WO1994003055A1 - Producing increased numbers of hematopoietic cells by administering inhibitors of dipeptidyl peptidase iv - Google Patents

Producing increased numbers of hematopoietic cells by administering inhibitors of dipeptidyl peptidase iv Download PDF

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WO1994003055A1
WO1994003055A1 PCT/US1993/007173 US9307173W WO9403055A1 WO 1994003055 A1 WO1994003055 A1 WO 1994003055A1 US 9307173 W US9307173 W US 9307173W WO 9403055 A1 WO9403055 A1 WO 9403055A1
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inhibitor
boropro
cells
pro
mammal
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PCT/US1993/007173
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French (fr)
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Laszlo Takacs
Lynn A. Bristol
William W. Bachovchin
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The Government Of The United States Of America, Asrepresented By The Secretary Of The Department Of Health And Human Services
Tufts University
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Priority to AU47943/93A priority Critical patent/AU4794393A/en
Publication of WO1994003055A1 publication Critical patent/WO1994003055A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes

Definitions

  • the present invention relates to methods of producin increased numbers of hematopoietic cells through th administration of an inhibitor of dipeptidyl peptidase IV.
  • Dipeptidyl peptidase IV (DP-IV, also known as CD26) is member of a class of proteolytic enzymes known as serin proteases. Found in humans and a variety of animal species, including yeasts, insects, frogs, pigs, and rats, DP-IV i expressed in mammals in the serum, the kidneys, th intestines, and some hematopoietic cells.
  • the hematopoieti cells on which DP-IV has been identified include granulocytes, macrophages, thymocytes, and T-cells.
  • the precise biologica function of this enzyme in mammalian systems has not been wel established. However, it has been shown to play a role i proteolyzing bioactive peptides in other animal systems.
  • DP-IV may play a role i regulating the activity of certain T-cell growth factors.
  • cytokines including IL-2, contain sites which may b susceptible to proteolysis by DP-IV. However, it has not bee shown that these cytokines are actually substrates for DP-IV, nor has it been demonstrated that the biological activity o such cytokines would be altered even after proteolysis by DP IV.
  • DP-IV is known to have a specificity for cleaving Xaa-Pr sequences from the N-terminus of a polypeptide, where Xa ' represents any amino acid used to build proteins in biological systems.
  • Peptides comprising boronic acid analogs of amino acids are also known to be inhibitors of the serine proteases.
  • the alpha-amino boronic acid Pro-boroPro has thus been found to be a potent inhibitor of DP-IV.
  • Inhibitors of DP-IV such as Pro-boroPro can be used to co-stimulate the proliferation of such cells, for example in combination with known cytokines. Such combinations stimulate cell growth more effectively than cytokines alone. Research into the immune system will be benefitted in particular, since many immune system functions are mediated by hematopoietic cells.
  • the present invention is also useful in treating a variety of disorders.
  • Patients with AIDS for example, suffer from depressed T-cell populations. These patients would therefore benefit from the administration of inhibitors of DP- IV, which can stimulate the proliferation of thymocytes such as T-cells.
  • the present invention will be of particular use in treating patients undergoing bone marrow transplants.
  • an inhibitor of DP-IV By administering an inhibitor of DP-IV, the population of hematopoietic cells transplanted in such a procedure can be expanded in the donor before being transplanted, in vitro after being removed from the donor, and in the recipient after transplantation. The chances that the procedure will be a success are thereby increased by the use of inhibitors of DP- IV.
  • One method of producing increased numbers of hematopoietic cells is to remove such cells from a mammal and administer to those cells an effective dose of an inhibitor o DP-IV, such as Pro-boroPro, along with other factors in vitro.
  • This method can be used to stimulate the growth of cells use in laboratory cultures.
  • Such cells can also be transplante into another mammal of the same species for experimental o therapeutic purposes.
  • hematopoiet c cells ar removed from a mammal in need of increased numbers o hematopoietic cells, co-stimulated with an effective dose o an inhibitor of DP-IV and a growth factor such as a cytokine, and then reintroduced into the same mammal. In this way, th hematopoietic and/or immune system of the mammal i strengthened.
  • a mammal with deficiency of hematopoietic cells is identified and a inhibitor of DP-IV, such as Pro-boroPro, is administered t that mammal directly.
  • the inhibitor of DP-IV is administered with an acceptable pharmaceutical carrier.
  • an inhibitor can be administered orally, intravenously, intraperitoneally, intramuscularly, or in any othe appropriate fashion.
  • Inhibitors of DP-IV are normally used in conjunction wit proliferative agents or growth factors such as cytokines t co-stimulate the proliferation of hematopoietic cells.
  • cytokines t co-stimulate the proliferation of hematopoietic cells.
  • IL-1, IL-2, IL-3, GM-CSF, or erythropoietin can be used along with inhibitors of DP-IV.
  • Pro-boroPro When Pro-boroPro is applied to cell cultures in vitro, it has been found that such growth factors are required to stimulate cell proliferation.
  • the use of a combination of a growth factor and an inhibito of DP-IV has the benefit of reducing the dosage of each agen required to produce increased numbers of cells compared t using any of these agents alone. Thus, the side-effects produced by using these agents alone can be reduced o avoided.
  • Figure 1 is a graph showing the results of an experimen testing the effect of Pro-boroPro on the number of thymocytes in a rat thymus, where Pro-boroPro is administered to th thymuses of embryonic rats in their 17th day of gestation an to the thymuses of newborn rats.
  • Figure 2 is a graph showing the results of an experimen testing the effect of Pro-boroPro on the hematopoieti precursors of granulocyte and macrophage cells from the bon marrows of rats.
