MXPA00010348A - Hematopoietic stimulation - Google Patents

Abstract

Methods and products for stimulating hematopoietic, preventing low levels of hematopoietic cells and producing increased numbers of hematopoietic and, mature blood cells are provided. The methods and products can be used both in vivo and in vitro.

Description

HEMATOPOYETIC STIMULATION Background of the Invention The present invention relates to methods and products to produce a greater number of hematopoietic cells, to restore the number of hematopoietic cells to pre-selected normal levels, with therapies to treat deficiencies in hematopoietic cells and with methodologies in vi to cultivate hematopoietic cells. 10 PT-100 is a dipeptide consisting of valin-prolinboronic acid (ValboroPro) designed to interact with the CD26 cell surface receptor. CD26, a transmembrane type II protein is expressed on the cell surface of numerous cell types, including lymphocytes (Marguet, D. et al., Advances in Neuroimmunol., 3: 209-215 (1993)), hematopoietic cells (Vivier, I. et al., J. Immunol., 147: 447-454 (1991) Bristol, et al., J. Immunol., 149: 367 (1992)) thymocytes (Dang, NH et al., J. Immunol 147: 2825-2832 (1991), Tanaka, T. et al., J. Immunol. 149: 481-486 1992), Darmoul, D. et al., J. Biol. Chem. 267: 4824-4833 (1991)), intestinal brush border membrane and endothelial cells. The CD26 associated with the cell surface is a sialoglycoprotein, with most of its mass on the outer side of the cell.

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CD26 has been better characterized on peripheral T cells where it functions as a powerful co-stimulatory signal for the activation of T cells. Its surface expression is up-regulated up to the activation of T cells (Dong, RP et al., Cell 9). : 153-162 (1996), Torimoto Y. et al., J. Immunol., 147: 2514 (1991), Mittrucker, HW et al., Eur. J. Immun. 25: 295-29"(1995), Hafler, DA et al, J. Immunol., 142: 2590-2596 (1989), Dang, NH et al., J. Immunol., 144: 409 (1990)). CD26 has also been identified in rodents as an important regulatory surface receptor in the development of hematopoiesis and lymphoid (Vivier, I. et al., J Immunol., 147: 447-454 (1991)). The primary structure of J CD26 is highly conserved among species (Ogata, S. et al., J. Biol. Chem. 264: 3596-3601 (1998)). In humans, CD26 appears to be involved in the regulation of the activation, differentiation and maturation of thymocytes (Dang, NH et al., J. Immunol 147: 2825-2832 (1991); Kameoka, J. et al. , Blood 85: 1132-1137 (1995)). We have evidence that CD26 is expressed within the human and murine hematopoietic systems. CD26 is an ectoenzyme with activity identical to that of dipeptidyl peptidase IV (DPP-IV), an exopeptidase of the serine type with high substrate specificity. This cleaves the N-terminal dipeptides of proteins if the penultimate amino acid is proline, or in some cases alanine (Fleischer, B. Immunol., Today, 15: 180 (1994)). PT-100 is a potent inhibitor of DPP-IV activity. Published PCT application O94 / 03055 of the prior art teaches methods for producing a greater number of hematopoietic cells by administering DPP-IV inhibitors. The teaching of this published application, however, is that doses of at least 1 mg / kg of body weight are necessary to achieve such increases in hematopoietic cells. This published application also teaches that inhibitors are administered to mammals that have an established deficiency of hematopoietic cells. The teaching also suggests that cytokines be administered in conjunction with the inhibitors to increase the production of hematopoietic cells in a subject.

BRIEF DESCRIPTION OF THE INVENTION The invention is based on a variety of surprising and unexpected findings. It has been discovered, unexpectedly, that the agents useful according to the invention stimulate the production of growth factor by the stromal cells. It has also been discovered, unexpectedly, that the useful agents according to the invention stimulate cell proliferation.b-FAMMS &K ^ primitive hematopoietic progenitors, but does not stimulate! directly the differentiation or proliferation of the involved progenitor cells. It has also been discovered, unexpectedly, that the agents useful according to the invention can be administered at much lower doses than would have been expected according to the teachings of the prior art. Another unexpected discovery is that the agents according to the invention can accelerate the time it takes for the hematopoietic cells to recover after treatment with a hematopoietic cell inhibitor. Another unexpected finding is that the agents useful according to the invention can, at relatively low doses, restore normal levels of neutrophils at least as fast as the most successful commercially available product used worldwide for this purpose, except that the agents Useful according to the invention can be used orally, while the commercially available product (which represents more than one trillion dollars in the market) must be injected. These unexpected results have important experimental research and therapeutic implications. According to one aspect of the invention, there is provided a method for treating a subject to stimulate hematopoiesis in the subject. The invention * < - *. J involves administering to a subject in need of such treatment an amount of an effective agent for increasing the number of hematopoietic cells or mature blood cells in the subject, where the amount is less than 1 mg / kg of body weight per day and wherein the agent is ur compound of Formula I. The useful agents according to the invention are the compounds of Formula I: Formula I where m is an integer between 0 and 10, inclusive; A and Ai are residual L-amino acids (for glycine there is no such distinction) so that the A in each repeated unit, enclosed in brackets, may be a different residual amino acid; the C bonded to B is in the L configuration; the bonds between A and N, Ai and C, and between Ai and N are peptide bonds; and each of Xi and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH. The "C attached to B is in the L configuration" means that the absolute configuration of the C is similar to that of an L-amino acid. In this way, the group \ has the same relation to C as the -COOH group of an L-amino acid has in its carbon a. In some embodiments, A and Ai are independently proline or alanine residues; m is 0; Xi and X2 are hydroxyl groups; the inhibitor is L-Ala-L-boroPro; and the inhibitor is L-Pro-L-boroPro. In an important aspect of the invention, the subject has an abnormally low level of hematopoietic cells or mature blood cells and the agent is administered in an amount effective to restore the levels of a type of hematopoietic cell or a mature blood cell type to a normal level or preselected guard. The agent is preferably administered to the subject in at least 2 doses in a period of 18 hours. The invention has particularly important applications in restoring normal or protective levels of neutrophils, erythrocytes and platelets. The most preferred agent is ValBoroPro. .. &"According to another aspect of the invention, there is provided a method for shortening or eliminating the time in which a subject has an abnormally low level of mature blood or hematopoietic cells resulting from treatment with an inhibitor of hematopoietic cells. . An agent is administered to a subject in need of such treatment in an amount effective to increase the number of hematopoietic cells or mature blood cells in the subject, where the administration of the agent begins before or substantially simultaneously with the administration of the inhibitor of the hematopoietic cells. The agents and preferred agent are as described above. In an important modality, the hematopoietic cell inhibitor produces an abnormally low level of hematopoietic cells or mature blood cells in the subject and the agent is administered in an amount effective to restore the levels of a hematopoietic cell type to a preselected normal or protective level. Preferably, the agent is administered to the subject in at least 2 doses in a period of 18 hours. In important embodiments, the agent is used to restore the normal or protective levels of neutrophils, erythrocytes or platelets of the subject. The preferred effective amount of the agent is as described above.

