Recent advances in bioaffinity strategies for preclinical and clinical drug discovery: Screening natural products, small molecules and antibodies.

Bioaffinity drug screening strategies have gained popularity in preclinical and clinical drug discovery for natural products, small molecules and antibodies owing to their superior selectivity, the large number of compounds to be screened and their ability to minimize the time and expenses of the drug discovery process. This paper provides a systematic summary of the principles of commonly used bioaffinity-based screening methods, elaborates on the success of bioaffinity in clinical drug development and summarizes the active compounds, preclinical drugs and marketed drugs obtained through affinity screening methods. Owing to the high demand for new drugs, bioaffinity-guided screening techniques will play a greater part in clinical drug development.

Tolerogenic nanoparticles mitigate the formation of anti-drug antibodies against pegylated uricase in patients with hyperuricemia.

Biologic drugs have transformed the standard of care for many diseases. However, many biologics induce the formation of anti-drug antibodies (ADAs), which can compromise their safety and efficacy. Preclinical studies demonstrate that biodegradable nanoparticles-encapsulating rapamycin (ImmTOR), but not free rapamycin, mitigate the immunogenicity of co-administered biologic drugs. Here we report the outcomes from two clinical trials for ImmTOR. In the first ascending dose, open-label study (NCT02464605), pegadricase, an immunogenic, pegylated uricase enzyme derived from Candida utilis, is assessed for safety and tolerability (primary endpoint) as well as activity and immunogenicity (secondary endpoint); in the second single ascending dose Phase 1b trial (NCT02648269) composed of both a double-blind and open-label parts, we evaluate the safety of ImmTOR (primary endpoint) and its ability to prevent the formation of anti-drug antibodies against pegadricase and enhance its pharmacodynamic activity (secondary endpoint) in patients with hyperuricemia. The combination of ImmTOR and pegadricase is well tolerated. ImmTOR inhibits the development of uricase-specific ADAs in a dose-dependent manner, thus enabling sustained enzyme activity and reduction in serum uric acid levels. ImmTOR may thus represent a feasible approach for preventing the formation of ADAs to a broad range of immunogenic biologic therapies.

Non-specificity as the sticky problem in therapeutic antibody development.

Antibodies are highly potent therapeutic scaffolds with more than a hundred different products approved on the market. Successful development of antibody-based drugs requires a trade-off between high target specificity and target binding affinity. In order to better understand this problem, we here review non-specific interactions and explore their fundamental physicochemical origins. We discuss the role of surface patches - clusters of surface-exposed amino acid residues with similar physicochemical properties - as inducers of non-specific interactions. These patches collectively drive interactions including dipole-dipole, π-stacking and hydrophobic interactions to complementary moieties. We elucidate links between these supramolecular assembly processes and macroscopic development issues, such as decreased physical stability and poor in vivo half-life. Finally, we highlight challenges and opportunities for optimizing protein binding specificity and minimizing non-specificity for future generations of therapeutics.

Cullin3 deficiency shapes tumor microenvironment and promotes cholangiocarcinoma in liver-specific Smad4/Pten mutant mice.

Cholangiocarcinoma (CC), the most lethal type of liver cancer, remains very difficult to treat due to an incomplete understanding of the cancer initiation and progression mechanisms and no effective therapeutic drugs. Thus, identification of genomic drivers and delineation of the underlying mechanisms are urgently needed. Here, we conducted a genome-wide CRISPR-Cas9 screening in liver-specific Smad4/Pten knockout mice (Smad4co/co;Ptenco/co;Alb-Cre, abbreviated as SPC), and identified 15 putative tumor suppressor genes, including Cullin3 (Cul3), whose deficiency increases protein levels of Nrf2 and Cyclin D1 that accelerate cholangiocytes expansion leading to the initiation of CC. Meanwhile, Cul3 deficiency also increases the secretion of Cxcl9 in stromal cells to attract T cells infiltration, and increases the production of Amphiregulin (Areg) mediated by Nrf2, which paracrinely induces inflammation in the liver, and promotes accumulation of exhausted PD1high CD8 T cells at the expenses of their cytotoxic activity, allowing CC progression. We demonstrate that the anti-PD1/PD-L1 blockade inhibits CC growth, and the effect is enhanced by combining with sorafenib selected from organoid mediated drug sensitive test. This model makes it possible to further identify more liver cancer suppressors, study molecular mechanisms, and develop effective therapeutic strategies.

