Chronic graft-vs-host disease: Current understanding of disease and treatment landscape.

Chronic graft-vs-host disease (cGVHD) is a common complication and a major cause of morbidity and mortality following allogeneic hematopoietic cell transplantation. It is primarily characterized by chronic inflammation and fibrosis involving multiple organs. Firstline treatment with steroids with or without calcineurin inhibitors for cGVHD is well established; however, a significant number of patients develop steroid-refractory/resistant cGVHD (SR-cGVHD). Subsequent treatment for SR-cGVHD varies widely, but recent advances include the approval of several medications specifically for this indication, offering new opportunities to improve the prognosis in this challenging-to-treat condition. Ongoing research in preemptive and treatment strategies, such as combinations with newer and better-tolerated agents, may inform future management of SR-cGVHD. DISCLOSURES: This supplement was written by Bridget Flavin, PharmD, Founder, Connected Content, Ltd. Connected Content, Ltd. received payment from AMC Media Group for the preparation of this manuscript. Flavin is also an adjunct associate professor at the University of Florida College of Pharmacy. This supplement was funded by Kadmon Pharmaceuticals, LLC.

Interplay of phosphate and carbonate ions with flavin photosensitizers in photodynamic inactivation of bacteria.

Photodynamic inactivation (PDI) of pathogenic bacteria is a promising technology in different applications. Thereby, a photosensitizer (PS) absorbs visible light and transfers the energy to oxygen yielding reactive oxygen species (ROS). The produced ROS are then capable of killing microorganisms via oxidative damage of cellular constituents. Among other PS, some flavins are capable of producing ROS and cationic flavins are already successfully applied in PDI. When PDI is used for example on tap water, PS like flavins will encounter various ions and other small organic molecules which might hamper the efficacy of PDI. Thus, the impact of carbonate and phosphate ions on PDI using two different cationic flavins (FLASH-02a, FLASH-06a) was investigated using Staphylococcus aureus and Pseudomonas aeruginosa as model organisms. Both were inactivated in vitro at a low light exposure of 0.72 J cm-2. Upon irradiation, FLASH-02a reacts to single substances in the presence of carbonate or phosphate, whereas the photochemical reaction for FLASH-06a was more unspecific. DPBF-assays indicated that carbonate and phosphate ions decreased the generation of singlet oxygen of both flavins. Both microorganisms could be easily inactivated by at least one PS with up to 6 log10 steps of cell counts in low ion concentrations. Using the constant radiation exposure of 0.72 J cm-2, the inactivation efficacy decreased somewhat at medium ion concentrations but reached almost zero for high ion concentrations. Depending on the application of PDI, the presence of carbonate and phosphate ions is unavoidable. Only upon light irradiation such ions may attack the PS molecule and reduce the efficacy of PDI. Our results indicate concentrations for carbonate and phosphate, in which PDI can still lead to efficient reduction of bacterial cells when using flavin based PS.

Neuroprotective Potential of Novel Multi-Targeted Isoalloxazine Derivatives in Rodent Models of Alzheimer's Disease Through Activation of Canonical Wnt/ß-Catenin Signalling Pathway.

Previous reports suggest that Alzheimer's disease is protected by cholinesterase inhibitors. We synthesized some isoalloxazine derivatives and evaluated them using in vitro cholinesterase inhibition assay. Two of the compounds (7m and 7q) were figured out as potent cholinesterase inhibitors. They further showed anti-Aß aggregatory activity in the in vitro assay. The current study deals with the evaluation of neuroprotective potentials of the potent compounds (7m and 7q) using different in vitro and in vivo experiments. The compounds were first assessed for their tendency to cross blood-brain barrier using in vitro permeation assay. They were evaluated using scopolamine-induced amnesic mice model. Additionally, ROS scavenging and anti-apoptotic properties of 7m and 7q were established against Aß1-42-induced toxicity in rat hippocampal neuronal cells. 7m and 7q were also evaluated using Aß1-42-induced Alzheimer's rat model. Lastly, their involvement in Wnt/ß-catenin pathway was also demonstrated. The results indicated good CNS penetration for 7m and 7q. The neuroprotective effects of 7m and 7q were evidenced by improved cognitive ability in both scopolamine and Aß1-42-induced Alzheimer's-like condition in rodents. The in vivo results also confirmed their anti-cholinesterase and anti-oxidant potential. Immunoblot results showed that treatment with 7m and 7q decreased Aß1-42, p-tau, cleaved caspase-3, and cleaved PARP levels in Aß1-42-induced Alzheimer's rat brain. Additionally, immunoblot results demonstrated that 7m and 7q activated the Wnt/ß-catenin pathway as evidenced by increased p-GSK-3, ß-catenin, and neuroD1 levels in Aß1-42-induced Alzheimer's rat brain. These findings have shown that isoalloxazine derivatives (7m and 7q) could be the potential leads for developing effective drugs for the treatment of AD.

