Bell-shaped Dose-response Curve Research Articles

Addition of Vitamin C Mitigates the Loss of Antioxidant Capacity, Vitality and DNA Integrity in Cryopreserved Human Semen Samples.

Cryopreservation of human spermatozoa is a necessity for males suffering from infertility who cannot produce fresh semen for insemination. However, current ART cryopreservation protocols are associated with losses of sperm motility, vitality and DNA integrity, which are thought to be linked to the induction of oxidative damage and the toxic properties of commercial cryoprotectants (CPAs). Preventing or mitigating these losses would be hugely beneficial to sperm survival during ART. Therefore, in this in vitro investigation, lipid peroxidation, production of reactive oxygen species, movement characteristics, antioxidant capacity, vitality, and DNA integrity were examined in semen samples both pre- and post-cryopreservation with CPA supplementation. The findings revealed a 50% reduction in antioxidant capacity with CPA addition, which was accompanied by significant increases in generation of reactive oxygen species and formation of lipid aldehydes. These changes were, in turn, correlated with reductions in sperm viability, motility and DNA integrity. Antioxidant supplementation generated bell-shaped dose-response curves with both resveratrol and vitamin C, emphasising the vulnerability of these cells to both oxidative and reductive stress. At the optimal dose, vitamin C was able to significantly enhance vitality and reduce DNA damage recorded in cryopreserved human spermatozoa. An improvement in sperm motility did not reach statistical significance, possibly because additional pathophysiological mechanisms limit the potential effectiveness of antioxidants in rescuing this aspect of sperm function. The vulnerability of human spermatozoa to reductive stress and the complex nature of sperm cryoinjury will present major challenges in creating the next generation of cryoprotective media.

Reconciling intrinsic properties of activating TNF receptors by native ligands versus synthetic agonists.

The extracellular domain of tumor necrosis factor receptors (TNFR) generally require assembly into a homotrimeric quaternary structure as a prerequisite for initiation of signaling via the cytoplasmic domains. TNF receptor homotrimers are natively activated by similarly homo-trimerized TNF ligands, but can also be activated by synthetic agonists including engineered antibodies and Fc-ligand fusion proteins. A large body of literature from pre-clinical models supports the hypothesis that synthetic agonists targeting a diverse range of TNF receptors (including 4-1BB, CD40, OX40, GITR, DR5, TNFRSF25, HVEM, LTβR, CD27, and CD30) could amplify immune responses to provide clinical benefit in patients with infectious diseases or cancer. Unfortunately, however, the pre-clinical attributes of synthetic TNF receptor agonists have not translated well in human clinical studies, and have instead raised fundamental questions regarding the intrinsic biology of TNF receptors. Clinical observations of bell-shaped dose response curves have led some to hypothesize that TNF receptor overstimulation is possible and can lead to anergy and/or activation induced cell death of target cells. Safety issues including liver toxicity and cytokine release syndrome have also been observed in humans, raising questions as to whether those toxicities are driven by overstimulation of the targeted TNF receptor, a non-TNF receptor related attribute of the synthetic agonist, or both. Together, these clinical findings have limited the development of many TNF receptor agonists, and may have prevented generation of clinical data which reflects the full potential of TNF receptor agonism. A number of recent studies have provided structural insights into how different TNF receptor agonists bind and cluster TNF receptors, and these insights aid in deconvoluting the intrinsic biology of TNF receptors with the mechanistic underpinnings of synthetic TNF receptor agonist therapeutics.

S1PR-1/5 modulator RP-101074 shows beneficial effects in a model of central nervous system degeneration.

In multiple sclerosis (MS), chronic disability primarily stems from axonal and neuronal degeneration, a condition resistant to conventional immunosuppressive or immunomodulatory treatments. Recent research has indicated that selective sphingosine-1-phosphate receptor S1PR-1 and -5 modulators yield positive effects in progressive MS and mechanistic models of inflammation-driven neurodegeneration and demyelination. In this study, the S1PR-1/-5 modulator RP-101074 was evaluated as a surrogate for ozanimod in the non-inflammatory, primary degenerative animal model of light-induced photoreceptor loss (LI-PRL) in CX3CR1-GFP mice to assess potential neuroprotective effects, independent of its immunomodulatory mechanism of action. Prophylactic administration of RP-101074 demonstrated protective effects in the preclinical, non-inflammatory LI-PRL animal model, following a bell-shaped dose-response curve. RP-101074 treatment also revealed activity-modulating effects on myeloid cells, specifically, CX3CR1+ cells, significantly reducing the marked infiltration occurring one week post-irradiation. Treatment with RP-101074 produced beneficial outcomes on both retinal layer thickness and visual function as evidenced by optical coherence tomography (OCT) and optomotor response (OMR) measurements, respectively. Additionally, the myelination status and the quantity of neural stem cells in the optic nerve suggest that RP-101074 may play a role in the activation and/or recruitment of neural stem cells and oligodendrocyte progenitor cells, respectively. The data from our study suggest that RP-101074 may have a broader role in MS treatment beyond immunomodulation, potentially offering a novel approach to mitigate neurodegeneration, a core contributor to chronic disability in MS.

