Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the plasmacytoma and hybridoma growth inhibitor cytokine restrictin-P, herein identified as being activin A of stromal origin and an antagonist of the cytokines interleukin(IL)-6 and/or IL-1 1, and in particular to pharmaceutical compositions and methods using said restrictin-P/activin A for modulation of the in vivo activity of IL-6 and/or IL-l 1.
• cytokine restrictin-P herein identified as being activin A of stromal origin and an antagonist of the cytokines interleukin(IL)-6 and/or IL-1
• CSFs colony-stimulating factors
• TNF tumor necrosis factor
• Restrictin-P has formerly been described by one of the present inventors as an inhibitor of plasmacytoma cell growth (Zipori et al., 1986; Honigwachs-Sha'anani et al,
• Factor(s) mediating the growth inhibition were found to be produced by the stromal cell line in minuscule amounts and thus, in order to isolate the active component, it was necessary to establish conditions for large scale production of the factor. It was found that the producer cell line MBA-2.1 could be propagated on a three-dimensional carrier of non- woven fabric of polyester loaded in a bioreactor system under complete protein-free conditions (Kadouri et al., 1992). The study of such bioreactors showed that the cells could be maintained under protein-free conditions for up to 10 months while producing restrictin-P activity along with transforming growth factor (TGF) ⁇ , macrophage (M)-CSF and EL-6.
• TGF transforming growth factor
• M macrophage
• Restrictin-P obtained from the bioreactor system induced in its target cells early Gi/Go arrest, morphological changes and signs of cell damage (Honigwachs-Sha'anani et al, 1991) accompanied by intracellular ionic changes (Malik et al., 1992).
• IL-6 is a pleiotropic cytokine that affects cells in different tissues and organs.
• IL-6 has growth-promoting effect on plasma-like cells and is involved, within the hemopoietic system, in processes such as induction of growth and differentiation as well as growth inhibition of a variety of hemopoietic cell types (Ikebuchi et al., 1987; Hoang et al., 1988; Bot et al, 1989). It is therefore expected that the activity of IL-6 would be tightly regulated. This may occur on the level of expression of the IL-6 gene as a result of activity of other cytokines or a variety of mediators. In the treatment of human patients with IL-6, e.g.
• Activins are members of a TGF ⁇ family and are involved in the regulation of many biological events.
• Activin A also known as FSH-releasing protein (FRP)
• FRP FSH-releasing protein
• EDF erythroid differentiation factor
• Activin A was found to be expressed by stromal cells (Yamashita et al., 1992). Activin A was further shown to kill the B9 plasmacytoma cells and the U266B1 myeloma cell line (Nishihara et al., 1993). The U266B1 cells are E -6 independent; they grow in medium supplemented with serum and do not require IL-6 for growth. In this same publication, the autors showed in short term experiments that B9 cells grown with or without IL-6, died to the same extent when exposed to activin A. It is further stated in said publication that EL-6 did not prevent apoptosis of B9 cells induced by activin.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising restrictin-P/activin A as active ingredient together with a pharmaceutically acceptable carrier, for modulation of the in vivo activity of DL-6 and/or LL-11.
• the composition is intended for blocking or reducing the deleterious effects of LL-6, for example in patients receiving IL-6 during tumor therapy or bone marrow transplantation.
• the invention relates to the use of the cytokine restrictin-P/activin
• the invention relates to a method of modulating the in vivo activity of IL-6 and/or EL- 11 , which comprises administering to a patient in need thereof an effective amount of restrictin-P/activin A.
• the method is suitable for blocking or reducing the deleterious effects of IL-6 in patients undergoing tumor therapy or bone marrow transplantation.
• Figs. 1A-D show a series of elution profiles constituting the final purification of restrictin-P from stromal cell conditioned medium as described in Example 2, wherein Fig.
• FIG. 1A is an elution profile on a Q-Sepharose anion exchange chromatography column; Fig. IB on a Superdex 75 gel filtration; Fig. 1 C on a C-8 Reversed Phase HPLC-I (RP-HPLC I), and
• Figs. 2 A-B show analysis of highly purified restrictin-P.