Novel riboswitch-binding flavin analog that protects mice against Clostridium difficile infection without inhibiting cecal flora.

Novel mechanisms of action and new chemical scaffolds are needed to rejuvenate antibacterial drug discovery, and riboswitch regulators of bacterial gene expression are a promising class of targets for the discovery of new leads. Herein, we report the characterization of 5-(3-(4-fluorophenyl)butyl)-7,8-dimethylpyrido[3,4-b]quinoxaline-1,3(2H,5H)-dione (5FDQD)-an analog of riboflavin that was designed to bind riboswitches that naturally recognize the essential coenzyme flavin mononucleotide (FMN) and regulate FMN and riboflavin homeostasis. In vitro, 5FDQD and FMN bind to and trigger the function of an FMN riboswitch with equipotent activity. MIC and time-kill studies demonstrated that 5FDQD has potent and rapidly bactericidal activity against Clostridium difficile. In C57BL/6 mice, 5FDQD completely prevented the onset of lethal antibiotic-induced C. difficile infection (CDI). Against a panel of bacteria representative of healthy bowel flora, the antibacterial selectivity of 5FDQD was superior to currently marketed CDI therapeutics, with very little activity against representative strains from the Bacteroides, Lactobacillus, Bifidobacterium, Actinomyces, and Prevotella genera. Accordingly, a single oral dose of 5FDQD caused less alteration of culturable cecal flora in mice than the comparators. Collectively, these data suggest that 5FDQD or closely related analogs could potentially provide a high rate of CDI cure with a low likelihood of infection recurrence. Future studies will seek to assess the role of FMN riboswitch binding to the mechanism of 5FDQD antibacterial action. In aggregate, our results indicate that riboswitch-binding antibacterial compounds can be discovered and optimized to exhibit activity profiles that merit preclinical and clinical development as potential antibacterial therapeutic agents.

Discovery of isoalloxazine derivatives as a new class of potential anti-Alzheimer agents and their synthesis.

This article describes discovery of a novel and new class of cholinesterase inhibitors as potential therapeutics for Alzheimer's disease. A series of novel isoalloxazine derivatives were synthesized and biologically evaluated for their potential inhibitory outcome for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These compounds exhibited high activity against both the enzymes AChE as well as BuChE. Of the synthesized compounds, the most potent isoalloxazine derivatives (7m and 7q) showed IC50 values of 4.72 µM and 5.22 µM respectively against AChE; and, 6.98 µM and 5.29 µM respectively against BuChE. These two compounds were further evaluated for their anti-aggregatory activity for ß-amyloid (Aß) in presence and absence of AChE by performing Thioflavin-T (ThT) assay and Congo red (CR) binding assay. In order to evaluate cytotoxic profile of these two potential compounds, cell viability assay of SH-SY5Y human neuroblastoma cells was performed. Further, to understand the binding behavior of these two compounds with AChE and BuChE enzymes, docking studies have been reported.

Treatment of metabolic syndrome with ankaflavin, a secondary metabolite isolated from the edible fungus Monascus spp.

Edible fungi of the Monascus species have been used as traditional Chinese medicine in eastern Asia for several centuries. Monascus-fermented products possess a number of functional secondary metabolites, including anti-inflammatory pigments (such as monascin and ankaflavin [AK]), monacolins, and dimerumic acid. These secondary metabolites have anti-inflammatory, anti-oxidative, and anti-tumor activities. We found that AK positively regulates several transcription factors associated with the prevention of metabolic syndrome and other diseases, including peroxisome proliferator-activated receptor (PPAR)-gamma, PPAR-alpha, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). AK reduced hyperglycemia and enhanced pancreatic function via PPAR-gamma activation and increased lipid metabolism due to PPAR-alpha activation. The compound also exerted antioxidant effects via activation of Nrf2. These results suggest that AK belongs to the class of selective peroxisome proliferator-activated receptor modulators (SPPARMs), which are associated with a good safety profile when used in patients suffering from metabolic syndrome. Together with our studies to determine how AK production can be increased during Monascus fermentation, these data demonstrate the great potential of AK as a nutraceutical or therapeutic agent.

