A multifunctional 5-aminolevulinic acid derivative induces erythroid differentiation of K562 human erythroleukemic cells.

Anemia is a major clinical symptom of a wide variety of pathological conditions a common related to reduced erythropoiesis. Whereas erythropoietin treatment showed an improvement in the patients' condition, it revealed increased risks of thromboembolic and cardiovascular events. Herein we describe stimulation of erythropoiesis by the multifunctional 1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu), a 5-aminolevulinic-acid (ALA) derivative, which undergoes metabolic hydrolysis yielding two erythroid differentiation inducers, ALA and butyric acid (BA), each acting through a different mechanism. ALA, the first precursor in the heme biosynthesis, accelerates heme synthesis and BA, a histone deacetylase inhibitor (HDACI) that activates the transcription of globin mRNA. Our results show that the AlaAcBu mutual prodrug is a potent chemical differentiation inducer of K562 human erythroleukemia cells manifested by augmentation of heme and globin synthesis and assembly of hemoglobin. Exposure of K-562 cells to AlaAcBu resulted in an increase in heme synthesis and globin expression. Stimulation of the heme pathway was evident by the over-expression of porphobilinogen deaminase (PBGD) and ferrochelatase. AlaAcBu promoted cellular erythroid differentiation depicted by the expression of the marker glycophorin A and cellular maturation characterized by cytoplasm hemoglobinization, polar arrangement of mitochondria and a developed central vacuolar system preceding nuclear extrusion. The ability of AlaAcBu to promote differentiation along the erythroid lineage and to dramatically induce hemoglobin synthesis presented in this report.

A histone deacetylase inhibitory prodrug - butyroyloxymethyl diethyl phosphate - protects the heart and cardiomyocytes against ischemia injury.

Butyroyloxymethyl diethylphosphate (AN-7) is a prodrug of butyric acid effective in reducing cardiotoxicity caused by chemotherapy. In this study, we tested whether AN-7 protects the heart and cardiomyocytes against ischemia injury. A single oral dose of AN-7 was given to mice or rats. Animals were sacrificed 1.5 or 24 h later and the hearts were subjected to ischemia and reperfusion ex-vivo (Langendorff). The mechanical performance was recorded throughout and the infarct size was measured at the end of reperfusion. Neonatal rat cardiomyocytes were subjected to 24-48 h hypoxia (1% O(2)) in the absence or presence of AN-7 and mitochondria damage and cell death were assessed. Proteins were analyzed by Western immunoblotting. In the two rodents, a single dose of AN-7 given in vivo preconditioned the hearts for improved functional recovery from ischemia and reperfusion performed ex-vivo. Both 1.5 h and 24 h treatments improved the pressure-related parameters whereas the coronary flow was ameliorated in the 24 h treatment only. Infarct size was smaller in the AN-7 treated hearts. In cardiomyocytes, AN-7 diminished the hypoxia induced dissipation of mitochondria membrane potential and cell death. Compared with untreated controls, AN-7-treated hearts recovering from global ischemia and cardiomyocytes undergoing hypoxia, displayed significantly higher levels of the cytoprotective heme oxygenase-1. Our findings indicate that AN-7 imparts cardioprotection against ischemia both in vivo and in vitro and emerges as a potential treatment modality for cardiac injury.

Anticancer prodrugs of butyric acid and formaldehyde protect against doxorubicin-induced cardiotoxicity.

Formaldehyde has been previously shown to play a dominant role in promoting synergy between doxorubicin (Dox) and formaldehyde-releasing butyric acid (BA) prodrugs in killing cancer cells. In this work, we report that these prodrugs also protect neonatal rat cardiomyocytes and adult mice against toxicity elicited by Dox. In cardiomyocytes treated with Dox, the formaldehyde releasing prodrugs butyroyloxymethyl diethylphosphate (AN-7) and butyroyloxymethyl butyrate (AN-1), but not the corresponding acetaldehyde-releasing butyroyloxydiethyl phosphate (AN-88) or butyroyloxyethyl butyrate (AN-11), reduced lactate dehydrogenase leakage, prevented loss of mitochondrial membrane potential (DeltaPsim) and attenuated upregulation of the proapoptotic gene Bax. In Dox-treated mice, AN-7 but not AN-88 attenuated weight-loss and mortality, and increase in serum lactate dehydrogenase. These findings show that BA prodrugs that release formaldehyde and augment Dox anticancer activity also protect against Dox cardiotoxicity. Based on these observations, clinical applications of these prodrugs for patients treated with Dox warrant further investigation.