  • th Xaa-boroPro compounds Lys-boroPro, Arg-boroPro, His-boroPro, Asp-boroPro, Glu-boroPro, Gly-boroPro, Asn-boroPro, Gln boroPro, Cys-boroProd, Ser-boroPro, Thr-boroPro, Tyr-boroPro, Ala-boroPro, Val-boroPro, Leu-boroPro, Ileu-boroPro, Phe boroPro, Met-boroPro, and Trp-boroPro are within the scope o the present invention.
  • Other inhibitors of DP-IV includedin other serine protease inhibitors, can be identified by one o skill in the art by screening such compounds, a matter o routine experimentation.
  • Pro-boroPro is relatively unstable at physiological pH, having a half-life of approximately 1.5 hours. However, i has been found that Pro-boroPro may be stored indefinitely a low pH. Preferably, Pro-boroPro is stored in solution at p 4.0.
  • the hematopoietic cells used in the methods of th present invention include thy us-derived cells, macrophages, granulocytes, and other hematopoietic cells. As shown i Figure 1, thymocytes isolated from newborn and 17-da embryonic rat thymuses exhibit marked proliferation i response to treatment with Pro-boroPro.
  • the thymuses of 17 day embryonic rats contain primarily CD4-/CD8- (double negative, or DN) thymocytes, while the thy ocytes in the thymuses of newborn rats contain a mixed population of DN cells, CD4+/CD8+ thymocytes, and mature CD4+/CD8- or CD8+/CD4- cells.
  • the preferred dose of Pro-boroPro in these cultures is approximately 10 " ° moles per liter in vitro, although Pro- boroPro in the range of 10 "7 moles per liter to 10 "6 moles per liter in vitro is efficacious.
  • Granulocyte and macrophage precursors also respond to the DP-IV inhibitor Pro-boroPro.
  • Bone marrow cells from Buffalo rats were treated in vitro with Pro-boroPro.
  • the results, as seen in Figure 2, show that the number of granulocyte and macrophage colonies increased in response to stimulation by Pro-boroPro and spleen supernatant, a source of growth factors.
  • the preferred dose of Pro-boroPro for macrophage colonies is 10 "6 moles per liter, although treatments in the range of 10 "8 moles per liter to 10 "7 moles per liter also stimulate proliferation.
  • 10 "7 to 10 "4 moles per liter of Pro-boroPro may be used, with a preferred range of 10 "5 to 10 "4 moles per liter.
  • granulocytes and macrophages have been found to require, in addition to Pro-boroPro, a growth factor such as a cytokine for proliferation to occur.
  • a growth factor such as a cytokine for proliferation.
  • the combination of Pro- boroPro and another growth factor stimulates proliferation to a significantly greater extent compared to the use of that growth factor alone (see Figure 2) .
  • a mammal in need of increased numbers of hematopoietic cells can be benefitted by the methods of the present invention.
  • a person with AIDS for example, is deficient in T- cells.
  • hematopoietic cells such as T-cells, and bone marrow-derived precursors can be removed, co-stimulated to proliferate by the administration of Pro- boroPro and other factors, and then reintroduced into the person from whom they were taken, thus strengthening that person's immune system and fostering hematopoiesis.
  • the removal of such cells can be accomplished by any of a number of techniques known to the art, depending on the cell type to be extracted. Once removed, the cells can be stimulated to proliferate according to the protocols of Examples 1 or 2. Cells grown up in this way can also be transplanted into another mammal of the same species in order to treat that mammal.
  • a mammal can also be treated for disease through the direct administration of inhibitors of DP-IV to that mammal.
  • inhibitors of DP-IV for example, Pro-boroPro or another suitable inhibitor can be injected directly into the bloodstream of a mammal in an appropriate pharmaceutical carrier, or can be administered orally.
  • a mammal suffering from a deficiency of hematopoietic cells can also be treated using the methods of the present invention.
  • Such a mammal must first be identified, which can be accomplished through any of a number of methods known to the art. For example, a blood sample can be taken and then tested to determine whether the subject is deficient in T- cells or other hematopoietic cells carried in the blood. If the subject is found to be deficient in such cells, Pro- boroPro may be administered to that subject by any of the methods described above.
  • Example 1 To investigate the effect of the DP-IV inhibitor Pro- boroPro on thymocytes, thymic lobes were removed from embryonic Buffalo rats (Charles River) on the 17th day of gestation and from newborn Buffalo rats. Single lobes were prepared by dissecting the two thymic lobes of each thymus.
  • the lobes from the embryonic rats were placed onto a 0.8cm x 0.8cm x 0.8cm triangular, tissue culture quality polycarbonat membrane (Neuroprobe) and the membrane was placed over culture medium comprising 45% RPMI 1640 (ABI) , 45% Click' medium (Irvine Scientific Products) , and 10% steroid fre fetal calf serum (Hyclone) with a bicarbonate buffer.
  • culture medium comprising 45% RPMI 1640 (ABI) , 45% Click' medium (Irvine Scientific Products) , and 10% steroid fre fetal calf serum (Hyclone) with a bicarbonate buffer.
  • the lobes were cultured in a humidified incubator at 37° with 5% C0 2 for 5 days.
  • Pro-boroPro was administered at th initiation of the culture and once each day thereafter for th five days of culture.
  • the Pro-boroPro was obtained from Dr. William Bachovchin at Tufts University (136 Harrison Avenue, Boston, MA 02111) and had a half life of about 1.5 hours at physiological pH.
  • the lobes were made into a suspension of single cells at the end of the culture and the number of cells in each individual lobe was counted.
