Effects of ON101 for Hard-to-Heal Diabetic Foot Ulcers in A Randomized Phase III Trial: A Post hoc Analysis.

OBJECTIVE: Hard-to-heal diabetic foot ulcers (DFUs) are associated with higher mortality rates and an increased medical burden for patients. ON101, a new topical cream, exhibited better healing efficacy than the control dressing in a Phase III trial. In this post-hoc analysis, we further identify whether ON101 can improve the healing of ulcers with hard-to-heal risk factors in this cohort of DFU patients. APPROACH: To compare the efficacy of ON101 with absorbent dressing among various hard-to-heal wounds in patients with DFU, a post hoc analysis of a randomized phase III trial included 276 DFU patients was performed by subgrouping those patients based on ulcer depth, location, size, duration, and patients' glycated hemoglobin (HbA1c) levels and body mass index (BMI). RESULTS: In the full analysis set, the proportion of patients achieving healing was 61.7% in the ON101 group and 37.0% in the comparator (P =0.0001). In sub-group analysis according to risk factors, ON101 demonstrated superior healing capacity on Wagner grade 2 ulcers (P < 0.0001); plantar ulcers (P = 0.0016), ulcers size ≥5 cm² (P = 0.0122), ulcers duration ≥3 months (P = 0.0043); for patients with HbA1c ≥9% (P = 0.0285); and patients with BMI ≥25 (P = 0.0005). INNOVATION: ON101, a novel therapeutic drug, can modulate the functions of macrophages and demonstrate superior healing rates to conventional absorbent dressing in patients with hard-to-heal DFUs. CONCLUSIONS: The results of this post hoc study suggest that ON101 is a better therapeutic option than conventional dressing used in treatment for DFU patients with higher HbA1c, BMI, or ulcers with complex conditions such as longer duration, deeper wounds, larger size, and plantar location.

Cost-effectiveness of Novel Macrophage-Regulating Treatment for Wound Healing in Patients With Diabetic Foot Ulcers From the Taiwan Health Care Sector Perspective.

Importance: Diabetic foot ulcers (DFUs) and subsequent amputation incur enormous health and economic burdens to patients, health care systems, and societies. As a novel macrophage-regulating drug, ON101 is a breakthrough treatment for DFUs, which demonstrated significant complete wound healing effects in a phase 3 randomized clinical trial, but its economic value remains unknown. Objective: To assess the cost-effectiveness of an ON101 cream added on to general wound care (GWC; ie, conventional treatments for DFUs, which comprised initial and regular foot examinations, ulcer management, comorbidity control, patient education, and multidisciplinary care) vs GWC alone for DFUs from the Taiwan health care sector perspective. Design, Setting, and Participants: This economic evaluation used a hypothetical cohort of patients with diabetes, with characteristics mirroring those of the participants in the ON101 trial. A Markov state-transition simulation model was constructed to estimate costs and health outcomes associated with the ON101 with GWC and GWC alone strategies over a 5-year time horizon, discounting costs and effectiveness at 3% annually. Costs were in 2021 US dollars. Data were sourced from the ON101 trial and supplemented from published literature. Deterministic and probabilistic sensitivity analyses were performed to assess the uncertainty of input parameters and study generalizability. The analysis was designed and conducted from September 1, 2020, to January 31, 2022. Exposures: ON101 with GWC vs GWC alone. Main Outcomes and Measures: DFU-related complications, costs, quality-adjusted life-years (QALYs), and incremental cost-effectiveness ratio. Results: Patients in the hypothetical cohort had a mean age of 57 years and an uninfected DFU of 1 to 25 cm2 that was present for 4 or more weeks with a Wagner grade of 1 or 2. Over 5 years, the ON101 with GWC group vs the GWC alone group experienced more healing events, stayed for a longer time in the healing state, and had fewer infected DFUs, gangrene, and amputations (eg, 2787 additional healing events and 2766 fewer infected DFU, 72 fewer amputation, and 7 fewer gangrene events in the ON101 with GWC group vs GWC alone group). The ON101 with GWC strategy vs GWC alone yielded an additional 0.038 QALYs at an incremental cost of $571, resulting in $14 922/QALY gained. Economic results were most sensitive to healing efficacy, drug cost, and health utility of the healing state. Cost-saving results were observed in patient subgroups with poor glycemic control, larger ulcer sizes, longer ulcer durations, and current smoking. The ON101 with GWC strategy was considered cost-effective in 60% to 82% of model iterations against willingness-to-pay thresholds of $32 787/QALY gained to $98 361/QALY gained. Conclusions and Relevance: In this economic evaluation study using a simulated patient cohort, the ON101 with GWC strategy represented good value compared with GWC alone for patients with DFUs from the Taiwan health care sector perspective and may be prioritized for those with high risks for disease progression of DFUs.

