Short-chain Fatty Acid Derivatives Research Articles

Sodium butyrate inhibits high glucose-induced inflammation by controlling the acetylation of NF-κB p65 in human monocytes.

Hyperglycemia is a major cause of diabetes and diabetes-related diseases. Sodium butyrate (NaB) is a short-chain fatty acid derivative that produces dietary fiber by anaerobic bacterial fermentation in the large intestine and occurs in foods, such as Parmesan cheese and butter. Butyrate has been shown to prevent obesity, improve insulin sensitivity, and ameliorate dyslipidemia in diet-induced obese mice. Therefore, this study examined the effects and mechanism of NaB on the secretion of inflammatory cytokines induced by high glucose (HG) in THP-1 cells. THP-1 cells were used as an in vitro model for HG-induced inflammation. The cells were cultured under normal glycemic or hyperglycemic conditions with or without NaB (0-25 μM). Western blotting and quantitative polymerase chain reaction were used to evaluate the protein and mRNA levels of nuclear factor-κB (NF-κB), interleukin-6, tumor necrosis factor-α, acetylated p65, acetyl CREB-binding protein/p300 (CBP/p300), and p300 using THP-1 cells. Histone acetyltransferase (HAT), histone deacetylase (HDAC), and pro-inflammatory cytokine secretion activity were analyzed using an enzyme-linked immunosorbent assay. HG significantly upregulated histone acetylation, acetylation levels of p300, NF-κB activation, and inflammatory cytokine release in THP-1 cells. Conversely, the NaB treatment reduced cytokine release and NF-κB activation in HG-treated cells. It also significantly reduced p65 acetylation, CBP/p300 HAT activity, and CBP/p300 gene expression. In addition, NaB decreased the interaction of p300 in acetylated NF-κB and TNF-α. These results suggest that NaB suppresses HG-induced inflammatory cytokine production through HAT/HDAC regulation in monocytes. NaB has the potential for preventing and treating diabetes and its related complications.

Chylomicron production is repressed by RPTOR knockdown, R-α-lipoic acid and 4-phenylbutyric acid in human enterocyte-like Caco-2 cells

Although the role of mechanistic target of rapamycin complex 1 (mTORC1) in lipid metabolism has been the subject of previous research, its function in chylomicron production is not known. In this study, we created three stable human colorectal adenocarcinoma Caco-2 cell lines exhibiting normal, low, or high mTORC1 kinase activity, and used these cells to investigate the consequences of manipulating mTORC1 activity on enterocyte differentiation and chylomicron-like particle production. Constitutively active mTORC1 induced Caco-2 cell proliferation and differentiation (as judged by alkaline phosphatase activity) but weakened transepithelial electrical resistance (TEER). Repressed mTORC1 activity due to the knockdown of RPTOR significantly decreased the expression of lipogenic genes FASN, DGAT1, and DGAT2, lipoprotein assembly genes APOB and MTTP, reduced protein expression of APOB, MTTP, and FASN, downregulated the gene expression of very long-chain fatty acyl-CoA ligase (FATP2), acyl-CoA binding protein (DBI), and prechylomicron transport vesicle-associated proteins VAMP7 (vesicle-associated membrane protein 7) and SAR1B (secretion associated Ras related GTPase 1B) resulting in the repression of apoB-containing triacylglycerol-rich lipoprotein secretion. Exposure of Caco-2 cells harboring a constitutively active mTORC1 to short-chain fatty acid derivatives, R-α-lipoic acid and 4-phenylbutyric acid, downregulated chylomicron-like particle secretion by interfering with the lipidation and assembly of the particles, and concomitantly repressed mTORC1 activity with no change to Raptor abundance or PRAS40 (Thr246) phosphorylation. R-α-lipoic acid and 4-phenylbutyric acid may be useful to mitigate intestinal lipoprotein overproduction and associated postprandial inflammation.

Valproic Acid Induced Thrombocytopenia and Dysmegakaryopoiesis in a Pediatric Patient

