Effects of oral administration of nonselective Trk inhibitor on bladder overactivity in rodent models of prostatic inflammation.

BACKGROUND: Our research focused on the assessment of the impact of systemic inhibition of Trk receptors, which bind to nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), on bladder hypersensitivity in two distinct rodent models of prostatic inflammation (PI). METHODS: Male Sprague-Dawley rats were divided into three groups (n = 6 each): the control group (no PI, vehicle administration), the untreated group (PI, vehicle administration), and the treated group (PI, nonselective Trk inhibitor, GNF 5837, administration). PI in rats was induced by a intraprostatic injection of 5% formalin. Posttreatment, we carried out conscious cystometry and a range of histological and molecular analyses. Moreover, the study additionally evaluated the effects of a nonselective Trk inhibitor on bladder overactivity in a mouse model of PI, which was induced by prostate epithelium-specific conditional deletion of E-cadherin. RESULTS: The rat model of PI showed upregulations of NGF and BDNF in both bladder and prostate tissues in association with bladder overactivity and inflammation in the ventral lobes of the prostate. GNF 5837 treatment effectively mitigated these PI-induced changes, along with reductions in TrkA, TrkB, TrkC, and TRPV1 mRNA expressions in L6-S1 dorsal root ganglia. Also, in the mouse PI model, GNF 5837 treatment similarly improved bladder overactivity. CONCLUSIONS: The findings of our study suggest that Trk receptor inhibition, which reduced bladder hypersensitivity and inflammatory responses in the prostate, along with a decrease in overexpression of Trk and TRPV1 receptors in sensory pathways, could be an effective treatment strategy for male lower urinary tract symptoms associated with PI and bladder overactivity.

Peripheral Nerve Safety of Nerve Growth Factor Inhibition by Tanezumab: Pooled Analyses of Phase III Clinical Studies in Over 5000 Patients with Osteoarthritis.

BACKGROUND: Tanezumab, a humanized anti-nerve growth factor antibody, was developed for the treatment of pain associated with osteoarthritis. Due to its mechanism of action, peripheral nerve safety was assessed in all clinical studies. OBJECTIVES: To summarize the neurological safety of intravenous (IV) and subcutaneous (SC) tanezumab versus placebo in patients with osteoarthritis. METHODS: Data were pooled from 3389 patients across seven studies that investigated IV administration, and from 1840 patients across three studies that investigated SC administration. The treatment period of each study ranged from 16 to 24 weeks, and follow-up periods ranged from 8 to 24 weeks. Neurological safety evaluations focused on adverse events (AEs) of abnormal peripheral sensation (APS), neurologic examinations, and consultations. RESULTS: Across datasets, the incidence of AEs of APS was higher in tanezumab groups versus placebo. Paresthesia and hypoesthesia were the most frequently reported AEs in tanezumab-treated patients, compared with placebo. In both datasets, most AEs were of mild severity, resolved, and rarely resulted in discontinuation. In all treatment groups in both IV and SC studies, over 90% of patients had no new or worsened neurological examination abnormalities at the last study visit. Across datasets, mononeuropathy was diagnosed more frequently in tanezumab groups compared with placebo. Polyneuropathy was diagnosed in ≤ 0.9% of patients in tanezumab and placebo groups. CONCLUSIONS: Tanezumab IV or SC had an increased incidence of AEs of APS, such as paresthesia and hypoesthesia, and diagnoses of mononeuropathy compared with placebo. However, tanezumab was not associated with generalized peripheral neuropathy. GOV IDENTIFIERS: NCT00733902, NCT00744471, NCT00830063, NCT00863304, NCT00863772, NCT01089725, NCT00985621, NCT02697773, and NCT02709486.

Preparation and characterization of a high-affinity monoclonal antibody against nerve growth factor.

Nerve growth factor (NGF) is produced and released in injured tissues or chronic pain tissues caused by other diseases. Studies have shown that monoclonal antibodies targeting NGF have a good efficacy in the treatment of osteoarthritis (OA), low back pain and chronic pain, which may be a promising therapy. In this study, DNA sequences of NGF-his and NGF-hFc were synthesized using eukaryotic expression system and subcloned into pTT5 expression vector. After that, NGF proteins were expressed by transient expression in HEK293E cells. We immunized mice with NGF-hFc protein and fused mouse spleen cells to prepare hybridomas. NGF-His protein was used to screen out the hybridoma supernatant that could directly bind to NGF. Antibodies were purified from hybridioma supernatant. Futhermore, via surface plasmon resonance (SPR) screening, six anti-NGF mAbs were screened to block the binding of NGF and TrkA receptor in the treatment of chronic pain. Among them, 58F10G10H showed high affinity (KD = 1.03 × 10-9 M) and even better than that of positive control antibody Tanezumab (KD = 1.53 × 10-9 M). Moreover, the specific reactivity of 58F10G10H was demonstrated by TF-1 cell proliferation activity experiments, competitive binding Enzyme-linked immunosorbent assay (ELISA) and the arthritis animal models in mice, respectively. In conclusion, in this study, a method for the preparation of high-yield NGF-HFC and NGF-His proteins was designed, and a high-affinity monoclonal antibody against NGF with potential for basic research and clinical application was prepared.

