Effect of Polar Organic Compounds on Leukemic Cells

Butyrate-Induced Partial Remission of Acute Myelogenous Leukemia in a' Child

ABRAHAM NOVOGRODSKY, MD, PHD, AMALIA DVIR, MD, AMIRAM RAVID, PHD, TAMAR SHKOLNIK, PHD, KURT H. STENZEL, MD, ALBERT L. RUBIN, MD, AND RlNA ZAIZOV, MD

Polar organic compounds, such as dimethylsulfoxide and butyric acid, are known to induce differen- tiation in Friend erythroleukemia cells as well as in other cell types. It has been found that many of the compounds that induce cellular differentiation, inhibit 3H-thymidine incorporation and induce cell damage when incubated with leukemic cells from patients with acute or chronic myelogenous or acute lymphocytic leukemia. These effects are time and dose dependent. Among the compounds tested, butyrate was the most potent. Parenteral administration of butyrate (500 mg/kg/day) for ten days to a child with acute myelogenous leukemia in relapse, and resistant to conventional therapy, resulted in elimination of myeloblasts from the peripheral blood, a n increase in mature myeloid cells and a reduction in 'H-thymidine uptake by the patient's peripheral blood cells. Bone marrow myeloblasts were reduced from 70430% to 20% following the course of intravenous butyrate. No impairment of liver or renal function and no coagulation abnormalities were observed during butyrate treatment. Organic agents that induce cell differentiation may provide additional reagents for the clinical management of selected cases of leukemia.

Cancer 51:9-14. 1983.

CUTE LEUKEMIA is characterized by the accumu- A lation of immature undifferentiated hemopoietic cells. Normal differentiation of these cells is controlled, at least in part, by naturally occurring proteins, such as erythropoietin and macrophage and granulocyte colony stimulating factor.' Since the discovery by Friend that dimethylsulfoxide induces differentiation in Friend erythroleukemic cells,2 additional compounds have been reported to share this effect394 and to induce differentia- tion in other mammalian cells as We recently found that the organic compounds that are effective in the induction of cell differentiation, inhibit mitogen-in- duced proliferation of normal human lymphocytes.",' ' Inducers of cellular differentiation may be considered as potential therapeutic agents in acute leukemia, since the proliferative potential of hemopoietic cells declines along the differentiation pathway. Butyrate is a potent inducer of erythroid differentiation in Friend leukemia

From the Rogoff-Wellcome Medical Research Institute and the Department of Pediatric Hematology-Oncology, Beilinson Medical Center, Petah-Tikva, Israel, Tel-Aviv University Sackler Medical School and the Rogosin Kidney Center, Departments of Biochemistry and Medicine, Cornell University Medical College, New York, New York.

Supported in part by The Rogosin Foundation. Address for reprints: A. Novogrodsky, MD, PhD, Beilinson Medical

Accepted for publication October 23, 198 I . Center, Petah-Tikva, Israel.

cells,3 is capable of inducing morphologic and metabolic alterations in other mammalian cells and inhibits DNA synthesis and cell division." The induction of terminal differentiation of a human promyelocytic leukemic cell line by butyric acid has recently been r e p ~ r t e d . ' ~

The authors investigated the effect of a variety of in- ducers of cellular differentiation on the proliferation of cells isolated from the blood of leukemic patients, and attempted to induce differentiation in vivo by admin- istration of butyrate to a child with acute myelogenous leukemia. We report here that these agents inhibit pro- liferation of leukemic cells from patients and induce cell damage. Furthermore, parenteral administration of bu- tyrate to a child with acute myelogenous leukemia in relapse resulted in the reduction, and finally the disap- pearance, of myeloblasts from the peripheral blood fol- lowed by the re-appearance of mature myeloid cells.

