cancer immunotherapy.

Photochemical targeting of antigens to the cytosol for stimulation of MHC class-I-restricted T-cell responses.

Tumour chemotherapy with drugs is typically associated with severe systemic and local side effects for which reason immunotherapy represents a safer alternative. However, vaccination often fails to generate the required cytotoxic CD8 T-cell responses due to insufficient access of antigens to the cytosol and the MHC class I pathway of antigen presentation. One important issue of tumour research is therefore to develop strategies that allow cytosolic targeting or endosomal escape of tumour antigens. The objective of the current study was to test whether endocytosed antigen could be delivered to MHC class I by means of photochemical internalisation (PCI). Briefly, the antigen and the photosensitiser Amphinex were loaded in vitro onto bone-marrow-derived murine dendritic cells (DCs). After light activation, which is supposed to cause disruption of OVA- and Amphinex-containing endosomes, the DCs were cultured with OVA-specific CD8 T cells or used for immunisation of mice. PCI facilitated CD8 T-cell responses as measured by IFN-γ secretion in vitro and CD8 T-cell proliferation in vivo. In conclusion, the current proof-of-concept study is the first to describe PCI-mediated immunisation and the results revealed the feasibility of this novel technology in autologous vaccination for stimulation of CD8 T-cell responses.

Tetraphenylporphyrin tethered chitosan based carriers for photochemical transfection.

Water-soluble amphiphilic chitosan nanocarriers tethered with the photosensitizer meso-tetraphenylporphyrin were synthesized in a seven-step procedure, starting from 3,6-di-O-tert-butyldimethylsilyl-chitosan and 5-(p-aminophenyl)-10,15,20-triphenylporphyrin. The lipophilic photosensitizer could be introduced in a quantitative and reproducible reaction to give either 0.1 or 0.25 degrees of substitution per glucosamine monomer. Fluorescence and NMR investigations revealed the dynamic structures of the carriers, which formed nanoparticles in aqueous solution with a core of π-stacked photosensitizers. These carriers can then unfold in the lipophilic environment, and the photosensitizer moiety can thus be inserted into the cell membrane. The efficacy of the carriers for photochemical internalization (PCI) mediated gene delivery was evaluated in vitro using the HCT116/LUC human colon carcinoma cell line. The efficacy of transfection was comparable to what could be achieved by the reference compound and current clinical candidate TPCS(2a).

In vitro pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107.

Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

Syntheses and structure-activity relationship (SAR) studies of 2,5-diazabicyclo[2.2.1]heptanes as novel alpha7 neuronal nicotinic receptor (NNR) ligands.

Biaryl substituted 2,5-diazabicyclo[2.2.1]heptanes have been synthesized and tested for their affinity toward alpha7 neuronal nicotinic receptors (NNRs). SAR studies established that 5-N-methyl substituent, heteroaryl linker and the nature of terminal aryl group are critical for the ligand to achieve potent alpha7 NNR agonist activity.

Evaluation of alpha7 nicotinic acetylcholine receptor agonists and positive allosteric modulators using the parallel oocyte electrophysiology test station.

Neuronal acetylcholine receptors (nAChRs) of the alpha7 subtype are ligand-gated ion channels that are widely distributed throughout the central nervous system and considered as attractive targets for the treatment of various neuropsychiatric and neurodegenerative diseases. Both agonists and positive allosteric modulators (PAMs) are being developed as means to enhance the function of alpha7 nAChRs. The in vitro characterization of alpha7 ligands, including agonists and PAMs, relies on multiple technologies, but only electrophysiological measurements assess the channel activity directly. Traditional electrophysiological approaches utilizing two-electrode voltage clamp or patch clamp in isolated cells have very low throughput to significantly impact drug discovery. Abbott (Abbott Park, IL) has developed a two-electrode voltage clamp-based system, the Parallel Oocyte Electrophysiology Test Station (POETs()), that allows for the investigation of ligand-gated ion channels such as alpha7 nAChRs in a higher-throughput manner. We describe the utility of this technology in the discovery of selective alpha7 agonists and PAMs. With alpha7 agonists, POETs experiments involved both single- and multiple-point concentration-response testing revealing diverse activation profiles (zero efficacy desensitizing, partial, and full agonists). In the characterization of alpha7 PAMs, POETs testing has served as a reliable primary or secondary screen identifying compounds that fall into distinct functional types depending on the manner in which current potentiation occurred. Type I PAMs (eg, genistein, NS1738, and 5-hydroxyindole) increase predominantly the peak amplitude response, type II PAMs affect the peak current and current decay (eg, PNU-120,596 and 4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), and anothertype slowing the current decay kinetics in the absence of increases in the peak current. In summary, POETs technology allows for significant impact on higher throughput in the testing of alpha7 agonists and PAMs and for identification of compounds with unique profiles that could prove valuable in identifying an optimum in vitro profile in the development of therapeutics for which the alpha7 subtype is considered.

