Halofuginone

Halofuginone is a plant derivative that has been shown to inhibit Th17 differentiation, and recently tested as a potential immunosuppressant. IC50 value: Target: In vitro: The addition of halofuginone can significantly inhibit the migratory and invasive trend induced by irradiation, and the TGF-β pathway was also inhibited [1]. In the study, we observed the strong inhibitory activity of halofuginone on HepG2 cell growth and the cell cycle and apoptosis assays showed that halofuginone arrested the cell cycle and inhibited the induction. And we found that halofuginone inhibits tumor cell cycle possibly by up-regulating p15 and p21 of expression. Then, we found that the proportion of cleaved PARP, caspase-3, 8 and 9 in HepG2 cells increased after halofuginone treatment. And the results showed that halofuginone down-regulated Mcl-1 and c-IAP1 expression. Finally, our results showed halofuginone regulated the activities of JNK and MEK/ERK signaling pathways in hepatocellular carcinoma cells [3]. In vivo: In animal xenograft model, the addition of halofuginone to irradiation inhibited the growth of subcutaneously implanted xenografts, reduced hepatic and pulmonary metastases, and improved survival of the mice [1]. C57BL/6 recipient mice received an orthotopic left lung transplant, and Lung transplant recipients received daily intraperitoneal injections of 2.5 μg halofuginone or vehicle alone. Lung grafts were assessed on Days 7, 14, and 28 post-transplant. Compared with control mice, on Day 28 post-transplant, lung grafts of mice treated with halofuginone showed a significant reduction in the percentage of obliterated airways (6.8 ± 4.7% vs 52.5 ± 13.8%, p < 0.01), as well as significantly reduced parenchymal fibrosis (5.5 ± 2.3% vs 35.9 ± 10.9%, p < 0.05) [2].

References:
[1]. Runlong Lin, et al. Inhibition of TGF-β signaling with halofuginone can enhance the antitumor effect of irradiation in Lewis lung cancer. Onco Targets Ther. 2015; 8: 3549–3559. [2]. Tony L. H. Chu, et al. Halofuginone Synergistically Enhances Anti-Proliferation of Rapamycin in T Cells and Reduces Cytotoxicity of Cyclosporine in Cultured Renal Tubular Epithelial Cells. PLoS One. 2015; 10(12): e0144735. [3]. Sun XH, et al. Halofuginone alleviates acute viral myocarditis in suckling BALB/c mice by inhibiting TGF-β1. Biochem Biophys Res Commun. 2016 Apr 29;473(2):558-64.

Treatment of cryptosporidiosis in captive green iguanas (Iguana iguana).[Pubmed:29559143]

Vet Parasitol. 2018 Mar 15;252:17-21.

There are no standard guidelines for the treatment of cryptosporidiosis in reptiles. The aim of this study was to evaluate the efficacy of two cryptosporidiosis therapies in captive green iguanas. Eight green iguanas aged 2-6 years, including 6 (1 male symbol and 5 female symbol) animals with chronic diarrhea, received treatment for cryptosporidiosis. The presence of Cryptosporidium sp. oocysts was determined in 8 iguanas (100%), Isospora sp. oocysts were detected in 3 animals (37.5%), and Oxyuridae eggs were observed in 5 iguanas (62.5%). The animals were divided into two therapeutic groups (A and B). Group A iguanas were administered Halofuginone (Halocur, 0,50mg/ml Intervet Productions S.A., France) at a dose of 110mg/kg body weight (BW) every 7days for 5 weeks. Group B animals were administered sulfadiazine and trimethoprim (Norodine Vet Oral Paste sulfadiazine 288,3mg/g, trimethoprim 58mg/g, ScanVet Animal Health A/S, Denmark) at 75mg/kg BW per os every 5days for 5 weeks and spiramycin and metronidazole (Stomorgyl, spiramycin 1500000 IU, metronidazole 250mg, Merial, France) at 200mg/kg BW every 5days for 5 weeks. Both groups received hyperimmune bovine colostrum and subcutaneous fluids. Before treatment, the average number of Cryptosporidium sp. oocysts in 1g of feces was determined at 1.71*10(5) (+/-313,262.44) in group A and 1.56*10(5) (+/-262,908.53) in group B; the average number of Isospora sp. oocysts was determined at 3.53*10(3) (+/-1747.38), and the average number of Oxyuridae eggs was determined at 810 (+/-496.74). Blood tests were performed once before treatment. The results of blood morphology and biochemistry tests before treatment revealed leukocytosis with a significant increase in heterophile and monocyte counts in all animals. Dehydration, elevated hematocrit values and low levels of Na(+), Ca(2+), PO4(-) and Cl(-) ions were observed in 6 iguanas. Two iguanas died during treatment. The gross necropsy revealed acute inflammation of gastric and duodenal mucosa, mucosal ecchymoses in the gastrointestinal tract, hepatomegaly and liver congestion, cholecystitis, enlarged kidneys and renal edema and congestion, cystitis, and an absence of fat bodies. Parasites were not detected in any developmental form after 40days of therapy and during an monthly 18-month follow-up period. Effective treatment of cryptosporidiosis in reptiles minimizes the adverse consequences of disease, improves the animals' well-being and decreases euthanasia rates.

