RU2376997C2 - Application of carbon oxide for improvement of tissue and organ transplantation issue and apoptosis suppression - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention concerns methods of transplantation of organs, tissues and individual cells, as well as to methods of cell maintenance in vitro and increase of survival rate and/or post-transplantation functionality of donor body, tissue or cell. Specified methods involve introduction carbon oxide in effective amount sufficient to increase survival rate and/or functionality of cell.

EFFECT: there is presented method for application carbon oxide to maintain animal cell prior to transplantation and to treat or prevent rejection of the transplanted organ or tissue.

69 cl, 3 ex, 4 tbl, 12 dwg

Description

Declared as a study that received financial support from federal authorities

The present invention was carried out at the National Institute of Health with Government support, Grant No. HL586688. The specified Government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to the field of enhancing cell viability.

BACKGROUND OF THE INVENTION

Gas carbon monoxide (CO) in high concentrations has a destructive effect. However, it is currently known as an important signaling molecule (Verma et al., Science 259: 381-384, 1993). It is also believed that carbon monoxide acts in the brain as an intermediary molecule (ibid.) And as a neuroendocrine modulator in the hypothalamus (Pozzoli et al., Endocrinology 735: 2314-2317, 1994). Like nitric oxide (NO), carbon monoxide is a smooth muscle relaxant (Utz et al., Biochem. Pharmacol. 47: 195-201, 1991; Christodoulides et al., Circulation 97: 2306-9, 1995) and inhibits platelet aggregation ( Mansouri et al., Tromb. Haemost. 48: 286-8, 1982). Inhalation with a small amount of CO has an anti-inflammatory effect, as shown in some animal models.

Islet cell transplantation stimulates the effective treatment of type I diabetes (Lacy et al., Annu. Rev. Immunol. 2: 183-98; Weir et al., J. Am. Optom. Assoc. 69: 727-32, 2000; Berney et al., Langenbechs Arch. Surg. 385: 378-8, 2000; Shapiro et al., N. Engl. J. Med. 343: 230-8, 2000). But to implement methods of clinical transplantation of islets for a number of reasons is difficult. One such reason is the primary nonfunctionality (PNF) of this transplant. Another reason is that a large number of donor islets are required for the successful treatment of diabetes (Shapiro et al., N. Engl. J. Med. 343: 230-8, 2000). Both situations reflect the same pathophysiology: mainly the loss of cells in the transplant occurs in the first weeks after transplantation. After transplantation, the islets are exposed to various stress factors, such as hypoxia before secondary vascularization (Carlsson et al., Diabetes 47: 1027-32, 1998), and are exposed to pre-inflammatory cytokines, as well as free radicals released from macrophages into the microenvironment transplant (Rabinovitch et al., Diabetes 48: 1223-9, 1999; Kaufman et al., J. Exp. Med. 772: 291-302, 1990; Corbett et al., Proc. Natl. Acad. Sci. USA 90 : 1731-5, 1993) and from macrophages of resident islets (Mandrup-Poulsen et al., J. Immunol. 739: 4077-82, 1987; Arnush et al., J. Clin. Invest. 702: 516-26, 1998 ) The toxic effects of immunosuppressive drugs, and in addition rejection (Weir et al., Diabetes 46: 1247-56, 1997) also contribute to the loss of islet cells. The existence of PNF after experimental transplantation of syngenic islets (Nagata et al., Transplant Proc. 22: 855-6, 1990; Arita et al., Transplantation 65: 1429-33, 1998) suggests that non-specific inflammation plays a major role in this. scripts.

It is believed that the viability of the transplanted organ is mainly associated with successful immunosuppression in the form of blocking the immune response, which leads to transplant rejection. However, it has been previously shown that transplanted organs themselves can protect themselves from vascular damage leading to rejection as a result of the expression of “protective genes” (see, for example, Bach et al., Nature Med. 3: 196-202 (1997); and Soares et al., Nature Med. 4: 1073-1077, 1998). One such gene, the hemoxygenase-1 (HO-1) gene, catabolizes heme to biliverdin, free iron, and CO (Tenhunen et al., Proc. Natl. Acad. Sci. USA 61: 748-755, 1968).

