CN1705439A - Modulation of stem and progenitor cell differentiation, assays, and uses thereof - Google Patents

Modulation of stem and progenitor cell differentiation, assays, and uses thereof Download PDF

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CN1705439A
CN1705439A CN 03813654 CN03813654A CN1705439A CN 1705439 A CN1705439 A CN 1705439A CN 03813654 CN03813654 CN 03813654 CN 03813654 A CN03813654 A CN 03813654A CN 1705439 A CN1705439 A CN 1705439A
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cell
cfu
compound
differentiation
stem cell
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R·J·哈里里
D·I·斯蒂尔林
K·W·H·陈
L·A·穆托-德帕塞瓦尔
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Celgene Corp
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Celgene Corp
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Abstract

The present invention relates to methods of modulating mammalian stem cell and progenitor cell differentiation. The methods of the invention can be employed to regulate and control the differentiation and maturation of mammalian, particularly human stem cells along specific cell and tissue lineages. The methods of the invention relate to the use of certain small organic molecules to modulate the differentiation of stem or progenitor cell populations along specific cell and tissue lineages, and in particular, to the differentiation of embryonic-like stem cells originating from a postpartum placenta or for the differentiation of early progenitor cells to a granulocytic lineage. Finally, the invention relates to the use of such differentiated stem or progenitor cells in transplantation and other medical treatments.

Description

Adjusting, evaluation and the application thereof of stem cell and CFU-GM differentiation
The application requires the U.S. Provisional Application submitted on April 12nd, 2002 number 60/372,348, the U.S. Provisional Application of submitting on May 30th, 2002 number 60/384,251, the U.S. Provisional Application of submitting on December 31st, 2002 number 60/437, the priority of the U.S. Provisional Application of submitting on December 31st, 348 and 2002 number 60/437350, the full content that this paper quotes each piece as a reference.
1. introduce
The present invention relates to regulate the method for mammalian stem cell and/or CFU-GM differentiation.Method of the present invention can be used for regulating and controlling mammal, especially people's stem cell along specific cells and differentiation and the maturation of organizing pedigree.Method of the present invention relates to uses some little organic molecule to regulate stem cell or progenitor cell populations along specific cells with organize the differentiation of pedigree, especially come from the placenta in postpartum embryonic-like stem cell differentiation or along specific differentiation pathway, the particularly adjusting of the early stage hemopoietic progenitor cell of granulocyte differentiation pathway.The invention still further relates to and use these organic molecules to regulate the particular lineage CFU-GM, as CD34 +, CD45 +And CD133 +The differentiation of CFU-GM.The invention still further relates to the time aspect that CFU-GM is grown, and based on these time-related external models.The invention further relates to these adjusted cells in prevention and methods of treatment, be included in the application in the pharmaceutical composition of this cell and/or little organic compound.At last, the present invention relates to the application of cell in transplanting and other therapeutic treatments of this differentiation.
2. background of invention
The evaluation of people's stem cell and CFU-GM, separate and propagation on very big concern is arranged.Stem cell is the all-round or multipotency precursor that can produce multiple mature cell pedigree, and precursor is the cell that can produce specific cells pedigree cell.These abilities are as for the essential cell differentiation of organ pipe and tissue development and the basis of specialization.
Recently provide new clinical tool with the marrow-reconstitution after being used for because of disease, being exposed to the marrow excision of toxic chemical substance and/or radiation and/or replenish transplanting success on stem cell and the CFU-GM.Further the existence of evidence proof stem cell can be used for rebuilding many, even be not whole tissues and repair function on physiological situation and physiology and the anatomy.The application of stem cell in organizational project, gene therapy transmission and cell therapy also just developing by leaps and bounds.
Many dissimilar mammalian stem cells and ancestral stem cell have obtained identifying.For example, known have embryonic stem cell, embryonic genital cell, adult stem cell or committed stem cell or a CFU-GM.The also not separated and evaluation of some stem cell, but allowing under the condition of restricted portion differentiation, to obtain cultivation.Yet surplus next basic problem is exactly that control and regulate stem cell and CFU-GM is difficult as the differentiation of hemopoietic progenitor cell.At present, the method for existing these cell differentiation of adjusting is jejune and uncontrollable, thereby cell is divided into the unwanted cells type in the unwanted time.And the productive rate of cell products is generally lower.
In addition, obtain sufficient amount be used for the treatment of or the human stem cell of research purpose still has problem.Partly and since in blood or tissue the limited quantity of the stem cell found or CFU-GM and with obtain the relevant remarkable discomfort of marrow aspirate, the stem cell or the progenitor cell populations that are separated into normal presence in the soma have technical difficulty, and the expense costliness.Generally speaking, be used for the treatment of or the stem cell of research purpose or CFU-GM effort very normally, comprise from substituting source results sufficient amount, for example, from donor or patient's harvesting or tissue, culture in vitro and/or propagate cell, dissociated cell etc., comprise obtaining these cells among the preborn especially from embryo or fetal tissue as for stem cell, risen to the misgivings of religion and ethics.Extensively people embryo who assert or the fetus conviction that just constitutes independent life has been facilitated at cell in source like this and has been used for all purposes, comprises forbidding of government on the purposes of medical research.Thereby the substituting source that does not need to use the cell that obtains from embryo or fetus just is desirably in further developing the stem cell clinical practice.Yet, the substituting source of available stem cell or CFU-GM, particularly human stem cell or CFU-GM is seldom arranged, therefore, supply is limited.
(WO 00/73421 for Hu etc., be entitled as " separation of people's amniotic epithelial cells, the method for freezing and therapeutical uses ", be disclosed in 2000-12-07) separation, cultivation of the people's amniotic epithelial cells that comes from when childbirth placenta disclosed, for the freezing that uses in the future or induce differentiation.Described according to Hu etc., divide the puerperium to obtain a placenta immediately and separate amnion, for example by dissecting from chorion.Amniotic epithelial cells separates from amnion according to the cell separation technology of standard.The cell of the disclosure can be cultivated in various medium, propagation, cryopreservation or quilt are induced differentiation in cultivation.It is multipotential hemopoietic stem cell (also may be pluripotential hemopoietic stem cell) that Hu etc. disclose amniotic epithelial cells, and can be divided into epithelial tissue, as cornea epidermal tissue or vagina tissue.Yet the shortcoming of this method is a labour intensive, and the productive rate of stem cell is very low.
The external expansion method of existing cell colony also is labor-intensive.For example, (Emerson etc. such as Emerson, U.S. Patent number 6326198, be entitled as " being used for the duplicating of stem cell in the body, the optimization that hemopoietic progenitor cell is cultivated and method and the component that strengthens metabolism, GM-CSF secretion and/or the IL-6 secretion of people's stroma cell ", be published in 2001-12-04) method of optimization of human stem cell division and/or artificial blood CFU-GM and the culture medium condition of culture in vitro disclosed.According to disclosed method, come from the human stem cell of marrow or the CFU-GM liquid medium within and cultivate, it can be replaced, preferred perfusion cultures, continuously or periodically, with the speed of 1 milliliter of medium of every milliliter of culture, every about 24 hours to 48 hours is the cycle.But on physiology, keep under the acceptable conditions when cultivating, remove metabolite, the nutrient of supplement consumed.
(Kraus etc. such as Kraus, U.S. Patent number 6338942, be entitled as " the selectivity expansion of targeted cell population ", be published in 2002-01-15) disclose predetermined targeted cell population and optionally expanded, come from the initial standard specimen cell of Cord blood or peripheral blood ground by in growth medium, introducing, cause the targeted cell population division, and in growth medium, use the selection component exposing cell, comprise the binding molecule (for example monoclone antibody of CD34 cell) that the cell colony of being scheduled to (as the CD34 cell) is had specific affinity, in growth medium, to select predetermined targeted cell population from other cells.
(U.S. Patent number 6335195 such as Rodgers, be entitled as " method that promotes hematopoiesis and mesenchymal cell propagation and differentiation " and be published in 2002-01-01) hematopoiesis and mesenchymal cell culture in vitro are disclosed and by proangiotensin, angiotensin I (AI), AI analogue, AI fragment and its analog, Angiotensin II (AII), AII analogue, AII fragment and its analog or AII AT are being arranged 2Type 2 receptor stimulating agents are united the existence special cell proliferation and the induced differentiation method of pedigree of growth down separately or with other growth factors and cell factor.As this stem cell source in marrow, peripheral blood or Cord blood.Yet as discussed above, the shortcoming of this method is that the method for this external evoked stem cells hyperplasia and differentiation is consuming time, has also caused the low yield of stem cell.
Stem cell and CFU-GM have the potential that is used for the treatment of various disease conditions, comprise malignant tumour, inborn error of metabolism, hemoglobinopathy and immune deficiency.Relate to a utilization of the stem cell that comes from Cord blood or placenta or a main field of research and be used for marrow and other relevant transplantations for using these cells produce small amounts of cells.Yet up to now, the no one proposes to produce a large amount of stem cells or CFU-GM, as people CD34 +Or CD133 +The method of CFU-GM.Specifically, a large amount of latter cell will use that the methods of treatment of CFU-GM becomes easily.At this, method disclosed by the invention relates to this demand.
Retinoid is as vitamin A and retinoic acid (RA), the known differentiation that influences stem cell.For example, retinoic acid has been used to suppress the propagation (Nadkarni etc. of irregular orientation (chronic myelogenous leukemia) candidate stem cell, 1984, Tumori 70:503-505) and in preceding marrow leukaemia, induce the differentiation and the forfeiture (Melchner etc. of self potential, 1985, Blood66 (6): 1469-1472).Retinoic acid also is used to induce the neuronic differentiation that comes from embryonic stem cell and suppresses spontaneous mesoderm differentiation (Slager etc., Dev.Genet.1993,14 (3): 212-24, Ray etc., 1997, J.Biol.Chem.272 (30): 18702-18708).Retinoic acid also further is used to induce the reproductive cell precursor (Damjanov etc. of conversion, 1993, Labor.Investig, 68 (2): 220-232), placenta cells precursor (Yan etc., 2001, Devel.Biol.235:422-432), reach the differentiation of endothelial cell precursor (Hatzopoulos etc., 1998, Development 125:1457-1468).Yet the effect of retinoid in differentiation very understood, and it can be used as the regulating measure of control stem cell differentiation and uses.
Folacin, the effect in the candidate stem cell differentiation has obtained research as aminopterin and amethopterin (methotrexate (MTX)).Folacin is used for acute lymphoblast anaemia and other blood proliferative disorders and cancers as chemotherapy reagent, and show that it influences the differentiation (DeLoia etc. of stem cell by killing certain class stem cell colony off, 1998, Human Reproduction13 (4): 1063-1069), therefore, can not become the effective tool of the differentiation that is used to regulate a large amount of stem cells that deliver medicine to the patient.
Some cell factors, as IL-1, IL-2, IL-3, IL-6, IL7, IL-11, also be shown with some albumen such as hematopoietin, Kit part, M-CSF and GM-CSF and instructed stem cell to be divided into particular cell types (Dushnik-Levinson etc. by hematopoietic lineage, 1995, Biol.Neonate 67:77-83), but these processes are not fully understood and still too coarse and inaccuracy and can not be as the regulating measure of control stem cell differentiation.
Up to now, the no one describes the application of for example hereinafter disclosed PDE IV inhibitor of compound in stem cell or precursor differentiation.Specifically, the no one proves such compound adjusting CFU-GM such as CD34 +CFU-GM, away from the purposes of dendritic cells pedigree differentiation, this differentiation is the useful ability that is used to improve the trnasplantion immunity tolerance.And the no one describes compound described herein in the purposes of breeding in order to produce the medical composition that contains this cell in the progenitor cell populations.The progenitor cell culture of this propagation is useful in the raising of treatment graft versus host disease(GVH disease) and immunological tolerance.Can produce effective cell colony in treatment because control the differentiation of stem cell and precursor, therefore, for control and adjusting marrow sample dendritic cells pedigree cell, or early progenitor cell, as people CD34 +Or CD133 +The ability of the differentiation of CFU-GM has required, with control dendritic cells and/or granulocytic generation.
3. summary of the invention
The invention provides the method for regulating mammal, particularly human stem cell or CFU-GM differentiation.Especially, method of the present invention can be used for regulating and controlling human stem cell along specific cell and differentiation and the maturation of organizing pedigree.The present invention includes PDE IV inhibitor, a compounds that particularly is known as SelCIDs (Celgene) influences the purposes of this adjusting and control.The invention further relates to the CFU-GM that delivers medicine to, to regulate its differentiation from particular approach at these compounds of special time.
Method of the present invention comprises that stem cell or CFU-GM are divided into the regulation and control of specific cells pedigree, these cell lineages include but not limited to, a matter, hematopoiesis, living fat, living liver, living neural pedigree that give birth to, collagen, that give birth to cartilage, angiogenic, myogenic, living cartilage or living bone.In specific embodiment, method of the present invention comprises that stem cell is divided into the regulation and control of hematopoietic lineage cell.
The present invention comprises that also committed cell becomes the regulation and control of particular cell types, and described cell type is for example mesenchymal cell, hematopoietic cell, adipocyte, liver cell, neuroblast, spongioblast, cartilage cell, endothelial cell (EC) CFU-GM, myocyte, cartilage cell or Gegenbaur's cell.In specific embodiment, the present invention includes directed hemopoietic progenitor cell and be divided into red blood cell, blood platelet or leucocyte (white blood corpuscle), as neutrophil cell, monocyte, macrophage, eosinophil, basophilic cell, mast cell, B cell, T cell or plasmacytic regulation and control.
In another embodiment, method of the present invention relates to regulates stem cell to hematopoietic lineage cell, specifically, and CD34 +, CD133 +And CD45 +The differentiation of hematopoietic lineage, and produce the prevention that contains this cell or treat the method that goes up useful pharmaceutical composition.In another embodiment, method of the present invention relates to the differentiation of adjusting early progenitor cell to dendritic cells pedigree or granulocyte pedigree, endothelial cell pedigree or cardiomyocyte lineage cell.
In another embodiment, the invention provides and regulate CFU-GM, especially the method for dendritic cells pedigree or granulocyte pedigree, endothelial cell pedigree, nerve cell pedigree or cardiomyocyte lineage cell differentiation to hematopoietic cell lineage.In specific embodiment, described CFU-GM is CD34 +Or CD133 +Cell.These regulation and control are by finishing with compound contact CFU-GM of the present invention in incubation.In one embodiment, described compound is a PDE IV activity inhibitor.In a more particular embodiment, described compound is a PDE IV inhibitor.More preferably, described PDE IV inhibitor is SelCID TM(referring to 4.3 following joints).
In another specific embodiment, method of the present invention comprises that CFU-GM is divided into the inhibition of dendritic cells.In another specific embodiment, the invention provides the method that is adjusted in the CFU-GM differentiation in the incubation in initial six days, its cultivation is used to produce the enrichment culture thing of this CFU-GM.In another embodiment, method of the present invention comprises early progenitor cell is developed into granulocytic promotion that it be effective that this granulocyte may infect for opposing.Granulocyte pedigree committed progenitor (CD15 +Being increased in cell) alleviates in neutrocytopenia and its infection complication subsequently and has potential purposes, and this illness has been represented the most general dose-limiting toxicity of cancer chemotherapy.In another embodiment, method of the present invention can be used for suppressing the differentiation of dendritic cells, and it is effective for the effect that relaxes graft versus host disease(GVH disease).
CFU-GM of the present invention, after compound adjusting of the present invention, for transplanting is effectively (promptly, and can be used as the renewable source that substitutes cell and tissue (as pancreas, heart, liver, kidney, liver, brain, lung, bladder, intestines or muscle cell) and be used for the treatment of regenerating medicine in normal aging, damage or disease such as cardiopathy, apoplexy, Parkinson's and the Alzheimer disease recovery of hematopoiesis).This cell also is useful in the biochemical route in the born of the same parents of the effect of measuring mediation compound of the present invention.These cells also are useful for new medicine and the toxin of screening, for example, are used for determining the potential of cancer therapy drug, understand the cause of inborn defect etc.
Method of the present invention can be used for suppressing specifically the generation of red blood cell or red blood cell colony (BFU-E and CFU-E), increases the output that leukocytic generation and blood platelet form colony (CFU-GM) and improve total colony forming unit simultaneously.Method of the present invention not only can be used for regulating stem cell and CFU-GM such as CD34 +The differentiation of CFU-GM also can be used for stimulating colony-forming efficiency, moves into speed and provides important help to HSCT by improving bone marrow graft.
Any mammalian stem cell can use according to method of the present invention, includes but not limited to the stem cell that separates from Cord blood, placenta and other sources.Stem cell can separate from any mammal species, for example mouse, rat, rabbit, cavy, dog, cat, pig, sheep, ox, horse, monkey etc., more preferably, the people.Stem cell can comprise pluripotent cell, that is, have and break up diversity, energy self completely and keep dormancy or static cell in tissue.Stem cell can also comprise multipotency cell or committed progenitor.In a preferred embodiment, the present invention utilizes stem cell alive, static, versatility, it is present in or results from subsequently the embryo of foot phase, in other words, this cell can be along with the childbirth and the placenta of success are given birth to, bloodletting and placenta perfusion and obtain reclaiming, cause the nearly generation and the recovery of 1,000,000,000 karyocytes, its karyocyte produces multipotency or the multipotent stem cells of 50-100 1,000,000.This cell is mentioned at this as people's placenta stem-cell or embryonic-like stem cell.
In a specific embodiment of the present invention, cell, for example the endogenous cell of the placenta of marrow or postpartum perfusion includes, but not limited to embryonic-like stem cell, CFU-GM such as CD34+ or CD133 +Cell, pluripotent cell or multipotential cell are exposed to compound of the present invention and are subjected to inducing differentiation.This endogenous cell can be in external breeding.In another embodiment, endogenous cell can be collected from placenta and medium and obtain, and cultivates under external suitable condition, and cultivation a period of time amount is enough to induce differentiation by cell type of wanting or pedigree.
In another embodiment of the invention, stem cell or CFU-GM are derived from other source as Cord blood, peripheral blood or adult blood, and are exposed in the compound of the present invention quilt and are induced differentiation.In a preferred embodiment, differentiation is carried out a period of time under external appropriate condition, and the described time is enough to induce to desired cell-line or cell type differentiation.Compound of the present invention is used for by adding, and produces in position or mode that other any permission stem cells or CFU-GM contact with compound of the present invention is used for breaking up the medium medium.
The time that has been found that compound administration of the present invention is to CD34 +The differentiation of CFU-GM has profound influence.Therefore, in an embodiment of the present invention, CD34 +CFU-GM is divided into dendritic cells and comprises that by a kind of the method that makes CFU-GM contact compound of the present invention when cultivating in first day obtains postponing or suppressing.In another embodiment, from CD34 +The CD1a that CFU-GM is come +Cells whose development is reduced or hinders by a kind of method that makes described CFU-GM contact compound of the present invention when first day cultivates that comprises.In another embodiment, come from CD34 +The CD1a of CFU-GM +The persistence of cell colony contacts this compound by the described CFU-GM of cultivation in the presence of no compound of the present invention and is improved after six days.
The present invention also comprises adjusting early progenitor cell such as people CD34 +And CD133 +The method of cell differentiation, comprise make CFU-GM propagation with the idiophase between different time contact with one or more compounds of the present invention.Therefore, in one embodiment, the present invention includes a kind of method of regulating CFU-GM differentiation, comprise described cell was only contacted with one or more compounds of the present invention cultivating in first day.In another embodiment, described cell contacts with dose with described compound any given day in first day to the 12nd day that cultivates.In another embodiment, described cell is at 0 to 12 day, comprise 0 day with 12 days different number of days in contact twice with described compound at least.In being still another embodiment, described cell propagation and/or differentiation contact with one or more compounds period one day twice, once a day or every once a day.In another embodiment, described contact is carried out external.Still in another embodiment, described contact is carried out in curee's body.In a more particular embodiment, described curee is people, inhuman mammal, birds or reptile.
Generally speaking, the endogenous that can cultivate in the placenta of perfusion in postpartum or exogenous stem cell or CFU-GM are exposed to compound of the present invention and can take place when these cells are incubated at placenta, or preferably can take place with these cells recovery or after removing from placenta external.
The present invention includes and have the active compound of PDE IV inhibition is grown conditioning agent as stem cell and/or CFU-GM purposes.In specific embodiment, this compound is that PDE IV inhibitor is as being known as SelCIDs TMA compounds (Celgene Corp., Warren, NJ).
The present invention also comprises through pretreated stem cell or CFU-GM and being used for the treatment of and prophylactic transplanting.In one embodiment, the patient that needs are transplanted before transplanting, middle and/or transplant after also administration compound of the present invention.
The present invention further comprises by the CFU-GM of method generation of the present invention or the purposes of specific cell type.In other words, the present invention includes the purposes of the leucocyte, granulocyte or the dendritic cells that are made by the hemopoietic progenitor cell differentiation, wherein said CFU-GM differentiation is regulated with compound of the present invention or is regulated and control.
In another embodiment, the present invention includes control or the regulation and control of stem cell, by in vivo with the patient's administration of stem cell and micromolecular compound of the present invention to needs.
In one embodiment, the invention provides and comprise CD34 +Or CD133 +The pharmaceutical composition of CFU-GM and pharmaceutically suitable carrier, wherein said CFU-GM promoting in preceding 6 days that cultivate under the condition of described progenitor cell proliferation and differentiation and compound of the present invention, especially suppress the compound contact of PEDIV activity.In one specific embodiment, pharmaceutical composition comprises the cell of cultivating back collection and cryopreservation in six days.In another specific embodiment, the cell in the pharmaceutical composition is CD34 +CD38 -CD34 -Or CD34 +CD38 -CD34 +Cell.In another specific embodiment, with the compound of cells contacting be PDE IV inhibitor of the present invention.In another specific embodiment, with the compound of cells contacting be SelCID TM
In another embodiment, the present invention also provides the method for pharmaceutical compositions, comprises with the compound contact CD34 that suppresses PED IV activity +Or CD133 +CFU-GM, wherein said CFU-GM was cultivated in medium six days under the condition of culture that allows described progenitor cell proliferation and differentiation; After cultivating six days, collect described cell; And accept the combination of carrier and described cell with medicinal.In a specific embodiments of this method, described contact was carried out in cultivation in first day.In another specific embodiments of this method, described contact is carried out twice in described six days cultivate at least.In another embodiment, the compound with cells contacting is a PDE IV inhibitor of the present invention.In another specific embodiment, with the compound of cells contacting be SelCID TMIn being still another specific embodiment of this method, described CFU-GM is separated from other haemocyte before described cultivation.In another specific embodiment of this method, described medium also comprises GM-CSF and TNF-α in addition.In another more particular embodiment of this method, described SelCID TMConcentration with 0.1 μ M to 10.0 μ M exists.In another more particular embodiment of this method, described SelCID TMConcentration with 1.0 μ M exists.In another specific embodiment of this method, described cell after collection by cryopreservation.
The present invention further provides the method for propagation progenitor cell populations in mammalian subject, comprise that administration is to treat the CD34 of effective dose +Or CD133 +CFU-GM and one or more SelCIDs TMIn described acceptor mammalian subject.In the specific embodiments of this method, described CFU-GM is broken up in the acceptor mammalian subject.In another specific embodiment of this method, described CFU-GM with the form administration of the cellular preparations of essentially no red blood cell in described curee.In another specific embodiment of this method, described CFU-GM with the cellular preparations form administration that comprises bone marrow cell, placenta cells, cord blood cell or PBMC in the acceptor mammalian subject.In another specific embodiments of this method, described CFU-GM with the form administration of carrier associating in the acceptor mammalian subject.In another specific embodiments of this method, described CFU-GM is CD34 +CD133 +CFU-GM.In another specific embodiments of this method, described CFU-GM is expressed the purpose genetic stocks that mixes.
The present invention also provides the cell as pharmaceutical composition by method for preparing.
Still in another embodiment, present invention resides in for example CD34 of cryopreservation and thawing back adjusting stem cell or CFU-GM +CFU-GM is to eliminate cryopreservation and to be exposed to the method for freezing agent to the adverse effect of stem cell.In certain embodiments, the invention provides at cryopreservation and melt the back and regulate stem cell, be exposed to the freezing agent (for example, DMSO) and to the method for the adverse effect of stem cells hyperplasia and transfer ability with elimination.
3.1 definition
As used herein, term " bio-reactor " is meant and is used for propagated cell, production or expresses the vitro system of biomaterial and growth or cultured cell tissue, organoid, virus, albumen, polynucleotides and microorganism.
As used herein, " DC cell " is meant dendritic cells.
As used herein, " early progenitor cell " is meant CD34 +CFU-GM, CD133 +Arbitrary equivalent in CFU-GM or mammal, bird or the reptiles.
As used herein, term " embryonic stem cell " is meant and comes from the cell of giving birth to cell mass in the blastula (pl blastulae) (as 4-5 days people embryo), and has versatility.
As used herein, term " embryonic-like stem cell " is meant the non-cell that comes from the blastula (pl blastulae) inner cell mass.As used herein, " embryonic-like stem cell " also may be meant " placenta stem-cell ".Preferred embryo sample stem cell is a versatility.Yet, can comprise embryonic-like stem cell, multipotency cell and committed progenitor from the stem cell that placenta obtains.According to method of the present invention, the embryonic-like stem cell that comes from placenta can be collected from the placenta that separates, and bloodletting and perfusion that it has passed through a period of time are enough to remove residual cell.Preferably, embryonic-like stem cell is the people source, though can derive from any mammal.
As used herein, term " bloodletting " or " bloodletting " when use relevant with placenta, are meant from placenta and remove and/or discharge all basically Cord bloods.According to the present invention, the placenta bloodletting can be passed through, and for example, but does not limit the outflow of discharge, gravity being induced, massage, extruding, suction or the like and finish thus.In a preferred embodiment, the bloodletting of placenta can be further by perfusion, rinsing or with containing or do not contain reagent, as the liquid wash placenta of anticoagulant, to help the bloodletting of placenta and finish.
