Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1988
The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a ... more The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a developmental clock-like mechanism and determined by the gestational age of the fetus. Prolonging fetal globin gene expression is of considerable interest for therapeutic potential in diseases caused by abnormal P-globin genes. Butyric acid, which is found in increased plasma concentrations in infants of diabetic mothers who have delayed globin gene switching, was infused into catheterized fetal lambs in utero during the time of the normal globin gene switch period. The globin gene switch was significantly delayed in three of four butyrate-treated fetuses compared with controls and was entirely prevented in one fetus in whom the infusion was begun before the globin switch was under way. These data provide a model for investigating and arresting the biologic clock of hemoglobin switchIng. The timing of the fetal (y-globin) to adult (,f-globin) hemo
In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of i... more In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition.
Advances in Experimental Medicine and Biology, 1989
The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a norm... more The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a normally set biologic clock which proceeds even if the adult (beta) globin genes are defective. Preventing or reversing the globin gene switch would be beneficial for subjects with abnormal beta globin genes. We have now identified a class of agents which, when present in elevated plasma concentrations during gestation, appears to inhibit the gamma beta globin gene switch in developing humans. Further investigation has shown that butyric acid and related compounds can increase gamma globin and decrease beta globin expression in erythroid cells cultured from subjects with diseases of abnormal beta globin. Butyrate compounds were therefore infused in an in vivo fetal animal model, and the globin switch was inhibited in most and reversed in some fetal lambs. These data suggest that inhibiting expression of abnormal beta globin genes may be possible in future generations. Histone modification may be a mechanism of action involved. The developmental switch from production of gamma globin to beta globin results in significant morbidity when the beta globin genes are defective. The globin switch has therefore been extensively studied, appearing to be set on a biologic clock and proceeding despite the site of blood production and solely on the basis of gestational age. We previously found that this developmental gene switch is delayed in human fetuses developing in the presence of maternal diabetes. A number of metabolites present in abnormal concentrations in these infants were therefore tested for effects on globin expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Lymphoproliferative disorders associated with the Epstein-Barr virus (EBV) include non-Hodgkin's ... more Lymphoproliferative disorders associated with the Epstein-Barr virus (EBV) include non-Hodgkin's lymphoma, Hodgkin's lymphoma, and ''post-transplant lymphoproliferative disorders'' (PTLD), which occur with immunosuppression after marrow and organ transplantation. PTLD is characterized by actively proliferating, latently infected EBV(π) B-lymphocytes, and often manifests a rapidly progressive fatal clinical course if the immunosuppression cannot be reversed. Lung transplant recipients are a subset of patients at special risk for developing PTLD. The incidence of PTLD development in these patients has been estimated at 5-10%. Whereas immunologic and antiviral therapy have been moderately effective for treating EBV-associated infections in the lytic phase, they have been less useful in the more common latent phase of the disease. One common treatment for herpesvirus infections has targeted the virus-specific enzyme thymidine kinase (TK). The lack of viral TK expression in EBV(π) tumor cells, due to viral latency, makes antiviral therapy alone ineffective as an anti-neoplastic therapy, however. We have developed a strategy for the treatment of EBV-associated lymphomas/ PTLD using pharmacologic induction of the latent viral TK gene and enzyme in the tumor cells, followed by treatment with ganciclovir. Arginine butyrate selectively activates the EBV TK gene in latently EBV-infected human lymphoid cells and tumor cells. A Phase I/II trial has been initiated, employing an intra-patient dose escalation of arginine butyrate combined with ganciclovir. In six patients with EBV-associated lymphomas or PTLD, all of which were resistant to conventional radiation and/or chemotherapy, this combination produced complete clinical responses in four of six patients, with a partial response occurring in a fifth patient. Pathologic examination in two of three patients demonstrated complete necrosis of the EBV lymphoma, with no residual disease, following a single three-week course of the combination therapy. Possible side-effects of the therapy included nausea and reversible lethargy at the highest doses. One patient suffered acute liver failure, thought to be secondary to release of FasL from the necrotic tumor. Analysis of patientderived tumor cells in culture demonstrated that arginine butyrate produced selective induction of the EBV TK gene, which then conferred sensitivity to ganciclovir, resulting in tumor apoptosis. Additional patient accrual is sought for further evaluation of this therapy. The original description of an Epstein-Barr virus (EBV)-associated disease was a nasopharyngeal tumor identified in African children