Abstract 2929: Modular peptide cargo delivery by targeting CD40 enables ligandome driven, precision immunotherapy via the Adaptable Drug Affinity Conjugate (ADAC™) technology

Abstract Agonistic anti-CD40 therapy has shown impressive efficacy in preclinical murine models, models inert to toxicity of high systemic drug exposure. Clinical data consist of pharmacodynamic responses reported as non-specific cytokine release, transient drop of CD40-expressing cells in blood and induction of co-stimulatory ligands/receptors on antigen-presenting cells, but so far clinically meaningful efficacy data on monotherapy use is lacking. Clinical data also indicate a bell-shaped dose-response curve, with excessive immune activation leading to immune exhaustion when combined with check-point inhibitors[1]. Intratumoral route of administration, bispecific antibodies for tumor drug localization or Antibody-Drug Conjugates (ADCs) carrying antigenic cargo are or have been assessed, but have associated clinical utility enigmas. Herein we present a novel solution ensuring modular peptide delivery along with an efficient T cell priming strategy; a bivalent anti-CD40 agonistic antibody equipped with a peptide-binding single chain variable fragment (scFv) that binds a short peptide tag (pTag) in the low nM range. The resulting drug strategy makes use of synthetic long peptide production of any tumor-associated antigen (TAA) of choice where the pTag itself ensures simple conjugate production to the protein, through affinity linkage, leading to a final rapid in-hospital mixing step. Data suggest that the novel candidate drug (STRIKE2001), now developed based on the ADAC concept[2], retains similar agonistic activity in its bispecific, IgG2 format and does not inhibit CD40L binding to the CD40 protein. Data further show impressive expansion of endogenous tumor-specific T cells (as measured by tetramer staining), along with effective anti-tumor responses in the TC1 model. In addition, by using the subcutaneous delivery strategy CD40 agonist exposure to liver and spleen is vastly reduced, limiting systemic immune toxicity/exhaustion risks[3]. [1] Padrón, L.J., et al. Sotigalimab and/or nivolumab with chemotherapy in first-line metastatic pancreatic cancer: clinical and immunologic analyses from the randomized phase 2 PRINCE trial. Nat Med 28, 1167-1177 (2022). https://doi.org/10.1038/s41591-022-01829-9 [2] Eltahir, M., et al. (2022), An Adaptable Antibody-Based Platform for Flexible Synthetic Peptide Delivery Built on Agonistic CD40 Antibodies. Adv. Therap., 5: 2200008. https://doi.org/10.1002/adtp.202200008 [3] Sandin, L., et al. Locally Delivered CD40 Agonist Antibody Accumulates in Secondary Lymphoid Organs and Eradicates Experimental Disseminated Bladder Cancer. Cancer Immunol Res 1 January 2014; 2 (1): 80-90. https://doi.org/10.1158/2326-6066.CIR-13-0067 Citation Format: Aman Mebrahtu, Ida Lauren, Rosanne Veerman, Alexandros Kostakis, Gözde Gucluler Akpinar, Oskar Andersson, Lindvi Gudmundsdotter, Tina Furebring, Helena Persson, Pierre Donnes, Johan Rockberg, Sara Mangsbo. Modular peptide cargo delivery by targeting CD40 enables ligandome driven, precision immunotherapy via the Adaptable Drug Affinity Conjugate (ADAC™) technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2929.

N-Formyl-L-aspartate mediates chemotaxis in sperm via the beta-2-adrenergic receptor.

Chemotaxis is a highly conserved physiological event required for directed sperm movement during fertilization. Recently, studies from our laboratory have identified N-formyl-L-aspartate (NFA) as a sperm chemoattractant. NFA is a known agonist for the beta-2-adrenergic receptor (β-2-AR) that regulates cAMP production and Ca2+ mobilization in somatic cells. As these downstream signaling molecules are also reported to be involved in sperm chemotaxis, in the present study we investigated the putative mechanism/s by which NFA may mediate chemotaxis. Toward this, the expression and localization of β-2-AR in sperm were studied by Western blot and indirect immunofluorescence, respectively. The responses of sperm to various concentration gradients of NFA and ICI-118,551, a β-2-AR specific antagonist, were evaluated using the microfluidics device-based chemotaxis assay. The intracellular concentration of Ca2+, on exposure to NFA, was analyzed using FURA-2 AM-based fluorimetric assay. Furthermore, the effect of NFA on sperm capacitation and acrosome reaction was evaluated using Western blot and immunofluorescence. NFA exhibited a bell-shaped dose-response curve typical of chemotaxis, with maximum response observed at 0.01M NFA, beyond which it was inhibitory; β-2-AR localization was seen on the sperm head and the mid-piece region of the flagella. Inhibition of sperm chemotaxis by ICI-118,551 confirms that sperm respond chemotactically to NFA via β-2-AR. Interestingly, at the concentration used for chemotaxis, NFA induced an increase in the intracellular Ca2+ but decreased cAMP in capacitating sperm. However, NFA per se did not induce capacitation as seen from the lack of effect on tyrosine phosphorylation and membrane potential of uncapacitated sperm. Acrosome evaluation of NFA-treated sperm using PSA-FITC staining showed no effect on the acrosome structure. Our data thus provide evidence indicating that NFA induces sperm chemotaxis and the chemotactic response of sperm to NFA from the ovulatory phase of oviductal fluid is mediated through the β-2-AR on sperm possibly via non-canonical signaling.