Abstract Introduction/Objective Valproic acid is a branched short chain fatty acid derivative that is used primarily to treat epilepsy as well as mood disorders, certain types of headaches, and neuropathic pain. It is commonly prescribed in the pediatric population and has shown to be effective for refractory epilepsy with adequate seizure control. Serious side effects may be prominent if the medication is not kept at the therapeutic range. A wide variety of known hematologic problems can be encountered including but not limited to anemia, thrombocytopenia, and leukopenia. Methods/Case Report We present a case of a pediatric patient with a past medical history significant for history of seizure disorder and who presented to Jackson Memorial Hospital for intermittent fevers and multiple unexplained bruises for 3 weeks as well as fatigue and weakness.The patient was recently started on valproic acid. Complete blood count (CBC) was obtained and showed a platelet count of 23 x10(3)/mcL with WBC of 3.5 x10(3)/mcL and hemoglobin of 9.7 g/dL.Serum valproate concentration was critically high (154 mg/L). Trephine biopsy showed a normocellular marrow (60%) showing maturing trilineage hematopoiesis and scattered atypical megakaryopoiesis characterized by small forms that are seen in relatively loose interstitial clusters (Figure 1). The marrow aspirate smears were characterized by cellular spicules with dysmegakaryopoiesis including numerous small hypolobated forms with frequent forms showing separated nuclei (Figure 2, 3, and 4). Blasts did not appear increased, comprising overall 1% of marrow cellularity. Karyotype studies revealed a normal female karyotype, 46, XX. FISH studies using probes commonly detected in MDS were negative. Next generation sequencing was negative for AML specific mutations including GATA1 and GATA2 mutations. Results (if a Case Study enter NA) N/A Conclusion This case report highlights the significant hematologic adverse effects of valproic acid, specifically pancytopenia with dysmegakaryopoiesis, raising the clinical suspicion of potential myelodysplastic syndrome. Critically high level of valproic acid (154 mg/L) and normalization of CBC after the stoppage of the medication strongly suggests that valproic acid can cause severe bone marrow suppression and specific morphologic atypia in the megakaryocytic lineage thus introducing a potential diagnostic pitfall. Because the CBC returned to normal, bone marrow biopsy was not repeated.

Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms

Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy.

스트렙토조토신으로 유도한 당뇨마우스에서 Sodium Butyrate의 혈당, 혈청 지질 성상 및 염증 억제에 미치는 영향

Sodium butyrate is a short-chain fatty acid derivative found in foods, such as Parmesan cheese and butter and is produced by anaerobic bacteria fermentation of dietary fibers in the large intestine. There have been reports that butyrate prevented obesity, protected insulin sensitivity, and ameliorated dyslipidemia in dietary obese mice. This study investigated the effects of sodium butyrate on fasting blood glucose level and serum lipid profile in streptozotocin(STZ)-induced diabetic mice. Male C57BL/6 mice were fed AIN-93G for four weeks prior to intraperitoneal injections with STZ (100 mg/kg body weight). Diabetic mice had supplements of 5% sodium butyrate for four weeks. The 5% sodium butyrate diet significantly improved fasting blood glucose level and lipid profile in STZ-induced diabetic mice. Inflammation has been recognized to decrease beta cell insulin secretion and increase insulin resistance. Circulating cytokines can directly affect beta cell function, leading to secretory dysfunction and increased apoptosis. Thus, anti-inflammatory therapies represented a potential approach for the therapy of diabetes and its complications. In this animal study, the 5% sodium butyrate supplementation also inhibited inflammatory cytokine production in STZ-induced diabetic mice. These results suggested that sodium butyrate can be a potential candidate for the prevention of diabetes and its complications.

Multiple Oral Therapeutics Suppress Repressors (LSD-1, HDACs, and BCL11A) of Gamma Globin Gene Expression

Induction of gamma globin expression to certain target levels reduces clinical severity in sickle cell disease, globin imbalance, and anemia in the beta thalassemias. However, achieving high level expression in diverse patients with variable baseline HbF levels is clinically challenging. To determine whether different therapeutics have complimentary molecular actions that could be combined for higher efficacy, we evaluated a panel of oral fetal globin-inducing therapeutics in clinical use or trials for effects on transcriptional suppressors of HBG, including components of the NuRD complex, LSD1 and HDACs 1-3, and the downstream repressor BCL11A, in erythroid progenitors cultured from sickle cell and beta thalassemia patients. Using chromatin immunoprecipitation assays, immunoblot, and qRT-PCR, we investigated two pan-HDAC inhibitors, MS-275 and SB939, a short chain fatty acid derivative without global histone deacetylation effects, sodium dimethylbutyrate (ST20), and PB-04, a PK-enhancing drug identified through high throughput screening (HTS), which has a long-standing benign clinical safety profile. These therapeutics induce fetal globin expression by 1.5 to 12-fold in different individuals’ progentiors over untreated controls and are active in 60-90% of patients’ progenitors. BCL11A mRNA and protein were suppressed by 3-7 fold and 5-10 fold, respectively, over untreated cells from the same subject by all agents tested. PB-04 treatment decreased LSD1 mRNA by 5-fold. MS-275 and ST20 suppressed KLF-1 mRNA by 3- and 2.5-fold respectively. ChIP analyses demonstrated that PB-04 reduced LSD1 and HDAC-3 occupancy on the gamma globin gene promoter by 4-fold and 3-fold, respectively, coincident with gamma globin induction; ST20 decreased HDAC-2 and LSD1 binding by 6- and 2-fold respectively. Further, histone marks associated with gene activation were enriched at the gamma-globin promoter by treatment with PB-04, including acetylated Histone H3K9 (AcH3K9), the target of HDAC3, and dimethylated H3K4 (H3K4me2), a target of LSD1. Collectively, these results identify oral clinical stage therapeutics which disrupt two major mechanisms of transcriptional repression. The findings provide a basis for targeting therapies in individuals and combining pharmacologic agents which induce HbF expression through complimentary molecular mechanisms to provide high level induction. DisclosuresFaller:Phoenicia BioSciences: Employment, Membership on an entity’s Board of Directors or advisory committees. Perrine:Phoenicia BioSciences: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Intra-Patient Variability in Fetal Hemoglobin Measurements over Time in Sickle Cell Disease Patients Not on Fetal Hemoglobin Inducing Agents