As used herein, term " perfusion " or " perfusion " are meant that seeing through organ or tissue flows out or discharge liquid, and preferably, the discharge that sees through the liquid of organ or tissue has enough strength or pressure to remove any residual cell, as, come from the non-adherent cell of organ or tissue.As used herein, term " perfusion liquid " is meant the liquid of collecting along with discharging from organ or tissue.In an embodiment preferred, perfusion liquid comprises one or more anticoagulant.
As used herein, term " endogenous cell " refers to a kind of " non-external source " cell, that is, a kind of self or autologous cell, it comes from placenta.
As used herein, term " foreign cell " is meant " external " cell, that is, heterogenous cell (promptly, come from " non-oneself " cell of non-placenta donor source) or come from the autologous cell (that is, coming from " the self-cell " of placenta donor) of the organ or tissue of non-placenta.
As used herein, term " PDE IV inhibitor " is meant the hereinafter disclosed compound of 4.3 joints.
As used herein, term " organoid " is meant with appearance form or as any organ or mammalian body, the preferably aggregation of one or more cell types of the practical structures form of the body of gland of human body assembling.
As used herein, term " multipotency cell " is meant the cell with the ability that is grown to the about 260 kinds of arbitrary subclass of cell type of mammalian body.Be different from multipotential cell, the multipotency cell does not have the ability that forms all cells type.
As used herein, term " multipotential cell " is meant to have and breaks up multifarious cell fully, that is, is grown to the ability of the about 260 kinds of cell types of mammalian body.But the multipotential cell self, and can in tissue, keep dormancy or static.Be different from totipotent cell (for example, the dliploid egg cell of fertilization), embryonic-like stem cell can not form new blastaea usually.
As used herein, term " CFU-GM " is meant that directed differentiation is particular cell types or the cell that forms particular organization.
As used herein, term " stem cell " is meant and can regenerates indefinitely and the standard cell lines of the qualification cell of formative tissue and organ.Stem cell is the cell that has versatility or multipotency on growing.Stem cell can be divided and produced two filial generation stem cells, or filial generation stem cell and an ancestral (transformations) cell, and it breeds cell maturation, Gestalt for organizing subsequently.
As used herein, term " totipotent cell " is meant the cell that can form a complete embryo (for example, blastula (pl blastulae)).
4. detailed Description Of The Invention
The present invention partly is based on unexpected discovery, be stem cell or CFU-GM be exposed to compound of the present invention cause produce a kind of control stem cell or CFU-GM to specific progenitor cell populations differentiation or CFU-GM to particular cell types, the regulatable method of breaking up as dendritic cells, granulocyte, endothelial cell or nerve cell.Specifically, stem cell or CFU-GM are exposed to compound of the present invention causes generation hematopoietic cell special group, comprises CD34 +, CD38 +And CD133 +The regulatable differentiation and the propagation of cell.The regulation and control of this differentiation are not because of cell death or be divided into the unwanted cells type or cell lineage causes under the situation of remarkable loss of output and finishes; In other words, compound of the present invention does not cause the apoptosis of one or more cell colonys.Further, hemopoietic progenitor cell is exposed to regulatable differentiation and the propagation that compound of the present invention causes particular cell types.
Thereby, the invention provides mediator stem cell differentiation, particularly CD34 +Hemopoietic progenitor cell and CD133 +The method of CFU-GM differentiation.Specifically, the invention provides use and suppress the little organic molecule adjusting CFU-GM of PDE IV activity along specific cells and the method for organizing the pedigree differentiation.In addition, the present invention includes the propagation early progenitor cell, as people CD133 +Or CD34 +Cell, especially CD34 +CD38 -Cell to be implanted into the method in mammal, birds or the reptile, comprises hemopoietic progenitor cell is exposed to PDE IV inhibitor or antagonist that wherein inhibitor or antagonist are small-molecule substances.The present invention also provides the method that produces the other types cell from these early progenitor cells, includes but not limited to brain cell, nephrocyte, intestinal cell and muscle cell.Compound of the present invention is also to suppressing from early progenitor cell, as people CD34 +The dendritic cell differentiation of CFU-GM and promote granulocytic differentiation to work.
The example of the micromolecular compound that uses includes but not limited to related to the present inventionly, suppresses the compound of PDE IV activity.The compound of Shi Yonging is described in detail at 4.3 joints in the method for the invention.In an especially preferred embodiment, this compound is SelCIDs TM(Celgene).
Method of the present invention comprises the regulation and control that stem cell or CFU-GM are broken up to the specific cells pedigree, these cell lineages include but not limited to, mesochymal, hematopoiesis, living fat, living liver, living neural, collagen, living cartilage, angiogenic, myogenic, living pedigree cartilage or skeletonization, it comprises preferably external causes the cytophyletic cell differentiation of cell to expectation with stem cell or CFU-GM and compound of the present invention one enough period of incubation together.In a specific embodiment, be that the differentiation of cell is regulated to a kind of hematopoiesis to stem cell or CFU-GM.Specifically, method of the present invention can be used for regulating the haemocyte colony by CD34 in the relevant mode of a kind of dosage +, CD133 +And CD45 +The generation of hemopoietic progenitor cell.
Method of the present invention also comprises CD34 +CFU-GM is to the adjusting of dendritic cell differentiation, and it comprises preferably external causes the cytophyletic cell differentiation of cell to expectation with CFU-GM and compound of the present invention incubation sufficiently long one period together.In a specific embodiment, this CFU-GM is passed through described cells contacting PDEIV inhibitor, particularly SelCID to the differentiation of dendritic cells pedigree cell TMOr this class inhibitor or SelCID TMAnalog or pro-drug and regulating.In another specific embodiment, CD34 +The differentiation of CFU-GM is adjusted to suppress along the differentiation of marrow sample pedigree and to support differentiation along the granulocyte pedigree.In a more particular embodiment, CD34 +CFU-GM to the differentiation of granulocyte pedigree cell by comprising with CD34 +CFU-GM is regulated in the method that contacted in the 1st day that CFU-GM is cultivated with compound of the present invention.
Any mammalian stem cell or CFU-GM can be used according to method of the present invention, include but not limited to the stem cell that separates from Cord blood (" CB " cell), placenta and other sources.Stem cell can comprise multipotential cell, that is, have and break up diversity, energy self completely and keep dormancy or static cell in tissue.Stem cell also can comprise multipotency cell and committed progenitor.In a preferred embodiment, the present invention utilizes stem cell alive, static, versatility, it is present among the embryo of foot phase, can be along with the childbirth and the placenta of the success of the recovery that causes multipotency and multipotent stem cells are given birth to, bloodletting and placenta perfusion and reclaimed.
In another preferred embodiment, CFU-GM is early progenitor cell, especially CD34 +Or CD133 +Cell.Preferably, CD34 +Or CD133 +CFU-GM derives from people's marrow, placenta or Cord blood.Also can use from the equivalent of these cells in other mammals source.For example, in mouse, Sca +CFU-GM can be used in the method for the invention.Also can use from the early progenitor cell of the equivalence in birds or reptile source.
In a specific embodiment of the present invention, placenta is endogenous or by pouring into the cell that placenta produces postpartum, include but not limited to, embryonic-like stem cell, CFU-GM, multipotential cell and multipotency cell, be exposed to compound of the present invention when in that separate and placenta perfusion, cultivating, and induce differentiation.The endogenous cell of breeding in the placenta of postpartum perfusion can reclaim, and/or bioactive molecule can reclaim from perfusion liquid, medium or from placenta cells self.
In another embodiment of the invention, with come from except postpartum placenta the stem cell in other source or CFU-GM be exposed to compound of the present invention and induce differentiation when the culture in vitro.Therefore, the present invention includes and make mammalian stem cell be divided into the method for specific CFU-GM, it comprises makes stem cell under the situation of the condition of the differentiation that is suitable for expecting and/or medium and under the situation that compound of the present invention exists stem cell is broken up.
Further, the present invention includes and regulate or regulate and control the method for specific progenitor cell populations to the particular cell types differentiation, it comprises makes described CFU-GM under the condition that is fit to described differentiation and break up in the presence of one or more compounds of the present invention.Selectively, stem cell or CFU-GM can be exposed to compound of the present invention, break up with appropriate condition subsequently.The example of appropraite condition comprises the nutrient medium preparation that replenishes with human serum and cell culture substrate, as the MATRIGEL that replenishes with growth factor
Method of the present invention also relates to different cell colonys, its can by with compound of the present invention at the different times of cultivating, no matter be propagation or idiophase to contact CFU-GM and produce.Referring to 4.4 joints, be specially following 4.4.2 joint.
In a specific embodiment, the invention provides and use little molecule, especially PDEIV inhibitor, the method for the hemopoietic in before hemopoietic progenitor cell is transplanted adjusting period is regulated and regulated and control to preferred SelCID or its prodrug.
The present invention also provides and has used small-molecule substance of the present invention to regulate and regulate and control the method that hemopoietic progenitor cell is regulated the hemopoietic in period in the body of formerly external back.Method of the present invention comprises the regulation and control of stem cell or CFU-GM vitro differentiation, is included in external incubation compound and stem cell or CFU-GM, subsequently the cell that breaks up to curee's directly transplanting.
The present invention also comprise by with stem cell or CFU-GM and compound administration of the present invention in its have demand the patient and in vivo to the control and the regulation and control of stem cell or CFU-GM.
The present invention comprises that further the transplanting of pretreated stem cell or CFU-GM is with treatment or prevent disease.In one embodiment, the patient that needs are transplanted before transplanting, transplant in and/or transplant the back administration with compound of the present invention.In another embodiment, also undressed stem cell of patient's administration or the CFU-GM that needs are transplanted, for example cord blood cell, adult blood cell, peripheral blood cells or bone marrow cell.In another embodiment, method of the present invention comprises that to curee's administration with compound, described curee is the acceptor of unadjusted stem cell or CFU-GM, to induce the adjusting effect to the stem cell of having transplanted.
In certain embodiments, the present invention includes bone-marrow transplantation, it comprises transplants Cord blood (or the stem cell that obtains from Cord blood), periphery (that is, adult) blood (or the stem cell that obtains from peripheral blood), and wherein said Cord blood or stem cell have used compound of the present invention pretreated.In addition, the present invention includes the leukocytic purposes that makes by the hemopoietic progenitor cell of in the presence of compound of the present invention, having broken up.For example, the leucocyte that produces by the differentiation hemopoietic progenitor cell can be used for transplanting or can mixing with Cord blood or cord blood stem cell before transplanting.
In other embodiment, the present invention includes bone-marrow transplantation, it comprises the early progenitor cell that obtains according to method of the present invention, as CD34 +Or CD133 +CFU-GM is transplanted, and wherein said CFU-GM has used compound of the present invention pretreated.In one embodiment of the invention, the precursor of described dendritic cells is CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Precursor.In addition, the present invention includes the CD34 that in the presence of compound of the present invention, has broken up by +The purposes of the cell that CFU-GM makes.For example, pass through CD34 +The differentiation of CFU-GM and the CD34 that produces with compound of the present invention +CD38 -CD33 +Precursor, CD34 +CD38 -CD33 -Precursor, granulocyte etc. are in can be used for transplanting.With compound of the present invention from CD133 +The cell that cell differentiation is come is also included for the present invention.
The present invention further is included in cryopreservation and melts the back and regulates stem cell, to eliminate cryopreservation and to be exposed to the method for freezing agent to the adverse effect of stem cell.In certain embodiments, the invention provides at cryopreservation and melt the back and regulate stem cell, be exposed to the freezing agent (for example, DMSO) to the method for the adverse effect of the propagation of stem cell and transfer ability with elimination.
4.1 stem cell and CD34 +Or CD133 +The regulation and control of the differentiation of CFU-GM
4.1.1 stem cell
The invention provides the method for mediator stem cell differentiation.In certain embodiments, method of the present invention is included in the regulation and control of external stem cell or CFU-GM differentiation, comprise external with stem cell with the compound incubation, subsequently to the cell of curee's directly transplanting differentiation.In another embodiment, method of the present invention comprises the regulation and control of interior stem cell of body or CFU-GM differentiation, comprises that the curee to unadjusted stem cell acceptor sends compound, directly gives drug compound to the curee subsequently.
The embryonic-like stem cell that obtains by method of the present invention can be induced the differentiation to the specific cells pedigree, and that these cell lineages include but not limited to is mesochymal, hematopoiesis, it is fat, livings liver to give birth to, it is neural, collagen to give birth to, give birth to cartilage, angiogenic, myogenic, living pedigree cartilage or skeletonization.
In certain embodiments, will induce differentiation, to be used for transplanting and the scheme of earlier external back interior therapeutic according to the embryonic-like stem cell that method of the present invention obtains.In certain embodiments, induce differentiation by the embryonic-like stem cell that method of the present invention obtains to specific cell type, and be subjected to genetic manipulation so that the product of gene therapy to be provided.In a specific embodiment, the embryonic-like stem cell that obtains by method of the present invention external with compound of inducing its differentiation such as little organic molecule incubation, subsequently to the cell of curee's directly transplanting differentiation.In a preferred embodiment, be used to control and the compound of regulating and control the stem cell differentiation is not polypeptide, peptide, albumen, hormone, cell factor, oligonucleotides or nucleic acid.
The stem cell of using according to the present invention comprises, but be not limited to, Cord blood (CB) cell, placenta cells, embryo do (ES) cell, embryonic-like stem cell, trophoderm stem cell, CFU-GM, stem cell and multipotency, versatility and totipotent cell.
Specifically, method of the present invention comprises the regulation and control that the stem cell colony except that mescenchymal stem cell breaks up to particular organization's pedigree.For example, the generation and the specific muscle tissue of regeneration by promoting the specific muscle bone and reparation, neovascularity, as cardiac muscle and skeletal muscle, and the revascularization of various organs and tissue, described organ and tissue include but not limited to brain, spinal cord, liver, lung, kidney and pancreas, and method of the present invention can be used for regulating and control the multipotency stem cell to giving birth to cartilage, angiogenic, myogenic meat and differentiation skeletonization pedigree cell.Method of the present invention can be used for regulating and control the differentiation of multipotency stem cell to the pedigree of giving birth to fat, living cartilage, skeletonization, living nerve or living liver.
The reagent that is used for regulating differentiation can import the embryo of perfusion in postpartum to induce the differentiation in embryo's cultured cells.Optionally, this reagent can be used for from placenta collecting or removes behind the cell in external adjusting differentiation.
Method of the present invention comprises the regulation and control of CFU-GM to hematopoietic lineage cell differentiation, is included in externally with CFU-GM and one enough period of compound incubation, causes the differentiation of cell to hematopoietic lineage.Specifically, method of the present invention can be used for regulating by CD34 in the relevant mode of dosage +, CD133 +And CD45 +Hemopoietic progenitor cell generates the generation (about the discussion of dosage, seeing 4.7 joints) of haemocyte colony.
Preferably, method of the present invention can be used for suppressing specifically the generation of red blood cell or red blood cell colony (BFU-E and CFU-E), increase simultaneously leucocyte and blood platelet colony form (CFU-GM) the two generation and the output that improves total colony forming unit.Method of the present invention not only can be used for regulating and control the differentiation of stem cell, also can stimulate colony-forming efficiency, the speed by improving the marrow implant and the recovery of leucocyte and/or platelet production and provide significant benefits to HSCT.
In other embodiment, method of the present invention can be used for regulating and control the differentiation to specific neural cell type such as sensory neuron (for example, cellula visualis, olfactory cell, mechanical sense are answered neuron, chemical co-ordination neuron etc.), motor neuron, cortical neuron or intrerneuron of neuronal precursor for example or neuroblast.In other embodiment, method of the present invention can be used for the differentiation that regulation and control include but not limited to cholinergic neuron, dopaminergic neuron, GABAergic neuron, Deiter's cells (comprise oligodendroglia, it produces myelin) and ependymocyte (it is arranged in ventricular system) cell type.Still in other embodiment, method of the present invention can be used for being regulated to the differentiation of the cell of organ component, includes but not limited to the pul base alunite cell of heart, the bile duct epithelial cell of liver, β islet cells, cortex renis or the myelocyte of pancreas and the visual impression photo-cell of eyes.
The evaluation of the stem cell differentiation state that obtains according to method of the present invention can be identified by the existence of cell surface marker.For example, embryonic-like stem cell of the present invention can pass through following cell surface marker: OCT -4 +And ABC -Pt and distinguishing.In addition, the present invention includes the embryonic-like stem cell that has following mark: CD10, CD29, CD44, CD54, CD90, SH2, SH3, SH4, OCT-4 and ABC-p or lack following cell surface marker: CD34, CD38, CD45, SSEA3 and SSEA4 as described.Such cell surface marker can be according to method conventional determining well known in the art, for example, by the flow cytometry method, follows washing and with the antibody staining of anti-cell surface markers.For example, for measuring the existence of CD34 or CD38, cell can wash in PBS, uses the phycocyanin of anti-CD34 and the fluorescein isothiocynate of anti-CD38 (Becton Dickinson, Mountain View, CA) two dyeing then.
4.1.2CD34 +And CD133 +Early progenitor cell
The present invention also provides mediator CD34 +Or CD133 +The method of cell differentiation.In certain embodiments, method of the present invention is included in the regulation and control of external stem cell or CFU-GM, comprises external incubation stem cell and compound, subsequently the cell directly transplanting of differentiation is gone into the curee.
Can induce differentiation along the specific cells pedigree by the CFU-GM that method of the present invention obtains, include, but not limited to for CD34 +CFU-GM is along marrow sample or granulocyte pedigree, for CD133 +Cell is along endothelium or nerve cell pedigree.In certain embodiments, CFU-GM is induced differentiation to be used for transplanting with elder generation external back interior therapeutic scheme.In certain embodiments, CFU-GM is induced differentiation to special cell type, and is subjected to genetic manipulation so that the product of gene therapy to be provided.In specific embodiment, with CFU-GM external with compound of inducing its differentiation such as little organic molecule incubation, subsequently to the cell of curee's directly transplanting differentiation.In a preferred embodiment, be used to control and the compound of regulating and control the stem cell differentiation is not polypeptide, peptide, albumen, hormone, cell factor, oligonucleotides or nucleic acid.In another preferred embodiment, CFU-GM is facilitated to CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -The CFU-GM differentiation.
Preferably, method of the present invention can be used for suppressing specifically the generation of red blood cell or red blood cell colony (BFU-E and CFU-E), increases leucocyte and blood platelet colony simultaneously and forms the generation of (CFU-GM) and the output that improves total colony forming unit.Method of the present invention not only can be used for regulating and control the differentiation of stem cell, also can stimulate colony-forming efficiency, the speed by improving the marrow implant and the recovery of leucocyte and/or platelet production and provide significant benefits to HSCT.
In other embodiment, method of the present invention can be used for reducing CD34 +CFU-GM is to CD1a +Cell, especially CD86 +CD1a +The differentiation of cell.In another embodiment, method of the present invention can be used for reducing or preventing CD34 +CFU-GM is to CD14 +CD1a -The differentiation of cell.CD14 +CD1a -Cell is epidermis dendritic cells or monokaryon/huge CFU-GM of biting.In another embodiment, method of the present invention can be used for reducing the CD34 in propagation +The expression of CD80 and CD86 costimulatory molecules on the CFU-GM.In another embodiment, method of the present invention can be used for reducing the CD34 of propagation +CFU-GM is to CD54 BrightThe differentiation of cell, and excitation is to CD54 DimThe differentiation of cell.In another embodiment, method of the present invention can be used for increasing CD133 +The quantity of cell, it is the endothelial cell CFU-GM.Still in another embodiment, method of the present invention can be used for reducing the CD34 of propagation +Cell is to CD11c -CD15 +The differentiation of cell, and increase to CD11c +CD15 -The differentiation of cell is broken up thus from marrow sample dendritic cells pedigree and is changed the granulocyte pedigree into.
The evaluation of the stem cell differentiation state that obtains according to method of the present invention can be identified by the existence of cell surface marker.For example, CFU-GM of the present invention can be passed through CD34 +Or CD133 +Cell surface marker and distinguishing.In addition, the present invention includes and have or show with respect to contrast high expressed one or more following mark: CD15, CD34, CD33, CD133 or CD54 as described DimThe CFU-GM of propagation.The present invention further comprises shortage or shows with respect to low one or more following mark: HLA-DR, CDla, CD11c, CD38, CD80, CD86, the CD54 of expressing of contrast BrightOr the CFU-GM of the propagation of CD14.In a preferred embodiment, the CFU-GM of propagation of the present invention presents CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Such cell surface marker can be according to method conventional determining well known in the art, for example, and by washing with the antibody staining of anti-cell surface markers, subsequently through flow cytometry method mensuration.For example, for measuring the existence of CD34 or CD38, cell can wash in PBS, uses the phycocyanin of anti-CD34 and the fluorescein isothiocynate of anti-CD38 (BectonDickinson, Mountain View, CA) two dyeing then.
In certain embodiments, the cell of differentiation can be tested and appraised the cytophagy ability of differentiation and characterize.For example, can be by measuring the phagocytosis that intake is estimated the cell that has broken up or breaking up with FITC mark dextran and with known method.The ability of cytositimulation T cell differentiation or that just breaking up can be measured in mixed lymphocyte reaction (MLP) (MLR), and wherein the cell of Jia Ding load antigen and T mixing with cells are measured T cell activity level then.
4.1.3 the evaluation of cell and sign
In certain embodiments, the cell that has broken up can be identified (difference that for example, shows gene pool with the gene pool of the cell that has broken up that comes from CFU-GM in undifferentiated CFU-GM source) by the gene of performance differential expression.For example, nucleic acid amplification method such as polymerase chain reaction (PCR) (PCR) or can be used for being presented in the gene expression in the different cell colonys based on the amplification method of transcribing (for example, in-vitro transcription (IVT)), for example, by using the polynucleotides microarray.This method that is used to represent differential gene expression be in the art known (see, for example, Wieland etc., Proc.Natl.Acad.Sci.USA 87:2720-2724 (1990); Lisitsyn etc., Science259:946-951 (1993); Meth.Enzymology 254:291-304 (1995) such as Lisitsyn; U.S. Patent number 5,436,142; U.S. Patent number 5,501,964; Lisitsyn etc., NatureGenetics 6:57-63 (1994); Hubank and Schatz, 1994, Nucleic Acids Research22:5640-5648; Zeng etc., 1994, Nucleic Acids Research 22:4381-4385; U.S. Patent number 5,525,471; Linsley etc., U.S. Patent number 6,271,002 is entitled as " RNA amplification method ", is disclosed in 2001-08-07; Van Gelder etc., U.S. Patent number 5,716,785 is entitled as " process of using promotor to carry out genetic manipulation ", is disclosed in 1998-02-10; Stoflet etc., Science 239:491-494,1988; Sarkarand Sommer, 1989, Science244:331-334; Mullis etc., U.S. Patent number 4,683,195; Malek etc., U.S. Patent number 5,130,238; Kacian and Fultz, U.S. Patent number 5,399,491; Burg etc., U.S. Patent number 5,437,990; R.N Van Gelder etc., (1990), Proc.Natl.Acad.Sci.USA87,1663; D.J.Lockhart etc., 1996, Nature Biotechnol.14,1675; Shannon U.S. Patent number 6,132,997; Lindemann etc., U.S. Patent number 6,235,503 is entitled as " method of cutting down hybridization and variance analysis ", is disclosed in 2001-05-22).
It is useful that commercial existing kit represents for gene, for example, shows PROFILE TM(CA), it uses the processing method based on gel to represent gene expression to the reagents series box for Qbiogene, Carlsbad.This kit utilizes restriction fragment difference to show that PCR (RFDD-PCR) compares gene expression pattern in eukaryotic.The PCR-selectivity is cut down kit (Clontech) and PCR-selectivity differential screening reagent box (Clontech) also can use, and it allows to cut down the evaluation of differential expression clone in the library.Cut down kit generation difference expression gene storehouse with the PCR-selectivity after, PCR-selectivity differential screening reagent box obtains using.With the direct probe and reduction library and hybridization that synthesizes by detection and driven element cluster, a kind of probe that originates from reduction cDNA, and the probe of a kind of system self-reversal reduction cDNA (reduction second time that is reversed).Obtain differential expression with the clone who detects son rather than driven element probe hybridization; Yet the probe of Xiao Jianing is not sensitive inadequately for detecting micro-information.The probe of cutting down is that differential expression cDNA obtains a large amount of enrichments, but may provide false-positive result.(Clontech) according to manufacturer instructs probe that use is cut down and that do not cut down to identify difference expression gene.
In another embodiment, the stem cell of differentiation or CFU-GM obtain identifying and characterize by the colony forming unit determination method, and it is known to usually, as MesenCuIt in the art TMMedium (stem cells technology, Inc., Vancouver British Columbia).
Stem cell or CFU-GM have been divided into the mensuration of special cell type and can have finished by means commonly known in the art, for example, (for example use as flow cytometry method or immunocytochemistry, with the dyeing of tissue specificity or cell marking specific antibody pair cell) variation of measuring morphologic and cell surface marker, by with optics or the morphological examination of Laser Scanning Confocal Microscope pair cell, or use technology well known in the art, and show as PCR and gene expression, measure the variation of gene expression.
4.2 stem cell and progenitor cell populations
The invention provides the method for mediator stem cell differentiation.Any mammalian stem cell can use in the method for the invention, includes, but not limited to separate the stem cell from Cord blood (CB cell), peripheral blood, adult blood, marrow, placenta, mescenchymal stem cell and other sources.In not preferred embodiment, stem cell is an embryonic stem cell or from the source isolated cells of non-placenta.
The source of mescenchymal stem cell comprises marrow, embryo's yolk sac, placenta, umbilical cord, fetus and teenager's skin and blood.For example, bone marrow cell can obtain from the cavity of iliac crest, leg section, tibia, rib or other marrows.
The stem cell of using according to method of the present invention can comprise multipotential cell, that is, have and break up diversity, energy self completely and keep dormancy or static cell in tissue.Stem cell also can comprise multipotency cell, committed progenitor and fibroblast.In a preferred embodiment, the stem cell that utilizes of the present invention be the stem cell of mazolytic work, static, the versatility of pouring into from the bloodletting of foot phase.