Therapeutic effects of nerve growth factor-targeting therapy on bladder overactivity in rats with prostatic inflammation.

BACKGROUND: The present study examined the effect of liposomes conjugated with antisense oligonucleotide of nerve growth factor (NGF-OND) on local overexpression of NGF and bladder overactivity using rats with prostatic inflammation (PI). METHODS: Male Sprague-Dawley rats were divided into three groups: (1) Control group; intact rats, (2) PI-NS group; rats with PI and intravesical instillation of normal saline (NS), (3) PI-OND group; rats with PI and intravesical instillation of NGF-OND. On Day 0, PI was induced by intraprostatic 5%-formalin injection. On Day 14, NGF-OND or NS was instilled directly into the bladder after laparotomy. On Day 28, therapeutic effects of NGF-OND were evaluated by awake cystometry and histological analysis as well as reverse-transcription polymerase chain reaction measurements of messenger RNA (mRNA) levels of NGF in the bladder and prostate, inflammatory markers in the prostate, C-fiber afferent markers, and an A-type K+ channel α-subunit (Kv 1.4) in L6-S1 dorsal root ganglia (DRG). RESULTS: Intravesical NFG-OND treatment reduced PI-induced overexpression of NGF in both bladder and prostate, and reduced PI-induced bladder overactivity evident as longer intercontraction intervals in association with reductions of TRPV1 and TRPA1 mRNA expression levels in DRG. mRNA expression of Kv1.4 in DRG was reduced after PI, but improved in the PI-OND group. CONCLUSIONS: These results indicate that NGF locally expressed in the bladder is an important mediator inducing bladder overactivity with upregulation of C-fiber afferent markers and downregulation of an A-type K+ channel subunit in DRG following PI, and that liposome-based, local NGF-targeting therapy could be effective for not only bladder overactivity and afferent sensitization, but also PI. Thus, local blockade of NGF in the bladder could be a therapeutic modality for male LUTS due to BPH with PI.

Nerve Growth Factor: A Potential Therapeutic Target for Lung Diseases.

The lungs play a very important role in the human respiratory system. However, many factors can destroy the structure of the lung, causing several lung diseases and, often, serious damage to people's health. Nerve growth factor (NGF) is a polypeptide which is widely expressed in lung tissues. Under different microenvironments, NGF participates in the occurrence and development of lung diseases by changing protein expression levels and mediating cell function. In this review, we summarize the functions of NGF as well as some potential underlying mechanisms in pulmonary fibrosis (PF), coronavirus disease 2019 (COVID-19), pulmonary hypertension (PH), asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. Furthermore, we highlight that anti-NGF may be used in future therapeutic strategies.

Fracture-induced pain-like behaviours in a femoral fracture mouse model.

This study is the first comprehensive characterisation of the pain phenotype after fracture using both evoked and naturalistic behaviours in adult male and ovariectomised female mice. It also shows that an anti-nerve growth factor (NGF) therapy could be considered to reduce pain after fracture surgery. INTRODUCTION: Bone fractures are common due to the ageing population and very painful even after healing. The phenotype of this pain is still poorly understood. We aimed to characterise it in a femoral fracture model in mice. METHODS: We employed both adult male, and female ovariectomised (OVX) mice to mimic osteoporotic fractures. Mice underwent a unilateral femoral fracture maintained by an external fixator or a sham surgery. Pain behaviours, including mechanical and thermal sensitivity, weight bearing and LABORAS, were measured from baseline to 6 weeks after fracture. The effect on pain of an antibody against nerve growth factor (anti-NGF) was assessed. Changes in nerve density at the fracture callus were analysed by immunohistochemistry. RESULTS: Following surgery, all groups exhibited high levels of invoked nociception. Mechanical and thermal hyperalgesia were observed from 1 week after surgery, with nociceptive sensitization in the fracture group maintained for the 6 weeks, whereas it resolved in the sham group after 3 weeks. OVX induced reduction in pain thresholds, which was maintained after fracture. The frequency of naturalistic behaviours did not change between groups. Anti-NGF administered before and weekly after surgery alleviated fracture-induced mechanical nociception. The density of nerve fibres in the fracture callus was similar in all groups 6 weeks after surgery. CONCLUSIONS: Fractures in rodent models are highly painful in both sexes. This pain-like phenotype is prolonged and should be routinely considered in fracture healing studies as it can affect the study outcome. The anti-NGF alleviates fracture-induced mechanical pain.

Local Administration of Low-Dose Nerve Growth Factor Antibody Reduced Pain in a Rat Osteoarthritis Model.