Memantine has a nicotinic neuroprotective pathway in acute hippocampal slices after an NMDA insult

Memantine is a non-competitive antagonist with a moderate affinity to the N-methyl-d-Aspartate (NMDA) receptor. The present study assessed memantine's neuroprotective activity using electrophysiology of ex-vivo hippocampal slices. Interestingly, a nicotinic component was necessary for memantine's neuroprotection (NP). Memantine demonstrated a bell-shaped dose-response curve of NP against NMDA. Memantine was neuroprotective at concentrations below 3 μM, but the NP declined at higher concentrations (>3 μM) when memantine inhibits the NMDA receptor. Additional evidence that memantine NP is mediated by an alternate mechanism independent of the inhibition of the NMDA receptor is supported by its ability to protect neurons when applied before or after the NMDA insult and in the presence of D(−)-2-Amino-5-phosphonopentanoic acid (APV), the standard NMDA receptor inhibitor. We found several similarities between the memantine NP mechanism and the neuroprotective nicotinic drug, the 4R cembranoid. Memantine's NP requires the release of acetylcholine, the activation of α4β2, and is independent of MEK/MAPK signaling. Both 4R and memantine require the activation of PI3K/AKT for NP against NMDA-mediated excitotoxicity, although at different concentrations. In conclusion, our studies show memantine is neuroprotective through a nicotinic pathway, similar to the nicotinic drug 4R. This information leads to a better understanding of memantine's mechanisms of action and explains its dose-dependent effectiveness in Alzheimer's and other neurological disorders.

Ataluren suppresses a premature termination codon in an MPS I-H mouse

AbstarctSuppressing translation termination at premature termination codons (PTCs), termed readthrough, is a potential therapy for genetic diseases caused by nonsense mutations. Ataluren is a compound that has shown promise for clinical use as a readthrough agent. However, some reports suggest that ataluren is ineffective at suppressing PTCs. To further evaluate the effectiveness of ataluren as a readthrough agent, we examined its ability to suppress PTCs in a variety of previously untested models. Using NanoLuc readthrough reporters expressed in two different cell types, we found that ataluren stimulated a significant level of readthrough. We also explored the ability of ataluren to suppress a nonsense mutation associated with Mucopolysaccharidosis I-Hurler (MPS I-H), a genetic disease that is caused by a deficiency of α-L-iduronidase that leads to lysosomal accumulation of glycosaminoglycans (GAGs). Using mouse embryonic fibroblasts (MEFs) derived from Idua-W402X mice, we found that ataluren partially rescued α-L-iduronidase function and significantly reduced GAG accumulation relative to controls. Two-week oral administration of ataluren to Idua-W402X mice led to significant GAG reductions in most tissues compared to controls. Together, these data reveal important details concerning the efficiency of ataluren as a readthrough agent and the mechanisms that govern its ability to suppress PTCs.Key messagesAtaluren promotes readthrough of PTCs in a wide variety of contexts.Ataluren reduces glycosaminoglyan storage in MPS I-H cell and mouse models.Ataluren has a bell-shaped dose–response curve and a narrow effective range.

P-568 The effect of recombinant hCG on FSH-induced ovarian stimulation in rats depends on the FSH dose and can be detrimental at high concentrations