Stem cell colony can be made up of the placenta stem-cell that obtains by commerce services, the LifeBank U.S. (Cedar Knolls for example, NJ), ViaCord (Boston MA), Cord BloodRegistry (San Bruno, CA) and Cryocell (Clearwater, FL).
Stem cell colony also comprises according to U. S. application, publication number US20020123141, be published in 2002-09-05, be entitled as " method of collecting placenta stem-cell " and U. S. application, publication number US20030032179 is published in 2003-02-13, is entitled as the placenta stem-cell that " postpartum mammal placenta; it is used and placenta stem-cell thus " (at this, the full content of quoting each piece as a reference) disclosed method is collected.
In one embodiment, can use stem cell from Cord blood.Primary collection from the blood of placenta mentions that as Cord blood it mainly comprises CD34 +And CD38 +Hemopoietic progenitor cell.In first 24 hours of perfusion in postpartum, the CD34 of high concentration +CD38 -Hemopoietic progenitor cell can be separated from placenta.After about 24 hours of perfusion, the CD34 of high concentration -CD38 -Cell can be along with aforesaid cell together separates from placenta.The placenta of the perfusion that the present invention separates provides a large amount of enrichment CD34 +CD38 -And CD34 -CD38 +The source of human stem cell of stem cell; Poured into 24 hours or the placenta of separation of longer time provides a large amount of enrichment CD34 -And CD38 -The source of human stem cell of stem cell.
The preferred cell that uses according to method of the present invention is the embryonic-like stem cell that comes from the placenta of bloodletting perfusion, or the cell that obtains from embryo's sample placenta stem-cell.Embryonic-like stem cell of the present invention can obtain characterizing by using the variation of measuring gene expression as round pcr as the variation and the use of flow cytometry method and immunocytochemical technique measurement morphology and cell surface marker.In one embodiment of the invention, this embryonic-like stem cell can obtain characterizing by the existence of following cell surface marker: CD10, CD29, CD44, CD54, CD90, SH2, SH3, SH4, OCT-4 and APC-p or the shortage of following cell surface marker: CD34, CD38, CD45, SSEA3 and SSEA4.In a preferred embodiment, such embryonic-like stem cell can obtain by the existence of cell surface marker OCT-4 and ABC-p characterizing.Such cell surface marker can for example, by the flow cytometry method, wash and use the antibody staining of anti-cell surface markers afterwards according to method conventional determining well known in the art.For example, for measuring the existence of CD34 or CD38, cell can wash in PBS, uses the phycocyanin of anti-CD34 and the fluorescein isothiocynate of anti-CD38 (Becton Dickinson, MountainView, CA) two dyeing then.
The embryonic-like stem cell that is derived from placenta has the characteristic of embryonic stem cell but is not to obtain from the embryo.In other words, the present invention includes OCT-4 +And ABC-p +The purposes of cell, it is the mazolytic undifferentiated stem cell from the perfusion in postpartum.This cell is the same with human embryo stem cell to be omnipotent (for example, versatility).As mentioned above, many different versatilities or multipotency stem cell can separate at the placenta of different time points from perfusion, for example, and CD34 +CD38 +, CD34 +CD38 -And CD34 -CD38 -Hematopoietic cell.According to method of the present invention, people's placenta is in the source of postpartum as embryonic-like stem cell.
For example, after giving birth to from the uterus, placenta by bloodletting as quickly as possible with prevention or reduce Apoptosis.Subsequently, placenta is just perfusion as soon as possible after a bloodletting, to remove any other material that exists in blood, residual cell, albumen, the factor and the organ.The material fragment is also removed from placenta.Perfusion continues at least 2 hours extremely above 24 hours usually with suitable perfusion liquid.In some other embodiments, placenta is subjected at least 4,6,8,10,12,14,16,18,20,22 hours perfusion.In other words, the present invention's discovery that can activate by bloodletting and perfusion time enough based on the cell in the postpartum placenta to small part.Therefore, placenta can be easy to the rich and abundant source as embryonic-like stem cell, and its cell can be used for research, comprises drug discovery, treatment of diseases and prevention, and especially surgery is transplanted and therapy, and the generation that is used for committed cell, tissue or organoid.See series number 10/004,942 co-pending application, submit in 2001-12-05, be entitled as the application of " method of collecting placenta stem-cell " and series number 10/076,180, submit in 2002-02-13, be entitled as " postpartum mammal placenta; it is used and placenta stem-cell thus ", at this, the full content of quoting each piece as a reference.
Embryonic-like stem cell can be from the placenta of drainage extracts by the method for the perfusion technique that utilizes arteria umbilicalis and umbilical vein.Placenta is preferably by bloodletting drainage (for example residual Cord blood) with collecting residual blood.Then, handle under the state of the placenta of drainage bio-reactor environment in setting up elder generation's external back body, natural, the embryonic-like stem cell that wherein resides at parenchyma and blood vessel external cavity obtains recruiting.This embryonic-like stem cell migrates in drainage, the empty microenvironment, according to method of the present invention it is collected, and preferably washes in the collecting pipe by perfusion.
That especially pay close attention to as part of the present invention is CD34 +And CD133 +CFU-GM is to myeloid cell, especially dendron or granulocytic adjusting.Nearest report shows that this cell is a versatility; Therefore, the present invention also pays close attention to the adjusting that these CFU-GM are grown to brain cell, nephrocyte, intestinal cell, liver cell or myocyte.
Any mammal, birds or reptiles CD34 +Or CD133 +Stem cell or CFU-GM can be used in company with method of the present invention, comprise, but be not limited to, the placenta that comprises perfusion from Cord blood (CB cell), peripheral blood, adult blood, marrow, placenta (is seen U. S. application publication number US20030032179, be published in 2003-02-13, be entitled as " postpartum mammal placenta, it is used and placenta stem-cell thus ", at this, it is whole in conjunction with as a reference), the stem cell that separates of mescenchymal stem cell and other sources.In a preferred embodiment, stem cell is candidate stem cell or isolated cells from marrow.This cell can obtain from other organ or tissues, but these sources are less preferred.
In one embodiment, can use from Cord blood or the next CFU-GM of placenta in postpartum.As above put down in writing, Cord blood mainly comprises CD34 +And CD38 +Hemopoietic progenitor cell.In first 24 hours of perfusion in postpartum, the CD34 of high concentration +CD38 -Hemopoietic progenitor cell can be separated from placenta that separated, perfusion.After about 24 hours of perfusion, the CD34 of high concentration -CD38 -Cell can be along with aforesaid cell together separates from placenta.In another embodiment, progenitor cell populations can obtain by commerce services, for example, the LifeBank U.S. (Cedar Knolls, NJ), ViaCord (Boston MA), Cord Blood Registry (San Bruno, CA) and Cryocell (Clearwater, FL).
4.3 compound of the present invention
Used in the present invention compound comprises selective cytokine inhibitory drugs racemic, that stereoisomer is pure or the stereoisomer enrichment, have selective cytokine and suppress the active stereoisomer or the compound of enantiomer-pure, and pharmaceutically useful salt, solvate, hydrate, stereoisomer, inclusion compound and prodrug thereof.Used in the present invention preferred compound is known as the selective cytokine inhibitory drugs (SelCIDs of Celgene company TM).
Unless it is as used herein and show used in the present invention term " SelCIDs in addition TM" comprise small-molecule drug, for example not peptide, protein, nucleic acid, oligosaccharides or other macromolecular organic molecule.Preferred compound suppresses the generation of TNF-α.In addition, this compound also can be induced the depression effect that has appropriateness on IL1 β and the IL12 at LPS.More preferably, compound of the present invention is effective PDE4 inhibitor.PDE4 is a kind of main phosphodiesterase isoenzyme of finding in human medullary or lymphoid cell.This enzyme plays a significant role regulating in the cytoactive by the degrade ubiquitous second messenger cAMP and the level that holds it in the low cell.Do not accept opinion institute and limit, the inhibition of PDE4 activity causes the raising of cAMP level, and this raising brings the adjusting of the cell factor that LPS induces, and is included in the inhibition of monocyte and the TNF-α in lymphocyte generation.
The object lesson of selective cytokine inhibitory drugs includes but not limited at U.S. Patent number 5,605, disclosed cyclic imides class in 914; Respectively at U.S. Patent number 5,728, disclosed cycloalkyl amide-type and cycloalkyl nitrile in 844 and 5,728,845; U.S. Patent number 5,801,195 and 5,736,570 aryl amides (for example, embodiment be N-benzoyl-3-amino-3-(3 ', 4 '-Dimethoxyphenyl)-propionamide); At U.S. Patent number 5,703, disclosed acid imide/amide ether and alcohols in 098 (for example 3-phthaloyl imino-3-(3 ', 4 '-Dimethoxyphenyl) third-1-alcohol); At U.S. Patent number 5,658, disclosed succinimide and maleimide in 940 (for example 3-(3 ', 4 ', 5 ', 6 '-the tetrahydrochysene phthaloyl imino)-3-(3 ", 4 " Dimethoxyphenyl) methyl propionate); The alkane hydroximic acid that disclosed acylimino and acylamino-replace in WO 99/06041 and at U.S. Patent number 6,020, the phenethyl sulfone class of disclosed replacement in 358; And at U.S. Patent number 6,046, disclosed aryl amides such as N-benzoyl-3-amino-3-in 221 (3 ', 4 '-Dimethoxyphenyl) propionamide.The full content that this paper quotes each these patent and patent application as a reference.
Other selective cytokine inhibitory drugs belongs to a kind of synthetic chemical compound family, wherein typical embodiment comprises 3-(1,3-dioxo benzo-[f] iso-indoles-2-yl)-3-(3-cyclopentyloxy-4-methoxyphenyl) propionamide and 3-(1,3-dioxo-4-azepine iso-indoles-2-yl)-3-(3, the 4-Dimethoxyphenyl)-propionamide.
Other concrete selective cytokine inhibitory drugs belongs at U.S. Patent number 5,698, the non-polypeptide cyclic amides of a disclosed class in 579 and 5,877,200, and this paper quotes this two patent.Representational cyclic amides comprises the compound of following formula:
Figure A0381365400321
Wherein n has 1,2 or 3 value;
R 5Be adjacent phenylene, described phenylene is not substituted or is replaced by 1-4 substituting group, and described substituting group is selected from nitro, cyano group, trifluoromethyl, carbethoxyl group, methoxycarbonyl group, the third oxygen carbonyl, acetyl group, carbamoyl, acetoxyl group, carboxyl, hydroxyl, amino, alkyl amino, dialkyl amido, acylamino-, the alkyl with 1-10 carbon atom, the alkyl with 1-10 carbon atom and halogen independently of one another;
R 7For (i) phenyl or by the phenyl of a plurality of substituting groups replacements, described substituting group is selected from nitro separately independently of each other, cyano group, trifluoromethyl, carbethoxyl group, methoxycarbonyl group, the third oxygen carbonyl, acetyl group, carbamoyl, acetoxyl group, carboxyl, hydroxyl, amino, alkyl with 1-10 carbon atom, alkoxyl and halogen with 1-10 carbon atom, (ii) be not substituted or by 1-3 the benzyl that substituting group replaced, described substituting group is selected from nitro, cyano group, trifluoromethyl, carbethoxyl group, methoxycarbonyl group, the third oxygen carbonyl, acetyl group, carbamoyl, acetoxyl group, carboxyl, hydroxyl, amino, alkyl with 1-10 carbon atom, alkoxyl and halogen with 1-10 carbon atom, (iii) naphthyl and (iv) benzyloxy;
R 12Be-OH, have the alkoxyl of 1-12 carbon atom, or
Figure A0381365400331
R 8For hydrogen or have the alkyl of 1-10 carbon atom;
R 9For hydrogen, have 1-10 carbon atom alkyl ,-COR 10Or-SO 2R 10, R wherein 10For hydrogen, have the alkyl or phenyl of 1-10 carbon atom.
The compound that this class is concrete includes, but are not limited to:
3-phenyl-2-(1-oxo isoindole quinoline-2-yl) propionic acid;
3-phenyl-2-(1-oxo isoindole quinoline-2-yl) propionamide;
3-phenyl-3-(1-oxo isoindole quinoline-2-yl) propionic acid;
3-phenyl-3-(1-oxo isoindole quinoline-2-yl) propionamide;
3-(the 4-methoxyphenyl)-3-(propionic acid of 1-oxo isoindole quinoline-yl);
3-(the 4-methoxyphenyl)-3-(propionamide of 1-oxo isoindole quinoline-yl);
3-(3, the 4-Dimethoxyphenyl)-3-(1-oxo isoindole quinoline-2-yl) propionic acid;
3-(3, the 4-Dimethoxyphenyl)-3-(1-oxo-1,3-xylylenimine-2-yl)-propionamide;
3-(3, the 4-Dimethoxyphenyl)-3-(1-oxo isoindole quinoline-2-yl) propionamide;
3-(3,4-diethoxy phenyl-3-(propionic acid of 1-oxo isoindole quinoline-yl);
3-(1-oxo isoindole quinoline-2-yl)-3-(3-ethyoxyl-4-methoxyphenyl) methyl propionate;
3-(1-oxo isoindole quinoline-2-yl)-3-(3-ethyoxyl-4-methoxyphenyl) propionic acid;
3-(1-oxo isoindole quinoline-2-yl)-3-(3-propoxyl group-4-methoxyphenyl) propionic acid;
3-(1-oxo isoindole quinoline-2-yl)-3-(3-butoxy-4-methoxyphenyl) propionic acid;
3-(1-oxo isoindole quinoline-2-yl)-3-(3-propoxyl group-4-methoxyphenyl) propionamide;
3-(1-oxo isoindole quinoline-2-yl)-3-(3-butoxy-4-methoxyphenyl) propionamide;
3-(1-oxo isoindole quinoline-2-yl)-3-(3-butoxy-4-methoxyphenyl) methyl propionate; With
3-(1-oxo isoindole quinoline-2-yl)-3-(3-propoxyl group-4-methoxyphenyl) methyl propionate.
Other concrete optionally cytokine inhibitory drugs is included in the alkane hydroximic acid that disclosed acylimino and acylamino-replace among the WO 99/06041, and this paper quotes it as a reference.This examples for compounds includes, but are not limited to:
Each R wherein 1And R 2, when value independently of each other, be hydrogen, low alkyl group, or when with the described carbon atom value of combination separately, R 1And R 2Be adjacent phenylene, adjacent naphthylene or cyclohexene-1,2-two bases, described group is not substituted or is replaced by 1-4 substituting group, and described substituting group is independently selected from nitro, cyano group, trifluoromethyl, carbethoxyl group, methoxycarbonyl group, the third oxygen carbonyl, acetyl group, carbamoyl, acetoxyl group, carboxyl, hydroxyl, amino, alkyl amino, dialkyl amido, acylamino-, the alkyl with 1-10 carbon atom, the alkoxyl with 1-10 carbon atom and halogen;
R 3For by 1-4 the phenyl that substituting group replaced, described substituting group is selected from nitro, cyano group, trifluoromethyl, carbethoxyl group, methoxycarbonyl group, the third oxygen carbonyl, acetyl group, carbamoyl, acetoxyl group, carboxyl, hydroxyl, amino, the alkyl with 1-10 carbon atom, the alkoxyl with 1-10 carbon atom, the alkylthio group with 1-10 carbon atom, benzyloxy, the cycloalkyloxy with 3-6 carbon atom, C 4-C 6Cycloalkylidene methyl, C 3-C 10Alkylidene methyl, indane oxygen base and halogen;
R 4Be hydrogen, alkyl, phenyl or benzyl with 1-6 carbon atom;
R 4 'For hydrogen or have the alkyl of 1-6 carbon atom;
R 5For-CH 2-,-CH 2-CO-,-SO 2-,-S-or-NHCO-;
N has 0,1 or 2 value; And
Comprising can be by the acid-addition salts of the described compound of protonated nitrogen-atoms.
Being used for other concrete selective cytokine inhibitory drugs of the present invention includes, but are not limited to:
3-(3-ethyoxyl-4-methoxyphenyl)-N-hydroxyl-3-(1-oxo isoindole quinoline base) propionamide;
3-(3-ethyoxyl-4-methoxyphenyl)-N-methoxyl group-3-(1-oxo isoindole quinoline base) propionamide;
N-benzyloxy-3-(3-ethyoxyl-4-methoxyphenyl)-3-phthaloyl imino propionamide;
N-benzyloxy-3-(3-ethyoxyl-4-methoxyphenyl)-3-(3-nitro phthaloyl imino) propionamide;
N-benzyloxy-3-(3-ethyoxyl-4-methoxyphenyl)-3-(1-oxo isoindole quinoline base) propionamide;
3-(3-ethyoxyl-4-methoxyphenyl)-N-hydroxyl-3-phthaloyl imino propionamide;
N-hydroxyl-3-(3, the 4-Dimethoxyphenyl)-3-phthaloyl imino propionamide;
3-(3-ethyoxyl-4-methoxyphenyl)-N-hydroxyl-3-(3-nitro phthaloyl imino) propionamide;
N-hydroxyl-3-(3, the 4-Dimethoxyphenyl)-3-(1-oxo isoindole quinoline base) propionamide;
3-(3-ethyoxyl-4-methoxyphenyl)-N-hydroxyl-3-(4-methyl-phthaloyl imino) propionamide;
3-(3-cyclopentyloxy-4-methoxyphenyl)-N-hydroxyl-3-phthaloyl imino propionamide;
3-(3-ethyoxyl-4-methoxyphenyl)-N-hydroxyl-3-(1,3-dioxo-2,3-dihydro-1H-benzo [f] iso-indoles-2-yl) propionamide;
N-hydroxyl-3-{3-(2-propoxyl group)-4-methoxyphenyl }-3-phthaloyl imino propionamide;
3-(3-ethyoxyl-4-methoxyphenyl)-3-(3,6-difluoro phthaloyl imino)-N-hydroxyl propionamide;
3-(the amino phthaloyl imino of 4-)-3-(3-ethyoxyl-4-methoxyphenyl)-N-hydroxyl propionamide;
3-(the amino phthaloyl imino of 3-)-3-(3-ethyoxyl-4-methoxyphenyl)-N-hydroxyl propionamide;
N-hydroxyl-3-(3, the 4-Dimethoxyphenyl)-3-(1-oxo isoindole quinoline base) propionamide;
3-(3-cyclopentyloxy-4-methoxyphenyl)-N-hydroxyl-3-(1-oxo isoindole quinoline base) propionamide; With
N-benzyloxy-3-(3-ethyoxyl-4-methoxyphenyl)-3-(3-nitro phthaloyl imino) propionamide.
Be used for other selective cytokine inhibitory drugs of the present invention and be included in the phenethyl sulfone class that is replaced by oxo isoindole quinoline base on the phenyl.Such examples for compounds include but not limited to in the U.S. Patent number 6,020,358 that this paper quoted disclosed those, they comprise following compounds:
The carbon atom that wherein indicates * constitutes a chiral centre;
Y is C=O, CH 2, SO 2Or CH 2C=O; Each R 1, R 2, R 3And R 4Separately independently of each other for hydrogen, halogen, alkyl, alkoxyl, nitro, cyano group, hydroxyl with 1-4 carbon atom with 1-4 carbon atom or-NR 8R 9Or the R on adjacent carbon atom 1, R 2, R 3And R 4Central any two is naphthylene with described phenylene ring;
Each R 5And R 6Be hydrogen, the alkyl with 1-4 carbon atom, the alkoxyl with 1-4 carbon atom, cyano group or have the cycloalkyloxy that is up to 18 carbon atoms separately independently of each other;
R 7Be hydroxyl, alkyl, phenyl, benzyl or NR with 1-8 carbon atom 8R 9
Each R 8And R 9Be hydrogen, alkyl, phenyl or benzyl separately independently of each other with 1-8 carbon atom, or R 8And R 9In the middle of have one to be hydrogen, and another is-COR 10Or-SO 2R 10, or R 8And R 9Be together tetramethylene, pentamethylene, hexa-methylene or-CH 2CH 2X 1CH 2CH 2-, X wherein 1For-O-,-S-or-NH-; And
Each R 8 'And R 9 'Be hydrogen, alkyl, phenyl or benzyl separately independently of each other with 1-8 carbon atom, or R 8 'And R 9 'In the middle of have one to be hydrogen, and another is-COR 10 'Or-SO 2R 10 ', or R 8 'And R 9 'Be together tetramethylene, pentamethylene, hexa-methylene or-CH 2CH 2X 2CH 2CH 2-, X wherein 2For-O-,-S-or-NH-.
Should be appreciated that for convenience's sake top compound being called phenethyl sulfone time-like, work as R 7Be NR 8 'R 9 'The time, they comprise sulfonamides.
Concrete group of such compound is C=O or CH for Y wherein 2Those.
The concrete group of another of such compound those: each R wherein for being defined as follows 1, R 2, R 3And R 4Be independently of each other separately hydrogen, halogen, methyl, ethyl, methoxyl group, ethyoxyl, nitro, hydroxyl or-NR 8R 9, each R wherein 8And R 9Be hydrogen or methyl independently of each other, or R 8And R 9In the middle of have one to be hydrogen, and another is-COCH 3
Specific compound is those that are defined as follows: R wherein 1, R 2, R 3And R 4In the middle of have one to be-NH 2, and all the other R 1, R 2, R 3And R 4Be hydrogen.
Specific compound is those that are defined as follows: R wherein 1, R 2, R 3And R 4In the middle of have one to be NHCOCH 3, and all the other R 1, R 2, R 3And R 4Be hydrogen.
Specific compound is those that are defined as follows: R wherein 1, R 2, R 3And R 4In the middle of have one to be N (CH 3) 2, and all the other R 1, R 2, R 3And R 4Be hydrogen.
The preferred such compound of another group is those that are defined as follows: R wherein 1, R 2, R 3And R 4In the middle of have one to be methyl, and all the other R 1, R 2, R 3And R 4Be hydrogen.
Specific compound is those that are defined as follows: R wherein 1, R 2, R 3And R 4In the middle of have one to be fluorine, and all the other R 1, R 2, R 3And R 4Be hydrogen.
Specific compound is those that are defined as follows: each R wherein 5And R 6Be hydrogen, methyl, ethyl, propyl group, methoxyl group, ethyoxyl, propoxyl group, cyclopentyloxy or cyclohexyloxy independently of each other.
Specific compound is those that are defined as follows: R wherein 5Be methoxyl group, and R 6Be monocycle alkoxyl, polynaphthene oxygen base and benzo cycloalkyloxy.
Specific compound is R wherein 5Be methoxyl group, and R 6Be those of ethyoxyl.
Specific compound is those that are defined as follows: R wherein 7Be hydroxyl, methyl, ethyl, phenyl, benzyl or NR 8 'R 9 ', each R wherein 8 'And R 9 'Be hydrogen or methyl independently of each other.
Specific compound is R wherein 7Be those of methyl.
Specific compound is those that are defined as follows: R wherein 7Be NR 8 'R 9 ', each R wherein 8 'And R 9 'Be hydrogen or methyl independently of each other.
Other concrete selective cytokine inhibitory drugs is included in U.S. Provisional Application that people such as Muller submits on December 30th, 2002 number 60/436,1 of the Fluoroalkyloxy replacement of putting down in writing in 975,3-dihydro-isoindolyl compounds, this paper are quoted its full content as a reference.1 of representational Fluoroalkyloxy replacement, 3-dihydro-isoindolyl compounds comprises following formula: compound:
Wherein:
Y is-C (O)-,-CH 2,-CH 2C (O)-,-C (O) CH 2-or SO 2
Z is-H ,-C (O) R 3,-(C 0-1-alkyl)-SO 2-(C 1-4-alkyl) ,-C 1-8-alkyl ,-CH 2OH, CH 2(O) (C 1-8-alkyl) or-CN;
R 1And R 2Be independently-CHF respectively 2,-C 1-8-alkyl ,-C 3-18-cycloalkyl or-(C 1-10-alkyl) (C 3-18And R-cycloalkyl), 1And R 2In have at least one to be CHF 2
R 3For-NR 4R 5,-alkyl ,-OH ,-O-alkyl, phenyl, benzyl, the phenyl of replacement or the benzyl of replacement;
R 4And R 5Be independently respectively-H ,-C 1-8-alkyl ,-OH ,-OC (O) R 6
R 6For-C 1-8-alkyl ,-amino (C 1-8-alkyl) ,-phenyl ,-benzyl or-aryl;
X 1, X 2, X 3And X 4Be independently respectively-H ,-halogen ,-nitro ,-NH 2,-CF 3,-C 1-6-alkyl ,-(C 0-4-alkyl)-(C 3-6-cycloalkyl), (C 0-4-alkyl)-NR 7R 8, (C 0-4-alkyl)-N (H) C (O)-(R 8), (C 0-4-alkyl)-N (H) C (O) N (R 7R 8), (C 0-4-alkyl)-N (H) C (O) O (R 7R 8), (C 0-4-alkyl)-OR 8, (C 0-4-alkyl)-imidazole radicals, (C 0-4-alkyl)-pyrrole radicals, (C 0-4-alkyl)-oxadiazole base or (C 0-4-alkyl)-triazolyl, perhaps X 1, X 2, X 3And X 4In the middle of two can be joined together to form cycloalkyl or heterocycloalkyl ring (X for example 1And X 2, X 2And X 3, X 3And X 4, X 1And X 3, X 2And X 4Or X 1And X 4Can form 3,4,5,6 or 7 yuan of rings, described ring can be an aromatic ring, forms bicyclic ring system with the isoindolyl ring thus); And
R 7And R 8Be H, C independently respectively 1-9-alkyl, C 3-6-cycloalkyl, (C 1-6-alkyl)-(C 3-6-cycloalkyl), (C 1-6-alkyl)-N (R 7R 8), (C 1-6-alkyl)-OR 8, phenyl, benzyl or aryl;
Or its officinal salt, solvate, hydrate, stereoisomer, inclusion compound or prodrug.