a-Lipoic acid is a naturally-occurring co-factor found in a number of multi-enzyme complexes regu... more a-Lipoic acid is a naturally-occurring co-factor found in a number of multi-enzyme complexes regulating metabolism. We report here that a-lipoic acid induces hyperacetylation of histones in vivo and has differential effects on the growth and viability of normal versus transformed cell lines. The human tumor cell lines FaDu and Jurkat, as well as a Ki-v-Ras-transformed Balb/c-3T3 murine mesenchymal cell line, all initiated apoptosis following exposure to a-lipoic acid. In contrast, treatment of non-transformed cell lines with a-lipoic acid resulted only in reversible cell cycle arrest in G 0 /G 1. Treatment with butyrate, another short-chain fatty acid, induced a G 0 /G 1 arrest in both transformed and non-transformed cell lines. a-Lipoic acid caused a post-translational elevation in the levels of the cyclin-dependent kinase inhibitor p27 Kip1. Studies using p27 Kip1-deficient MEF cells demonstrated that p27 Kip1 was required for the a-lipoic acid-mediated cell cycle arrest. The mechanism of apoptosis was independent of Fas-mediated signaling, as a-lipoic acid-treated Jurkat cell mutants deficient in Fas or FADD retained sensitivity to apoptosis. The differential selectivity of the pro-apoptotic effects of a-lipoic acid for transformed cells supports its potential use in the treatment of neoplastic disorders.
Development of non-cytotoxic, orally-active therapeutics, which induce fetal globin production, w... more Development of non-cytotoxic, orally-active therapeutics, which induce fetal globin production, would be beneficial for treatment of beta-thalassemias and hemoglobinopathies. The short-chain fatty acids (SCFA) butyrate and phenylbutyrate are active fetal globin inducers, but their clinical application has been limited by inhibition of erythropoiesis and unfavorable PK profiles. HQK-1001 is a SCFA which stimulates fetal globin gene expression in reporter gene assays, erythroid progenitors, transgenic mice, and anemic baboons. HQK-1001 also stimulates human erythroid progenitor (BFU-E) proliferation by a mean of 30% above controls, and increased red blood cell counts in phlebotomized baboons. HQK-1001 has demonstrated a wide safety margin in pre-clinical toxicology testing, with no significant adverse effects in sub-chronic (6-month) studies and negative mutagenicity testing. Based on these pre-clinical data, a placebo-controlled, randomized, blinded, single-dose escalating Phase 1 trial was conducted to evaluate HQK-1001 at 4 dose levels ranging from 2 to 20 mg/kg in 32 normal volunteers. The drug was welltolerated, with no significant adverse events. Pharmacokinetic analyses showed low intersubject variability, a linear increase in Cmax and AUC with increasing dose, and T½ of 11 hours, indicating suitability for once-daily dosing. Administration under fed conditions demonstrated equivalent AUC to fasting conditions. In a subsequent 14-day Phase 1 dosing study in 41 normal subjects, the drug was also well-tolerated, with no significant adverse effects at the studied dose levels (5, 10 and 15 mg/kg). F-reticulocytes increased in subjects treated with 5 to 10 mg/kg doses. Absolute reticulocytes increased significantly above baseline (p <0.02 – 0.002) following 14-day dosing at all dose levels, while no significant change occurred in placebo controls. These first-in-human clinical studies demonstrating safety of HQK-1001 in 73 normal volunteer subjects and an improved pharmacologic profile over prior SCFAs indicate that HQK-1001 merits further clinical investigation in anemic patients with beta-globin gene disorders.
Summary. Polycythaemia in the neonate is a serious pathologic entity which occurs particularly in... more Summary. Polycythaemia in the neonate is a serious pathologic entity which occurs particularly in infants of diabetic mothers (IDM) and srnall‐for‐gestational age (SGA) infants. Both of these conditions are associated with fetal hyperinsulinaemia. Cultures of cord blood mononuclear cells from polycythae‐mic IDM showed increased growth of late erythroid progenitor colonies, compared to cord blood mononuclear cells from non‐polycythaemic infants, reflecting a possible expansion of this progenitor population in the polycythae‐mic fetus. No changes were observed in early erythroid progenitor populations. Biosynthetic human insulin at physiological levels characteristic of IDM stimulated growth in culture of late erythroid progenitors in cord blood from premature, term and IDM infants. Three out of five polycythaemic infants had elevated cord blood plasma levels of insulin C‐peptide at birth, whereas no infant with a haematocrit of less than 65% had high insulin C‐peptide measurements. These data suggest that the polycythaemia noted in infants of diabetic mothers may be secondary, in large part, to a stimulatory effect on erythroid progenitor growth by the hyperinsulinaemic environment in which they develop in utero.