Abstract Study question What is the effect of choriogonadotropin beta (CG beta) on FSH-induced ovarian stimulation and multifollicular development in a rat model? Summary answer CG beta dose-dependently potentiates effects of low-to-mid FSH doses but has inhibitory effects at high concentrations: optimal CG beta/FSH ratio depends on the FSH dose. What is known already Similarly to follitropin delta (rFSH), CG beta (FE 999302) is a novel recombinant hCG purified from the human PER.C6®cell line. A recent placebo-controlled trial in women undergoing ovarian stimulation with follitropin delta demonstrated that the addition of 1 to 12 µg CG beta reduced the number of intermediate follicles and related hormones. This observation required further preclinical research to (1) evaluate whether the pharmacology of CG beta at LH/CGR was different than other hCG forms used in the clinic and/or (2) assess the effect of high concentrations of hCG and different hCG/FSH ratios on multiple follicular development and follicle atresia. Study design, size, duration Signaling properties of CG beta and other LH/hCG forms were compared at downstream pathways of LH/CGR in recombinant systems and human granulosa cells. To evaluate the effects of FSH±hCG in vivo, juvenile female rats were injected subcutaneously twice daily with follitropin delta ± CG beta/alfa for three days followed by an ovulatory dose of hCG. Oviducts were then collected for oocyte enumeration, ovaries and uteri were weighed, and ovaries were fixed for histological analysis. Participants/materials, setting, methods The pharmacology of CG beta and other LH/hCG forms was evaluated in a cAMP assay in human granulosa cells from follicular fluid from IVF patients and in recombinant systems, at the Gs, Gq and arrestin pathways. In the rat model, a dose response of follitropin delta (Rekovelle) was first evaluated, followed by evaluation of the dose-dependent effects of CG beta (0.00117-2.4 µg/kg), or CG alfa (Ovidrel/Ovitrelle), in combination with 1, 3 or 10 µg/kg rFSH. Main results and the role of chance The in vitro pharmacology (potency and efficacy) of CG beta was similar to recombinant LH, urinary hCG and recombinant hCG (CG alfa) tested at all proximal pathways evaluated downstream of LH/CGR as well as in human granulosa cells. In vivo, treatment with follitropin delta induced a bell-shaped dose-response curve for oocyte release with a maximum response of 40-50 oocytes at 8-10 µg/kg follitropin delta dose. The addition of CG beta dose-dependently potentiated the effects at low-to-mid follitropin delta doses but had inhibitory effects on the number of ovulated oocytes at high CG beta concentrations. The lowest CG beta dose that clearly reduced the number of ovulated oocytes was 2.4, 0.6 and 0.3 µg/kg in combination with a fixed dose of 1, 3 and 10 µg/kg follitropin delta, respectively, which indicated that the optimal hCG/FSH ratio and corresponding hCG efficacious dose was inversely related to the FSH dose. There was no difference between CG beta and CG alfa for the dose effect on the number of ovulated oocytes or ovarian weight. Histology data indicated many cystic follicles following high CG beta exposure which may represent atretic follicles prior to triggering follicular maturation and ovulation. Limitations, reasons for caution This is the first study demonstrating that the FSH dose in combination with the hCG dose determines the effect on multiple follicle growth, ovulation, and atresia. These observations need to be confirmed in clinical research, as doses and ratios applied in the rat cannot be extrapolated to the clinical setting. Wider implications of the findings A better understanding of the effect of different FSH to hCG ratios will help to improve current mixed protocols and design future recombinant combination products providing the optimal treatment outcome for each individual patient. Trial registration number not applicable

Nanotherapeutics for hydrogen sulfide-involved treatment: An emerging approach for cancer therapy

Abstract Hydrogen sulfide (H2S), as the third gasotransmitter, plays important roles in promoting the growth, proliferation, and metastasis of cancer cells. Recently, emerging nanotherapeutics have been developed for cancer therapy by regulating the H2S concentration at tumor sites. The action of H2S in cancer therapy can be demonstrated by a bell-shaped dose-response curve. In addition to the H2S donation from gas therapy, reducing the level of intratumoral H2S can also exert an antitumor effect by disturbing the redox balance of tumor cells. On top of that, the integration of nanotechnology further enhances the diversity of H2S-involved cancer treatments. As a result, phototherapy, chemotherapy, immunotherapy, and ultrasonic therapy can all be realized by designing nanotherapeutics based on the physicochemical properties of H2S. Furthermore, the active/passive targeting effects and onsite H2S-regulating ability of nanotherapeutics greatly improve the safety of treatment. To track the development and promote the clinical transformation of H2S-involved nanotherapeutics, this review introduced the antitumor-related properties of H2S gas and the evolution of H2S-involved cancer therapy. Then, we discussed the design concepts and mechanisms of H2S-involved nanotherapeutics in depth. Finally, the future expectations and challenges were spotlighted in the concept of translational medicine.

Abstract 415: The characterization of in vitro models demonstrating a significant delay of acquired resistance to ulixertinib (ERK1/2)

Abstract Ulixertinib (BVD-523) is a first-in-class small molecule inhibitor of ERK1/2 currently being investigated in several oncology clinical trials, both as a single agent and in combination with other anti-cancer therapeutics. Clinical acquired resistance has been described by others for numerous efficacious small molecule agents. Relevant to the MAPK pathway, emergence of resistance to BRAF and MEK inhibitors limits their clinical efficacy. In vitro models of ulixertinib resistance were generated in an endeavor to characterize and predict potential clinical mechanisms of resistance and guide rational combination therapies. The BRAF-V600E mutant melanoma cell line, A375, was cultured in progressively increasing concentrations of ulixertinib or other MAPK pathway inhibitors (dabrafenib and/or trametinib). Drug-resistant A375 clones were readily obtained following growth in high concentrations of dabrafenib or trametinib. In contrast, developing resistance to ulixertinib proved challenging. Eventually, ulixertinib resistant clones emerged and demonstrated varying degrees of cross-resistance to other MAPK pathway inhibitors and displayed bell-shaped dose-response curves indicative of drug addiction for optimal proliferation. To elucidate potential mechanisms of resistance, Reverse Phase Protein Array profiling of 306 proteins was performed on parental A375 and ulixertinib-resistant clones following treatment with ulixertinib at varying concentrations and treatment times. Principal component analysis revealed clustering of resistant clones by ulixertinib treatment conditions (concentrations and duration). Differences between parental A375 and ulixertinib resistant clones were revealed, including components of MAPK, HER2, and autophagy markers. This work begins to tease out potential mechanisms of resistance to ulixertinib and guide potential combination partners that could circumvent acquired resistance. Citation Format: Deborah Knoerzer, Anupama Reddy, David Sorrell, Caroline M. Emery. The characterization of in vitro models demonstrating a significant delay of acquired resistance to ulixertinib (ERK1/2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 415.