Recent advances in understanding and exploiting the activation of anthracyclines by formaldehyde.

The anthracycline group of compounds are amongst the most effective chemotherapy agents currently in use for cancer treatment. They are generally classified as topoisomerase II inhibitors but also have a variety of other targets in cells. It has been known for some years that the anthracyclines are capable of forming DNA adducts, but the relevance and extent of these DNA adducts in cells and their role in causing cell death has remained obscure. When the adduct structure was solved, it became clear that formaldehyde was an absolute requirement for adduct formation. This led to a renewed interest in the capacity of anthracyclines to form DNA adducts, and there are now several ways in which adduct formation can be facilitated in cells. These involve strategies to provide the requisite formaldehyde in the form of anthracycline-formaldehyde conjugates, and the use of formaldehyde-releasing drugs in combination with anthracyclines. Of particular interest is the new therapeutic compound AN-9 that releases both butyric acid and formaldehyde, leading to efficient anthracycline-DNA adduct formation, and synergy between the two compounds. Targeted formation of adducts using anthracycline-formaldehyde conjugates tethered to cell surface targeted molecules is now also possible. Some of the cellular consequences of these adducts have now been studied, and it appears that their formation can overcome anthracycline-resistance mechanisms, and that they are more efficient at inducing apoptosis than when functioning primarily through impairment of topoisomerase II. The clinical application of the use of anthracyclines as DNA adduct forming agents is now being explored.

Molecular basis for the synergistic interaction of adriamycin with the formaldehyde-releasing prodrug pivaloyloxymethyl butyrate (AN-9).

The interaction of Adriamycin and pivaloyloxymethyl butyrate (AN-9) was investigated in IMR-32 neuroblastoma and MCF-7 breast adenocarcinoma cells. Adriamycin is a widely used anticancer drug, whereas AN-9 is an anticancer agent presently undergoing Phase II clinical trials. The anticancer activity of AN-9 has been attributed to its ability to act as a butyric acid prodrug, although it also releases formaldehyde and pivalic acid. Adriamycin and AN-9 in combination display synergy when exposed simultaneously to cells or when AN-9 treatment is up to 18 h after Adriamycin administration. However, the reverse order of addition results in antagonism. These interactions have been established using cell viability assays and classical isobologram analysis. To understand the molecular basis of this synergy, the relative levels of Adriamycin-DNA adducts were determined using various treatment combinations. Levels of Adriamycin-DNA adducts were enhanced when treatment combinations known to be synergistic were used and were diminished using those treatments known to be antagonistic. The relative timing of the addition of Adriamycin and AN-9 was critical, with a 20-fold enhancement of Adriamycin-DNA adducts occurring when AN-9 was administered 2 h after the exposure of cells to Adriamycin. The enhanced levels of these adducts and the accompanying decreased cell viability were directly related to the esterase-dependent release of formaldehyde from AN-9, providing evidence for the formaldehyde-mediated activation of Adriamycin.

Prodrugs of butyric acid. Novel derivatives possessing increased aqueous solubility and potential for treating cancer and blood diseases.