Validation of Fespixon in Postoperative Scar Cosmesis Using Quantitative Digital Photography Analysis.

BACKGROUND: Unsightly scarring after surgery remains a dilemma. One of the challenges is the lack of objective scar assessment tools. OBJECTIVES: This study aimed to evaluate the efficacy of a novel medicine, Fespixon, for prevention and/or alleviation of post-skin incision scarring. A second aim was to demonstrate the practicality of our digital image analysis system to see if this could serve as a sensitive tool to assess scar improvement. METHODS: A prospective, placebo-controlled trial involving patients with postoperative transverse scars was conducted. Each patient received a topical formulation of Fespixon on the left part of the scar and placebo cream on the right. In addition to recording the subjective modified Vancouver Scar Scale and visual analog scale scores, we utilized digital photography for monthly scar analysis, with CIELAB and hue serving as the colorimetric information, and with contrast, correlation, homogeneity, and entropy providing texture information. RESULTS: Forty-six participants (mean age, 52 years) were enrolled in the trial. All the parameters of subjective assessment showed superior results for the Fespixon-treated side, with significant differences in pigmentation, vascularity, pliability, height, itchiness, and patient satisfaction (P = .043, .013, .026, .002, .039, .012, respectively). The trends in color and texture showed increased relative difference ratios, with significant differences in most of the eigenvalues towards the Fespixon-treated side, including CIELAB-L* (P = .000), hue-R,G,B (red, blue, green) values (P = .034, .001, .011), contrast (P = .000), homogeneity (P = .000), correlation (P = .011), and entropy (P = .000). CONCLUSIONS: We validated the efficacy of Fespixon for postoperative scar healing based not only on subjective assessments but also on objective quantitative analyses. The results also indicated that our digital photography quantitative analysis system is an ideal tool for quantification of scar appearance.

New Horizons of Macrophage Immunomodulation in the Healing of Diabetic Foot Ulcers.

Diabetic foot ulcers (DFUs) are one of the most costly and troublesome complications of diabetes mellitus. The wound chronicity of DFUs remains the main challenge in the current and future treatment of this condition. Persistent inflammation results in chronic wounds characterized by dysregulation of immune cells, such as M1 macrophages, and impairs the polarization of M2 macrophages and the subsequent healing process of DFUs. The interactive regulation of M1 and M2 macrophages during DFU healing is critical and seems manageable. This review details how cytokines and signalling pathways are co-ordinately regulated to control the functions of M1 and M2 macrophages in normal wound repair. DFUs are defective in the M1-to-M2 transition, which halts the whole wound-healing machinery. Many pre-clinical and clinical innovative approaches, including the application of topical insulin, CCL chemokines, micro RNAs, stem cells, stem-cell-derived exosomes, skin substitutes, antioxidants, and the most recent Phase III-approved ON101 topical cream, have been shown to modulate the activity of M1 and M2 macrophages in DFUs. ON101, the newest clinically approved product in this setting, is designed specifically to down-regulate M1 macrophages and further modulate the wound microenvironment to favour M2 emergence and expansion. Finally, the recent evolution of macrophage modulation therapies and techniques will improve the effectiveness of the treatment of diverse DFUs.