Patients a n d Methods

Patients

Five patients with acute leukemia were studied. The type of leukemia was determined by standard hemato- logic and serologic assays. Three were children, two with acute T-cell lymphocytic leukemia and one with acute myelogenous leukemia. The latter patient is described in detail in the case report. The remaining two patients

0008-543X/83/010l/0009 $1.10 (c) American Cancer Society

9

CANCER January 1 1983 VOl. 51 10

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FIG. 1. In vitro effect of polar organic compounds on 'H-thymidine incorporation in five patients with leukemia. Each point represents the mean of triplicate determination in each patient. Three patients had acute T-cell lymphocytic leukemia (0, 0, A), one chronic my- elogenous leukemia (A), and one acute myelogenous leukemia (0). Concentrations of the compounds used were as noted in the figure. Results are expressed as ?6 of 'H-thymidine incorporation in control cultures, without the organic compounds. )H-Thymidine pulse was on either day 2 or 3 of the cultures. Values of 'H-thymidine incor- poration in control cultures of the different patients' cells were: 2880 cpm (0), 10,170 cpm (O), 26,690 cpm (A), 10,520 cpm (A) and 55 10 cpm (0).

were adults, one with acute T-cell lymphocytic leukemia and one with chronic myelogenous leukemia in blast crisis.

Isolation of Cells and Cell Culture

Cells were separated from heparinized peripheral blood by Ficoll-Hypaque gradient centrifugation. Cells ( 1 X 106/ml) suspended in RPMI 1640 medium con- taining heat-inactivated pooled human AB serum (5%) or fetal calf serum (5%) and supplemented with peni- cillin (100 units/ml) and streptomycin (100 pg/ml) were cultured (0.2 ml aliquots) in flat bottom microwells at 37OC in a humidified 95% air-5% COz incubator. The polar organic compounds tested were added to replicate wells, cultures were incubated for different times (as specified in results) and five hours before termination of the incubation, 1 pCi methyl-3H-thymidine (2 Ci/ mmole) was added to each culture well and incorpo- ration into DNA was measured.I4 The organic com- pounds used did not cause cell death in normal lym- phocytes following three days exposure to the highest concentration evaluated, as assessed by trypan blue ex-

clusion and mitogenic response to PHA." Cells were also cultured for morphologic studies, and assayed for Fc and C3 receptors, as de~cribed.'~

Butyrate Solution for Intravenous Infusion

Butyric acid (2%) was neutralized with sodium bicar- bonate (final concentration 2%). The final pH of the solution was 6.5 and it was sterilized by filtering through 0.2 p Millipore filters.

Case Report

A five-year-old boy was referred to the Pediatric Hematol- ogy-Oncology Department, Beilinson Medical Center, at the age of three and three quarters years because of acute my- elogenous leukemia, characterized by myelornonoblasts posi- tive for peroxidase and Sudan black, and elevated serum mur- amidase. Remission was induced with combination chemo- therapy, consisting of seven day courses of vincristine, Adriamycin (doxorubicin), cytosine arabinoside, 6-thiogua- nine and prednisone, repeated every ten days for three courses. Thereafter, he was maintained with that combination admin- istered every 5-6 weeks, along with daily 6-mercaptopurine. In addition, daily thymic humoral factor (THF) was given for two weeks, following a course of combination chemotherapy.16 A complete remission was maintained for eight months. A second remission, which lasted for three months, was attained with the same combination chemotherapy. A combination of weekly methotrexate, L-asparaginase and hydroxyurea failed to induce a third remission. A trial with daily small doses of cytosine arabinoside, aimed at induction of differentiation of the myeloid leukemic cells," resulted in a transitory fall in the number of peripheral blood myeloblasts without the appear- ance of mature forms of the myeloid series. As a possible ther- apeutic approach, the patient was placed on daily continuous infusion of 2% butyrate 500 m1/24 hours (500 mg/kg/24 hours) for ten days.