The synthesis and biological activities of acidic, basic and neutral types of butyric acid (BA) prodrugs possessing increased aqueous solubility are described. The compounds are butyroyloxyalkyl derivatives of carboxylic acids, which possess functionalities suitable for aqueous solubilization. The anticancer activity of the prodrugs in vitro was evaluated by examining their effect on the growth of human colon, breast and pancreatic carcinoma cell lines, and their solubility in aqueous media was determined. The most promising compounds, with respect to activity and solubility, were found to be the butyroyloxymethyl esters of glutaric 2a and nicotinic acids 4a and phosphoric acid as its diethyl ester 10a, which displayed IC(50) values of 100 microM or lower. These prodrugs are expected to release formaldehyde upon metabolic hydrolysis. The corresponding butyroyloxyethyl esters (2b, 4b and 10b) that release acetaldehyde upon metabolism were significantly less potent. A similar correlation was observed for growth inhibition of the human prostate carcinoma cell lines PC-3 and LnCap and for induction of differentiation and apoptosis in the human myeloid leukemia cell line HL-60. The higher biological activity of the formaldehyde-releasing prodrugs 2a and 10a was further confirmed when induction of hemoglobin (Hb) synthesis in the human erythroleukemic cell line K562 was measured. Moreover, a therapeutic index (IC(50)/ED(50)) of ca. 5 was observed. The acute i.p. toxicity LD(50) in mice for 2a, 2b, 10a and 10b was similar and in the range of 400-600 mg kg(-1). The results obtained support the potential use of the butyric acid prodrugs for the treatment of neoplastic diseases and beta-globin disorders.

Novel mutual prodrug of retinoic and butyric acids with enhanced anticancer activity.

Acyloxylalkyl esters of retinoic acid and small carboxylic acids (C3-5) were evaluated for anticancer activity. The derivative of butyric acid (BA) and all-trans-retinoic acid (ATRA)-retinoyloxymethyl butyrate (RN1)-acting as a mutual prodrug was a more potent inducer of cancer cell differentiation and inhibitor of proliferation than the parent acids. ED50 of RN1 for differentiation induction in HL-60 was over 40-fold lower than that of ATRA. The differentiating activity of ATRA compared to that of the acyloxylalkyl esters derived from butyric (RN1), propionic (RN2), isobutyric (RN3), and pivalic (RN4) acids was found to be: RN1 > RN2 > RN3 > ATRA approximately RN4. This observation implies that the activity of the prodrugs depends on the specific acyl fragment attached to the retinoyl moiety, and the butyroyl fragment conferred the highest potency. The IC50 values for inhibition of Lewis lung (3LLD122) and pancreatic (PaCa2) carcinoma cell line colony formation elicited by RN1 were significantly higher than those of ATRA. In addition to its superiority over ATRA or BA as growth inhibitors of the above cell lines, RN1 was also able to overcome the resistance to ATRA in 3LLD122 cells.

Uptake of pivaloyloxymethyl butyrate into leukemic cells and its intracellular esterase-catalyzed hydrolysis.

UNLABELLED: Pivaloyloxymethyl butyrate (AN-9), a butyric acid (BA) prodrug, exhibited low toxicity and significant anticancer activity in vitro and in vivo. The purpose of this study was to elucidate the basis for AN-9 increased anticancer activity compared to BA, by studying the uptake of BA and AN-9 into the cells. METHODS: The uptake rate and level of [14C]-AN-9 and [14C]-BA, labeled on the carboxylic moiety of BA, into HL-60 and MEL leukemic cell lines was measured. The cells were filtered and the retained radioactivity was determined. The dependence of the uptake on the activity of cellular esterases and membrane fluidity was investigated. RESULTS: The uptake level in cells incubated with [14C]-AN-9 increased rapidly, peaked after 30 min in MEL and 1 h in HL-60 cells, and declined thereafter. This decline could be attributed to the hydrolysis of AN-9 by cellular esterases and catabolism of the released BA to CO2. In cells pretreated with an esterase inhibitor and incubated with [14C]-AN-9, the reduction of radioactivity was less precipitous. In cells exposed to [14C]-BA, the intracellular radioactivity level was low and unaffected by treatment with an esterase inhibitor. The uptake of [14C]-AN-9 decreased significantly at 4 degrees C compared to that at 37 degrees C. CONCLUSION: The higher potency of AN-9 compared to BA could be at least partially attributed to the more rapid uptake of the lipophilic AN-9 and the release of BA in the cells.

Activity of pivaloyloxymethyl butyrate, a novel anticancer agent, on primary human tumor colony-forming units.