Modern management of hemorrhoidal disease.

Complaints secondary to hemorrhoidal disease have been treated by health care providers for centuries. Most symptoms referable to hemorrhoidal disease can be managed nonoperatively. When symptoms do not respond to medical therapy, procedural intervention is recommended. Surgical hemorrhoidectomy is usually reserved for patients who are refractory to or unable to tolerate office procedures. This article reviews the pathophysiology of hemorrhoidal disease and the most commonly used techniques for the nonoperative and operative palliation of hemorrhoidal complaints.

Irradiated riboflavin diminishes the aggressiveness of melanoma in vitro and in vivo.

Melanoma is one of the most aggressive skin cancers due to its high capacity to metastasize. Treatment of metastatic melanomas is challenging for clinicians, as most therapeutic agents have failed to demonstrate improved survival. Thus, new candidates with antimetastatic activity are much needed. Riboavin (RF) is a component of the vitamin B complex and a potent photosensitizer. Previously, our group showed that the RF photoproducts (iRF) have potential as an antitumoral agent. Hence, we investigated the capacity of iRF on modulating melanoma B16F10 cells aggressiveness in vitro and in vivo. iRF decreases B16F10 cells survival by inhibiting mTOR as well as Src kinase. Moreover, melanoma cell migration was disrupted after treatment with iRF, mainly by inhibition of metalloproteinase (MMP) activity and expression, and by increasing TIMP expression. Interestingly, we observed that the Hedgehog (HH) pathway was inhibited by iRF. Two mediators of HH signaling, GLI1 and PTCH, were downregulated, while SUFU expression (an inhibitor of this cascade) was enhanced. Furthermore, inhibition of HH pathway signaling by cyclopamine and Gant 61 potentiated the antiproliferative action of RF. Accordingly, when a HH ligand was applied, the effect of iRF was almost completely abrogated. Our findings indicate that Hedgehog pathway is involved on the modulation of melanoma cell aggressiveness by iRF. Moreover, iRF treatment decreased pulmonary tumor formation in a murine experimental metastasis model. Research to clarify the molecular action of flavins, in vivo, is currently in progress. Taken together, the present data provides evidence that riboflavin photoproducts may provide potential candidates for improving the efficiency of melanoma treatment.

Monascin and ankaflavin have more anti-atherosclerosis effect and less side effect involving increasing creatinine phosphokinase activity than monacolin K under the same dosages.

Monacolin K has long been considered a major effective component in the hypolipidemic functions of Monascus . Monacolin K also serves as a well-known hypolipidemic medication, but its side effect myopathy is a concern. Monascin and ankaflavin, the yellow pigments produced by Monascus species, have been proven to possess hypolipidemic functions; however, no studies have compared the hypolipidemic effects of monascin, ankaflavin, and monacolin K under the same dosages. In this study, the equal dosages of monascin, ankaflavin, and monacolin K were oral administrated to hamsters fed a high cholesterol diet for 6 weeks. Comparison of the displayed hypolipidemic and anti-atherosclerosis effects was performed, in addition to an investigation into the inducement of side effect. The results indicated that monascin and ankaflavin were similar to monacolin K in significantly reducing total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels in serum and lipid plaque (p < 0.05) in the heart aorta. In addition, ankaflavin achieved the effects of serum TC and TG reduction, with no significant difference as compared to those effects of monacolin K (p > 0.05). However, as compared to monacolin K, ankaflavin possessed more significant effects on the prevention of fatty liver and lipid plaque accumulation in heart aorta. More importantly, monascin significantly enhanced high-density lipoprotein cholesterol (HDL-C) concentrations, while monacolin K displayed the opposite effect. Regarding the side effect, monacolin K also raised elevated creatinine phosphokinase (CPK) activity, which was highly correlated with rhabdomyolysis development, while monascin and ankaflavin did not induce such a side effect. In conclusion, MS and AK had the potential to be developed as hypolipidemic agents without rhabdomyolysis development.

Ankaflavin: a natural novel PPARγ agonist upregulates Nrf2 to attenuate methylglyoxal-induced diabetes in vivo.