  • the results, plotted in Figure 1 show that the stimulated thymus lobes treated with Pro-boroPro contained almost double the number of thymocytes in the untreated lobes.
  • the optimal dose of Pro-boroPro was 10 " ° moles per liter for both newborn and I7th-day gestational rat thymocytes under the conditions described above.
  • DP-IV inhibitors were administered to bone marrow cells containing granulocyte and macrophage precursor cells.
  • Unfractionated bone marrow cells were removed from Buffalo rats (Charles River) and plated at a density of 5 x 10 4 cells per 2 ml medium (QBSF-56, Quality Biologicals, Inc.) containing 0.8% methylcellulo ⁇ e (Fluka) , 3% conditioned spleen supernatant (as a source of growth factors) , and 20% fetal calf serum (FCS, Hyclone) in 3.5 cm plastic petri dishes.
  • the spleen cell supernatant was produced by stimulating Buffalo rat spleen cells (5 x 10 6 / ⁇ .l) with 5 micrograms/ml Con A (Sigma) in RPMI 1640 (ABI) medium -
  • the bone marrow cells were cultured both with and withou Pro-boroPro.
  • Pro-boroPro was administered to th cultures, only one treatment was given, and varyin concentrations of Pro-boroPro were used. Colonies were grow at 37°C in a humidified incubator with 5% C0 2 and then counte on the eighth day of culture. The results are shown in Figur 2 in terms of the mean number of colonies in triplicat plates. As may be seen, the number of macrophage colonie increased by about 70% at the optimal dosage of Pro-boroPr for acrophages, while the number of granulocyte cells mor than doubled at the optimal dosage for granulocytes.
  • the optimal dose of Pro-boroPro under the above conditions is 10 "6 moles per liter, although doses of 10 "8 t 10 "7 moles per liter are also effective in producin proliferation compared to the administration of no Pro- boroPro.
  • the preferred range of Pro-boroPro is 10 "5 to 10 "4 moles per liter administered under the conditions described above, although some increase in the number of colonies was observed at a dosage of as little as 10 "7 moles per liter.
  • Example 3 The stimulated bone marrow-derived precursor cells produced in Example 2 above are separated from other bone marrow components through centrifugation at 1200 rpm for 5 minutes and then resuspended in 0.15 M saline solution.
  • the major histocompatibility (MHC) antigens of these cells are cross-matched to the MHC antigens of rats which are possible transplantation recipients of such cells.
  • a compatible recipient is selected, and the stimulated bone marrow derived cells are then transplanted into the selected rat through intravenous injection.
  • Example 4 The stimulated bone marrow-derived precursor cells produced in Example 2 above are separated from other bone marrow components through centrifugation at 1200 rpm for 5 minutes and then resuspended in 0.15 M saline solution.
  • MHC major histocompatibility
  • Hematopoietic cells in the blood ar removed from a human in need of increased numbers of suc cells. Such cells are separated from the blood an concentrated through centrifugation, and are then put i suspension in an appropriate buffer, such as 0.15 M saline. An amount of Pro-boroPro which is effective to co stimulate the proliferation of such cells in combination wit a growth factor such as a cytokine is then administered to th cell suspension.
  • an amount of Pro-boroPro which is effective to co stimulate the proliferation of such cells in combination wit a growth factor such as a cytokine is then administered to th cell suspension.
  • enough Pro boroPro is added to bring the concentration of Pro-boroPro i the suspension to between 10 "8 and 10 "A moles per liter.
  • the concentration of Pro-boroPro is about 10 ' moles per liter.
  • a cytokine such as IL-2 or GM-CSF is als added to the suspension at a concentration of about 1,000 BRM units/ml.
  • the suspension is treated once with Pro-boroPro an then cultured for up to eight days at 37 °C in a humidifie incubator with 5% C0 2 to allow the cells to proliferate.
  • the cells thus stimulated are the reintroduced into the subject through the injection of th cells intravenously.
  • the number of DP-IV-expressing cells i the subject is thereby increased compared to the number o such cells in the subject before the foregoing procedure.
  • Example 5 Thymocytes are removed from a child with a functional thymus. Pro-boroPro is then administered to these cells as i Example 1 along with a growth factor such as a cytokine i order to stimulate the proliferation of such cells.
  • a growth factor such as a cytokine i order to stimulate the proliferation of such cells.
  • the cells are suspended in a solution suitabl for administration to the child and then reintroduced into th child through injection.
  • Example 7 Hematopoietic cells are directly stimulated to proliferate in a human subject through the administration of Pro-boroPro. Between 1 mg/kg and 10 mg/kg of Pro-boroPro is suspended in an appropriate volume of a pharmaceutically acceptable solution. This solution is then administered intravenously at least once daily in order to allow the production of increased numbers of such cells. An oral formulation of Pro-boroPro as described above can also be administered.
  • Example 8 An agent suspected of being an inhibitor of DP-IV is screened for its ability to inhibit that enzyme, as described in "Thymocyte Co-stimulating Antigen is CD26 (Dipeptidyl- peptidase IV) : Co-stimulation of Granulocyte, Macrophage and T-lineage Cell Proliferation via CD26," J. Immunol. , 149:367 (1992) .
  • DP-IV is first purified from bone marrow cells harvested by aspirating the femurs and tibias of rats with HBSS using an 18 gauge needle.
  • DP-IV can be purified from the lamina limbal, the kidney, or the liver.