Systemic injection of a nerve growth factor (NGF) antibody has been proven to have a significant relevance in relieving osteoarthritis (OA) pain, while its adverse effects remain a safety concern for patients. A local low-dose injection is thought to minimize adverse effects. In this study, OA was induced in an 8-week-old male Sprague-Dawley (SD) rat joint by monoiodoacetate (MIA) injection for 2 weeks, and the effect of weekly injections of low-dose (1, 10, and 100 µg) NGF antibody or saline (control) was evaluated. Behavioral tests were performed, and at the end of week 6, all rats were sacrificed and their knee joints were collected for macroscopic and histological evaluations. Results showed that 100 µg NGF antibody injection relieved pain in OA rats, as evidenced from improved weight-bearing performance but not allodynia. In contrast, no significant differences were observed in macroscopic and histological scores between rats from different groups, demonstrating that intra-articular treatment does not worsen OA progression. These results suggest that local administration yielded a low effective NGF antibody dose that may serve as an alternative approach to systemic injection for the treatment of patients with OA.

A Quantitative Bioassay to Determine the Inhibitory Potency of NGF-TrkA Antagonists.

In this article, we demonstrate and validate a new bioassay named the NTAB [NGF-TrkA (nerve growth factor-tropomyosin receptor kinase A) antagonist bioassay] for the determination of the inhibitory potency of NGF-TrkA antagonists, based on the inhibition of NGF-dependent proliferation of the human TF1 erythroleukemic cell line.It is well known that NGF holds great therapeutic potential due to its neurotrophic and neuroprotective properties. NGF is also involved in some pathways, however, principally driven by TrkA that, if not correctly regulated, can lead to unwanted pathological outcomes linked to pain, angiogenesis, and cancer.Indeed, there is an increasing interest, from a therapeutic perspective, in designing new effective molecules (antibodies, antibody fragments, or small molecules) able to inhibit the undesired NGF-TrkA pathway. For these reasons, there is an interest to develop functional cell-based assays for determination of the inhibition potency of compounds inhibiting the NGF-TrkA axis. The NTAB presents significant advantages over other published NGF-TrkA functional bioassays, for these reasons: (1) It is quantitative, (2) it measures a pure TrkA response, (3) it is simpler, (4) it is based on a natural biological response, and (5) it is easily scalable from a lab scale to an automated industrial assay.The NTAB assay was validated with a panel of well-characterized NGF-TrkA inhibitors, yielding characteristic dose-response curves, from which the relative strength of the inhibitors was quantitatively determined and used for comparisons. This new bioassay will be very useful to assist in the validation and prioritization of the best inhibitors among a large number of candidates.

Long-Term Safety and Efficacy of Subcutaneous Tanezumab Versus Nonsteroidal Antiinflammatory Drugs for Hip or Knee Osteoarthritis: A Randomized Trial.

OBJECTIVE: To assess the long-term safety and 16-week efficacy of subcutaneous tanezumab in patients with hip or knee osteoarthritis (OA). METHODS: This was a phase III randomized, double-blind, active treatment-controlled (using nonsteroidal antiinflammatory drugs [NSAIDs] as the active treatment control) safety trial of tanezumab (56-week treatment/24-week posttreatment follow-up) in adults who were receiving stable-dose NSAID therapy at the time of screening and who had Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain and physical function scores of ≥5; patient global assessment (PtGA) of OA of fair, poor, or very poor; history of inadequate pain relief with standard analgesics; and no history or radiographic evidence of prespecified bone/joint conditions beyond OA. Patients received oral naproxen, celecoxib, or diclofenac twice daily (NSAID group; n = 996) or tanezumab 2.5 mg (n = 1,002) or 5 mg (n = 998) subcutaneously every 8 weeks. Coprimary efficacy end points at week 16 were changes in WOMAC pain and physical function scores and changes in PtGA. The primary joint safety end point over 80 weeks comprised adjudicated rapidly progressive OA type 1 or 2, primary osteonecrosis, subchondral insufficiency fracture, or pathologic fracture. Mean values, least squares mean values, and least squares mean differences between groups (with 95% confidence intervals [95% CIs]) were calculated. RESULTS: Of 3,021 randomized patients, 2,996 received ≥1 treatment dose. Adverse events (AEs) were similar between patients treated with tanezumab 2.5 mg and those treated with NSAIDs, and were more prevalent in those treated with tanezumab 5 mg. Composite joint safety events were significantly more prevalent with tanezumab 2.5 mg and tanezumab 5 mg than with NSAIDs (observation time-adjusted rate/1,000 patient-years 38.3 [95% CI 28.0, 52.5] and 71.5 [95% CI 56.7, 90.2], respectively, versus 14.8 [95% CI 8.9, 24.6]; P = 0.001 for tanezumab 2.5 mg versus NSAIDs; P < 0.001 for tanezumab 5 mg versus NSAIDs). Tanezumab 5 mg significantly improved pain and physical function but did not improve PtGA at week 16 when compared to NSAIDs; corresponding differences between the tanezumab 2.5 mg and NSAID groups were not statistically significant. CONCLUSION: In patients previously receiving a stable dose of NSAIDs, tanezumab administered subcutaneously resulted in more joint safety events than continued NSAIDs, with differences being dose dependent. Pain and physical function improved with both doses of tanezumab compared to NSAIDs, reaching statistical significance with tanezumab 5 mg at 16 weeks.