Journal of Laboratory and Clinical Medicine, Dec 1, 1998
The exposure of endothelial cells to hypoxic environments regulates the expression of a number of... more The exposure of endothelial cells to hypoxic environments regulates the expression of a number of genes with products that are vasoactive or mitogenic for vascular tissue, including platelet-derived growth factor, endothelin-1, and endothelial nitric oxide synthase. Hypoxia is also known to alter the adhesive properties of endothelium toward a variety of blood cell types. Thrombospondin-1 (TSP-1) is a glycoprotein with major roles in cellular adhesion and vascular smooth muscle proliferation and migration. We report here that hypoxia induces TSP-1 gene and protein expression. Oxygen tensions of ≤30 torr resulted in TSP-1 transcript induction initially apparent at 1 to 6 hours, with maximal induction (6.5-fold ± 1.2-fold) within 24 to 48 hours in both human and bovine endothelial cells. TSP-1 protein levels remain elevated after 72 hours of continuous hypoxic exposure. The induction of TSP-1 steady-state transcript levels is caused in large part, if not entirely, by post-transcriptional stabilization of the TSP-1 mRNA. The TSP-1 induction by hypoxia is a graded and reversible physiologic response and can be mimicked by the use of cobalt chloride or the inhibition of nitric oxide production, suggesting both the involvement of a heme-containing oxygen sensor and a role for the endogenous production of nitric oxide in TSP-1 regulation. The effects of hypoxia both on the stabilization of the TSP-1 transcript and the stimulation of TSP-1 protein production are completely inhibited by arginine butyrate.
Increasing the expression of the 'y globin genes is considered a useful therapeutic approach to t... more Increasing the expression of the 'y globin genes is considered a useful therapeutic approach to the fi globin diseases. Because butyrate and a-amino-n-butyric acid (ABA) augment 'V globin expression in normal neonatal and adult erythroid progenitors. we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with fi thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of
In the normal fetus, a switch from production of hemoglobin F (alpha 2 gamma 2) to hemoglobin A (... more In the normal fetus, a switch from production of hemoglobin F (alpha 2 gamma 2) to hemoglobin A (alpha 2 beta 2) occurs at 28 to 34 weeks of gestation. In the fetus with beta-hemoglobinopathy or beta-thalassemia, this switch proceeds despite the morbidity that results when production of beta-globin is abnormal or reduced. Since insulin has recently been shown to induce renewed expression of some inactive genes, we studied globin biosynthesis during the natural evolution of the fetal globin switch under conditions of hyperinsulinemia, which occurs in infants of diabetic mothers. Such infants develop in a hyperglycemic environment, which produces reactive hyperinsulinemia. The normal increase in beta-globin production from pre-switch levels did not occur in 9 of 10 such infants at term, as compared with 11 normal infants, in whom the switch occurred by 36 to 39 weeks of gestation (P less than 0.0001). The delay in the switch from gamma-globin to beta-globin in this unique clinical setting may allow identification of physiologic factors that can modulate developmental gene suppression.