According to another aspect of the invention, there is provided a method for preparing a subject with treatment with an inhibitor of hematopoietic cells. The method involves administering to the subject before the subject receives the hematopoietic cell inhibitor an agent in an amount effective to stimulate in the subject the production of growth factors. In one embodiment the agent stimulates the production of the growth factor of the stromal cells. The agents and preferred agent are as described above. In an important modality, the growth factor is the stimulating factor of the granulocyte colony. In other embodiments, the growth factor is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-6, IL-11, IL-17, TPO, EPO, MCSF, GMCSF, ligand FLT-3 and undifferentiated cell factor. Preferably, the amount administered to the subject is less than 1 mg / kg of body weight per day. It is also preferred that the administration of the agent be in at least 2 doses of the agent in a period of 18 hours. According to another aspect of the invention there is provided a method for treating a subject to increase the number of hematopoietic cells or mature blood cells in the subject. An agent is administered to a subject in need of such treatment in an amount effective to increase the hematopoietic cells or mature blood cells in the subject, where the agent is administered in a first regimen consisting of 2 doses or 3 doses over a period of time. 18 hours. The agents and preferred agent are as described above. In an important embodiment, the agent is administered in a second regimen consisting of 2 doses or 3 doses in a period of 18 hours, where the second regimen is separated at the time of the first regimen. In another embodiment, the agent is administered in the third regimen consisting of 2 doses or 3 doses in a period of 18 hours, where the third regimen is separated at the time of the first and second regimens. In other embodiments, the agent is optionally administered in a fourth regimen, a fifth regimen, a sixth regimen, or a seventh regimen, wherein each of such regimens consists of 2 doses or 3 doses in a period of 18 hours, wherein the regimes are separated in time from each other by previous regimes. In an important embodiment, the subject has an abnormally low neutrophil count and the amount is effective to reestablish a preselected level of neutrophils in the subject. In other important modalities the subject has abnormally low levels of erythrocytes and platelets. The preferred doses, agents and the like are as described above. In important modalities, the dose is not more than six regimes, no more than five regimes, no more than four regimes, no more than three regimes, and no more than two regimes. According to another aspect of the invention, there is provided a method for preparing the subject ur cells to be reintroduced into a subject. The method involves treating the subject with an agent in an amount effective to stimulate the hematopoietic cells in the subject, then collect the hematopoietic cells of the subject. The collected cells are subsequently reintroduced into the subject. The harvested cells can optionally be cultured ex vivo. The agents and preferred agent are as described above. In one embodiment the ex vivo culture is carried out in the presence of an amount of agent effective to stimulate the proliferation of the harvested cells. In another embodiment, the concentration of the agent in the medium surrounding the harvested cells is less than 10 ~ 8 moles per liter, and less than 10 ~ 9 moles per liter and even less than 10"1C per liter. A method for stimulating the production of growth factor by stromal cells is provided by the method of the invention The method involves contacting the stromal cells with an agent in an amount effective to stimulate the production of growth factor by the stromal cells. agents and the preferred agent are as described above., the stromal cells are in an in vi tro layer of stromal cells to support initial progenitor cell growth and further comprises culturing undifferentiated cells in the presence of those stromal cells. In another embodiment, the stromal cells are in vivo in a subject. In another modality, the growth factor is the stimulating factor of the granulocyte colony. In other embodiments, the growth factor is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-6, IL-11, IL-17, TPO, EPO, MCSF, GMCSF, ligand FLT-3 and Factor of Non Differentiated Cells. In an in vivo embodiment, the agent is administered to a subject in an amount of less than 1 mg / kg of body weight per day. In yet another embodiment, undifferentiated cells are grown in an exogenously added granulocyte colony-stimulating factor-free environment. In important modalities the stromal cells are bone marrow or thymic stromal cells. According to another aspect of the invention, there is provided a kit for treating a subject having an abnormally low level of hematopoietic cells resulting from treatment by the hematopoietic cell inhibitor or to prophylactically treat a subject being treated with a inhibitor of hematopoietic cells to prevent the decrease or loss of mature hematopoietic and / or blood cells. The kit is a package containing a first dose and instructions for treating a subject substantially simultaneously with or prior to treatment with the hematopoietic cell inhibitor. The package also contains a second dose and instructions for treating a subject only after treatment with the hematopoietic cell inhibitor. The doses are in effective amounts and the preferred agents and agent are as described above. In one embodiment, the second dose is between 2 and 5 regimens, each of the regimens consisting of 2 or 3 doses per day of agent. In one embodiment, the dose combination is less than 1 mg / kg of body weight per day. A preferred device is for the treatment of neutropenia. Other preferred equipment are for the treatment of an abnormally low level of erythrocytes or platelets. According to yet another aspect of the invention, equipment is provided for treating a subject having an abnormally low level of hematopoietic cells. The kit is a package that contains a full dose to restore normal levels of a hematopoietic cell type. The package consists essentially of: (1) a first dose in an effective amount to be administered to the subject during a first day, (2) a second dose in an amount effective to be administered to the subject during a second day, (3) optionally, a third dose in an effective amount to be administered to the subject for a third day, (4) optionally, a fourth dose in an effective amount to be administered to the subject for a fourth day, (5) optionally, a fifth dose in an effective amount to be administered when subjected for a fifth day, (6) optionally, a sixth dose in an effective amount to be administered to the subject during a sixth day and, (7) optionally, a seventh dose in an effective amount to be administered to the subject during a seventh day. The agents and preferred agent are as described above. In an important embodiment, each of the doses consists of 2 or 3 doses of the agent to be administered each day. The preferred doses and doses are as described above. In important modalities, the equipment consists essentially of less than 5, less than 4 and less than 3 and even less than 2 doses. These and other aspects of the invention will be described in more detail below. na * Brief Description of the Drawings Figure 1 is a medical package to administer a 5-day medicinal course of treatment to treat myelosuppression or anemia resulting from cancer chemotherapy. Figure 2 is a graph describing the regeneration of neutrophils in mice treated with cyclophosphamide. The PT-100 at the indicated doses or administered in saline solution per dose. Absolute neutrophil counts in mice not treated with cyclophosphamide are above the average of 190 x 10 '' cells / ml as indicated by the discontinuous horizontal line. Figure 3 is a graph describing the regeneration of neutrophils in mice treated with cyclophosphamide in response to subcutaneous administration of PT-100. The saline solution or PT-100 is administered b.i.d. for 5 consecutive days. The absolute neutrophil count in mice treated with cyclophosphamide was 185 x 10 4 cells / ml as indicated by the horizontal dashed line. Figure 4 is a graph describing the regeneration of neutrophils in mice treated with cyclophosphamide in response to PT-100 and the granulocyte colony stimulating factor. The PT-100 in saline was administered per intake and the GSCF by subcutaneous injections, for 5 days. Absolute nitrophil counts in mice not treated with cyclophosphamide average 190 x 104 cells / ml as indicated by the dashed horizontal line. Figure 5 is a graph describing the effect of the dose number of PT-100 on neutrophil regeneration in mice treated with cyclophosphamide. PT-100, at the indicated concentrations, was administered once or twice a day subcutaneously for 5 days. The absolute average neutrophil count for mice not treated with cyclophosphamide was 200 x 10 4 cells / ml as indicated by the dashed horizontal line. Figure 6 is a graph describing the duration of the administration effect of PT-100 on absolute neutrophil count and neutrophil recovery rate in mice treated with cyclophosphamide. PT-100 (5 μg / b.i.d.) was administered to mice treated with cyclophosphamide per dose for an indicated period of time. The discontinuous horizontal line indicates the absolute average neutrophil count for mice not treated with cyclophosphamide. Figure 7 is a graph showing the duration of the effect of PT-100 treatment on neutrophil regeneration in mice treated with cyclophosphamide. The PT-100 (2 μg / b.i.d.), or saline solution were administered per intake for the indicated time. The absolute average neutrophil count for mice not treated with cyclophosphamide was 194 x 104 cells / ml according to or shown by the dotted line. Figure 8 is a graph describing the colony forming ability of cells in response to PT-100 in a long-term culture (LTC) assay. Human bone marrow cells were incubated in LTC for 4 weeks in the absence or presence of the indicated amounts of PT-100, followed by a 2 week culture in semi-solid medium. Figure 9 is a graph showing that PT-100 stimulates hematopoiesis in the spleen of normal mice. Figure 10 is a graph showing that PT-100 stimulates the projection of G-GSF by human stromal cells.