Abstract 4159: Lower doses of self-amplifying mRNA drive superior neoantigen-specific CD8 T cell responses in cancer patients versus high doses

Abstract The immunogenicity and efficacy of RNA-based vaccine platforms has been abundantly shown through their application in prophylactic SARS-CoV2 vaccines. Contrasting to mRNA based vectors, self amplifying mRNA platforms may offer dose-sparing and superior induction of T cell responses, and may also trigger distinct innate immune pathways, which may exert adjuvanting or inhibiting effects on vaccine-induced immunity. Optimal dosing for a novel self-amplifying mRNA (SAM) in a heterologous prime-boost vaccination approach consisting of Chimpanzee Adenovirus (ChAd) prime and SAM boosts was evaluated in two first-in-human phase 1/2 clinical trials assessing personalized neoantigen vaccines in patients with metastatic cancer (NCT03639714, NCT03953235). SAM vaccine dose escalation was performed to assess safety, tolerability, and immunogenicity, including administration of up to 8 SAM doses at 30, 100, or 300µg following a fixed dose of ChAd (1012 vp) over the course of a year. SAM was safe and well tolerated at all 3 dose levels, with no evidence of increasing reactogenicity with sequential doses. However, while immune monitoring via IFNγ ELISpot revealed that the 30µg SAM dose boosted T cell responses induced by the ChAd prime, the 100µg and 300µg SAM doses resulted in maintenance of T cell levels, without a clear T cell boost, suggesting a non-linear and likely bell-shaped dose-response curve to SAM in humans. Follow-up studies in non-human primates (NHPs) using a model antigen revealed dose-dependent increases in serum IFNa levels following administration of increasing SAM doses. Similarly, while multiple inflammatory cytokines were transiently increased following both ChAd and SAM administration in patients, serum IFNa levels were only increased 24h post SAM administration and correlated positively with SAM dose. Increased IFNa levels post SAM dosing suggested activation of mRNA-sensing innate immune pathways that may reduce the amplification of, and/or antigen expression by, the SAM vector and thus blunt T cell boosting at higher SAM doses. In addition, analysis of T cell responses in patients and NHPs showed increased boosting of T cell responses with longer intervals. These data lead to a reduction of the SAM dose to 30µg and adjusting SAM dosing intervals to 8 weeks in the Phase 2 portion of these clinical studies. Multiple patients have been dosed with the adjusted vaccine regimen, and preliminary data suggest robust boosting of ChAd-induced neoantigen-specific T cell responses with the selected SAM dosing regimen and the 30µg dose. We anticipate that this translational approach of adjusting clinical vaccine regimens based on strong translational immune data will increase the potency of our heterologous neoantigen vaccine, and subsequently provide more durable clinical benefit to patients with cancer. Citation Format: Christine D. Palmer, Amy R. Rappaport, Meghan G. Hart, Lauren D. Kraemer, Sonia Kounlavouth, Martina Marrali, Jason R. Jaroslavsky, Charmaine N. Nganje, Annie Shen, Gregory R. Boucher, Melissa A. Kachura, Ciaran D. Scallan, Sue-Jean Hong, Leonid Gitlin, Alexander I. Spira, Chrisann Kyi, Daniel V. Catenacci, Raphael Rousseau, Andrew Ferguson, Karin Jooss. Lower doses of self-amplifying mRNA drive superior neoantigen-specific CD8 T cell responses in cancer patients versus high doses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4159.

Increased Remyelination and Proregenerative Microglia Under Siponimod Therapy in Mechanistic Models.

Background and ObjectivesSiponimod is an oral, selective sphingosine-1-phosphate receptor-1/5 modulator approved for treatment of multiple sclerosis.MethodsMouse MRI was used to investigate remyelination in the cuprizone model. We then used a conditional demyelination Xenopus laevis model to assess the dose-response of siponimod on remyelination. In experimental autoimmune encephalomyelitis–optic neuritis (EAEON) in C57Bl/6J mice, we monitored the retinal thickness and the visual acuity using optical coherence tomography and optomotor response. Optic nerve inflammatory infiltrates, demyelination, and microglial and oligodendroglial differentiation were assessed by immunohistochemistry, quantitative real-time PCR, and bulk RNA sequencing.ResultsAn increased remyelination was observed in the cuprizone model. Siponimod treatment of demyelinated tadpoles improved remyelination in comparison to control in a bell-shaped dose-response curve. Siponimod in the EAEON model attenuated the clinical score, reduced the retinal degeneration, and improved the visual function after prophylactic and therapeutic treatment, also in a bell-shaped manner. Inflammatory infiltrates and demyelination of the optic nerve were reduced, the latter even after therapeutic treatment, which also shifted microglial differentiation to a promyelinating phenotype.DiscussionThese results confirm the immunomodulatory effects of siponimod and suggest additional regenerative and promyelinating effects, which follow the dynamics of a bell-shaped curve with high being less efficient than low concentrations.