Annals of the New York Academy of Sciences, Feb 26, 2008
Erythroid differentiation is presumably orchestrated by a combination of transcription factors th... more Erythroid differentiation is presumably orchestrated by a combination of transcription factors that activate lineage-specific genes, such as globin, genes involved in heme synthesis, and red cell cytoskeletal proteins. Some of the erythroidspecific transcription factors characterized to date include GATA1 and NF-E2. Recently, a number of laboratories have provided evidence suggesting that the HOX family of homeobox genes are regulators of erythroid development (reviewed in ref. 1). A large body of data has emerged on the role of homeobox genes in embryogenesis (reviewed in refs. 2 and 3). These genes, which share a 183-nt homeodomain, encode DNA-binding proteins that play a profound role in specifying relative positions and tissue fate in the developing In mammals, 38 class I or HOX homeobox genes are organized into four clusters, each containing 8-11 individual genes, and each localized to a different chromosome (see FIG. l).6.7 These HOX loci also show homology to the HOM-C cluster of homeobox genes in Drosophila. Individual HOX genes within a locus show overlapping zones of expression in the embryonic spinal cord from head to tail, with progressively more anterior rostra1 boundaries of expression as one moves from 5' to 3' in the Depsite the explosion of evidence linking HOX genes to embryogenesis, neither the target genes nor the precise mechanism of action has been established for any HOX gene. The nomenclature for the HOX genes has been confusing, and a new naming system has recently been introduced. In FIGURE 1 we show both the old and new system. The new nomenclature assigns each cluster a letter designation ( i . e . ,
For the majority of children with beta- hemoglobinopathies and -thalassemias who do not have a tr... more For the majority of children with beta- hemoglobinopathies and -thalassemias who do not have a transplant donor, survival is shortened and morbidity is high. Hydroxyurea, EPO preparations, sodium phenylbutyrate, arginine butyrate, and 5-azacytidine/decitabine have shown efficacy in approximately 40% to 70% of sickle cell and beta-thalassemia patients. Many responses, although significant, were not completely ameliorating of symptoms or pathology, and trials of new agents with dual actions, or drug combinations, are needed. Ideally, limiting chemotherapeutic exposure is desirable for long-term treatment of children, and an oral therapeutic at tolerable doses is necessary for practical use. A new oral therapeutic candidate that induces fetal hemoglobin production and also stimulates erythropoiesis is entering clinical evaluation. Use of agents that should have additive or synergistic effects in combination, such as EPO and hydroxyurea or a short-chain fatty acid derivative (SCFAD), offer better therapeutic potential than hydroxyurea alone. Childhood is an optimal time to introduce such therapies, particularly the non-mutagenic SCFADs, while the erythroid marrow reserve is preserved and before organ damage has become widespread. A challenge for successful application of these therapies is to define patient subsets that are most likely to respond to a particular agent, or which require combination therapies, and to develop optimal dose regimens in thalassemias with rapid erythroid apoptosis. Development of this therapeutic avenue will require close collaboration among treating and academic physicians, families and patients, funding agencies, and researchers.
Lymphocytes from three patients with the Wiskott-Aldrich syndrome were subjected to surface radio... more Lymphocytes from three patients with the Wiskott-Aldrich syndrome were subjected to surface radioiodination and their 125I-labelled surface proteins were analysed by SDS-polyacrylamide gel electrophoresis and autoradiography. Autoradiographs demonstrated the absence in all three patients of an 125I-labelled protein, molecular weight of 115 000, that was present in normal individuals. In addition, one patient had an additional labelled protein, molecular weight 135 000, not found in normal individuals. The platelets from one patient were radioiodinated and the 125I-labelled membrane proteins were analysed. Glycoproteins Ia and Ib were reduced in amount and restricted in heterogeneity while glycoproteins IIb and IIIa were normal. These results suggest that the primary defect in the Wiskott-Aldrich syndrome may be abnormalities of (glyco)proteins normally present on the surface of lymphocytes and platelets.
2,2-Dimethylbutyrate (DMB) is a potential treatment for thalassemia and hemoglobinopathies. To fa... more 2,2-Dimethylbutyrate (DMB) is a potential treatment for thalassemia and hemoglobinopathies. To facilitate pharmacokinetic evaluation of DMB, we developed an LC-MS assay and quantitated DMB in plasma of rats after an oral dose of 500 mg/kg. After acetonitrile protein precipitation, DMB and dimethylvaleric acid (DMV) internal standard were derivatized to benzylamides, chromatographed on a Hydro-RP column with acetonitrile, water, and 0.1% formic acid, and detected by electrospray positive-mode ionization mass spectrometry. The assay was accurate (97-107%) and precise (3.4-6.2%) between 100 and 10,000 ng/mL. Recovery from plasma was >62%. Plasma freeze-thaw and room temperature stability were acceptable.