Low-dose ferrous bisglycinate chelate supplementation in chronic kidney disease and hemodialysis patients.

BACKGROUND: Provision of parenteral or oral iron supplementation can restore iron stores and maintain stable hemoglobin levels in chronic kidney disease (CKD) and hemodialysis (HD) patients. The route for oral or intravenous (IV) administration of iron depends on the acuity of anemia, costs, and patient tolerance. IV iron can restore iron stores rapidly but also carries higher risks for allergy and infection. Oral iron supplementation is limited by high gastrointestinal adverse effects. METHODS: We conducted an open-label trial to study the efficiency of a film-coated iron supplementation tablet, which contains ferrous bisglycinate chelate, vitamin C, and folic acid, in CKD stage 3b to 4 and HD patients. RESULTS: Twenty-seven HD patients and 20 CKD patients participated this study. After a 16-week intervention, low-dose ferrous bisglycinate chelate improved serum iron concentration (67.8 vs 87.2 mg/dL, p = 0.04) and transferrin saturation (24.7% vs 31.3%, p = 0.03) in stage 3 to 4 CKD patients, restored iron loss, and maintained stable hemoglobin levels in HD patients. No GI upset events were reported. CONCLUSION: Ferrous bisglycinate chelate is a well-tolerated oral iron supplementation for CKD and HD patients.

Extract From Plectranthus amboinicus Inhibit Maturation and Release of Interleukin 1ß Through Inhibition of NF-κB Nuclear Translocation and NLRP3 Inflammasome Activation.

Uncontrolled inflammation may produce massive inflammatory cytokines, in which interleukin 1ß (IL-1ß) plays a key role, resulting in tissue damage and serious disorders. The activation of NLRP3 inflammasome is one of the major mechanisms in maturation and release of IL-1ß. Plectranthus amboinicus is a perennial herb. Several pharmacological activities of natural components and crude extracts from P. amboinicus have been reported including anti-inflammation; however, the underlying mechanism is not clear. Phorbol-12-myristate 13-acetate-differentiated THP-1 monocytic leukemia cells were used as a reliable model in this study to examine the effect on inflammasome signaling pathway by PA-F4, an extract from Plectranthus amboinicus. PA-F4 inhibited ATP-induced release of caspase-1, IL-1ß, and IL-18 from lipopolysaccharides (LPS)-primed cells. PA-F4 induced a concentration-dependent inhibition of both ASC dimerization and oligomerization in cells under LPS priming plus ATP stimulation. Co-immunoprecipitation of NLRP3 and ASC demonstrated that PA-F4 significantly blunted the interaction between NLRP3 and ASC. Furthermore, PA-F4 completely abolished ATP-induced K+ efflux reaction in LPS-primed cells. Taken together, PA-F4 displayed an inhibitory activity on NLRP3 inflammasome activation. Moreover, PA-F4 also inhibited LPS-induced p65 NF-κB activation, suggesting an inhibitory activity on LPS priming step. Further identification showed that rosmarinic acid, cirsimaritin, salvigenin, and carvacrol, four constituents in PA-F4, inhibited LPS-induced IL-6 release. In contrast, rosmarinic acid, cirsimaritin and carvacrol but not salvigenin inhibited ATP-induced caspase-1 release from LPS-primed cells. In conclusion, PA-F4 displayed an inhibitory activity on activation of NLRP3 inflammasome. PA-F4 inhibited LPS priming step through block of p65 NF-κB activation. It also inhibited ATP-induced signaling pathways in LPS-primed cells including the inhibition of both ASC dimerization and oligomerization, K+ efflux reaction, and the release reaction of caspase-1, IL-1ß, and IL-18. Rosmarinic acid, cirsimaritin, salvigenin, and carvacrol could partly explain PA-F4-mediated inhibitory activity on blocking the activation of NLRP3 inflammasome.