Results

The effect of several short chain fatty acids and or- ganic solvents on the incorporation of 'H-thymidine into peripheral blood cells obtained from five patients with different types of leukemia is illustrated in Figure 1. Butyric acid, DMSO, dimethyl acetamide, and di- methyl formamide inhibited 3H-thymidine incorpora- tion, whereas propionic, isobutyric, and B-OH-butyric acids had little effect. However, the pattern of inhibition by the different organic compounds was not uniform among the five patients. Additional results obtained from culutres of peripheral blood cells from the child with acute myelogenous leukemia (Case Report), are depicted in Figure 2. In addition to the compounds listed in Figure 1, caproic and valeric acids were studied and found to have little effect on 3H-thymidine incorpora- tion. On a molar basis, butyric acid was the most potent inhibitor among the compounds tested. The extent of

No. I POLAR ORGANIC COMPOUND EFFECTS ON LEUKEMIA - Novogrodsky et al. 11

inhibition of 'H-thymidine uptake increased with the time of exposure to butyric acid in the culture medium (Fig. 3).

Decreased 3H-thymidine uptake following incubation of leukemic cells from several of the patients with the polar organic compounds, was associated with cell dam- age. Cells were examined, following incubation in con- trol medium, or in medium containing the organic agents listed in Figure 1, after deposition on glass slides in a Shandon cytofuge and staining by the Wright- Geimsa method. Cell damage was manifested initially by vacuolization of the cytoplasm and nucleus and fol- lowed by disintegration of the cells. These morphologic changes were induced only by the agents that inhibited 3H-thymidine uptake (Fig. 1) and they were time de-

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incorporation in leukemic cells obtained from the child with acute myelogenous leukemia prior to butyric acid treatment. Cells (60% myeloblasts) were incubated for 1-4 days with dimethylsulfoxide, 140 mM (W), dimethylacetamide, 20 mM (O), dimethylformamide, 110 mM (V), propionic acid (x), butyric acid (0). 8-hydroxybutyric acid (0), valenc acid (A), and caproic acid (A) ( I mM each). 'H-Thymidine incorporation was determined on days I , 2, and 4 after initiation of the cultures. Results are presented as percent of 'H-thymidine incor- poration in control cultures without the organic compounds. 'H-Thy- midine incorporation in control cultures for days 1, 2, and 4 were 2480, 55 10 and 6400 cpm/culture respectively.

0

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midine incorporation in peripheral blood cells of a patient with chronic myelogenous leukemia in blast crisis. Incorporation was determined on days 1-4 following initiation of the cultures.

pendent. The cytotoxic effect of the inducers of differ- entiation on leukemic cells is in marked contrast to their effect on normal cells. Incubation of normal cells with these agents for the same duration and at the maximal concentrations used, either with or without mitogenic stimulation, did not affect cell viability.''

The potency of the organic compounds to inhibit 3H- thymidine incorporation and to induce morphologic changes in leukemic cells closely paralleled both their potency to induce differentiation in Friend erythroleu- kemia cells2*' and to inhibit mitogen-induced lympho- cyte responses.' ' Inhibition of 'H-thymidine incorpo- ration and cell damage caused by the chemical inducers were not prevented by either tetracaine (0.1 mM) or by the lysosomotrophic agents butylamine (10 mM) or NH4CL (10 mM). Thus, the antiproliferative effects of

12 CANCER January I 1983 VOl. 5 1

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Cytosine , arabinoside Butyr ic acid

A

I 4 8 12 I8 26 36 40 44 48 Days

FIG. 4. Changes in peripheral blood cells in the patient who received intravenous butyric acid. Cytosine arabinoside was administered sub- cutaneously at 10 mg/day for nine days. Twenty-six days following discontinuation of cytosine arabinoside therapy, butyric acid was ad- ministered intravenously at 500 mg/kg/day for ten days.

the polar organic compounds are probably not related to release of lysosomal enzymes.