  • lysis buffer 0.1 M Tris, pH 7.2, 0.15 M NaCl, 2% volume/volume Triton X-100 and 1.8 mg/ml iodoacetamide
  • lysis buffer 0.1 M Tris, pH 7.2, 0.15 M NaCl, 2% volume/volume Triton X-100 and 1.8 mg/ml iodoacetamide
  • the lysate is collected and diluted in 0.1 M Tris, pH 7.2 , 0.15 M NaCl to a final concentration of 0.5% Triton X-100.
  • ConA-Sepharose supplied by Pharmacia, Inc., Piscataway, New Jersey
  • 20 ml ConA-Sepharose supplied by Pharmacia, Inc., Piscataway, New Jersey
  • ConA-Sepharose column is washed extensively with a solution of 0.1 M Tris, pH 7.2, 0.5% volume/volume Triton X-100, and 0.15 M NaCl at 4°C, and the material bound to the column is eluted with 5 mg/ml alpha- methyl mannoside in 0.5% Triton X-100 lysis buffer.
  • the eluted material in solution which contains the DP-IV enzyme, can be used in a competition assay, as described below, by adding a substrate of DP-IV and a suspected DP-IV inhibitor to this solution and then detecting the rate of substrate cleavage. If it is desired to further purify the DP-IV from the material eluted from the Sepharose column, however, the eluted material can be be purified by any of a number of methods known to the art. One way to further purify the DP-IV in solution is to pass the solution over a Sepharose immunoaffinity column conjugated with an antibody specific for DP-IV. Obtaining such antibodies is known to those of skill in the art.
  • any of a number of competition assays can be performed, as is known to those of skill in the art (see, e.g., "Inhibition of dipeptidyl aminopeptidase IV (DP-IV) b Xaa-boroPro dipeptides and use of these inhibitors to examin the role of DP-IV in T-cell function", Proc. Natl. Acad. Sci USA, February, 1991, vol. 88, pp.
  • variou concentrations of the suspected DP-IV inhibitor e.g., 0.0 mM 0.01 mM, 0.02 mM, 0.04 mM, 0.1 mM, and 0.2 mM
  • the degree of inhibition of substrate cleavage by th suspected DP-IV inhibitor is then determined by comparing th rate of substrate cleavage by DP-IV in the presence of th suspected inhibitor to the rate of substrate cleavage when n inhibitor is present. If the rates of substrate cleavage b DP-IV in the solutions containing the suspected DP-I inhibitor are not statistically different from the rate o cleavage in the solution containing no inhibitor, then th screened compound is not a DP-IV inhibitor.

Abstract

Methods of producing increased numbers of hematopoietic cells by administering an inhibitor of dipeptidyl peptidase IV to such cells.

Description

PRODUCING INCREASED NUMBERS OF HEMATOPOIETIC CELLS BY ADMINISTERING INHIBITORS OF DIPEPTIDYL PEPTIDASE IV
Field of the Invention The present invention relates to methods of producin increased numbers of hematopoietic cells through th administration of an inhibitor of dipeptidyl peptidase IV.
Background of the Invention Dipeptidyl peptidase IV (DP-IV, also known as CD26) is member of a class of proteolytic enzymes known as serin proteases. Found in humans and a variety of animal species, including yeasts, insects, frogs, pigs, and rats, DP-IV i expressed in mammals in the serum, the kidneys, th intestines, and some hematopoietic cells. The hematopoieti cells on which DP-IV has been identified include granulocytes, macrophages, thymocytes, and T-cells. The precise biologica function of this enzyme in mammalian systems has not been wel established. However, it has been shown to play a role i proteolyzing bioactive peptides in other animal systems.
The presence of DP-IV on the surface of som hematopoietic cells, and in particular on T-cells, has led t speculation that this enzyme might be involved in th functioning of the immune system. In one study, it wa reported that administering an inhibitor of DP-IV to murine T cells inhibits the antigen-induced proliferation of suc cells, as well as the interleukin-2 (IL-2) production of thos cells. (Flentke, George R. , et al., Proc. Natl. Acad. Sci. , USA 88, 1556-1559 (1991)).
It has also been suggested that DP-IV may play a role i regulating the activity of certain T-cell growth factors. Several cytokines, including IL-2, contain sites which may b susceptible to proteolysis by DP-IV. However, it has not bee shown that these cytokines are actually substrates for DP-IV, nor has it been demonstrated that the biological activity o such cytokines would be altered even after proteolysis by DP IV.
DP-IV is known to have a specificity for cleaving Xaa-Pr sequences from the N-terminus of a polypeptide, where Xa 'represents any amino acid used to build proteins in biological systems. Peptides comprising boronic acid analogs of amino acids are also known to be inhibitors of the serine proteases.
The alpha-amino boronic acid Pro-boroPro has thus been found to be a potent inhibitor of DP-IV.
Summary of the Invention In spite of prior art teachings that inhibitors of DP-IV suppress the proliferation of T-cells, the surprising discovery has been made that such inhibitors can in fact contribute to the production of increased numbers of hematopoietic cells (cells derived from multipotent hematopoietic stem cells) .
This discovery will facilitate research involving hematopoietic cells. Inhibitors of DP-IV such as Pro-boroPro can be used to co-stimulate the proliferation of such cells, for example in combination with known cytokines. Such combinations stimulate cell growth more effectively than cytokines alone. Research into the immune system will be benefitted in particular, since many immune system functions are mediated by hematopoietic cells.
The present invention is also useful in treating a variety of disorders. Patients with AIDS, for example, suffer from depressed T-cell populations. These patients would therefore benefit from the administration of inhibitors of DP- IV, which can stimulate the proliferation of thymocytes such as T-cells.