Inhibition of hyperactivity and impulsivity by carbonic anhydrase inhibitors in spontaneously hypertensive rats, an animal model of ADHD.

RATIONALE: Dysregulation of noradrenergic and dopaminergic systems is involved in the pathology of attention deficit hyperactivity disorder (ADHD). Carbonic anhydrase (CA) has been reported to affect monoamine transmission in the central nervous system. OBJECTIVES: The aim of this study is to investigate the effect of CA inhibitors on the hyperactivity and impulsivity of the spontaneously hypertensive rat (SHR), which is currently the best-validated animal model of ADHD. METHODS: SHRs and Wistar Kyoto rats at 6 to 8 weeks of age were pretreated with intraperitoneal injections of acetazolamide and methazolamide, both carbonic anhydrase inhibitors, before the behavior tests. The open-field locomotion test and the electro-foot shock aversive water drinking test were then applied to quantify their hyperactivity and impulsivity, respectively. The Morris water maze test, on the other hand, monitored their spatial learning. RESULTS: Acetazolamide and methazolamide significantly inhibited the hyperactivity of SHRs but had no effects in Wistar Kyoto rats. Acetazolamide also inhibited the impulsivity of SHRs. Low doses of acetazolamide had the greater inhibitory effects on the hyperactivity and impulsivity, but did not impair the spatial learning of SHRs. CONCLUSIONS: This is the first study to show that carbonic anhydrase inhibitors can strain-specifically antagonize the hyperactivity and impulsivity of SHRs. Under a low dose of acetazolamide, there was no cognition impairment in SHRs. Carbonic anhydrase inhibitors may be the novel drugs for treatment for patients with ADHD.

CCN3 increases cell motility and MMP-13 expression in human chondrosarcoma through integrin-dependent pathway.

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN3, also called nephroblastoma overexpressed gene (NOV), regulates proliferation and differentiation of cancer cells. However, the effect of CCN3 on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells (JJ012 cells). αvß3 or αvß5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase (PI3K) inhibitors (Ly294002 and wortmannin) and Akt inhibitor inhibited the CCN3-induced increase of the migration and MMP-13 upregulation of chondrosarcoma cells. CCN3 stimulation increased the phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. In addition, NF-κB inhibitors also suppressed the cell migration and MMP-13 expression enhanced by CCN3. Moreover, CCN3 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-13 promoter. Taken together, our results indicate that CCN3 enhances the migration of chondrosarcoma cells by increasing MMP-13 expression through the αvß3/αvß5 integrin receptor, FAK, PI3K, Akt, p65, and NF-κB signal transduction pathway.

HMG-CoA reductase inhibitors activate the unfolded protein response and induce cytoprotective GRP78 expression.

AIMS: Since apoptosis of macrophages induced by stress to the endoplasmic reticulum (ER) contributes to advanced atherosclerotic lesions, we sought to understand the effects of statins on the unfolded protein response (UPR). METHODS AND RESULTS: We used pharmacological, biochemical, and siRNA (small interfering ribonucleic acid) approaches to determine the signalling cascades of statin-induced 78 kDa glucose-regulated protein (GRP78) gene transcription and its role in cytoprotection. Exposure of RAW264.7 macrophages to statins increased the expression of GRP78, activating transcription factor 6, X box protein-1, and phosphorylated eukaryotic translation initiation factor 2alpha, while it had no effect on CCAAT/enhancer binding protein-homologous protein. GRP78 induction was abolished by co-treatment with mevalonate and 1,2-bis(o-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid, indicating the involvement of both 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase-dependent and -independent mechanisms. Studies on promoter activity measurements indicated that phosphoinositide turnover, cellular homologue of v-src (c-Src), protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and p38 are involved in upregulating GRP78 gene transcription. We also observed that elevation of intracellular Ca(2+) and interruption of small G proteins are two bifurcated but cooperative signalling pathways for c-Src activation, leading to downstream activation of phospholipase C, PKC, ERK, and p38. Functionally we demonstrated that fluvastatin could protect macrophages from hypoxia-induced cell death through GRP78 induction. CONCLUSION: We demonstrate a novel action of statins of inducing a cytoprotective UPR, providing new insights into the clinical potential of statins for ameliorating ER stress-related diseases.