Fabry disease-associated globotriaosylceramide induces mechanical allodynia via activation of signaling through proNGF-p75NTR but not mature NGF-TrkA.

Fabry disease (FD) is an X-linked metabolic storage disorder arising from the deficiency of lysosomal α-galactosidase A, which leads to the gradual accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), throughout the body. Pain in the extremities is an early symptom of FD; however, the underlying pathophysiological mechanisms remain unknown. α-Galactosidase A knockout animals exhibit nociceptive behaviors, with enhanced expression levels of several ion channels. These characteristics are observed in animals treated with nerve growth factor (NGF). Here, we aimed to elucidate the potential of NGF signaling as a cause of FD-associated pain, using intraplantar Gb3-treated mice displaying mechanical allodynia. Treatment with a neutralizing antibody against a precursor of NGF (proNGF) or its receptor, p75 neurotrophin receptor (p75NTR), resulted in the recovery from Gb3-induced pain. Conversely, anti-NGF and anti-tropomyosin receptor kinase A antibodies failed to exert analgesic effects. Gb3 injection had no effects on the expression levels of proNGF and p75NTR in the plantar skin and dorsal root ganglia, suggesting that Gb3 activates the pain pathway, possibly mediated through functional up-regulation of proNGF-p75NTR signaling. Furthermore, by pharmacological approaches using a protein kinase A (PKA) inhibitor and a cholesterol-removing agent, we found that p75NTR-phosphorylating PKA and lipid rafts for phosphorylated p75NTR translocation were required for Gb3-induced pain. These results suggest that acute exposure to Gb3 induces mechanical allodynia via activation of the proNGF-p75NTR pathway, which involves lipid rafts and PKA. Our findings provide new pathological insights into FD-associated pain, and suggest the need to develop therapeutic interventions targeting proNGF-p75NTR signaling.

The evolution of nerve growth factor inhibition in clinical medicine.

Nerve growth factor (NGF) is a neurotrophin that activates nociceptive neurons to transmit pain signals from the peripheral to the central nervous system and that exerts its effects on neurons by signalling through tyrosine kinase receptors. Antibodies that inhibit the function of NGF and small molecule inhibitors of NGF receptors have been developed and tested in clinical studies to evaluate the efficacy of NGF inhibition as a form of analgesia in chronic pain states including osteoarthritis and chronic low back pain. Clinical studies in individuals with painful knee and hip osteoarthritis have revealed that NGF inhibitors substantially reduce joint pain and improve function compared with NSAIDs for a duration of up to 8 weeks. However, the higher tested doses of NGF inhibitors also increased the risk of rapidly progressive osteoarthritis in a small percentage of those treated. This Review recaps the biology of NGF and the studies that have been performed to evaluate the efficacy of NGF inhibition for chronic musculoskeletal pain states. The adverse events associated with NGF inhibition and the current state of knowledge about the mechanisms involved in rapidly progressive osteoarthritis are also discussed and future studies proposed to improve understanding of this rare but serious adverse event.

Proteomic Analysis of Uterine Tissues During Peri-Implantation Period in Mice with Experimentally Induced Adenomyosis that Treated with anti-Ngf: Implications for Cell-Cell Adhesion and Metabolic Processes.

Nerve growth factor (NGF) has been verified to be expressed with higher level in adenomyosis uteri, and its neutralizing antibody has a strong inhibitory influence on inflammation. The present study aimed to explore the effect of anti-NGF on the expression of proteins in uteri of mice-induced adenomyosis and assessed its potential role in improving pregnancy rate. In this study, we established a mouse model of adenomyosis and administrated NGF-neutralizing antibody into mice. The mass spectrometry (MS) analysis of the uteri during the implantation window was performed to explore the essential proteins participating in therapy. Besides, embryos of healthy mice were transferred into the uteri to assess the implantation rate. The results of MS analysis demonstrated that 119 proteins were changed in the adenomyosis group compared with control group, and 126 proteins were differentially expressed in the anti-NGF group compared with the adenomyosis group (fold change > 1.5, P < 0.05. After performing cluster analysis using Mfuzz package, we found that a group of proteins participated in cell-cell adhesion and metabolic processes, which were attenuated in the adenomyosis group, while those were successfully recovered by anti-NGF treatment. Western blotting confirmed that the expression levels of integrin alpha-1 (ITGA1), integrin beta-1 (ITGB1), laminin subunit gamma-1 (LAMC1), and creatine kinase M-type (CKM) were decreased in adenomyosis group, whereas those levels were elevated after anti-NGF treatment. Embryo implantation rate in the adenomyosis group was significantly decreased compared with that in the control group (2.31% vs. 26.15%, P < 0.001) and anti-NGF treatment slightly enhanced the embryo implantation rate in mice with experimentally induced adenomyosis (9.23% vs. 2.31%, P = 0.017). Our results revealed that anti-NGF therapy can improve fertility of mice with experimentally induced adenomyosis, possibly through promoting integrin-related proteins.