• Fig. 2A depicts the sequence that was obtained for restrictin-P as compared to the known sequence of activin A.
• the Xs at positions 4, 1 1 and 12 are the expected positions of cystein residues which are usually undetectable in the sequencing machine, and the X in position 25 corresponds to a
• Fig. 2B shows SDS-PAGE analysis of highly purified restrictin-P obtained from the RP-HPLC II column, as described in Example 2.
• Fig. 3 shows inhibition of MPC-1 1 plasmacytoma cell growth by purified restrictin-P
• Figs. 4A-B show that restrictin-P antagonizes IL-6 (4 A) and IL-1 1 (4B) induced proliferation of B9 cells, as described in Example 4.
• B9 cells were treated with the indicated dilutions of recombinant human IL-6 (4A, open symbols) or recombinant human IL-l 1 (4B, open symbols) or with purified restrictin-P and IL-6 (4 A, closed symbols) or IL-l 1 (4B, closed symbols.
• Figs. 5A-B show that restrictin-P does not interfere with the binding of IL-6 to B9 cells, in an experiment as described in Example 5.
• Figs. 6 depicts a Western blot showing that restrictin-P interferes with the IL-6 induced secretion of the acute phase proteins ⁇ -acid glycoprotein (AGP) and haptoglobin
• Fig. 7 shows that in HepG2 hepatoma cells restrictin-P did not interfere with STAT activation involved in EL-6 signaling.
• the amounts of either Stat3 and Statl ⁇ homodimers or Stat3 /Statl ⁇ heterodimers were followed under different incubation conditions as described in Example 7 Detailed Description of the Invention
• Activin A was found to be expressed by stromal cells (Yamashita et al., 1992). Since the molecular weight of monomeric restrictin-P, as deduced from PAGE, was 15 kDa, a size similar to that of monomeric activin A ( ⁇ A inhibin), it was concluded that these two molecules are identical. Indeed, recombinant activin A is shown hereinafter to be inhibitory to the MPC-11 plasmacytoma to the same extent as restrictin-P.
• Restrictin-P in its purified form killed the factor dependent hybridoma cell line B9 by competing with externally added IL-6 or IL-l 1.
• IL-6 factor dependent hybridoma cell line B9
• IL-l 1 IL-6
• Fig. 5 A a 270 fold excess of partially purified restrictin-P to compete with r-Mu-[ ⁇ - ⁇ I)-IL-6 binding
• Fig. 5B a 340 fold excess of highly purified restrictin-P to compete with r-Hu-[ ⁇ I]-EL-6 Viuteine
• restrictin-P The antagonistic effect of restrictin-P is specific to IL-6 and IL- 11 since restrictin-P did not affect the growth of other cytokine-dependent cell lines such as 14M1.4 macrophages that depend on M-CSF for growth, MC/9 mastocvtoma which are IL-3 dependent or NFS-60, GM-CSF dependent cells (results not shown).
• restrictin-P The strict specificity of killing by restrictin-P of plasmacytomas and hybridomas suggested that the factor detects some molecular machinery characteristic to this cell type.
• the growth dependence on EL-6 is a characteristic of many plasmacytomas and hybridomas. Indeed we show here that restrictin-P inhibits the growth of B9 cells by competing with the growth factors obligatory for the survival of the hybridoma. However, some cells like the MPC-11 clone, are cytokine independent, but their growth is nonetheless inhibited by restrictin-P.
• Plasmacytomas are but one target cell type that responds to IL-6 signalling.
• the HepG2 hepatoma cells release acute phase proteins under the influence of IL-6 (Gauldie et al., 1987). an activity that can be inhibited by restrictin-P.
• restrictin-P can abolish both the high mitochondrial and growth inhibition activity of Ml myeloblastic cells and their differentiation into adherent monocytes, effects that are induced by IL-6.
• Restrictin-P is thus an universal antagonist to IL-6, inhibiting IL-6 activity in all systems in which IL-6 is active.
• Restrictin-P herein identified as a stromal activin A, is an antagonist of IL-6 and/or IL-l 1, which are growth factors for plasma-like cells, but it is not restrictive to other cells that depend on alternative growth factors.