Introduction:We previously reported results of the placebo-controlled phase II study of the short-chain fatty acid derivative 2,2-dimethylbutyrate in inducing fetal hemoglobin (Hb F) in 76 patients with sickle cell disease (SCD). The primary endpoint was a comparison of Hb F levels in the treatment versus the placebo arms. Week 24 interim analyses revealed no statistically significant difference in change in Hb F levels between the 2 groups. We examined the placebo arm in order to assess untreated, intra-patient variability of Hb F%.Methods:Only Hb F values performed by HPLC at the central reference laboratory (Georgia Health Science University) were included. Any Hb F values determined within a 3 month period after a blood transfusion were excluded. Baseline and at least one subsequent Hb F levels were available in 37 of the 38 patients randomized to the placebo arm, which were included in the analysis. Serial determinations were performed at 4 week intervals, resulting in a total of 348 determinations and a median of 9 values per patient (range 2 - 15). Mean and standard deviation of repeat measures of Hb F% in each individual patient were calculated. A mean ± SD of the individual coefficients of variation (SD/mean of repeat measures for each individual) was calculated. Peak-to-trough (maximum - minimum) ranges of repeated measures were also calculated per individual and quartiles for the group determined. To assess the potential for regression to the mean from baseline, we calculated the median peak-to-trough within quartiles of baseline Hb F. We assessed the difference between maximum and minimum values as a percentage of the maximum or percentage deviation from personal best Hb F%, and finally we examined the association of baseline Hb F, age, gender and race with the degree of variability.Results:Patient median (range) age was 25.9 years (12 – 46), 24 (63%) were female, the genotype was Hb SS in 30 patients (79%) and Hb S/β0 thalassemia in 8 (21%), and no patient was treated with hydroxycarbamide at enrollment. Median (range) values of Hb F percentage at baseline were 7.5% (0.5 – 23.4). The mean coefficient of variation of all Hb F values was 13%, with a peak to trough median (range) of 1.8 (0.1 - 9.8). The mean percentage Hb F % variability over time was 31.8% (S.D +/- 18) and median (range) value of 26.1 (7.2-80) with quartiles depicted in Table 1. Baseline Hb F% negatively correlated with the percentage variability and this association was highly statistically significant (Spearman rho –0.34, p = 0.04).Table I:Intra-patient variability in successive Hb F% levels expressed as a percentage deviation from peak valuesQuartilesRange of Values (% difference between maximum and minimum HbF)n< 25th7.2-20.6925-4920.7-26.11050-7426.2- 39.2975-10039.3-809Discussion:There is substantial Hb F variability among patients not on any Hb F inducer. This variability has not been previously reported and influences both the standard deviation and standard error, and would likely lower the statistical power of any comparative analysis in a clinical trial. Thus, sample sizes should be larger to be able to detect a sizeable difference between experimental and control groups.Variability among treated patients is harder to assess because it is difficult to separate out intrinsic variability from treatment effects and medication adherence. We have not attempted to assess the intra-patient variability in Hb F among patients given study drug in this study. However, it seems reasonable to assume that the variability seen in untreated patients would also be seen, to some degree, within treated patients. Thus, using Hb F percentage alone as a measure of medication compliance (with a Hb F inducing agent) may not be reliable.Possible explanations for this variability over time include artifacts of laboratory technique, increased hemolysis with increased erythropoietic drive, worsening renal function (and subsequent decrease in erythropoietin production), and bone marrow infarction, leading to disrupted hematopoiesis. DisclosuresKutlar:NIH/NIMHD: Research Funding. Ghalie:HemaQuest Pharmaceuticals, Inc.: Employment.