The anti-proliferative effects of pivaloyloxymethyl butyrate (AN-9), a butyric acid (BA) derivative with potent tumor-differentiating properties both in vitro and in vivo, was evaluated against colorectal, breast, lung, ovarian, renal cell, bladder, and other types of tumor colony-forming units in a human tumor cloning assay. A total of 76 evaluable specimens were exposed to AN-9 continuously, 48 of these were also exposed to BA continuously for direct comparison of the two agents, and 20 specimens were exposed to AN-9 for two hours. An in vitro inhibitory response was defined as a > or = 50% decrease in tumor colony formation in treated cells compared to untreated controls. Superior anti-tumor activity was observed with the continuous exposure to AN-9 (39% in vitro response at 100 microM and 70% at 200 microM) than with the two-hour exposure (20% at 100 microM and 25% at 200 microM). At a continuous concentration of 200 microM, AN-9 demonstrated greater tumor-specific activity than BA against melanoma (100% vs. 67%), ovarian (67% vs. 40%), breast (63% vs. 0%), non-small cell lung (60% vs. 10%), and colorectal tumor colony-forming units (62% vs. 20%). AN-9 is a novel differentiating agent with activity against colony-forming units derived from a variety of primary human tumors, including those that are considered relatively chemoresistant, and may thus provide a therapeutic alternative or addition to standard cytotoxic agents, if appropriate drug concentrations can be achieved in patients.

Butyric acid and pivaloyloxymethyl butyrate, AN-9, a novel butyric acid derivative, induce apoptosis in HL-60 cells.

A novel butyric acid derivative, pivaloyloxymethyl butyrate, AN-9, was previously shown to be a potent differentiating agent. AN-9 exerts a significant anticancer activity in vitro and in vivo. In all the activities examined, AN-9 was more potent than butyric acid. Here we show that AN-9 and butyric acid induce cell death by apoptosis. Exposure of HL-60 cells to butyric acid and AN-9 decreased cell numbers and induced cell differentiation and the appearance of typical apoptotic features. Induction of apoptosis and/or differentiation by AN-9 and butyric acid was dependent on the concentration and the time of exposure to the drugs. The advantage of AN-9 over butyric acid was further confirmed. Apoptosis induced by AN-9 occurred after a shorter exposure and at lower drug concentrations than that induced by butyric acid. Apoptosis by AN-9 was accompanied by reduction in Bcl-2 expression. Preincubation with antioxidants did not protect HL-60 cells from apoptosis induced by AN-9. HL-60 cells that were induced to differentiate by preincubation with retinoic acid or low AN-9 concentrations were more resistant to apoptosis, induced later by high concentrations of AN-9, than were undifferentiated cells.

Effect of the cytostatic butyric acid pro-drug, pivaloyloxymethyl butyrate, on the tumorigenicity of cancer cells.

Previously we have shown that pivaloyloxymethyl butyrate (AN-9), a pro-drug of butyric acid (BA), is a differentiation-inducing agent in a variety of cells. In this report, we demonstrate that AN-9 is a cytostatic but not cytotoxic agent in a myelomonocytic cell line (WEHI); thus, the cells were growth-arrested and differentiated. These late changes in the cells were preceded by changes in the expression of the early regulatory genes, c-myc and c-jun. Although initiation of all these events had already occurred after 1 h exposure to AN-9, the tumorigenicity of these cells tested in Balb/c mice was not affected. A marked reduction in the tumorigenicity of AN-9-treated cells was observed after 4 h of exposure. Exposure of the highly metastatic subclone of Lewis lung carcinoma (3LLD122) to AN-9 resulted in a very pronounced effect on the tumorigenicity of these cells tested in C57BL mice. Unlike WEHI cells, the tumorigenicity of 3LLD122 was almost completely diminished after 1 h of exposure. In both cell types a 10-fold higher concentration of BA did not affect the tumorigenicity of the cells as did AN-9.

An anti-cancer derivative of butyric acid (pivalyloxmethyl buterate) and daunorubicin cooperatively prolong survival of mice inoculated with monocytic leukaemia cells.