The present invention will be of particular use in treating patients undergoing bone marrow transplants. By administering an inhibitor of DP-IV, the population of hematopoietic cells transplanted in such a procedure can be expanded in the donor before being transplanted, in vitro after being removed from the donor, and in the recipient after transplantation. The chances that the procedure will be a success are thereby increased by the use of inhibitors of DP- IV.
One method of producing increased numbers of hematopoietic cells is to remove such cells from a mammal and administer to those cells an effective dose of an inhibitor o DP-IV, such as Pro-boroPro, along with other factors in vitro. This method can be used to stimulate the growth of cells use in laboratory cultures. Such cells can also be transplante into another mammal of the same species for experimental o therapeutic purposes.
In a preferred embodiment, hematopoiet c cells ar removed from a mammal in need of increased numbers o hematopoietic cells, co-stimulated with an effective dose o an inhibitor of DP-IV and a growth factor such as a cytokine, and then reintroduced into the same mammal. In this way, th hematopoietic and/or immune system of the mammal i strengthened.
In another preferred embodiment, a mammal with deficiency of hematopoietic cells is identified and a inhibitor of DP-IV, such as Pro-boroPro, is administered t that mammal directly. Preferably, the inhibitor of DP-IV is administered with an acceptable pharmaceutical carrier. Suc an inhibitor can be administered orally, intravenously, intraperitoneally, intramuscularly, or in any othe appropriate fashion.
Inhibitors of DP-IV are normally used in conjunction wit proliferative agents or growth factors such as cytokines t co-stimulate the proliferation of hematopoietic cells. Fo example, IL-1, IL-2, IL-3, GM-CSF, or erythropoietin can be used along with inhibitors of DP-IV. When Pro-boroPro is applied to cell cultures in vitro, it has been found that such growth factors are required to stimulate cell proliferation. The use of a combination of a growth factor and an inhibito of DP-IV has the benefit of reducing the dosage of each agen required to produce increased numbers of cells compared t using any of these agents alone. Thus, the side-effects produced by using these agents alone can be reduced o avoided. Description of the Drawings
Figure 1 is a graph showing the results of an experimen testing the effect of Pro-boroPro on the number of thymocytes in a rat thymus, where Pro-boroPro is administered to th thymuses of embryonic rats in their 17th day of gestation an to the thymuses of newborn rats.
Figure 2 is a graph showing the results of an experimen testing the effect of Pro-boroPro on the hematopoieti precursors of granulocyte and macrophage cells from the bon marrows of rats.
Detailed Description of the Invention The methods of the present invention involv administering inhibitors of DP-IV to hematopoietic cells i order to produce increased numbers of such cells. It has bee found that Pro-boroPro, a highly specific transition stag inhibitor of DP-IV, works well in the present methods. An other inhibitor of DP-IV, however, is also within the scope o the present invention, including other amino-boronic acid with the formula Xaa-boroPro, where Xaa is any amino acid use in biological systems to build proteins. For example, th Xaa-boroPro compounds Lys-boroPro, Arg-boroPro, His-boroPro, Asp-boroPro, Glu-boroPro, Gly-boroPro, Asn-boroPro, Gln boroPro, Cys-boroProd, Ser-boroPro, Thr-boroPro, Tyr-boroPro, Ala-boroPro, Val-boroPro, Leu-boroPro, Ileu-boroPro, Phe boroPro, Met-boroPro, and Trp-boroPro are within the scope o the present invention. Other inhibitors of DP-IV, includin other serine protease inhibitors, can be identified by one o skill in the art by screening such compounds, a matter o routine experimentation.
Pro-boroPro is relatively unstable at physiological pH, having a half-life of approximately 1.5 hours. However, i has been found that Pro-boroPro may be stored indefinitely a low pH. Preferably, Pro-boroPro is stored in solution at p 4.0.
The hematopoietic cells used in the methods of th present invention include thy us-derived cells, macrophages, granulocytes, and other hematopoietic cells. As shown i Figure 1, thymocytes isolated from newborn and 17-da embryonic rat thymuses exhibit marked proliferation i response to treatment with Pro-boroPro. The thymuses of 17 day embryonic rats contain primarily CD4-/CD8- (double negative, or DN) thymocytes, while the thy ocytes in the thymuses of newborn rats contain a mixed population of DN cells, CD4+/CD8+ thymocytes, and mature CD4+/CD8- or CD8+/CD4- cells. The number of thymocytes recovered from both types of thymic lobes, when exposed to Pro-boroPro once every day for five days, increased compared to the number of thymocytes recovered from lobes not exposed to Pro-boroPro. The preferred dose of Pro-boroPro in these cultures is approximately 10"° moles per liter in vitro, although Pro- boroPro in the range of 10"7 moles per liter to 10"6 moles per liter in vitro is efficacious.
Granulocyte and macrophage precursors also respond to the DP-IV inhibitor Pro-boroPro. Bone marrow cells from Buffalo rats were treated in vitro with Pro-boroPro. The results, as seen in Figure 2, show that the number of granulocyte and macrophage colonies increased in response to stimulation by Pro-boroPro and spleen supernatant, a source of growth factors. When treated once with Pro-boroPro, the preferred dose of Pro-boroPro for macrophage colonies is 10"6 moles per liter, although treatments in the range of 10"8 moles per liter to 10"7 moles per liter also stimulate proliferation. For granulocytes, 10"7 to 10"4 moles per liter of Pro-boroPro may be used, with a preferred range of 10"5 to 10"4 moles per liter.
Unlike the hematopoietic cells in thymic lobes, granulocytes and macrophages have been found to require, in addition to Pro-boroPro, a growth factor such as a cytokine for proliferation to occur. However, the combination of Pro- boroPro and another growth factor stimulates proliferation to a significantly greater extent compared to the use of that growth factor alone (see Figure 2) .