Neutralizing antibody to proNGF rescues erectile function by regulating the expression of neurotrophic and angiogenic factors in a mouse model of cavernous nerve injury.

BACKGROUND: Radical prostatectomy induces some degree of cavernous nerve injury (CNI) and causes denervation-induced pathologic changes in cavernous vasculature, regardless of the advances in surgical techniques and robotic procedures. The precursor for nerve growth factor (proNGF) is known to be involved in neuronal cell apoptosis and microvascular dysfunction through its receptor p75NTR . OBJECTIVES: To determine the expression of proNGF/p75NTR and the efficacy of proNGF neutralizing antibody (anti-proNGF-Ab) in a mouse model of ED induced by CNI. MATERIALS AND METHODS: Age-matched 12-week-old C57BL/6 mice were distributed into three groups: sham group and bilateral CNI group treated with intracavernous injections of PBS (20 µL) or of anti-proNGF-Ab (20 µg in 20 µL of PBS) on days -3 and 0. Two weeks after treatment, erectile function was measured by electrical stimulation of cavernous nerve. Penis tissues from a separate group of animals were harvested for further analysis. We also determined the efficacy of anti-proNGF-Ab on neural preservation in major pelvic ganglion (MPG) ex vivo. RESULTS: We observed increased penile expression of proNGF and p75NTR after CNI. Intracavernous administration of anti-proNGF-Ab increased nNOS and neurofilament expression probably by enhancing the production of neurotrophic factors, such as neurotrophin-3, NGF, and brain-derived neurotrophic factor. Anti-proNGF-Ab preserved the integrity of cavernous sinusoids, such as pericytes, endothelial cells, and endothelial cell-to-cell junctions, possibly by controlling angiogenic factors (angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor) and induced endogenous eNOS phosphorylation in CNI mice. And finally, treatment with anti-proNGF-Ab rescued erectile function in CNI mice. Anti-proNGF-Ab also enhanced neurite sprouting from MPG exposed to lipopolysaccharide. DISCUSSION AND CONCLUSION: The preservation of damaged cavernous neurovasculature through inhibition of the proNGF/p75NTR pathway may be a novel strategy to treat radical prostatectomy-induced erectile dysfunction.

Targeting nerve growth factor, a new option for treatment of osteoarthritis: a network meta-analysis of comparative efficacy and safety with traditional drugs.

OBJECTIVE: Osteoarthritis (OA) is the most common joint disease and leading cause of pain and disability in the elderly population. Most guidelines recommend the use of non-steroidal anti-inflammatory drugs (NSAIDs) and opioids for the non-operative treatment of OA. Monoclonal nerve growth factor (NGF) antibodies are new drugs with the potential to provide pain relief and functional improvement in OA. We compared the efficacy (pain reduction and functional improvement), and safety of monoclonal NGF antibodies with NSAIDs and opioids in the treatment of OA with a Bayesian network meta-analysis. RESULTS: 38 articles, comprising 41 trials and 20489 patients with OA were included. Overall from the network meta-analysis, anti-NGFs were the most effective drugs for pain relief (Standardized Mean Difference or SMD compared with placebo 4.25, 95% CI 2.87 to 5.63, Surface Under the Cumulative RAnking curve or SUCRA=93.7%) and for functional improvement (SMD 4.90, 95% CI 3.46 to 6.33, SUCRA=98.3%). Although anti-NGFs were associated with higher risk of peripheral sensation abnormality (paresthesia and pruritus), they were not associated with higher risk of other AEs (headaches and nausea) or with higher withdrawal rates related to AEs. CONCLUSIONS: Monoclonal NGF antibodies provide significantly greater pain relief and functional improvement in OA compared to NSAIDs and opioids. Monoclonal NGF antibodies are not associated with severe AEs. More studies are needed to confirm these findings. METHODS: PubMed, CNKI, Web of Science, Scopus, Embase and Cochrane Library databases were searched for relevant studies (OA treated with anti-NGFs, opioids, selective COX-2 inhibitors or NSAIDs) published between January 1999 to January 2020. Bayesian network and conventional meta-analyses were conducted. Pain relief, functional improvement and AEs were assessed.

Antagonism of proNGF or its receptor p75NTR reverses remodelling and improves bladder function in a mouse model of diabetic voiding dysfunction.