The antiproliferative properties of butyrate, its ability

to induce differentiation of different cell types in vitro and our finding of its selective cytotoxic effect on human leukemic cells prompted us to determine possible in vivo antileukemic effects of butyrate in one of the patients, the child with acute myelogenous leukemia. This patient was in relapse and resistant to conventional therapy. ’H- Thymidine incorporation was markedly suppressed and morphologic changes were induced in this patient’s pe- ripheral blood cells in vitro by l mM butyrate (Fig. 2). Butyrate is a normal intermediate of fatty acid metab- olism and was reported to cause no apparent side effects upon parenteral administration to a patient with neu- roblastoma. ’ * However, this information is insufficiently documented and there apparently has been no formal Phase I testing of butyrate. It should be noted that an- imal studies indicate that butyrate has a low order of toxicity (LD 50 is 8.79 g/kg orally in rats).” The ad- ministration of butyrate to our patient had prior ap- proval of the Human Rights in Research Committee at Beilinson Medical Center and the informed consent of the patient’s parents. We administered butyrate as a con- tinuous intravenous infusion of a 2% solution of butyric acid, neutralized with NaHC03, at a dose of 500 mg/ kg/day for ten days, at a time when the patient was receiving no other chemotherapy. Butyrate administra- tion resulted in remarkable changes in the peripheral blood cells (Fig. 4). The number of myeloblasts gradually decreased, and by day 15 following initiation of intra- venous butyrate were no longer detectable in the pe- ripheral blood. The reduction in myeloblasts was fol-

FIGS. 5A-5C. Butyric acid-induced maturation of leukemic myeloblasts. Cells were separated from peripheral blood by Ficoll-Hypaque gradient centrifugation, stained by the Giemsa method, and observed at an original magnification of X1000. (A) Prior to butyric acid treatment. The predominant cell is a leukemic myeloblast. (B AND C) Ten days after initiation of butyric acid treatment. (B) Different forms of mature myeloid cells. (C) A cluster of mature giant granulocytes.

No. 1 POLAR ORGANIC COMPOUND EFFECTS ON LEUKEMIA . Nuvogrodsky et al. 13

lowed by the appearance of mature myeloid cells, (Figs. 5B and 5C), the most prominent being giant granulo- cytes, along with an increase in monocytes. Repopula- tion of the peripheral blood by mature cells was also indicated by an increase in cells bearing Fc and C3 re- ceptors. Cells bearing Fc receptors were undetectable prior to butyrate infusion and increased to 5.2% on day 13 following the initiation of butyrate. Those bearing C3 receptors increased from 1.3% prior to butyrate to 17.2% on day 13 following butyrate treatment. 3H-Thy- midine incorporation by the patient’s peripheral blood cells in vitro was gradually reduced during butyrate treat- ment (Fig. 6). This reduction in general paralleled the reduction in peripheral blood myeloblasts. Prior to bu- tyrate treatment, bone marrow aspirates revealed 70- 80% myeloblasts. Twelve days following initiation of intravenous butyrate, when myeloblasts were no longer present in the peripheral blood, bone marrow aspirates (eight smears were evaluated) revealed rich cellularity with absence of megakaryocytes. A striking feature was hyperplasia of the myeloid series in various stages of maturation, with approximately 20% myeloblasts. In the erythroid series, early signs of erythropoiesis were ob- served with a M/E ratio of 4:l (M-2 marrow). We in- terpreted these bone marrow findings as evidence for a partial remission. There were no changes in clinical lab- oratory assays of hepatic or renal function and no co- agulation abnormalities were detected following intra- venous butyrate. Specifically, the SGOT and bilirubin remained in a normal range during and following bu- tyrate infusion. LDH was elevated to 630 u prior to butyrate and remained at this level during the ten day period of butyrate infusion and decreased to 349 u one week following cessation of butyrate. Total protein re- mained at 5.9 g/dl and albumin at 3.8 g/dl Blood urea nitrogen (BUN), uric acid, serum creatinine levels, glu- cose, sodium, potassium, chloride and bicarbonate and serum amylase were in the normal range prior to bu- tyrate and remained in the normal range during and following the ten day course of butyrate. Coagulation studies including prothrombin time, fibrinogen levels, and PTT were within the normal range before, during and after butyrate. The hemoglobin value prior to bu- tyrate infusion was 5.9 g/dl and increased to 8.2 g/dl after five days of butyrate infusion, and after ten days was 6.5 g/dl. The platelet levels at these times were: 10,000/mm3. 14,000/mm3 and 9000/mm3.