Stimulation of dopamine release by nicotinic acetylcholine receptor ligands in rat brain slices correlates with the profile of high, but not low, sensitivity alpha4beta2 subunit combination.

alpha4beta2 neuronal nicotinic receptors (nAChRs) can exist in high and low sensitivity states possibly due to distinct stoichiometries during subunit assembly: (alpha4)(2)(beta2)(3) pentamer (high sensitivity, HS) and (alpha4)(3)(beta2)(2) pentamer (low sensitivity, LS). To determine if there is a linkage between HS or LS states and receptor-mediated responses in brain, we profiled several clinically studied alpha4beta2* nAChR agonists for the displacement of radioligand binding to alpha4beta2 [(3)H]-cytisine sites in rat brain membranes, effects on stimulation of [(3)H]-dopamine release from slices of rat prefrontal cortex and striatum, and activation of HS and LS human alpha4beta2 nAChRs expressed in Xenopus laevis oocytes. Binding affinities (pK(i)) and potency (pEC(50)) values for [(3)H]-dopamine release closely correlated with a rank order: varenicline>(-)-nicotine>AZD3480>dianicline congruent with ABT-089. Further, a good correlation was observed between [(3)H]-dopamine release and HS alpha4beta2 pEC(50) values, but not between [(3)H]-dopamine release and LS alpha4beta2. The relative efficacies of the agonists ranged from full to partial agonists. Varenicline behaved as a partial agonist in stimulating [(3)H]-dopamine release and activating both HS and LS alpha4beta2 nAChRs expressed in oocytes. Conversely, ABT-089, AZD3480 and dianicline exhibited little efficacy at LS alpha4beta2 (<5%), were more effective at HS alpha4beta2 nAChRs, and in stimulating cortical and striatal [(3)H]-dopamine release >or=30%. In the presence of alpha-conotoxin MII to block alpha6beta2* nAChRs, the alpha4beta2* alpha-conotoxin-insensitive [(3)H]-dopamine release stimulated by these ligands correlates well with their interactions at HS, but not LS. In summary, this study provides support for HS alpha4beta2* nAChR involvement in neurotransmitter release.

In vitro pharmacological characterization of a novel allosteric modulator of alpha 7 neuronal acetylcholine receptor, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), exhibiting unique pharmacological profile.

Targeting alpha7 neuronal acetylcholine receptors (nAChRs) with selective agonists and positive allosteric modulators (PAMs) is considered a therapeutic approach for managing cognitive deficits in schizophrenia and Alzheimer's disease. In this study, we describe a novel type II alpha7 PAM, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), that exhibits a unique pharmacological profile. In oocytes expressing alpha7 nAChRs, A-867744 potentiated acetylcholine (ACh)-evoked currents, with an EC(50) value of approximately 1 microM. At highest concentrations of A-867744 tested, ACh-evoked currents were essentially nondecaying. At lower concentrations, no evidence of a distinct secondary component was evident in contrast to 4-naphthalen-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), another type II alpha7 PAM. In the presence of A-867744, ACh concentration responses were potentiated by increases in potency, Hill slope, and maximal efficacy. When examined in rat hippocampus CA1 stratum radiatum interneurons or dentate gyrus granule cells, A-867744 (10 microM) increased choline-evoked alpha7 currents and recovery from inhibition/desensitization, and enhanced spontaneous inhibitory postsynaptic current activity. A-867744, like other alpha7 PAMs tested [1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)urea (NS1738), TQS, and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596)], did not displace the binding of [(3)H]methyllycaconitine to rat cortex alpha7(*) nAChRs. However, unlike these PAMs, A-867744 displaced the binding of the agonist [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539) in rat cortex, with a K(i) value of 23 nM. A-867744 neither increased agonist-evoked responses nor displaced the binding of [(3)H]A-585539 in an alpha7/5-hydroxytryptamine(3) (alpha7/5-HT(3)) chimera, suggesting an interaction distinct from the alpha7 N terminus or M2-3 loop. In addition, A-867744 failed to potentiate responses mediated by 5-HT(3A) or alpha3beta4 and alpha4beta2 nAChRs. In summary, this study identifies a novel and selective alpha7 PAM showing activity at recombinant and native alpha7 nAChRs exhibiting a unique pharmacological interaction with the receptor.

Preclinical characterization of A-582941: a novel alpha7 neuronal nicotinic receptor agonist with broad spectrum cognition-enhancing properties.

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the alpha7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine alpha7 nAChR agonist, A-582941. A-582941 was found to exhibit high-affinity binding and partial agonism at alpha7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A-582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A-582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short-term recognition memory, memory consolidation, and sensory gating deficit. A-582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that alpha7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes.

Selective modulation of alpha7 nicotinic acetylcholine receptors (nAChRs) is thought to regulate processes impaired in schizophrenia, Alzheimer's disease, and other dementias. One approach to target alpha7 nAChRs is by positive allosteric modulation. Structurally diverse compounds, including PNU-120596, 4-naphthalene-1-yl-3a,4,5,9b-tetrahydro-3-H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), and 5-hydroxyindole (5-HI) have been identified as positive allosteric modulators (PAMs), but their receptor interactions and pharmacological profiles remain to be fully elucidated. In this study, we investigated interactions of these compounds at human alpha7 nAChRs, expressed in Xenopus laevis oocytes, along with genistein, a tyrosine kinase inhibitor. Genistein was found to function as a PAM. Two types of PAM profiles were observed. 5-HI and genistein predominantly affected the apparent peak current (type I) whereas PNU-120596 and TQS increased the apparent peak current and evoked a distinct weakly decaying current (type II). Concentration-responses to agonists [ACh, 3-[(3E)-3-[(2,4-dimethoxyphenyl)methylidene]-5,6-dihydro-4H-pyridin-2-yl]pyridine dihydrochloride (GTS-21), and N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)] were potentiated by both types, although type II PAMs had greater effects. When applied after alpha7 nAChRs were desensitized, type II, but not type I, PAMs could reactivate alpha7 currents. Both types of PAMs also increased the ACh-evoked alpha7 window currents, with type II PAMs generally showing larger potentiation. None of the PAMs tested increased nicotine-evoked Ca(2+) transients in human embryonic kidney 293 cells expressing human alpha4beta2 or alpha3beta4 nAChRs, although some inhibition was noted for 5-HI, genistein, and TQS. In summary, our studies reveal two distinct alpha7 PAM profiles, which could offer unique opportunities for modulating alpha7 nAChRs in vivo and in the development of novel therapeutics for central nervous system indications.