SUMMARY OF THE BACKGROUND OF THE INVENTION

The present invention is based in part on the observation that CO promotes the survival and / or functionality of an organ, tissue, and individual cell transplants.

Accordingly, a first object of the present invention is to provide a method for administering to a transplant donor a pharmaceutical composition comprising carbon monoxide, obtaining an organ, tissue or cells from this donor, and transplanting said organ, tissue or cells to a recipient, wherein the amount of carbon monoxide administered to the donor is sufficient to enhance the viability or function of a given organ, tissue or cells after transplantation to a recipient.

Said pharmaceutical composition may be administered to a living donor, a donor whose brain has lost biological activity, or to a donor before or after the brain has lost biological activity.

Optionally, a donor organ may be treated in situ and / or ex vivo with a pharmaceutical composition comprising carbon monoxide.

The method may also include or alternatively include the step of administering to the recipient a second pharmaceutical composition that contains carbon monoxide, before and / or during and / or after the organ or tissue transplantation step to the recipient.

In this or any of the methods described herein, an organ or tissue can be any organ or tissue that can be transplanted, for example, a liver, kidney, heart, pancreas, lung, small intestine, and / or skin, and the donor used can the species is different from the recipient, or the donor and recipient may be of the same species. The donor and recipient may relate to either non-human animals or be human. Alternatively, the donor may refer to animals that do not belong to the human race, such as a pig, and the recipient may be a human.

In another aspect, the present invention provides a method for transplanting an organ, tissue or cell, which involves receiving an organ, tissue or cell from a donor, introducing ex vivo or in situ into an organ, tissue or cell a pharmaceutical composition that contains carbon monoxide, and transplanting the treated an organ, tissue, or cell to the recipient, the amount of carbon monoxide being sufficient to increase the viability or function of the organ, tissue, or cells in the recipient. In one embodiment of the present invention, the pharmaceutical composition is administered by perfusion of an organ or tissue in situ while the organ or tissue is held by a donor.

Optionally, the method may include the step of administering to the recipient a second pharmaceutical composition comprising carbon monoxide before and / or during and / or after transplantation of the organ or tissue to the recipient.

Another aspect of the present invention relates to the creation of a method for transplanting an organ, tissue or cells, which includes the steps of obtaining an organ, tissue or donor cells, transplanting the resulting organ, tissue or cells to a recipient, and before and / or during and / or after stage of transplantation of the specified organ, tissue or cells to the recipient, the introduction of the same recipient the amount of the pharmaceutical composition containing carbon monoxide sufficient to increase the survival and / or functionality of the transplanted organ, t cani or cells in the recipient.

In one of the embodiments of the present invention, the pharmaceutical composition can be administered to the recipient in the range from 0 to 20 days, for example, on 1, 2, 4, 6, 8, 10, 12, 14, 18, or the 20th day, after as indicated to the recipient, the indicated organ was transplanted. In another embodiment of the present invention, the pharmaceutical composition is administered to the recipient at least once, for example, several times or continuously, starting from the 21st day after the organ or tissue transplantation stage to the recipient until the transplant survives. The pharmaceutical composition can be administered to the recipient to determine whether the transplanted organ or tissue is rejected or the graft is close to rejection, for example, chronic rejection or acute rejection.

Optionally, the method according to the invention may further include the step of administering to the donor a second pharmaceutical composition comprising carbon monoxide before receiving the organ, tissue or cells of that donor. The second pharmaceutical composition can be administered to a living donor or a donor whose brain has lost biological activity.

The method according to the invention may include the step of introducing into the organ of the donor in situ and / or ex vivo a second pharmaceutical composition comprising carbon monoxide.

In another aspect, the present invention relates to a method for increasing the viability and / or functioning of a donor organ, tissue or cell, which includes receiving an organ, tissue or cell from a donor in a marginal state, and exposing the resulting organ, tissue or cell with an amount containing carbon monoxide of the pharmaceutical composition sufficient to increase the viability and / or functioning of the resulting donor organ, tissue or cell.

In another aspect, the present invention relates to a method for maintaining an animal cell in vitro, which comprises providing a vessel containing a pressurized gas that contains carbon monoxide gas, producing an isolated in vitro cell, wherein the cell is a primary cell or stem cell, releasing compressed gas from said vessel for forming an atmosphere that includes carbon monoxide gas, and maintaining said animal cell in vitro in the presence of an atmosphere that includes carbon monoxide gas ode.

If necessary, the cell can then be transplanted to the recipient. A cell can be obtained from a donor that is not a recipient, or it can be obtained from a specified recipient. In addition, the carbon monoxide composition can be administered to the recipient before and / or during and / or after the transplantation step. Such a composition is typically presented as respirable gas.

In another embodiment of the present invention, said animal cell is obtained from a donor by a method that includes administering to the donor a composition comprising carbon monoxide and obtaining a cell from the tissue of this donor.

The present invention also provides a method for maintaining an in vitro animal cell, which comprises creating a culture medium containing an effective amount of carbon monoxide, for example at least 0.0001 g CO / 100 g medium, and maintaining the isolated cell in the medium. This medium may contain, for example, at least 0.0002, 0.0004, 0.0006, 0.0008, 0.0010, 0.0013, 0.0014, 0.0015, 0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028, 0.0030, 0.0032, 0.0035, 0.0037, 0.0040, 0.0042, or 0 , 0044 g CO / 100 g medium.

In addition, the present invention provides a method for increasing the survival of an animal cell after removing it from a donor, which includes administering to a living donor or donor after the death of his brain, a pharmaceutical composition containing carbon monoxide, and obtaining a cell isolated from the donor. The pharmaceutical composition may be presented, for example, in the form of a sealed gas suitable for inhalation by a donor.

The method may further include the step of maintaining in vitro said cell in the presence of a second composition comprising carbon monoxide.

However, an in vitro cell can be placed in a liquid medium. In this case, the step of exposing this cell to a second composition containing carbon monoxide can be accomplished by providing a source of pressurized carbon monoxide gas and contacting the liquid medium with carbon monoxide gas released from this source. The liquid medium itself can also be obtained as a carbon monoxide composition, i.e. dissolved in it carbon monoxide.

Further, the present invention provides a method for transplanting an animal cell, which includes the steps of administering to a living donor or donor after brain death, a pharmaceutical composition containing carbon monoxide, obtaining a cell isolated from the donor and transplanting the cell to the recipient. An animal cell can be obtained from a donor that is not a recipient, or it can be obtained from a recipient. If necessary, the carbon monoxide composition may be administered to the recipient before and / or during and / or after the transplantation step.

The present invention also provides a method for increasing the survival or functionality of an animal cell transplanted to a recipient, which includes the stage of transplanting the animal cell to the recipient before, during and / or after the stage of transplantation, wherein the recipient must inhale a volume of carbon monoxide gas sufficient to increase in the recipient of the survival or functionality of the transplanted cell. Carbon monoxide gas may be supplied in a container containing a pressurized gas that contains carbon monoxide. In one embodiment of the invention, the isolated cell is maintained in vitro in a gas medium containing carbon monoxide prior to the transplantation step. For example, an isolated animal cell can be maintained in a liquid medium that includes at least 0.0001 g carbon monoxide / 100 g medium (for example, at least about 0.0002, 0.0004, 0.0006, 0, 0008, 0.0010, 0.0013, 0.0014, 0.0015, 0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028, 0.0030, 0.0032, 0.0035, 0.0037, 0.0040, 0.0042, or 0.0044 g of CO / 100 g of medium).

The method may optionally include, prior to the transplantation step, the step of exposing the isolated cell to an ex vivo carbon monoxide composition.

Carbon monoxide gas can be introduced to the recipient before and / or during and / or after the transplantation step. This animal cell can be obtained from a donor who is not a recipient, or it can be obtained from a recipient. A pharmaceutical composition comprising carbon monoxide may be administered to a donor before and / or during removal of said cell from the donor.

Another aspect of the present invention relates to a method for improving the survival of a transplanted cell in a recipient, comprising administering to the recipient before and / or during and / or after transplanting said cell an effective amount of a pharmaceutical composition comprising carbon monoxide gas.

In any of the above methods according to the invention, the survival effect can be enhanced by inducing in the donor or recipient the enzyme hemoxygenase-1 (HO-1), for example, induced by heme, heavy metals, cytokines, hormones, nitric oxide, endotoxins, UV radiation, depletion glutathione; or by heat shock. In a donor, such induction may occur before or during removal of an organ, tissue, or cell. In the recipient, such induction can occur before, during, or after transplantation. Alternatively, said enzyme can be induced in an organ, tissue, or ex vivo cells before being transplanted to a recipient.

The present invention also provides an industrial product that includes a container containing a pressurized gas that contains at least 0.001 ppm, for example at least about 1, 10, 50, 100, 150, 200, 250, 300, 500 , 1000, 2000, 5000, 10000, 100000, 200000, 300000, 400000, 500000 and up to 1,000,000 ppm of carbon monoxide, and a label that describes the use of gas to increase the viability of isolated animal cells, tissues or islets before, during or after transplantation of the specified cells, tissues or islets to the patient.

In addition, the present invention provides a sterile cell medium that contains nutrients suitable for maintaining an animal cell in culture, and at least about 0.0001 g carbon monoxide / 100 g medium, for example at least 0.0002, 0 , 0004, 0.0006, 0.0008, 0.0010, 0.0013, 0.0014, 0.0015, 0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024 , 0.0026, 0.0028, 0.0030, 0.0032, 0.0035, 0.0037, 0.0040, 0.0042, or 0.0044 g of CO / 100 g of medium. It may also contain animal cells.

A method has also been created for maintaining an animal cell in vitro and its subsequent transplantation. This method includes the steps of providing a container containing a pressurized gas containing carbon monoxide gas; obtaining a single animal cell in vitro, and the specified cell is placed in a medium that contains dissolved carbon monoxide; the release of the pressurized gas from the tank to form a gaseous atmosphere containing carbon monoxide gas; maintaining the animal cell in the presence of a gaseous medium and transplanting this cell to the recipient.

In any of the above aspects or embodiments of the present invention, the cell in question may be any cell. For example, the cell may be an animal cell, such as a primary, cell, secondary cell, or cell line. As another example, the cell in question may be part of an islet of the pancreas, for example, a β-cell. The cell in question may also be, for example, a liver cell, fibroblast, bone marrow cell, neural cell, myocyte, lymphocyte, or stem cell. In each of the ex vivo methods of the invention, the tissue is preferably not blood and contains a small amount, if any, of whole blood, and the cells are preferably not red blood cells and do not contain a significant amount of red blood cells.

Unless otherwise noted, all technical and scientific terms used herein have the same meaning as those generally accepted by those skilled in the art to which the present invention relates. Although methods and materials similar or equivalent to the methods and materials described herein can be practically used and tested in the present invention, the methods and materials used herein are described below. All publications, patent applications, patents, and other references mentioned herein are considered to be included in their entirety in the present description. In the event of a conflict, the present description, including definitions, will be justified. The presented materials, methods and examples serve only to illustrate the invention and do not imply its limitations.

Other characteristic features and advantages of the present invention are apparent from the following detailed description, as well as from the claims.

DESCRIPTION OF DRAWINGS

1A is a bar graph that illustrates the effect of treating βTC3 cells with increasing concentrations of TNF-α.

FIG. 1B is a graphical illustration of a FACScan ^™ analysis of DNA fragmentation in βTC3 cells after TNF-α treatment.

On Figs presents a histogram that illustrates the effect of cotransfection of βTC3 cells with β-gal expression vector (pcDNA3 / β-gal), and control (pcDNA3), as well as their processing or caspase-3 inhibitor Z-DEVD-FMK (C3- i) or a caspase-8 inhibitor of IETD-CHO (C8-i). The gray histograms correspond to untreated β-cells, and the black histograms correspond to β-cells treated for 24 hours with TNF-α. The results obtained are the average values for repeated wells ± standard deviation, corresponding to one of three representative experiments.

FIG. 2A is a bar graph showing that exogenous carbon monoxide can be replaced with HO-1 (hemoxygenase-1) when HO-1 activity is blocked. The gray histograms correspond to untreated β-cells, and the black histograms correspond to β-cells treated with TNF-α or etoposide, or subjected to serum deprivation. The results obtained are the average values for repeated wells ± standard deviation, corresponding to one of three representative experiments.

2B is a graphical representation of a FACScan ^™ analysis of DNA fragmentation in βTC3 cells after 24-hour treatment with carbon monoxide after treatment with TNF-α.

2C is a bar graph showing that exogenous carbon monoxide protects β cells from apoptosis in the absence of HO-1. βTC3 cells were transfected with β-gal expression vectors and exposed to exogenous carbon monoxide. The gray histograms correspond to untreated β-cells, and the black histograms correspond to β-cells treated with TNF-α or etoposide, or subjected, as indicated, to serum deprivation. The results obtained are the average values obtained in repeated wells ± standard deviation, corresponding to one of three representative experiments.

Figure 3 presents the results of the analysis of DNA fragmentation using FACScan ^™ , which indicate that exogenous carbon monoxide protects the islets of Langerhans in mice from apoptosis. CHX = cycloheximide.

Fig. 4A is a bar graph showing that the antiapoptotic effect of exogenous carbon monoxide is mediated by activation of guanylate cyclase. ODQ = guanylyl cyclase ODQ inhibitor.

4B is a bar graph showing that the cGMP analogue can be replaced with carbon monoxide to protect cells from apoptosis. 8-Br-cGMP = 8-Br-cGMP analogue.

4C is a bar graph explaining that cGMP-dependent protein kinase (cGK) mediates the anti-apoptotic effect of carbon monoxide. ΒTC3 cells were cotransfected with β-gal expression vector. In Figures 4A-C, gray histograms correspond to untreated β-cells, black histograms correspond to β-cells treated with TNF-α. The results obtained are the average values for repeated wells ± standard deviation, corresponding to one of three representative experiments. KT = KT5823, protein kinase G inhibitor.

Fig. 5A is a bar graph showing that exposure for one hour to carbon monoxide is sufficient to prevent apoptosis.

5B is a bar graph showing that carbon monoxide protects β cells after induction of apoptosis.

Fig. 5C is a bar graph showing that preincubation with carbon monoxide prevents apoptosis of β cells. In Figures 5A-C, gray histograms correspond to untreated β-cells, and black histograms correspond to β-cells treated with TNF-α. The results obtained are the average values for repeated wells ± standard deviation, corresponding to one of three representative experiments.

6 is a line graph showing that exposure of murine carbon monoxide islands enhances their survival and functionality after transplantation.

Fig. 6B is a line graph that shows the likelihood of recovery (blood glucose below 200 mg / dl) of animals receiving pre-exposed carbon monoxide islets or control islets. * P = 0.001 against control.

7 is a histogram that illustrates the expression of HO-1 in mouse hearts transplanted into rats treated with CVF and CsA. Mouse hearts were transplanted into rats treated with cobra venom factor (CVF) during transplantation and daily treated with cyclosporin A (CsA) after transplantation. The expression of HO-1 mRNA and β-actin was determined using RT-PCR. The - / - symbol indicates HO-1 RNA of mouse hearts used as a negative control. Histograms correspond to the relative level of HO-1 mRNA expression with respect to β-actin mRNA expression.

On Fig presents a group of histograms indicating that SnPPIX inhibits in vivo enzymatic activity of HO-1. Mouse hearts were transplanted into untreated rats (II) or rats treated with CVF and CsA (III) plus FePPIX (IV) or SnPPIX (V). The total HO activity was measured in the cardiac tissue of the donor and recipient 2 days after transplantation and compared with the initial HO activity in the cardiac tissue, respectively, of a normal mouse and normal rat (I). The results obtained are the average of three animals per treatment ± standard deviation (SD). Statistical analysis was performed using the unpaired Welsh t-test.

Figure 9 presents a series of curves indicating that SnPPIX and FePPIX do not interfere with the formation of antibodies to transplants (AT). Mouse hearts were transplanted into rats treated with CVF and CsA as described above. Grade IgM antiserum levels were assessed using a cell ELISA. The binding of the C3 component of complement to murine endothelial cells was evaluated using a cell ELISA. Hemolytic complement activity (CH50) was evaluated using a standard hemolysis assay. The results obtained are mean ± SD (n = 3).

10A is a series of histograms showing that exogenous CO does not affect the ability of SnPPIX to suppress HO-1 enzymatic activity. Mouse hearts were transplanted into rats treated with CVF plus CsA plus SnPPIX (II) or SNPPIX and CO (III). Total HO activity was measured in the cardiac tissue of donors and recipients, as well as in the liver of recipients 2 days after transplantation. The HO activity of different samples was compared with the initial HO activity of normal mouse hearts (I), rat hearts (I) or liver (I), in accordance with the analyzed sample. The results obtained are the average of three animals at each treatment ± SD. Statistical analysis was performed using the Welsh t-test.

10B is a bar graph showing that exogenous CO does not affect the ability of SnPPIX to suppress HO-1 activity. In animals used to obtain the data of Fig. 10A, the carboxyhemoglobin content was determined 2 days after transplantation. The results obtained are mean values ± SD (n = 3).

11 is a line graph showing that increased HO-1 in endothelial cells inhibits platelet activation. Endothelial cells of 2F-2B mice were left untreated (NT) or treated with CoPPIX (50 μM, 16 h) to increase HO-1 activity, SnPPIX - to suppress HO-1 activity (50 μM, 16 h) or CoPPIX (50 μM, 12 h) plus SnPPIX (50 μM, 4 h) - to control the specificity of CoPPIX in increasing HO-1 activity. Rat platelets were isolated, superimposed on mouse endothelial cells for 5 min, and aggregation was tested after stimulation with 2 μM adenosine diphosphate (ADP).

On Fig presents a histogram indicating that carbon monoxide causes suppression of apoptosis of endothelial cells. Gray histograms correspond to cells treated with Act.D alone, and black histograms correspond to cells treated with Act.D plus TNF-α. Where indicated, endothelial cells were treated with SnPPIX (50 μM) and exposed to exogenous CO (10,000 ppm (ppm)).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As used herein, the term “carbon monoxide” (or “CO”) refers to molecular carbon monoxide in its gaseous state, compressed to a liquid form, or dissolved in an aqueous solution. As used throughout the specification, the terms “carbon monoxide composition” and “pharmaceutical composition containing carbon monoxide” refer to a gaseous, liquid, solid or semi-liquid composition containing carbon monoxide that can be administered to a patient donor, corpse or animal; to any body; or into a part of an organ, for example, in an organ tissue or in a single cell (s) constituting that organ, for example, neurons, hepatocytes, myocytes, islets, or an islet cell, such as a pancreatic β-cell. It will be apparent to practitioners which form of pharmaceutical composition, for example, a gaseous, liquid, or both a gaseous and a liquid form, is preferred for a particular application.

As used herein, the terms “effective amount” and “processing effective” refer to the amount or concentration of carbon monoxide used over a period of time (including acute or chronic administration, as well as periodic or continuous administration) that is effective when administered to produce the intended effect or physiological result. Effective amounts of carbon monoxide for use in the present invention include, for example, amounts that are effective for enhancing survival and / or improving organ or cell function in vivo and / or in vitro. In the case of transplantation of individual cells or a collection of cells, for example, a donor and / or recipient transplant, the effective amount of carbon monoxide is the amount introduced into the transplant of the donor and / or recipient, sufficient to increase the viability of the given cell or set of cells, for example, to reduce losses cells, or a combination of cells, and / or to improve the performance of the transplanted cell or combination of cells. In the case of processing cells outside the body, for example, cultured and / or islet cells used for transplantation, the effective amount of carbon monoxide corresponds to the amount in which these cells are incubated or stored in order to increase the preservation of cells and / or reduce cell loss, for example, loss during apoptosis, and / or to enhance function. In case of organ or tissue transplantation, for example, donor and / or recipient transplants, the effective amount of carbon monoxide corresponds to the amount which, when a donor and / or recipient is introduced into the transplant, is sufficient to increase the viability of the organ, tissue or cells of interest, for example, to reduce loss of cells that make up an organ or tissue, and / or improving the functional properties of any organ. In the case of treating organs, tissues or cells ex vivo for storage or use for transplantation, an effective amount of carbon monoxide is sufficient to increase the viability and / or functionality of the organ or tissues. As used herein, the term "inhibition" includes delaying the onset, reducing, preventing, or attenuating a biological process, such as apoptosis. In the case of gases, an effective amount of carbon monoxide is generally comprised in the range of from about 0.0000001 mass% to 0.3 mass%, for example, from about 0.0001 mass% to 0.25 mass%, preferably at least about 0.001%, for example, at least 0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0, 10%, 0.15%, 0.20%, 0.22%, or 0.24% of the mass. carbon monoxide. Preferred ranges of carbon monoxide are from about 0.001% to 0.24%, from about 0.005% to 0.22%, from about 0.01% to 0.20%, and from about 0.01% to 0.1% by weight . Other preferred ranges are from about 0.005% to 0.24%, from about 0.01% to 0.22%, from about 0.15% to 0.20%, and from about 0.025% to 0.1% by weight. In the case of liquid solutions of CO, effective amounts are usually from about 0.0001 to 0.0044 g CO / 100 g of liquid, for example, at least 0.0002, 0.0004, 0.0006, 0.0008, 0.0010, 0.0013, 0.0014, 0.0015, 0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028, 0.0030, 0, 0032, 0.0035, 0.0037, 0.0040, or 0.0042 g of CO / 100 g of an aqueous solution. Preferred ranges are, for example, from about 0.0010 to 0.0030 g of CO / 100 g of liquid, from about 0.0015 to 0.0026 g of CO / 100 g of liquid, or from about 0.0018 to 0.0024 g of CO / 100 g of liquid. Practitioners should take into account that, depending on the application, quantities beyond these ranges may be used.

As used herein, the term “patient” refers to an animal that is, or is not, a human being that is treated in accordance with the methods of the invention. The present invention implies the use of this term also in veterinary medicine. This term includes, but is not limited to, birds, reptiles, amphibians and mammals, for example, humans, other primates, pigs, rodents, such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats. As used herein, the term "donor" or "donor patient" refers to an animal (human or non-human) from which an organ, tissue or individual cells are obtained for storage and / or transplantation to a recipient patient. As used herein, the term “recipient” or “recipient patient” refers to an animal (human or non-human) into which an organ, tissue, a collection of cells or individual cells can be transferred.

The term "diabetes" is a general term for diabetes-related disorders recognized in the art, for example, diabetes. Diabetes mellitus is characterized by an inability to regulate blood glucose levels. The two most prevalent types of diabetes are known as type I diabetes and type II diabetes. In the case of type I, or insulin-dependent diabetes mellitus (IDDM), the pancreas produces little or no insulin because of the destruction of the insulin-producing beta cells. In the case of type II diabetes, or non-insulin-dependent diabetes (NIDDM), the pancreas produces some insulin, but this insulin is ineffective. The term also includes countless secondary disorders caused by diabetes, both acute and chronic, such as diabetes-related complications, such as hypoglycemia and hyperglycemia, retinopathy, angiopathy, neuropathy and nephropathy.