Direct evidence of the impact of aqueous self-assembly on biological behavior of amphiphilic molecules: The case study of molecular immunomodulators Sulfavants

Sulfavant A and Sulfavant R, sulfoquinovoside-glycerol lipids under study as vaccine adjuvants, structurally differ only for the configuration of glyceridic carbon, R/S and R respectively. The in vitro activity of these substances follows a bell-shaped dose–response curve, but Sulfavant A gave the best response around 20 µM, while Sulfavant R at 10 nM. Characterization of aqueous self-assembly of these molecules by a multi-technique approach clarified the divergent and controversial biological outcome. Supramolecular structures were present at concentrations much lower than critical aggregation concentration for both products. The kind and size of these aggregates varied as a function of the concentration differently for Sulfavant A and Sulfavant R. At nanomolar range, Sulfavant A formed cohesive vesicles, while Sulfavant R arranged in spherical micellar particles whose reduced stability was probably responsible for an increase of monomer concentration in accordance with immunomodulatory profile. Instead, at micromolar concentrations transition from micellar to vesicular state of Sulfavant R occurred and thermodynamic stability of the aggregates, assessed by surface tensiometry, correlated with the bioactivity of Sulfavant A at 20 µM and the complete loss of efficacy of Sulfavant R. The study of Sulfavants provides clear evidence of how self-aggregation, often neglected, and the equilibria between monomers and aqueous supramolecular forms of lipophilic molecules deeply determine the overall bio-response.

DNA Mediated Blood Coagulation: Extracellular DNA Accelerates Fibrinogen Polymerization By Thrombin Independent of Contact Pathway

Introduction- Several in vivo studies and clinical studies have demonstrated extracellular DNA as a mediator of coagulation and this effect was reversed by the administration of DNA degrading enzyme DNase I. However, there is no clear understanding of the mechanism by which extracellular DNA activates coagulation in vitro. Conventionally, it was thought to be the activator of the contact pathway. But recent studies have shown that extracellular DNA isolated without contaminants like silica particles is a weak activator of the contact pathway. In this study, we have investigated the mechanism by which extracellular DNA is contributing to coagulation. Corroborating with recent results, we show that extracellular genomic DNA is a weak activator contact pathway. We determined that extracellular DNA accelerate fibrinogen polymerization by thrombin via a possible template mechanism. Our biophysical studies corroborate the interaction of DNA, thrombin, and fibrinogen. Understanding the mechanism of DNA induced blood coagulation will help address the gaps in the literature as well as develop inhibitors against DNA- mediated thrombosis.Methods- Silica-free extracellular DNA was purified with the PAXgene™ Blood DNA Kit. Contact activation in plasma was measured by monitoring the cleavage of the substrate S2302. To study the contact independent activation of plasma clotting by extracellular DNA, 1.5 µM Corn Trypsin Inhibitor (CTI) was applied to the plasma. Next, acceleration of fibrinogen polymerization by thrombin in presence of extracellular DNA was measured by monitoring the absorbance of 350 nm. Interaction of DNA with fibrinogen and thrombin in phosphate buffer was determined by CD spectroscopy.Results- Our results show that silica-free extracellular genomic DNA is a weak activator of the contact pathway of coagulation [Fig-A]. Moreover, genomic DNA accelerated the plasma clotting even when the contact pathway was inhibited with CTI indicating a contact independent mechanism of the procoagulant activity of extracellular DNA. Interestingly, the presence of extracellular DNA accelerated the polymerization of fibrinogen in presence of thrombin [Fig-B]. A bell-shaped dose-response curve for extracellular DNA indicates a likely template mechanism in which both thrombin and fibrinogen could assemble on the DNA molecule. These results are supported by the results from the CD spectroscopy studies where an alteration of the structure of fibrinogen and thrombin can be noticed in presence of extracellular DNA. Confocal studies further corroborate this observation. Our results also show different nucleic acids activate coagulation via different pathways.Significance- Procoagulant activity of extracellular DNA is demonstrated in several mouse models. However, a clear understanding of the mechanism of procoagulant activity of DNA in vitro has been challenging due to the caveats in the isolation of extracellular DNA where it is often contaminated with silica particles. Here we show a novel procoagulant mechanism of cell- free DNA where it augments the polymerization of fibrinogen by thrombin. These results provide insights into the mechanism of procoagulant activity of DNA which is key to develop therapeutics against procoagulant DNA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Assessment of the Anti-Allodynic and Anti-Hyperalgesic Efficacy of a Glycine Transporter 2 Inhibitor Relative to Pregabalin, Duloxetine and Indomethacin in a Rat Model of Cisplatin-Induced Peripheral Neuropathy.