HMG-CoA reductase inhibitors upregulate heme oxygenase-1 expression in murine RAW264.7 macrophages via ERK, p38 MAPK and protein kinase G pathways.

Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme catabolism, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. HMG-CoA reductase inhibitors (statins) possess several anti-inflammatory mechanisms and may be beneficial in the treatment of inflammatory diseases. Our previous study has shown that statins can inhibit iNOS gene expression in murine RAW264.7 macrophages. In this study, we showed that lovastatin, fluvastatin, atorvastatin, simvastatin, mevastatin and pravastatin are able to upregulate the mRNA expression of HO-1 gene. This effect of lovastatin was attenuated by farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), a protein kinase G (PKG) inhibitor (KT5823), a soluble guanylyl cyclase inhibitor (ODQ), a p38 MAPK inhibitor (SB203580), and MEK inhibitors (U0126 and PD98059), but not by inhibitors of protein kinase C (PKC), protein kinase A (PKA), c-jun N-terminal kinase (JNK) and Rho kinase. Consistent with this notion, our previous study has reported the ability of statins to activate ERK and p38 MAPK in RAW264.7 macrophages. Here we further found the participation of cyclic guanosine monophosphate (cGMP)/PKG pathway for ERK activation in cells stimulated with statin and the ability of statin to induce AP-1 activity, which is an essential transcription factor in the regulation of HO-1 gene expression. In addition, a Ras inhibitor (manumycin A) treatment also caused a marked induction of HO-1 mRNA followed by a corresponding increase in HO-1 protein; instead, inhibition of Rho activity by toxin B only led to a transient and weak induction of HO-1. The involvement of signal pathways in manumycin A-induced HO-1 gene expression was associated with p38 MAPK, JNK and ERK activation. Taken together, these results demonstrate for the first time that statins might activate PKG to elicit activations of ERK and p38 MAPK pathways and finally induce HO-1 gene expression, which provides a novel anti-inflammatory mechanism in the therapeutic validity.

Inhibition of iNOS gene expression by quercetin is mediated by the inhibition of IkappaB kinase, nuclear factor-kappa B and STAT1, and depends on heme oxygenase-1 induction in mouse BV-2 microglia.

In the present study, experiments were performed to explore the action of quercetin, the most widely distributed flavonoids, and its major metabolite, quercetin-3'-sulfate, on lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-induced nitric oxide (NO) production in BV-2 microglia. Quercetin could suppress LPS- and IFN-gamma-induced NO production and inducible nitric oxide synthase (iNOS) gene transcription, while quercetin-3'-sulfate had no effect. LPS-induced IkappaB kinase (IKK), nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1) activation, and IFN-gamma-induced NF-kappaB, signal transducer and activator of transcription-1 (STAT1) and interferon regulatory factor-1 (IRF-1) activation were reduced by quercetin. Moreover quercetin was able to induce heme oxygenase-1 expression. To address the involvement of heme oxygenase-1 induction in iNOS inhibition, heme oxygenase-1 antisense oligodeoxynucleotide was used. Quercetin-mediated inhibition of NO production and iNOS protein expression were partially reversed by heme oxygenase-1 antisense oligodeoxynucleotide, but was mimicked by hemin, a heme oxygenase-1 inducer. The involvement of signal pathways in quercetin-induced heme oxygenase-1 gene expression was associated with tyrosine kinase and mitogen-activated protein kinases activation. All these results suggest quercetin should provide therapeutic benefits for suppression of inflammatory-related neuronal injury in neurodegenerative diseases.