AIMS/HYPOTHESIS: Although 80% of diabetic patients will suffer from voiding difficulties and urinary symptoms, defined as diabetic voiding dysfunction (DVD), therapeutic targets and treatment options are limited. We hypothesise that the blockade of the pro-nerve growth factor (NGF)/p75 neurotrophin receptor (p75NTR) axis by an anti-proNGF monoclonal antibody or by a small molecule p75NTR antagonist (THX-B) can restore bladder remodelling (represented by bladder weight) in an animal model of DVD. Secondary outcomes of the study include improvements in bladder compliance, contractility and morphology, as well as in voiding behaviour, proNGF/NGF balance and TNF-α expression. METHODS: In a streptozotocin-induced mouse model of diabetes, diabetic mice received either a blocking anti-proNGF monoclonal antibody or a p75NTR antagonist small molecule as weekly systemic injections for 4 weeks. Animals were tested at baseline (at 2 weeks of diabetes induction), and after 2 and 4 weeks of treatment. Outcomes measured were voiding function with voiding spot assays and cystometry. Bladders were assessed by histological, contractility and protein expression assays. RESULTS: Diabetic mice showed features of DVD as early as 2 weeks after diabetes diagnosis (baseline) presented by hypertrophy, reduced contractility and abnormal cystometric parameters. Following treatment initiation, a twofold increase (p < 0.05) in untreated diabetic mouse bladder weight and thickness compared with non-diabetic controls was observed, and this change was reversed by p75NTR antagonism (37% reduction in bladder weight compared with untreated diabetic mice [95% CI 14%, 60%]) after 4 weeks of treatment. However, blocking proNGF did not help to reverse bladder hypertrophy. While diabetic mice had significantly worse cystometric parameters and contractile responses than non-diabetic controls, proNGF antagonism normalised bladder compliance (0.007 [Q1-Q3; 0.006-0.009] vs 0.015 [Q1-Q3; 0.014-0.029] ml/cmH2O in untreated diabetic mice, representing 62% reduction [95% CI 8%, 110%], p < 0.05) and contractility to KCl, carbachol and electrical field stimulation (p < 0.05 compared with the diabetic group) after 2 weeks of treatment. These effects were not observed after 4 weeks of treatment with proNGF antagonist. p75NTR antagonism did not show important improvements in cystometric parameters after 2 weeks of treatment. Slightly improved bladder compliance (0.01 [Q1-Q3; 0.009-0.012] vs 0.013 [Q1-Q3; 0.011-0.016] ml/cmH2O for untreated diabetic mice) was seen in the p75NTR antagonist-treated group after 4 weeks of treatment with significantly stabilised contractile responses to KCl, carbachol and electric field stimulation (p < 0.05 for each) compared with diabetic mice. Bladder dysfunction observed in diabetic mice was associated with a significant increase in bladder proNGF/NGF ratio (3.1 [±1.2] vs 0.26 [±0.04] ng/pg in control group, p < 0.05 at week 2 of treatment) and TNF-α (p < 0.05). The proNGF/NGF ratio was partially reduced (about 60% reduction) with both treatments (1.03 [±0.6] ng/pg for proNGF antibody-treated group and 1.4 [±0.76] ng/pg for p75NTR blocker-treated group after 2 weeks of treatment), concomitant with a significant decrease in the bladder levels of TNF-α (p < 0.05), despite persistent hyperglycaemia. CONCLUSIONS/INTERPRETATION: Our findings indicate that blockade of proNGF and the p75NTR receptor in diabetes can impede the development and progression of DVD. The reported improvements in morphological and functional features in our DVD model validates the proNGF/p75NTR axis as a potential therapeutic target in this pathology. Graphical abstract.

Patient preferences for osteoarthritis pain and chronic low back pain treatments in the United States: a discrete-choice experiment.

OBJECTIVE: To quantify preferences for attributes of potential analgesic treatments for moderate-to-severe pain associated with osteoarthritis (OA) and/or chronic low back pain (CLBP) as relevant to injectable nerve growth factor (NGF)-inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), and opioids. METHODS: We used a discrete-choice experiment (DCE) to elicit preferences for attributes of OA and CLBP pharmaceutical treatments, and a best-worst scaling (BWS) exercise to further characterize the relative importance of treatment-related side-effect risks. The survey was completed online by 602 US residents with self-reported chronic, moderate-to-severe OA pain and/or CLBP who had tried, had contraindications for, or were unwilling to take currently available pharmaceutical therapies. In the DCE, respondents repeatedly chose between two hypothetical treatments defined by six attributes (symptom control; treatment-related risks of (1) severe joint problems, (2) heart attack, and (3) physical dependence; mode/frequency of administration; and cost). In the BWS exercise, respondents evaluated ten side-effect risks. Random-parameters logit models were estimated; conditional relative attribute importance, maximum acceptable risks, and willingness to pay were calculated. RESULTS: The most important DCE attributes were improving symptom control (scaled conditional relative importance, 10.00) and reducing risk of physical dependence (6.99). The three most important BWS attributes were, in rank order, risks of stroke, physical dependence, and heart attack. Respondents were willing to accept a > 4% treatment-related risk of severe joint problems for even modest symptom improvement. CONCLUSION: A pharmaceutical treatment with a risk of severe joint problems was viewed as an acceptable alternative to other treatments with comparable efficacy but risks associated with NSAIDs or opioids.