• Restrictin-P/activin A can thus be used, for example, in the treatment of immune disorders.
• restrictin-P/activin A is recommended in each case where it is required to kill plasma cells without harming other cell types, thus avoiding the use of cytotoxic drugs and immunosuppressive agents with a wide spectrum of target cells.
• the following categories of immune disorders can be treated with restrictin P/activin A: a.
• Hypersensitivity type I IgE antibodies sensitized mast cells produce an inflammatory reaction with symptoms such as asthma or rhinitis.
• the cytokine restrictin-P/ activin A may be used to reduce the load of viable antibody producing plasma cell.
• Hypersensitivity type II antibody-dependent cytotoxicity by phagocytosis, killer cell activity or complement-mediated lysis.
• antibodies to the cell surface of erythrocytes cause hemolytic disease of the newborn (anti Rh) and autoimmune hemolytic anemias of adults.
• Hyperacute graft rejection is also mediated by antibodies.
• the disease myasthenia gravis is due to antibodies to acetylchoiine receptors.
• restrictin-P should reduce in these cases the burden of plasma cells.
• Hypersensitivity type III immune complex diseases.
• Restrictin-P may be used in these cases to reduce the number of complex-producing plasma cells without the need of using cytotoxic drugs that would affect other lineages.
• Some autoimmune diseases, like thyroiditis, are known to be mediated by autoantibodies and restrictin-P/activin A may be used in these cases as well.
• restrictin-P/activin A may be used to treat abnormal immuno- globulin synthesis-plasma cell dyscrasias.
• a series of diseases are associated with accumulation of a monoclonal immunoglobulin. Some of these situations are controlled and the amount of the monoclonal protein is tolerated well. Other situations result in severe diseases and cytotoxic drugs are used to suppress plasma cells.
• Restrictin-P/activin A may be a much better approach due to its killing specificity to plasma-like cells.
• the conditions that may be treated include chronic cold agglutinin syndrome, heavy-chain diseases, papular mucinosis, pyoderma gangrenosum. Waldenstrom macroglobulinemia, and immunoglobulin- related amyloidosis.
• restrictin-P/activin A will be used for modulation of the in vivo activity of EL-6, a pleiotropic cytokine known to be a regulator of hemopoietic stem cell proliferation and differentiation, and affecting many other cell types.
• EL-6 a pleiotropic cytokine known to be a regulator of hemopoietic stem cell proliferation and differentiation, and affecting many other cell types.
• Treatment of human patients with IL-6 faces therefore the problem of side effects caused by EL-6 activity on cells in which the treatment is not directed to.
• the use of restrictin-P/activin A is meant to block or reduce the effect of IL-6 upon in vivo administration, in cases such as treatment with LL-6 in the course of bone marrow transplantation and tumor therapy.
• restrictin-P/activin A may be useful in immunomodulation in the case of the immune disorders described in a-d above.
• the present invention encompasses the use of any activin A molecule, native as well as recombinant activin A from different species, such as human, porcine, bovine and rat activins. There is 100% amino acid conservation among these activins. Native and recombinant activins of different species are described in US 4,798,885 (Mason et al., assigned to Genentech), US 4,973, 577 (Vale, Jr.
• compositions to be used in the present invention will be formulated and dosed according to standard procedures, for example as described in US 5,102, 868, herein incorporated by reference.
• the daily dose will depend from the disease to be treated and the condition of the patient and will be in the range of about l ⁇ g/kg body weight to about lmg/kg body weight.
• the restrictin-P/activin A is formulated with a parenteral pharmaceutically acceptable carrier, e.g a solution that is isotonic with the patient's blood, such as phosphate-buffered saline, and is administered by injection,
• a parenteral pharmaceutically acceptable carrier e.g a solution that isotonic with the patient's blood, such as phosphate-buffered saline, and is administered by injection.
• MPC-11 (Laskov and Scharff, 1970), SP-2, NSO, X-24, X-63 and P3.1 (Kohler and Milstein, 1976) plasmacytoma cell lines were grown in Roswell Park Memorial Institute (RPMI) 1640 (Gibco) with 10% FCS.