Detailed Description of the Invention The invention involves the stimulation of the proliferation, differentiation and mobilization of hematopoietic cells. The invention is useful when it is desirable to stimulate the proliferation or differentiation, or to mobilize, hematopoietic cells. The mobilization of hematopoietic cells is characterized by the enrichment of early progenitor cells in the bone marrow and the recruitment of those cells in the periphery in response to a mobilizing agent (eg G-GSF, GM-CSF, etc.) . The agents useful according to the invention can be used to inhibit haematopoietic cell deficiencies or to re-establish the hematopoietic or mature blood cell count in subjects with such deficiencies. Such agents can also be used in combination with hematopoietic cell transplants, such as transplants of bone marrow or peripheral blood, when they are used to resupply or create an immune system in a subject. The agents can also be used as an immune enhancer. The agents are also useful in vivo in connection with a cell culture for therapeutic and research uses. As used here, subject means humans, non-human primates, dogs, cats, sheep, goats, horses, cows, pigs and rodents. An important aspect of the invention involves restoring or preventing a deficiency in the number of hematopoietic cells in a subject. Such deficiencies may arise, for example, from genetic abnormalities, from diseases, from tension, from chemotherapy (for example treatment with cytotoxic drugs, treatment with steroid drugs, treatments with immunosuppressive drugs, etc.) and from radiation treatment. The invention is useful in general for restoring deficiencies created by hematopoietic cell inhibitors. An inhibitor of hematopoietic cells is an exogenously applied agent (such as drug treatment or radiation) that produces: a decrease in the subject of hematopoietic cells and / or mature blood cells. Hematopoietic cells as used here, se >; refers to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, and basophils), erythrocytes, reticulocytes, thrombocytes (e.g., megakaryoblasts, plaque-platelet megakaryocytes), lymphocytes, monocytes, dendritic cells, and macrophages. Mature blood cells consist of mature lymphocytes, platelets, erythrocytes, reticulocytes, granulocytes and macrophages. In certain important aspects of the invention, the agents useful according to the invention increase the number of neutrophils, erythrocytes and platelets. In connection with neutrophils, the agents can be used to treat inter- teria, drug-induced neutropenia or radiation, chronic idiopathic neutropenia and cyclic neutropenia. . ^^^ á ^ ^ ^^^ ái? ^^^ j ^^^ M ^ í An important aspect of the invention is to restore levels of "normal" or "protective" hematopoietic cells in a subject. A "normal" level as used herein may be a level in a control population, which preferably includes subjects that have characteristics similar to those of the treated individuals, such as age. The "normal" level can also be a range, for example, where a population is used to obtain a baseline interval for a particular group in which the subject falls. The population can also be divided into groups, such as quadrants, with the lowest quadrant being individuals with the lowest levels of hematopoietic cells and the highest quadrant being individuals that have the highest levels of hematopoietic cells. In this way, the "normal" value may depend on a particular selected population. Preferably, normal levels are those of apparently healthy subjects, who do not have a prior history of hematopoietic cell disorders. Such "normal" levels can then be established as pre-selected values, taking into account the category in which the individual falls. The appropriate ranges and categories can be selected with no more than routine experimentation by those skilled in the art. Any of the mean or other preselected number within the range can be set as the normal pre-selected value. Similarly, the level in a subject before treatment with a hematopoietic cell inhibitor can be used as the predetermined value. In general, the normal range for neutrophils is approximately 1800-7250 per μl (mean -3650); for basophils 0-150 per μl (mean -30); for eosinophils 0-700 per μl (mean -150); for the macrophages and monocytes 200-950 per μl (mean -430); for lymphocytes 1500-4000 per μl (mean -2500); for erythrocytes 4.2 x 10 ° - 6.1 x 106 per μl; and for platelets of 133 x 103 - 333 x 103 μl. The above intervals are at the 95% confidence level. 15 In relation to certain conditions, the medical community has established certain pre-selected values. For example, mean neutropenia is characterized by having a count between 100 and 200 per μl, moderate neutropenia between 500 and 1000 per μl and severe neutropenia below of 500 per μl. Similarly, in adults, a lymphocyte count less than 1500 is considered a medically undesirable condition. In children the value is less than 3000 Other preselected values will be readily known to those skilled in the art. The useful agents of according to the invention can be used for establish or reset such pre-selected values, including normal levels. The protective levels of hematopoietic cells are the number of cells required to confer clinical benefits to the patient. The required levels can be equal to or less than the "normal levels". Such levels are well known to those skilled in the art. For example, a protective level of neutrophils is above 1000, preferably at least 1500. According to another aspect of the invention, the agents useful herein can be applied at lower doses than those described in the prior art. In particular, it has been unexpectedly discovered that the agents of the invention can be administered in doses of less than 1 mg / kg of body weight per day. In particular, the agents of the invention have been used successfully at levels of 0.1 mg / kg of body weight per day, which are of the order of 10 times of magnitude lower than the teachings of the prior art. As will be readily recognized by those skilled in the art, this has the advantage that less material is required for the treatment, thereby decreasing any risk of side effects. Likewise, this has advantages in relation to the manufacturing costs of the pharmaceutical products of the invention.