Suppression of peripheral NGF attenuates neuropathic pain induced by chronic constriction injury through the TAK1-MAPK/NF-κB signaling pathways.

BACKGROUND: Anti-nerve growth factor (NGF) monoclonal antibodies (anti-NGF mAbs) have been reported to significantly attenuate pain, but the mechanism involved has not been fully elucidated, and the serious adverse events associated with mAbs seriously limit their clinical use. This study further investigated the mechanism by which peripheral NGF is involved in neuropathic pain and found safe, natural compounds that target NGF to attenuate neuropathic pain. METHODS: Nociception was assessed by the Von Frey hair and Hargreaves' methods. Western-blotting, qPCR and immunofluorescence were used to detect the cell signaling pathway. RAW264.7 macrophages and RSC96 Schwann cells were cultured for in vitro evaluation. RESULTS: Intraplantar administration of anti-NGF mAbs suppressed the expression of phosphorylated transforming growth factor-ß-activated kinase 1 (TAK1) in the dorsal root ganglion (DRG) and sciatic nerve. Intraplantar administration of a TAK1 inhibitor attenuated CCI-induced neuropathic pain and suppressed the expression of phosphorylated mitogen-activated protein kinases (MAPKs) in the DRG and sciatic nerve. Perisciatic nerve administration of levo-corydalmine (l-CDL) on the operated side obviously attenuated CCI-induced neuropathic pain and suppressed the expression of mNGF and proNGF. In addition, l-CDL-induced antinociception was reversed by intraplantar administration of NGF. Further results indicated that l-CDL-induced suppression of phosphorylated TAK1, MAPKs, and p65 and expression of the proinflammatory cytokines TNF-α and IL-1ß in the DRG and sciatic nerve were all abolished by NGF. In addition, in vitro experiments indicated that l-CDL suppressed the secretion of NGF and proNGF in RAW264.7 macrophages and RSC96 Schwann cells, which was abolished by AP-1 and CREB agonists, respectively. CONCLUSIONS: This study showed NGF inhibition suppressed TAK1 in the periphery to attenuate CCI-induced neuropathic pain through inhibition of downstream MAPK and p65 signaling. The natural compound l-CDL inhibited NGF secretion by macrophages and Schwann cells and downstream TAK1-MAPK/NF-κB signaling in the periphery to attenuate CCI-induced neuropathic pain. Video abstract Proposed mechanisms underlying the effect of l-CDL in periphery of CCI rats. In CCI rats, macropahages and Schwann cells could secret NGF to act on the receptors in the periphery to activate TAK1-MAPK/NF-κB axis and promote the release of proinflammatory cytokines, including TNF-α and IL-1ß to promote neuropathic pain. l-CDL decreased the secretion of NGF through inhibiting AP-1 and CREB respectively in RAW264.7 and RSC96 Schwann cells to attenuate CCI-induced neuropathic pain by inhibiting the TAK1-p38 MAPK/NF-κB signaling pathway.

Pharmacology of nerve growth factor and discovery of tanezumab, an anti-nerve growth factor antibody and pain therapeutic.

The pharmacology of Nerve Growth Factor (NGF) and the discovery and development of tanezumab, a monoclonal anti-NGF antibody for the treatment of pain illustrate the complex and unpredictable nature of modern drug development. Initial efforts attempted to use NGF agonistically for Alzheimer's disease and neuropathies. Most unexpectedly, clinical studies unmasked hyperalgesic effects. These observations together with new data emerging from molecular and animal model studies stimulated the idea of using an NGF antagonist for chronic pain. These events also reflect the advances of neuropharmacology from classical small molecule efforts directed at neurotransmitter receptors to modern biotechnology with significant integration in molecular biology, biochemistry, and protein engineering.

Effect and mechanisms of sacral nerve stimulation on visceral hypersensitivity mediated by nerve growth factor.