Cartilage-Hair Hypoplasia (CHH) is an autosomal recessive form The Wiskott-Aldrich syndrome (WAS)... more Cartilage-Hair Hypoplasia (CHH) is an autosomal recessive form The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder
Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1988
The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a ... more The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a developmental clock-like mechanism and determined by the gestational age of the fetus. Prolonging fetal globin gene expression is of considerable interest for therapeutic potential in diseases caused by abnormal P-globin genes. Butyric acid, which is found in increased plasma concentrations in infants of diabetic mothers who have delayed globin gene switching, was infused into catheterized fetal lambs in utero during the time of the normal globin gene switch period. The globin gene switch was significantly delayed in three of four butyrate-treated fetuses compared with controls and was entirely prevented in one fetus in whom the infusion was begun before the globin switch was under way. These data provide a model for investigating and arresting the biologic clock of hemoglobin switchIng. The timing of the fetal (y-globin) to adult (,f-globin) hemo
In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of i... more In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition.
Advances in Experimental Medicine and Biology, 1989
The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a norm... more The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a normally set biologic clock which proceeds even if the adult (beta) globin genes are defective. Preventing or reversing the globin gene switch would be beneficial for subjects with abnormal beta globin genes. We have now identified a class of agents which, when present in elevated plasma concentrations during gestation, appears to inhibit the gamma beta globin gene switch in developing humans. Further investigation has shown that butyric acid and related compounds can increase gamma globin and decrease beta globin expression in erythroid cells cultured from subjects with diseases of abnormal beta globin. Butyrate compounds were therefore infused in an in vivo fetal animal model, and the globin switch was inhibited in most and reversed in some fetal lambs. These data suggest that inhibiting expression of abnormal beta globin genes may be possible in future generations. Histone modification may be a mechanism of action involved. The developmental switch from production of gamma globin to beta globin results in significant morbidity when the beta globin genes are defective. The globin switch has therefore been extensively studied, appearing to be set on a biologic clock and proceeding despite the site of blood production and solely on the basis of gestational age. We previously found that this developmental gene switch is delayed in human fetuses developing in the presence of maternal diabetes. A number of metabolites present in abnormal concentrations in these infants were therefore tested for effects on globin expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Lymphoproliferative disorders associated with the Epstein-Barr virus (EBV) include non-Hodgkin's ... more Lymphoproliferative disorders associated with the Epstein-Barr virus (EBV) include non-Hodgkin's lymphoma, Hodgkin's lymphoma, and ''post-transplant lymphoproliferative disorders'' (PTLD), which occur with immunosuppression after marrow and organ transplantation. PTLD is characterized by actively proliferating, latently infected EBV(π) B-lymphocytes, and often manifests a rapidly progressive fatal clinical course if the immunosuppression cannot be reversed. Lung transplant recipients are a subset of patients at special risk for developing PTLD. The incidence of PTLD development in these patients has been estimated at 5-10%. Whereas immunologic and antiviral therapy have been moderately effective for treating EBV-associated infections in the lytic phase, they have been less useful in the more common latent phase of the disease. One common treatment for herpesvirus infections has targeted the virus-specific enzyme thymidine kinase (TK). The lack of viral TK expression in EBV(π) tumor cells, due to viral latency, makes antiviral therapy alone ineffective as an anti-neoplastic therapy, however. We have developed a strategy for the treatment of EBV-associated lymphomas/ PTLD using pharmacologic induction of the latent viral TK gene and enzyme in the tumor cells, followed by treatment with ganciclovir. Arginine butyrate selectively activates the EBV TK gene in latently EBV-infected human lymphoid cells and tumor cells. A Phase I/II trial has been initiated, employing an intra-patient dose escalation of arginine butyrate combined with ganciclovir. In six patients with EBV-associated lymphomas or PTLD, all of which were resistant to conventional radiation and/or chemotherapy, this combination produced complete clinical responses in four of six patients, with a partial response occurring in a fifth patient. Pathologic examination in two of three patients demonstrated complete necrosis of the EBV lymphoma, with no residual disease, following a single three-week course of the combination therapy. Possible side-effects of the therapy included nausea and reversible lethargy at the highest doses. One patient suffered acute liver failure, thought to be secondary to release of FasL from the necrotic tumor. Analysis of patientderived tumor cells in culture demonstrated that arginine butyrate produced selective induction of the EBV TK gene, which then conferred sensitivity to ganciclovir, resulting in tumor apoptosis. Additional patient accrual is sought for further evaluation of this therapy. The original description of an Epstein-Barr virus (EBV)-associated disease was a nasopharyngeal tumor identified in African children