Biological Therapies in the Treatment of Cancer-Update and New Directions.

Biological therapies have changed the face of oncology by targeting cancerous cells while reducing the effect on normal tissue. This publication focuses mainly on new therapies that have contributed to the advances in treatment of certain malignancies. Immunotherapy, which has repeatedly proven to be a breakthrough therapy in melanoma, as well as B-ALL therapy with CAR T cells, are of great merit in this progress. These therapies are currently being developed by modifying bispecific antibodies and CAR T cells to improve their efficiency and bioavailability. Work on improving the therapy with oncolytic viruses is also progressing, and efforts are being made to improve the immunogenicity and stability of cancer vaccines. Combining various biological therapies, immunotherapy with oncolytic viruses or cancer vaccines is gaining importance in cancer therapy. New therapeutic targets are intensively sought among neoantigens, which are not immunocompromised, or antigens associated with tumor stroma cells. An example is fibroblast activation protein α (FAPα), the overexpression of which is observed in the case of tumor progression. Universal therapeutic targets are also sought, such as the neurotrophic receptor tyrosine kinase (NTRK) gene fusion, a key genetic driver present in many types of cancer. This review also raises the problem of the tumor microenvironment. Stromal cells can protect tumor cells from chemotherapy and contribute to relapse and progression. This publication also addresses the problem of cancer stem cells resistance to treatment and presents attempts to avoid this phenomenon. This review focuses on the most important strategies used to improve the selectivity of biological therapies.

The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus.

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.

Development of natural products for anti-PD-1/PD-L1 immunotherapy against cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is one of the most promising therapeutic targets for cancer immunotherapy, but several challenges remain in current anti-PD-1/PD-L1 therapy. Natural products, mainly derived from traditional medicine, could improve and expand anti-PD-1/PD-L1 therapy because of their advantages such as large diversity and multi-target effects. AIM OF THE STUDY: This review summarize natural products, raw extracts, and traditional medicines with pharmacological effects associated with the PD-1/PD-L1 axis, particularly PD-L1. MATERIALS AND METHODS: Electronic literature databases, including Web of Science, PubMed, and ScienceDirect, and online drugs and chemicals databases, including DrugBank, ZINC, PubChem, STITCH, and CTD, were searched without date limitation by February 2021. 'Natural product or herb or herbal plant or traditional medicine' and 'PD-L1' and 'Cancer immunotherapy' were used as the search keywords. Among 112 articles identified in database searching, 54 articles are full text articles, reporting in silico, in vitro, in vivo and clinical trials. 68 articles included are review articles and grey literature such as thesis and congress abstracts. RESULTS: Several natural products and traditional medicines have exhibited diverse and multi-functional effects including direct blockade of PD-1/PD-L1 interactions, modulation of PD-L1 expression, and cooperation with PD-1/PD-L1 inhibitors. CONCLUSION: Natural products and traditional medicines can facilitate the development of more effective and acceptable diverse strategies for anti-PD-1/PD-L1 therapy, but further exploration of natural products and pharmaceutical techniques is required.

Virtual Screening and In Vitro Evaluation of PD-1 Dimer Stabilizers for Uncoupling PD-1/PD-L1 Interaction from Natural Products.

Genetic mutations accumulated overtime could generate many growth and survival advantages for cancer cells, but these mutations also mark cancer cells as targets to be eliminated by the immune system. To evade immune surveillance, cancer cells adopted different intrinsic molecules to suppress immune response. PD-L1 is frequently overexpressed in many cancer cells, and its engagement with PD-1 on T cells diminishes the extent of cytotoxicity from the immune system. To resume immunity for fighting cancer, several therapeutic antibodies disrupting the PD-1/PD-L1 interaction have been introduced in clinical practice. However, their immunogenicity, low tissue penetrance, and high production costs rendered these antibodies beneficial to only a limited number of patients. PD-L1 dimer formation shields the interaction interface for PD-1 binding; hence, screening for small molecule compounds stabilizing the PD-L1 dimer may make immune therapy more effective and widely affordable. In the current study, 111 candidates were selected from over 180,000 natural compound structures through virtual screening, contact fingerprint analysis, and pharmacological property prediction. Twenty-two representative candidates were further evaluated in vitro. Two compounds were found capable of inhibiting the PD-1/PD-L1 interaction and promoting PD-L1 dimer formation. Further structure optimization and clinical development of these lead inhibitors will eventually lead to more effective and affordable immunotherapeutic drugs for cancer patients.