A mammal in need of increased numbers of hematopoietic cells can be benefitted by the methods of the present invention. A person with AIDS, for example, is deficient in T- cells. Using the present methods, hematopoietic cells, such as T-cells, and bone marrow-derived precursors can be removed, co-stimulated to proliferate by the administration of Pro- boroPro and other factors, and then reintroduced into the person from whom they were taken, thus strengthening that person's immune system and fostering hematopoiesis. The removal of such cells can be accomplished by any of a number of techniques known to the art, depending on the cell type to be extracted. Once removed, the cells can be stimulated to proliferate according to the protocols of Examples 1 or 2. Cells grown up in this way can also be transplanted into another mammal of the same species in order to treat that mammal.
A mammal can also be treated for disease through the direct administration of inhibitors of DP-IV to that mammal. For example, Pro-boroPro or another suitable inhibitor can be injected directly into the bloodstream of a mammal in an appropriate pharmaceutical carrier, or can be administered orally.
A mammal suffering from a deficiency of hematopoietic cells can also be treated using the methods of the present invention. Such a mammal must first be identified, which can be accomplished through any of a number of methods known to the art. For example, a blood sample can be taken and then tested to determine whether the subject is deficient in T- cells or other hematopoietic cells carried in the blood. If the subject is found to be deficient in such cells, Pro- boroPro may be administered to that subject by any of the methods described above.
Detailed protocols for the above procedures, as well as other examples of how to practice the present invention, are given in the examples below.
Example 1 To investigate the effect of the DP-IV inhibitor Pro- boroPro on thymocytes, thymic lobes were removed from embryonic Buffalo rats (Charles River) on the 17th day of gestation and from newborn Buffalo rats. Single lobes were prepared by dissecting the two thymic lobes of each thymus. The lobes from the embryonic rats were placed onto a 0.8cm x 0.8cm x 0.8cm triangular, tissue culture quality polycarbonat membrane (Neuroprobe) and the membrane was placed over culture medium comprising 45% RPMI 1640 (ABI) , 45% Click' medium (Irvine Scientific Products) , and 10% steroid fre fetal calf serum (Hyclone) with a bicarbonate buffer. Sinc there may be variations in the degree of development of th various lobes due to differences in the individual ra embryos, the two lobes from each of the thymuses were groupe to reduce such variation. The same procedure was the followed with the thymic lobes from the newborn rats.
The lobes were cultured in a humidified incubator at 37° with 5% C02 for 5 days. Pro-boroPro was administered at th initiation of the culture and once each day thereafter for th five days of culture. The Pro-boroPro was obtained from Dr. William Bachovchin at Tufts University (136 Harrison Avenue, Boston, MA 02111) and had a half life of about 1.5 hours at physiological pH. The lobes were made into a suspension of single cells at the end of the culture and the number of cells in each individual lobe was counted. The results, plotted in Figure 1, show that the stimulated thymus lobes treated with Pro-boroPro contained almost double the number of thymocytes in the untreated lobes. The optimal dose of Pro-boroPro was 10"° moles per liter for both newborn and I7th-day gestational rat thymocytes under the conditions described above. Example 2
To investigate the effect of DP-IV inhibitors on hematopoietic cells which are not thymus-derived, Pro-boroPro was administered to bone marrow cells containing granulocyte and macrophage precursor cells. Unfractionated bone marrow cells were removed from Buffalo rats (Charles River) and plated at a density of 5 x 104 cells per 2 ml medium (QBSF-56, Quality Biologicals, Inc.) containing 0.8% methylcelluloεe (Fluka) , 3% conditioned spleen supernatant (as a source of growth factors) , and 20% fetal calf serum (FCS, Hyclone) in 3.5 cm plastic petri dishes. The spleen cell supernatant was produced by stimulating Buffalo rat spleen cells (5 x 106/κ.l) with 5 micrograms/ml Con A (Sigma) in RPMI 1640 (ABI) medium -
for 48 hours at 37°C. The Con A was removed by repeate passage of the supernatant over thyroglobulin-agarose column (Sigma) .
The bone marrow cells were cultured both with and withou Pro-boroPro. When Pro-boroPro was administered to th cultures, only one treatment was given, and varyin concentrations of Pro-boroPro were used. Colonies were grow at 37°C in a humidified incubator with 5% C02 and then counte on the eighth day of culture. The results are shown in Figur 2 in terms of the mean number of colonies in triplicat plates. As may be seen, the number of macrophage colonie increased by about 70% at the optimal dosage of Pro-boroPr for acrophages, while the number of granulocyte cells mor than doubled at the optimal dosage for granulocytes. Fo macrophages, the optimal dose of Pro-boroPro under the above conditions is 10"6 moles per liter, although doses of 10"8 t 10"7 moles per liter are also effective in producin proliferation compared to the administration of no Pro- boroPro. For granulocytes, the preferred range of Pro-boroPro is 10"5 to 10"4 moles per liter administered under the conditions described above, although some increase in the number of colonies was observed at a dosage of as little as 10"7 moles per liter.