According to another aspect of the invention, better therapeutic results can be achieved when the agents are applied in multiple doses per day. This discovery is unexpected and, additionally, it has been found that there is no additional medically useful effect when the agents useful according to the invention are administered for prolonged periods of time. In this way, it has been discovered, unexpectedly, that only very short treatment periods are necessary to achieve the established therapeutic goals. As described in the examples below, the subjects treated with the agents useful according to the invention in 2 doses per day against one dose per day achieve a recovery of the hematopoietic cells almost 33% faster than the subjects who received only 1 dose per day. Surprisingly, this result did not depend on the absolute amount of drug given to the subject, but was related to the number of times the drug was administered to the subject. In other words, as shown below, giving twice as much drug, but only once a day, did not accelerate the recovery of the number of hematopoietic cells. Thus, one aspect of the invention involves giving the useful agents according to the invention in 2 or 3 doses over a period of 18 hours. As used herein, a period of 18 refers generally to the time during which a subject is awake in any 24-hour period; it was intended to indicate 2 doses per day, 3 doses per day, and the like. According to another aspect of the invention, it has been discovered, unexpectedly, that the agents useful according to the invention need to be administered less days than expected according to the prior art. In particular, in the mouse models used, there was very little difference in the acceleration of; the recovery of hematopoietic cell counts and the ability to restore normal levels of: hematopoietic cells when the treatment was 3 days, against 4 days, against 5 days. It is believed, therefore, that when applied to humans, a complete drug treatment will involve 7 days or less, more preferably 1 6 days or less, more preferably 5 days or less, more preferably 4 days or less. less, and even more preferably 3 days or less. As a result, the invention therefore provides equipment which contains complete treatment packages for restoring the hematopoietic cell count, which devices are described in more detail below. According to another aspect of the invention, the time a subject has an abnormally low level of hematopoietic cells resulting from treatment with a hematopoietic cell inhibitor is shortened. It has been discovered, unexpectedly, that the agents used according to the invention stimulate the production of the growth factor by the stromal cells. For example, the production of granulocytic colony stimulating factor (GCSF) by the stromal cells is stimulated. The GCSF acts to specifically direct the differentiation of the neutrophilic lineage. This does not affect the differentiation or proliferation of other compromised hematopoietic cells, including other granulocytes, such as eosinophils, basophils, mast cells and macrophages. (It is known how it acts synergistically, however, in vi tro with other cytokines to affect the proliferation of pluripotent mast cells, although the importance in vivo of this observation is not known). Because the stromal cells are not rapidly dividing cells and are generally not adversely impacted by the hematopoietic cell inhibitors, the agents useful according to the invention can be applied to subjects substantially simultaneously with or even earlier of treatment with an inhibitor of hematopoietic cells to stimulate stromal cells to produce growth factor, which will be easily * ^ ^ ^: ^ TsX ^ abundant and will help to generate hematopoietic cells after treatment by the hematopoietic cell inhibitor. In the prior art, such treatment has been delayed until substantially after treatment with the hematopoietic cell inhibitor. Substantially simultaneously, as used herein, means within 24 hours of treatment with the hematopoietic cell inhibitor. Preferably, the agents useful according to the invention are administered within 2 hours of treatment with the hematopoietic cell inhibitor, if administered after treatment with the hematopoietic cell inhibitor. If they are administered before treatment with the hematopoietic cell inhibitor, then they are administered sufficiently close in time to the treatment with the inhibitor, so that the production of the growth factor by the stromal cells increases in the days immediately after treatment with the inhibitor of hematopoietic cells. Another aspect of the invention involves the treatment of a subject to prepare a subject for further treatment with other agents. It has been discovered, unexpectedly, that the agents useful according to the invention stimulate the proliferation of hematopoietic progenitor cells, primitive, not compromised, but not directly the differentiation of committed progenitor cells. It is also known in the art that such cells may or may not include CD34 + cells. CD34 + are immature cells present in blood products, express the cell surface marker CD34, and is believed to include an overpopulation of cells with the ability to self-renew and differentiate into all types of mature blood cells. Because the agents useful according to the invention stimulate the proliferation of such self-renewing cells, the invention is useful for preparing a subject for treatment with other exogenous growth factors and cytokines, which in turn results in differentiation of such progenitor cells not committed to committed progenitor cells. Similarly, the agents useful according to the invention can be administered to a subject to expand the target hematopoietic cells and to mobilize such cells, before extracting the cells from the subject for transplantation or reinfusion. Such cells can be used for research purposes or can be treated ex vivo or reintroduced into the subject with or without in vitro expansion. »-, - ^ - .-- - - ^ - ^^ -; fc ..... - • -" The agents useful according to the invention can be administered in conjunction with growth factors. exogenous and cytokines, which are specifically selected to achieve a particular result. For example, if it is desired to stimulate a particular type of hematopoietic cell, then growth factors and cytokines are used that stimulate the proliferation and differentiation of such cell type. Thus, it is known that interleukins 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, 13 and 17 are involved in the differentiation of lymphocytes. Interleukins 3 and 4 are involved in the differentiation of mast cells. The stimulating factor of the granulocytic macrophage colony (GMCSF), interleukin 3 and interleukin 5 are involved in the differentiation of eosinophils. GMSCF, the macrophage colony stimulating factor (MCSF) and IL-3 are involved in the differentiation of macrophages. The GMSCF, GCSF and IL-3 are involved in the differentiation of neutrophils. GMCSF, IL-3, IL-6, IL-11 and TPO are involved in the differentiation of platelets. Ligand Flt3 is involved in the growth of dendritic cells. The GMCSF, and IL-3, and erythropoietin are involved in the differentiation of erythrocytes. Finally, the self-renewal of pluripotent progenitor cells, primitive, capable of sustaining hematopoiesis requires CFS, ligand Flt3, G-CSF, IL-3, IL-6 and IL-11. Various combinations to achieve a desired result will be apparent to those skilled in the art. Because the agents useful according to the invention stimulate the primed, non-compromised hematopoietic progenitor cells, agents can be used in connection with any of the foregoing categories to specifically stimulate the proliferation of a particular type of hematopoietic cell. The above factors are well known to those skilled in the art, and most are commercially available. The invention also provides a variety of uses in vi tro. Hematopoietic progenitor cells are preserved or expanded, or their colony-forming unit potential increases in vi tro. A benefit that can be obtained according to the invention is the stimulation of the hematopoietic progenitor cells by the agents useful according to the invention. Another benefit that can be obtained is the effect that the agent can have on the stromal cells used in vitro by culturing hematopoietic progenitor cells. In vitro culture of the hematopoietic cells is often carried out in the presence of stromal cells. Hematopoietic progenitor cells will typically not survive, proliferate or differentiate for very long periods of time without the support of the appropriate growth factor. Stromal cell layers are used to deliver such growth agents to cultured hematopoietic cells, either by culturing the hematopoietic progenitor cells in vi tro with such stromal cells or by supplying the hematopoietic progenitor cells with conditioned medium with stromal cells. The agents useful according to the present invention can be used to treat such stromal cells or cause the stromal cells to manufacture or release growth factors. The incubation of the stromal cells with the useful agents according to the invention and in the medium that is for a period of time sufficient to allow the stromal cells to secrete factors in the medium. The medium can then be supplied to supplement the culture of hematopoietic progenitor cells and other hematopoietic cells. The culture of hematopoietic cells is with means which are conventional for culturing cells. Examples include RPMI, DM, ISCOVES, etc. The conditions for such cultivation are also known to those skilled in the art. Conditions typically refer to a combination of parameters (for example, temperature, C02 content, and 02, nutritive media, etc.). Sufficient time to increase the number of cells is a time that can be easily determined by one skilled in the art, and can vary depending on the original number of cells seeded and the aggregate amount of growth factors and agents useful according to the invention . The potential for colony formation of non-compromised hematopoietic progenitor cells can be increased by the culture of hematopoietic cells. The cells can be obtained from any blood product or organ that contains cells of hematopoietic origin. Crude or nonseparated blood products may be enriched with cells having characteristics of hematopoietic progenitor cells in forms well known to those skilled in the art, before or after culture with agents useful in accordance with the invention. A particularly important aspect of the invention is the use of the agents for the treatment of nopenia. A combination of unexpected results makes the invention particularly useful in the treatment of nopenia. First, the agents according to the invention can stimulate the proliferation of uncommitted progenitor cells. Secondly, 1 ¿I ií níi ---! -----!, -. • - the agents according to the invention also stimulate the stromal cells to produce GCSF, which is the critical growth factor in the differentiation and production of nophils per se. In this way, the patient has the double benefit of stimulating the progenitor cells and differentiating those cells into nophils using the agents useful according to the invention. Similar effects are found with erythrocytes and platelets. Thus, the treatment for restoring nophils, erythrocytes and platelets forms an indistinct independent aspect of the invention, based on the unexpected discoveries described above. The invention also involves equipment for housing a complete medicinal treatment course for a deficiency of hematopoietic cells such as nopenia. As discussed above, it has been surprisingly discovered that the number of doses per day and the total number of doses favorably affects the recovery of the hematopoietic cells after treatment with a hematopoietic cell inhibitor. These unexpected discoveries in themselves help the development of a dispensary or medicinal dispenser, which houses a complete medical treatment course using the agents useful according to the invention. The compliance * of the patient will therefore improve, and a complete prescription can be contained in a single package. In common, a pharmacist individually fills a distribution unit with ur medication once the pharmacist receives the prescription from the doctor. Because the dispenser of the invention includes a complete medicinal treatment course and can always include a specific number of solid oral dosage forms, the package can be pre-filled with the appropriate number of units of medicament for treatment for a particular medical purpose. . The dispensary or medicinal dispenser is a package that defines a plurality of medicinal storage compartments, each compartment serving to house an individual unit of medication. A complete medicinal treatment course is housed in a plurality of medicinal storage compartments. A package defining a plurality of medicinal storage compartments can be any type of disposable pharmaccal pack or card containing drugs in individual compartments. Preferably, the pack is a pack of ampoules constructed from a card, which can be made from a rigid paper material, a sheet of ampoules and a support sheet. Such cards are well known to those skilled in the art. Figure 1 shows a medicinal dispenser (1) for housing a complete medicinal treatment course for neutropenia. The indication of the day (2) indicates on which day the individual units of the medicine should be consumed. Those are marked along a first side of the medicinal package. The indication of the dose (3) is marked along a second side of the medicinal package perpendicular to the first side of the medicinal package and indicates the time at which the individual unit of the medication should be taken. The unit doses (4) are contained in the dispenser which is a pack of ampoules. This particular package shows a course of treatment for 5 days with 2 doses per day. The pharmaceutical preparations, as described above, are administered in effective amounts. The effective amount will depend on the mode of administration, the particular condition that is being treated and the desired result. It will also depend, as discussed above, on the stage of the condition, the age and physical condition of the subject, the nature of the concurrent therapy, if any, and similar factors well known to the practicing physician. For therapeutic applications, this is that amount sufficient to ¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿? In some cases, there is some increase in the hematopoietic cell count and the counting of mature blood cells. In other cases, there will be an increase to a preselected level. The invention is useful in one aspect to alleviate the effects of treatment with a hematopoietic cell inhibitor. If the agents are used prophylactically, they can decrease the amount of hematopoietic cells that would be lost in the subject against the amount lost if the subject were treated with the inhibitor but not with the agent. If used prophylactically or acutely, agents can shorten the recovery time of a type of hematopoietic cell to a more or less protective level, preferably at normal levels, against the period of time that would pass before the levels were reached. protective or normal if the subject was treated with the inhibitor, but not with the agent. Generally, the doses of active compounds of the present invention would be from about 0.01 mg / kg per day to less than 1 mg / kg per day. A variety of administration routes are available. The methods of the invention, generally speaking, can be practiced using any mode of administration which is medically acceptable, which means any mode that produces effective levels of the active compounds without producing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, intrathermal or palenteral routes. The term "palenteral" includes the subcutaneous, intravenous, intramuscular or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. She could, however, be preferred in emergency situations. Oral administration is preferred by the convenience of the patients, as well as the dosage program. (See Remigton's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712, incorporated here as reference). Those skilled in the art can easily determine the different parameters and conditions for producing doses without resorting to undue experimentation. The compositions suitable for Oral administration can be presented as discrete units, such as capsules, tablets, pills, each containing a predetermined amount of active agent. Other compositions include suspensions in aqueous or non-aqueous liquids such as a syrup, elixir or an emulsion. - "-" "*» - "» - ~~ ^ - ^ j ^ * - ^ - ~~~. .. ~ * ^ - - - - »» .. -? - * i ---- Bia¡? > ? - * - ^^ Preparations for the parenteral administrator include aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Palenteral vehicles include a solution of sodium chloride, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