A derivative of butyric acid, pivalyloxymethyl butyrate (AN-9), inhibited the proliferation and induced apoptosis of mouse monocytic leukaemia Mm-A cells, although sodium butyrate, but not AN-9, induced differentiation of the cells. AN-9 and DNA-specific antineoplastic agents synergistically inhibited the growth of Mm-A cells, and the simultaneous treatment was required to evoke the maximum growth-inhibitory effect. On the other hand, there was no synergy between butyrate and the drugs, or AN-9 and anti-metabolic agents in inhibiting the growth of the cells, suggesting that the synergistic effect is specific to AN-9 and DNA-reacting agents. AN-9 as a single agent prolonged the survival of mice inoculated with Mm-A cells in a dose-dependent manner. Moreover, administration of AN-9 plus daunorubicin (DNR) markedly prolonged their survival. These results suggest that combination with AN-9 and DNR entails an obvious therapeutic potential.

Esterase inhibitors diminish the modulation of gene expression by butyric acid derivative, pivaloyloxymethyl butyrate (AN-9).

Pivaloyloxymethyl butyrate (AN-9) belongs to the family of acyloxyalkyl ester prodrugs of carboxylic acids which undergo intracellular hydrolysis to yield butyric acid (BA). We have previously shown that AN-9 and BA reduce the level of c-myc and enhance c-jun transcripts in HL-60 cells, and that the differentiation of these cells, induced by AN-9, is dependent on the presence of intracellular esterases. In this study we show that esterase inhibitors abolish the changes induced by AN-9 on c-myc and c-jun expression. In contrast, esterase inhibitors do not change the effects of BA on c-myc or c-jun. Interestingly, these inhibitors affect the modulation induced by both AN-9 and BA on the retinoblastoma tumor suppressor gene. These data suggest that AN-9 is indeed a prodrug of BA and that prior intracellular hydrolysis by esterases is material for AN-9 activity.

Comparison between the effect of butyric acid and its prodrug pivaloyloxymethylbutyrate on histones hyperacetylation in an HL-60 leukemic cell line.

A butyric acid pro-drug, pivaloyloxymethyl butyrate, AN-9, developed in our laboratory, was previously shown to act as a differentiation-inducing and an anti-cancer agent. In this study we have shown that both AN-9 and butyric acid caused a transient hyperacetylation of histones, which returned to basal levels after 6 and 12 hr, respectively. This activity precedes the induction of differentiation elicited by both agents. AN-9 induced acetylation of histones at a concentration one order of magnitude lower than butyric acid. Pre-treatment of the cells with esterase(s) inhibitors diminished the ability of AN-9 to inhibit proliferation and induce differentiation. The above suggests that the intracellular release of butyric acid fragment, from the pro-drug, is catalyzed by cellular esterase(s).

Butyrate-induced differentiation in leukemic myeloid cells - in-vitro and in-vivo studies.

A study of the effects of three differentiating agents, butyric acid, retinoic acid and cytosine arabinoside on proliferation and differentiation of primary cultures, obtained from sixteen patients with myelo-proliferative disorder was conducted. The results showed that BA was an effective inhibitor of cell proliferation and inducer of cytodifferentiation. An acute non-lymphoblastic leukemia patient was treated with sodium butyrate. A temporary increase in differentiation-associated parameters were noted. However, the effects of SB were short-lived. The lack of clinical response led to the development of a BA prodrug pivaloyloxymethylbutyrate (AN-9). This prodrug was more potent in vitro than BA in the induction of cytodifferentiation and inhibition of cell proliferation.

Cytodifferentiation inducers as anticancer agents (review).

Several successful applications of differentiating agents in leukemic diseases, led to a regained interest in differentiation therapy. In this report we focus on novel classes of natural and synthetic compounds, which were not extensively reviewed and have a promising potential as anticancer agents e.g. protein kinase analogues, butyric acid, vitamin D-3 and purine and pyrimidine derivatives. The biological activities, preclinical and clinical studies of these agents are described.

Rapid alteration of c-myc and c-jun expression in leukemic cells induced to differentiate by a butyric acid prodrug.