Example 3 The stimulated bone marrow-derived precursor cells produced in Example 2 above are separated from other bone marrow components through centrifugation at 1200 rpm for 5 minutes and then resuspended in 0.15 M saline solution. The major histocompatibility (MHC) antigens of these cells are cross-matched to the MHC antigens of rats which are possible transplantation recipients of such cells. A compatible recipient is selected, and the stimulated bone marrow derived cells are then transplanted into the selected rat through intravenous injection. Example 4
Hematopoietic cells in the blood, such as T-cells, ar removed from a human in need of increased numbers of suc cells. Such cells are separated from the blood an concentrated through centrifugation, and are then put i suspension in an appropriate buffer, such as 0.15 M saline. An amount of Pro-boroPro which is effective to co stimulate the proliferation of such cells in combination wit a growth factor such as a cytokine is then administered to th cell suspension. In a preferred embodiment, enough Pro boroPro is added to bring the concentration of Pro-boroPro i the suspension to between 10"8 and 10"A moles per liter. Preferably, the concentration of Pro-boroPro is about 10' moles per liter. A cytokine such as IL-2 or GM-CSF is als added to the suspension at a concentration of about 1,000 BRM units/ml. The suspension is treated once with Pro-boroPro an then cultured for up to eight days at 37 °C in a humidifie incubator with 5% C02 to allow the cells to proliferate.
After proliferation, the cells thus stimulated are the reintroduced into the subject through the injection of th cells intravenously. The number of DP-IV-expressing cells i the subject is thereby increased compared to the number o such cells in the subject before the foregoing procedure.
Example 5 Thymocytes are removed from a child with a functional thymus. Pro-boroPro is then administered to these cells as i Example 1 along with a growth factor such as a cytokine i order to stimulate the proliferation of such cells. Followin this treatment, the cells are suspended in a solution suitabl for administration to the child and then reintroduced into th child through injection.
Example 6 Human subjects suffering from any disease or diseas state, or from a deficiency of hematopoietic cells, for who increased hematopoietic activity or increased T-cell production would be beneficial are first identified. Example of such subjects include AIDS patients, patients undergoin chemotherapy treatment, or patients undergoing radiotherap for hematological or other cancers. These subjects are the treated with an inhibitor of DP-IV. For example, they can b treated through the intravenous administration of Pro-boroPr once daily in an appropriate pharmaceutical carrier at a dosage of about 1-10 mg/kg. Alternatively, Pro-boroPro can be administered orally once or several times daily in an appropriate pharmaceutical formulation at a dosage of 1-10 mg/kg per day. Cytokines can also be administered in conjunction with Pro-boroPro to increase the production of hematopoietic cells in a subject.
Example 7 , Hematopoietic cells are directly stimulated to proliferate in a human subject through the administration of Pro-boroPro. Between 1 mg/kg and 10 mg/kg of Pro-boroPro is suspended in an appropriate volume of a pharmaceutically acceptable solution. This solution is then administered intravenously at least once daily in order to allow the production of increased numbers of such cells. An oral formulation of Pro-boroPro as described above can also be administered.
Example 8 An agent suspected of being an inhibitor of DP-IV is screened for its ability to inhibit that enzyme, as described in "Thymocyte Co-stimulating Antigen is CD26 (Dipeptidyl- peptidase IV) : Co-stimulation of Granulocyte, Macrophage and T-lineage Cell Proliferation via CD26," J. Immunol. , 149:367 (1992) . DP-IV is first purified from bone marrow cells harvested by aspirating the femurs and tibias of rats with HBSS using an 18 gauge needle. Alternatively, DP-IV can be purified from the lamina propria of the small intestine, the kidney, or the liver. Many ways of purifying proteins from cells known to the art can be used. One such way is to homogenize the collected cells in lysis buffer (0.1 M Tris, pH 7.2, 0.15 M NaCl, 2% volume/volume Triton X-100 and 1.8 mg/ml iodoacetamide) , incubate the mixture at 4°C for 30 minutes, and then centrifuge it at 400 g for 30 minutes at 4°C. The lysate is collected and diluted in 0.1 M Tris, pH 7.2 , 0.15 M NaCl to a final concentration of 0.5% Triton X-100. The lysate and 20 ml ConA-Sepharose (supplied by Pharmacia, Inc., Piscataway, New Jersey) are then incubated overnight with shaking. Following incubation the ConA-Sepharose column is washed extensively with a solution of 0.1 M Tris, pH 7.2, 0.5% volume/volume Triton X-100, and 0.15 M NaCl at 4°C, and the material bound to the column is eluted with 5 mg/ml alpha- methyl mannoside in 0.5% Triton X-100 lysis buffer.
The eluted material in solution, which contains the DP-IV enzyme, can be used in a competition assay, as described below, by adding a substrate of DP-IV and a suspected DP-IV inhibitor to this solution and then detecting the rate of substrate cleavage. If it is desired to further purify the DP-IV from the material eluted from the Sepharose column, however, the eluted material can be be purified by any of a number of methods known to the art. One way to further purify the DP-IV in solution is to pass the solution over a Sepharose immunoaffinity column conjugated with an antibody specific for DP-IV. Obtaining such antibodies is known to those of skill in the art. After extensive washing of the column with 0.5% Triton X-100 lysis buffer, the antibody specific for DP-IV is eluted from the column with 0.2 M glycine, pH 2.0 containing 0.1% Triton X-100 in 5 ml fractions. The fractions are neutralized to pH 7.6 by the addition of 1 M Tris, pH 7.6. After concentrating the fractions, aliquots from each fraction are analyzed on a 7.5% SDS-PAGE gel. The gel is silver stained and analyzed to determine which fractions contained the DP-IV enzyme. The enzyme-containing fractions are then pooled, concentrated to 200 μl and made to 20% glycerol for storage at -70°C until needed.