Ankaflavin (AK) is an active compound having anti-inflammatory, anti-cancer, antiatherosclerotic, and hypolipidemic effects. We have previously reported that AK acts as an antioxidant and antidiabetic drug; however, the mechanism by which AK prevents diabetes remains unknown. Hyperglycemia is associated with protein glycation, which produces advanced glycation end-products (AGEs). Methylglyoxal (MG)-a metabolite of carbohydrates-is believed to cause insulin resistance by inducing inflammation and pancreas damage. In this work, diabetes was induced in Wistar rats (4 weeks of age) by treating them with MG (600 mg/kg bw) for 4 weeks. We observed that AK (10mg/kg bw) exerted peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity, thereby enhancing insulin sensitivity (as indicated by hepatic GLUT2 translocation, PTP1B suppression, and glucose uptake) by downregulating blood glucose and upregulating pancreatic and duodenal homeobox-1 and Maf-A expression and increasing insulin production in MG-induced rats. However, these effects were abolished by the administration of GW9662 (PPARγ antagonist), but the expression of hepatic heme oxygenase-1 (HO-1) and glutamate-cysteine ligase (GCL) was not suppressed in MG-induced rats. Therefore, the nuclear factor erythroid-related factor-2 (Nrf2) activation was investigated. AK did not affect hepatic Nrf2 mRNA or protein expression but significantly increased Nrf2 phosphorylation (serine 40), which was accompanied by increased transcriptional activation of hepatic HO-1 and GCL. These data indicated that AK protected rats from oxidative stress resulting from MG-induced insulin resistance. In contrast, these effects were not detected when the rats were treated with the antidiabetic drug rosiglitazone (10mg/kg bw). Moreover, we found that AK did not inhibit the generation of AGEs in vitro; however, the glutathione (GSH) levels in liver and pancreas of MG-induced rats were elevated in rats administered AK. Therefore, we believe that GSH may lower the MG level, which attenuates the formation of AGEs in the serum, kidney, liver, and pancreas of MG-induced rats. We also found that AK treatment reduced the production of inflammatory factors, such as tumor necrosis factor-α and interleukin-1ß. Taken together, the results of our mechanistic study of MG-induced rats suggest that the protective effects of AK against diabetes are mediated by the upregulation of the signaling pathway of Nrf2, which enhances antioxidant activity and serves as a PPARγ agonist to enhance insulin sensitivity.

Ankaflavin, a novel Nrf-2 activator for attenuating allergic airway inflammation.

The role of inflammation-induced oxidative stress in the pathogenesis and progression of chronic inflammatory airways diseases has received increasing attention in recent years. Nuclear factor-erythroid 2 related factor 2 (Nrf-2) is the primary transcription factor that regulates the expression of antioxidant and detoxifying enzymes. In this study, yellow pigment ankaflavin (AK), derived from Monascus-fermented products, elevated nuclear Nrf-2 protein translocation in both the A549 lung cell line and the lungs of ovalbumin (OVA)-challenged mice. Furthermore, AK increased the mRNA expression of antioxidant enzymes regulated by Nrf-2, leading to a reduction in allergen-driven airway inflammation, mucus cell hyperplasia, and eosinophilia in OVA-challenged mice. Additionally, AK prevented T-cell infiltration and Th2 cytokines including interleukin (IL)-4, IL-5, and IL-13 generation in bronchial alveolar lavage fluid. The adhesion molecules ICAM-1, VCAM-1, and eotaxin were substantially reduced by AK treatment. Importantly, the inhibitory effect of AK on adhesion molecule production and immune cell infiltration was abolished by Nrf-2 small interfering RNA. This is the first study to illustrate that AK acts as a novel Nrf-2 activator for modulating the oxidative stress pathway to improve the lung injury and ameliorate the development of airway inflammation.

A new type of tyrosinase inhibitors from natural products as potential treatments for hyperpigmentation.

Selected flavonoids were evaluated for their effects on melanin biosynthesis by using mushroom-tyrosinase assay. Out of 27 tested flavonoids, only six showed a potential inhibitory activity on melanin biosynthesis. Flavonoids containing an alpha-keto group showed to be the active compounds in this assay. This may be explained in terms of similarity between the dihydroxyphenyl group in L-DOPA and the alpha-ketol group in flavonoids. The results of this study revealed a new type of tyrosinase inhibitor from natural origin. These compounds will be further examined for their application for treatment of hyperpigmentation problems e.g. melasma and ephelides.