Platelet-derived nanomotor coated balloon for atherosclerosis combination therapy.

Atherosclerosis can lead to thrombosis, blood supply disorders, and even serious consequences such as lumen occlusion or wall rupture and bleeding, so it is urgent to develop an effective comprehensive therapy. Here, a novel kind of drug-coated balloon, where drug-loaded porous nanomotors with autonomous motion ability are used as the coating of the balloon, is reported. The drug-loaded porous nanomotors based on Janus aminated mesoporous silica (JAMS) that was obtained by asymmetric modification of platinum (Pt) nanoparticles are prepared and characterized. The platelet membrane is used to wrap the nanomotors to reduce the leakage of drugs before reaching the plaque. The motion ability of the nanomotor under the irradiation of near-infrared light, the sustained release behavior and effect of the loaded drugs (anti-proliferative drug paclitaxel and the anti-vascular cell adhesion molecule-1 antibody) are investigated in detail. The biomimetic effect and encapsulation effect on drug loading of the platelet membrane, and the elimination of inflammatory macrophages under the photothermal effect produced by Pt are also characterized. The results indicate that the drug-loaded porous nanomotors proposed for drug balloon coating in this work can penetrate into the plaque and enhance the drug retention efficiency, realizing short-term photothermal elimination of inflammatory macrophages and long-term anti-proliferation effect of the drug, providing a possible choice for drug balloon coating with high efficiency in the treatment of atherosclerosis.

Astragalus membranaceus-Derived Anti-Programmed Death-1 Monoclonal Antibodies with Immunomodulatory Therapeutic Effects against Tumors.

Astragalus membranaceus polysaccharide (APS) components are main ingredients of TCM and have proven efficacy to activate T cells and B cells, enhancing immunity in humans. In this study, elevated cytokine and anti-PD-1 antibody titers were found in mice after immunization with APS. Therefore, phage-display technology was utilized to isolate specific anti-programmed death-1 (PD-1) antibodies from mice stimulated by APS and to confirm whether the isolated anti-PD-1 antibody could inhibit the interaction of PD-1 with the programmed death-ligand 1 (PD-L1), resulting in tumor growth inhibition. The isolated single-chain fragment variable (scFv) S12 exhibited the highest binding affinity of 20 nM to PD-1, completed the interaction between PD-1 and PD-L1, and blocked the effect of PD-L1-induced T cell exhaustion in peripheral blood mononuclear cells in vitro. In the animal model, the tumor growth inhibition effect after scFv S12 treatment was approximately 48%. However, meaningful synergistic effects were not observed when scFv S12 was used as a cotreatment with ixabepilone. Moreover, this treatment caused a reduction in the number of tumor-associated macrophages in the tumor tissue. These experimental results indirectly indicate the ability of APS to induce specific antibodies associated with the immune checkpoint system and the potential benefits for improving immunity in humans.

Multifunctional Magnetic Nanoplatform Eliminates Cancer Stem Cells via Inhibiting the Secretion of Extracellular Heat Shock Protein 90.

Cancer stem cells (CSCs) are responsible for malignant tumor initiation, recurrences, and metastasis. Therefore, targeting CSCs is a promising strategy for the development of cancer therapies. A big challenge for CSC-based cancer therapy is the overexpression of therapeutic stress protein, heat shock protein 90 (Hsp90), which protects CSCs from further therapeutic-induced damage, leading to the failure of treatment. Thus, efficient strategies to target CSCs are urgently needed for cancer therapy. To this end, a multifunctional nanoparticle (MNP) for CSC-based combined thermotherapy and chemotherapy is reported. This strategy dramatically suppresses tumor growth in breast CSC xenograft-bearing mice. Furthermore, a new mechanism is present that the MNP exerts its striking effects on CSCs by inhibiting the secretion of extracellular Hsp90 (eHsp90), resulting in the interruption of several key signaling pathways. These findings open new perspectives on the use of an MNP for effective CSC-based cancer treatment by inhibiting the function of eHsp90.

[Immunotoxins and immunocytokines]. / Immunotoxines et immunocytokines.