The patient was subsequently discharged from the hospital, but returned two weeks later with a rise in blood myeloblasts. Peripheral leukocyte count was 1 100 with 54% lymphocytes, 23% monocytes and 23% my- eloblasts. He was placed on chemotherapy (vincristine 1.5 mg/m2; cytosine arabinoside, 200 mg/m*/day for four days and 6-thioguanine 75 mg/m2/day for ten days) along with butyrate infusion (500 mg/kg/24 hr). He had

Butyr ic acid

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rc)

01 I I I 0 5 10 15

Days fol lowing treatment w i th butyric a c i d

FIG. 6 . ’H-Thymidine incorporation in cells obtained at different times after initiation of butyric acid treatment. Incorporation was de- termined immediately after isolation of the cells.

E. coli sepsis and subsequently developed pseudomonas sepsis. During three weeks of butyrate infusion he re- mained neutropenic, with 30-40s myeloblasts in the peripheral blood. During this period, BUN, electrolytes, SGOT, creatinine, prothrombin time, fibrinogen and uric acid remained within normal limits. Despite che- motherapy and intensive supportive care, the child died with sepsis two months following the initial trial of bu- tyrate.

Discussion

Previous studies have shown that a variety of agents, including DMSO and butyric acid,20.2’ as well as retinoic acid” and tumor promoting phorbol ester^'^-'^ induce differentiation of human myelogenous leukemic cell lines. Our studies indicate that treatment of leukemic cells from patients, with polar organic compounds at concentrations that are effective in the induction of ery- throid differentiation in Friend leukemia cells, results in decreased proliferation and cytotoxicity. The data available do not resolve the question of whether cell damage follows inhibition of proliferation, or whether the cytotoxic effect of these drugs is unrelated to inhi-

14 CANCER Junuary 1 1983 Vol. 51

bition of cell proliferation. Parenteral administration of butyrate was effective in vivo in eliminating peripheral blood myeloblasts and decreasing those in the bone marrow.

The authors do not have data regarding serum levels of butyrate in the patients. We have initiated studies to determine pharmacokinetics of butyrate in dogs. Prelim- inary data indicate that a steady state level of 0.28 mM is rapidly achieved with an infusion of 2 g/kg/24 hour as a 3% solution. Following cessation of the infusion the plasma half life of butyrate is extremely rapid, less than five minutes.

Mechanisms involved in the induction of cell differ- entiation by polar organic compounds are unknown as are mechanisms involved in our findings of decreased proliferation and cytotoxicity induced by these agents in human leukemic cells. That they are related phenom- enon is suggested by the similar structural specificity and potency of the compounds that are effective in inducing both differentiation in cultured cell lines and cytotox- icity in patients’ leukemic cells. The in vivo effect of butyrate could have resulted from a selective cytotoxic effect of butyrate on leukemic cells, as demonstrated in vitro. In addition, the induction of terminal differentia- tion by butyrate may have played a role, although we have no direct evidence for this. The repopulation of the peripheral blood with monocytes seven days after ini- tiation of butyrate treatment is commonly observed in patients recovering from myelosuppression.

The authors have thus found that the polar organic compounds that induce differentiation in Friend eryth- roleukemia cells, inhibit proliferation and induce cyto- toxicity in cells isolated from the peripheral blood of patients with leukemia. In addition, one of these agents, butyrate, decreased peripheral blood and bone marrow myeloblasts in vivo. The polar organic compounds that induce cell differentiation may provide additional agents for induction of remissions in leukemia. Their possible synergy with other chemotherapeutic agents should also be evaluated. We chose butyrate to administer to the patient because of its low toxicity. However, its rapid half-life may hamper its clinical use. We are currently evaluating additional agents, known to induce erythroid differentiation in Friend leukemia cells, in experimental models in vitro and in vivo.

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