Protective effects of riboflavin and its derivatives against ischemic reperfused damage of rat heart.

The effects of riboflavin and its derivatives such as FAD, FMN and lumichrome on the levels of high energy phosphate compounds (ATP and creatine phosphate) and intracellular pH in ischemic reperfused rat hearts were investigated using a Langendorff perfusion technique. 31P-NMR study showed a decrease in the levels of high energy phosphate compounds and pH values in myocardium after 30 min global ischemia and a slight recovery of these levels after a 30 min reperfusion following ischemia. However, in all the hearts perfused with riboflavin and its derivatives during ischemia-reperfusion, a marked recovery of high energy phosphate compounds and pH values were observed. In addition, the cardiac mitochondrial respiratory function was protected from ischemia-reperfusion injury. These results suggest that riboflavin, FAD, FMN, and lumichrome have a protective effect against ischemia-reperfusion injury to rat myocardium in vitro. It is assumed that these substances exert their effect directly in the extracellular space.

Antihypertensive effect of riboflavin analogs in rats with mineralocorticoid-induced hypertension.

This study investigated whether the riboflavin analogs, 7,8-dimethyl-10-formylmethyl isoalloxazine (FMI) and 7,8-dimethyl-10-(2'-hydroxyethyl) isoalloxazine (HEI), are effective antihypertensive agents in mineralocorticoid-induced or deoxycorticosterone acetate (DOCA)-salt hypertension. These studies are based on our previous observation tht aldosterone enhances the biosynthesis of renal flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) from riboflavin, and that FMI and HEI competitively inhibit conversion of riboflavin to FMN and reabsorption of Na+ in the kidney of adrenalectomized rats. When 1.6 mg of FMI or HEI were administered simultaneously with 3.0 mg of DOCA, the tail systolic blood pressure (SBP) of unanesthetized rats rose only to 136 +/- 5 mm Hg (standard error of the mean, SEM) compared to 163 +/- 5 mm Hg during DOCA therapy alone (p less than 0.0005). This hypotensive effect of FMI or HEI was noted after the fourth week of treatment and persisted through the ninth week. The rats tolerated the medication well and had no signs of riboflavin deficiency. DOCA administration alone resulted in a 24% increase in iliopsoas muscle Na+ concentration (p less than 0.0005), and a 0.8% increase in the water content of the muscle (p less than 0.025), suggesting a positive Na+ balance. Administration of FMI or HEI blunted the ability of DOCA to increase muscle Na+ concentration (p less than 0.025), water content (p less than 0.01). HEI treatment of the Kyoto strain of spontaneously hypertensive rats (SHR) did not lower their mean SBP. Thus it appears that the hypotensive actions of FMI or HEI are closely associated with their ability to modify the effects of mineralocorticoids on NA+ balance.

[Light and electron microscopy of rhinoscleroma (author's transl)]. / Die licht- und elektronenmikroskopische Untersuchung des Rhinoskleroms

The authors report the case of a 50 year old male patient whose rhinoscleroma, localized to the upper respiratory tract, was demonstrated by the isolation of Klebsiella bacilli and histologically. Electron microscopically the Mikulicz cells were characterized by fused vacuoles occupying the largest portion of the cytoplasm and displacing the damaged cytoplasmic organelles. Phagosomes and dense bodies reminiscent of Russel bodies also occurred in the Mikulicz cells, in the vacuoles of which formations representing Klebsiella rhinoscleromatis were demonstrated. A light halo was visible around some of these formations. It could not be, however, decided whether these halos represented the mucous sheath of the bacillus or an artifact only. In the plasmacells the authors observed the bag-like dilatation of the ergastoplasm and the presence of Russel bodies. Transitory forms were not seen among the plasma and Mikulicz cells. As a result of the treatment, Klebsiella disappeared from the nasal mucosa of the patient. The authors wish to follow by means of electron microscopy the changes of the granulation tissue and pathogens following antibiotic therapy.

Photodynamic inactivation with proflavine: quantitative comparison with iodo-deoxyuridine.

Photodynamic inactivation utilizing proflavine and light was quantitatively compared with the therapeutic antiviral effect of iodo-deoxyuridine in experimental herpetic keratitis in the rabbit. This rabbit model, which has been shown to be highly predictive for antiviral therapy in man, indicates that the photodynamic effect, though definite, is minimal.