a-Lipoic acid is a naturally-occurring co-factor found in a number of multi-enzyme complexes regu... more a-Lipoic acid is a naturally-occurring co-factor found in a number of multi-enzyme complexes regulating metabolism. We report here that a-lipoic acid induces hyperacetylation of histones in vivo and has differential effects on the growth and viability of normal versus transformed cell lines. The human tumor cell lines FaDu and Jurkat, as well as a Ki-v-Ras-transformed Balb/c-3T3 murine mesenchymal cell line, all initiated apoptosis following exposure to a-lipoic acid. In contrast, treatment of non-transformed cell lines with a-lipoic acid resulted only in reversible cell cycle arrest in G 0 /G 1. Treatment with butyrate, another short-chain fatty acid, induced a G 0 /G 1 arrest in both transformed and non-transformed cell lines. a-Lipoic acid caused a post-translational elevation in the levels of the cyclin-dependent kinase inhibitor p27 Kip1. Studies using p27 Kip1-deficient MEF cells demonstrated that p27 Kip1 was required for the a-lipoic acid-mediated cell cycle arrest. The mechanism of apoptosis was independent of Fas-mediated signaling, as a-lipoic acid-treated Jurkat cell mutants deficient in Fas or FADD retained sensitivity to apoptosis. The differential selectivity of the pro-apoptotic effects of a-lipoic acid for transformed cells supports its potential use in the treatment of neoplastic disorders.
Development of non-cytotoxic, orally-active therapeutics, which induce fetal globin production, w... more Development of non-cytotoxic, orally-active therapeutics, which induce fetal globin production, would be beneficial for treatment of beta-thalassemias and hemoglobinopathies. The short-chain fatty acids (SCFA) butyrate and phenylbutyrate are active fetal globin inducers, but their clinical application has been limited by inhibition of erythropoiesis and unfavorable PK profiles. HQK-1001 is a SCFA which stimulates fetal globin gene expression in reporter gene assays, erythroid progenitors, transgenic mice, and anemic baboons. HQK-1001 also stimulates human erythroid progenitor (BFU-E) proliferation by a mean of 30% above controls, and increased red blood cell counts in phlebotomized baboons. HQK-1001 has demonstrated a wide safety margin in pre-clinical toxicology testing, with no significant adverse effects in sub-chronic (6-month) studies and negative mutagenicity testing. Based on these pre-clinical data, a placebo-controlled, randomized, blinded, single-dose escalating Phase 1 trial was conducted to evaluate HQK-1001 at 4 dose levels ranging from 2 to 20 mg/kg in 32 normal volunteers. The drug was welltolerated, with no significant adverse events. Pharmacokinetic analyses showed low intersubject variability, a linear increase in Cmax and AUC with increasing dose, and T½ of 11 hours, indicating suitability for once-daily dosing. Administration under fed conditions demonstrated equivalent AUC to fasting conditions. In a subsequent 14-day Phase 1 dosing study in 41 normal subjects, the drug was also well-tolerated, with no significant adverse effects at the studied dose levels (5, 10 and 15 mg/kg). F-reticulocytes increased in subjects treated with 5 to 10 mg/kg doses. Absolute reticulocytes increased significantly above baseline (p <0.02 – 0.002) following 14-day dosing at all dose levels, while no significant change occurred in placebo controls. These first-in-human clinical studies demonstrating safety of HQK-1001 in 73 normal volunteer subjects and an improved pharmacologic profile over prior SCFAs indicate that HQK-1001 merits further clinical investigation in anemic patients with beta-globin gene disorders.
Summary. Polycythaemia in the neonate is a serious pathologic entity which occurs particularly in... more Summary. Polycythaemia in the neonate is a serious pathologic entity which occurs particularly in infants of diabetic mothers (IDM) and srnall‐for‐gestational age (SGA) infants. Both of these conditions are associated with fetal hyperinsulinaemia. Cultures of cord blood mononuclear cells from polycythae‐mic IDM showed increased growth of late erythroid progenitor colonies, compared to cord blood mononuclear cells from non‐polycythaemic infants, reflecting a possible expansion of this progenitor population in the polycythae‐mic fetus. No changes were observed in early erythroid progenitor populations. Biosynthetic human insulin at physiological levels characteristic of IDM stimulated growth in culture of late erythroid progenitors in cord blood from premature, term and IDM infants. Three out of five polycythaemic infants had elevated cord blood plasma levels of insulin C‐peptide at birth, whereas no infant with a haematocrit of less than 65% had high insulin C‐peptide measurements. These data suggest that the polycythaemia noted in infants of diabetic mothers may be secondary, in large part, to a stimulatory effect on erythroid progenitor growth by the hyperinsulinaemic environment in which they develop in utero.