Preferred compound includes but not limited to:
3-(4-acetyl-amino-1,3-dioxo-1,3-dihydro-iso-indoles-2-yl)-3-(3-cyclo propyl methoxy-4-difluoro-methoxy-phenyl)-propionic acid;
3-(4-acetyl-amino-1,3-dioxo-1,3-dihydro-iso-indoles-2-yl)-3-(3-cyclo propyl methoxy-4-difluoro-methoxy-phenyl)-N, N-dimethyl-propionamide;
3-(4-acetyl-amino-1,3-dioxo-1,3-dihydro-iso-indoles-2-yl)-3-(3-cyclo propyl methoxy-4-difluoro-methoxy-phenyl)-propionamide;
3-(3-cyclo propyl methoxy-4-difluoro-methoxy-phenyl)-3-(1,3-dioxo-1,3-dihydro-iso-indoles-2-yl)-propionic acid;
3-(3-cyclo propyl methoxy-4-difluoro-methoxy-phenyl)-3-(1,3-dioxo-1,3-dihydro-iso-indoles-2-yl)-N-hydroxyl-propionamide;
3-(3-cyclo propyl methoxy-4-difluoro-methoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-iso-indoles-2-yl)-methyl propionate;
3-(3-cyclo propyl methoxy-4-difluoro-methoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-iso-indoles-2-yl)-propionic acid;
3-(3-cyclo propyl methoxy-4-difluoro-methoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-iso-indoles-2-yl)-N, N-dimethyl-propionamide;
3-(7-amino-1-oxo-1,3-dihydro-iso-indoles-2-yl)-3-(3-cyclo propyl methoxy-4-difluoro-methoxy-phenyl)-N, N-dimethyl-propionamide;
3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-iso-indoles-2-yl) methyl propionate;
3-(7-amino-1-oxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-methyl propionate;
3-[7-(cyclopropane carbonyl-amino)-1-oxo-1,3-dihydro-iso-indoles-2-yl]-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-methyl propionate;
3-(7-acetyl-amino-1-oxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-methyl propionate;
3-(7-acetyl-amino-1-oxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-propionic acid;
3-[7-(cyclopropane carbonyl-amino)-1-oxo-1,3-dihydro-iso-indoles-2-yl]-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-propionic acid;
Cyclopropane-carboxylic acid 2-[2-carbamoyl-1-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-ethyl]-3-oxo-2,3-dihydro-1H-iso-indoles-4-yl }-acid amides;
Cyclopropane-carboxylic acid 2-[1-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-2-formyl-dimethylamino-ethyl]-3-oxo-2,3-dihydro-1H-iso-indoles-4-yl }-;
Cyclopropane-carboxylic acid 2-[1-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-2-hydroxyl amino formoxyl-ethyl]-3-oxo-2,3-dihydro-1H-iso-indoles-4-yl }-acid amides;
3-(7-acetyl-amino-1-oxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-propionamide;
3-(7-acetyl-amino-1-oxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-N, N-dimethyl-propionamide;
3-(7-acetyl-amino-1-oxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-N-hydroxyl-propionamide;
3-(4-acetyl-amino-1,3-dioxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-propionic acid;
3-(4-acetyl-amino-1,3-dioxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-propionamide;
3-(4-acetyl-amino-1,3-dioxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-N, N-dimethyl-propionamide;
3-(4-acetyl-amino-1,3-dioxo-1,3-dihydro-iso-indoles-2-yl)-3-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-N-hydroxyl-propionamide;
Cyclopropane-carboxylic acid 2-[1-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-2-mesyl-ethyl]-3-oxo-2,3-dihydro-1H-iso-indoles-4-yl }-acid amides;
N-{2-[1-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-2-mesyl-ethyl]-1,3-dioxo-2,3-dihydro-1H-iso-indoles-4-yl }-acetamide; With
Cyclopropane-carboxylic acid 2-[2-carbamoyl-1-(4-difluoro-methoxy-3-ethyoxyl-phenyl)-ethyl [7-chloro-3-oxo-2,3-dihydro-1H-iso-indoles-4-yl }-acid amides.
Other optionally cytokine inhibitory drugs be included in the isoindolyl compounds that the 7-acylamino-of record in the U.S. Provisional Application that people such as Muller submits on March 12nd, 2002 number 60/454,155 replaces, this paper quotes its full content as a reference.
The isoindolyl compounds that representational 7-acylamino-replaces comprises following formula: compound:
Wherein:
Y is-C (O)-,-CH 2,-CH 2C (O)-or-SO 2
X is H,
Z is (C 0-4-alkyl)-C (O) R 3, C 1-4-alkyl, (C 0-4-alkyl)-OH, (C 1-4-alkyl)-O (C 1-4-alkyl), (C 1-4-alkyl)-SO 2(C 1-4-alkyl), (C 0-4-alkyl)-SO (C 1-4-alkyl), (C 0-4-alkyl)-NH 2, (C 0-4-alkyl)-N (C 1-8-alkyl) 2, (C 0-4-alkyl)-N (H) (OH), CH 2NSO 2(C 1-4-alkyl);
R 1And R 2Be C independently 1-8-alkyl, cycloalkyl or (C 1-4-alkyl) cycloalkyl;
R 3Be NR 4R 5, OH or O-(C 1-8-alkyl);
R 4Be H;
R 5For-OH or-OC (O) R 6
R 6Be C 1-8-alkyl, amino-(C 1-8-alkyl), (C 1-8-alkyl)-(C 3-6-cycloalkyl), C 3-6-cycloalkyl, phenyl, benzyl or aryl;
Or its pharmaceutically useful salt, solvate, hydrate, stereoisomer, inclusion compound or prodrug; Or compound shown in the following formula
Figure A0381365400411
Wherein:
Y is-C (O)-,-CH 2,-CH 2C (O)-or SO 2
X be halogen ,-CN ,-NR 7R 8,-NO 2Or-CF 3,
W is
Or
Z is (C 0-4-alkyl)-SO 2(C 1-4-alkyl) ,-(C 0-4-alkyl)-CN ,-(C 0-4-alkyl)-C (O) R 3, C 1-4-alkyl, (C 0-4-alkyl) OH, (C 0-4-alkyl) O (C 1-4-alkyl), (C 0-4-alkyl) SO (C 1-4-alkyl), (C 0-4-alkyl) NH 2, (C 0-4-alkyl) N (C 1-8-alkyl) 2, (C 0-4-alkyl) N (H) (OH) or (C 0-4-alkyl) NSO 2(C 1-4-alkyl);
W is-C 3-6-cycloalkyl ,-(C 1-8-alkyl)-(C 3-6-cycloalkyl) ,-(C 0-8-alkyl)-(C 3-6-cycloalkyl)-NR 7R 8, (C 0-8-alkyl)-NR 7R 8, (C 0-4-alkyl)-CHR 9-(C 0-4-alkyl)-NR 7R 8,
R 1And R 2Be C independently 1-8-alkyl, cycloalkyl or (C 1-4-alkyl) cycloalkyl;
R 3Be C 1-8-alkyl, NR 4R 5, OH or O-(C 1-8-alkyl);
R 4And R 5Be H, C independently of each other 1-8-alkyl, (C 0-8-alkyl)-(C 3-6-cycloalkyl), OH or-OC (O) R 6
R 6Be C 1-8-alkyl, (C 0-8-alkyl)-(C 3-6-cycloalkyl), amino-(C 1-8-alkyl), phenyl, benzyl or aryl;
R 7And R 8Be hydrogen, C independently of one another 1-8-alkyl, (C 0-8-alkyl)-(C 3-6-cycloalkyl), phenyl, benzyl or aryl, or can form 3-7 unit's Heterocyclylalkyl or heteroaryl ring with the atom that connects them;
R 9Be C 1-4-alkyl, (C 0-4-alkyl) aryl, (C 0-4-alkyl)-(C 3-6-cycloalkyl), (C 0-4-alkyl) heterocycle;
Or its officinal salt, solvate, hydrate, stereoisomer, inclusion compound or prodrug.
Other optionally cytokine inhibitory drugs also be included in the N-alkyl-hydroximic acid-isoindolyl compounds of record in the U.S. Provisional Application that people such as Muller submits on March 12nd, 2003 number 60/454,149, this paper quotes its full content as a reference.Representational N-alkyl-hydroximic acid-isoindolyl compounds comprises following formula: compound:
Figure A0381365400431
Wherein:
Y is-C (O)-,-CH 2,-CH 2C (O)-or-SO 2
R 1And R 2Be C independently 1-8-alkyl, CF 3, CH 2CHF 2, cycloalkyl or (C 1-8-alkyl) cycloalkyl;
Z 1Be H, C 1-6Alkyl ,-NH 2-NR 3R 4Or OR 5
Z 2Be H or C (O) R 5
X 1, X 2, X 3And X 4Respectively be hydrogen, halogen, NO independently 2, OR 3, CF 3, C 1-6-alkyl, (C 0-4-alkyl)-(C 3-6-cycloalkyl), (C 0-4-alkyl)-N-(R 8R 9), (C 0-4-alkyl)-NHC (O)-(R 8), (C 0-4-alkyl)-NHC (O) CH (R 8) (R 9), (C 0-4-alkyl)-NHC (O) N (R 8R 9), (C 0-4-alkyl)-NHC (O) O (R 8), (C 0-4-alkyl)-O-R 8, (C 0-4-alkyl)-imidazole radicals, (C 0-4-alkyl)-pyrrole radicals, (C 0-4-alkyl)-oxadiazole bases, (C 0-4-alkyl)-triazolyl or (C 0-4-alkyl)-heterocycle;
R 3, R 4And R 5Be H, C independently of one another 1-6-alkyl, O-C 1-6-alkyl, phenyl, benzyl or aryl;
R 6And R 7Be H or C independently of one another 1-6-alkyl;
R 8And R 9Be H, C independently of one another 1-9-alkyl, C 3-6-cycloalkyl, (C 1-6-alkyl)-(C 3-6-cycloalkyl), (C 0-6-alkyl)-N (R 4R 5), (C 0-6-alkyl)-OR 5, phenyl, benzyl, aryl, piperidyl, piperazinyl, pyrrolidinyl, morpholino or C 3-7-Heterocyclylalkyl;
Or its officinal salt, solvate, hydrate, stereoisomer, inclusion compound or its prodrug.
Concrete selective cytokine inhibitory drugs includes but not limited to:
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl-sulfonyl ethyl] 1-isoindolinone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-(N, N-dimethyl-amino-sulfonyl) ethyl] 1-isoindolinone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl-sulfonyl ethyl] isoindoline-1, the 3-diketone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl-sulfonyl ethyl]-5-nitro-isoindoline-1, the 3-diketone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl-sulfonyl ethyl]-4-nitro isoindoline-1, the 3-diketone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl sulphonyl ethyl]-4-aminoisoindoline-1, the 3-diketone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl sulphonyl ethyl]-5-methyl isoindoline-1, the 3-diketone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl sulphonyl ethyl]-5-acetylamino isoindoline-1, the 3-diketone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl sulphonyl ethyl]-4-dimethylamino isoindoline-1, the 3-diketone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl sulphonyl ethyl]-5-dimethylamino isoindoline-1, the 3-diketone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl sulphonyl ethyl] benzo [e] isoindoline-1, the 3-diketone;
2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl sulphonyl ethyl]-4-methoxyl group isoindoline-1, the 3-diketone;
1-(3-cyclopentyloxy-4-methoxyphenyl)-2-methyl sulphonyl ethyl-amine;
2-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-methyl sulphonyl ethyl] isoindoline-1, the 3-diketone; With
2-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-methyl sulphonyl ethyl]-4-dimethylamino isoindoline-1, the 3-diketone.
Other selective cytokine inhibitory drugs is included in U.S. Provisional Patent Application that people such as G.Muller submits on March 20th, 2002 number 60/366,515 and 60/366,516 and the U.S. Provisional Patent Application submitted on January 7th, 2003 of people such as G.Muller number 60/438,450 and 60/438, the compound of disclosed enantiomer-pure in 448, this paper quotes all these patents as a reference.Preferred compound comprises 2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-methyl sulphonyl ethyl]-4-acetyl-amino isoindoline-1; the enantiomer of 3-diketone and 3-(3; 4-dimethoxy-phenyl)-enantiomer of 3-(1-oxo-1,3-dihydro-iso-indoles-2-yl)-propionamide.
Selective cytokine inhibitory drugs preferred for the present invention is 3-(3; 4-dimethoxy-phenyl)-3-(1-oxo-1; 3-dihydro-iso-indoles-2-yl)-propionamide and cyclopropane-carboxylic acid 2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-mesyl ethyl]-3-oxo-2; 3-dihydro-1H-iso-indoles-4-yl }-acid amides; it can be from the Warren of Celgene company, and NJ obtains.3-(3,4-dimethoxy-phenyl)-3-(1-oxo-1,3-dihydro-iso-indoles-2-yl)-propionamide has following chemical constitution:
Figure A0381365400451
Cyclopropane-carboxylic acid 2-[1-(3-ethyoxyl-4-methoxyphenyl)-2-mesyl ethyl]-3-oxo-2,3-dihydro-1H-iso-indoles-4-yl }-acid amides has following chemical constitution:
Figure A0381365400452
Compound of the present invention also includes but not limited to suppress the compound of PDE IV activity, as cilomilast, theophylline, Zardaverine, rolipram, oxpentifylline, Enoximone, the iso-indoles acid imide, phenethyl sulfone class, the alkane hydroximic acid, non-polypeptide cyclic amides class, the oxo isoindole class, the isoindoline class, the indazole class, the assorted pyridines that replaces, the diphenyl pyridines, the aryl thiophene class, the aryl furans, the indenes class, the trisubstd phenyl class, the benzodiazine ketone, benzene sulfonamide, tetracyclic compound and salt thereof, solvate, isomer, inclusion compound, prodrug, hydrate and derivative thereof.In one embodiment, this compound is not polypeptide, peptide, protein, hormone, cell factor, oligonucleotides or nucleic acid.
In another embodiment, compound of the present invention has following structure (I):
Comprise its isomer, prodrug and officinal salt, hydrate, solvate and inclusion compound, wherein:
Y represents N or N-oxide;
R 1And R 2Be independently selected from:
H, C 1-6Alkyl and halo C 1-6Alkyl;
R 3And R 4Be independently selected from H and C 1-6Alkyl perhaps is connected the R on the same carbon atom 3And R 4Represent carbonylic oxygen atom together, perhaps be connected the R on the different carbon atoms 3And R 4Represent saturated 5,6 or 7 yuan of carbocyclic rings with carbon atom and any intervenient atom that they connected;
R 5And R 6Representative is selected from H, C independently 1-6Alkyl, halo C 1-6The group of alkyl and CN;
N represents the integer of 0-6;
Ar 1Be selected from thienyl, thiazolyl, pyridine radicals, phenyl and naphthyl; Described Ar 1Can choose wantonly by 1-3 and be selected from following group replacement: halogen, C 1-6Alkoxyl, C 1-7Alkylthio group, CN, C 1-6Alkyl, hydroxyl C 1-6Alkyl ,-C (O) C 1-6Alkyl ,-CO 2H ,-CO 2C 1-6Alkyl, NH (SO 2Me), N (SO 2Me) 2, SO 2Me, SO 2NH 2, SO 2NHC 1-6Alkyl, SO 2N (C 1-6Alkyl) 2NO 2, C 2-6Thiazolinyl, C 1-6Alkyl and NH 2
And work as Ar 1When representative had two or three substituent phenyl or naphthyls, these two substituting groups can be represented 5 or 6 yuan together and condense lactonic ring.
This embodiment also comprises as U.S. Patent number 6,316, the compound of record in 472, and this paper quotes its full content as a reference.
In another embodiment, compound of the present invention has following structure (II):
Comprise its isomer, prodrug and officinal salt, hydrate, solvate and inclusion compound, wherein:
R 1And R 2Represent C 1-C 4Alkyl or C 3-C 10Cycloalkyl;
R 3And R 4Represent C independently 1-C 4Alkyl, cycloalkyl, has the C of two keys 2-C 4Thiazolinyl, has the C of a triple bond 2-C 4Alkynyl, (CH 2) nCO (CH 2) mCH 3, (CH 2) PCN, (CH 2) PCO 2Me, or form 3-10 unit ring with the nitrogen-atoms that they connected;
N and m are 0-3;
P is 1-3.
This embodiment also is included in U.S. Patent number 6,162, the compound of record in 830, and this paper quotes its full content as a reference.
In another embodiment, compound of the present invention has following structure (III):
Figure A0381365400472
Comprise its isomer, prodrug and officinal salt, hydrate, solvate and inclusion compound, wherein:
R 1Be independently selected from hydrogen, halogen, lower alkoxy, hydroxyl, low alkyl group, low alkyl group sulfydryl, low alkyl group sulfonyl, low-grade alkyl amino, two-low-grade alkyl amino, amino, nitro, nitrile, low alkyl group carboxyl (carboxylate) ,-CO 2H and sulfonamido;
R 2Be selected from hydrogen and low alkyl group;
R 3Be selected from hydrogen, low alkyl group, hydroxyl and amino;
R 4Be selected from-COM and CH 2OH, wherein M is selected from lower alkoxy, amino, alkyl amino, dialkyl amido, N-morpholino, hydroxy alkyl amino, polyhydroxy amino, Diaminoalkyl amino, aminoalkyl amino and the OMe of hydroxyl, replacement, and wherein Me is a cation;
R 5Be alkyl sulphonyl; And
N is the integer of 0-4.
This embodiment also is included in U.S. Patent number 6,177, the compound of record in 471, this paper this with its full content as a reference.
In another embodiment, compound of the present invention has following structure (IV):
Comprise its isomer, prodrug and officinal salt, hydrate, solvate and inclusion compound, wherein:
R 0Represent hydrogen, halogen or C 1-6-alkyl;
R 1Be selected from: hydrogen; C 1-6-alkyl, described alkyl is optional to be selected from following substituting group and to replace by one or more: phenyl, halogen ,-CO 2R a,-NR aR bC 3-6-cycloalkyl, phenyl and be selected from 5 or 6 yuan of heterocycles of pyridine radicals, morpholinyl, piperazinyl, pyrrolidinyl and piperidyl, described group can be chosen wantonly by one or more C 1-6-alkyl replaces, and the optional R that is connected 1Pass through C 1-6On the nitrogen-atoms that-alkyl connected;
R 2Be selected from: phenyl, described phenyl is optional by one or more following substituting group replacement :-OR that are selected from a,-NR a, R b, halogen, hydroxyl, trifluoromethyl, cyano group and nitro;
And R aAnd R bRepresent hydrogen or C independently 1-6-alkyl,
Comprise its isomer, prodrug and officinal salt.
This embodiment also is included in U.S. Patent number 6,218, the compound of record in 400, this paper this with its full content as a reference.
In another embodiment, compound of the present invention has following structure (V):
Comprise its isomer, prodrug and officinal salt, hydrate, solvate and inclusion compound, wherein:
X is S or O;
Ar 1For being selected from the aromatic ring of phenyl, pyridine radicals or furyl, the optional quilt of described aromatic ring is up to two substituting groups and is replaced, and each substituting group is independently: C 1-6Alkyl, described alkyl choose wantonly quilt-OH ,-CO 2H, CO 2C 1-3Alkyl or CN replace; C 1-6Alkoxyl; C 1-3Alkylthio group, C 1-3Alkyl sulphonyl, C 1-3Fluoroalkyl, the optional quilt-OH of described group replaces; Halogen ,-OH ,-CO 2H or CO 2C 1-3Alkyl;
R 2Be hydrogen or C 1-3Alkyl; And
R 3Be phenyl, pyridine radicals, quinolyl or furyl, the optional quilt of described group is up to two substituting groups and is replaced, and each substituting group is independently: C 1-3Alkyl, C 1-3Fluoroalkyl, C 1-6Alkoxyl, C 1-3Fluoroalkyloxy, C 1-3Alkylthio group, halogen or-OH.
This embodiment also is included in U.S. Patent number 6,034,089 and U.S. Patent number 6,020,339 in the compound of record, the full content that this paper quotes them is as a reference.
In another embodiment, compound of the present invention has following structure (VI):
Comprise its isomer, prodrug and officinal salt, hydrate, solvate and inclusion compound, wherein:
Y be hydrogen or alkyl or-XR aGroup;
Z is-O-,-S (O) p-or-N (R b)-, wherein p is 0 or integer 1 or 2;
L is-XR ,-C (R 11) C (R 1) (R 2) or-(CHR 11) nCH (R 1) (R 2), wherein n is 0 or integer 1;
Each R aAnd R bBe hydrogen or optional substituted alkyl independently;
R is optional substituted alkyl, thiazolinyl, cycloalkyl or cycloalkenyl group;
Each R 1And R 2Can be identical or different, and be hydrogen, fluorine ,-CN ,-NO 2, or optional substituted alkyl, thiazolinyl, alkynyl, alkoxyl, alkylthio group ,-CO 2R 8,-CONR 9R 10Or-CSNR 9R 10, or R 1And R 2Connect to form optional substituted cycloalkyl or cycloalkenyl group with the carbon atom that they connected;
R 3Be hydrogen, fluorine, hydroxyl or optional substituted straight or branched alkyl;
R 4For hydrogen ,-(CH 2) tAr or-(CH 2) t-Ar-(L 1) n-Ar 1, wherein t is 0 or integer 1,2 or 3;
R 5For-(CH 2) tAr or-(CH 2) t-Ar-(L 1) n-Ar ';
R 6Be hydrogen, fluorine or optional substituted alkyl;
R 7For hydrogen, fluorine, optional substituted straight or branched alkyl ,-ORc, wherein Rc is hydrogen or optional substituted alkyl or alkenyl, or formoxyl, alkoxyalkyl, alkanoyl, formamido group (carboxamido) or sulfo-formamido group (thiocarboxamido);
Each R 8, R 9And R 10Be hydrogen or optional substituted alkyl, aralkyl or aryl independently; And
R 11Be hydrogen, fluorine or methyl.
This embodiment also is included in U.S. Patent number 5,798, and the full content that the compound of record in 373, this paper are quoted them as a reference.
In a preferred embodiment, compound has structure (VII):
Or its officinal salt, hydrate, solvate, inclusion compound, enantiomer, diastereomer, racemic modification or stereoisomer mixture.
In another preferred embodiment, compound has structure (VIII):
Figure A0381365400502
Comprise its isomer, salt, inclusion compound, solvate, hydrate, prodrug and officinal salt.
Some of these compounds can be by Celgene, Inc., Warren, the commercial acquisition in New Jersey.Compound above other can make by means commonly known in the art, comprises that those are disclosed in the method in the above-cited patent, and the full content that this paper quotes them as a reference.
Effectively the example of other PDE IV inhibitor is included in GB2 063 249 A in the method for the invention, EP 0 607 439 A1, U.S. Patent number 6,333,354, U.S. Patent number 6,300,335, U.S. Patent number 6,166,041, U.S. Patent number 6,069,156, U.S. Patent number 6,011,060, U.S. Patent number 5,891,896, U.S. Patent number 5,849,770, U.S. Patent number 5,710,170, U.S. Patent number 4,101,548, U.S. Patent number 4,001,238, U.S. Patent number 4,001,237, U.S. Patent number 3,920,636, U.S. Patent number 4,060,615, WO 97/03985, EP 0 607 439 A1, U.S. Patent number 4,101,548, U.S. Patent number 4,001,238, U.S. Patent number 4,001,237, U.S. Patent number 3,920,636, U.S. Patent number 4,060,615, WO97/03985, EP 0 395 328, U.S. Patent number 4,209,623, EP 0 395 328, U.S. Patent number 4,209,623, U.S. Patent number 5,354,571, EP 0 428 268 A2, U.S. Patent number 5,354,571, EP 0 428 268 A2,807,826, U.S. Patent number 3,031,450, U.S. Patent number 3,322,755, U.S. Patent number 5,401,774,807,826, U.S. Patent number 3,031,450, U.S. Patent number 3,322,755, U.S. Patent number 5,401,774, U.S. Patent number 5,147,875, PCT WO 93/12095, U.S. Patent number 5,147,875, PCT WO 93/12095, U.S. Patent number 4,885,301, WO 93/07149, EP 0 349 239 A2, EP 0 352 960 A2, EP 0,526 004 A1, EP 0,463 756 A1, U.S. Patent number 4,885,301, WO 93/07149, EP 0 349 239 A2, EP 0,352 960 A2, EP 0,526 004 A1, EP 0 463 756 A1, EP 0 607 439 A1, EP 0 607 439 A1, WO 94/05661, EP 0,351 058,, U.S. Patent number 4,162,316, EP 0347146, U.S. Patent number 4,047,404, U.S. Patent number 5,614,530, U.S. Patent number 5,488,055, WO 97/03985, WO 97/03675, WO95/19978, U.S. Patent number 4,880,810, WO 98/08848, U.S. Patent number 5,439,895, U.S. Patent number 5,614,627, PCT US94/01728, WO 98/16521, EP 0 722943 A1, EP 0 722 937 A1, EP 0 722 944 A1, WO 98/17668, WO 97/24334, WO 97/24334, WO 97/24334, WO 97/24334, WO 97/24334, WO98/06722, PCT/JP97/03592, WO 98/23597, WO 94/29277, WO98/14448, WO 97/03070, WO 98/38168, among WO 96/32379 and the PCT/GB98/03712 disclosed those, the full content that this paper quotes them is as a reference.
Many compounds as part of the present invention can use standard fractionation well known in the art or asymmetric syntheses and the optical antipode of the described compound of enrichment.Referring to, for example, Shealy etc., Chem.Indus.1030 (1965); With Casini etc., Farmaco Ed.Sci.19:563 (1964).
The present invention also relates to the physiologically acceptable non-toxicity acid-addition salts of these compounds.Such salt comprises those salt that are derived from organic and inorganic acid well known in the art or alkali: such acid comprises for example hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, acetate, tartaric acid, lactic acid, succinic acid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid, salicylic acid, phthalic acid, embelin (embolicacid), enanthic acid etc.