• RPMI Roswell Park Memorial Institute
• ABLS-8 (Sklar et al., 1975) and AVRij-1 (Zipori and Bol, 1978) pre-B lymphoma cells and the T lymphomas BW-5147 (Hyman and Stallings, 1974), the myeloid tumor cell line WEHI-265.1 (Warner et al., 1965), the F4N Friend erythroleukemia cell line and the Pu-5-lR macrophage cell line (Ralph et al., 1976) were all grown in RPMI 1640 with 10%) FCS and 50 mM ⁇ -mercaptoethanol.
• B9 B cell hybridoma cells (Helle et al., 1988) were grown in RPMI 1640 supplemented with 10 % FCS, 50 mM ⁇ - mercaptoethanol and 10 IU/ml human recombinant IL-6.
• M1-S6 (a subclone of a mouse myelomonocytic leukemia Ml cell line (Ichikawa, 1969) was grown in RPMI 1640 supplemented with 10% FCS.
• HepG2 hepatoma cells were grown in DMEM supplemented with 8% FCS.
• Viable cell number was determined following 4 days of incubation using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, which measures cell viability via mitochondrial activity.
• MTT (Sigma) (10 ⁇ l 100 ⁇ l medium; stock solution of 5 mg/ml in PBS) was added to each well, and plates were incubated at 37°C for 3 h. After incubation, 150 ⁇ l acid-isopropanol was added to each well.
• Optical density (OD) was then determined in an enzyme-linked immunosorbent assay (ELISA) reader (Titertek Twinreader; FLAB, Helsinki, Finland), using a test wavelength of 570 nm and a reference wavelength of 630 nm.
• ELISA enzyme-linked immunosorbent assay
• One unit of activity was designated as the amount of protein which, under the above conditions, caused 50% growth inhibition relative to the control.
• the assay was essentially the same using B9 cells except that the culture conditions were as indicated above for the B9 hybridoma. The latter was also used to titrate IL-6 and IL-l 1 levels. Briefly, B9 cells (5xl0 3 cells/200 ⁇ l) were cultured in 96 well plates in the presence of test samples and recombinant human IL-6 and
• IU IL-6 and IL-l 1 were determined by relating to standard curves of human recombinant IL-6 and IL- 11 , respectively.
• Cytokines and Corresponding Neutralizing Antibodies The antibodies to TGF- ⁇ used were rabbit anti-native porcine platelet TGF- ⁇ 1. neutralizing for both TGF- ⁇ 1 and
• IFN- ⁇ antibodies were from Genzyme Corporation (Boston, MA).
• IL-3 was purchased from Peprotec.
• Recombinant human, and monoclonal rat anti-mouse neutralizing antibodies to mouse IL-6 were purchased from Genzyme Corporation (Boston, MA).
• Recombinant, N- terminally truncated, human EL-6 (Muteine) and basic fibroblast growth factor (bFGF) were kindly provided by Pharmacia Biocenter (Nerviano, Italy).
• Crude concentrated murine IL-6 was kindly provided by Dr. J. Lotem, Weizmann Institute, and murine EL-6 was obtained from Dr. J. Van Snizk, Ludwig Institute for Cancer Research, Brussels.
• Goat anti-M-CSF neutralizing antiserum was kindly provided by Dr. R.E. Stanley from the Albert Einstein
• EL- 11 and PDGF were purchased from Genzyme Corporation (Boston, MA). Human G-
• Hu-[l 2 5i]_iL-6 used was 30-40% of the amount that gave saturation binding. Under these conditions there were 2,070+530 high affinity binding sites and the Mu-IL-6 and Hu-IL-6 bound with apparent Kd's of 5.5x10 " 10 and 1.1x10 " ⁇ M. respectively, as determined by the LIGAND program (Munson and Rodbard.1980). Specific high affinity binding was competed with varying concentrations of crude concentrated mouse IL-6, r-Hu-IL-6 Muteine, partially purified or highly purified restrictin-P preparations as indicated. The results of the competition binding studies were plotted as a function of fold excess where one unit of restrictin-P inhibits one unit of EL-6 by 50% on B9 cells.