Cisplatin, which is a chemotherapy drug listed on the World Health Organisation’s List of Essential Medicines, commonly induces dose-limiting side effects including chemotherapy-induced peripheral neuropathy (CIPN) that has a major negative impact on quality of life in cancer survivors. Although adjuvant drugs including anticonvulsants and antidepressants are used for the relief of CIPN, analgesia is often unsatisfactory. Herein, we used a rat model of CIPN (cisplatin) to assess the effect of a glycine transporter 2 (GlyT2) inhibitor, relative to pregabalin, duloxetine, indomethacin and vehicle. Male Sprague-Dawley rats with cisplatin-induced mechanical allodynia and mechanical hyperalgesia in the bilateral hindpaws received oral bolus doses of the GlyT2 inhibitor (3–30 mg/kg), pregabalin (3–100 mg/kg), duloxetine (3–100 mg/kg), indomethacin (1–10 mg/kg) or vehicle. The GlyT2 inhibitor alleviated both mechanical allodynia and hyperalgesia in the bilateral hindpaws at a dose of 10 mg/kg, but not at higher or lower doses. Pregabalin and indomethacin induced dose-dependent relief of mechanical allodynia but duloxetine lacked efficacy. Pregabalin and duloxetine alleviated mechanical hyperalgesia in the bilateral hindpaws while indomethacin lacked efficacy. The mechanism underpinning pain relief induced by the GlyT2 inhibitor at 10 mg/kg is likely due to increased glycinergic inhibition in the lumbar spinal cord, although the bell-shaped dose–response curve warrants further translational considerations.

Oxytocin Receptor Activation Rescues Opioid-Induced Respiratory Depression by Systemic Fentanyl in the Rat.

Opioid overdose intervention by naloxone, a high affinity receptor antagonist, reverses opioid-induced respiratory depression (OIRD) and analgesia by displacing opioids. Systemic naloxone stimulates release of the hypothalamic neuropeptide oxytocin, which has analgesic properties and participates in cardiorespiratory homeostasis. To test the hypothesis that oxytocin can reverse OIRD, we assessed the rescue potential of graded doses (0, 0.1, 2, 5, 10, 50 nmol/kg, i.v.) of oxytocin to counter fentanyl (60 nmol/kg, i.v.)-induced depression of neural inspiration indexed by recording phrenic nerve activity (PNA) in anesthetized (urethane/α-chloralose), vagotomized, and artificially ventilated rats. Oxytocin dose-dependently rescued fentanyl OIRD by almost immediately reversing PNA burst arrest (P = 0.0057) and restoring baseline burst frequency (P = 0.0016) and amplitude (P = 0.0025) at low but not high doses, resulting in inverted bell-shaped dose-response curves. Oxytocin receptor antagonism (40 nmol/kg, i.v.) prevented oxytocin reversal of OIRD (arrest: P = 0.0066, frequency: P = 0.0207, amplitude: P = 0.0022). Vasopressin 1A receptor (V1aR) antagonism restored high-dose oxytocin efficacy to rescue OIRD (P = 0.0170 to P < 0.0001), resulting in classic sigmoidal dose-response curves, and prevented (P = 0.0135) transient hypertension from V1aR cross-activation (P = 0.0275). Alone, vasopressin (5 nmol/kg, i.v.) failed to reverse fentanyl respiratory arrest (P = 0.6184). The nonpeptide oxytocin receptor agonist WAY-267464 (75 nmol/kg, i.v.), which has V1aR antagonist properties, quickly reversed fentanyl OIRD (P < 0.0001), with rapid recovery of PNA frequency (P = 0.0011) and amplitude (P = 0.0044) without adverse hemodynamic consequences (P = 0.9991). Findings indicate that peptide and nonpeptide agonist activation of oxytocin receptors without V1aR cross-activation rescues fentanyl OIRD. Oxytocin receptor agonists could be lifesaving resuscitation agents that enhance rather than interrupt opioid analgesia. SIGNIFICANCE STATEMENT: Oxytocin receptor activation produces analgesia. Here, we demonstrate that activation by the US Food and Drug Administration-approved agonist oxytocin and the nonpeptide partial agonist WAY-267464 can each reverse fentanyl cardiorespiratory depression. Selective targeting of oxytocin receptors for resuscitation from opioid overdose, alone or in combination with an opioid antagonist, could eliminate or attenuate negative side effects associated with traditional opioid receptor antagonism.

Modulation of the Cholesterol-Dependent Activity of Macrophages IC-21 by CRAC Peptides with Substituted Motif-Forming Amino Acids.