Methods of screening inhibitors of DP-IV are known to the art, and any of these can be used in searching for inhibitors of DP-IV (see, e.g., "Diprotins A and B, Inhibitors of Dipeptidyl Aminopeptidase IV, Produced by Bacteria", J. Antibiotics, April, 1984, vol. 37, no. 4, pp. 422-425). In order to test a suspected inhibitor of DP-IV for inhibitory activity, any of a number of competition assays can be performed, as is known to those of skill in the art (see, e.g., "Inhibition of dipeptidyl aminopeptidase IV (DP-IV) b Xaa-boroPro dipeptides and use of these inhibitors to examin the role of DP-IV in T-cell function", Proc. Natl. Acad. Sci USA, February, 1991, vol. 88, pp. 1556-1559) variou concentrations of the suspected DP-IV inhibitor (e.g., 0.0 mM 0.01 mM, 0.02 mM, 0.04 mM, 0.1 mM, and 0.2 mM) are then adde to a solution containing 50 μM sodium Hepes (pH 7.8) , 10 μ Ala-Pro-4-nitroanilide (a substrate of DP-IV) , 6 milliunits o the DP-IV collected as described above, and 2% (volume/volume) dimethylformamide brought to a total volume of 1.0 ml wit water. These solutions are incubated at approximately 25° and are sampled approximately every minute for about 1 minutes to monitor the amount of substrate (Ala-Pro-4 nitroanilide) cleaved by DP-IV. When Ala-Pro-4-nitroanalid is cleaved by DP-IV, para-nitroanaline is formed. Thi compound appears yellow in solution, so the amount of Ala-Pro 4-nitroanilide cleaved can be measured by measuring the colo change of the solution with a spectrometer at approximatel 410 nanometers. The degree of inhibition of substrate cleavage by th suspected DP-IV inhibitor is then determined by comparing th rate of substrate cleavage by DP-IV in the presence of th suspected inhibitor to the rate of substrate cleavage when n inhibitor is present. If the rates of substrate cleavage b DP-IV in the solutions containing the suspected DP-I inhibitor are not statistically different from the rate o cleavage in the solution containing no inhibitor, then th screened compound is not a DP-IV inhibitor.
The foregoing embodiments of the present invention ar illustrative only and do not limit the present invention. Suitable inhibitors of DP-IV and suitable hematopoietic cell not explicitly mentioned herein may be easily identifie through routine experimentation by those skilled in the art.

Claims

WHAT I CLAIM IS :
1. A method of producing increased numbers of hematopoietic cells by administering an inhibitor of dipeptidyl peptidase IV to such cells, comprising the steps of: removing hematopoietic cells from a mammal ; and administering an effective amount of an inhibitor of dipeptidyl peptidase IV to said cells.
2. The method of Claim 1, including the step of transplanting said cells into another mammal of the same species.
3. The method of Claim 1, wherein said inhibitor is Xaa-boroPro, Xaa being any amino acid used to build proteins in biological systems.
4. The method of Claim 3, wherein said inhibitor is Pro-boroPro.
5. The method of Claim 1 above, wherein a growth factor is administered with the inhibitor of DP-IV.
6. The method of Claim 5, wherein said growth factor is selected from the group consisting of IL-1, IL-2, IL-3, Con A, erythropoietin, and GM-CSF.
7. A method of treating a mammal in need of increased numbers of hematopoietic cells, comprising the steps of: removing hematopoietic cells from a mammal; administering an effective amount of an inhibitor of dipeptidyl peptidase IV to said cells; and reintroducing said cells into said mammal.
8. The method of Claim 7, wherein said inhibitor is
Xaa-boroPro, Xaa being any amino acid used to build proteins in biological systems.
9. The method of Claim 8, wherein said inhibitor is Pro-boroPro.
10. The method of Claim 7, wherein a growth factor is administered with the inhibitor of DP-IV.
11. The method of Claim 10, wherein said growth factor is selected from the group consisting of IL-1, IL-2, IL-3, Con A, erythropoietin, and GM-CSF.
12. The method of Claim 7, wherein said mammal is suffering from an immunodeficiency disease such as AIDS.
13. A method of treating a mammal having a deficiency of hematopoietic cells, comprising the steps of: identifying a mammal with a deficiency of hematopoietic cells; and administering to said mammal an effective dose of an inhibitor of dipeptidyl peptidase IV.
14. The method of Claim 13, wherein said inhibitor is Xaa-boroPro, Xaa being any amino acid used to build proteins in biological systems.
15. The method of Claim 14, wherein said inhibitor is
Pro-boroPro.
16. The method of Claim 13, wherein said inhibitor is carried in a pharmaceutically acceptable carrier.
17. The method of Claim 13, wherein a growth factor is administered with said inhibitor.
18. The method of Claim 17, wherein said growth factor is selected from the group consisting of IL-1, IL-2, IL-3, Con A, erythropoietin, and GM-CSF.
19. The method of Claim 13, wherein said administration step is selected from the group consisting of intravenous injection, intraperitoneal injection, and intramuscular injection.
20. A method of producing increased numbers of hematopoietic cells in a mammal, comprising the step of administering to said mammal an effective dose of an inhibitor of dipeptidyl peptidase IV.
21. The method of Claim 20, wherein said inhibitor is Xaa-boroPro, Xaa being any amino acid used to build proteins in biological systems.
22. The method of Claim 20, wherein said inhibitor is
Pro-boroPro.
23. The method of Claim 20, wherein said inhibitor is carried in a pharmaceutically acceptable carrier.
24. The method of Claim 20, wherein a growth factor is administered with said inhibitor.
25. The method of Claim 24, wherein said growth factor is selected from the group consisting of IL-1, IL-2, IL-3, Con A, erythropoietin, and GM-CSF.
26. The method of Claim 20, wherein said administration step is selected from the group consisting of intravenous injection, intraperitoneal injection, and intramuscular injection.
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