• Restrictin-P was eluted with 400 ml of 0.05 M NaCl in the initial buffer, at the same flow rate. Elution of proteins was followed by their absorbency at 280 nm.
• the salt-eluted material was filter- sterilized and then desalted and concentrated using reversed phase high performance liquid chromatography (RP-HPLC, the system consisted of Model SP8700/SP8750 pump, Spectra- Physics, CA, and model 441 detector from Waters Associates, MA). Each sample (450-500 ml) was loaded onto an Aquapore RP-300 column (7 ⁇ , 4.6 x 100 mm + 4.6 x 30 mm pre- column, Applied Biosystems, CA), at a flow rate of 1-3 ml/min.
• the loaded column was washed with 30 ml of an aqueous solution of acetonitrile (ACN, 5%) and trifluoroacetic acid (TFA. 0.1%1 Proteins were then eluted with a three step gradient of aqueous ACN in 0.1% TFA at a flow rate of 0.5 ml min: 5-35% ACN/10 min followed by an isocratic step of 35% ACN/20 min and finally 35-80% ACN/20min. Fractions of 1 ml were collected. Elution of proteins was followed by their absorbency at 214 nm. Protein content was determined according to Bradford reagent (Bio-Rad, Kunststoff) and restrictin-P content was assayed as described above.
• ACN acetonitrile
• TFA trifluoroacetic acid
• Fractions were vacuum dried in a Savant SpeedVac centrifuge and kept at -20°C. Fractions with restrictin-P activity were dissolved in 20 mM Hepes (pH 7.8), pooled, centrifiiged, filtered and loaded onto a Superdex 75 gel filtration column (20/60, Pharmacia) in batches of about 10 mg protein in 5 ml each. The column was washed with 360 ml of 2 x PBS at a flow rate of 2 ml/minute/tube. Elution pattern was followed at 280 nm. Protein and restrictin-P content was measured as described above. Protein samples were further purified by RP-HPLC.
• the biologically active fractions from the gel filtration step (3.0-3.5 mg protein) were pooled and loaded onto an RP-300 column (7 ⁇ , 4.6 x 100 mm) and the column was washed with an aqueous solution of 5% ACN in 0.1% TFA, at a flow rate of 0.5 ml/min. Proteins were then eluted with a multi-step linear gradient of aqueous ACN in 0.1% TFA. The gradient composition was 5-25%> ACN/5 minutes followed by 25-40%o ACN/30 min, and then 40-80% ACN/2 min and an isocratic step of 80% ACN. Fractions of 0.5 ml were collected every minute. Elution of proteins was followed by their absorbency in 214 nm.
• Biologically active fractions were pooled, vacuum dried and rechromatographed (RP-HPLC) under essentially identical conditions, except for the following minor modification: when the protein peak started to elute (usually at ca. 36% ACN) the gradient program was put on "hold". When the protein peak was completely eluted from the column (within ca. 5-8 min) the original gradient program was resumed.
• Table 1 Target cell range of restrictin-P activity in conditioned media of the MBA-2.1 stromal cell line
• Partially purified conditioned medium from MBA-2.1 cells was added in the concentration indicated. Cultures were incubated for four days and viability was measured by the MTT colorimetric assay. Values represent the mean of triplicates + SD.
• IL-7, H-10, IL-1 1, M-CSF, G-CSF, TGF ⁇ l, PDGF, bFGF, JTN- ⁇ and LU were tested over a range of concentrations.
• These cytokines were found to be devoid of the ability to inhibit the growth of the MPC-1 1 plasmacytoma which is highly sensitive to restrictin-P (Zipori et al., 1986).
• neutralizing antibodies to TGF ⁇ l and ⁇ 2, TNF, IL6, DrN- ⁇ and M-CSF did not reduce restrictin-P -like activity in media conditioned by MBA-2.1 cells (results not shown).
• the above conditioned media were used in an attempt to obtain some clue as to the mechanism by which the inhibition of plasma-like cells is mediated.
• the MBA-2.1 cells conditioned medium inhibited the growth of the MPC-1 1 plasmacytoma and, as detailed below, it also interfered with the growth promoting effect of IL-6 on B9 cells.