The activity of many membrane proteins, such as receptors, ionic channels, transporters, and enzymes, is cholesterol dependent; however, mechanisms of the cholesterol-dependent regulation of protein functions remain obscure. Recent studies suggest that membrane proteins can directly interact with cholesterol owing to the presence of the cholesterol-recognizing amino-acid consensus (CRAC) motifs. One of the ways to verify and further develop this notion is a design of CRAC-containing peptides and investigation of their effects on cholesterol-dependent cell functions. Previously we showed that a newly constructed peptide RTKLWEMLVELGNMDKAVKLWRKLKR (peptide P4) containing two CRAC motifs modulates cholesterol-dependent interactions of cultured macrophages IC-21 with 2-μm particles. In this work, in order to clarify the role of CRAC-forming amino acids, we employed the same experimental system to test the activity of peptides closely related to P4 but with modified CRAC motifs. We found that peptide STKLSEMLSELGNMDKASKLSRKLSR (Mut2) analogous to P4, except that all CRAC-forming amino acids (V, W, K/R) were substituted by serine, did not produce any effect in the concentration range 0.5–50 μM corresponding to the range of the P4 activity. Neither was effective peptide RTKLSEMLVELGNMDKAVKLSRKLKR (Mut3), in which only aromatic amino acids (W) of the CRAC motifs were substituted. Peptide STKLWEMLVELGNMDKAVKLWRKLSR (Mut4), in which only cationic amino acids (R/K) in the CRAC motifs were changed, produced almost the same effect as that of peptide P4 with a bell-shape dose–response curve. At low concentrations (1–4 μM) Mut4 notably increased the number of beads per cell, at higher concentrations this parameter diminished, and at 50 μM Mut4 produced a robust toxic effect. Finally, peptide EWGMAVLWERNRKLKKDLKVLKMLRT (Mut1) composed of the same amino acid residues as P4 but in a random order (“scramble”) and possessing one CRAC motif, different from that in P4, produced a moderate stimulation at 4–10 μM but was not toxic at 50 μM. As in the case of peptide P4, the effects of Mut4 and Mut1 depended on the cholesterol content in the cell membrane: after the incubation of cells with cholesterol-extracting agent methyl-β-cyclodextrin stimulatory effects produced by Mut4 and Mut1 at low doses were suppressed. Our results indicate that CRAC motifs play an important role in the mechanisms of the peptide-induced modulations of cholesterol-dependent cell functions in the experimental system used and that of the three motif-forming amino acids, critical is the presence of the aromatic amino acid (W). Further research is required to comprehend the molecular mechanisms of interactions of CRAC-containing peptides with cell membrane components that lead to modulation of cell functions. We anticipate that CRAC-containing peptides may provide a basis for the development of new tools for directed regulation of the activity of target cholesterol-dependent membrane proteins and for the design of new antimicrobial and immunomodulating drugs in particular.

Biased Opioid Antagonists as Modulators of Opioid Dependence: Opportunities to Improve Pain Therapy and Opioid Use Management.

Opioid analgesics are effective pain therapeutics but they cause various adverse effects and addiction. For safer pain therapy, biased opioid agonists selectively target distinct μ opioid receptor (MOR) conformations, while the potential of biased opioid antagonists has been neglected. Agonists convert a dormant receptor form (MOR-μ) to a ligand-free active form (MOR-μ*), which mediates MOR signaling. Moreover, MOR-μ converts spontaneously to MOR-μ* (basal signaling). Persistent upregulation of MOR-μ* has been invoked as a hallmark of opioid dependence. Contrasting interactions with both MOR-μ and MOR-μ* can account for distinct pharmacological characteristics of inverse agonists (naltrexone), neutral antagonists (6β-naltrexol), and mixed opioid agonist-antagonists (buprenorphine). Upon binding to MOR-μ*, naltrexone but not 6β-naltrexol suppresses MOR-μ*signaling. Naltrexone blocks opioid analgesia non-competitively at MOR-μ*with high potency, whereas 6β-naltrexol must compete with agonists at MOR-μ, accounting for ~100-fold lower in vivo potency. Buprenorphine’s bell-shaped dose–response curve may also result from opposing effects on MOR-μ and MOR-μ*. In contrast, we find that 6β-naltrexol potently prevents dependence, below doses affecting analgesia or causing withdrawal, possibly binding to MOR conformations relevant to opioid dependence. We propose that 6β-naltrexol is a biased opioid antagonist modulating opioid dependence at low doses, opening novel avenues for opioid pain therapy and use management.

Bell-shaped dose-response curve of antipsychotic drugs and dopaminergic auto-receptors: a hypothesis

Bell-shaped dose-response curve of antipsychotic drugs and dopaminergic auto-receptors: a hypothesis

Is the bell-shaped dose-response curve of the selective serotonin reuptake inhibitor due to 5-HT1A auto-receptors?

A dose-response curve is a plot of drug efficacy versus dose. Interestingly, some antidepressants show a bell-shaped dose-response curve where increasing dose leads to increasing efficacy only up to a point, whereupon further increases lead to decreasing efficacy. Here, we propose that the first part of the curve reflects the basic, uncomplicated dose-response relationship of these antidepressants whereas the second, decreasing part remains to be explained. Our hypothesis is that a negative feedback pathway through 5-HT1A auto-receptors decreases the efficacy of selective serotonin reuptake inhibitors with increasing dose, thereby creating the second, anomalous part of the dose-response curve. This effect can also account for the so-called therapeutic window of such antidepressants.