A double‐blind, placebo‐controlled phase II study of the efficacy and safety of 2,2‐dimethylbutyrate (HQK‐1001), an oral fetal globin inducer, in sickle cell disease

This placebo-controlled phase II study evaluated the pharmacodynamics, efficacy and safety of 2,2-dimethylbutyrate (HQK-1001), a fetal globin gene-inducing short-chain fatty acid derivative, administered orally at 15 mg/kg twice daily for 48 weeks in 76 subjects with sickle cell disease (SCD). The median age was 26 years (range: 12-55 years) and 37 subjects (49%) were treated previously with hydroxycarbamide. Sixty subjects (79%) had Hb SS and 16 (21%) had S/β(0) thalassemia. The study was terminated after a planned interim analysis showed no significant increase in fetal hemoglobin (Hb F) and a trend for more pain crises in the HQK-1001 group. For 54 subjects with Week 24 data, the mean absolute increase in Hb F was 0.9% (95% confidence interval (CI): 0.1-1.6%) with HQK-1001 and 0.2% (95% CI: -0.7-1.1%) with placebo. Absolute increases in Hb F greater than 3% were noted in 9 of 38 subjects (24%) administered HQK-1001 and 1 of 38 subjects (3%) administered placebo. The mean changes in hemoglobin at Week 24 were comparable between the two groups. The mean annualized rate of pain crises was 3.5 with HQK-1001 and 1.7 with placebo. The most common adverse events in the HQK-1001 group, usually graded as mild or moderate, consisted of nausea, headache, vomiting, abdominal pain, and fatigue. Additional studies of HQK-1001 at this dose and schedule are not recommended in SCD. Intermittent HQK-1001 administration, rather than a daily regimen, may be better tolerated and more effective, as shown previously with arginine butyrate, and warrants further evaluation.

A key role for lipoic acid synthesis duringPlasmodiumliver stage development

The successful navigation of malaria parasites through their life cycle, which alternates between vertebrate hosts and mosquito vectors, requires a complex interplay of metabolite synthesis and salvage pathways. Using the rodent parasite Plasmodium berghei, we have explored the synthesis and scavenging pathways for lipoic acid, a short-chain fatty acid derivative that regulates the activity of α-ketoacid dehydrogenases including pyruvate dehydrogenase. In Plasmodium, lipoic acid is either synthesized de novo in the apicoplast or is scavenged from the host into the mitochondrion. Our data show that sporozoites lacking the apicoplast lipoic acid protein ligase LipB are markedly attenuated in their infectivity for mice, and in vitro studies document a very late liver stage arrest shortly before the final phase of intra-hepaticparasite maturation. LipB-deficient asexual blood stage parasites show unimpaired rates of growth in normal in vitro or in vivo conditions. However, these parasites showed reduced growth in lipid-restricted conditions induced by treatment with the lipoic acid analogue 8-bromo-octanoate or with the lipid-reducing agent clofibrate. This finding has implications for understanding Plasmodium pathogenesis in malnourished children that bear the brunt of malarial disease. This study also highlights the potential of exploiting lipid metabolism pathways for the design of genetically attenuated sporozoite vaccines.

A randomized phase I/II trial of HQK‐1001, an oral fetal globin gene inducer, in β‐thalassaemia intermedia and HbE/β‐thalassaemia

β-thalassaemia intermedia (BTI) syndromes cause haemolytic anaemia, ineffective erythropoiesis, and widespread complications. Higher fetal globin expression within genotypes reduces globin imbalance and ameliorates anaemia. Sodium 2,2 dimethylbutyrate (HQK-1001), an orally bioavailable short-chain fatty acid derivative, induces γ-globin expression experimentally and is well-tolerated in normal subjects. Accordingly, a randomized, blinded, placebo-controlled, Phase I/II trial was performed in 21 adult BTI patients (14 with HbE/β(0) thalassaemia and seven with β(+)/β(0) thalassaemia intermedia, to determine effective doses for fetal globin induction, safety, and tolerability. HQK-1001 or placebo were administered once daily for 8 weeks at four dose levels (10, 20, 30, or 40 mg/kg per day), and subjects were monitored for laboratory and clinical events. Pharmacokinetic profiles demonstrated a t(1/2) of 10-12 h. Adverse events with HQK-1001 treatment were not significantly different from placebo treatment. The 20 mg/kg treatment doses increased median HbF above baseline levels by 6·6% and 4·4 g/l (P < 0·01) in 8/9 subjects; total haemoglobin (Hb) increased by a mean of 11 g/l in 4/9 subjects. These findings identified a safe oral therapeutic which induces fetal globin in BTI. Further investigation of HQK-1001 with longer dosing to definitively evaluate its haematological potential appears warranted.