• FIGs. 1A-D broken lines indicate the absorption at 280 nm (Figs. 1A and IB) or 214 nm (Figs. 1C and ID); open circles and thick lines show the biological activity of restrictin-P as measured by the MPC-11 assay and thin lines indicate percentage of acetonitrile (Figs. 1C and D).
• EXAMPLE 3 Inhibition of MPC-11 plasmacytoma cell growth bv purified restrictin- P and bv recombinant activin A.
• a similar experiment was set with recombinant human EL-11. B9 cells were seeded with serial dilutions of this cytokine (Fig. 4B. open symbols) or with EL-1 1 and 0.018 units/well of restrictin-P (Fig. 4B, closed svmbols). This low concentration of restrictin-P is was used since IL-l 1 was a poor
• SUBSTITUTE SHEET (RULE 2G stimulator of B9 cells and a ' relatively low concentration of restrictin-P was sufficient to cause complete growth inhibition. Cells were incubated for 48 hours and then pulsed with
• IL-l 1 is an additional stimulator of plasma-like cells (Burger and Gramatzaki, 1993).
• restrictin-P also inhibited the growth of IL-l 1 stimulated B9 cells and this inhibition was competed out by increasing the titer of IL-l 1.
• restrictin-P counteracted the growth stimulating effect of IL-6 and IL-l 1 and these cytokines at high titers overcame the effect of restrictin-P. It therefore seems that the growth factors EL-6 and IL-l 1 and restrictin-P are competing on some target machinery used to generate a signaling pathway.
• a candidate target molecule for restrictin-P action was the IL-6 receptor complex.
• restrictin-P is a receptor antagonist by testing its ability to compete with radiolabled IL-6 for binding to its receptor on the surface of B9 cells.
• the amount of r-Mu-[ 125 I]-IL-6 or r-Hu-[ 125 I]-IL-6 Muteine used (4.8xl0 4 and l. lxlO 5 cpm, respectively) was 30-40%) of the amount that gave saturation binding.
• B9 cells were weaned from IL-6 in the growth medium 24 hours before binding assay. Under these conditions, the cells have 2070+530 high affinity binding sites and both r-Mu-E -6 and r-Hu- L-6 bound with high affinity (apparent Kd's of 5.5x10" ⁇ and l. lxl0 ⁇ l° M, respectively).
• Fig. 5 A shows specific high affinity binding of a constant amount of r-Mu-[ ⁇ 2 ⁇ I]-IL-
• Fig. 5B shows specific high affinity binding of a constant amount of r-Hu-[ 12 ⁇ I]-IL-6
• EXAMPLE 6 Restrictin-P interferes with the IL-6 induced secretion of the acute phase proteins ⁇ -acid glvcoprotein (AGP) and haptoglobin (HG).
• AGP ⁇ -acid glvcoprotein
• HG haptoglobin
• HepG2 hepatoma releases acute phase proteins under the influence of IL-6.
• HepG2 cells were grown to confluence and stimulated with 100 units/ml of IL-6, or an equal amount of IL-6 with 312 units/ml restrictin-P.
• Conditioned media were collected at 24 hr, subjected to PAGE (10%) and 7% gels for AGP and HG. respectively) and were tested by Western blotting for the acute phase proteins using the corresponding antibodies (purchased from Sigma, Israel).
• the secretion of both AGP and HG, induced in HepG2 cells by IL-6 was markedly reduced by addition of restrictin-P.
• JAK Janus Kinases
• STAT signal transducers and activators of transcription
• EXAMPLE 8 Restrictin-P interferes with IL-6 augmented mitochondrial activity in Ml veloma cells.
• Ml cells were seeded at 5x10 4 /ml in microliter plates with the addition of restrictin- P and/or JX-6 as ndicated in Table 2.
• Restrictin-P was added at 130 units/well and EL-6 at 50 units/well. Under these conditions, no net change in cell number occurred at day 3 when cultures were tested by the MTT colorimetric assay (Materials and Methods, section (ii)). Data shown are from one out of 3 experiments performed, all showing similar results. The experiments indicate that restrictin-P interferes with the IL-6 mediated mitochondrial activity of Ml myeloblasts.

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