Have acid compound of the present invention and can form salt with various pharmaceutically useful alkali.These alkali that can be used in the pharmaceutically acceptable base addition salts of preparation these acid compounds of the present invention are that those form non-toxic bases addition salts, that is, contain the alkali of pharmaceutically acceptable cationic salt, as, but be not limited to alkali metal or alkali salt, particularly calcium, magnesium, sodium or sylvite.The organic base that is fit to includes but not limited to N, N-dibenzyl-ethylenediamin, chloroprocanine, choline, diethanol amine, ethylenediamine, meglumine (N-methyl glucoside amine), lysine and procaine.
Can it suppress the ability of PDE IV to compound determination of the present invention with methods known in the art, for example, in U.S. Patent number 6,316,472; U.S. Patent number 6,204,275; (2000) " Comparison of phosphodiesterase inhibitorsof differing isoenzyme selectivity added to St.Thomas ' hospitalcardioplegic solution used for hypothermic preservation of rat lungs " such as Featherstone R.L, Am.J.Respir Crit.Care Med.162:850-6; With (1995) " Design and synthesis of conformationally constrained analoguesof4-(3-butoxy-4-methoxybenzyl) imidazolidin-2-one (Ro 20-1724) aspotent inhibitorsof cAMP-specific phosphodiesterase " such as Brackeen M.F, disclosed those tests among the J Med.Clieni.38:4848-54, the full content that this paper quotes them as a reference.
Compound of the present invention both can be obtained also can be prepared according to the method for describing in patent disclosed herein or the patent publication by commerce.In addition, optically pure composition can synthesize asymmetricly or utilize known resolving agent or chiral column and other standard synthesis of organic to learn a skill to split.
4.4 stem cell culture method
In certain embodiments of the invention, with stem cell or CFU-GM, include but not limited to pour in embryonic stem cell, embryonic-like stem cell, CFU-GM, multipotential cell, totipotent cell, multipotential cell, postpartum placenta interior living cell, cord blood cell, derive from the stem cell or the CFU-GM of peripheral blood or adult blood, or bone marrow cell is exposed to compound of the present invention and induces differentiation.These cells can be bred in external use method known in the field, or optionally, also can breed in the placenta of perfusion in postpartum.
In certain embodiments, the interior living cell that pours into placenta postpartum can be by collecting in placenta or the medium, and under appropriate condition external cultivation time enough, to induce differentiation to the cell type or the pedigree of expectation.Referring to U. S. application publication number US20030032179, be published in 2003-02-13, be entitled as " postpartum the mammal placenta use and later placenta stem-cell ", quote its full content here.
In another embodiment of the invention, stem cell or CFU-GM do not derive from pours into placenta postpartum, but on the contrary, separates from other sources, for example Cord blood, marrow, peripheral blood or adult blood are exposed to compound of the present invention and induce differentiation.In a preferred embodiment, differentiation is carried out external under appropriate condition, through adequate time, to induce differentiation to the cell lineage or the cell type of expectation.Compound of the present invention is by adding, produce in position, or makes stem cell or CFU-GM can touch any alternate manner of compound of the present invention and use in differentiation medium/medium.
In another embodiment, the stem cell of cultivating, for example, irriate in the process that the stem cell of culture in vitro or the stem cell of cultivating in the placenta of perfusion in postpartum are all being cultivated and breeding, for example, be subjected to the stimulation of following material: erythropoietin(EPO), cell factor, lymphokine, interferon, colony stimulating factor (CSF), interferon, chemokines, interleukin, the recombined human hematopoietic factor comprises part, stem cell factor, TPO (Tpo), interleukin, granulocyte colony stimulating factor (G-CSF) or other growth factor etc.
Cultured cells can detect test by colony forming unit and obtain identifying that it is being known in the art, as Mesen CuIt after collecting and/or separating TMMedium (stem cells technology, Inc., Vancouver British Columbia).
Culture in vitro stem cell or the method for CFU-GM is well known in the art, for example, referring to Thomson etc., 1998, Blood, 93 (4): 1253-63, with Hatzopoulos etc., 1998, Development 125:1457-1468 (endothelial cell CFU-GM); Slager etc., 1993, Dev.Genet.14 (3): 212-24 (nerve cell or muscle cell CFU-GM); Genbachev etc., 1995, Reprod.Toxicol.9 (3): 245-55 (cytotrophoblast such as placenta epithelial cell CFU-GM); .1984 such as Nadkarni, Tumori 70:503-505, Melehner etc., 1985, Blood 66 (6): 1469-1472, International PCT announces that WO publishes Himori etc., 1,984 00,/27,999 2000 on May 18,, Intl.J.Cell Cloning 2:254-262, with Douay etc., 1995, Bone Marrow Transplantation 15:769-775 (hemopoietic progenitor cell); Shamblott etc., 1998, Proc.Natl.Acad.Sci.USA 95:13726-31 (spermatogonium); Yan etc., 2001, Devel.Biol.235:422-432 (embryo's trophoderm stem cell).These methods can be used in the method for the invention through simple improvement, as long as the cultivation of CFU-GM is to be cultivated in the effect of compound of the present invention next step or multistep by cultured cell, at preset time, just can produce the colony of required noble cells.
4.4.1 external stem cell is cultivated
The inventive method comprises the adjusting in vitro differentiation of stem cell or CFU-GM, comprise cell and compound, at external incubation together, induce them to be divided into the cell lineage of specific expectation as little organic compound of the present invention, then the cell directly transplanting of differentiation is gone among the curee.In a preferred embodiment, cell induction is divided into hematopoietic cell lineage.
In certain embodiments, with the purpose stem cell cultivated at the external compound of the present invention that is exposed to 0.1 μ g/ml, 0.2 μ g/ml, 0.3 μ g/ml, 0.4 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 5 μ g/ml or 10 μ g/ml concentration.Preferably, the purpose cellular exposure is the PDE IV inhibitor of about 0.005 μ g/ml to about 5mg/ml in concentration, or concentration is the SelCID of about 0.005 μ g/ml to 5mg/ml TM(Celgene Corp., Warren, NJ) (also referring to 4.7 joints, " pharmaceutical composition ").
In certain embodiments, by some nutriments, hormone, vitamin, growth factor or its combination are incorporated in the primer solution embryonic-like stem cell is induced propagation in the placenta bio-reactor.Serum and other growth factor also can be added in propagation primer solution or the medium.Growth factor is protein normally, includes but not limited to: cell factor, lymphokine, interferon, colony stimulating factor (CSF), interferon, chemokines and interleukin.Also can use other growth factors, comprise the recombined human hemopoieticgrowth factor, the growth factor and the epidermal growth factor in part, stem cell factor, TPO (Tpo), granulocyte colony stimulating factor (G-CSF), leukaemia inhibitory factor, trofermin, placenta source wherein arranged.
Add that growth factor in the primer solution can stimulate undifferentiated embryonic-like stem cell, committed progenitor or the propagation of the cell (for example Fen Hua hematopoietic cell) that broken up.But the generation of the molecule of growth factor stimulating organism material and biologic activity includes but not limited to some growth factors of having described in immunoglobulin, hormone, enzyme or front.The placenta of cultivating regularly " supply " reduces the cell that discharges to remove the medium that has consumed, replenishes fresh culture.Cultivate placenta and should deposit under aseptic condition with the minimizing contamination of heavy, and keep growth to create a such environment under supercharging condition intermittently, regular, it keeps the sufficient nutrition supply to placenta cells.Should be realized that the perfusion of placenta and cultivation can realize that all automation and computerization are to obtain high efficient and capacity.
4.4.2 external CFU-GM is cultivated
The inventive method also comprises CFU-GM, especially CD34 +And CD133 +Adjusting and regulation and control that CFU-GM is grown.In one embodiment of the invention, CFU-GM is induced to differentiate into a kind of hematopoietic cell lineage.In a specific embodiment, this cell lineage is the granulocyte pedigree.In another embodiment, CD133 +Cell induction is divided into endothelial cell, brain cell, nephrocyte, liver cell or intestinal cell.
CFU-GM can be cultivated by standard method above-mentioned.In addition, the cultivation of CFU-GM is handled cell in a plurality of times of cultivating or the time of setting possibly, so that CFU-GM is divided into different cell lineage.
Therefore, cultivating CD34 +Or CD133 +In the method for CFU-GM, cell was implanted in the time of 0 day and is contained in the medium of stem cell factor (SCF), Flt-3L, GM-CSF and TINT-A, cultivated 6 days.In the 6th day, cell is implanted in the medium that contains GM-CSF and TNF-α again, continues to continue to cultivate six days.This method can produce dendritic cells.In the changing method of this method, cell is implanted in the medium that contains GM-CSF and IL-4 at the beginning, changes the medium (referring to Steinman etc., international publication number WO 97/29182) of monocyte condition of culture then in the 6th day into.In order to produce CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Progenitor cell contacted The compounds of this invention with CFU-GM with compound of the present invention at the 0th day, and collected CD34 at the 6th day +CD38 -CD33 +Or CD34 +CD38 -CD33 -CFU-GM.
Estimate to add compound of the present invention, especially SelCID TMTime for CD34 +Cell is to the differentiation pathway of specific cells pedigree, and CD133 +The differentiation of cell has crucial influence.CD34 +CFU-GM is cultivated under standard conditions, is along the approach of development of bone marrow or pedigree, just, becomes dendritic cells in beginning to implant 12 days of back (just beginning to cultivate the back).Yet, beginning to cultivate in first six days, the time in several specific times is added compound of the present invention, has changed this approach basically.For example, if CD34 +The especially myeloid CD34 of cell +Cell was at first day that cultivates and compound of the present invention, especially SelCID TMContact, the differentiation along the bone marrow cell pedigree will be suppressed so, is not cultivating the 6th day CD34 as be exposed to compound of the present invention (just being exposed to DMSO) with respect to contrast +CD38 -The increase of cell quantity and CDla +CD14 -Cell quantity minimizing proved.And, contacting the cells whose development that can cause expressing surface markers with compound of the present invention and be suppressed, surface markers is by dendritic cells system, expresses as CD80 and CD86.At first day that cultivates or cultivate any moment in back three days of the beginning, allow cell contact with compound of the present invention, all can cause CD34 +This growth of CFU-GM is regulated.If between the 0th day and the 6th day, giving repeatedly taking medicine of compound of the present invention, for example, administration in the 0th and the 2nd day, administration in the 0th and the 4th day, administration or the 2nd, the 4th in the 3rd and the 6th day, administration in the 6th day can aggravate CD34 +The increase of cell quantity.
One of the present invention be particularly useful aspect, at CD34 +Added compound of the present invention in first day of the CFU-GM cultivation, continued to expose in 12 days, can cause the growth of unique CFU-GM, this CFU-GM is expressed the recombinant cell surface markers of uniqueness: CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Cell mass has been represented an intermediateness of differentiation.This cell mass is very useful as the bred group of CFU-GM, and they can be implanted on one's body the patient of the hematopoietic cell lineage that needs fast breeding easily, for example granulocyte.In another embodiment, CD34 +Can propagation in the phase (about 6 days) (just be not exposed to PDE IV inhibitor, at standard medium as SelCID TMOr other analogs) implant in and cultivate, it is forwarded to comprise SelCID then TMOr in the same or analogous medium of analog, continued to cultivate the 12nd day always.In this embodiment, the cell that is breaking up generally shows the minimizing of CD80, CD86 and CD14 expression, but causes CDla +The cell comparison is according to but continuing to increase.Cell in this differentiation can be divided into dendritic cells by obstruction.In another embodiment, CD34 +Cell was used SelCID in lasting three days at least in the propagation phase (implanting the back 1-6 days) TMOr other compound treatment of the present invention.Still in another embodiment, CD34 +Or CD133 +In CFU-GM first six days after implantation with the SelCID of twice or more multiples TMOr other compound treatment of the present invention.This multiple processing method can increase CD34 +And CD133 +The propagation of cell mass.Use SelCID TMOr other compound several different methods processing of the present invention, can cause CD34 +The transformation of CFU-GM differentiation is not divided into CDlc +CD15 -Cell lineage but be divided into CDlc -CD15 +Cell lineage just is not divided into marrow dendritic cells pedigree, and becomes granulocyte pedigree (Fig. 6 B).
The 0th day processing CFU-GM from cultivating especially adopted multidose in the 0th day and the 6th day, also can cause CD133 +The increase of CFU-GM quantity, especially CD34 +CD133 +The increase of progenitor cell.CD133 is the mark of a hematopoiesis, can be used as the alternative of CD34 separator, because CD133 +Cell can be with CD34 +The same mode of subclass increases, and has kept the ability (referring to Kobariv etc., J.Hematother.Stem CellRes.10 (2): 273-81 (2001)) of many cells pedigrees simultaneously.Reported CD133 +CD34 in the human fetal brain tissue source -Occur in the cell, and when it is injected in the newborn mice, demonstrate potential transplanting, propagation, migration and Neural Differentiation (referring to Proc.Natl.Acad.Sci.U.S.A.19:97 (26): 14720-5 (2000)).CD133 +Candidate stem cell shown and has been imbued with the CFU-GM activity, has by the colony that strengthens to form ability and at NOD-SCID mouse higher transfer ability on one's body.
Although more than, if the CD34 in the propagation +CFU-GM (any times in just beginning to cultivate 3-6 days) after cultivating three days contact with compound of the present invention, and the CFU-GM that has begun in the propagation of express cell surface markers CDla can continue to increase this mark of expression with the contrast of DMSO processing relatively.What deserves to be mentioned is that this lasting increase can't cause cytotoxicity.In other words, with PDE IV inhibitor, as SelCID TMHandle the apoptosis that can not cause other cell masses.Pure effect is exactly the maintenance of existing immunocompetence and the development of new immunocompetence.
Therefore, in an embodiment of the inventive method, CD34 +It is modulated (just being suppressed) that cell differentiation becomes dendritic cells, and this is to pass through CD34 +CFU-GM realizes with compound treatment of the present invention the 0th day that cultivates (just first day of cultivation).In another embodiment, CD34 +The cell differentiation granuloblast passes through CD34 +CFU-GM contacts with compound of the present invention and strengthens the 0th day that cultivates (just cultivate first day).In another embodiment, CD34 +Cell differentiation becomes CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Progenitor cell contacts with compound of the present invention in first three days of cultivating by CFU-GM and strengthens.In another embodiment, CD34 +CD133 +The group contacts with the compound of the present invention of multidose by CFU-GM in the 0th day to the 6th day process of cultivation and strengthens or increase.In another embodiment, CD1a +Cell mass is by CD34 when cultivating the 6th day +CFU-GM contacts with compound of the present invention and strengthens or increase, wherein said CD34 +Cell differentiation becomes to present the cell of CD1a surface markers, and wherein said cultivation comprises handling without above-claimed cpd and reaches six days most.
In the superincumbent embodiment, be appreciated that at SelCID TMOr the difference in the administration of relevant compound can be applied to CFU-GM in the body, for example, transplanted or implanted among the patient of this cell at one, as being applied to CFU-GM external.
Method of the present invention comprises the adjusting in vitro differentiation of stem cell or CFU-GM, be included in cell in vitro and inducing cell and be divided into specific cytophyletic compound, as little organic molecule of the present invention incubation together, then the cell directly transplanting of differentiation is gone into the curee.In a preferred embodiment, cell is induced and is divided into a kind of hematopoietic cell lineage.In a selectable embodiment, with CD133 +Cell induction is divided into endothelial cell, brain cell, nephrocyte, liver cell or intestinal cell.
What deserves to be mentioned is, consider method as herein described and mammal, preferred people's CD34 +Or CD133 +CFU-GM is used together, and also consideration is used with birds or reptiles CFU-GM.But also there is different effects in compound of the present invention, and this depends on the species in CFU-GM source.Aspect cultural method, exist some differences, especially, also considered about used compound concentrations.For example, the CFU-GM in mouse source is to compound of the present invention, for example SelCID TMMore insensitive, need higher concentration just can reach the effect of people source CFU-GM under 1 μ M.It will be appreciated by those skilled in the art that this optimization is routinely.
4.5 the genetic engineering of stem cell and CFU-GM
In another embodiment of the invention, use, for example, viral vectors such as adenovirus or retrovirus vector, or by using the DNA picked-up of mechanical means such as liposome or chemistry mediation, will according to the stem cell of method differentiation of the present invention or CFU-GM before or after compound of the present invention contacts, carried out genetically engineered.In specific embodiment, CD34 +CFU-GM is used compound treatment of the present invention then through genetically engineered; In more specific embodiments, described compound is SelCID TMOr its analog.In another embodiment, described cell compound treatment of the present invention is carried out genetically engineered then.
By certain methods well known in the art, for example transfection, conversion, electroporation, infection, microinjection, cytomixis, DEAE-glucan, calcium phosphate precipitation, liposome, LIPOFECTIN TM, lysosome merges, synthetic cation lipid, the use or dna vector transhipment of particle gun, can import in the purpose cell containing genetically modified carrier, like this transgenosis is delivered in the daughter cell, as filial generation embryonic-like stem cell or CFU-GM by the embryonic-like stem cell division.Be used to transform or the distinct methods of transfection mammalian cell, referring to Keown etc., 1990, MethodsEnzymol.185:527-37; Sambrook etc., 2001, MolecularCloning, A Laboratory Manual, the third edition, cold spring harbor laboratory publishes, New York.
Preferably, utilize any technology that transgenosis is imported, only otherwise destroy nuclear membrane or other the already present cell or the genetic structure of cell.In certain embodiments, can transgenosis be inserted in the interior genetic material of nuclear by microinjection.Cell and cyto-architectural microinjection are known in this field and skilled.
Mammalian cell for stable transfection is cultivated as cultivating the cell in placenta, has only the sub-fraction cell foreign DNA can be incorporated on its genome.The efficient of integrating depends on carrier and used rotaring dyeing technology.For identifying and filter out integron, generally can change the gene that has a selection markers (for example, antibiotic resistance) over to embryonic-like stem cell together with the sequence of genes of interest.Preferred selection markers comprises that those have the antiradiation drug resistance, as G418, hygromycin and amethopterin.Stable transfection the cell of exogenous nucleic acid can identify (cell that just is integrated with the selected marker can be survived, and other cells are all dead) by drug screening.Particularly useful during the homologous recombination of this method before recombinant mammalian cells (for example embryonic-like stem cell) imports or be transplanted to main body or patient.
Many screening systems can be used for screening the host transformed stem cell, as embryonic-like stem cell or CFU-GM, resemble CD34 +Or CD133 +CFU-GM.Especially, this carrier can comprise some observable or selectable mark.Other system of selection includes but not limited to screen another mark as herpes simplex virus thymidine kinase (Wigler etc., 1977, Cell, 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc.Natl.Acad.Sci USA 48:2026), adenine phosphoribosyl transferase (Lowy etc., 1980, Cell 22:817), these genes can use in tk-, hgprt-or aprt-cell respectively.Equally, the antimetabolite resistance also can be given amethopterin resistance (Wigler etc., 1980, Proc.Natl.Acad.Sci.USA 77:3567 as the basis of the following gene of screening: dhfr; O ' Hare etc., 1981, Proc.Natl.Acad.Sci.USA 78:1527); Gpt gives mould fen acid resistance (Mulligan and Berg, 1981, Proc.Natl.Acad.Sci.USA 78:2072); Neo, give aminoglycoside G-418 resistance (Colberre-Garapin etc., 1981, J.Mol.Biol.150:1); And hygro, give hygromycin resistance (Santerre etc., 1984, Gene 30:147).
Transgenosis can be incorporated on the genome of purpose cell, preferably passes through random integration.In other embodiment, transgenosis can be integrated by direct method, as by direct homologous recombination (just in the purpose cell, " knock in " or " knock out " genes of interest), Chappel, the U.S.. the patent No. 5,272,071; PCT publication number WO91/06667 is published in 1991-05-16; United States Patent (USP) 5,464,764, Capecchi etc. are disclosed in 1995-11-07; United States Patent (USP) 5,627,059, Capecchi etc. are disclosed in 1997-05-06; United States Patent (USP) 5,487,992, Capecchiet etc. are disclosed in 1996-01-30).
By the homologous recombination modified target dna method that it changes cell over to is well known in the art.This construct will comprise the part of the genes of interest that passes through genetic modification at least, and will comprise the zone with the target site homology, the just copy of endogenic genes of interest on host genome.The DNA construct of random integration needn't comprise that homologous region is with the mediation reorganization with respect to those for homologous recombination.Mark can be included in the target construct or at random in the construct, to form just screening or the negative screening that transgenosis is inserted.
For producing the cell of homologous recombination, the for example embryonic-like stem cell of homologous recombination, endogenous placenta cells or the foreign cell of in placenta, cultivating, the carrier for preparing a kind of homologous recombination, wherein genes of interest is positioned at 5 of the endogenous gene order of target cell genome ' and 3 ' end, so that between genes of interest that is carried by carrier and the target cell genome endogenous gene homologous recombination takes place.Remaining nucleic acid of both sides is wanted long enough so that with the target cell genome on the homologous recombination of endogenous gene success.Usually, the DNA of both sides on the carrier (5 ' and 3 ' end) has several thousand base-pairs.Make up homologous recombination vector and be known in the art by the method that the reorganization stem cell obtains the homologous recombination animal.(referring to, for example Thomas and Capecchi, 1987, Cell 51:03; Bradley, 1991, Curr.Opin.Bio/Technol.2:823-29; PCT publication No. WO90/11354, WO 91/01140 and WO 93/04169).
In a specific embodiment, (U.S. Patent number 5,942,496 is entitled as method and compound that multiple gene changes osteocyte over to, is disclosed in 1999-08-24 to utilize the method for Bonadio etc.; PCT W095/22611 is entitled as the method and the compound that stimulate osteocyte, is disclosed in 1995-08-24) nucleic acid is imported in the purpose cell, as stem cell, CFU-GM or the foreign cell in placenta, cultivated, for example osteoprogenitor cells.
4.6. the stem cell of being adjusted that is used to break up and the purposes of CFU-GM
4.6.1. general service
Stem cell of the present invention, CD34 +And CD133 +CFU-GM can induce differentiation to be used for transplanting with elder generation external back interior therapeutic scheme.In one embodiment, population of stem cells is divided into specific cell type, and through the genetically engineered gene therapy product that provides.In another embodiment, progenitor cell propagation is early progenitor cell, and through genetic engineering modified so that the gene therapy product to be provided.In another embodiment, progenitor cell is divided into specific cell type such as granulocyte, and through genetic engineering modified so that the gene therapy product to be provided.
Compound of the present invention also is applied clinically, and transplanting has at present become and recovers the elementary object that the marrow white blood corpuscle produces, as the neutropenia that caused by disease and/or the excision of clinical marrow and the counter-rotating of leukopenia.These compounds also are used in the recovery of early progenitor cell or granulocyte generation, and it is by disease, and various known treatment side effects or marrow excise and causes.Compound of the present invention also is applied in some cases, and the wherein inhibition that preferably red blood cell is generated does not produce and can not suppress marrow.
In certain embodiments, with the stem cell and the untreated cell of compound treatment of the present invention, as the stem cell co-administered that comes from Cord blood or peripheral blood is in its required patient.In other schemes, used the CD34 of compound treatment of the present invention +Or CD133 +Cell and untreated cell, as the stem cell co-administered that comes from Cord blood or peripheral blood is in its required patient.In a scheme, from first day the CD34 that cultivates with compound treatment of the present invention +CFU-GM and untreated cell co-administered are in its required patient.At one more specifically in the scheme, the CFU-GM of transfer is CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -CFU-GM.
Stem cell, for example embryonic-like stem cell or candidate stem cell, or CFU-GM, its differentiation is regulated according to method of the present invention, can be made into injectable preparation (referring to PCT WO96/39101, quoting its full content herein as a reference).In another possibility, cell and tissue, its differentiation is regulated according to method of the present invention, can be as U.S. Patent number 5,709,854; 5,516,532; Or 5,654,381 describe, and makes preparation with the crosslinked hydrogel of polymerization, and the full content of quoting each piece here as a reference.
Embryonic-like stem cell can be used for substituting treatment or the specific CFU-GM monoid in the research method (for example cartilage cell, liver cell, hematopoietic cell, pancreas parenchyma, neuroblast, muscle CFU-GM etc.) that uses CFU-GM general.
4.6.2. tissue is replaced or is increased
Stem cell, embryonic-like stem cell especially of the present invention, and CFU-GM, its differentiation is regulated according to method of the present invention, can be used for a variety of methods of treatments, and described method is pointed to the transplanting or the fusion of required cell colony, as stem cell or progenitor cell.Stem cell or CFU-GM can be used to replace or increase existing tissue, import new or tissues replaced or connect biological tissue or structure.
In an embodiment preferred of the present invention, stem cell is as the embryonic-like stem cell that comes from placenta, or CFU-GM such as hemopoietic progenitor cell, its differentiation is regulated according to method of the present invention, can use from body homology or allos, comprise coupling or unmatched HLA type, hematopoiesis is transplanted.The operating position of transplanting as allos hematopoiesis with embryonic-like stem cell is consistent, and it can treat the host better, reduces the immunological rejection to donorcells, and as U.S. Patent number 5,800, on September 1st, 539,1998 announced; U.S. Patent number 5,806 described in announcing on September 15th, 529,1998, is quoted each piece of writing here as a reference.
For example, embryonic-like stem cell, its differentiation is regulated and control according to method of the present invention, in the transplanting scheme that can be used for treating, for example increase or replace the stem cell or the CFU-GM of liver, pancreas, kidney, lung, nervous system, musculature, bone, marrow, thymus gland, spleen, mucosal tissue, sexual gland or hair.Similarly, hemopoietic progenitor cell, its differentiation is regulated and control according to method of the present invention, can be used for replacing marrow or endothelial progenitor cells.
Stem cell, embryonic-like stem cell for example, its differentiation is regulated and control according to method of the present invention, can be used for the increase of cartilage, tendon or ligament, repairs or replaces.For example, in certain embodiment, false organ (for example false stern) is the replacement cartilaginous tissue structure that grows up at the embryonic-like stem cell that surface coverage one deck is cultivated by the inventive method.In other embodiment, joint (for example knee) can be reconstituted by the cartilaginous tissue structure that embryonic-like stem cell grows up to.The cartilaginous tissue structure also can be applicable in the reconstruction operations in dissimilar joints (as for scheme, referring to as Resnick, D., and Niwayama, G., eels., 1988, bone and disorder of joint diagnosis, second edition, W.B.Saunders Co.).