To investigate the efficacy of sacral nerve stimulation (SNS) on nerve growth factor (NGF) mediated visceral sensitivity in normal rat and visceral hypersensitivity model rats. 120 male newborn rats were randomly divided into 6 groups: group A was normal model group; group B ~ F were all sensitized with acetic acid enema and grouped again. Group c2 was given NGF antagonist, d2 group was given NGF agonist, e2 group was given PI3K inhibitor, and f2 group was given PLC-γ inhibitor. After treatment, the expression of NGF, TrKA, PI3K, AKT, PLC-γ, NF-κB, TRPV1, pTRPV1 and intracellular Ca2+ content were detected. The expression of protein TRPV1 and pTRPV1 was increased, and Ca2+ was increased in the visceral hypersensitive group. NGF, TrKA in NGF antagonist group, PI3K, AKT, NF-κB in PI3K inhibitor group, PLC-γ in PLC-γ inhibitor group were all almost not expressed. The relative expression of NGF, TrKA, PI3K, AKT, PLC-γ and NF-κB in NGF antagonist group was lower than that in visceral hypersensitivity group and NGF activator group (P < .01). The relative expression of NGF, TrKA, PI3K and AKT mRNA in NGF antagonist group was lower than that in the normal model group (P < .01). There was no significant difference in the relative expression of PLC-γ and NF-κB mRNA (P > .05). The expression level of MAPK, ERK1 and ERK2 in visceral hypersensitivity group was higher than that in PI3K inhibitor group and PLC-γ inhibitor group. The normal group Ca2+ curve was flat, and the NGF agonist group had the highest Ca2+ curve peak. Calcium concentration in visceral hypersensitivity group was higher than that in PI3K inhibitor group and that in PLC-γ inhibitor group was higher than that in NGF antagonist group. The binding of TrkA receptor to NGF activates the MAPK/ERK pathway, the PI3K/Akt pathway and the PLC-γ pathway, causing changes in the fluidity of intracellular and extracellular Ca2+ , resulting in increased sensitivity of visceral tissues and organs.

Effect of Tanezumab on Joint Pain, Physical Function, and Patient Global Assessment of Osteoarthritis Among Patients With Osteoarthritis of the Hip or Knee: A Randomized Clinical Trial.

Importance: Patients with osteoarthritis (OA) may remain symptomatic with traditional OA treatments. Objective: To assess 2 subcutaneous tanezumab dosing regimens for OA. Design, Setting, and Participants: A randomized, double-blind, multicenter trial from January 2016 to May 14, 2018 (last patient visit). Patients enrolled were 18 years or older with hip or knee OA, inadequate response to OA analgesics, and no radiographic evidence of prespecified joint safety conditions. Interventions: Patients received by subcutaneous administration either tanezumab, 2.5 mg, at day 1 and week 8 (n = 231); tanezumab, 2.5 mg at day 1 and 5 mg at week 8 (ie, tanezumab, 2.5/5 mg; n = 233); or placebo at day 1 and week 8 (n = 232). Main Outcomes and Measures: Co-primary end points were change from baseline to week 16 in Western Ontario and McMasters Universities Osteoarthritis Index (WOMAC) Pain (0-10, no to extreme pain), WOMAC Physical Function (0-10, no to extreme difficulty), and patient global assessment of osteoarthritis (PGA-OA) (1-5, very good to very poor) scores. Results: Among 698 patients randomized, 696 received 1 or more treatment doses (mean [SD] age, 60.8 [9.6] years; 65.1% women), and 582 (83.6%) completed the trial. From baseline to 16 weeks, mean WOMAC Pain scores decreased from 7.1 to 3.6 in the tanezumab, 2.5 mg, group; 7.3 to 3.6 in the tanezumab, 2.5/5 mg, group; and 7.3 to 4.4 in the placebo group (least squares mean differences [95% CI] vs placebo were -0.60 [-1.07 to -0.13; P = .01] for tanezumab, 2.5 mg, and -0.73 [-1.20 to -0.26; P = .002] for tanezumab, 2.5/5 mg). Mean WOMAC Physical Function scores decreased from 7.2 to 3.7 in the 2.5-mg group, 7.4 to 3.6 in the 2.5/5-mg group, and 7.4 to 4.5 with placebo (differences vs placebo, -0.66 [-1.14 to -0.19; P = .007] for tanezumab, 2.5 mg, and -0.89 [-1.37 to -0.42; P < .001] for tanezumab, 2.5/5 mg). Mean PGA-OA scores decreased from 3.4 to 2.4 in the 2.5-mg group, 3.5 to 2.4 in the 2.5/5-mg group, and 3.5 to 2.7 with placebo (differences vs placebo, -0.22 [-0.39 to -0.05; P = .01] for tanezumab, 2.5 mg, and -0.25 [-0.41 to -0.08; P = .004] for tanezumab, 2.5/5 mg). Rapidly progressive OA occurred only in tanezumab-treated patients (2.5 mg: n = 5, 2.2%; 2.5/5 mg: n = 1, 0.4%). The incidence of total joint replacements was 8 (3.5%), 16 (6.9%), and 4 (1.7%) in the tanezumab, 2.5 mg; tanezumab, 2.5/5 mg; and placebo groups, respectively. Conclusions and Relevance: Among patients with moderate to severe OA of the knee or hip and inadequate response to standard analgesics, tanezumab, compared with placebo, resulted in statistically significant improvements in scores assessing pain and physical function, and in PGA-OA, although the improvements were modest and tanezumab-treated patients had more joint safety events and total joint replacements. Further research is needed to determine the clinical importance of these efficacy and adverse event findings. Trial Registration: ClinicalTrials.gov Identifier: NCT02697773.