Journal of Laboratory and Clinical Medicine, Dec 1, 1998
The exposure of endothelial cells to hypoxic environments regulates the expression of a number of... more The exposure of endothelial cells to hypoxic environments regulates the expression of a number of genes with products that are vasoactive or mitogenic for vascular tissue, including platelet-derived growth factor, endothelin-1, and endothelial nitric oxide synthase. Hypoxia is also known to alter the adhesive properties of endothelium toward a variety of blood cell types. Thrombospondin-1 (TSP-1) is a glycoprotein with major roles in cellular adhesion and vascular smooth muscle proliferation and migration. We report here that hypoxia induces TSP-1 gene and protein expression. Oxygen tensions of ≤30 torr resulted in TSP-1 transcript induction initially apparent at 1 to 6 hours, with maximal induction (6.5-fold ± 1.2-fold) within 24 to 48 hours in both human and bovine endothelial cells. TSP-1 protein levels remain elevated after 72 hours of continuous hypoxic exposure. The induction of TSP-1 steady-state transcript levels is caused in large part, if not entirely, by post-transcriptional stabilization of the TSP-1 mRNA. The TSP-1 induction by hypoxia is a graded and reversible physiologic response and can be mimicked by the use of cobalt chloride or the inhibition of nitric oxide production, suggesting both the involvement of a heme-containing oxygen sensor and a role for the endogenous production of nitric oxide in TSP-1 regulation. The effects of hypoxia both on the stabilization of the TSP-1 transcript and the stimulation of TSP-1 protein production are completely inhibited by arginine butyrate.
Increasing the expression of the 'y globin genes is considered a useful therapeutic approach to t... more Increasing the expression of the 'y globin genes is considered a useful therapeutic approach to the fi globin diseases. Because butyrate and a-amino-n-butyric acid (ABA) augment 'V globin expression in normal neonatal and adult erythroid progenitors. we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with fi thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of
In the normal fetus, a switch from production of hemoglobin F (alpha 2 gamma 2) to hemoglobin A (... more In the normal fetus, a switch from production of hemoglobin F (alpha 2 gamma 2) to hemoglobin A (alpha 2 beta 2) occurs at 28 to 34 weeks of gestation. In the fetus with beta-hemoglobinopathy or beta-thalassemia, this switch proceeds despite the morbidity that results when production of beta-globin is abnormal or reduced. Since insulin has recently been shown to induce renewed expression of some inactive genes, we studied globin biosynthesis during the natural evolution of the fetal globin switch under conditions of hyperinsulinemia, which occurs in infants of diabetic mothers. Such infants develop in a hyperglycemic environment, which produces reactive hyperinsulinemia. The normal increase in beta-globin production from pre-switch levels did not occur in 9 of 10 such infants at term, as compared with 11 normal infants, in whom the switch occurred by 36 to 39 weeks of gestation (P less than 0.0001). The delay in the switch from gamma-globin to beta-globin in this unique clinical setting may allow identification of physiologic factors that can modulate developmental gene suppression.