The novel prodrug of butyric acid (BA), pivaloyloxymethyl butyrate, has been shown, in vitro, to induce differentiation and inhibit leukemic cell proliferation. The prodrug affects the cells in vitro at lower concentration and at least 100 times faster than does (BA). We have compared the ability of BA with that of its prodrug AN-9 to modulate the expression of the early regulating genes, c-myc and c-jun, in HL-60 cells. Exposure of HL-60 cells to the prodrug resulted in a decrease of c-myc and an increase of c-jun expression. The prodrug elicited this effect at lower concentrations and at least 100 times faster than BA. Since changes in the expression of c-myc and c-jun occur minutes after exposure of the cells to the prodrug, these genes are likely to play a major role in the early stages of the differentiation pathway.

Novel anticancer prodrugs of butyric acid. 2.

The antitumor activity of novel prodrugs butyric acid was examined. The in vitro effect of the compounds on induction of cytodifferentiation and on inhibition of proliferation and clonogenicity showed that (pivaloyloxy)methyl butyrate (1a) (labeled AN-9) was the most active agent. SAR's suggested that its activity stemmed from hydrolytically released butyric acid. In vivo, 1a displayed antitumor activity in B16F0 melanoma primary cancer model, manifested by a significant increase in the life span of the treated animals. Murine lung tumor burden, induced by injection of the highly metastatic melanoma cells (B16F10.9), was decreased by 1a. It also displayed a significant therapeutic activity against spontaneous metastases which were induced by 3LL Lewis lung carcinoma cells. Moreover, 1a has the advantage of low toxicity, with an acute LD50 = 1.36 +/- 0.1 g/kg (n = 5). These results suggest that 1a is a potential antineoplastic agent.

Derivatives of butyric acid as potential anti-neoplastic agents.

A novel derivative of butyric acid, pivalyloxymethyl butyrate (AN-9) has been shown, in vitro, to: (a) induce cytodifferentiation and inhibit the proliferation of leukemic cells; (b) inhibit the growth and formation of Lewis lung carcinoma colonies in semi-solid agar. AN-9 affect cells at about 10-fold lower concentration and at a faster rate than does butyric acid. The pivalyloxymethyl esters of propionic, isobutyric and valeric acids do not elicit effects similar to those of AN-9, while the isobutyryloxymethyl butyrate does, which strongly suggests that the activity of AN-9 stems from intracellular metabolic degradation of the pro-drug to butyric acid. In vivo, AN-9, increased the survival of mice in Lewis lung carcinoma primary cancer model and significantly decreased the number of lung lesions of the animals inoculated with highly metastatic cells, but did not affect their life span. Acute LD50 studies have shown that AN-9 possesses low toxicity. These results suggest that AN-9 is a potential anti-neoplastic agent as well as a tool for investigation of the differentiation induction mechanism.

The role of increased calcium influx rate in receptor mediated function of differentiating HL-60 cells.

In this study we have investigated the link between increased Ca2+ influx rate, acquired upon the differentiation of HL-60 cells, to changes in cytosolic free Ca2+ ([Ca2+]i], evoked by the chemotactic peptide-FMLP and the mitogen Con-A. Although differentiating and undifferentiated HL-60 cells exhibited similar steady-state levels of [Ca2+]i, cells induced to differentiate by dibutyryl-cAMP, at 48 h, exhibited enhanced Ca2+ influx rate, measured by non-steady state 45Ca2+ uptake, and augmentation of FMLP-stimulated Ca2+ influx. At 120 h the above cells responded to FMLP but not to Con-A, by a marked augmentation of Ca2+ influx, and elevated levels of [Ca2+]i. On the other hand HL-60 cells induced to differentiate by retinoic acid responded, as described above, to Con-A but not to FMLP. HL-60 cells grown in the presence of cholera-toxin, were reported to express high levels of FMLP-receptors without expressing cell differentiation. We have demonstrated that, in these cells the Ca2+ influx rate was unchanged, moreover, FMLP-stimulated Ca2+ influx and [Ca2+]i rise were low. These findings strongly suggest that the presence of receptor is not sufficient for FMLP-mediated changes in [Ca2+]i. A link between increased Ca2+ influx rate, acquired upon induction of differentiation, and receptor mediated response in these cells is proposed.