Condoms and other additives may also be present such as, for example, antimicrobial agents, antioxidants, chelants, and inert gases and the like. The lower doses will be the result of other forms of administration, such as intravenous administration. In In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively larger doses per a more localized, different release route) may be used to the extent that patient tolerance allows. Doses were contemplated multiple per day.

. - - • * • »= * -« --- - A ^^ »» "-» ^ - * - - ^ ~ a? LM * JM > »M ifflfcrfflr r **" • * '- - * "•" The agent can be combined, optionally, with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein means one or more solid or liquid fillers or fillers, diluents or encapsulating substances that are suitable for administration to a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient s' combines to facilitate the application. The components of the pharmaceutical compositions are also capable of being commingled with molecules of the present invention, and with each other, in such a form that there is no interaction that would substantially damage the pharmaceutical efficacy. When administered, the pharmaceutical spreads of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts ** 4 ^ - ^ = »i ^ iV ^ include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic and the like. Also, pharmaceutically acceptable salts can be prepared as alkali metal or alkaline earth metal salts, such as the sodium, potassium or calcium salts. Other delivery systems may include distribution systems of temporary release, delayed release or sustained release. Such system can avoid repeated administrations of the agent, increasing the convenience to the subject and the doctor. Many types of delivery distribution systems are available and are known to those skilled in the art. They include polymer-based systems such as poly (lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid and polyanhydrides. Capsules of the above drug-containing polymers are described in, for example, U.S. Patent 5,075,109. The distribution systems also include non-polymeric systems which are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral acids such as mono, di and triglycerides; hydrogel distribution systems; silastic systems; systems based on peptides; wax coatings; compressed tablets using conventional binders and excipients; partially cast implants; and similar. Specific examples include, but are not limited to: (a) erosional systems in which the agent is contained in a form within a matrix, such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 or 5,239,660 and ( b) diffusion systems in which the active components are filtered or permeated at a controlled rate from a polymer as described in US Pat. Nos. 3,832,253 and 3,854,480. In addition, distribution systems can be used with pump-based equipment, some of which are adapted for implantation. The use of a long-term sustained release implant may be particularly suitable for the treatment of chronic conditions. The long-term release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well known to those skilled in the art and include some of the delivery systems described above.

Examples We have shown a series of in vivo studies that the agent ValboroPro (PT-100), has the ability to shorten the myelosuppression caused by chemotherapy in mice. In these studies, mice were injected intraperitoneally with a sublethal dose of 220 mg / kg cyclophosphamide (Day 1). This treatment reproducibly induced a nadir in blood cell counts on Day 4. 72 hours later (Day 3), the mice were divided into 3 groups. One group received PT-100, at the indicated concentrations, per dose or by subcutaneous administration (s.c.), one group received the G-CSF by s.c. injections. and the third group received alkaline solution as control, either by oral intake or s.c. injections. G-CSF was used at 0.04 ug / dose (4 μg / kg / day) which is the dose frequently used in published reports that study the effects of G-CSF in mice and is also the equivalent dose used in cancer patients . All administrations were performed twice a day (b.i.d.) for 5 consecutive days or as indicated. Blood samples were taken from individual mice on Days 4-8, and in some experiments on Days 13 or 17. At each time 4 or 5 test animals were sampled. The g ^ g ^ l ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ M ^^^^^^^^^^^^^ ^ M ^. ^^^^^ counts of total white blood cells and differential blood smears stained with Gimsa.

Response to PT-100 Dose For let Neutrophil Regeneration For the data presented in Figure 2, mice treated with cyclophosphamide received 0.1 μg, 2 μg or 5 μg / b.i.d. of PT-100 or saline by oral intake twice daily for 5 consecutive days beginning on Day 3 after treatment with cyclophosphamide and continuing until Day 7. In mice receiving 2 or μg / b.i.d. of PT-100 the reproducibility of the recovery of neutrophils presided over the recovery of mice treated with saline in 1 or 2 days, while: with 0.1 μg / b.i.d. of PT-100 there was no significantly better recovery of: neutrophils over saline. Normal levels of absolute neutrophil counts (ANC) were reached at Day 5 for mice that received 2 μg or 5 μg / b.i.d. of PT-100, while: mice treated with saline did not reach normal levels until Day 7. At Day 5 the mice; They had received a total of 4 doses of PT-100 (in Days 3 and 4). The additional administration of PT-100 in Day 5, Day 6, and Day 7 produced a further increase in ANC. .-¿- ^ saa - ^ - »^ ^^^^ mi The effect of PT-100 on the recovery of neutrophils when administered by the s.c. it was very similar to that observed when it was administered orally. For the data shown in Figure 3, the mice were injected s.c. with doses of PT-100 that fluctuate from 1 to 20 μg / b.i.d. for 5 days and blood cell counts were determined on Days 4 through 8, and on Day 17. For mice that received 5 μg, 10 μg, or 20 μg / b.i.d. of PT-10, the recovery of neutrophils was accelerated over that observed in mice treated with saline. A dose of 1 μg / b.i.d. of PT-100 did not show much effect. After finishing treatment with PT-100, it was concluded that PT-100 accelerates the regeneration of neutrophils in mice treated with cyclophosphamide.