HMG-CoA reductase inhibitors induce COX-2 gene expression in murine macrophages: role of MAPK cascades and promoter elements for CREB and C/EBPbeta.

Except functioning as lipid-lowering agents, HMG-CoA inhibitors, statins, are good tools to clarify the signaling role of small G proteins. In this study, we found in murine RAW264.7 macrophages, statins within 1-30 microM stimulated COX-2 gene transcription and PGE(2) formation, displaying potencies as lovastatin > fluvastatin > atorvastatin >> pravastatin. Transfection experiments with COX-2 promoter construct showed the necessity of C/EBPbeta and CRE promoter sites, but not NF-kappaB promoter site. Effects of statins on the activation of COX-2 promoter, induction of COX-2 protein, and PGE(2) production were all prevented by mevalonate and prenylated metabolites, FPP and GGPP. Consistent with the effect of statins, manumycin A, farnesyltransferase inhibitor, and geranylgeranyltransferase inhibitor increased PGE(2) production and COX-2 induction. Likewise, toxin B, an inhibitor of Rho family members, caused a prominent COX-2 induction. Results also indicated that tyrosine kinase, ERK, and p38 MAPK play essential roles in statin action. Taken together, these results not only demonstrate a unique action of statins in the upregulation of COX-2 expression in macrophages, but also suggest a negative role controlled by small G proteins in COX-2 gene regulation. Removal of this negative control by impairing G protein prenylation with statins leads to MAPKs activation and promotes COX-2 gene expression through the activation at CRE and C/EBPbeta sites.

Statins induce suppressor of cytokine signaling-3 in macrophages.

Our previous study has shown that lipophilic 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors of statins can inhibit interferon-gamma-induced inducible nitric oxide synthase gene expression in RAW264.7 macrophages. In this study, we showed that lovastatin and fluvastatin are able to upregulate the mRNA expression of the suppressor of cytokine signaling-3 (SOCS-3) gene. This effect is specific for SOCS-3 and could be blocked by mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, while it was not affected by inhibitors of protein kinase C and A, mitogen-activated protein/extracellular signal-regulated kinase kinase, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, Src, Raf and Rho kinase. SOCS-3 expression results in the inhibition of interferon-gamma-, interleukin-6- and macrophage colony-stimulating factor-elicited signal transducer and activator of transcription phosphorylation, suggesting a novel anti-inflammatory mechanism of statins to down-modulate the functions of interferon-gamma-activated macrophages.

HMG-CoA reductase inhibitors inhibit inducible nitric oxide synthase gene expression in macrophages.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are potent inhibitors of cholesterol synthesis and have wide therapeutic use in cardiovascular diseases. Recent evidence, however, suggests that the beneficial effects of statins may extend beyond their action on serum cholesterol levels. In this study, we investigated the effects of lovastatin, pravastatin, atorvastatin and fluvastatin on macrophage formation of nitric oxide (NO) in murine RAW 264.7 cells. Stimulation of macrophages with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) resulted in inducible NO synthase (iNOS) expression, which was accompanied by a large amount of NO formation. At concentrations of 0.1-30 microM, statins can inhibit stimuli-induced NO formation and iNOS induction to different extents. This inhibition occurs at the transcriptional level, and displays potency in the order of lovastatin > atorvastatin > fluvastatin >> pravastatin. We found that LPS-induced I kappa B kinase and nuclear factor-kappa B (NF-kappa B) activation, as well as IFN-gamma-induced signal transducer and activator of transcription 1 (STAT1) phosphorylation, were reduced by lovastatin. Moreover, inhibition by lovastatin of NO production and kappa B activation was reversed by mevalonate, geranylgeranyl pyrophosphate and farnesyl pyrophosphate. All these results suggest that inhibition of iNOS gene expression by statins can be attributed to interference with protein isoprenylation, which mediates both NF-kappa B and STAT1 activation in the upstream signaling pathways for iNOS gene transcription.