Cytokines and biological toxins represent two potent classes of biomolecules that have long been explored for their potential as therapeutics. Considerable side effects and poor pharmacokinetics frequently observed with both have limited their broad application. Recombinant protein engineering has allowed the construction of immunocytokines and immunotoxins that seek to exploit the advantageous properties of immunoglobulins to address these issues. Whole antibodies, antibody fragments, constant domains and derivatives have been fused genetically to a range of cytokines and toxins. This review considers the strategies that have been employed and the problems sought to be resolved in the clinical evaluation of this class of biotherapeutic.

TITLE: Immunotoxines et immunocytokines. ABSTRACT: Les cytokines et les toxines biologiques représentent deux classes de biomolécules qui ont longtemps été explorées pour leur potentiel thérapeutique. Des effets secondaires considérables et des mauvaises propriétés pharmacocinétiques sont fréquemment observés chez chacune d'elles, ce qui limite leur application. L'ingénierie des protéines recombinantes a permis la création d'immunocytokines et d'immunotoxines qui visent à utiliser les propriétés avantageuses des immunoglobulines, pour résoudre ces problèmes. Des anticorps entiers, des fragments d'anticorps, des domaines constants et des dérivés ont été génétiquement fusionnés à une gamme de cytokines et de toxines. Cette revue présente les stratégies déployées et les problèmes à résoudre au cours de l'évaluation clinique pour cette classe de biothérapeutiques.

Recent advances in understanding colorectal cancer.

The achievements in the treatment of metastatic colorectal cancer during recent years are based on a better understanding of the disease and individualized regimen planning. In adjuvant treatment, the highly important IDEA (International Duration Evaluation of Adjuvant Chemotherapy) study has shown that treatment duration can safely be reduced in selected patient populations. In patients with pN1 and pT1-pT3 tumors, 3 months of treatment with 5-fluorouracil and oxaliplatin is comparable with respect to 3-year survival rate to 6 months of treatment. For patients with N2 tumors, 6 months of treatment should stay the standard of care. The limitation of the duration of the adjuvant treatment is significantly reducing the chemotherapy-induced morbidity. New studies will explore the use of immune-checkpoint inhibitors in the adjuvant setting in microsatellite-instable (MSI) tumors. In metastatic disease, next to the required molecular testing for RAS and BRAF mutations, MSI testing is recommended. In the rare group of patients with a MSI tumor, immune-checkpoint inhibition is changing the course of the disease dramatically. Therefore, it is important to identify those patients early. For the RAS-mutant cases, no new and targeted treatment options have been identified yet. An optimal treatment strategy for those patients is urgently needed. RAS wild-type patients with tumors derived from the left side of the colon (splenic flexure to rectum) should be treated in first line with epithelial growth factor receptor (EGFR) antibodies. This selection by a molecular and a clinical marker increased the benefit derived by EGFR antibodies dramatically and defined the most effective treatment option for those patients. New selection criteria based on gene expression, methylation, and other molecular changes are explored and will further influence our therapeutic strategies in the future.

161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS.

Myelodysplastic syndrome (MDS) is a clonal heterogeneous stem cell disorder driven by multiple genetic and epigenetic alterations resulting in ineffective hematopoiesis. MDS has a high frequency of immune suppressors, including myeloid-derived suppressor cells (MDSCs), that collectively result in a poor immune response. MDSCs in MDS patients express CD155 that ligates the T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) and delivers an inhibitory signal to natural killer (NK) cells. To mediate a productive immune response against MDS, negative regulatory checkpoints, like TIGIT, expressed on MDS NK cells must be overcome. NK cells can be directed to lyse MDS cells by bispecific killer engagers (BiKEs) that ligate CD16 on NK cells and CD33 on MDS cells. However, such CD16 × CD33 (1633) BiKEs do not induce the proliferative response in MDS NK cells needed to sustain their function. Here, we show that the addition of an NK stimulatory cytokine, interleukin-15 (IL-15), into the BiKE platform leads to productive IL-15 signaling without TIGIT upregulation on NK cells from MDS patients. Lower TIGIT expression allowed NK cells to resist MDSC inhibition. When compared with 1633 BiKE, 161533 trispecific killer engager (TriKE)-treated NK cells demonstrated superior killing kinetics associated with increased STAT5 phosphorylation. Furthermore, 161533 TriKE-treated MDS NK cells had higher proliferation and enhanced NK-cell function than 1633 BiKE-treated cells without the IL-15 linker. Collectively, our data demonstrate novel characteristics of the 161533 TriKE that support its application as an immunotherapeutic agent for MDS patients.