Induction of HBF

The role of HbF inducers in ameliorating the pathophysiology and severity associated with hemoglobinopathies like sickle cell disease and thalassemia is well demonstrated. So many studies have focused on explain the pathways that involved in the re-activation of γ-globin gene expression. The HbF inducers are mainly includes chemotherapeutic agents (e.g. 5-Azacytidine and hydroxyurea), short-chain fatty acid derivatives (e.g. butyrates, valporate, sodium phenylbutyrate) and other HbF inducers like stem cell factor and synthetic gene-targeted transcription factors. These HbF inducers have significant effects on improve clinical manifestations of hemoglobinopathies and decreasing transfusion dependency. But regarding some limitations of these agents, more studies for the investigation of new agents with more safety and effectiveness is needed.

Do Large Intestinal Events Explain the Protective Effects of Whole Grain Foods Against Type 2 Diabetes?

Consumption of whole grain foods has been associated with decreased risk of type 2 diabetes. Insulin sensitivity and inflammation are key mechanisms in the development of type 2 diabetes, but the cause of the protective effects of whole grains is not known. In this review, we search for evidence to support the hypothesis of a link between whole grains, large intestinal events, and peripheral inflammation. Due to the unique structure and composition of the grain fiber complex, fermentation in the large intestine is probably an important mediator of the effects of whole grains. Fermentation of grain fiber takes place throughout the large intestine affecting beneficially the composition of gut microbiota, hence decreasing the permeability of gut barrier. Improved gut barrier function reduces leaking of endotoxic bacterial lipopolysaccharides (LPS) into the circulation. Lower concentration of LPS in blood seems to alleviate peripheral inflammation. Fermentation of grain fiber also leads to continuous supply and absorption of metabolites such as short chain fatty acids and ferulic acid derivatives which may have anti-inflammatory effects. These phenomena, mainly based on in vitro and animal studies, are associated with fermentation of grain fiber and improve insulin sensitivity, which over time may decrease the risk of type 2 diabetes. To test these mechanisms, more well-designed human studies are needed.

A Phase 2 Trial of HQK-1001 (sodium 2,2-dimethylbutyrate), an Oral Fetal Globin Gene Inducer, in HbE-Beta Thalassemia in Thailand

AbstractAbstract 251Beta thalassemia intermedia (BTI) syndromes are characterized by globin chain imbalance, ineffective erythropoiesis, hemolytic anemia, and have no therapeutic approved for the underlying pathology. Higher fetal globin (HbF) expression can compensate for beta globin deficiency, reduce globin chain imbalance, and ameliorate phenotype within the same genotypes. HQK-1001 (HemaQuest Pharmaceuticals, San Diego, CA) is an orally bioavailable, non-cytotoxic, short-chain fatty acid derivative which induces fetal globin expression in multiple experimental models. In thalassemic erythroid progenitors in vitro, HQK-1001 prolongs STAT-5 phosphorylation and increases expression of the pro-survival protein Bcl-xL. In a Phase I/II dose-escalation trial in BTI, HQK-1001 administered at 10, 20, 30, and 40 mg/kg/day for 8 weeks was well–tolerated, demonstrated a t½ of 9–11 hours, and treatment resulted in an increase in HbF and total hemoglobin (Hgb), with best results observed at 20 mg/kg.This open-label Phase 2 trial evaluated HQK-1001 administered orally at 20 mg/kg once daily for 26 weeks in adult patients with HbE-β0 thalassemia (NCT01609595). Ten subjects were enrolled, ages 20–36 years, including 7/10 female and 5/10 splenectomized subjects. The beta globin molecular mutations of the subjects, in addition to Codon 26 (G-A), included Codon 41/42 (-TTCT), Codon 17 (A-T), Codon 110 (T-C), and IVS I-I (G-T). At the time of this analysis, 9 of 10 subjects have completed at least 20 weeks of dosing. HbF has increased in all subjects above baseline levels by a mean of 10% (range 4.3% to 20.9%) and total HbF has increased by a mean of 1.07 g/dL, (range 0.52–2.38 g/dL, with positive changes in total HbF observed in 9/10 subjects. Total Hgb has increased by >0.5 g/dL above baseline (mean of 1.27 g/dL, range 0.7 to 2.2 g/dL) in 3 subjects after 3 to 5 months of dosing. Treatment was well-tolerated. There have been no severe drug-related adverse events, no serious adverse events, and no clinically relevant laboratory abnormalities. One subject developed transient proteinuria and edema without changes in renal function 3 weeks following an episode of pharyngitis. This ongoing trial demonstrates consistent induction of fetal globin expression by HQK-1001 at a well-tolerated dose level in subjects with HbE-beta thalassemia. Disclosures:Fucharoen:HemaQuest Pharmaceuticals: Research Funding. Perrine:HemaQuest Pharmaceuticals: Equity Ownership, Patents & Royalties.