Stem cell and the CFU-GM handled according to the inventive method can be used in tissue and the organ damage that reparation is caused by disease.In this embodiment, the patient can regenerate with embryonic-like stem cell or repairs tissue or the organ damage that is caused by disease by administration, for example strengthens functions of immune system after chemotherapy or the radiotherapy, repairs the heart tissue after the miocardial infarction.According to this method and stem cell and/or the CFU-GM handled with PDE IV inhibitor of the present invention, or, can implant in the individuality that needs, to repair or replacement liver, pancreas or heart tissue with the stem cell or the CFU-GM of PDEIV inhibitor of the present invention administration.
Stem cell and the CFU-GM handled according to the inventive method also can be used for increasing or replacing bone marrow cell in bone-marrow transplantation.The people is in body homology and allos bone-marrow transplantation just are being applied to treat disease as leukemia, lymphoma and some other life threatening at present.Yet the shortcoming of these methods need to be a large amount of donor bone marrows, to guarantee having enough cells to be used for transplanting.
The embryonic-like stem cell of collecting according to method of the present invention can provide stem cell and CFU-GM, can reduce necessity of a large amount of marrow donations.Certainly, also can obtain a spot of donations marrow, before injecting or being transplanted to acceptor, in placenta, cultivate expansion, obtain a large amount of stem cells and CFU-GM according to method of the present invention.
By a large amount of embryonic-like stem cells and/or the CFU-GM that this method obtained, in certain embodiments, can reduce the necessity of a large amount of marrow donations.In bone-marrow transplantation, approximately need every kg of patient body weight 1 * 10 8To 2 * 10 8Myelomonocyte is used for transplanting (just, needing about 70 milliliters marrow for heavy 70 kilograms contributor).The marrow that obtains 70 milliliters needs highdensity beneficence, and a large amount of losses of blood are arranged in the donations process.In specific embodiment, the cell (for example 7-10 milliliter) that is obtained by a small amount of marrow donations can obtain enlarging by propagation, for example in the placenta bio-reactor before being injected into acceptor.Stem cell and CFU-GM, particularly CD34 +Or CD133 +CFU-GM, its differentiation is regulated according to method of the present invention, also can provide stem cell and/or CFU-GM, to eliminate the necessity of a large amount of marrow donations.
Also can be used in the specific embodiment by mazolytic embryonic-like stem cell, replace some special disease of therapy for treating with homology or isodynamic enzyme, include but not limited to lysosomal storage disease, as Tay-Sach disease (Jay-Sachs), Niemann's disease (Niemarm-Pick), fabry disease (Fabry ' s), familial splenic anemia (Gaucher ' s), Heng Teshi (Hunter ' s) syndrome, what Le Shi (Hurler ' s) syndrome, and other gangliosides deposition disease, mucopolysaccharidosis and glycogen storage disease.
In other embodiment, cell can use in gene therapy, correct some inborn errors of metabolisms as homology or heterologous transgene carrier, as adrenoleukodystrophy, gallbladder cystic fibrosis, glycogen storage disease, hypothyroidism, sickle cell anemia disease, pendant Ademilson (Pearson) syndrome, other Pa Shi (Pompe ' s) disease, phenylketonuria (PKU), Tay-Sach disease, porpharia, maple syrup urine disease, homocystinuria, mucopolysaccharidosis, granulomatous disease and tyrosinemia, or treatment cancer, tumour or other illness.
In other embodiment, cell can use in homology or allos regeneration or replacement therapy, include but not limited to that corneal epithelium defective, repair of cartilage, face go scar, mucous membrane, eardrum, intestines internal layer, nerve fiber (for example olfactory nerves unit of the auditory neuron at retina, basilar memebrane place, olfactory sensory epithelium), also can be used in the burn and trauma repair of skin injury, during scalp (hair) is transplanted, or other are impaired, in the reconstruction of the organ of disease, tissue.
And a spot of stem cell and CFU-GM normally circulate in blood flow.In other embodiments, ectogenic stem cell or CFU-GM are collected by the separation property blood transfusion, and in this program, blood is drawn out of, and one or more compositions are optionally separated, and remaining blood is still refilled in the donor body.The foreign cell that obtains by the separation property blood transfusion increases with the inventive method, can get rid of the necessity of donating marrow fully.
In another embodiment, be used for replacement therapy after the chemotherapy with the hemopoietic progenitor cell of the inventive method amplification.Most of chemotherapeutics is a target and tumoricidal, can kill the cell in all propagation, just is in the cell in the cell division.Because marrow is one of most active tissue of proliferation in vivo, candidate stem cell just often is subjected to the damage and the destruction of chemotherapeutics, and therefore the generation of haemocyte reduces or stop.Chemotherapy needs to stop interimly so that patient's hemopoietic system can the supply haemocyte before chemotherapy once more.This may need one month or the longer time make before static stem cells hyperplasia, make leukocyte count reach once more the acceptable level of chemotherapy (when chemotherapy once more, bone marrow cell wound in damaged condition).
When haemocyte when chemotherapy is regenerated interval, tumour is also grown up if having time certainly, and because natural selection and chemotherapeutics become have more resistance.Therefore, the time of chemotherapy is long more, and blanking time is short more, and the probability of kill tumor is just big more.For shortening the interval of chemotherapy, the embryonic-like stem cell or the CFU-GM of breaking up according to the inventive method can be injected in patient's body.This therapy can reduce the time that the patient is in low quantity of leucocyte, can be therefore from chemotherapy, recovering more early.
In another embodiment, people's placenta stem-cell can be used to treatment or prevention genetic disease, as the chronic granuloma disease.
4.6.3. inflammation is improved
Stem cell and CFU-GM, its differentiation is regulated according to method of the present invention, can be used as common anti-inflammatory preparation.The inventor finds, when the stem cell in for example Cord blood source and CFU-GM are in being transplanted to patient's body, can reduce or the reaction that diminishes inflammation fully.Therefore, in one embodiment, method of the present invention comprises that it is broken up the stem cell or the CFU-GM that have been subjected to one or more compounds adjustings of the present invention delivers medicine to a patient who produces inflammatory reaction, or among the patient of the reaction that may be inflamed.In specific embodiment, stem cell is an embryonic-like stem cell, and CFU-GM is a candidate stem cell, particularly CD34 +Or CD133 +CFU-GM.
The inventor also finds to use compound of the present invention, and SelCIDs treatment just is individual, has stimulated those to regulate, improve or reduce the cells whose development and the differentiation of inflammatory reaction.Therefore, another embodiment of the invention comprises the patient that treatment produces inflammatory reaction, or the patient's of the reaction that may be inflamed method, comprises that the effective dose with one or more compounds of the present invention delivers medicine to described individuality.In another embodiment, this method contacts stem cell or CFU-GM with compound of the present invention before being included in and delivering medicine to described individuality, and the dose therapeutically effective with described cell delivers medicine to described individuality then.Still in another embodiment, the cell of so handling can deliver medicine to described individuality simultaneously with the dose therapeutically effective of one or more compounds of the present invention.
In other embodiment, other compounds are combined administration with compound of the present invention and/or cell also can reduce inflammation.For example, these additional compounds comprise steroids, as prednisone, or other on-steroidal anti-inflammatory agent, suppress acetylsalicylic acid (aspirin), brufen, acetaminophen, the special inhibitor of cox-1 as cox-1/cox-2, or the derivative of any of these compound.These additional anti-inflammatory agents can be by the method transmission of standard, as vein, part, intracutaneous or by inhalation, also can be simultaneously and compound of the present invention and/or cell transmission, or in the different time.
Above method can be used for treating that those are relevant with inflammation, by its some diseases that causes or cause.For example, this method can be used to treat the inflammation that is caused by the outer damage of trauma, and this method also can be used for treating the especially blood vessel relevant with operation relevant operation such as normal structure transplanting, artificial blood vessel's transplanting, heart valve or caused inflammation of angioplasty or damage.This method also can be used for preventing the narrow or ISR behind the cardiac valve procedure.This method also can be used for treating the inflammation that any disease causes, and includes but are not limited to heart disease, arteriosclerosis, sensitivity or hypersensitization disease, immune disorders, autonomous immune disorders such as arthritis, or by infecting caused inflammation.Except treating already present inflammation, cell of the present invention and/or compound also deliver medicine to preventability individuality, to reduce the generation of inflammation.Its form as the preceding treatment of operation is particularly useful, and can reduce postoperative inflammatory reaction thus, improves the chance that the patient successfully recovers, and shortens hospital stays and sick and wounded phase.
The monitoring of the effect of above anti-inflammatory therapy can be finished by a lot of known methods, as visual inspection MRI or cat scan, and system or local temperature mensuration etc.Because a kind of protein of c reactive protein by name is a mark of inflammation, the minimizing of the amount of the c reactive protein that the validity of above methods of treatment just can be by individuality detects, particularly the part before the inflammation generation.
4.6.4. the generation of dendritic cells and granulocyte colony
Compound of the present invention can carry out administration specifically, to regulate stem cell and/or CFU-GM with respect to the dendritic cells development pathway and along the differentiation of granulocytic development pathway.Similarly, cell of the present invention also can be in vivo or external adjusting to produce the dendritic cells or the granulocyte group of propagation.
Dendritic cells can be used as the medicament based on the treatment of immunity.For example, can cause the in vitro activation of the T cell of antigen-specific with dendritic cells and T lymphocyte and antigen protein in external co-incubation.Activated T cells is carried out immediately from the immune response (WO97/24438) of body administration to cause antigen-specific in vivo.In another embodiment, the T cell can be activated external, contacts with dendritic cells from the direct antigen expressed albumen of recombinant precursor by the T lymphocyte.Activated T cells can be used as from body infusion (WO97/29183).
Become anti-albumen, cell or biological immunizing agent with specific polypeptide or protein fragments activated T cells, and they are sources of special peptide or protein.For example, dendritic cells may have the peptide of tumour-specific.The concrete application of external t cell activation that comes from dendritic cells, is disclosed in 963 and has carried out claim at U.S. Patent number 5,788 with the treatment prostate cancer.The dendritic cells of demonstration derived from bone marrow such as Mayordomo carry out pulse with synthetic tumour peptide and bring out protectiveness and curative antineoplastic immune (Nature Medicine 1:1297-1302 (1995); J.Exp.Med., 183:1357-1365 (1996)).U.S. Patent number 5,698,679 have described and in vivo antigenic peptide have been passed to the domain-immunoglobulin fusion proteins that the targeting antigen that comprises dendritic cells presents cell (APCs).This identical method can be used to produce virus, bacterium or parasitic vaccine to be derived from virus, cell or parasitic peptide or antigen.
Dendritic cells also are for the therapeutic interventional target in the treatment of various immunologically mediated diseases.For example, dendritic cells have been implied to be the pathogenesis of acquired immune deficiency syndrome (AIDS) and a kind of important factor in the Pathological Physiology person of possessing of HIV virus (for example, as).Referring to Zoeteweij etc., J.Biomed.Sci.5 (4): 253-259 (1998); Grouard etc., Curr.Opin.Immunol.9 (4): 563-567 (1997); Weissman etc., Clin.Microbiol.Rev.10 (2): 358-367 (1997).At U.S. Patent number 5,627, the in-vitro method that the drug candidate that the HIV of dendritic cells infects is eliminated in screening has been described in 025.In another embodiment, can use the dendritic cells inducing T cell to donor tissue or organ the anergy (referring to U.S. Patent number 6,375,950) in acceptor.
Granulocyte can be used for the granulocyte blood transfusion in treatment of infecting or prevention, the infection that for example bacillary newborn septicemia, the neutrophil leucocyte in the cancer patient are relevant and move latent infection among the patient who grows accepting marrow.Granulocyte also can be used in hypersensitive prevention and treatment.For example, can carry out deactivation with the granulocyte (that is, with the granulocyte of IgE antibody sandwich, some of them have specificity for anaphylactogen) of the inflammation-related of IgE mediation and be used to relax set up at the immunoreactive symptom of anaphylactogen (referring to U.S. Patent number 6,383,489).
Thereby, in one embodiment of the invention, granulocyte population in individuality is bred by CFU-GM of the present invention by a kind of method, this method comprises that wherein said amount is enough to induce described individuality by endogenous CD34 to the compound of the present invention of effective dose on the described individual drug treatment +Cell produces various granulocytes.In another embodiment, the granulocyte population is bred by a kind of method in individuality, this method comprises described individual administration CD34 +Or CD133 +CFU-GM, wherein said cell contacts at least 3 days with compound of the present invention, and to the described cell population of described individual administration.In another embodiment, by the amplification in individual of a kind of method, this method comprises with CD34 with the granulocyte population +Or CD133 +CFU-GM population and compound of the present invention deliver medicine to above-mentioned individuality together, and the dosage of wherein said compound of the present invention is enough to cause that various described cell populations are divided into granulocyte.In the concrete scheme in above embodiment, described CD34 +CFU-GM is CD34 +CD38 -CD33 +Cell.
4.6.5. other diseases and treatment of conditions
Stem cell that the present invention has broken up and CFU-GM, or compound of the present invention, also can be separately or use in conjunction to treatment or prevent in the various other diseases.In certain embodiments, for example, disease or illness include but not limited to blood vessel or angiocardiopathy, arteriosclerosis, diabetes, alpastic anemia, myelodysplasia, miocardial infarction, epilepsy, multiple sclerosis, apoplexy, hypopiesia, heartbeat stops, amyotrophy, inflammation, the forfeiture of the cognitive function relevant with the age, radioactive damage, cerebral paralysis, nerve retrograde affection, Alzheimer's, Parkinson's disease, Lay Fu Shi disease, the AIDS dementia, the loss of memory, amyotrophic lateral sclerosis (ALS), the kidney ischaemic, brain or chorda dorsalis injury, cardiopulmonary bypass, glaucoma, retinal ischemia, retinal damage, lysosomal storage disease such as Tay-Sach disease, niemann-Pick disease, fabry disease, familial splenic anemia, mucopolysacchari dosis H, what reins in syndrome, resemble other gangliosides deposition disease, mucopolysaccharidosis, glycogen storage disease, inborn errors of metabolism such as adrenoleukodystrophy, gallbladder cystic fibrosis, glycogen storage disease, hypothyroidism, sickle cell anemia disease, pendant Ademilson syndrome, other Pa Shi disease, phenylketonuria (PKU), porpharia, maple syrup urine disease, homocystinuria, mucopolysaccharidosis, chronic granuloma, tyrosinemia, Tay-Sach disease, cancer, situation tumour or other pathology or that become knurl.
In other embodiments, cell of the present invention (for example, being exposed to the cell of compound of the present invention) can be used to treat any by the wound caused wound of wound of inflammation particularly.These diseases relevant with wound comprise central nervous system (CNS) damage, comprise the damage, CNS surrounding tissue of brain, the notochord damage to peripheral neverous system (PNS); Or the damage of health remainder.This wound may be caused by accident, maybe may be normal and abnormal result of therapy such as operation or angioplasty.This wound may with blood vessel break or inaccessible relevant, as apoplexy or phlebitis.In specific embodiment, cell can be used for from body or allos regeneration or replaces in treatment or the scheme, include but not limited to that corneal epithelium defective, repair of cartilage, face remove scar, mucous membrane, eardrum, intestines internal layer, nerve fiber (retina for example, the auditory neuron at basilar memebrane place, the olfactory nerves unit of olfactory sensory epithelium) treatment, also can be used in the burn and trauma repair of skin injury, or other are impaired, in the reconstruction of the organ of pathology, tissue.
In a specific embodiment, disease or illness are alpastic anemia, myelodysplasia, leukemia, marrow illness or hematopoietic disease or illness.In another embodiment, the curee is the people.
4.7. pharmaceutical composition
The present invention includes pharmaceutical composition, it comprises potion and/or multi-agent one or more compounds of the present invention, and wherein said potion or multi-agent are effectively for single or multiple administering modes, and it is with adjusted or unadjusted people CD34 +Or CD133 +CFU-GM or stem cell transplantation in the individuality before or after, to suppressing, regulate and/or regulate and control these stem cells and/or CFU-GM is divided into specific cell type, for example, hematopoietic lineage cell, particularly marrow pedigree cell can play good effect.At this, just as other places of the context of the invention, " individuality " represents any individuality of medicine or cell administration, for example, and mammal, birds or reptile.
Therefore, in specific embodiment, described a times or the many multiple doses that delivers medicine to individual compound of the present invention regulated endogenous CD34 +CFU-GM is divided into dendritic cells.In scheme more specifically, one times or many multiple doses have improved granulocytic quantity in the described individuality, this body and function described one times or the administration of many multiple doses.At another more specifically in the scheme, one times or many multiple doses have improved CD34 in the mammal +CD38 -CD33 +Or CD34 +CD38 -CD33 -The quantity of CFU-GM, this mammal is with described one times or the administration of many multiple doses.
In other embodiment, purpose CD34 +Or CD133 +CFU-GM or stem cell transplantation go into to have in this people curee or patient's body who needs.After the transplanting, compound administration of the present invention is in people curee or patient, to regulate the differentiation of the purpose cell of implanting in vivo.In specific embodiment, these cells are divided into granulocyte in vivo.Purpose CFU-GM in the embodiment that also has, people curee or patient's body or the differentiation of stem cell are regulated by the administration of compound of the present invention in position.
Still in other embodiment, the invention provides pharmaceutical composition, it comprises the cord blood stem cell or the progenitor cell of separation, and they obtain amplification be exposed to the compound that suppresses PDE IV activity according to method of the present invention the hemopoietic progenitor cell of having broken up.In another embodiment, the invention provides the pharmaceutical composition that comprises Cord blood, described Cord blood is replenished stem cell or the CFU-GM with compound treatment of the present invention; In a concrete scheme, described stem cell or CFU-GM are by described compound differentiation.
In another embodiment, the invention provides pharmaceutical composition, it comprises one or more PDE IV inhibitor of the present invention and stem cell of the present invention and/or CFU-GM.Said composition can be before administration preparation in 1,2,3,4,5,6,7,8,9,10,11 or 12 day, to regulate the differentiation of stem cell and/or CFU-GM along different growth/differentiation pathway.
Still in another embodiment, pharmaceutical composition of the present invention also comprises stem cell or CFU-GM self, and wherein said cell obtains differentiation according to method disclosed herein.Therefore, the invention provides a pharmaceutical composition that comprises a plurality of stem cells and/or CFU-GM, wherein said a plurality of stem cell or CFU-GM contact with one or more PDE IV inhibitor compounds of the present invention, and the differentiation that its concentration and duration are regulated and control described cell for described compound is sufficient.
Therefore, pharmaceutical composition of the present invention comprises the compound of the present invention that delivers medicine to individuality; With compound of the present invention or individually dosed in the cell of the present invention of individuality; And contact with compound of the present invention, deliver medicine to individual cell of the present invention.
The invention provides the method for treatment and prevent disease or illness, compound of the present invention by the drug treatment effective dose or composition be to mammal, preferred people, and the curee is can effectively regulate the CD34 that transplants or reside among the curee +Or CD133 +Propagation of CFU-GM or stem cell and/or differentiation.In one embodiment, the invention provides adjusting CD34 +And CD133 +CFU-GM or stem cell differentiation are to increase the method for the granulocytic quantity in the mammalian body.In another embodiment, any CD34 that derives from +And/or CD133 +The cell lineage of CFU-GM or stem cell all can be by delivering medicine to mammal, and especially people's compound of the present invention is regulated.Term used herein " mammal " comprises any mammal.Preferred mammal needs this treatment and prevention.Mammiferous embodiment includes but not limited to, ox, horse, sheep, pig, cat, dog, mouse, rat, rabbit, cavy, monkey etc., more preferably people.
The administration of compound of the present invention can be system or local.In most of the cases, will make system of compounds of the present invention ground discharge (that is, entering blood flow) to the mammal administration.The method of administration comprises the enteron aisle approach, as per os, cheek, hypogloeeis and rectum; Topical is as through skin and intracutaneous; Also has the parenteral approach.The outer approach of the stomach and intestine that are fit to comprises via hypodermic needle or tube injection, as the injection in intravenous, muscle, subcutaneous, intracutaneous, endoperitoneal, endarterial, intraventricular, the sheath, intraocular, the anterior chamber, and non-injecting pathway, as vagina, rectum or nasal administration approach.Preferably, compound of the present invention or composition are with oral administration.In specific embodiment, the zone that one or more compound administrations of the present invention need be treated in the part preferably.This can reach, for example, by the local infusion in the operation, overall applicability for example, is united use with wound dressing after operation, mode by injection, conduit, suppository, implantation, said implant is the material of porous, atresia or gel, comprises film, as ptyalectasis film or tunica fibrosa.
Compound of the present invention can for example be used parcels such as liposome, microparticle, microcapsules, capsule by general and off-gauge transmission system administration.For example, compound of the present invention and composition can place in the folliculus, particularly liposome and transmitted (referring to Langer, 1990, Science 249:1527-1533; Treat communicable disease and cancer with liposome, Lopez-Berestein and Fidler (editor) Liss, New York, pp.353-365 (1989); Lopez-Berestein, the same, the 317-327 page or leaf; Usually referring to as preceding).In another example, compound of the present invention and composition are transmitted in an in check delivery system.In one embodiment, can use pump (referring to Langer, supra; Sefton, 1987, CRC Crit.Ref Biomed.Eng.14:201; Buchwald etc., 1980, Surgery 88:507 Saudek etc., 1989, N.Engl.J.Med.3:574).In another example, can use polymeric material (referring to, the medical application of in check delivery system, Langer and Wise (editor), CRC Press, BocaRaton, Florida (1974)); In check medicine biological effectiveness, the design of drug products and characteristic, Smolen and Ball (editor), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol.Sci.Rev.Macromol.Chem.23:61; Be also shown in Levy etc., 1985, Science 228:190; During etc., 1989, Ann.Neurol.25:351; Howard etc., 1989, J.Neurosurg 71:105).Still in another example, in check delivery system can place near the target region that needs treatment, for example, and liver, therefore also just only need part system's dosage (see, for example, Goodson, the application of in check delivery system in medical science, supra, vol.2,115-138 page or leaf (1984)).Also can use other the delivery system of in summary, discussing (Langer, 1990, Science 249:1527-1533).When as the composition administration, compound of the present invention can be prepared with an amount of pharmaceutically useful excipient or carrier, can provide for the suitable administering mode of mammal.Term " pharmaceutically useful " is meant through federation management mechanism or state government to be checked and approved, or is listed in American Pharmacopeia or other suitable mammals especially in people's the generally acknowledged pharmacopeia.Term " excipient " is meant thinner, auxiliary agent, excipient or carrier, and compound of the present invention can be made them and deliver medicine to mammal.These excipient pharmaceutically can be liquid, and Ru Shui and oil comprise the oil that derives from oil, animal, plant or synthetic, as peanut oil, soybean oil, mineral oil, sesame wet goods analog.Drug excipient can be analogs such as salt, gum Arabic, gel, gelatinized corn starch, talcum, keratin, colloidal silica, urea.In addition, adjuvant, stabilizing agent, thickener, lubricant, colouring agent etc. also can use.Preferably, when delivering medicine to mammal, compound of the present invention and composition and pharmaceutically useful carrier, excipient or thinner are aseptic.When compound intravenous administration of the present invention, aqueous medium is preferred excipient, as water, saline solution, D/W and glycerite.
Compound of the present invention can be made capsule, tablet, pill, pilule, lozenge, pulvis, granule, syrup, ingredients, solution, suspension, emulsion, suppository or its extended release preparation, or any other suitable delivers medicine to mammiferous form.In a preferred embodiment, compound of the present invention and composition prepare with conventional program, just as the program of preparation human oral or intravenous (IV) drug.In one embodiment, pharmaceutically useful excipient is a hard gelatin capsule.The suitable pharmaceutically useful excipient and the embodiment of its preparation method are as describing in Remington: medicament science and practice, Alfonso R.Gennaro coding, the .Easton of Mack publishing company, PA, the 19th edition, 1995,86,87,88,91 and 92 chapters are incorporated herein by reference.
The medicament forms of capsule, tablet, pill or any compression is preferably made in the oral medicine that compound of the present invention and composition are prepared.And when tablet or pill, but compound and composition need of coating continue the activated time to postpone them in GI degraded and absorption thereby can exceed or prolong.Osmotically active around the permoselective membrane drives the oral administration that compound also is fit to compound of the present invention and composition.In the platform of these back, absorbed by advancing compound at the liquid of capsule surrounding environment, described compound replaces the composition of activating agent or activating agent by the slit.These transmit platform can provide a kind of unordered basically transfer curve opposite with the description of rapid release prepared product.Time delay material such as glyceryl monostearate or stearine also can use.Orally administered composition comprises standard excipients, excipient and thinner, as dolomol, sodium saccharinate, cellulose, magnesium carbonate, lactose, glucose, sucrose, sorbierite, mannitol, starch, gum Arabic, calcium silicates, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup and methylcellulose, preparation also can comprise lubricant, as talcum, dolomol and mineral oil, wetting agent, emulsifier and suspending agent, preservative such as methyl hydroxybenzoate and propyl ester.The preferred pharmacy level of these excipient.Oral administration of compound of the present invention and composition can arbitrarily comprise one or more sweeteners, as fructose, aspartame or asccharin; Or one or more flavor enhancements such as peppermint oil, methyl salicylate or cherry flavor enhancement; Or one or more colouring agents, thereby provide pharmaceutically good to eat preparation.