Blockade of sustained tumor necrosis factor in a transgenic model of progressive autoimmune encephalomyelitis limits oligodendrocyte apoptosis and promotes oligodendrocyte maturation.

BACKGROUND: Tumor necrosis factor (TNF) is associated with several neurodegenerative disorders including multiple sclerosis (MS). Although TNF-targeted therapies have been largely unsuccessful in MS, recent preclinical data suggests selective soluble TNF inhibition can promote remyelination. This has renewed interest in regulation of TNF signaling in demyelinating disease, especially given the limited treatment options for progressive MS. Using a mouse model of progressive MS, this study evaluates the effects of sustained TNF on oligodendrocyte (OLG) apoptosis and OLG precursor cell (OPC) differentiation. METHODS: Induction of experimental autoimmune encephalomyelitis (EAE) in transgenic mice expressing a dominant-negative interferon-γ receptor under the human glial fibrillary acidic protein promoter (GFAPγR1Δ) causes severe non-remitting disease associated with sustained TNF. Therapeutic effects in GFAPγR1Δ mice treated with anti-TNF compared to control antibody during acute EAE were evaluated by assessing demyelinating lesion size, remyelination, OLG apoptosis, and OPC differentiation. RESULTS: More severe and enlarged demyelinating lesions in GFAPγR1Δ compared to wild-type (WT) mice were associated with increased OLG apoptosis and reduced differentiated CC1+Olig2+ OLG within lesions, as well as impaired upregulation of TNF receptor-2, suggesting impaired OPC differentiation. TNF blockade during acute EAE in GFAPγR1Δ both limited OLG apoptosis and enhanced OPC differentiation consistent with reduced lesion size and clinical recovery. TNF neutralization further limited increasing endothelin-1 (ET-1) expression in astrocytes and myeloid cells noted in lesions during disease progression in GFAPγR1Δ mice, supporting inhibitory effects of ET-1 on OPC maturation. CONCLUSION: Our data implicate that IFNγ signaling to astrocytes is essential to limit a detrimental positive feedback loop of TNF and ET-1 production, which increases OLG apoptosis and impairs OPC differentiation. Interference of this cycle by TNF blockade promotes repair independent of TNFR2 and supports selective TNF targeting to mitigate progressive forms of MS.

In vitro-engineered non-antibody protein therapeutics.

Antibodies have proved to be a valuable mode of therapy for numerous diseases, mainly owing to their high target binding affinity and specificity. Unfortunately, antibodies are also limited in several respects, chief amongst those being the extremely high cost of manufacture. Therefore, non-antibody binding proteins have long been sought after as alternative therapies. New binding protein scaffolds are constantly being designed or discovered with some already approved for human use by the FDA. This review focuses on protein scaffolds that are either already being used in humans or are currently being evaluated in clinical trials. Although not all are expected to be approved, the significant benefits ensure that these molecules will continue to be investigated and developed as therapeutic alternatives to antibodies. Based on the location of the amino acids that mediate ligand binding, we place all the protein scaffolds under clinical development into two general categories: scaffolds with ligand-binding residues located in exposed flexible loops, and those with the binding residues located in protein secondary structures, such as α-helices. Scaffolds that fall under the first category include adnectins, anticalins, avimers, Fynomers, Kunitz domains, and knottins, while those belonging to the second category include affibodies, ß-hairpin mimetics, and designed ankyrin repeat proteins (DARPins). Most of these scaffolds are thermostable and can be easily produced in microorganisms or completely synthesized chemically. In addition, many of these scaffolds derive from human proteins and thus possess very low immunogenic potential. Additional advantages and limitations of these protein scaffolds as therapeutics compared to antibodies will be discussed.

Anti­IL­39 (IL­23p19/Ebi3) polyclonal antibodies ameliorate autoimmune symptoms in lupus­like mice.