Comparison of the Effects of PT-100 and G-SCF on the Regeneration of Neutrophils G-SCF is currently used to accelerate the recovery of neutrophils in cancer patients undergoing chemotherapy. The effects of G-SCF in mice are well established and can be used as a reference to elucidate the mechanism by means of PT-100 stimulates hematopoiesis in mice. Figure 4 shows data from an experiment in which they were compared the effects of PT-100 and G-SCF on neutrophil regeneration. Mice treated with cyclophosphamide were administered with 2 μg / b.i.d. of PT-100 per tap or 0.04 μg / b.i.d. of G-CSF (the equivalent dose used in patients and most commonly used in published reports for murine studies) by subcutaneous injections for 5 consecutive days beginning on Day 3. Blood cell counts were performed on Days 4-8 and on Day 13. Mice treated with PT-100 and G-SCF stimulated the regeneration of neutrophils at a similar level during the treatment period. After stopping treatment, ANC decreased to normal counts on Day 13. Although PT-100 has a very similar effect on the reconstitution of neutrophils, the mechanism of action is different from that of G-SCF. PT-100 not only targets different cells in a receptor (CD 26), but has also been shown to stimulate early human hematopoietic progenitor cells, which are not affected by G-SCF.

Numbering the Administration Dosage of PT-100 To determine the numbering of the administration dose for optimal recovery of the neutrophils, s.c. PT-100, at the indicated doses, to mice treated with cyclophosphamide, either once or twice a day for a period of 5 days, beginning on Day 3 after treatment with cyclophosphamide. As shown in Figure 5, for both doses, administration twice daily resulted in a faster recovery rate of neutrophils at higher neutrophil levels than a once-a-day administration.

Duration of Administration of PT-100 In the experiments described above, the mice had been treated with PT-100 for 5 consecutive days. To determine if a shorter treatment period with PT-100 was sufficient for the recovery of: the neutrophils were administered 5 μg, 2 μg, or 1 μg / b.i.d. (with six hours of separation) of PT-100 to mice treated with cyclophosphamide per dose for 1, 2, 3, or 5 days beginning on Day 3 after treatment with cyclophosphamide. Blood counts were obtained on Days 4 to 8. The administration of PT-100 for one day was sufficient to produce an accelerated reconstitution of neutrophils on animals treated with saline. However, additional administrations of PT-100 for 2 or 3 days increased the speed of recovery even more. The data for the 5 μg dose are shown in Figure 6. Continuous administration of PT-100 for a total of 4 or 5 days does not significantly increase the recovery rate of neutrophils or ANCs over what has been achieved with 3-day administrations. (data for 2 μg / bid are shown in Figure 7). The results shown in Figures 6 and 7 indicate that the effect of PT-100 on neutrophil regeneration occurs initially during treatment and continues until ANC of between 1000 and 1400 are reached. Repeated administrations affect the restoration kinetics of the neutrophils. neutrophils during the initial period but did not significantly alter the ANC reached after 3 days of administration. In conclusion, the PT-100 accelerates the reconstitution of neutrophils over what is observed with saline solution even after one day of treatment. Accelerated reconstitution of the neutrophils is obtained with each additional day of treatment up to three days. A fourth or fifth day of treatment does not significantly increase ANC or reconstitution kinetics.

Responses of Human Hematopoietic Cells In Vitro Hematopoiesis is supported by a set of undifferentiated hematopoietic cells (HSC) that can self-renew and differentiate into hematopoietic progenitor cells (HPC). HPCs are committed to specific lineages, which can be identified on the basis of their colony morphology when they grow in semi-solid media in vi tro, typically over a period of 2 weeks. Colonies that grow in the semisolid colony assay are functionally defined as colonies or units that form bursts and include the BFU-E and CFU-E (cells committed to the erythroid lineage), CFU-GM (cells committed to the granulocytic lineage). / monocytic), BFU-MK and CFU-MK (cells committed to the megakaryocytic lineage) and CFU-GEMM (multipotent progenitors). Although the semi-solid colony assay is a valuable tool for identifying factors, such as G-CSF, which affects terminal differentiation, it does not evaluate the proliferative potential or self-renewal properties of primitive hematopoietic progenitor cells (PHPC) (Dexter, TA et al., Hema Act 62: 299-305 (1979); Chen, BP et al., Immunological Reviews: 157: 41-51 (1997)). g $. »^ jgp An assay to evaluate the effect of a compound or growth factors on PHPC was first described by Dexter (Dexter TM et al., J. cell. Physiol. 91: 335-344 (1977)), and combines Long Term Cultivation (LTC) with the semi-solid colony assay. The CTL is initiated on a preformed stromal cell layer that provides the necessary hematopoietic growth factors. This has been widely used for the in vitro examination of murine and human hematopoiesis and to evaluate the ability of the test compounds to generate LTC-IC. The effect of PT-100 on the growth of human hematopoietic cells was examined in the CFU of 2 weeks and the LTC trial of 4 and 5 weeks, using human bone marrow, affected peripheral blood or umbilical cord blood cells. PT-100 did not stimulate the generation of CFUs in the semisolid 2-week assay, indicating that PT-100 does not affect the differentiation of compromised progenitor cells into mature blood cells. It also suggests that the mechanism and cellular targets for PT-100 for the stimulation of neutrophil regeneration in vivo is different from G-CSF, which has been shown to stimulate CFU formation in this assay. In the LTC assays, which test the effects on early progenitor cells, the PT-100 significantly increases the growth of very early progenitor cells from all three cellular sources. In addition, the data suggest that the effect of PT-100 is on the PHPC since increases were observed; in LTC-IC at 4 weeks (Figure 8), 5 weeks and 6 weeks; (data not shown) in the crop. At this time, the less primitive hematopoietic progenitor cells have undergone terminal differentiation and lose the ability to form colonies in semisolid cultures. For CTL assays, CD34 + cells were isolated by positive selection of cells from human bone marrow, affected peripheral blood or umbilical cord blood using a MAC separation system. To establish a stromal feeder layer, human bone marrow cells were cultured in Myelocult long-term culture medium for 2 weeks. One day before use, adherent stromal cells were cultured overnight at the indicated concentrations of PT-100 in LTC medium and irradiated. The isolated CD34 + cells were coated on the layer of stromal cells and incubated for 30 days in the absence or presence of the indicated amounts of PT-100. The medium and the PT-100 were exchanged every three days thereafter. At the end of the culture period, the culture was tested to determine progenitor cells by culturing in semi-solid medium : V .-- (methylcellulose) supplemented with growth factors (undifferentiated cell factor, GM-CSF, IL-3 and erythropoietin). The total number of myeloid progenitors. Erythroids, blast and chlorogenic formers of multiple lineages (CFU-GM, CFU-E, BFU-E and CFU-GEMM colonies, respectively) were determined 14 days later in culture in methylcellulose. The data shown in Figure 8 for a human bone marrow culture indicate that during a 4 week LTC trial, the PT-100 increased, in a dose-dependent manner, the number of clonogenic progenitors, which are capable of form colonies in semi-solid media. This suggests that PT-100 stimulates the growth of primitive hematopoietic progenitor cells. In a similar manner, CD34-purified cells from affected peripheral blood or umbilical cord blood were cultured on primary stromal cells irradiated for 30 days. As it will be observed with the bone marrow cells, the PT-100 increased the number of 4 and 5 weeks LTC-IC of peripheral blood and of: umbilical cord at very similar levels, indicating that the PT-100 is able to stimulate the growth of the cells; primitive hematopoietic progenitors from those sources; cell phones too (data not shown).