Mainly depend on its characteristic and seriousness for specific illness or the treatment of diseases effective dose of treatment, also can decide by the standard clinical techniques that the foundation practitioner has been done judgement.In addition, the interior detection of external or body can be used to help to identify optimal dosage.Certainly, can constitute the amount for the treatment of the compound of the present invention of going up effective dose and also depend on method of administration.In general, arrive about 20mg compound of the present invention about the about 0.001mg of per kilogram of body weight every day greatly for the oral administration suitable dosage ranges, preferably about 0.7mg is to about 6mg, and more preferably from about 1.5mg is to about 4.5mg.In a preferred embodiment, particularly human oral compound of the present invention about 0.01mg every day is to about 1000mg for mammal, and more preferably from about 0.1mg is to about 300mg, and perhaps about 1mg takes separately or separately to about 250mg.Dosage as described herein refers to the total amount of taking medicine; That is to say that if more than a kind of compound of the present invention of taking, so preferred taking dose is suitable with the total amount that compound of the present invention is taken.Orally administered composition preferably comprises the compound of the present invention of 10% to 95% weight.Preferred oral unit dosage form comprises pill, tablet and capsule, more preferably capsule.Usually these unit dosage forms comprise the about 0.01mg of compound of the present invention, 0.1mg, 1mg, 5mg, 10mg, 15mg, 20mg, 50mg, 100mg, 250mg or 500mg, preferably, each dosage unit contains the about 5mg of compound of the present invention to about 200mg.
In another embodiment, compound of the present invention and composition can parenterals (for example, by in the muscle, in the sheath, intravenous and intra-arterial approach), preferred intravenous.Usually, the compound of the present invention and the composition that are used for intravenous injection are the aqueous solution that is dissolved in sterile isotonic, as water, salting liquid, Ringer's solution, glucose solution.If need, composition also can comprise cosolvent.The intravenous injection composition comprises that optionally local anesthetic such as lignocaine alleviate the pain of injection site.For intravenous injection, the concentrating agents that dewaters in freeze-dried powder that compound of the present invention and composition can be sterilized or the closed container and providing, as the injection or the anther sac of ampoule, reservoir shows the amount of activating agent.Powder or concentrating agents dissolve with the suitable aqueous solution earlier before intravenous injection.One ampoule disinfectant, saline solution or other suitable aqueous solution can dissolve before injection for powder or concentrating agents.Perhaps composition can provide and administration with the premixed form.Compound of the present invention or composition will be used as intravenous words, use the injection bottle packing that comprises pharmaceutical grade water, saline solution or other suitable media to it so long.
Rectally can be subjected to the influence of suppository, and suppository is prepared from by vegetable oil and other lipid substrates of traditional sucrose such as cocoa butter, modification.Suppository is prepared by known method with known form, for example referring to Remington: medicament science and practice, Alfonso R.Gennaroed., the .Easton of Mack publishing company, PA, the 19th edition, 1995, the 1591-1597 page or leaf is quoted as a reference at this.
In order to prepare and the administration topical dosage forms, can use known through skin and intracutaneous transmission medium such as washing lotion, emulsifiable paste, ointment and transdermal delivery device such as patch.(Ghosh, T.K.; Pfister, W.R.; Yum, S.I. be through skin and local drug delivery system, InterpharmPress, the Inc.249-297 page or leaf is quoted as a reference at this).For example, storage type patch design can be made up of the substrate film that is coated with layer of adhesive, includes the storage crack of compound of the present invention or composition, can separate (for example, U.S. Patent number 4,615,699 is quoted as a reference at this) by semipermeable membrane and skin.The substrate layer that covers adhesive can extend providing one to seal with the concentric of skin along storage edge, and the maintenance storage is adjacent with skin.
The present invention also provides pharmacy packing material or medicine box, comprises that one or more is filled with the container of one or more compounds of the present invention.What preferably be associated with this container can be a kind of bulletin of the form of leaving with government organs, production, use or the sale of these bulletin regulation pharmacy or biology product, this bulletin has reflected the approval of production, use or marketing organization for human administration.In one embodiment, medicine box is made up of multiple compound of the present invention.In another scheme, medicine box comprises compound of the present invention and other biological is learned activating agent.
Compound of the present invention was preferably detected before being used for human body in vitro and in vivo, to obtain required treatment or prophylactic activity.For example, vitro detection can be more effective with specific administration that determines whether compound of the present invention or compound administering drug combinations of the present invention.Also can be by using animal model system to measure compound of the present invention and whether medicine is effective.Other method is understood by some masterful technique persons, and is also contained in the scope of the invention.
4.8. use the inventive method to detect
Methodology described above checks that just SelCIDs is to early progenitor cell such as CD34 +The influence of cell differentiation can be applied to any its purpose compound to the influence of differentiation of wanting to know.This can finish in several ways.
In one embodiment, compound can substitute SelCID or other compounds of any the present invention simply.Here, CD34 +CFU-GM and/or CD133 +CFU-GM can allow CFU-GM to contact with various concentration with the purpose compound under the condition of cell proliferation orientation and/or differentiation fully and/or differentiation.Can use cultural method disclosed herein, particularly at the disclosed cultural method of 4.4 joints.Decide even if the effect of purpose compound is the variation of the cell mass that come by CFU-GM differentiation by assessment, this variation is by the change monitoring of any phenotype, but preferably by measuring having or not having and assess of cell surface marker.Resemble the inventive method, any time of having cultivated from the beginning all can adopt the purpose compound administration of single dose to obtain the cell of final differentiation.Alternatively, the purpose compound also can be in the propagation phase, idiophase or two phase administrations.The phenotype of CFU-GM variation cell preferred and contrast culture comes comparison in the proliferation/differentiation, as the cell of handling with DMSO.Specific purpose will influence propagation and differentiation, as, but be not limited to: the adjusting of growth fraction; The adjusting of differentiation ratio; CFU-GM is to the adjusting of specific direction-sense precursor differentiation; Prevent differentiation to specific cell type; And promotion is to the differentiation of particular cell types.
In another embodiment, cultivate, breed and as above generation of differentiation, but purpose compound and CFU-GM are together with PDE IV inhibitor such as SelCID TMContact together.In this mode, the polyvoltine compound may be that synergitic effect can obtain measuring.It should be noted that especially any compound may not have or only have a bit the effect of propagation or differentiation separately, but and SelCID TMTime spent has but had obvious effects together.In another embodiment, any two kinds of purpose compounds all can contact with CFU-GM under condition of culture, and as above, this condition allows progenitor cell proliferation and differentiation usually.Here, a preferred experiment, wherein precursor contacts with two kinds of purpose compounds, comprises a kind of a contact that contrasts in the wherein CFU-GM and various described compounds; One contrasts wherein cell and PDE IV inhibitor such as SelCID TMContact; Contrast wherein with one that cell does not contact with compound, or contact with DMSO.Again, in dosage and the variation on action time all as described in 4.4 joints.
5. research embodiment:
5.1. embodiment 1:PDE IV inhibitor is at CD34 +Effect in the CFU-GM differentiation
Test below using is used for measuring PDE IV inhibitor for CD34 +Effect on differentiation of (hematopoiesis ancestral) cell and colony forming unit produce.Noteworthyly be, this evidence when increasing that leucocyte produces and blood platelet forms colony (CFU-GM) and improving whole colony-forming units (CFU-total) PDE IV inhibitor suppress the ability of red blood cell colony number (BFU-E and CFU-E) generation specifically.Therefore method of the present invention can be used for regulating the differentiation of stem cell, and also can be used for stimulating the formation speed of colony, provide for the HSCT obvious benefit by the speed that improves bone-marrow transplantation, leucocyte recovery and/or blood platelet generation.
With cord blood CD 34 +Hemopoietic progenitor cell places 96 hole culture dish adding 20% hyclone and cell factor (IL-3, G-CSF and kit-ligand (R ﹠amp with the density of every hole 1000 cells; D Systems is Inc) among) the IMDM.These cellular exposure in one or more PDE IV inhibitor, or under the DMSO (a kind of control compound), and were made it to cultivate 6 days.With cord blood CD 34 +Cell places 96 hole culture dish adding 20% hyclone and cell factor (IL-3, G-CSF and kit-ligand (KL) (R﹠amp with the density of every hole 1000 cells; DSystems is Inc) among) the IMDM.After the cultivation, carry out sorting with cell dyeing and with fluorescence-activated cell sorter (FACS).Collect the cell of 400 μ L dyeing and be diluted to 1.0ml with the phosphate buffered saline (PBS) (PBS) that has added 1% hyclone.Pair cell is counted the effect to determine that the stem cell differentiation is regulated.The result will show the inhibition of the generation of red blood cell or erythrocyte colony (BFU-E and CFU-E), and leucocyte and blood platelet form the enhancing of the generation of colony (CFU-GM), and the raising of the generation of whole colony forming unit.Therefore method of the present invention can be used to regulate the differentiation of stem cell, and also can be used to stimulate special colony to form speed, come to provide obvious benefit to the transplanting of candidate stem cell by improving the speed that marrow moves into and the recovery of leucocyte and/or blood platelet production, described improvement leucocyte and/or blood platelet production are to grow the initial stem cell of pedigree and carry out by being oriented to moving of expectation.
5.2. embodiment 2:PDE IV inhibitor is for human cord blood CD 34 +The effect of CFU-GM differentiation
In the following embodiments, to PDE IV inhibitor for the monocytic propagation of Cord blood (CB) and be divided into CD34 +The effect of (hematopoiesis ancestral) cell is studied.The Cord blood monocyte is a kind of a small set of hematopoiesis ancestral (CD34 that comprises +) mixed cell population of cell.The CD34 that this is little +The subclass of cell colony comprises a group CD34 +CD38 +Cell (being approximately whole C B monocytic 1%) and even littler a group CD34 +CD38 -Cell (being less than whole CB monocytic 1%).Significantly, compare with negative control with positive, PDE IV inhibitor will cause CD34 +The rise of cell (differentiation of raising), and suppress and delay the differentiation of candidate stem cell and CFU-GM.
5.2.1. materials and methods
CB CD34 +Cell is with 4 * 10 4The concentration of cell/ml originates in has added cell factor (IL-3, G-CSF and kit-ligand) (R﹠amp; D Systems is Inc) in 24 orifice plates of. 20%FCS IMDM (hyclone/Iscove ' s Modified Dulbecco ' s Medium).PDE IV inhibitor joins in the medium with different concentration.The DMSO of equal volume is with comparing.Also use the negative control that does not add any compound.Cell in moistening incubator with 37 ℃ and 5%CO 2Cultivated 7 days.Collect the cell in every hole then.
Total cell number in every hole is determined by go up counting at CELL-DYN1700 (Abbott Diaghostic), and is used FACS (fluorescence-activated cell sorting) staining analysis CXCR4, CD45, CD34, CD38, the expression of CD11b and the expression of Gly-A.
5.3. embodiment 3:PDE IV inhibitor is to the effect of human cord blood MNC cell
The Ficoll separating method of use standard separates the preservation of profound hypothermia temperature with the Cord blood MNC that melted and with 0.5 * 10 6/ ml in 24 orifice plates that contain the 20%FCS IMDM that added cell factor (IL-3, every kind of 10ng/ml of KL and G-CSF) (hyclone/Iscove ' s ModifiedDulbecco ' s Medium) to cultivate in triplicate.Experimental group is None (cell factor is only arranged), DMSO (1.7 μ l) and the PDEIV inhibitor of variable concentrations in DMSO.Collect cultured cells and use FACS dyeing to analyze after cultivating a week.
5.4. the influence that embodiment 4:PDE IV inhibitor is produced monocyte
The human cord blood CD 34 of purifying +Cell (surpasses 90% CD34 +) with 4 * 10 4Cell/ml is with 5% CO of the 20% FCS IMDM that has added cell factor (IL3, IL6, G-CSF, KL and Epo) at humidification 2Cultivated 14 days for 37 ℃ in the incubator.Experimental group comprises that (i) do not have DMSO or compound to add (" None "), (ii) has only DMSO, and (iii) is dissolved in the PDE IV inhibitor among the DMSO.Collecting the monoclone antibody that the cell of equal portions and the monoclone antibody of puting together with CD34-PE and CD14-FITC put together dyes.
5.5. embodiment 5:PDE IV inhibitor is to the influence of the transplanting karyocyte that comes from Cord blood and placenta
This experiment shows that the preliminary treatment of PDE IV inhibitor has improved surviving of the placenta karyocyte (PLNC) of transplanting, Cord blood karyocyte (UCBNC) and bone marrow cell (BMNC).
Placenta karyocyte (PLNC), Cord blood karyocyte (UCBNC) and bone marrow cell (BMNC) are available from people's donor.PLNC and UCBNC are available from placenta and umbilical cord.
By it is carried out incubation 24 hours in having added the DMEM of 2% people CB serum these cells are carried out preliminary treatment, described serum contains the PDE IV inhibitor of 10 μ g/ml.Washed cell carries out resuspension in self blood plasma subsequently, and has the adult SJL/L mouse (Jackson laboratory) of acceptor that marrow melts by the vein injection, and described marrow melts according to standard method and produces by the radiation (900cGy) that causes death.Such radiation is better than through 90% after the radiation in 50 days deadly, (Ende etc., 2001, Life Science 69 (13): 1531-1539; Chen and Ende, 2000, J.Med.31:21-30; Ende etc., 2000, Life Sci.67 (1): 53-9; Ende and Chen, 2000, Am.J.Clin.Pathol.114:89).
5.6. embodiment 6:SelCIDs TMFor CD34 +The influence of CFU-GM differentiation
The following examples have been analyzed SelCIDs TMFor CD34 +The influence that differentiation of (hematopoiesis ancestral) cell and colony forming unit (CFU) produce.Significantly, the result shows SelCIDs TMCan be used for suppressing the generation that erythrocyte generates colony (BFU-E and CFU-E) specifically, increase the generation that leucocyte and blood platelet form colony (CFU-GM) simultaneously, and improve total colony forming unit (CFU-Total) generation.
Therefore method of the present invention can be used to regulate the differentiation of stem cell, and also can be used to stimulate the speed of colony formation, provides obvious benefit by the speed of improvement marrow immigration and the recovery of leucocyte and/or blood platelet production to HSCT.
With cord blood CD 34 +Hemopoietic progenitor cell places 96 hole culture dish adding 20% hyclone and cell factor (IL-3, G-CSF, and kit-ligand (R﹠amp with the density of every hole 1000 cells; D System cultivates among IMDM Inc.).With these cellular exposure in SelCIDs TMPerhaps DMSO (control compound), and cultivated 6 days.With cord blood CD 34 +Hemopoietic progenitor cell places 96 hole culture dish adding 20% hyclone and cell factor (IL-3, G-CSF, and kit-ligand (R ﹠amp with the density of every hole 1000 cells; D System cultivates among IMDM Inc.).After cultivation, with cell dyeing, and with fluorescence-activated cell sorter (FACS carries out sorting.Collect 400 μ L staining cells, and be diluted to 1.0ml with the phosphate buffered saline (PBS) (PBS) that contains 1% hyclone.Pair cell is counted to measure the effect that the stem cell differentiation is regulated.
Compound among the present invention is effective in the adjusting of the pedigree orientation of hematopoiesis ancestral stem cell.Thereby, SelCIDs TMCan be used to suppress specifically the generation of red blood cell or erythrocyte colony (BFU-E and CFU-E), increase leukocytic generation and blood platelet simultaneously and form colony (CFU-GM) and improve total colony formation production of units.Therefore method of the present invention can be used to regulate the differentiation of stem cell, and also can be used to stimulate special colony to form speed, come to provide obvious benefit to the transplanting of candidate stem cell by improving the speed that marrow moves into and the recovery of leucocyte and/or blood platelet production, described improvement leucocyte and/or blood platelet production are to grow the initial stem cell of pedigree and carry out by being oriented to moving of expectation.
5.7. embodiment 7:SelCIDs TMIn the propagation of human cord blood CD 34+cell and the effect in the differentiation
Embodiment below is to SelCIDs TMFor the monocytic propagation of Cord blood and be divided into CD34 +The influence of (hematopoiesis ancestral) cell is studied.The Cord blood monocyte is a kind of a small set of hemopoietic progenitor cell (CD34 that comprises +) mixed cellularity group.This little CD34 +The subclass of cell mass comprise a group (approximately all CB monocytic 1%) CD34+CD38+ cell and even littler a group (being less than whole CB monocytic 1%) CD34+CD38-cell, and compare with negative control with positive, can suppress and delay the differentiation of candidate stem cell and CFU-GM significantly.
5.7.1. material and method
CB CD34+ cell is adding cell factor (IL3, GCSF and Kit-ligand) (R ﹠amp; DSystems originates in every milliliter 4 * 10 among 20% FCS IMDM Inc.) (fetal calf serum/Iscove ' s ModifiedDulbecco ' s Medium) in 24 well culture plates 4Cell.SelCIDs TMWith three variable concentrations: 5 μ g/ml, 1 μ g/ml and 0.3 μ g/ml join in the cultivation.The DMSO of equal volume is used for contrast.Do not add any compound in the negative control.Cell in the incubator of humidification with 37 ℃ and 5% CO 2Cultivated 7 days.Collect the cell in every hole then.
Total cell number in every hole is measured by going up counting at CELL-DYN1700 (Abbott Diagnostic), and uses FACS (fluorescence-activated cell sorter) staining analysis CXCR4, CD45, CD34, the expression of CD38, CD11b and the expression of Gly-A.
To cultivating respectively, measure and analyze from the CB cell of two different donors (CB2276 and CB2417).
5.7.2. result and discussion
To SelCIDs TMFor CD34 +The effect of the propagation that the cell factor of cell stimulates detects.Compare SelCIDs with negative control TMTo IL-3 being arranged, the CD34 that cultivates when Kit-ligand (KL) and G-CSF exist +The propagation of cell is significantly influence not.But compare expection SelCIDs with the DMSO contrast TMInduce the more cell yield of more number.
Facs analysis by surface protein CXCR4 and CD34 is analyzed SelCIDs TMEffect in the expression of cell differentiation.Expection SelCIDs TMIn the expression of CXCR4, show and suppress effect.
About surface protein CD34 +, expection SelCIDs TMCan cause CD34 +The rise of cell (differentiation of raising).At SelCIDs TMIn the cell of handling, most CD34 +And CD34 -Cell will be CD38 -, and contrast and DMSO processed group will mainly be CD38 +This illustrates SelCIDs TMCan be used to suppress specifically the generation of red blood cell or erythrocyte colony (BFU-E and CFU-E), increase leukocytic generation and blood platelet simultaneously and form colony (CFU-GM) and improve total colony forming unit generation.Therefore method of the present invention can be used to regulate the differentiation of stem cell, and also can be used to stimulate special colony to form speed, comes to provide obvious benefit to the transplanting of candidate stem cell by the speed of raising marrow immigration and the recovery of leucocyte and/or blood platelet production.
Pass through CD34+CD38 -To CD34 +CD38 +Or CD11b +The surface protein facs analysis of cell is analyzed SelCIDs TMThe influence of pair cell expression of differentiation.As average immunofluorescence (MIF) is measured, SelCIDs TMCD11b expression in the cell of handling reduces, and the expression of prompting CD11b is suppressed.Therefore the CD11b+ cell is when SelCIDs is being arranged TMWhen cultivating under existing, be in a kind of more weak differentiation state.
5.8. embodiment 8:SelCIDs TMInfluence to human cord blood MNC cell
In above embodiment, expection SelCIDs TMReduce cord blood CD 34 significantly +The expression of cell CXCR4 and increase CD34 +The CD38-cell mass.In this embodiment, show SelCIDs TM(MNC) has similar activity for the Cord blood monocyte.
Separate the Cord blood MNCs that uses standard method cryopreservation and thawing and with 0.5 * 10 with the Ficoll separating method of standard 6/ ml cultivates in 24 orifice plates that contain the 20% FCS IMDM that added 3 kinds of cell factors (IL-3, each 10ng/ml of KL and G-CSF) (hyclone/Iscove ' sModified Dulbecco ' s Medium).Experimental group is None (cell factor is only arranged), DMSO (1.7 μ l) and SelCIDs TM(5.0 μ g are at 1.7 μ l DMSO).Cultivate one week back collecting cell and analyze by FACS dyeing.
Use DMSO, SelCIDs TMTotal cell number expection of the MNC that cultivates is than control group low (" None " only has cell factor).Shown higher CD34 with MID1 cultured cells culture than other all groups +The percentage of cell, and CD34 +The total number of cell should be similar in all groups.CD34 +CD38 -The quantity of cell is apparently higher than SelCIDs TMHandle cell, this and CD34 with these compound treatment purifying +The result of cell is consistent.That make us abundant acceptance is CD34 +The CD38-cell is the hemopoietic progenitor cell of less differentiation, they after transplanting with than CD34 +CD38 +Higher efficient moves into and propagation (Dao etc., 1998, Blood91 (4): 1243-55; Huang etc., 1994, Blood83 (6): 1515-26).
At SelCIDs TMMost CXCR4 in the culture of the cell of handling +Cell is the CD45 feminine gender.This cell mass is higher than SelCIDs significantly TMThe cell of handling.
This results suggest SelCIDs TMIs useful to offset cryopreservation, melt or to be exposed to the refrigeration agent regulating stem cell on the harmful effect on the stem cell.This result also further points out CD34 +And CD14 +Cells produce can be by SelCIDs by the inhibition of DMSO TMProcessing offset.
5.9. embodiment 9:SelCIDs TMInfluence to monocyte production
The human cord blood CD 34 of purifying +Cell (surpasses 90% CD34 +) with 4 * 10 4Cell/ml under in the 20% FCS IMDM medium that has added cell factor (IL3, IL6, G-CSF, KL and Epo) 37 ℃ at 5% CO of humidification 2Cultivated 14 days in the incubator.Experimental group comprises: (i) do not add one group (" None ") of DMSO or synthetics, a group of DMSO is (ii) only arranged, (iii) SelCIDs TMBe dissolved in a group among the DMSO.Collecting the monoclone antibody that the cell of equal portions and the monoclone antibody of puting together with CD34-PE and CD14-FITC put together dyes.Expection SelCIDs TMProcessed group obviously shows than the more a high proportion of CD34 of control group +Cell.And, as on the CD14+ cell quantity minimizing proved, monocyte production reduced.Because SelCIDs TMProcessed group also is exposed to DMSO, can infer SelCIDs TMProcessing overcome the monocytic production that is suppressed by DMSO.
5.10. embodiment 10:SelCIDs TMPreliminary treatment is to the influence from the transplanting karyocyte in Cord blood and the placenta
This experiment shows SelCIDs TMPreliminary treatment has improved the survival of transplanting placenta karyocyte (PLNC), Cord blood karyocyte (UCBNC) and bone marrow cell (BMNC).
Placenta karyocyte (PLNC), Cord blood karyocyte (UCBNC) and bone marrow cell (BMNC) are available from people's donor.PLNC and UCBNC are available from placenta and Cord blood.
By it is carried out incubation 24 hours in having added the DMEM of 2% people CB serum these cells are carried out preliminary treatment, described serum contains the PDE IV inhibitor of 10 μ g/ml.Washed cell carries out resuspension in self blood plasma subsequently, and has the adult SJL/L mouse (Jackson laboratory) of acceptor that marrow melts by the vein injection, and described marrow melts according to standard method and produces by the radiation (900cGy) that causes death.Such radiation is better than through 90% after the radiation in 50 days deadly, (Ende etc., 2001, Life Science 69 (13): 1531-1539; Chen and Ende, 2000, J.Med.31:21-30; Ende etc., 2000, Life Sci.67 (1): 53-9; Ende and Chen, 2000, Am.J.Clin.Pathol.114:89).
SelCIDs TMPreliminary treatment has improved the survival of transplanting placenta karyocyte (PLNC), Cord blood karyocyte (UCBNC) and bone marrow cell (BMNC).
5.11. the adjusting of embodiment 11:CD34+ CFU-GM differentiation
Marrow and cord blood CD 34 +CFU-GM was cultivated 6 days in the presence of SCF, Flt-3L, GM-CSF and TNF-α by Clonetics and in the Iscove ' s of tool BIT95000 (stem cells technology) MDM, cultivated 6 days in the presence of GM-CSF and TNF-α again.
Cell surface phenotype analytical: use monoclone antibody that FITC and PE put together to carry out double staining (30 minutes in 4 ℃) at the 6th day and the 12nd day pair cell.Used antibody is from BDPharmnigen:CD34 (PE), CD38 (FITC), CD33 (FITC), CD1a (FITC), CD86 (PE), CD14 (PE), CD83 (PE), CD54 (PE), CD11b (PE), CD11c (PE), HLA-DR (PE), CD15 (FITC) with from the CD133 (PE) of Miltenyi.Fluorometric analysis is carried out after gathering 10,000 results (Coulter) on the FACScan flow cytometry.
Apoptotic detection: use Annexin V-FITC to dye according to operating guidance in conjunction with iodate third ingot (BD Pharmingen apoptosis test regent box I) and measure the exposure of phosphorylation serine.
Phagocytosis: the endocytosis activity of cell is analyzed by measuring the absorption of FITC-glucan.Cell was cultivated 1 hour at 37 ℃ in the perfect medium that contains 1mg/ml glucan-FITC (Sigma), and to cultivate 1 hour as negative control at 4 ℃.
T cell proliferation test: after having cultivated 13 days, collect the DC cell in CD34+ source, with mitomycin C (50 μ g/ml, Sigma) handle after, the allos adult CD3 that obtains as peripheral blood lymphocytes (PBMCs) purifying by healthy contributor +The irritation cell of T cell.CD3 +The T responsive cell is with 5 * 10 4The concentration of cells/well is used.Irritation cell adds in the T cell of black 96 orifice plates in flat with the dosage of classification, and cleaning bottom tissue culturing plate is to carry out chemiluminescence detection.In the RPMI1640 medium that has added 10% heat-inactivated FBS, glutamine and penicillin-streptomycin, cultivate.Cultivate after 6 days, according to operating guidance, (Roche, Nutley NJ) measures cell proliferation with the BrdU chemiluminescence analysis.The result provides with the mean value ± SD of triplicate cultivation.
SelCIDs TMCan obviously change from CD34 +The growth of the DC of CFU-GM.In order to study SelCIDs TMFor the effect that DC produces, CD34 +CFU-GM is having or is not having SelCIDs TMWhen (1 μ M) in propagation with the maturing stage (1-12 days) is cultivated 12 days time or in 6 days time of cultivation maturing stage (6-12 days).The SelCIDs that expection was added from 1-12 days TMSuppressing the acquisition of DC phenotype and the more important thing is increases CD34 +CD38 -The group changes CD34 +CD38 -To CD34 +CD38 +The normal differentiation of cell.But, expection SelCIDs TMThe CD34 that handles +Cell obtains CD33 marrow sample mark, and these cells will present CD34 at the 6th day +CD38 -CD33 +Phenotype.SelCIDs TMCan almost stop CD1a up hill and dale +Cell is the 6th day generation, particularly two positive CD86 +CD1a +The generation of cell.This two positive group is considered to the precursor of epidermis Lang Shi DC.SelCIDs TMAlso can reduce the CD14 that can produce corium DC and monocyte/macrophage +CD1a -The generation of cell.This early progenitor cell group (CD34 +CD38 -Cell) increase and marrow sample DC CFU-GM (CD1a +CD14 -And CD1a -CD14 +Cell) prevent may be dose dependent and at SelCIDs TMArrive maximum when being 1 μ M.If this effect be reversible and the interference of CD34 differentiation pathway only at CD34 +CFU-GM is being used SelCIDs TMCultivating at least 3 talent's observations obtains.
SelCIDs after 0 day between 6 days TMMultiple dosage will be strengthened CD34 +Group's increase.
At SelCIDs TMThe CD34 that cultivates when existing +CFU-GM also showed costimulatory molecules (CD86, the weakening of expression CD80) at the 12nd day.The CD54 adhesion molecule is with CD54 BrightThe minimizing and the CD54 that express DimThe increase that the group expresses and changing.The HLA-DR molecule be expressed in SelCIDs TMThe CD34 that handles +Weakened in the CFU-GM.
After non-processor is cultivated 0-6 days, when added one or more SelCIDs at the 6th day TMThe time, and work as CD1a +When the group has produced, SelCIDs TMStrengthen CD1a +Group's persistence.SelCIDs TMThe culture of handling comprised at the 12nd day than the more relatively CD1a of DMSO contrast +Precursor.At the 6th day, add CD34 to +SelCIDs in the noble cells TMAlso weakened CD14 significantly +The generation of precursor and costimulatory molecules (CD86, expression CD80).
SelCIDs TMPromote the granulocyte differentiation: whether relevant for the inhibition of measuring the DC generation with the change of different marrow differentiation pathway, can monitor D15 granulocyte mark.SelCIDs is being arranged TMThe CD34 that cultivates when existing +The expression of CD15 surface molecular is increased in the CFU-GM.When having the cytokine mixture that makes the DC differentiation to exist, SelCIDs TMAdding make the propagation/maturation of CFU-GM turn to class granulocyte phenotype more.Trend in the marrow sample differentiation can be studied by the expression of monitoring 2 marks: by the DC CFU-GM to the expressed CD11c of langerhans cell and interstitial cell with by the expressed CD15 of granulocyte CFU-GM.CD11c+CD15-group's minimizing and CD11c-CD15 +Increase relevant in the time of the granulocyte group.It should be noted that SelCIDs TMMultiple dosage promote conversion to the granulocyte pedigree.
The obstruction that DC produces not is that specific killing for the DC CFU-GM mediates: for whether the minimizing of definite DC CFU-GM is mediated by specific killing, with CD34 +CFU-GM is cultivated 6 days time under the situation that has SCF, Flt-3L, GM-CSF and TNF-α to exist.At the 6th day, by magnetic cell sorter (Miltenyi) separation of C D1a +CD14 -And CD1a -CD14 +Cell (DC CFU-GM).The purifying group is having GM-CSF and TNF-α to have or is not having SelCIDs TMCultivated in addition again 2 days when (1 μ M).At SelCIDs TMHandle the back annexin V +-PI -(early stage Apoptosis) group and annexin V +-PI +(later stage Apoptosis) group's level is not significantly increased.
By CD34 +The functional activity of the DC that CFU-GM produces obtains changing: with there being or not having SelCIDs TMThe CD34 that cultivates of cell factor +The phagocytic activity of CFU-GM derived cell was analyzed by the endocytosis of the glucan-FITC of mannose receptor mediation at the 12nd day.When added one or more SelCIDs at the 1st day to the 12nd day TMThe time, compare with the DMSO contrast, tangible minimizing has been arranged on phagocytic activity.When added SelCIDs at the 6th and the 12nd day TMThe time, phagocytic activity and DMSO contrast are quite.
Bring out CD3 by in mixed lymphocyte reaction (MLP) (MLR) test, measuring it +The ability of allos T cell proliferation was analyzed with there being or not having SelCIDs at the 12nd day TMThe CD34 that cultivates of cell factor +The antigen presentation ability (APC) of cell.When added one or more SelCIDs at the 1st day to the 12nd day TMThe time, compare the ability that the CD34+ cell has shown the stimulation T cell proliferation that weakens with the DMSO contrast.In contrast, added one or more SelCIDs at the 6th day to the 12nd day TMThe time, stimulate the ability of T cell proliferation suitable with the DMSO control cells.
SelCIDs TMCan sharply weaken CD34 +CFU-GM is divided into dendritic cell.As a result, SelCIDs TMThe APC ability that the cell of handling can present low phagocytic activity and weaken.SelCIDs TMAlso can increase early stage hemopoietic progenitor cell, CD34 +CD38 -Cell.Those early stage hemopoietic progenitor cell have shown in NOD-SCID mouse model (Tamaki etc., J.Neurosci.Res.69 (6): 976-86 (2002)) provides better immigration and repopulate.And, SelCIDs TMBy turning to the granulocyte pedigree to influence CD34 the differentiation of bone sample +The differentiation of cell, even work as the differentiation phase that cell factor pressure helps dendritic cell.In addition, find SelCIDs TMTo CD34 +Cell does not have toxic action, and does not weaken the hyperplasia ability.The adjusting of this DC function and the promotion of granulocytic differentiation can be for the treatments of various cancers, immune disorders and communicable disease, and have the treatment ability in organ transplant and reviviscence medicine.
5.12. embodiment 12:SelCIDs TMRegulate CD133 +The differentiation of CFU-GM
SelCIDs TMMultiple dosage, except strengthening at CD34 +Outside the increase on the group, also increased usually by CD34 BrightHemopoietic progenitor cell and some original CD34 -The expression of the CD133 of Expression of Subsets.By enrichment CD34 +CD133 +Primitive hematopoietic cell, SelCIDs TMShould have the clinical meaning that after stem cell transplantation, recovers for hematopoiesis.In addition, CD133 +Stem cell also can make the endothelium pedigree take place, and contributes to some extent aspect wound healing.SelCIDs TMMultiple dosage do not aggravate the inhibition of the generation of Lang Shi DC precursor.
5.13. embodiment 13: by marrow (BM) Sca +Hemopoietic progenitor cell and the generation of the mouse dendritic cell that comes
From the mouse bone marrow cells of inbrde C57BL/6 mouse available from Clonetics.Hematopoiesis Sca+Lin-CFU-GM uses SpinSep mouse CFU-GM enrichment mixture (StemCellTechnologies) to carry out enrichment, and in Iscove ' the s MDM of tool BIT95000 (StemCell Technologies), in the presence of mouse growth factor SCF, Flt3L, GM-GSF and M-CSF, cultivated 9 days, to promote the propagation of Sca+ cell and DC precursor phenotype, in the presence of GM-CSF and TNF-α, cultivate 3 days then again to impel these cells to become immature DC phenotype.The Sca+Lin-cell of enrichment had DMSO (0.1%), 10 μ M SelCIDs from 0 day TMOr cultivate under the situation of 10 μ M all-trans retinoic acid (ATRA) (ICN Biomedicals) existence.Compound was added to the cell from the 0th day and the 9th day.
Mouse cell surface phenotype analytical: the monoclone antibody that the mouse cell uses FITC and PE to put together the 9th day and the 12nd day is carried out double staining (14 minutes in room temperature).Used antibody is from BDPharmingen:Sca (PE), CD11b (FITC), Gr-1 (FITC), CD86 (PE), CD14 (PE), CD80 (PE), I-A b(PE) with from the CD32.1/16.1 of Miltenyi.Fluorometric analysis is carried out after gathering 10,000 results (Coulter) on the FACScan flow cytometry.
SelCIDs TMCan change from Sca +The growth of the mouse DC of CFU-GM.At the 9th day, cell will present height surface expression and the I-A with tree-shaped/marrow sample mark CD32/16 (Fc acceptor), CD11b, CD80 bThe DC precursor phenotype that resembles the expression of CD14 and Gr-1 pedigree mark with low expression and the shortage of CD86.By the 9th day, SelCIDs TMCan show that the expression of pair cell surface markers does not have positive effect, and ATAR can show to CD80, I-A bTangible downward modulation (data not shown) with the Sca+ expression.But by the 12nd day, SelCIDs TMCan show CD86 and bright I-A bThe rise that the following mediation CD11b that expresses expresses.ATRA shows similar but compares SelCIDs TMMore significant effect.In addition, SelCIDs TMDo not show that in the expression of CD40 and CD80 obvious effects ATRA then shows the tangible downward modulation to these molecules.
SelCIDs TMBy the expression of downward modulation CD86 and MHC II, suppress the DC precursor and be divided into immature DC.Expect that the effect of this compound does not have those observed violent like that in the artificial blood CFU-GM.SelCIDs TMEffect do not have mouse teratogens ATRA remarkable like that.
5.14. the application of the fractional analysis of the compound beyond the embodiment 14:SelCIDs
Above-mentioned method, that is, SelCIDs is for early progenitor cell such as CD34 +The influence of the differentiation of cell can apply on any target compound, and its effect in differentiation is the desired understanding of behaving.Extend to the example of other compound as this analytical method, we have compared with respect to contrast (DMSO-handles) cell, retinoic acid (ATRA) and aspirin and SelCIDs TMFor CD34 +Cell differentiation is the effect of DC pedigree.The research retinoic acid is because its known effect, the therapeutic action in certain cancers and its known teratogenesis effect in cell proliferation and differentiation.On the contrary, the effect of research aspirin is because it is the general antiinflammatory agent that does not have immune modulating characteristic that uses.CD34 +CFU-GM is cultivated 6 days time when having or do not have compound when having SCF, Flt-3L, GM-CSF and TNF-α to exist, can obtain the 6th day result.
Shown other medicines adjusting cell differentiation in the literature, for example, one piece of recent paper report is from CD34 +The generation of the DC of CFU-GM is by the adjusting of corticosteroid.Adjustment curve is at CD1a +Group's increase and CD14 +Be different from SelCIDs in group's the minimizing TM
The invention is not restricted to the scope of the specific embodiment of art that this paper retouches.In fact, from the description of front, except those of the present invention various improvement of describing in this article will be conspicuous for those skilled in the art.Such improvement will fall within the scope of appended claim.
6. document
The full content degree as a reference of quoting each other publication, patent or patent application with this paper for all purposes is the same, and the full content that this paper quotes all lists of references for all purposes as a reference.
Quote any publication and all be since its open day early than the application's day and can not be interpreted as a kind of permission, i.e. the present invention can not do sth. in advance this open date owing to the effect of invention formerly.

Claims (101)

1. method of regulating the differentiation of mammalian stem cell or CFU-GM, be included under the appropriate condition and under the situation that the compound that suppresses PDE IV activity exists and break up described stem cell or CFU-GM, wherein said compound is not polypeptide, peptide, protein, hormone, cell factor, oligonucleotides or nucleic acid.
2. the described method of claim 1, wherein said stem cell is divided into hematopoietic cell.
3. the described method of claim 1, wherein said stem cell is selected from embryonic stem cell, placenta stem-cell, cord blood stem cell, peripheral hematopoietic stem cells and stem cell.
4. the described method of claim 1, wherein said PDE IV inhibitor is SelCID TMOr its prodrug.
5. the described method of claim 1, wherein said differentiation is carried out in cell culture.
6. the described method of claim 1, wherein said differentiation is carried out in individuality.
7. the described method of claim 1, wherein compound concentrations is that about 0.005 μ g/ml is to about 5mg/ml.
8. the described method of claim 1, wherein stem cell is a human stem cell.
9. regulate mammal CD34 for one kind +Or CD133 +The propagation of CFU-GM or the method for differentiation, be included under the condition that is fit to propagation or differentiation and under the situation that the compound that suppresses PDE IV activity exists propagation or break up described cell, wherein said compound is not polypeptide, peptide, protein, hormone, cell factor, oligonucleotides or nucleic acid.
10. the described method of claim 9, wherein said CFU-GM is selected from CD34 +CFU-GM and CD133 +CFU-GM.
11. the described method of claim 9, wherein said CFU-GM is divided into CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Cell.The described method of claim 9, wherein said compound are a kind of SelCID TMOr its prodrug.
12. the described method of claim 9, wherein said propagation or differentiation are carried out in cell culture.
13. the described method of claim 9, wherein said propagation or differentiation are carried out in individuality.
14. the described method of claim 13, wherein said CFU-GM is for being implanted into the cell in the described individuality.
15. the described method of claim 9, wherein said compound be enough to compared with the control in described differentiation or propagation, to cause can detected difference amount exist.
16. the described method of claim 9, wherein said CD34 +Or CD133 +CFU-GM had been carried out cryopreservation and thawing before described differentiation.
17. the method for a propagation progenitor cell populations in mammalian subject comprises the CD34 to effective dose on the described mammalian subject administering therapeutic +CFU-GM and the compound that suppresses the PDEIN activity, wherein said compound is not polypeptide, peptide, protein, hormone, cell factor, oligonucleotides or nucleic acid.
18. the described method of claim 17, wherein said CD34 +CFU-GM is broken up in described mammalian subject.
19. the described method of claim 17 is wherein with described CD34 +CFU-GM is applied to described mammalian subject with the cellular preparations that does not have red blood cell substantially.
20. the described method of claim 17 is wherein with described CD34 +CFU-GM is applied to described mammalian subject with the cellular preparations that contains bone marrow cell, placenta cells or cord blood cell.
21. the described method of claim 17 is wherein with described CD34 +CFU-GM and carrier are co-administered in described mammalian subject.
22. the described method of claim 17, wherein said CD34 +CFU-GM is CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -CFU-GM.
23. the described method of claim 17, wherein said CD34 +CFU-GM is CD34 +CD133 +CFU-GM.
24. the described method of claim 17, wherein said CFU-GM is expressed the target genetic material of integrating.
25. pharmaceutical composition that comprises mammalian stem cell and pharmaceutically useful carrier, wherein said stem cell has contacted the time of the adjusting of the differentiation of the described stem cell of enough initiations or propagation with the compound that suppresses PDE IV activity, and wherein said compound is not polypeptide, peptide, protein, hormone, cell factor, oligonucleotides or nucleic acid.
26. the described pharmaceutical composition of claim 25, wherein stem cell is selected from embryonic stem cell, placenta stem-cell, cord blood stem cell, peripheral hematopoietic stem cells and stem cell.
27. the described pharmaceutical composition of claim 25, wherein said compound are SelCID TMOr its prodrug.
28. the described pharmaceutical composition of claim 25, wherein said contact procedure is carried out in cell culture.
29. the described pharmaceutical composition of claim 25, wherein said compound concentrations are that about 0.005 μ g/ml is to about 5mg/ml.
30. the described pharmaceutical composition of claim 25, wherein stem cell is a human stem cell.
31. the described pharmaceutical composition of claim 25, wherein differentiation is the differentiation to hematopoietic cell.
32. the described pharmaceutical composition of claim 25, wherein said hematopoietic cell are CD34 +Or CD38 +Hematopoietic cell.
33. the described pharmaceutical composition of claim 25, wherein said hematopoietic cell are CD11b +Cell.
34. one kind contains the cord blood cell of separation and the pharmaceutical composition of the white blood corpuscle colony that separates, wherein said white blood corpuscle produces by a kind of method, this method is included under the appropriate condition and differentiated stem cells under the situation that the compound that suppresses PDE IV activity exists, condition is that this compound is not polypeptide, peptide, protein, hormone, cell factor, oligonucleotides or nucleic acid, and separates the white blood corpuscle of differentiation thus.
35. the described pharmaceutical composition of claim 34, wherein compound is acid imide or amides compound.
36. the described pharmaceutical composition of claim 34, wherein differentiation step is carried out in cell culture.
37. the described pharmaceutical composition of claim 34, wherein compound concentrations is that about 0.005 μ g/ml is to about 5mg/ml.
38. the described pharmaceutical composition of claim 34, wherein stem cell is a human stem cell.
39. the described pharmaceutical composition of claim 34, wherein stem cell is a CFU-GM.
40. the described pharmaceutical composition of claim 39, wherein CFU-GM is limited to a kind of concrete cell-line.
41. the described pharmaceutical composition of claim 39, wherein CFU-GM is a hemopoietic progenitor cell.
42. CD34 who comprises cultivation +Or CD133 +The pharmaceutical composition of CFU-GM and pharmaceutically useful carrier, wherein said CFU-GM under the condition of propagation that promotes described CFU-GM and differentiation, contact with the compound that suppresses PDE IV activity in preceding 6 days cultivation.
43. the described pharmaceutical composition of claim 42 is wherein collected and the described CFU-GM of cryopreservation after 6 days cultivation.
44. the described pharmaceutical composition of claim 42, wherein said CFU-GM are CD34 +CD38 -CD34 +Or CD34 +CD38 -CD34 -Cell.
45. the described pharmaceutical composition of claim 42, wherein said compound are SelCID TM
46. a method of transplanting mammalian stem cell, it comprises:
(a) described stem cell is contacted to produce the stem cell of treated mistake with PDE IV inhibition compound, wherein said contact is enough to regulate the differentiation of described stem cell; With
(b) individuality is used the stem cell of described processing.
47. the described method of claim 46, wherein step (b) comprises the co-administered untreated cell and the stem cell of described processing.
48. the described method of claim 46, wherein untreated cell is selected from embryonic stem cell, placenta cells, cord blood cell, peripheral blood cells and bone marrow cell.
49. the described method of claim 46, wherein said stem cell had been carried out cryopreservation and thawing before described using.
50. a method of transplanting the mammal CFU-GM, it comprises:
(a) described CFU-GM is contacted to produce the CFU-GM of treated mistake with PDE IV inhibition compound, wherein said contact is enough to regulate the differentiation of described CFU-GM; With
(b) individuality is used the CFU-GM of described processing.
51. the described method of claim 50, wherein step (b) comprises the co-administered untreated cell and the CFU-GM of described processing.
52. the described method of claim 50, wherein untreated cell is selected from embryonic stem cell, placenta cells, cord blood cell, peripheral blood cells and bone marrow cell.
53. the described method of claim 50, wherein said stem cell had been carried out cryopreservation and thawing before described using.
54. a treatment suffers from the method for the individuality of illness, comprises described individual administration is selected from following activating agent:
(a) compound of inhibition PDE IV activity, wherein said compound is not polypeptide, peptide, protein, hormone, cell factor, oligonucleotides or nucleic acid;
(b) stem cell that under the situation that described compound exists, breaks up; With
(c) the wherein said activating agent of CFU-GM that breaks up under the situation that described compound exists can alleviate with detecting or improve described illness.
55. the method for claim 54, wherein said illness is selected from inflammation, cardiopathy, angiosis, amyotrophic lateral sclerosis, lysosomal storage disease and diabetes.
56. the method for claim 54, wherein said activating agent had both comprised stem cell and also comprised the compound that suppresses PDE IV activity, wherein said compound is not polypeptide, peptide, protein, hormone, cell factor, oligonucleotides or nucleic acid.
57. treat individual method for one kind, comprise white blood corpuscle to effective dose on the described acceptor mammalian subject administering therapeutic, wherein being that white blood corpuscle is described produces by a kind of method, this method be included under the appropriate condition and under the situation that the compound that suppresses PDE IV activity exists differentiated stem cells, condition is that this compound is not polypeptide, peptide, protein, hormone, cell factor, oligonucleotides or nucleic acid.
58. the described method of claim 57, wherein stem cell breaks up external.
59. the described method of claim 57, wherein stem cell is to break up in the placenta of perfusion in postpartum.
60. the described method of claim 57 is wherein used with the cellular preparations that does not have red blood cell substantially white blood corpuscle to individuality.
61. the described method of claim 57 is wherein used with the cellular preparations that comprises cord blood cell white blood corpuscle to individuality.
62. the described method of claim 57 is wherein united white blood corpuscle and carrier individuality is used.
63. the described method of claim 57, wherein the administration white blood corpuscle is to treat or to repair the defective in the acceptor mammalian subject.
64. the described method of claim 63, wherein defective is hematopoietic cell or blood cell proliferation defective.
65. the described method of claim 63, wherein hematopoietic cell or blood cell proliferation defective are neutrocytopenia or leukopenia.
66. the described method of claim 63, wherein white blood corpuscle is the whole body administration.
67. the described method of claim 63, wherein white blood corpuscle is an intravenous administration.
68. the described method of claim 63, wherein white blood corpuscle is expressed the target genetic material of integrating.
69. claim 57 described method, wherein white blood corpuscle is an allos.
70. the described method of claim 57, wherein the acceptor mammalian subject is behaved.
71. the method for a pharmaceutical compositions comprises:
(a) with CD34 +Or CD133 +CFU-GM contacts with the compound that suppresses PDE IV activity, and wherein said CFU-GM was cultivated 6 days under the condition of culture of propagation that allows described CFU-GM and differentiation;
(b) cultivate the described cell of collection after 6 days; With
(c) described cell is placed pharmaceutically useful carrier.
72. the described method of claim 71, wherein said contact was carried out at first day that cultivates.
73. the described method of claim 71, wherein said contact was carried out twice in 6 days that cultivate at least.
74. the described method of claim 71, wherein said compound is SelCID TMOr its prodrug.
75. the described method of claim 71 was wherein separated described CFU-GM before described cultivation from other haemocyte.
76. the described method of claim 71, wherein said medium also comprise GM-CSF and TNF-α in addition.
77. the described method of claim 74, wherein said SelCID TMOr its prodrug exists with the concentration of 0.1 μ M to 10.0 μ M.
78. the described method of claim 74, wherein said SelCID TMOr its prodrug exists with the concentration of 1.0 μ M.
79. the described method of claim 74, wherein said cell are carried out cryopreservation after described collection.
80. pharmaceutical composition by the method preparation of claim 74.
81. regulate CD34 for one kind +Or CD133 +The method of CFU-GM differentiation comprises:
(a) breaking up the colony that described CFU-GM is provided under the condition that can take place;
(b) described CFU-GM is contacted with compound, wherein said compound be PDE IV inhibitor and
(c) under the condition that is suitable for breaking up, described CFU-GM is broken up, wherein described compound is contacted the time that makes described CFU-GM differentiation to small part with described CFU-GM.
82. the described method of claim 81, wherein at step (b), the random time of described contact between 0 to 6 day that cultivates carried out.
83. the described method of claim 81, wherein at step (b), described contact is carried out beginning of cultivating of described CFU-GM.
84. the described method of claim 81, wherein at step (b), described contact is carried out at least after described CFU-GM has been bred 2 days.
85. the described method of claim 81, wherein at step (b), described contact is carried out at least after described CFU-GM has been bred 6 days.
86. the described method of claim 81, wherein said CFU-GM are CD34 +CFU-GM.
87. being divided into, the described method of claim 81, wherein said CFU-GM present the cell that is selected from following cell surface marker characteristic:
With respect to reducing to some extent to impinging upon in the CD11c expression;
With respect to reducing to some extent to impinging upon in the CD38 expression;
With respect to reducing to some extent to impinging upon in the CD80 expression;
With respect to reducing to some extent to impinging upon in the CD86 expression;
With respect to reducing to some extent to impinging upon in the CD1a expression;
With respect to reducing to some extent to impinging upon in the CD14 expression;
With respect to impinging upon CD54 BrightReduce to some extent in the expression;
With respect to reducing to some extent to impinging upon in the HLA-DR expression;
With respect to reducing to some extent to impinging upon in the CD15 expression;
With respect to reducing to some extent to impinging upon in the CD33 expression;
With respect to impinging upon CD54 DimReduce to some extent in the expression;
With respect to reducing to some extent to impinging upon in the CD133 expression; With
The combination of the mark characteristic above any;
The CD34 of wherein said contrast for when not having described compound to exist, under the condition identical, cultivating with described CFU-GM +CFU-GM.
88. the described method of claim 81, wherein said CFU-GM is divided into CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -CFU-GM.
89. the described method of claim 81, wherein said PDE IV inhibitor is SelCID TMOr its prodrug.
90. one kind by CD34 +CFU-GM produces the method for the cell of differentiation, and be included in the medium that allows propagation and differentiation and cultivate described cell, and with described CFU-GM and SelCID TMOr its prodrug contacts.
91. the described method of claim 90, wherein said contact is carried out first day of described cultivation.
92. the described method of claim 90, wherein said contact is carried out twice during preceding 6 days of described cultivation at least.
93. being no earlier than the 1st day of cultivation, the described method of claim 90, wherein said contact carry out.
94. the described method of claim 90, the cell of wherein said differentiation are dendritic cells, granulocyte, CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Cell.
95. the described method of claim 90, wherein said CD34 +CFU-GM is CD34 +CD133 +CFU-GM.
96. the described method of claim 90, the cell of wherein said differentiation separated at the 6th day that cultivates.
97. the described method of claim 90, the cell of wherein said differentiation separated at the 12nd day that cultivates.
98. the described method of claim 90, wherein said CD34 +Cell separated from other haemocyte before described cultivation.
99. the described method of claim 90, wherein said medium also comprise GM-CSF and TNF-α in addition.
100. the described method of claim 90, wherein said SelCID TMOr its prodrug exists with the concentration of 0.1 μ M to 10.0 μ M.
101. the described method of claim 86, wherein said SelCID TMOr its prodrug exists with the concentration of 1.0 μ M.
CN 03813654 2002-04-12 2003-04-11 Modulation of stem and progenitor cell differentiation, assays, and uses thereof Pending CN1705439A (en)

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CN107410288A (en) * 2017-08-26 2017-12-01 刘劼 A kind of storing liquid of human umbilical cord mesenchymal stem cells
CN107410288B (en) * 2017-08-26 2020-07-31 刘劼 Storage liquid of human umbilical cord mesenchymal stem cells

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