Annals of the New York Academy of Sciences, Feb 26, 2008
Erythroid differentiation is presumably orchestrated by a combination of transcription factors th... more Erythroid differentiation is presumably orchestrated by a combination of transcription factors that activate lineage-specific genes, such as globin, genes involved in heme synthesis, and red cell cytoskeletal proteins. Some of the erythroidspecific transcription factors characterized to date include GATA1 and NF-E2. Recently, a number of laboratories have provided evidence suggesting that the HOX family of homeobox genes are regulators of erythroid development (reviewed in ref. 1). A large body of data has emerged on the role of homeobox genes in embryogenesis (reviewed in refs. 2 and 3). These genes, which share a 183-nt homeodomain, encode DNA-binding proteins that play a profound role in specifying relative positions and tissue fate in the developing In mammals, 38 class I or HOX homeobox genes are organized into four clusters, each containing 8-11 individual genes, and each localized to a different chromosome (see FIG. l).6.7 These HOX loci also show homology to the HOM-C cluster of homeobox genes in Drosophila. Individual HOX genes within a locus show overlapping zones of expression in the embryonic spinal cord from head to tail, with progressively more anterior rostra1 boundaries of expression as one moves from 5' to 3' in the Depsite the explosion of evidence linking HOX genes to embryogenesis, neither the target genes nor the precise mechanism of action has been established for any HOX gene. The nomenclature for the HOX genes has been confusing, and a new naming system has recently been introduced. In FIGURE 1 we show both the old and new system. The new nomenclature assigns each cluster a letter designation ( i . e . ,
For the majority of children with beta- hemoglobinopathies and -thalassemias who do not have a tr... more For the majority of children with beta- hemoglobinopathies and -thalassemias who do not have a transplant donor, survival is shortened and morbidity is high. Hydroxyurea, EPO preparations, sodium phenylbutyrate, arginine butyrate, and 5-azacytidine/decitabine have shown efficacy in approximately 40% to 70% of sickle cell and beta-thalassemia patients. Many responses, although significant, were not completely ameliorating of symptoms or pathology, and trials of new agents with dual actions, or drug combinations, are needed. Ideally, limiting chemotherapeutic exposure is desirable for long-term treatment of children, and an oral therapeutic at tolerable doses is necessary for practical use. A new oral therapeutic candidate that induces fetal hemoglobin production and also stimulates erythropoiesis is entering clinical evaluation. Use of agents that should have additive or synergistic effects in combination, such as EPO and hydroxyurea or a short-chain fatty acid derivative (SCFAD), offer better therapeutic potential than hydroxyurea alone. Childhood is an optimal time to introduce such therapies, particularly the non-mutagenic SCFADs, while the erythroid marrow reserve is preserved and before organ damage has become widespread. A challenge for successful application of these therapies is to define patient subsets that are most likely to respond to a particular agent, or which require combination therapies, and to develop optimal dose regimens in thalassemias with rapid erythroid apoptosis. Development of this therapeutic avenue will require close collaboration among treating and academic physicians, families and patients, funding agencies, and researchers.
Lymphocytes from three patients with the Wiskott-Aldrich syndrome were subjected to surface radio... more Lymphocytes from three patients with the Wiskott-Aldrich syndrome were subjected to surface radioiodination and their 125I-labelled surface proteins were analysed by SDS-polyacrylamide gel electrophoresis and autoradiography. Autoradiographs demonstrated the absence in all three patients of an 125I-labelled protein, molecular weight of 115 000, that was present in normal individuals. In addition, one patient had an additional labelled protein, molecular weight 135 000, not found in normal individuals. The platelets from one patient were radioiodinated and the 125I-labelled membrane proteins were analysed. Glycoproteins Ia and Ib were reduced in amount and restricted in heterogeneity while glycoproteins IIb and IIIa were normal. These results suggest that the primary defect in the Wiskott-Aldrich syndrome may be abnormalities of (glyco)proteins normally present on the surface of lymphocytes and platelets.
2,2-Dimethylbutyrate (DMB) is a potential treatment for thalassemia and hemoglobinopathies. To fa... more 2,2-Dimethylbutyrate (DMB) is a potential treatment for thalassemia and hemoglobinopathies. To facilitate pharmacokinetic evaluation of DMB, we developed an LC-MS assay and quantitated DMB in plasma of rats after an oral dose of 500 mg/kg. After acetonitrile protein precipitation, DMB and dimethylvaleric acid (DMV) internal standard were derivatized to benzylamides, chromatographed on a Hydro-RP column with acetonitrile, water, and 0.1% formic acid, and detected by electrospray positive-mode ionization mass spectrometry. The assay was accurate (97-107%) and precise (3.4-6.2%) between 100 and 10,000 ng/mL. Recovery from plasma was >62%. Plasma freeze-thaw and room temperature stability were acceptable.
Cartilage-Hair Hypoplasia (CHH) is an autosomal recessive form The Wiskott-Aldrich syndrome (WAS)... more Cartilage-Hair Hypoplasia (CHH) is an autosomal recessive form The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder
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