The emerging role of fetal hemoglobin induction in non-transfusion-dependent thalassemia

Patients with beta (β)-thalassemia who have high levels of fetal hemoglobin (HbF) have less severe anemia and are often transfusion-independent. Therefore, augmentation of HbF production has been a longstanding therapeutic objective. Three classes of HbF-inducing agents have been investigated for the treatment of β-thalassemia including chemotherapeutics, short-chain fatty acid derivatives, and recombinant erythropoietin. These agents have several different mechanisms of action and have been shown to increase total hemoglobin levels by 1–5 g/dL above baseline, but none has been able to sustain the therapeutic levels needed to maintain transfusion independence. Recent findings have provided new insights regarding HbF regulatory pathways, providing new opportunities for derepression of fetal globin gene expression and HbF induction.

The histone deacetylase inhibitor, sodium butyrate, alleviates cognitive deficits in pre-motor stage PD

Parkinson's disease (PD) patients often times experience impairment in their cognitive abilities early on in the progression of the disease. The reported deficits appear to mainly involve functions that are associated with frontal lobe and frontal-striatal pathways subserving attentional set-shifting, working memory and executive function.The current study explored executive function deficits in a rat model of PD in the pre-motor deficit stage. The rats were lesioned with 12 μg of 6-hydroxydonpamine (6-OHDA) in the striatum in a two step process (10 μg/μl followed by 2 μg/μl) 48 hours apart. Executive function was tested at 3 weeks post-surgery using a rat analogue of Wisconsin card sorting test called the Extra Dimensional/Intra Dimensional (ED/ID) set-shifting task. The results demonstrated that performance by the pre-motor rat model of PD was equivalent to that of the control groups in the simple and the compound discriminations as well as the intra-dimensional set-shifting. However the PD group exhibited attentional set-shifting deficits similar to those observed in PD patients.Additionally, sodium butyrate, a short chain fatty acid derivative and inhibitor of class I and II histone deacetylase (HDACi), was tested as a potential therapeutic agent to mitigate the pre-motor cognitive deficits in PD. The results indicated that the sodium butyrate treatment not only effectively alleviated the set-shifting deficits, but also improved the attentional set formation in the treated rats.

Evaluation of Safety and Pharmacokinetics of Sodium 2,2 Dimethylbutyrate, a Novel Short Chain Fatty Acid Derivative, in a Phase 1, Double-Blind, Placebo-Controlled, Single-Dose, and Repeat-Dose Studies in Healthy Volunteers

Pharmacologic induction of fetal globin synthesis is an accepted therapeutic strategy for treatment of the beta hemoglobinopathies and thalassemias, as even small increases in hemoglobin F (HbF) levels reduce clinical severity in sickle cell disease (SCD) and reduce anemia in beta thalassemia. Prior generation short chain fatty acid therapeutics, arginine butyrate (AB), and phenylbutyrate, increased fetal and total hemoglobin levels in patients, but were limited by high doses or intravenous (IV) infusion. A fetal globin-inducing therapeutic with convenient oral dosing would be an advance for these classic molecular diseases. Healthy adult human subjects were treated with a novel short chain fatty acids (SCFA) derivative, sodium 2,2 dimethylbutyrate (SDMB), or placebo, with 1 of 4 single dose levels (2, 5, 10, and 20 mg/kg) or daily doses (5, 10, or 15 mg/kg) over 14 days, and monitored for adverse clinical and laboratory events, drug levels, reticulocytes, and HbF assays. SDMB was well-tolerated with no clinically significant adverse events related to study medication. The terminal half-life ranged from 9 to 15 hours. Increases in mean absolute reticulocytes were observed at all dose levels in the 14-day study. The favorable pharmacokinetics (PK) profiles and safety findings indicate that SDMB warrants further investigation for treatment of anemic subjects with beta hemoglobinopathies.