Coccidiostats in milk: development of a multi-residue method and transfer of salinomycin and lasalocid from contaminated feed.[Pubmed:29648988]

Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2018 Aug;35(8):1508-1518.

A confirmatory multi-residue method was developed for the determination in milk of 19 coccidiostats (amprolium, arprinocid, clazuril, clopidol, decoquinate, diclazuril, ethopabate, Halofuginone, lasalocid, maduramicin, monensin, narasin, nicarbazin, nequinate, robenidine, salinomycin, semduramicin, toltrazuril sulfone and toltrazuril sulfoxide). Sample preparation utilising extraction with organic solvent and clean up by SPE and freezing was found reliable and time-efficient. Optimised chromatography and MS conditions with positive and negative ESI achieved sufficient sensitivity and selectivity. Validation experiments has proven method usefulness for routine analysis of coccidiostats in milk samples. An on-farm study conducted on dairy cows fed with experimentally contaminated feed with salinomycin and lasalocid showed negligible transfer to milk. No residues of lasalocid were found in collected samples. Salinomycin was found only in 5 of 168 samples analysed, while the concentrations of salinomycin in those samples (0.119-0.179 microg kg(-)(1)) was significantly below the limit of salinomycin in milk set by European Union legislation. Such low concentrations of both coccidiostats cannot be explained by conjugation during dairy cows' metabolism, as shown by experiments with enzymatic hydrolysis.

Halofuginone Attenuates Osteoarthritis by Rescuing Bone Remodeling in Subchondral Bone Through Oral Gavage.[Pubmed:29636687]

Front Pharmacol. 2018 Mar 27;9:269.

Osteoarthritis (OA) is a common debilitating joint disorder worldwide without effective medical therapy. Articular cartilage and subchondral bone act in concert as a functional unit with the onset of OA. Halofuginone is an analog of the alkaloid febrifugine extracted from the plant Dichroa febrifuga, which has been demonstrated to exert inhibition of SMAD 2/3 phosphorylation downstream of the TGF-beta signaling pathway and osteoclastogenesis. To investigate whether Halofuginone (HF) alleviates OA after administration by oral gavage, 3-month-old male mice were allocated to the Sham group, vehicle-treated anterior cruciate ligament transection (ACLT) group, and HF-treated ACLT group. The immunostaining analysis indicated that HF reduced the number of matrix metalloproteinase 13 (MMP-13) and collagen X (Col X) positive cells in the articular cartilage. Moreover, HF lowered histologic OA score and prevented articular cartilage degeneration. The micro-computed tomography (muCT) scan showed that HF maintained the subchondral bone microarchitecture, demonstrated by the restoration of bone volume fraction (BV/TV), subchondral bone plate thickness (SBP.Th.), and trabecular pattern factor (Tb.Pf) to a level comparable to that of the Sham group. Immunostaining for CD31 and muCT based angiography showed that the number and volume of vessels in subchondral bone was restored by HF. HF administered by oral gavage recoupled bone remodeling and inhibited aberrant angiogenesis in the subchondral bone, further slowed the progression of OA. Therefore, HF administered by oral gavage could be a potential therapy for OA.

Halofuginone attenuates intervertebral discs degeneration by suppressing collagen I production and inactivating TGFbeta and NF-small ka, CyrillicB pathway.[Pubmed:29524883]

Biomed Pharmacother. 2018 May;101:745-753.

Most low back pain is caused by intervertebral discs (IVD) degeneration, a disease that prevalence is increasing with age. Halofuginone, an analog of ferbrifugine isolated from plant Dichroa febrifuga, has drawn much attention in recent years for the wide range of bioactivities in malaria, cancer, fibrotic and autoimmune diseases. In this study, we evaluated the benefit effects of Halofuginone in IVD degeneration treatment in a validated rabbit puncture model. Halofuginone treatment could attenuate disc degeneration by suppressing the decrease of discs height and nucleus pulposus signal strength. Besides, Halofuginone treatment could suppress mRNA and protein expression of collagen I in nucleus pulposus. This might possibly due to the inactivation of transform growth factor-beta (TGFbeta) signal pathway by down-regulating p-Samd3 and up-regulating inhibitory Smad7. Then, we evaluated the effects of Halofuginone treatment on nuclear factor of kappa B (NF-kappaB) signal pathway and its downstream pro-inflammatory cytokines. The level of p-p65 and p-IkappaBalpha was down-regulated in Halofuginone treated group, indicating the inactivation of NF-kappaB signal pathway. The mRNA expression of interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and interleukin 8 (IL-8) was decreased in nucleus pulposus too, indicating the down-regulation of pro-inflammatory cytokines. In conclusion, Halofuginone treatment could attenuate IVD degeneration and this was possibly due to suppressing of collagen I production and inactivation of TGFbeta and NF-kappaB signal pathway in nucleus pulposus of degenerated discs. These results suggest that Halofuginone has the potential for IVD degeneration treatment, but more research is needed to validate this.

Amino acid starvation sensing dampens IL-1beta production by activating riboclustering and autophagy.[Pubmed:29621237]

PLoS Biol. 2018 Apr 5;16(4):e2005317.

Activation of the amino acid starvation response (AAR) increases lifespan and acute stress resistance as well as regulates inflammation. However, the underlying mechanisms remain unclear. Here, we show that activation of AAR pharmacologically by Halofuginone (HF) significantly inhibits production of the proinflammatory cytokine interleukin 1beta (IL-1beta) and provides protection from intestinal inflammation in mice. HF inhibits IL-1beta through general control nonderepressible 2 kinase (GCN2)-dependent activation of the cytoprotective integrated stress response (ISR) pathway, resulting in rerouting of IL-1beta mRNA from translationally active polysomes to inactive ribocluster complexes-such as stress granules (SGs)-via recruitment of RNA-binding proteins (RBPs) T cell-restricted intracellular antigen-1(TIA-1)/TIA-1-related (TIAR), which are further cleared through induction of autophagy. GCN2 ablation resulted in reduced autophagy and SG formation, which is inversely correlated with IL-1beta production. Furthermore, HF diminishes inflammasome activation through suppression of reactive oxygen species (ROS) production. Our study unveils a novel mechanism by which IL-1beta is regulated by AAR and further suggests that administration of HF might offer an effective therapeutic intervention against inflammatory diseases.

Chemical synthesis of febrifugine and analogues.[Pubmed:29681487]

Bioorg Med Chem. 2018 May 15;26(9):2199-2220.

The quinazolinone-containing 2,3-disubstituted piperidines febrifugine and isofebrifugine have been the subject of significant research efforts since their occurrence in Dichroa febrifuga and their anti-malarial actions were first described in the late 1940s. Subsequently they have also been shown to be present in other plants belonging to the hydrangea family and various analogues of febrifugine have been prepared in attempts to tune biological properties. The most notable analogue is termed Halofuginone and a substantial body of work now demonstrates that this compound possesses potent human disease relevant activities. This review focuses on the literature associated with efforts dedicated towards uncovering the structures of febrifugine and isofebrifugine, the development of practical methods for their synthesis and the syntheses of structural analogues.

Development and comparison of HPLC-MS/MS and UPLC-MS/MS methods for determining eight coccidiostats in beef.[Pubmed:29730532]

J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Jun 15;1087-1088:98-107.

A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining eight coccidiostat (Halofuginone, lasalocid, maduramicin, monensin, narasin, nigericin, robenidine and salinomycin) residues in beef were developed and compared. Samples were extracted with a mixture of acetic acid, acetonitrile and ethyl acetate and were then purified on a C18 solid-phase extraction (SPE) column. The purified samples were analyzed by HPLC-MS/MS and UPLC-MS/MS, using 0.1% formic acid-water solution (A) and pure methanol (B) as the mobile phase. The samples were fractionated on a C18 column using different gradient elution procedures, followed by qualitative analysis using a mass spectrometer operated in multiple reaction monitoring (MRM) mode with positive electrospray ionization; the external standard method was used for quantitation. At spiked levels that ranged from the limit of quantification (LOQ) to 100mug/kg, the average recoveries were 71.96%-100.32% and 71.24%-89.24%, with relative standard deviations (RSDs) of 2.65%-12.38% and 2.98%-14.86% for UPLC-MS/MS and HPLC-MS/MS, respectively. The limits of detection (LODs) and LOQs of the eight coccidiostats were 0.14-0.32mug/kg and 0.43-1.21mug/kg, respectively, for UPLC-MS/MS analysis and 0.16-0.58mug/kg and 0.53-1.92mug/kg, respectively, for HPLC-MS/MS analysis. Both methods had good accuracy and precision, but UPLC-MS/MS had higher sensitivity than HPLC-MS/MS.

Halofuginone (RU-19110) is a less-toxic form of Febrifugine, which is isolated from the plant Dichroa febrifuga. Halofuginone inhibits prolyl-tRNA synthetase in an ATP-dependent manner with a Ki of 18.3 nM. Halofuginone attenuates osteoarthritis (OA) by inhibition of TGF-β activity.

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