PT-100 Does Not Stimulate Differentiation of Compromised Progenitor Cells Human bone marrow cells were enriched in CD34 + cells and 200 CD34 + cells per well 5 and incubated in serum free x-vivo 15 medium (Biohittaker) with or without the indicated concentrations of PT-100 for hours at 37 ° C. The preincubated CD34 + cells were added to 0.9% methylcellulose in MDM Iscove containing suboptimal concentrations of growth factors Human recombinants (5 ng / ml undifferentiated cell factor, 1 ng / ml GM-CSF, 1 ng / ml 11-3, 0.3 units / ml erythropoietin (Stem Cell Technologies, Vancouver, BC). PT-100 was added to the medium at the same concentrations used for pre-incubation. methylcellulose was cultured in duplicate on 35 mm discs and incubated for 14 days at 37 ° C. Progenitor colonies (CFU-E, CFU-GM, CFU-GEMM and BFU-E) were counted under an inverted microscope. The PT-100 nc stimulates the differentiation of these progenitorae cells committed.

Stimulation of Hematopoiesis in the Spleen of Normal Mice Female BALB / c mice 6-8 weeks of age 5 were administered with either saline or PT-100 twice daily for 5 days at the indicated doses via subcutaneous injection or oral intake. On the sixth day, the animals were sacrificed and their spleens were extirpated using sterile procedures. The spleens were crushed to produce unique cell suspensions, which were then treated with a solution of Tris ammonium chloride (pH 7.2) to lyse the erythrocytes. The resulting splenocyte populations were measured on a hemocytometer and resuspended at 5 x 10 D cells / ml in Iscove's modified Eagle's medium (IMDM) supplemented with 2% heat inactivated fetal sheep serum. 0.3 ml of each splenocyte solution was added to 3 ml of Methocult MRGF M3434 (Stem Cell Technologies, Vancouver, BC, Canada), a methylcellulose medium containing recombinant cytokines used for murine progenitor cell colony assays. The medium was mixed vigorously and then 1.1 mL of the mixture was placed in duplicate on sterile culture discs of 35 mm in diameter, resulting in 5 x 105 splenocytes / plate. Cultured cells were incubated at 37 ° C under humid conditions in 95% air / 5% C0 for 7 days. The CFU-E were listed according to the manufacturers' specifications after 2 days, while the CFU-GM BFU-E, and CFU-GEMM were listed 7 days later. For each mouse, the CFU / spleen were calculated using the total splenocyte count determined in the hemocytometer. The data shown in Figure 9 represent the mean ± SE of CFU / spleen of 3 mice in each dose group. The PT-10D stimulated hematopoiesis for all types of progenitor colonies tested.

PT-100 Induces the Production of G-CSF from Human Bone Marrow Stromal Cells Bone marrow mononuclear cells were purified and cultured in long-term culture medium (Stem Cell Technologies, Inc., Vancouver, BC) for 2 weeks , with a single feeding of fresh medium L week later. The stromal cells were removed by digestion with trypsin-EDTA seeded in a 35 mm tissue culture dish at 10 6 cells per well in 'ml of medium containing 10 5M PT-100 or media alone as control. The supernatants were assayed for human G-CSF using a Quantikine high sensitivity immuno assay kit (R + D Systems, Minneapolis, MN) Figure 10 describes the effect of PT-100 on the production of G-CSF by cultured human stromal cells The PT-100 stimulates the production of G-CSF by such cells.

* The manufacture of L-VAL-R-boroPro is described in a number of published procedures (Kelly, TA, et al., J. Am. Chem. Soc. 1993. 115: 12537-12638; Couts, SJ. , et al., J. Med. Chem. 1996. 39: 2087-2094; Beak, P., et al., Tetrahedon Letters, 1989, 30: 1197; Bean, FR, et al., J. Am. Chem. Soc. 1932. 54: 4415). Pure isomers are preferred. See also U.S. Patents 4,935,493 and 5,462,928, the descriptions of which are incorporated herein by reference. Although the invention has been described with respect to certain embodiments, it should be appreciated that those skilled in the art can make many modifications and changes in the art without departing from the spirit of the invention. It is intended that such modifications, changes and equivalents fall within the scope of the following claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Jis-ftá- - ^ .-.,. ^, D3 ^^

Claims (63)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for treating a subject to stimulate hematopoiesis in the subject, characterized in that it comprises: administering to a subject in need of such treatment an amount of an agent effective to increase the number of hematopoietic cells or mature blood cells in the subject, where a amount is less than 1 mg / kg of body weight and where the agent is a compound of Formula I.

2. The method according to claim 1, characterized in that the subject has an abnormally low level of hematopoietic cells and where the agent administered in an effective amount to restore the levels of a type of hematopoietic cell to a preselected normal or protective level.

3. The method according to claim 1, characterized in that the subject is administered at least two doses of the agent in a period of 18 hours.

4. The method according to claim 2, characterized in that the subject is neutrogenic and where the amount is effective to restore a preselected normal or protective level of neutrophils.

5. The method according to claim 2, characterized in that the subject has abnormally low levels of erythrocytes and where the amount is effective to restore a preset normal or protective level of erythrocytes.

The method according to claim 2, characterized in that the subject has: abnormally low levels of platelets and where the amount is effective to restore a normal or preselected level of platelets.

7. The method of compliance with the. claims 1, 2, 3, 4, 5 or 6, characterized in that the agent is ValBoroPro.

A method for shortening the time in which the subject has an abnormally low level of mature blood or hematopoietic cells resulting from treatment with a hematopoietic cell inhibitor, characterized in that it comprises: administering to a subject in need of such treatment an agent in an amount effective to increase the number of hematopoietic cells or mature blood cells in the subject, wherein the administration of the agent is before or substantially simultaneously with the administration of the hematopoietic cell inhibitor, and wherein the agent is a compound of Formula I.

The method according to claim 8, characterized in that the administration of the hematopoietic cell inhibitor produces an abnormally low level of hematopoietic cells in the subject and where the agent is administered with an effective amount to restore the levels of a type of hematopoietic cell a a normal or protective level: preselected.

The method according to claim 9, characterized in that the subject is administered with at least two doses of the agent in a period of 18 hours.

11. The method according to claim 9, characterized in that the inhibitor of hematopoietic cells reduces neutrophils in the subject and where; the amount is effective to restore a normal or preselected level of neutrophils in the subject.

12. The method according to claim 9, characterized in that the inhibitor of the hematopoietic cells reduces erythrocytes in the subject; and where the amount is effective to restore a normal or protective preselected level of erythrocytes in the subject.

13. The method according to claim 9, characterized in that the inhibitor of the hematopoietic cells reduces in the subject the platelets and where the amount is effective to re-establish in the subject, a normal or preselected level of platelets.

The method according to claims 8, 9, 10, 11, 12 or 13, characterized in that the amount is less than 1 mg / kg of body weight per day.

15. The method according to claim 8, 9, 10, 11, 12 or 13, characterized in that the agent is ValBoroPro.

16. A method for preparing a subject for treatment with a hematopoietic cell inhibitor, characterized in that it comprises: administering to the subject before the subject receives the hematopoietic cell inhibitor an agent in an amount effective to stimulate the subject the production of growth factor, where the agent is a compound of Formula I.

17. The method according to claim 16, characterized in that the factor of -s »* growth is the stimulating factor of the colony of: granulocytes.

18. The method according to claim 16, characterized in that the amount is less than 1 mg / kg of body weight per day.

The method according to claim 16, characterized in that the agent is administered to the subject by administering at least two doses of the agent in a period of 18 hours.

20. The method according to claim 16, characterized in that the agent stimulates the production of growth factor by the stromal cells.

21. The method according to claims 16, 17, 18, 19 or 20, characterized in that the agent is ValBoroPro.

22. A method for treating a subject to increase the number of hematopoietic cells in the subject, characterized in that it comprises: administering to a subject in need of such treatment an amount of an agent, effective to increase the hematopoietic cells in the subject, where the agent is administered in a first regimen consisting of 2 doses or 3 doses in a period of 18 hours, and wherein the agent is a compound of Formula I.

23. The method according to claim 22, characterized in that the agent is; administered in a second regimen consisting of 2 doses or 3 doses in a period of 18 hours, and where the second regimen is separated at the time of the first regimen.