Transcriptional changes associated with recovery from heart failure in the SHR

To identify biological pathways associated with myocardial recovery from heart failure (HF), gene profiling and gene set enrichment analysis (GSEA) were examined in left ventricle of spontaneously hypertensive rats with HF (SHR-F) with no treatment, following treatment with the angiotensin converting enzyme inhibitor captopril, and treatment with captopril combined with the short chain fatty acid derivative phenylbutyrate. Failing hearts demonstrated depressed left ventricular ejection fraction, while ventricular volume and mass increased. Captopril treatment alone prevented further deterioration but did not improve myocardial function; relatively few transcripts were differentially expressed relative to untreated SHR-F. Gene sets identified by GSEA as downregulated with captopril treatment compared to SHR-F group included those related to hypoxia and reactive oxygen species, while upregulated gene sets included G protein signaling. Treatment with phenylbutyrate alone did not improve survival (no animals in this group survived the 30 day treatment period), while phenylbutyrate combined with captopril increased survival and significantly improved cardiac function in vivo and in vitro. Normalized microarray data identified 780 genes that demonstrated a combined treatment effect of which 258 genes were modified with HF. Fatty acid metabolism and ion transport were among the most significantly upregulated pathways in the combined treatment group compared to untreated SHR with HF, whereas those related to oxidative stress, growth, inflammation, protein degradation, and TGF-β signaling were downregulated. These findings demonstrate improved myocardial function and regression of cardiac hypertrophy, and identify many HF related gene sets altered with phenylbutyrate and captopril treatment, but not captopril alone. These results characterize gene sets associated with recovery from HF, and suggest that phenylbutyrate may be a potentially effective adjunctive treatment, together with captopril, by synergistically modulating pathways that contribute to restoration of contractile function of the failing SHR heart.

X-Ray and UV Irradiation and Heat Shock Induce γ-Globin Gene Expression in Erythroid Cells Via P38 MAPK Signaling.

Abstract 459We have recently presented a cell stress signaling model to explain the ability of a wide variety of mechanistically distinct drugs to induce fetal hemoglobin (HbF) production (Mabaera et al, Exp Hematol 2008). This model proposes that HbF inducing agents, including DNMT and HDAC inhibitors, short-chain fatty acid derivatives and cytotoxic agents induce HbF production by activating intracellular cell stress signaling pathways and that the p38 MAPK (mitogen-activated protein kinase) pathway plays a central role in the induction process. The major alternative to this model is that each class of drugs works through a distinct mechanism, such as global DNA hypomethylation or histone hyperacetylation. If our model is correct, then non-pharmacologic inducers of p38 MAPK signaling, such as X-ray and UV irradiation, heat shock, and osmotic shock should also increase γ-globin gene expression. If these physical stresses do not up-regulate γ-globin gene expression, then our model is likely to be incorrect. To mimic our previous experiments in which we treated differentiating human primary erythroid cells with inducing agents over several days, we exposed K562 cells to physical stresses daily for 5 days at doses that did not cause cell death. To test whether X-irradiation induces the transcription of γ-globin mRNA, cells were irradiated at doses ranging from 0 to 1.0 cGy/day, in the absence and presence of SB203580 (a p38 MAPK inhibitor). Experiments were first performed on K562 cells, and then during in vitro erythroid differentiation of primary human CD34+ cells. These treatments stimulated a strong, dose-dependent increase of γ-globin mRNA in both K562 cells (up to 6-fold over untreated cells) and in primary erythroid cells (up to 5-fold) as measured by quantitative RT-PCR. SB203580 abolished this effect in both cell types. The inhibitor also caused a decrease in γ-globin mRNA expression in untreated control cells, suggesting that p38 MAPK signaling plays a role in basal γ-globin gene expression. The same X-ray doses also induced phosphorylation of the down-stream p38 MAPK target, HSP-27 and up-regulated expression of stress-induced transcription factor genes including GADD34, CHOP (GADD153), and ATF3. Expression of GADD34 and CHOP genes and HSP-27 phosphorylation were inhibited by SB203580. We next tested the ability of UV light (254 nm) administered daily for 5 days to induce γ-globin mRNA in K562 cells. A dose-dependent increase in γ-globin mRNA was observed following exposure to doses as low as 10 J/m2. Cells treated with 35 J/m2 had 2.7-fold higher levels of steady state γ-globin mRNA compared to untreated control cells. This induction was also inhibited by SB203580. Preliminary experiments with heat shock have yielded similar results. Following a single 180-minute exposure to 42°C, K562 cells showed a 2.2-fold increase in γ-globin mRNA compared to untreated cells. Shorter exposures to higher temperatures (e.g., 50°C for 15 minutes) caused an approximately 3-fold increase in γ-globin steady-state mRNA after 24 hours. These 2- to 3-fold increases in expression are similar to those we have previously observed in primary human erythroid cells with 5-azacytidine and butyrate. In none of our experiments did the K562 cells become benzidine positive, indicating that increased γ-globin expression was not the result of activating an erythroid differentiation program. Together with other published studies, these data support the hypothesis that p38 MAPK pathway signaling, whether caused by drugs or physical stress, is a key component of γ-globin gene induction. This in turn suggests that the components of the p38 MAPK pathway could serve as novel targets for the development of new HbF inducing agents. Disclosures:No relevant conflicts of interest to declare.