The interleukin (IL)­12 family cytokines have been examined as therapeutic targets in the treatment of several autoimmune diseases. Our previous study showed that a novel IL­12 family cytokine, IL­39 (IL­23p19/Ebi3) mediates inflammation in lupus­like mice. In the present study, the effect of anti­mouse IL­39 polyclonal antibodies on autoimmune symptoms in lupus­like mice was investigated. Rabbit anti­mouse IL­39 polyclonal antibodies were produced by immunization with recombinant mouse IL­39, and purified using protein A chromatography. These antibodies were subsequently used to treat lupus­like mice. Flow cytometry, captured images, ELISA and H&E staining were used to determine the effect of anti­IL­39 polyclonal antibodies on inflammatory cells, autoantibody titers, proteinuria, infiltrating inflammatory cells and the structure of the glomerular region. The anti­IL­39 polyclonal antibodies effectively reduced the numbers of inflammatory cells, splenomegaly, autoantibody titers, proteinuria, infiltrating inflammatory cells, and restored the structure of the glomerular region in MRL/lpr mice. Taken together, these results suggested that anti­IL­39 polyclonal antibodies ameliorated autoimmune symptoms in lupus­like mice. Therefore, IL­39 may be used as a possible target for the treatment of systemic lupus erythematosus.

Improved functional outcome after chronic stroke with delayed anti-Nogo-A therapy: A clinically relevant intention-to-treat analysis.

Many preclinical treatment strategies for stroke have failed when tested in human trials. Although the reasons for these translation failures are multifactorial, one potential concern is the statistical analysis of the preclinical data. One way to rigorously evaluate new therapies is to use an intention-to-treat analysis in preclinical studies. Therefore, in this study, we set out to evaluate the treatment efficacy of a potential clinically relevant therapeutic agent for stroke, i.e., anti-Nogo-A immunotherapy, using an intention-to-treat analysis. Adult rats were trained on the skilled forelimb reaching task and subsequently underwent an ischemic stroke. Nine weeks later, the rats either received intracerebroventricular anti-Nogo-A antibody, control antibody, or no treatment. Skilled reaching performance was assessed by a non-linear model using both an intention-to-treat and per-protocol analysis. Following testing, dendritic complexity was evaluated in the contralesional and perilesional sensorimotor cortex. Both intention-to-treat and per-protocol analysis showed that anti-Nogo-A immunotherapy resulted in statistically significant improved recovery on the skilled forelimb reaching task, although treatment effect was less (though statistically significant) in the intention-to-treat group. Improved functional performance was not shown to be associated with dendritic changes. In conclusion, this study provides evidence for the importance of using intention-to-treat paradigms in testing preclinical therapeutic strategies.

Efficacy of nerve growth factor antibody in a knee osteoarthritis pain model in mice.

BACKGROUND: Nerve growth factor (NGF) is not only an important factor in nerve growth but also a major contributor to the production of inflammation. It has been reported that inhibiting NGF could reduce several types of pain in several animal models. Here, we aimed to clarify the efficacy of NGF antibody in a knee osteoarthritis (OA) pain model in mice. METHOD: Six-week-old male C57BR/J mice were used (n = 30). Ten mice comprised the control group, which received saline injection into the right knee joints; the other 20 mice comprised the experimental group, which received monoiodoacetate (MIA) injection into the right knee joints. Three weeks after surgery, the 20 experimental mice were randomly placed into treatment groups which received either sterile saline (non-treat group: 10 mg/kg, i.p.) or an anti-NGF antibody (anti-NGF group: 10 mg/kg, i.p.). Simultaneously, all mice received fluorogold (FG) retrograde neurotracer injection into their right joints. In a behavioral study, we evaluated gait using the CatWalk quantitative gait analysis system before surgery, 3 weeks after surgery (before treatment), 4 weeks after surgery (one week after surgery), and 5 weeks after surgery (2 weeks after surgery). In immunohistochemical analysis, the right dorsal root ganglia (DRGs) from the L4-L6 levels were resected 5 weeks after surgery (2 weeks after surgery). They were immunostained for calcitonin gene-related peptide (CGRP), and the number of FG-labeled or CGRP-immunoreactive (IR) DRG neurons was counted. RESULTS: On gait analysis using the CatWalk system, duty cycle, swing speed, and print area were decreased in non-treat group compared with those in control group and improved in the anti-NGF group compared with those in non-treat group. CGRP expression in DRGs was up-regulated in non-treat group compared with that in control group and suppressed in the anti-NGF group compared with that in non-treat group (both p < 0.05). CONCLUSIONS: MIA injection into the knee joint induced gait impairment and the up-regulation of CGRP in DRG neurons in a knee OA pain model in mice. Intraperitoneal injection of anti-NGF antibody suppressed this impairment of gait and up-regulation of CGRP in DRG neurons. These finding suggest that anti-NGF therapy might be valuable in the treatment of OA pain in the knee.