24. The method according to claim 23, characterized in that the agent is; administered in a third regimen consisting of 2 doses or 3 doses in a period of 18 hours, and where, the third regimen is separated in time from the first and second regimens.

25. The method according to claim 24, characterized in that the agent is administered in a fourth regime consisting of 2 doses c 3 doses in a period of 18 hours, and where, the fourth regime is separated in the time of the first, second and third regimes.

26. The method according to claim 25, characterized in that the agent is administered in a fifth regimen consisting of 2 doses or 3 doses in a period of 18 hours, and where the fifth regimen is separated at the time of the first, second , third and fourth regimes.

27. The method according to claim 22, 23, 24, 25 or 26, characterized in that - * , j «Hri --- ugly» - ¿- ^ .t? - - »- ~ - - - -. a- .. ^ - < s¿íÉBáL¿trttÍMftt a? ¿?? 4? * t *. ... ^ ^? á? ^? Ji ^ M ?? inß ^^ i ^ the subject has abnormally low neutrophils and, where the amount is effective to restore a normal or preselected level of neutrophils.

28. The method according to claims 22, 23, 24, 25 or 26, characterized in that each regimen is less than 1 mg / kg of body weight per day.

29. The method according to claims 22, 23, 24, 25 or 26, characterized in that the agent is ValBoroPro.

30. A method for preparing the cells of a subject to be reintroduced into the subject, characterized in that it comprises: administering to the subject an agent in an amount effective to stimulate the hematopoietic cells, then collecting the hematopoietic cells of the subject, and reintroducing the cells collected from the subject, wherein the agent is a compound of Formula I.

31. The method according to claim 30, characterized in that it further comprises culturing ex vivo the collected cells in the presence of an amount of agent effective to stimulate the proliferation of the collected cells.

32. The method according to claim 30, characterized in that the concentration of the agent in the medium surrounding the harvested cells is less than 10"8 moles per liter

33. The method according to claim 30, 31 or 32, characterized in that the agent is ValBoroPro

34. A method for stimulating the production of growth factor by the stromal cells, characterized in that it comprises: contacting the stromal cells with an agent in an amount effective to stimulate the production of growth factor by the cells, stromal, where the agent is a compound of Formula I.

35. The method according to claim 34, characterized in that the stromal cells are an in vi tro layer of stromal cells to support cell growth and that further comprises cultivating undifferentiated cells in the presence of stromal cells.

36. The method according to claim 34, characterized in that the stromal cells are in vivo in a subject.

37. The method according to claim 34, characterized in that the growth factor is the stimulating factor of the granulocyte colony.

38. The method according to claim 36, characterized in that the agent is administered to the subject in an amount less than 1 mg / kg per day.

39. The method according to claims 34, 35, 36, 37 or 38, characterized in that the agent is ValBoroPro.

40. The method according to claim 35, characterized in that the undifferentiated cells are cultured in an environment free of exogenously added growth factors.

41. The method according to claim 35, characterized in that the undifferentiated cells are cultured in an exogenously added granulocyte colony-stimulating factor-free environment.

42. A kit for treating a subject to alleviate the effects resulting from treatment with a hematopoietic cell inhibitor, characterized in that it comprises a package containing: a first dose of an agent and instructions for treating the subject with the agent, in a manner substantially simultaneously with or prior to treatment with the hematopoietic cell inhibitor, and a second dose of the agent and instructions for treating the subject with the agent, only after the És- ^ saii ..at-ai - ^ - aaaBaa-fafc--. treatment with the inhibitor of the cells; hematopoietic agents, wherein the doses are in effective amounts and, where the agent is a compound of Formula I.

43. The equipment according to claim 42, characterized in that the second dose is between 2 and 7 regimens, each of the regimens consisting of two or three doses of the agent for administration in a period of 18 hours.

44. The equipment according to claim 44, characterized in that the second dose is less than 1 mg / kg of body weight per day.

45. The equipment according to claims 42, 43 or 44, characterized in that the agent is ValBoroPro.

46. The equipment according to claims 42, 43 or 44, characterized in that the equipment is for treating or inhibiting neutropenia.

47. The equipment according to claim 45, characterized in that the equipment is for treating or inhibiting neutropenia.

48. The equipment according to claims 42, 43 or 44, characterized in that the equipment is for treating or inhibiting a platelet deficiency.

49. The equipment according to claim 45, characterized in that the equipment is for treating or inhibiting a platelet deficiency.

50. The equipment according to claims 42, 43 or 44, characterized in that the equipment is for treating or inhibiting a deficiency of erythrocytes.

51. The equipment according to claim 45, characterized in that the equipment is for treating or inhibiting a deficiency of erythrocytes.

52. A kit for treating a subject having an abnormally low level of hematopoietic cells or mature blood cells, characterized in that it comprises: a package containing a complete dose to restore at least the protective levels of a hematopoietic cell or a cell type mature blood, the package essentially consists of a first dose of an agent in an effective amount to be administered to the subject during a first period of 18 hours of a first day, a second dose of an agent in an effective amount to be administered to the subject during a second 18-hour period of a second day, optionally a third dose of the agent in an amount effective to be administered to the subject during a third period of 18 hours on a third day, optionally, a fourth dose of the agent in an effective amount to be administered at subject during a fourth period of 18 hours of a fourth day, optionally, a fifth dose s of the agent in an effective amount to be administered to the subject during a fifth period of 18 hours of a fifth day, optionally, a sixth dose of the agent in an amount effective to be administered to the subject during a sixth period of 18 hours of a sixth day, optionally, a seventh dose of the agent in an amount effective to be administered to the subject during a seventh period of 18 hours of a seventh day, wherein the agent is a compound of Formula I and wherein the agent is present in an effective amount to restore a normal or preselected level of a type of mature hematopoietic or blood cells.

53. The equipment according to claim 52, characterized in that it consists essentially of only the first, second, third, fourth and fifth doses.

54. The equipment according to claim 52, characterized in that it consists of essentially only the first, second, third and fourth doses.

55. The equipment according to claims 52, 53 or 54, characterized in that each of the doses consists of 2 or 3 doses of the agent for each respective period of 18 hours.

56. The equipment according to claims 52, 53 or 54, characterized in that the amount of each dose is less than 1 mg / kg of body weight per day.

57. The equipment according to claims 52, 53, 54 or 55, characterized in that the agent is ValBoroPro.

58. A device for alleviating in a subject the effects of a hematopoietic cell inhibitor, characterized in that it comprises a package containing: a first dose of an agent to be administered to a subject during a first day, and a second dose of to be administered to a subject during a second day, optionally, a third dose of the agent in an effective amount to be administered to the subject during a third period of 18 hours of a third day, optionally, a fourth dose of the agent in an effective amount to be administered to the subject during a fourth period of 18 hours of a fourth day, optionally, a fifth dose of the agent in an effective amount to be administered to the subject during a fifth period of 18 hours of a fifth day, optionally, a sixth dose of the agent in an effective amount to be administered to the subject during a sixth period of 18 hours of a sixth day, optionally, a seventh dose of the agent in an amount effective to be administered to the subject during a seventh period 18 hours of a seventh day, where the dose is combined in effective amounts to alleviate the effects on the subject of the hematopoietic cell inhibitor and where each dose is less than 1 mg / kg of body weight per day.

59. The equipment according to claim 58, characterized in that it consists essentially of only the first, second, third, fourth and fifth doses.

60. The equipment according to claim 58, characterized in that it consists essentially of only the first, second, third and fourth doses.

61. The equipment according to claims 58, 59 or 60, characterized in that each of the doses consists of two or three doses of the agent for each respective day.

62. The equipment according to claims 58, 59 or 60 the agent is ValBoroPro.

63. The equipment according to claim 61, characterized in that the agent is ValBoroPro.