A Multi-Lineage Oral Hemokine™ HQK-1002 Enhances Neutrophil Recovery in Sub-Lethally Irradiated Mice and in Baboons.

Abstract 2509Poster Board II-486Acute radiation syndrome and therapeutic radiation are associated with cytopenias, notably severe neutropenia, particularly when large regions of bone marrow are exposed. An orally-active neutrophil stimulant which reduces the duration or magnitude of neutropenia would offer therapeutic benefit in radiation therapy and would be useful for acute radiation exposure. Orally-bioavailable short-chain fatty acid derivatives (SCFAD, HemokinesTM HQK-1002 and its S-enantiomer HQK-1002S), which stimulate proliferation of growth factor-dependent hematopoietic cell lines and CFU-GM cultures, were evaluated for in vivo activity in irradiated and 5-FU-treated mice and in normal baboons. In sub-lethal irradiation studies, mice were irradiated (6 Gy single dose) and then treated with saline or HQK-1002 once daily, 5 days/week for 2 weeks, beginning on day 1 after irradiation, and absolute neutrophil counts (ANC) were determined. The mean nadir of absolute neutrophil counts (ANC) <500/mm3 was 6 days in sub-lethally-irradiated mice treated with HQK-1002, and 18 days in the saline treated controls. In HQK-1002 treated mice, ANC did not decline below 200/mm3, while saline-treated animals experienced 10 days of severe neutropenia with ANC <200/mm3. In lethally-irradiated (LD90) mice, either saline or the (S) enantiomer of HQK-1002 was administered daily. ANC and marrow pathology were compared between saline and HQK-1002S-treated animals. The duration of peripheral neutropenia was similar in controls and HQK-1002S-treated animals, but bone marrow pathology demonstrated profound ablation of all mature myeloid series (10-20% cellularity) in the saline controls, while HQK-1002S-treated animals had normal bone marrow cellularity (55–75%) with complete neutrophil maturation within 2 weeks following radiation. In 5FU-treated mice, neutrophil recovery was compared between HQK-1002, saline, or G-CSF treatment for 3 weeks. Recovery from profound neutropenia (nadir at Day 9) occurred by Day 13 in G-CSF- and HQK-1002 treated animals, 5 days before recovery was observed in saline controls. HQK-1002 and HQK-1002S were also administered to non-irradiated, normal juvenile baboons, once daily for 5 days, and CBCs and neutrophil counts were assayed. HQK-1002 or HQK-1002S treatment in 3 unchallenged baboons resulted in a two-fold to three-fold rises in ANC within 7 days. Collectively, these studies strongly suggest that HQK-1002 and HQK-1002S, orally-bioavailable SCFADs, stimulate myelopoiesis in vivo in normal animals and during sub-lethal radiation-induced bone marrow suppression, and merit further evaluation in neutropenia associated with marrow suppression. Disclosures:Perrine:HemaQuest Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Boosalis:HemaQuest Pharmaceuticals: Equity Ownership. Thomson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Berenson:hemaQuest Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Faller:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Erythroid Kruppel‐like factor (EKLF) is recruited to the γ‐globin gene promoter as a co‐activator and is required for γ‐globin gene induction by short‐chain fatty acid derivatives

The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for beta-type globin gene switching, and specifically activates transcription of the adult beta-globin gene promoter. We sought to determine if EKLF is also required for activation of the gamma-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials. The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators. Knockdown of EKLF prevents SCFA-induced expression of the gamma-globin promoter in a stably expressed microLCRbeta(pr)R(luc) (A)gamma(pr)F(luc) cassette, and prevents induction of the endogenous gamma-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous gamma-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for gamma-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous gamma-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes.