The present invention provides a method of isolating, maintaining, and/or enriching for stem or progenitor cells derived from diverse organ or tissue sources. The invention specifically teaches that these can be accomplished by the controlled use of subatmospheric oxygen culture, and that the precise oxygen level or levels must be determined empirically and/or by reference to physiologic levels within intact functioning organ or tissue.

Regeneration after injury in post-natal organ systems, in many ways, recapitulates developmental processes during embryogenesis. Though many interesting and crucial individual genes that are important for embryogenesis and organogenesis have been discovered over the past decade, the integrated regulation of the process is in many ways unknown (Barinaga, 1994), as are the similarities and differences between embryonic development and regeneration/healing of post-natal cells, tissues and organs. In embryonic mice and man, the various tissue systems develop in parallel and use both inter- and intra-tissue signaling, while the environment around the tissue progresses from one dependent on diffusion of oxygen to one in which oxygen is supplied via the developing vascular system. In the embryo over time, oxygenation to tissues increases as the blood supply is laid down and extended, but this delivery of oxygen is not homogenous throughout any tissue. And though oxygenation becomes richer as the embryo grows, levels of oxygen present in the embryo are generally considered insufficient for normal adult tissue functioning.
Each tissue and organ develops by an exquisitely organized progression in which relatively unspecialized or xe2x80x9cundifferentiatedxe2x80x9d progenitor or stem cells give rise to progeny that ultimately assume distinctive, differentiated identities and functions. Mature tissues and organs are composed of many types of differentiated cells, with each cell type expressing a particular subset of genes that in turn specifies that cell""s distinctive structure, specialized function, and capacity to interact with and respond to environmental signals and nutrients. These molecular, structural and functional capacities and properties comprise the cell phenotype. A similar course of coupled cell proliferation and differentiation in the presence of changing local O2 supply occurs when an injured or degenerating adult tissue undergoes repair and regeneration. The level of oxygen is especially pertinent in many regeneration paradigms in which normal blood supply is reduced or even transiently stopped by trauma or embolic events (myocardial infarction, stroke).
The hypothesis that O2 levels have significant differential impact on different cell types or states has so far received little explicit attention in the literature, with the exception of formation of the vasculature itself. In particular it is important to note that the vast preponderance of studies of regeneration in vitro are performed in laboratories using room air oxygen levels. In room air, 20-21% of atmospheric gas is oxygen (at sea level, depending on humidity), which translates into an oxygen partial pressure of 160 mm Hg [0.21(760 mm Hg)]. The most highly oxygenated tissue in the human body is the arterial blood supply with an oxygen partial pressure of 90 mm Hg. Normal venous oxygenation is 40 mm Hg, and mean tissue oxygen level is 26 mm Hg. However, the vast majority of regeneration research or research on the culture of progenitor cells, stem cells, or differentiating products ignores the importance of oxygenation. The average tissue culture condition is 21% oxygen and 5% carbon dioxide which the remainder being nitrogen.
Herein, the inventors demonstrate that regulated oxygen levels, particularly subatmospheric levels of oxygen (i.e. levels below 21% oxygen and 5% carbon dioxide), can be used to exploit responses of stem and progenitor cells that differ from the response of other cells as a simple and general pathway for their isolation, maintenance, proliferation, enrichment, and/or selective developmental progression and differentiation. This work has important implications for clinical tissue and organ transplantation.
In a time of critical shortages of donor organs, efforts to bring cellular transplantation into the clinical arena are urgently needed (Neelakanta and Csete, 1996). For example, in the case of the liver, a stem cell has not been rigorously identified, and animal models of transplantation of fully-differentiated liver cells (normally quiescent and difficult to force into division experimentally) are not yet successful enough to warrant clinical trials. However, a liver stem cell would represent the ideal cellular transplant because of the potential to regenerate substantial organ function from a tiny rudiment. Thus, there remains a need for methods to identify cells (progenitors and stems) which can be used to regenerate tissue. The present invention is directed at these goals.
The present invention provides a method of isolating, maintaining, and/or enriching for stem or progenitor cells derived from diverse organ or tissue sources. The method can also be used to influence specific differentiation pathways, to alter the kinetics of developmental progression or differentiation or to alter the yield of one cell phenotype over other possible types via the use of subatmospheric or sub-physiologic oxygen levels. The invention specifically teaches that these can be accomplished by the controlled use of subatmospheric oxygen culture, and that the precise oxygen level or levels must be determined empirically and/or by reference to physiologic levels within intact functioning organ or tissue, or that tissue following injury, regeneration, or disease.
The inventors have discovered that when adult skeletal muscle fibers are cultured under physiologically hypoxic conditions, progenitor cells (which represent a source of regenerated new muscle) develop in greater numbers than when fibers are grown under traditional room air conditions. Furthermore, this subatmospheric oxygen-induced enrichment of the skeletal muscle progenitor population is followed by earlier regeneration of new skeletal muscle in culture.
Intentional hypoxia has been used in some culture systems, usually differentiated systems, to mimic pathologic conditions such as stroke (for example, Papadopoulos et al, 1996). In some circumstances, whole embryos have been cultured under subatmospheric oxygen conditions to mimic gestation (for example, Giles and Foote, 1997). In other circumstances reduced oxygen conditions are used as part of a broad range of tests of many environmental parameters on culture integrity (for example, Berthelot and Terqui, 1996). In a few cases oxygen conditions have been used to assess proliferation of particular cells as a function of oxygen levels (for example, Matsuda et al., 1998). However, such examples do not teach the general principle that an intentional reduction of oxygen surrounding cultures can be used to selectively promote survival, proliferation, enrichment or particular developmental or differentiation pathways from stem cell and/or progenitor cells. Furthermore, such reports have not had an effect on the general state or practice of the art, in that almost all tissue culture continues to be conducted in room air.
A method for isolating, maintaining, propagating or enriching progenitor or stem cells, and/or for influencing the differentiation outcome or differentiation kinetics of such cells into particular tissue types, comprising the steps of:
a) obtaining cells derived from mammalian tissue containing at least one progenitor cell or stem cell capable of producing progeny that can assume or produce cells with one or more differentiated phenotypes, and
b) culturing the cells derived from such mammalian tissue (various organ and tissue types) in suitable medium under empirically determined subatmospheric oxygen conditions for a time sufficient to promote the survival, proliferation, or enrichment of the stem or progenitor population, or to cause or influence entry into one or more differentiation pathways.
Definitions
A xe2x80x9cstem cellxe2x80x9d is a relatively undifferentiated cell that can be induced to proliferate and that can produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. In many biological instances, stem cells are also xe2x80x9cmultipotentxe2x80x9d because they can produce progeny of more than one distinct cell type, but this is not required for xe2x80x9cstem-ness.xe2x80x9d Self-renewal is the other classical part of the stem cell definition, and it is essential as used in this document. In theory, self-renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Formally, it is possible that cells that begin as stem cells might proceed toward a differentiated phenotype, but then xe2x80x9creversexe2x80x9d and re-express the stem cell phenotype.
xe2x80x9cProgenitor cellsxe2x80x9d have a cellular phenotype that is more primitive (i.e., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell). Often, progenitor cells also have significant or very high proliferative potential. Progenitor cells may give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate. Like stem cells, it is possible that cells that begin as progenitor cells might proceed toward a differentiated phenotype, but then xe2x80x9creversexe2x80x9d and re-express the progenitor cell phenotype.
xe2x80x9cDifferentiationxe2x80x9d refers to the developmental process whereby cells assume a specialized phenotype, i.e., acquire one or more characteristics or functions distinct from other cell types. In most uses, the differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway. In many but not all tissues, the process of differentiation is coupled with exit from the cell cyclexe2x80x94in these cases, the cells lose or greatly restrict their capacity to proliferate when they differentiate.
xe2x80x9cSubatmosphericxe2x80x9d conditions mean any oxygen concentration below about 20%, preferably below about 12%, more preferably below about 8%, at sea level.
xe2x80x9cPhysiologicxe2x80x9d oxygen levels are the range of oxygen levels normally found in healthy tissues and organs. These levels vary depending on tissue type.
xe2x80x9cHypoxiaxe2x80x9d occurs when the normal physiologic levels of oxygen are not supplied to a cell or tissue. xe2x80x9cNormoxiaxe2x80x9d refers to normal physiologic levels of oxygen for the particular cell type, cell state or tissue in question. xe2x80x9cAnoxiaxe2x80x9d is the absence of oxygen. xe2x80x9cHypoxic conditionsxe2x80x9d are those leading to cellular hypoxia. These conditions depend on cell type, and on the specific architecture or position of a cell within a tissue or organ, as well as the metabolic status of the cell. A critical point is that in most cell biology research of the past 25 years, ambient atmospheric oxygen levels of 20-21% are routinely called and experimentally taken to be xe2x80x9cnormoxic,xe2x80x9d but this assumption is physiologically erroneous. In this historic context, much cell culture literature refers to any condition with oxygen lower than ambient atmospheric as xe2x80x9chypoxic,xe2x80x9d but this usage is also physiologically incorrect.
xe2x80x9cAcidosisxe2x80x9d means that the pH is below normal physiologic levels.
xe2x80x9cEnrichingxe2x80x9d of cells means that the yield (fraction) of cells of one type is increased over the fraction of cells of that type in the starting culture or preparation.
xe2x80x9cProliferationxe2x80x9d refers to an increase in the number of cells in a population (growth) by means of cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to the environment, including growth factors and other mitogens. Cell proliferation may also be promoted by release from the actions of intra- or extracellular signals and mechanisms that block or negatively affect cell proliferation.
xe2x80x9cRegenerationxe2x80x9d means regrowth of a cell population, organ or tissue after disease or trauma.
Methods for Manipulating the Yield of Progenitor/Stem Cell Cultures or Preparations by Manipulation of Sub-atmospheric Oxygen Levels
The present invention provides a method for manipulating the yield of progenitor/stem cell cultures. This method has several embodiments, including
A. Subatmospheric/physiologic oxygen conditions used to culture or enrich stem cell and/or progenitor cells
B. Subatmospheric/physiologic oxygen conditions used to isolate/identify stem or progenitor cells that are not yet identified
C. Subatmospheric/physiologic oxygen conditions used to alter the kinetics of developmental progression or accelerate differentiation from stem cell and/or progenitor populations
D. Subatmospheric/physiologic oxygen conditions used to enhance regeneration
E. Subatmospheric/physiologic oxygen conditions used to select, promote or reinforce a specific cell fate or fates
A. Subatmospheric/Physiologic Oxygen Conditions Used to Culture or Enrich Stem Cells and/or Progenitor Cells
A method of enriching progenitor or stem cells in a population of cells comprising progenitors or stems, progeny thereof, and other xe2x80x9ccontaminatingxe2x80x9d cells, comprising the steps of:
a) obtaining a population of mammalian cells (adult or embryonic, from one or more solid tissue type) containing at least one stem cell or progenitor cell capable of producing differentiated progeny, and
b) culturing the cells derived from mammalian tissue in suitable medium under oxygen levels reflecting physiologic oxygenation for the tissue from which they were derived or under hypoxic conditions (below normal physiologic oxygenation) for a time sufficient to enrich the population of progenitor/stem cells in said culture relative to one or more other cell types in the population.
The low oxygen or physiologic oxygen conditions are part of the enrichment process from either the time of isolation or some time after (such as after attachment of cells to tissue culture plates) or throughout the process, depending on the specific cell type to be enriched and depending on the particular starting cell population and its physiologic status. Enrichment of stems/progenitors by physiologic or low levels of oxygen may be the result of one or more mechanisms that include (1) increase in the absolute number of stem cell and/or progenitor cells (2) enrichment by selective survival of progenitor/stem cells, or (3) enrichment of stems/progenitors by their selective proliferation
Any increase in the number of stems/progenitors is significant in that more cells are then available to regenerate a greater volume of new tissue. Any enrichment, even without increase in number, is important in applications where limitations on total cell number are pertinent or when the effects of the non-stem/non-progenitor contaminants are negative for the desired outcome or for defining the material adequately. Any enhancement of survival of the stem or progenitor state, even without increase in stem cell and/or progenitor cell number or any enrichment of stem cell and/or progenitor cell types is valuable in settings where culture is required (i.e., to handle tissue before administration of cell therapy, or to permit any other procedure during which the cells must survive such as transfection of genes, drug treatment, or enrichment by cell sorting or other additional procedures).
Suitable solid tissue from which cells can be obtained includes any organ or tissue from adult, post-natal, fetal or embryonic mammalian tissue. Any mammalian tissue or organ can be used in this invention, including but not limited to those obtained from mice, cattle, sheep, goat, pigs, dogs, rats, rabbits, and primates (including human). Specific examples of suitable solid tissues include skeletal muscle, brain and central nervous system tissue from which neurons and other supporting cells are derived, skin derived from cultured keratinocytes, germ cells or embryonic stem cells or cells from other organs (liver, pancreas, spleen, kidney, thyroid, etc.). Stem cells and progenitor cells isolated from any other solid organ are also amenable candidates for culturing under physiologic or hypoxic conditions.
Suitable medium and conditions for generating primary cultures are well known in the art and vary depending on cell type. For example, skeletal muscle, bone, neurons, skin, liver, and embryonic stem cells are all grown in media differing in their specific contents. Furthermore, media for one cell type may differ significantly from lab to lab and institution to institution. As a general principle, when the goal of culturing is to keep cells dividing, serum is added to the medium in relatively large quantities (10-20% by volume). Specific purified growth factors or cocktails of multiple growth factors can also be added or sometimes used in lieu of serum. As a general principle, when the goal of culturing is to reinforce differentiation, serum with its mitogens is generally limited (about 1-2% by volume). Specific factors or hormones that promote differentiation and/or promote cell cycle arrest can also be used.
Physiologic oxygen and hypoxic oxygen conditions can be used at any time during the growth and differentiation of cells in culture, as a critical adjunct to selection of specific cell phenotypes, growth and proliferation of specific cell types, or differentiation of specific cell types. In general, physiologic or hypoxic oxygen-level culturing is accompanied by methods that limit acidosis of the cultures, such as addition of strong buffer to medium (such as Hepes), and frequent medium changes.
Cells can be exposed to hypoxic/physiologic conditions using a variety of means. Specialized laboratory facilities may have completely enclosed environments in which the oxygen levels are controlled throughout a dedicated, isolated room. In such specialized areas, low oxygen levels can be maintained throughout the isolation, growth and differentiation of cells without interruption Very few laboratories have such specialized areas. Physiologic or low oxygen culturing conditions can be maintained by using commercially-available chambers which are flushed with a pre-determined gas mixture (Billups-Rothenberg, San Diego Calif.). As an adjunct, medium can be flushed with the same gas mixture prior to cell feeding. In general, it is not possible to maintain physiologic or hypoxic oxygen conditions during cell feeding and passaging using these smaller enclosed units, and so, the time for these manipulations should be minimized as much as possible. Any sealed unit can be used for physiologic oxygen or hypoxic culturing provided that adequate humidification, temperature, and carbon dioxide are provided.
Physiologic oxygen conditions range widely depending on the cell type, energy expenditure, and location within an organ structure. Physiologic conditions range from about 12% at the high extreme to less than 1% oxygen, but preferably in the range of 1-5% (about 8-40 mm Hg). (Guyton and Hall, 1996) Hypoxic conditions are generally less than 1% oxygen, but some cells may be hypoxic at 1% oxygen or higher.
In addition to oxygen, the other gases for culture are typically 5% carbon dioxide and the remainder is nitrogen, but optionally may contain varying amounts of nitric oxide (starting as low as 3 ppm), carbon monoxide and other gases, both inert and biologically active. Carbon dioxide concentrations typically range around 5% as noted above, but may vary between 2-10%. Both nitric oxide and carbon monoxide are typically administered in very small amounts (i.e. in the ppm range), determined empirically or from the literature.
The optimal physiologic or hypoxic conditions for any given progenitor/stem cell type will vary. A skilled artisan could determine suitable subatmospheric conditions by generating an oxygen dose response curve, in which carbon dioxide is kept constant, and oxygen levels are varied (with nitrogen as the remaining gas). For example, to determine the optimal ambient oxygen culturing conditions for expansion of a stem cell and/or progenitor, one would establish cultures from an organ system. The initial culture is mixed, consisting of some differentiated cells, cells of other developmental lineages or pathways, as well as stem cell and/or progenitor cells. After exposure to the various oxygen levels (e.g. 1%, 2%, 5%, 10% and 15%), the number and function of stem cell and/or progenitor cells is assessed by methods appropriate to the system. In some cases, a constellation of molecular markers is available to rapidly identify the stem cell population. But in other cases, a single marker coupled with proliferation assays is appropriate, while in other cases proliferation assays alone are appropriate. In some cases all or some of the above assays are coupled with bioassays to follow the differentiation potential of the presumed stem cells. Overall, the precise assays used to determine stem cell and/or progenitor response to oxygen levels are dependent on the nature of the system examined as well as available markers and techniques specific to that system.
The timing of physiologic or low oxygen conditions is also part of the oxygen dose response curve. Some cells may be more or less sensitive to oxygen immediately after isolation while some cells may respond only after some time in culture. The timing of physiologic or low oxygen conditions absolutely and in relation to other manipulations of the cultures is part of assessing the optimal oxygen culturing conditions. Furthermore, the mitogenic effects of other gases may be synergistic with physiologic or low oxygen conditions. Different gene regulatory networks may be induced by low/physiologic oxygen culturing during different phases of culture. During expansion of stems/progenitors, low oxygen may induce gene expression distinct from that induced by low oxygen during differentiation.
The cells are typically exposed to hypoxic conditions for a time sufficient to enrich the population of progenitor/stem cells compared to other cell types. Typically this is for 1 or more hours, preferably 3 or more hours, more preferably 6 or more hours, and most preferably 12 or more hours, and may be continuous. The temperature during the culture is typically reflective of core body temperature, or 37xc2x0 C., but may vary between 32 and 40xc2x0 C. Other important embodiments may simply achieve an increase in stem cell or progenitor cell absolute number or promote the survival of stem cells or progenitor cells.
Following an initial exposure to low or physiologic oxygen culturing conditions, cells can be maintained in these conditions or returned to normal laboratory oxygen conditions, depending on the desired outcome. Stem cell and/or progenitors may be maintained continuously in low or physiologic oxygen culture conditions.
It is understood that the initial medium for isolating stems/progenitors, the medium for proliferation of these cells, and the medium for differentiation of these cells can be the same or different. All can be used in conjunction with low or physiologic oxygen level culturing. The medium can be supplemented with a variety of growth factors, cytokines, serum, etc. Examples of suitable growth factors are basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factors (TGFxcex1 and TGFxcex2), platelet derived growth factors (PDGF""s), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), insulin, erythropoietin (EPO), and colony stimulating factor (CSF). Examples of suitable hormone medium additives are estrogen, progesterone or glucocorticoids such as dexamethasone. Examples of cytokine medium additives are interferons, interleukins, or tumor necrosis factor-xcex1 (TNFxcex1). One skilled in the art will test additives and culture components at varied oxygen levels, as the oxygen level may alter cell response to, active lifetime of additives or other features affecting their bioactivity.
Stem cells and progenitor cells isolated from solid tissues (the exception to solid tissue is whole blood, including blood, plasma and bone marrow) which were previously unidentified in the literature are also within the scope of this invention.
B. Subatmospheric/Physiologic Oxygen Conditions Used to Isolate/Identify Stem or Progenitor Cells that are not Yet Identified
In this embodiment, a culture derived from an organ or tissue system is established, consisting of a variety of cell types, both stem cell and/or progenitor and differentiated cells. Oxygen dose-response experiments are performed, in which parallel cultures are exposed to progressively decreasing levels of oxygen, starting with 12% (approximately adult arterial levels). The goal of these experiments is to identify the oxygen level at which stem cell and/or progenitor cells increase in absolute number, or survive in preference to other cells in the culture, or selectively proliferate, or a combination of these effects. In any of these cases, subatmospheric oxygen is used to facilitate identification and characterization of the usually rare stem cell and/or progenitor cell population.
Following culturing in physiologic or low oxygen conditions, progenitor/stem cells can be identified by a variety of methods. Progenitor/stem cells with defined morphology can often be identified visually, but their precise identification is made with specific markers such as antibodies, or by their expression of specific genes at the mRNA level (using PCR). Following culturing in physiologic or low oxygen conditions, one can determine if stems/progenitors are proliferating by a variety of means including incorporation of radionucleotides (such as 3H-thymidine), or by uptake of BrdU (a thymidine analog) (Assy and Minuk, 1997) Some progenitor/stem cells will be identified by their ability to differentiate into specific cell types, and this assay may be used alone or in combination with other assays as above, depending on the availability of suitable markers.
C. Subatmospheric/Physiologic Oxygen Conditions Used to Alter the Kinetics of Developmental Progression or Accelerate Differentiation from Stem Cell and/or Progenitor Populations
Another embodiment of the present invention involves sifting the kinetics or the yield of differentiation of progenitor cells and/or stem cells into various cell types by exposing them to physiologic/subatmospheric oxygen levels. In this embodiment, subatmospheric oxygen is used to alter the kinetics of progression from an undifferentiated stem cell and/or progenitor state to cells with a differentiated phenotype. This change in kinetics, depending on the cell type and culture conditions may be an acceleration or deceleration. Moreover, the use subatmospheric oxygen may increase or decrease the fraction of stem cells or progenitor cells that differentiate. Depending on the application, such increase or decrease might be desirable.
The steps involved in this embodiment are identical to those described above for enriching the population of stem cells. However, the conditions may differ as to absolute timing of physiologic/subatmospheric oxygen added to the culture, and timing of physiologic/subatmospheric oxygen culturing relative to other culture manipulations. Differentiation medium is generally different than medium supplemented to encourage growth and proliferation. In general, differentiation medium is low in mitogens from serum additives.
In some stem cell and/or progenitor cultures, physiologic/subatmospheric oxygen conditions dramatically increase the rate of differentiation in culture. The inventors have shown, for example, that when rat embryonic brain stem cells are placed in 1% oxygen cultures (for 7-10 days), more differentiated neurons, astrocytes, and oligodendrocytes are produced per plated stem cell than under normal laboratory oxygen conditions. This is an example of kinetic enhancement of differentiation which is also applicable to other systems. For example, the inventors have also shown an increase in kinetics of progression and initiation of differentiation in skeletal muscle progenitor cells.
Following differentiation, the specific differentiated cell types are identified by a variety of means including fluorescence activated cell sorting (FACS), protein-conjugated magnetic bead separation, morphologic criteria, specific gene expression patterns (using RT-PCR), or specific antibody staining The gene products expressed between two or more given differentiated cell types will vary. For example, following differentiation of skeletal muscle satellite cells, the transcription factors myf5, MyoD, myogenin, and mrf4 are expressed. It is understood that developmental pathways often involve more than one step or stage for differentiation and any of these steps or stages may be affected by subatmospheric oxygen.
D. Subatmospheric/Physiologic Oxygen Conditions Used to Enhance Regeneration
In this embodiment, subatmospheric oxygen is used to first expand the stem cell and/or progenitor pool, from which regeneration is then allowed to proceed. Since the initial pool from which tissue/organ can be regenerated is increased in number, the ultimate amount of regeneration into differentiated cells is also increased.
In skeletal muscle primary cultures, the inventors showed that the number of progenitor satellite cells on parental fibers increases twice as fast in culture for the first 60 hours when the fibers are cultured under 1% oxygen versus the normal room air oxygen levels. This is an example of more tissue generated for differentiation by hypoxic culturing conditions.
E. Subatmospheric/Physiologic Oxygen Conditions Used to Select, Promote or Reinforce or Promote a Specific Cell Fate or Fates
When a particular stem or progenitor cell is capable of differentiating into a number of developmentally distinct cell, tissue, or organ types, the cell is said to be pluripotent. By incubating a pluripotent cell line under hypoxic conditions in vitro, the inventors were able to manipulate or skew the direction of differentiation of the cell population.
The result of this technique is essentially an enrichment of one or more cell type but this may also be considered a selection against other cell types. In this example, growth of the cells under hypoxic conditions enriched for muscle cells and, at the same time, selected against fat cells. The inventors contemplate that oxygen concentrations may be manipulated to direct differentiation of other pluripotent stem or progenitor cells as well.
Specifically, the mouse cell line 10T1/2 cells can be pharmacologically (5-azacytidine) induced to generate cartilage, skeletal muscle and fat (adipocytes) (Taylor and Jones, 1979). Previous work subjecting these cells to 2% oxygen suggested that the numbers of skeletal muscle cells generated was increased by low oxygen conditions (Storch T G, 1996). The present inventors have shown that culturing of 10T1/2 cells under 21% oxygen conditions in conjunction with 5-azacytidine treatment results in skeletal muscle and adipocyte differentiation within 2 weeks. Notably, adipocyte differentiation in this cell line is blocked under 1% oxygen conditions in the presence (or absence) of azacytidine. This is an example of physiologic/subatmospheric oxygen conditions playing a role in the precise differentiation fate taken by pluripotent cells. The identification of gene networks up- or down-regulated by the change in oxygen environment could, in turn, provide pharmacologic targets for enhancement or blocking of specific differentiation pathways, in this case fat development.
In this embodiment, subatmospheric oxygen conditions can be used to exploit expression of particular genes characteristic of certain differentiated cell types. For example, the inventors have shown that culture of embryonic rat brain stem cells in 1% oxygen (vs. 20-21%) leads to increased expression of the gene tyrosine hydroxylase after 10 days in culture, when neuronal differentiation has occurred. Tyrosine hydroxylase is the rate-limiting enzyme in the synthetic pathway to dopamine. Deficiency of dopamine in certain areas of the brain is the cause of Parkinson""s disease. Thus culturing, expansion, and differentiation of central nervous system stem cells under low O2 may be used to maximize the efficiency of production of a therapeutic gene by differentiated progeny of the cultured cells (i.e. specific genes are regulated by low O2 conditions or specific gene products are expressed by specific differentiated progeny).
Techniques for Screening the Effect of Various Pharmacological Agents on Differentiation and/or Regeneration
To determine whether a test compound modulates cell proliferation and/or differentiation, a cell is identified as a progenitor cell (e.g., by using one of the above-described methods), the progenitor cell (typically a culture of progenitor cells) is contacted with the test compound, and the progenitor cell is monitored to assess the effect, if any, of the test compound on proliferation, differentiation and/or regeneration. Any compound of interest can be used as the test compound in this method.
The compound can be contacted with the progenitor/stem cells at any desired concentration, preferably, a wide range of concentrations. The cells are monitored for changes in the rates or patterns of proliferation and/or differentiation of the progenitor cell in order to determine which test compounds modulate proliferation and/or differentiation. Typically, such assays are performed in vitro.
Stem cell and/or progenitor cells subjected to physiologic/subatmospheric oxygen conditions will express different genes than those cultured under routine conditions. It is well known, for example, that physiologic hypoxia elicits expression of HIF-1 (hypoxia inducible factor-1) in a variety of tissues (Iyer et al., 1998). This gene, in turn, causes increased expression of an array of other genes required for the cells to respond to hypoxia, such as erythropoietin and VEGF. Other genes have increased expression under hypoxic conditions, apparently independent of HIF-1. These genes include tyrosine hydroxylase (Ramsey et al., 1996), which is the rate limiting enzyme in the pathway to dopamine synthesis.
The kinetic differences in stem cell and/or progenitor proliferation demonstrated by the inventors, and the differences in type of differentiation in some cells shown by the inventors, are all clear indications that other genetic regulatory networks are dramatically changed by physiologic/subatmospheric culture conditions.
Methods for Determining the Biochemical Effect of Hypoxia
In light of the present disclosure, one of skill in the art would understand that the ability to isolate, culture, and differentiate progenitor cells allows for the analysis or discovery of genes that contribute to the phenotype of the progenitor cell and cells differentiated therefrom. The ability to detect genes that are differentially expressed in two cell types or populations combined with advances of rapid gene detection and sequencing technologies has led to the discovery of many novel genes that contribute to the difference in the cell types or populations. Such technologies may be used to compare gene expression in cells cultured under varying oxygen concentrations, in progenitor cells of different origin, between cells of distinct differentiation states, and in cells contacted with a compound or environmental stimulus that may affect the ability of the cells to react to a variation in oxygen concentration.
Methods of differential display have been used to elucidate the genes responsible for a difference in phenotypes between two relatively similar cell types or during sequential changes of a cell from one state to another. For example, using the differential display technique, Kocher et al. (1995) selected for genes that were up-regulated in renal cell carcinoma compared with normal renal parenchyma. Through this method, Kocher et al. (1995) were able to isolate a gene (DD96) that was rarely expressed in normal epithelial cell populations, expressed diffusely in malignant epithelial cells of the wide majority of carcinomas, and markedly overexpressed in carcinomas originating from the colon, breast, and lung. A similar technique may be used to compare gene expression in cells incubated under normal or hypoxic conditions. Genes up-regulated in one population over the other then may be used as a probe to screen for expression of that gene in other cell populations or the same cell population under different culturing conditions (i.e., in the presence of compounds or environmental stimuli that may affect the expression of the gene).
Kang et al. (1998) have developed a reciprocal subtraction differential RNA display (RSDD) method that permits the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes. The technology was used to analyze gene expression alterations resulting during cancer progression as adenovirus-transformed rodent cells developed an aggressive transformed state (Kang et al, 1998). The approach resulted in the identification and cloning of known and unknown sequences that displayed expression as a function of progression and suppressed expression as a function of progression (Kang et al., 1998). The RSDD technique may be used to compare gene expression between cells during maintenance, proliferation and/or differentiation of the cells from progenitor or stem cells to fully differentiated cells in room air versus subatmospheric conditions.
The methods of differential display may be used in conjunction with rapid DNA sequencing and detection methods, allowing for the ability to screen for or sequence a large number of genes in a relatively short amount of time. U.S. Pat. No. 5,800,992 provides methods for detecting the differential expression of a plurality of genes between two cell types using complimentary polynucleotides in an array. Such technology is commonly referred to as xe2x80x9cDNA chipxe2x80x9d technology because the polynucleotides are deposited on a substrate that resemble computer microprocessor chips. Also described are methods of sequencing genes using DNA chips.
U.S. Pat. No. 5,834,181 utilizes similar technology to detect minor alterations in genes such as single nucleotide substitution, allowing detection of mutations in genes that lead to a change in the phenotype of a cell. Such methods may be used to determine if the inability of cells from some individual patients or animals or in cells carrying somatic mutations such as tumors to react to oxygen is caused by a mutation in one or more genes involved in oxygen-mediated/altered cell processes. In one embodiment of the present invention, cells that have an altered ability to react to oxygen concentration are analyzed using the methods of U.S. Pat. No. 5,834,181 In another embodiment of the present invention, cell cultures exposed to various levels of oxygen are analyzed using the methods of U.S. Pat. No. 5,834,18.
Methods for Cellular Transplant Therapies
Stem cell and/or progenitors isolated, expanded, or activated by physiologic/subatmospheric oxygen culturing conditions may be used in cell/organ replacement therapies. Currently in clinical practice, pancreatic islet transplants are the only common cell transplant therapy for solid organs. In this case, pancreatic islets (differentiated cells) cultured from cadaveric donors, are transplanted to replace diseased islets, usually in patients with end-stage diabetes.
For other organs, major surgical transplant procedures are the only other real option to replace damaged or diseased organs. In theory, cellular transplant therapies could be used to replace many of these high-risk surgical procedures. But cell transplants are limited by an insufficient identification of stems/progenitors, and insufficient supply of stems/progenitors for the various organ systems. Once a sufficient supply of stems/progenitors could be generated (either from autologous or allogeneic sources) they could, in theory, be used to regenerate a sufficient volume of cells to replace a key missing organ/tissue function(s). The regeneration could be carried out in part in the laboratory prior to transplantation of the cells and regenerated organ, or after transplantation of the cells into an orthotopic or heterotopic location in the body. The major impediment to such therapies is the availability of suitable stems/progenitors that generate sufficient tissue to perform physiologic function and withstand immune attack. The inventors propose that physiologic/subatmospheric culturing conditions can be used to identify specific populations of stem cell and/or progenitors useful for transplantation, and to expand the number of available stems/progenitors derived from a variety of culture systems.
Methods for Gene Therapy
Optionally, the progenitor cells obtained using the method of the present invention can be manipulated to express desired gene products. Gene therapy can be used to either modify a cell to replace a gene product, to facilitate regeneration of tissue, to treat disease, or to improve survival of the cells following implantation into a patient (i.e. prevent rejection).
In this embodiment, the progenitor cells are transfected prior to expansion and differentiation. Techniques for transfecting cells are known in the art.
A skilled artisan could envision a multitude of genes which would convey beneficial properties to the transfected cell or, more indirectly, to the recipient patient/animal. The added gene may ultimately remain in the recipient cell and all its progeny, or may only remain transiently, depending on the embodiment. For example, genes encoding angiogenic factors could be transfected into progenitor cells isolated from smooth muscle. Such genes would be useful for inducing collateral blood vessel formation as the smooth muscle tissue is regenerated. It some situations, it may be desirable to transfect the cell with more than one gene.
In some instances, it is desirable to have the gene product secreted. In such cases, the gene product preferably contains a secretory signal sequence that facilitates secretion of the protein. For example, if the desired gene product is an angiogenic protein, a skilled artisan could either select an angiogenic protein with a native signal sequence, e.g. VEGF, or can modify the gene product to contain such a sequence using routine genetic manipulation (See Nabel et al., 1993).
The desired gene can be transfected into the cell using a variety of techniques. Preferably, the gene is transfected into the cell using an expression vector. Suitable expression vectors include plasmid vectors (such as those available from Stratagene, Madison Wis.), viral vectors (such as replication defective retroviral vectors, herpes virus, adenovirus, adeno-virus associated virus, and lentivirus), and non-viral vectors (such as liposomes or receptor ligands).
The desired gene is usually operably linked to its own promoter or to a foreign promoter which, in either case, mediates transcription of the gene product Promoters are chosen based on their ability to drive expression in restricted or in general tissue types, or on the level of expression they promote, or how they respond to added chemicals, drugs or hormones. Other genetic regulatory sequences that alter expression of a gene may be co-transfected. In some embodiments, the host cell DNA may provide the promoter and/or additional regulatory sequences.
Other elements that can enhance expression can also be included such as an enhancer or a system that results in high levels of expression.
Methods of targeting genes in mammalian cells are well known to those of skill in the art (U.S. Pat. Nos. 5,830,698; 5,789,215; 5,721,367 and 5,612,205). By xe2x80x9ctargeting genesxe2x80x9d it is meant that the entire or a portion of a gene residing in the chromosome of a cell is replaced by a heterologous nucleotide fragment. The fragment may contain primary the targeted gene sequence with specific mutations to the gene or may contain a second gene. The second gene may be operably linked to a promoter or may be dependent for transcription on a promoter contained within the genome of the cell. In a preferred embodiment, the second gene confers resistance to a compound that is toxic to cells lacking the gene. Such genes are typically referred to as antibiotic-resistance genes. Cells containing the gene may then be selected for by culturing the cells in the presence of the toxic compound.
Methods of gene targeting in mammals are commonly used in the product of transgenic xe2x80x9cknockoutxe2x80x9d mice (U.S. Pat. Nos. 5,616,491; 5,614,396). These techniques take advantage of the ability of mouse embryonic stem cells to promote homologous recombination, an event that is rare in differentiated mammalian cells. Recent advances in human embryonic stem cell culture may provide a needed component to applying the technology to human systems (Thomson; 1998). Furthermore, the methods of the present invention may used to isolate and enrich for stem cells or progenitors cells that are capable of homologous recombination and, therefore, subject to gene targeting technology. Indeed, the ability to isolate and grow somatic stem cells and progenitor cells has been viewed as impeding progress in human gene targeting (Yanez and Porter, 1998).
Skeletal Muscle
In adult skeletal muscle, the progenitor cell is referred to as a satellite cell. Normally, satellite cells are dormant, but when muscle is traumatized, these cells divide and differentiate, and so are the source of regenerated skeletal muscle. As disclosed herein, subatmospheric oxygen culture conditions (1% oxygen) significantly increase the number of dividing satellite cells associated with myofibers over the first few days of culture. Using BrdU labeling and morphologic criteria, the number of satellites per unit length of muscle were quantified in 12 hour intervals. (The unit length of fiber was a 20xc3x97 power diameter microscope field.) At each interval for the first few days, the number of satellites dividing under hypoxic conditions was about twice that under traditional room air cultures:
Methods of isolating, identifying, culturing and differentiating satellite cells are well known to those of skill in the art. For example, in U.S. Pat. No. 5,328,695, Lucas et al. describe a myogenic protein isolate from mammalian bone that stimulates lineage commitment and differentiation of skeletal muscle stem cells. Primary cultures of muscle progenitor cells were obtained from chicken embryos, cultured and caused to differentiate in vitro. The inventors contemplate that the hypoxic culturing conditions they describe herein, used in conjunction with the methods of U.S. Pat. No. 5,328,695, will further increase the isolation, activation, and differentiation of such stem cells as well as satellites derived from mammalian systems.
Cornelison and Wold (1997) isolated satellite cells from adult murine skeletal muscle and characterized the expression of certain genes in quiescent and activated satellite cells. Traditionally, quiescent satellite cells have been hard to distinguish from contaminating fibroblasts because there were no known molecular markers that could be used to distinguish the two. Using a single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) technique, Cornelison and Wold (1997) demonstrated that c-met is expressed in quiescent satellite cells but not in muscle-derived fibroblasts or other mononucleated cells from healthy muscle explants. Furthermore, c-met was expressed throughout activation, proliferation, and differentiation of satellite cells. Therefore, c-met may be used as a molecular marker to detect satellite cells and cells differentiated therefrom (Cornelison and Wold, 1997).
Using the single-cell RT-PCR technique, Cornelison and Wold (1997) went on to show that, upon activation, the satellite cells showed a distinct progression of MyoD family regulators of muscle determination and differentiation (mrf""s) gene expression. Activated satellite cells began to express either c-met and MyoD or c-met and myf5 first among the mrf""s (Cornelison and Wold, 1997). Most cells then expressed c-met and both myf5 and MyoD simultaneously (Cornelison and Wold, 1997). This state was followed by one in which the cells expressed c-met, myf5, MyoD, and myogenin (Cornelison and Wold, 1997). Although rare cells later expressed c-met and myogenin, others expressed all the MRFs of the previous state plus MRF4 (Cornelison and Wold, 1997). In the next state, myf5 and MyoD expression is turned off leaving expression of c-met, myogenin and MRF4 (Cornelison and Wold, 1997). Thus, whereas quiescent satellite cells may be determined by c-met expression, activation and differentiation of satellite cells may be determined by the expression of c-met, myf5, MyoD, myogenin, and mrf4.
The single cell RT-PCR technique of Cornelison and Wold allows determination of expression of all the above genes at one time and may be used to identify satellite cells and determine their activation state when incubated in the hypoxic conditions of the present invention. The inventors showed, in particular, that expression of mrf group muscle transcriptional factors is accelerated when skeletal muscle fibers are cultured under 1% oxygen levels. For example, myogenin and mrf4 are not detected in satellites when cultured under normal laboratory oxygen (21%) conditions for 24 hours. However, some satellites cultured with 1% oxygen express mrf4 and some express myogenin after just 24 hours of culture. In addition, a greater percentage of satellites cultured under 1% oxygen conditions express MyoD at 24 hours when compared to the normal culture conditions. This is an example of changes in the timing of differentiation genetic pathways as a consequence of low oxygen culturing conditions.
Differentiated skeletal muscle has a distinctive appearance: myotubes are large fused cells with multiple nuclei aligned coordinately. The myofiber has distinctive, patterned striations. These features are used to define the appearance of newly regenerated muscle from satellites in culture. In addition, specific proteins, such as myosin heavy chain, are expressed by the differentiated fused myotube and are detected using antibody staining.
The methods of this present invention may be utilized to produce both slow- and fast-twitch myofibers (Cornelison and Wold, 1997). Regardless of muscle fiber type, in another embodiment, the muscle produced by the methods of the present invention are used to produce muscle satellites and/or fibers for the purpose of clinical transplantation. In theory satellites and or their progeny could be transplanted to treat muscular diseases such as the muscular dystrophies, or atrophy due to trauma, nerve damage, or disuse. For muscular dystrophies, the satellites and progeny would provide missing gene product (dystrophin, for example) necessary for normal muscle strength. In other cases, missing normal muscle mass would be regenerated from transplanted satellites and progeny. Satellite cells activated under hypoxic conditions may be transplanted into a patient without further differentiation. This protocol may be particularly useful in patients who lack functional satellites to activate because they lack muscle mass The protocol may also be useful in patients who have satellites that do not regenerate normal muscle (muscular dystrophies), if the donor cells express corrective gene product.
Central Nervous System Cells
The hypoxic culturing conditions of the present invention may also be used in methods comprising cells of the nervous system. Neural stem cells and stem cell progeny (daughter stem cells and progenitor cells) proliferate and differentiate. Undifferentiated neural progenitor cells differentiate into neuroblasts and glioblasts which give rise to neurons and glial cells. During development, cells that are derived from the neural tube give rise to neurons and glia of the CNS. Certain factors present during development, such as nerve growth factor (NGF), promote the growth of neural cells.
Methods of isolating and culturing neural stem cells and progenitor cells are well known to those of skill in the art (Hazel and Muller, 1997; U.S. Pat. No. 5,750,376).
Hazel and Muller describe methods of isolating, culturing, and differentiating rat brain neuroepithelial stem cells from both fetal and adult rat brains. Briefly, neural precursors are removed from desired regions of the fetal rat central nervous system by dissection, dissociated to a single-cell suspension, and plated on tissue culture dishes in medium containing the mitogen basic fibroblast growth factor (bFGF). Initially, many of the differentiated neurons die. Proliferating cells are then harvested in a buffered solution. The passaged cells are relatively homogenous for multipotent precursors. To induce differentiation to neurons and glia, the media containing bFGF is removed and replaced with media lacking bFGF.
Subatmospheric culturing conditions can be used in such a protocol from the start of stem cell isolation, in order to enrich the stem cell pool and enhance differentiation into a greater number of cells. Subatmospheric/physiologic culture conditions can also be used after initial plating and division, to up-regulate certain gene products in the more differentiated brain cells. Subatmospheric/physiologic culture conditions can also be used throughout the process to enhance the function of the entire population for transplantation.
Detection of neural stem cell derivatives can be determined by antibody staining. For example, central nervous system multipotential stems are marked by high level expression of the intermediate filament, nestin (Hazel and Muller, 1997). The differentiated neurons are marked by the antibody TUJ1 (O""Rourke et al., 1997), oligodendrocytes by GalC (Bosio et al., 1996), and astrocytes by GFAP antibodies (Rutka et al., 1997).
The methods of the present invention may be used to produce neural cells containing a heterologous gene. Methods of producing cells of neural origin comprising a heterologous gene and uses of such cells are described in U.S. Pat. No. 5,750,376 (incorporated herein by reference).
The hypoxic culturing conditions of the present invention may also be used in transplantation protocols for cells of the central nervous system or cells of the peripheral nervous system.
Skin Cells
In another embodiment, the hypoxic culturing methods of the present invention may be used in the culturing of keratinocytes (progenitor cells for skin). Methods of isolating, culturing, and differentiating keratinocytes are well known to those of skill in the art (Jones et al, 1995; Di Cunto et al., 1998). Although keratin 19 is widely considered to be the best available overall marker for keratinocytes (Michel et al., 1996), in preferred embodiments the progenitor cells are distinguished by morphologic criteria because a reliable antibody is not available. Hypoxic/physiologic oxygen conditions can be used from the time of isolation of both fetal and post-natal keratinocytes, in order to facilitate isolation, expansion, and differentiation of these cells.
The skin cells produced by the methods of the present invention are used to produce skin progenitors or tissue for the purpose of clinical transplantation. Transplantation may be used to treat skin injuries such as delayed wound healing, thermal or chemical burns, or severe allergic reactions, or after massive resection of skin for malignancy.
Embryonic Stem Cells
Embryonic stem cells have been used widely in the generation of experimental gene knockout mice. The isolation of embryonic stem cells from human tissue has also been reported (Thomson, 1998). However, as disclosed herein, oxygen concentration has a significant effect on proliferation and differentiation of developmentally primitive cells. Therefore, variation of ambient oxygen in the culture conditions for embryonic stem cells may be an important parameter in the development of specific tissue from these lines for therapeutic purposes. Methods of varying oxygen concentration and assaying for the effect of physiologic/subatmospheric oxygen culture conditions are disclosed herein.

Methods for isolation and activation of, and control of differentiation from, stem and progenitor cells


The present invention is directed to a gelatin matrix which contains a pharmaceutically active ingredient. In a preferred embodiment, a hydrocolloid is added to the gelatin matrix to improve the physical properties, taste and mouthfeel of the gelatin matrix. A method for making the gelatin matrix containing the hydrocolloid is also disclosed.

The present invention is directed at a soft, chewable pharmaceutical dosage form. More particularly, the present invention is directed to an oral delivery system for administering a pharmaceutical agent, medicament or other active ingredient by employing a soft, chewable matrix to incorporate the active ingredient.
Pharmaceutical and nutritional supplement dosage forms intended for oral administration are typically provided in solid form as tablets, capsules, pills, lozenges, or granules. The tablet form is swallowed whole, chewed in the mouth, or dissolved sublingually. Absorption of the active moiety depends upon its release from the dosage form and may be controlled by several different technologies.
Chewable systems are often employed in the administration of pharmaceuticals, where it is impractical to provide a tablet for swallowing whole. The act of chewing helps to break up the tablet particles as the tablet disintegrates and may increase the rate of absorption by the digestive tract. Chewable systems are also advantageous where it is desirable to make an active ingredient available topically in the mouth or throat for both local effects or systemic absorption. Chewable dosage forms are also utilized to improve drug administration in pediatric and geriatric patients.
Palatability and xe2x80x9cmouthfeelxe2x80x9d are important characteristics to be considered in providing a dosage form, or matrix, for an active pharmaceutical medicament. Unfortunately, many pharmaceuticals and other active ingredients have a bitter or otherwise unpalatable taste, or an unacceptable mouthfeel, due to the grittiness or chalkiness of the compound, or both. These characteristics make it difficult to prepare acceptable dosage forms using the current state of the art for chewable dosage forms, since the objectionable taste and/or mouthfeel make it less likely to obtain compliance by the user.
As a result, several approaches have been tried in attempting to overcome these problems. The poor taste of a pharmaceutical or other active ingredient may be masked by using suitable flavoring compounds, and/or sweeteners. Encapsulation of the active ingredient may also serve to mask bitterness and other undesirable tastes. These approaches do not effect the physical state of the dosage from currently employed in the art. For example, chewable vitamin tablets are typically prepared as a compressed tablet, incorporating one or more active ingredients (e.g., vitamins), a sweetener and flavoring agent to mask the taste of the active ingredient, and a binder, typically microcrystalline cellulose.
Generally, chewable tablets are made by direct compression of a mixture of tabulating compounds including the active ingredient, flavoring, binders, etc. The mixture is fed into a die cavity of a tablet press and a tablet is formed by applying pressure. Hardness of the resulting tablet is a direct function of the compression pressure employed. A softer tablet, having an easier bite-through, may be prepared by adding a disintegrant, such as alginic acid, to the tablet mix. Alternatively, a softer tablet may be formed by employing reduced compression pressures. In either case, the resulting tablet is softer, fragile, brittle, and easily chipped.
Attempts have been made to reduce the grittiness and/or chalkiness of the compressed tablet by coating particles of the active ingredient with oils or fats prior to incorporation into the delivery system. See U.S. Pat. Nos. 4,327,076 and 4,609,543, incorporated herein by reference. In this way, the grittiness or chalkiness of the particles in the mouth is reduced. After swallowing, the oil or fat is digested and the drug particle can dissolve in the gastric contents. However, the addition of particles coated with fats or oils to the tablet mix can decrease the binding of the tableting ingredients and cause a reduction in the tablet hardness.
Other techniques for providing a chewable delivery system involve the use of a gum base. Gum bases are insoluble elastomers which form the essential element for chewing gum. The gum base is typically blended with one or more sweeteners to obtain a confectionery gum. A coating containing the active ingredient is then applied over the confectionery gum. As the dosage form is chewed, the coating fractures and/or dissolves in the mouth and is swallowed. Despite these disclosures there is an ongoing need for a chewable delivery system, particularly for children, which is pleasant tasting and preferably in a form which is easy to chew and swallow.
The present invention provides a chewable, gelatin based matrix which contains pharmaceutically active ingredients. More particularly, the present invention provides gelatin based matrices that can be chewed and swallowed easily. In preferred embodiments of the invention, hydrocolloid ingredients are added to the gelatin matrix in order to enhance the physical properties of the gelatin matrix and increase the user acceptance. The addition of a hydrocolloid to the gelatin typically reduces the rubbery nature of the gelatin, improves the mold release characteristics, decreases sticking to the teeth, improves the mouthfeel, and improves the ability to chew and swallow the product in the desired period of time.
The gelatin used in the present invention is selected from a wide variety of sources that are pharmaceutically acceptable types, including gelatin-glycerin, pure gelatin, or sugar gelatins. These and other suitable gelatins are disclosed in Remington""s Practice of Pharmacy, Martin and Cook, 17th edition, p. 1298. The level of gelatin used in the present invention typically comprises from about 1 to about 20 weight percent, preferably from about 3 to about 15 and most preferably from about 4 to about 10 weight percent on a dry solids basis. Typically, the gelatin is employed in combination with hydrocolloid such that the gelatin/hydrocolloid is up to about 40 weight percent, generally from about 2 to about 30 weight percent, and preferably from about 6 to about 20 weight percent on a dry solids basis. In a highly preferred embodiment the hydrocolloid is about 10 weight percent on a dry solids basis of the gel-forming matrix.
The level of gelatin employed in the invention is related to bloom strength. Those with skill in the art will recognize that bloom strength is a measure of the gelling property of a particular gelatin, with higher bloom strengths relating to the ability of the gelatin to more strongly crosslink at an equal concentration of gelatin. The bloom strengths of the gelatins employed in the present invention are from about 150 to about 350 with a preferred value of about 250. A bloom strength of about 250 provides the proper mouthfeel, texture and chewing characteristics to the gelatin matrix of the invention.
In preferred embodiments of the invention, hydrocolloids are added to the gelatin matrix in order to improve the properties of the gelatin matrix. As used herein, hydrocolloid is understood to mean any water soluble material that swells when contacted with water. Suitable hydrocolloids include natural and modified gums, cellulosics, modified cellulosics, pectins, mucillages, modified starches, noncellulosic polysaccharides, algal polysaccharides and mixtures thereof. More specifically the hydrocolloids include starch, agar-agar, microcrystalline cellulose, methylcellulose, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), xanthan gum, carrageenan gum, locust bean gum, alginates, acacia, carboxymethylcellulose (CMC), karaya gum, acacia gum, sodium alginate, sodium CMC, guar gum, tragacanth, mixtures of the hydrocolloids and the like.
When using various hydrocolloid ingredients, various ratios of gelatin/hydrocolloids have been discovered which have been found to possess beneficial mouthfeel, texture and chewing characteristics. For example, when using starch, the weight ratio of from about 1:1.3 to about 1:1.8 possesses desirable characteristics. More preferably the gelatin/starch weight ratio is from 1:1.4 to about 1:1.55 and most preferably the weight ratio is about 1:1.50. When agaragar is used the weight ratio is about 1:0.35 to about 1: 1.5, preferably from 1:0.6 to about 1:1.2, and most preferably about 1:0.75. When employing hydroxypropylcellulose the ratio is from about 1:0.2 to about 1:0.8, preferably the HPC/starch weight ratio is from about 1:0.3 to about 1:0.6 and most preferably the weight ratio is about 1:0.45. Those with skill in the art will recognize that other hydrocolloids can be used in combination with the specified hydrocolloids in the ratios specified, i.e., a mixture of two or more hydrocolloids, without departing from the present invention.
The present invention incorporates an effective amount of the pharmaceutically active ingredient in the gelatin matrix. Typically the pharmaceutically active ingredient is employed in the gelatin matrix at a level depending on the dosage strength desired and the particular active used. Suitable categories of pharmaceutically acceptable materials that may be employed in the present invention include any stable drug/excipient combination. Illustrative categories and specific examples include antitussives, such as dextromethorphan, dextromethorphan hydrobromide, noscapine, carbetapentane citrate, and chlophedianol hydrochloride; antihistamines, such as chlorpheniramine maleate, phenindamine tartrate, pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate; decongestants, such as phenylephrine hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine, hydrochloride ephedrine; alkaloids, such as codeine phosphate, codeine sulfate and morphine; mineral supplements such as potassium chloride and calcium carbonates, magnesium oxide, and other alkali metal and alkali earth metal salts; laxatives, antacids; ion exchange resins such as cholestyramine; anti-cholesterolemic and anti-lipid agents such as gemfibrozil; H2 antagonists such as famotidine; antiarrhythmics such as N-acetyl-procainamide; antipyretics and analgesics such as acetaminophen, aspirin, ibuprofen and naproxen; appetite suppressant such as phenylpropanolamine hydrochloride or caffeine; expectorants such as guaifenesin; antibiotics such as penicillin, cephalosporins and macrolide antibiotics; oral nitrates such as isosorbide dinitrate, isosobide mononitrate, and nitroglycerin; and vitamins, alone or in combination, as multi-vitamins, antioxidants, herbal medicaments such as melatonin, cholestin, goldenrod, garlics, St. John""s wort and other DSHEA (dietary supplement health and safety act) ingredients or compounds. Additional useful active medicaments include anti-inflammatory substances, coronary dilators, cerebral dilators, peripheral vasodilators, anti-infectives psychotropics, antimanics, stimulants, gastro-intestinal sedatives, anti-diarrheal preparations, anti-anginal drugs, vasodilators, anti-hypertensive drugs, vasoconstrictors and migraine treatments, antibiotics, tranquilizers, antipsychotics, antitumor drugs, anticoagulants and anti-thrombotic drugs, hypnotics, sedatives antiemetics, anti-nauseants, anticonvulsants, neuromuscular drugs, hyper and hypoallergenic agents, thyroid and antithyroid preparations, diuretics, antispasmodics, uterine relaxants, nutritional additives, antiobesity drugs, anabolic drugs, erythropoietic drugs, antiasthmatic, cough suppressants, mucolytics, anti-uricemic drugs, and the like. Mixtures of the drug and medicaments may also be used. The dry solids weight basis of the pharmaceutically active ingredients is typically from about 2 to about 30 weight percent, preferably from about 5 to about 15 weight percent and most preferably about 10 weight percent.
The pharmaceutically active ingredients may be in solid or liquid form. In a highly preferred embodiment the pharmaceutically active ingredient is preferably coated or encapsulated with materials appropriate to survive temperature and water for processing and stability. This allows the gelatin mixture to be chewed while simultaneously tastemasking the drug or medicament. Suitable taste-masking technologies are known in the art, and include cellulose acetate, cellulose acetate butyrate, polyvinylpyrrolidone, hydroxymethylcellulose, dimethylamino methacrylate, cellulose triacetate and 2-vinyl pyridine. Especially preferred are the cellulose esters and methyl methacrylate coatings set forth in U.S. Pat. No. 5,489,436, hereby incorporated by reference.
The chewable, solid matrices of the present invention have a texture and hardness such that the matrix is chewed and swallowed in less than about 20 seconds, preferably in less than about 15 seconds and most preferably less than about 10 seconds. While the matrices of the present invention are rapidly chewed and swallowed, the texture of the matrices are such that they do not appreciably dissolve in the mouth in the times recited above. More specifically, the matrices have a texture such that they must be chewed.
One advantage of the present invention is that the gelatin matrix has a texture and mouthfeel similar to candy, the matrix is soft and is easily chewed and swallowed, particularly by children. Testing of the dosage form with children has indicated that children prefer this form and it is expected that because children prefer the gelatin matrix they will more readily take the drugs at the prescribed intervals and will also complete the prescribed treatments.
Without wishing to be bound by any theory, it is believed that the reason why the gelatin matrix is preferred, particularly by children, is because of its pleasant taste properties and soft moist texture. Since the matrix is soft and readily chewed, the drug remains within gelatin fragments, sequestered in the gelatin matrix. Because the drug is sequestered in the gelatin matrix, the unpleasant drug taste in the mouth is reduced. It is believed that the addition of the hydrocolloids to the gelatin matrix decreases the chewiness of the gelatin matrix, resulting in enhanced texture, break-up in the mouth, softness and mouthfeel. The present invention provides a convenient dosage form, particularly for children.
If the gelatin matrix is too chewy, the drug will have sufficient time to dissolve because it remains in the mouth for a longer period of time, and the unpleasant taste associated with drugs will become apparent to the user. Also if the drug is provided in an encapsulated form, an excessive amount of chewing will increase the likelihood that the drug coating will be broken or breached. This breaking of the coating will also increase the likelihood that the taste of the drug will become apparent to the user.
The other major ingredients used in the present invention are water, sugar or sugar-substitutes. Since the present invention provides a candy-like product, a large percentage of the final product is made up of sweeteners such as sugar, corn syrup, maltodextrose, polydextrose, ISOMALT as well as mixtures thereof. Artificial sweeteners known in the art, including saccharin, aspartame and Acesulfame-K, may also be used. Preferably a combination of sugar and artificial sweeteners are used in the present invention. Sweetener levels can be as high as about 60 weight percent dry solids, but more typically is from about 30 to about 50 weight percent.
Water is used to hydrate both the gelatin and hydrocolloid, and makes up the remainder of the dry product weight. Water is present in the final product at levels of from about 10 to 30 weight percent, more typically, water is present at a level at about 20 to about 25 weight percent.
For dietary and health reasons it may be desirable to provide a sugarless formulation in which the sweeteners are be replaced with polyhydric alcohols and mixtures of these alcohols, including but not limited to, sorbitol, xylitol, erythritol, maltitol and the like.
The present invention is preferably prepared by the following method. Gelatin is added to water at approximately 100xc2x0 C. to hydrate the gelatin, then maintaining the hydrated gelatin at a temperature of about 60 to about 70xc2x0 C. If a hydrocolloid is employed, the hydrocolloid is hydrated, also preferably while being heated to above boiling temperature in a separate container along with the soluble solids such as sugar and corn syrup. After the water fraction of the hydrocolloid/sugar solids suspension is reduced to desired solids content and cooled down to approximately 90xc2x0 C., the gelatin solution is added to the hydrocolloid/sugar solids mixture. At a cooler temperature that assures flowability of the mixture without compromising the integrity of the flavorings, it is appropriate to add flavorings, artificial sweeteners, preservatives, colorings, food acids and the like. The pharmaceutically active materials are then added to the mixture. The contents of the mixture are then mixed to achieve a uniform concentration.
In order to form the desired final shape of the matrix, at least three different options are possible. First the mixture could be allowed to cool sufficiently such that the mixture could be extruded and cut into the desired length pieces. Alternatively, the mixture could be deposited in starch molds and then removed from the starch molds by removing the starch from the solidified mixture. In the a preferred method the warm mixture is poured into molds. The molds are preferably coated with a lubricant to aid in the release of the gelatin matrix from the mold. The addition of the hydrocolloid to the gelatin matrix also improves the mold release properties of the gelatin matrices.


In order to minimize the potential for damage to bio-compatible and/or therapeutic-containing stent coatings, there is provided a compliant elastic sheath over layer between a non-compliant stent expansion balloon and an unexpanded stent, which is secured over the balloon with a securing crimp. This stent deployment assembly, which is mounted on a catheter, is maneuvered through a patient's body to a desired deployment site and then inflated to expand the stent to the desired diameter. During expansion, the compliant elastic material between the stent and the non-compliant balloon prevents non-compliant balloon-induced damage to the stent's coating, as well as preventing degradation of the non-compliant balloon by, for example, stent-caused punctures. The deflated non-compliant stent expansion balloon and compliant elastic sheath are then withdrawn from the patient's body, leaving the deployed stent at the desired implantation site with its coating substantially intact.

The positioning and deployment of medical implants is a common often-repeated procedure of modern medicine. Medical implants may be used for innumerable medical purposes, including the reinforcement of recently re-enlarged lumens, the replacement of ruptured vessels, and the treatment of disease such as vascular disease by local pharmacotherapy, i.e., delivering therapeutic drug doses to target tissues while minimizing systemic side effects.
Such localized delivery of therapeutic agents has been proposed or achieved using medical devices such as catheters, needle devices and various coated implantable devices such as stents. These implants may be delivered by securing them to the distal end of a delivery device, positioning the distal end of the device near a target delivery site, and then deploying the implant from the device to its desired position. The implant may be deployed by inflating the distal end of the device or through other forces that urge the implant from the device's distal end. When the implant has been coated this coating is susceptible to being damaged or completely removed from the implant during the deployment process—an unwanted result.
The mechanical process of deploying the implant often exerts significant shearing and adhesion forces on and against the surface of the coating of the implant. These forces can strip, damage or otherwise deplete the amount of coating on the implant. When the amount of coating is depleted the implant's effectiveness may be compromised and additional risks may be inured into the procedure. For example, when the coating of the implant includes a therapeutic, if some of the coating were removed during deployment, the therapeutic may no longer be able to be administered to the target site in a uniform and homogenous manner. Thus, some areas of the target site may receive high quantities of therapeutic while others may receive low quantities of therapeutic. Similarly, if the therapeutic is ripped from the implant it can reduce or slow down the blood flowing past it, thereby, increasing the threat of thrombosis or, if it becomes dislodged, the risk of embolisms.
The delivery of expandable stents is a specific example of a medical procedure that involves the deployment of coated implants. Expandable stents are tube-like medical devices, typically made from stainless steel, Tantalum, Platinum or Nitinol alloys, designed to support the inner walls of a lumen within the body of a patient. These stents are typically positioned within a lumen of the body and, then expanded to provide internal support for the lumen. They may be self-expanding or, alternatively, may require external forces to expand them, such as by an inflating a balloon within the stent's inner diameter. In either case they are typically deployed through the use of a catheter of some kind. These catheters will typically carry the stent at their distal end.
Because of the direct contact of the stent with the inner walls of the lumen, stents have been coated with various compounds and therapeutics to enhance their effectiveness. These coatings may, among other things, be designed to facilitate the acceptance of the stent into its applied surroundings. Such coatings may also be designed to facilitate the delivery of a therapeutic to the target site for treating, preventing, or otherwise affecting the course of a disease or tissue or organ dysfunction.
The term "therapeutic agent" as used herein includes one or more "therapeutic agents" or "drugs". The terms "therapeutic agents" and "drugs" are used interchangeably herein and include pharmaceutically active compounds, nucleic acids with and without carrier vectors such as lipids, compacting agents (such as histones), virus (such as adenovirus, andenoassociated virus, retrovirus, lentivirus and α-virus), polymers, hyaluronic acid, proteins, cells and the like, with or without targeting sequences.
Specific examples of therapeutic agents used in conjunction with the present invention include, for example, pharmaceutically active compounds, proteins, cells, oligonucleotides, ribozymes, anti-sense oligonucleotides, DNA compacting agents, gene/vector systems (i.e., any vehicle that allows for the uptake and expression of nucleic acids), nucleic acids (including, for example, recombinant nucleic acids; naked DNA, cDNA, RNA; genomic DNA, cDNA or RNA in a non-infectious vector or in a viral vector and which further may have attached peptide targeting sequences; antisense nucleic acid (RNA or DNA); and DNA chimeras which include gene sequences and encoding for ferry proteins such as membrane translocating sequences ("MTS") and herpes simplex virus-1 ("VP22")), and viral, liposomes and cationic and anionic polymers and neutral polymers that are selected from a number of types depending on the desired application. Non-limiting examples of virus vectors or vectors derived from viral sources include adenoviral vectors, herpes simplex vectors, papilloma vectors, adeno-associated vectors, retroviral vectors, and the like. Non-limiting examples of biologically active solutes include anti-thrombogenic agents such as heparin, heparin derivatives, urokinase, and PPACK (dextrophenylalanine proline arginine chloromethylketone); antioxidants such as probucol and retinoic acid; angiogenic and anti-angiogenic agents and factors; agents blocking smooth muscle cell proliferation such as rapamycin, angiopeptin, and monoclonal antibodies capable of blocking smooth muscle cell proliferation; anti-inflammatory agents such as dexamethasone, prednisolone, corticosterone, budesonide, estrogen, sulfasalazine, acetyl salicylic acid, and mesalamine; calcium entry blockers such as verapamil, diltiazem and nifedipine; antineoplastic/antiproliferative/anti-mitotic agents such as paclitaxel, 5-fluorouracil, methotrexate, doxorubicin, daunorubicin, cyclosporine, cisplatin, vinblastine, vincristine, epothilones, endostatin, angiostatin and thymidine kinase inhibitors; antimicrobials such as triclosan, cephalosporins, aminoglycosides, and nitorfurantoin; anesthetic agents such as lidocaine, bupivacaine, and ropivacaine; nitric oxide (NO) donors such as lisidomine, molsidomine, L-arginine, NO-protein adducts, NO-carbohydrate adducts, polymeric or oligomeric NO adducts; anticoagulants such as D-Phe-Pro-Arg chloromethyl ketone, an RGD peptide-containing compound, heparin, antithrombin compounds, platelet receptor antagonists, anti-thrombin antibodies, anti-platelet receptor antibodies, enoxaparin, hirudin, Warafin sodium, Dicumarol, aspirin, prostaglandin inhibitors, platelet inhibitors and tick antiplatelet factors; vascular cell growth promotors such as growth factors, growth factor receptor antagonists, transcriptional activators, and translational promotors; vascular cell growth inhibitors such as growth factor inhibitors, growth factor receptor antagonists, transcriptional repressors, translational repressors, replication inhibitors, inhibitory antibodies, antibodies directed against growth factors, bifunctional molecules consisting of a growth factor and a cytotoxin, bifunctional molecules consisting of an antibody and a cytotoxin; cholesterol-lowering agents; vasodilating agents; agents which interfere with endogeneus vascoactive mechanisms; survival genes which protect against cell death, such as anti-apoptotic Bcl-2 family factors and Akt kinase; and combinations thereof. Cells can be of human origin (autologous or allogenic) or from an animal source (xenogeneic), genetically engineered if desired to deliver proteins of interest at the insertion site. Any modifications are routinely made by one skilled in the art.
Polynucleotide sequences useful in practice of the invention include DNA or RNA sequences having a therapeutic effect after being taken up by a cell. Examples of therapeutic polynucleotides include anti-sense DNA and RNA; DNA coding for an anti-sense RNA; or DNA coding for tRNA or rRNA to replace defective or deficient endogenous molecules. The polynucleotides can also code for therapeutic proteins or polypeptides. A polypeptide is understood to be any translation product of a polynucleotide regardless of size, and whether glycosylated or not. Therapeutic proteins and polypeptides include as a primary example, those proteins or polypeptides that can compensate for defective or deficient species in an animal, or those that act through toxic effects to limit or remove harmful cells from the body. In addition, the polypeptides or proteins that can be injected, or whose DNA can be incorporated, include without limitation, angiogenic factors and other molecules competent to induce angiogenesis, including acidic and basic fibroblast growth factors, vascular endothelial growth factor, hif-1, epidermal growth factor, transforming growth factor α and β, platelet-derived endothelial growth factor, platelet-derived growth factor, tumor necrosis factor α, hepatocyte growth factor and insulin like growth factor; growth factors; cell cycle inhibitors including CDK inhibitors; anti-restenosis agents, including p15, p16, p18, p19, p21, p27, p53, p57, Rb, nFkB and E2F decoys, thymidine kinase ("TK") and combinations thereof and other agents useful for interfering with cell proliferation, including agents for treating malignancies; and combinations thereof. Still other useful factors, which can be provided as polypeptides or as DNA encoding these polypeptides, include monocyte chemoattractant protein ("MCP-1"), and the family of bone morphogenic proteins ("BMP's"). The known proteins include BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7 (OP1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, and BMP-16. Currently preferred BMP's are any of BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7. These dimeric proteins can be provided as homodimers, heterodimers, or combinations thereof, alone or together with other molecules. Alternatively or, in addition, molecules capable of inducing an upstream or downstream effect of a BMP can be provided. Such molecules include any of the "hedgehog" proteins, or the DNA's encoding them.
When a polymer and/or therapeutic coating is haphazardly applied or has somehow been removed during the stent's manufacture or delivery, the stent's effectiveness can be compromised. In certain circumstances faulty or ineffectively deployed stents can require the removal and reinsertion of the stent through a second medical procedure. For example, as the balloon at the distal end of the stent is inflated, to expand and position the stent, frictional shear forces are created between the surface of the catheter and the stent coating. These frictional surface shear forces, as well as the adhesion forces between the coating and the stent, act to tear away or unevenly redistribute the stent coating. Thus, the physical forces used to deliver the stent can create an abating result that reduces the overall effectiveness of a deployed coated stent.
During manufacture of assemblies of non-self-expanding stents and expansion balloons over the distal end of a catheter, the stent may be placed over the outer diameter of the unexpanded balloon and crimped onto the balloon. Frequently, the stent expansion balloon is composed of a non-compliant material, such as Polyimide, PET, HDPE or Pebax. Further, in order to minimize the profile of the stent assembly, it is common practice to form a number of wings in the wall of the non-compliant balloon and fold the wings down along the side of the balloon prior to crimping the stent over the balloon. The folding of wings in the non-compliant balloon minimizes the diameter of the uninflated balloon, and hence the diameter of the final crimped-on stent assembly. Moreover, when the stent expansion balloon is composed of a non-compliant material, the diameter of the expanded balloon, and therefore also the expanded stent, is more or less independent of the balloon inner pressure.
There are some disadvantages associated with the use of non-compliant balloons in stent assemblies, both during manufacture of the assembly and during inflation of the balloon and expansion of the stent. For example, during manufacture the hardness of the non-compliant balloon may increase the difficulty in securing the stent over the balloon, such that often high crimping forces must be applied in order to adequately secure the stent to the non-compliant balloon. These high securement forces increase the risk for stent-caused punctures. Likewise, during expansion of the stent during implantation, the wings of the non-compliant balloon move relative to the stent in a tangential direction. If the stent being implanted has a coating on its surface, the non-compliant balloon's sliding tangential motion may abrade or otherwise damage the stent coating.
Alternatively, a compliant balloon (i.e., a balloon composed of well-known elastic materials, such as Latex or silicone rubber) may be used for expanding a stent. Compliant balloons are softer than non-compliant balloons, and thus permit the stent to obtain a better "grip" on the balloon, which in turn permits the stent to be more easily secured to the stent using lower crimping forces. In addition, compliant balloons need not be formed and folded into a preferred pre-expansion shape like non-compliant balloons, as compliant balloons already have a minimal diameter in their unexpanded state. Compliant balloons also expand principally in the radial direction, hence there is no tangential sliding motion relative to the expanding stent, which in turn may reduce the chances of probability of stent coating damage during stent expansion.
Compliant balloons have their own disadvantages, however. For example, the expanded diameter of a compliant balloon depends directly on the applied pressure, requiring exacting control of inflation pressures to ensure a stent is properly expanded. In addition, compliant balloons sometimes exhibit what is sometimes referred to as a "dog-bone effect" during expansion, wherein the portions of the balloon outside the length of the stent expand more that the portion of the balloon within the length of the stent. Accordingly, use of compliant balloons to inflate non-self-expanding stents may not be preferred in some applications.

Non-compliant balloon with compliant top-layer to protect coated stents during expansion


An electrochromic device comprising a counter electrode layer comprised of lithium metal oxide which provides a high transmission in the fully intercalated state and which is capable of long-term stability, is disclosed. Methods of making an electrochromic device comprising such a counter electrode are also disclosed.

Certain materials, referred to as electrochromic materials, are known to change their optical properties in response to the application of an electrical potential. This property has been taken advantage of to produce electrochromic devices which can be controlled to transmit optical energy selectively.
A number of factors affect the operation of an electrochromic device. One limitation on how dark an electrochromic device can become is how much charge can be stored in the counter electrode layer. In this context, the term, "charge," refers to the amount of electronic charge, or quantity of electrons per unit area, and the equivalent, corresponding quantity of charge balancing lithium ions per unit area, which may be conveniently expressed in units of milliCoulombs per square centimeter (mC/cm2). There have been many different approaches for producing a charge storage medium, but most attention has focused on a thin film deposited parallel to the electrochromic material layer, and separated by an ionically conductive layer.
To date, most counter electrode layers have been made using NiO, LiNiO, or doped variants thereof. One advantage of using NiO and LiNiO materials is that under careful preparation conditions, the counter electrode can be made so that it displays anodic electrochromism with good electrochromic efficiency and a high bleached state light transmission. Here, the term, "electrochromic efficiency" refers to the modulation of optical density per amount of charge transferred per unit area. Unfortunately, it has been difficult to intercalate lithium into NiO based materials as a result of the material's compact crystalline structure. As such, higher voltages must be applied to such materials to intercalate lithium, in order to drive the electrochromic response at a reasonably fast rate, which leads to undesirable side reactions.
Other methods employ protons, or hydrogen ions instead of lithium ions, as the charge balancing counter ion for the coloration mechanism. These methods may use counter electrode layers comprised of nickel hydroxides, or iridium oxides and other mixtures containing iridium. Typically an aqueous medium is also required to provide a suitable source of protons. Although it may be relatively easy to manufacture a counter electrode layer capable of coloring anodically in an aqueous medium, it is difficult to produce a complete device capable of long-term stability. It is, therefore, more advantageous to use lithium intercalation based systems.
A typical material used for counter electrode applications with lithium is vanadium oxide, which is a material that forms crystal structures similar to those seen in tungsten oxide systems. The open crystalline lattice of vanadium oxide allows lithium intercalation more readily than in NiO based structures. However, the presence of vanadium ions leads to the generation of a strong yellow color. This yellow color is only slightly modulated by lithium intercalation, and shows a reasonable cathodic electrochromic effect throughout the majority of the visible region, thus limiting the maximum transmission that can be achieved using this material as a counter electrode layer. Attempts to reduce the degree of coloration by doping vanadium oxides with other components result in a reduced electrochromic efficiency by reduction of the charge capacity of the counter electrode layer. Such doping results in a device with a higher bleached state transmission at the cost of decreased range of modulation of optical density.
There remains a need for improved electrochromic coatings, and in particular electrochromic coatings that comprise solid state, inorganic thin films, and metal oxide or metal oxide-containing thin films.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.

This patent is subject to a terminal disclaimer.

Electrochromic nickel oxide simultaneously doped with lithium and a metal dopant