As used herein, the term "cell (s)" or "animal (s) cell (s)" refers to any type of animal cell, including cells suitable for transplantation. These cells, as a rule, are cells obtained from an animal donor, but can also be derived from them (secondary) cells or even cells of an established cell line. They are optionally transfected ex vivo with an expression vector that in some way alters their function. Said cells include, but are not limited to, islet cells, for example, cells that make up part of the pancreatic islet, as well as liver cells, fibroblasts, bone marrow cells, myocytes, as well as stem cells, and cells (for example, neurons) of the central nervous systems, including the spinal cord. As used herein, the term "islet cell (s)" as a general term refers to groups of cells in the pancreas known as islets of Langerhans. Langerhans islands contain several types of cells, which include, for example, β-cells (which produce insulin), α-cells (which produce glucagon), γ-cells (which produce somatostatin), F-cells (which produce pancreatic polypeptide) , intestinal argentaffinocytes (which produce serotonin), PP cells and D1 cells. The term "stem cell" is a generally accepted term that refers to cells with the ability to unlimited division in culture and giving rise to specialized cells. This term includes, for example, totipotent, pluripotent, multipotent and unipotent stem cells, for example, neuron, liver, muscle and hematopoietic stem cells.

By "isolated cell" is meant a cell that is removed from a particular tissue or organ in which it (or its predecessor) is found in nature. Any cell can only be partially cleared of its natural environment, considering it "isolated". For example, an intact islet of Langerhans is considered to be a component of "isolated" cells after the islet is removed from the pancreas and it is physically separated from other islets. Cells of any intact organ, such as a kidney or heart, or an incomplete organ, such as a fragment of a blood vessel, are not considered "isolated cells" because they are still part of this organ.

The term "organ (s)" is used herein as a general term to describe any anatomical site or member that has a specific function in an animal. Additionally included in this meaning of this term are essential parts of organs, for example, cohesive tissues derived from any organ. Such organs include, but are not limited to, the kidneys, liver, heart, intestines, for example, the large or small intestines, pancreas, and lungs. Additionally included in this definition are bones and blood vessels, for example, aortic transplants.

As used herein, the term “transplantation” is used as a generic term to refer to an implantation procedure for an organ, tissue, cell population or individual cells. The term "transplantation" is defined in this area as the transfer of living tissue or cells from a donor to a recipient, in order to preserve the functional integrity of the transplanted tissue or cells in the recipient (see, for example, The Merck Manual, Berkow, Fletcher, and Beers, Eds., Merck Research Laboratories, Rahway, NJ, 1992). In the present description, the term "cell transplantation" is used as a general term to refer to the procedure for moving at least one cell, for example, islet (s) cell (s) to a patient. For example, such transplantation can be accomplished by removing β-cells (or intact islets) from the donor pancreas and replanting them to a recipient whose pancreas cannot produce enough insulin. This term includes all categories of transplants known in the art, with the exception of blood transfusion. Transplants are divided into categories, depending on the location and genetic relationship between the donor and the recipient. This term includes, for example, autotransplantation (removal and transfer of cells or tissue from one particular place in a patient to the same or another place in the same patient), allograft (transplantation between representatives of the same species) and xenotransplantation (transplantation between representatives of different species).

The terms “organ rejection”, “transplant rejection” or “rejection” are commonly used in the art and are used throughout the present description as a general term for the rejection of an organ, tissue, or cell in a recipient. This definition includes, for example, three main types of rejection that are commonly identified in clinical practice: extremely acute rejection, acute rejection and chronic rejection (see, for example, Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994).

The terms “marginal donor (s)” and “marginal organ” are used here as general terms to describe a problematic donor or organ, which makes their use for transplantation operations not optimal. For example, the concept of a marginal donor may include a donor who is over 50 years old or who is affected by a chronic disease that may affect transplant function, such as diabetes, HTA, and alcohol consumption. A marginal organ is, for example, (1) an organ from such a donor or (2) an organ that has been exposed to prolonged exposure to heat or ischemia as a result of repeated cooling, or (3) an organ with anatomical abnormalities (e.g., small and numerous vessels, e.g. in the kidney), which complicates the anastomosis of the vessels, or with obvious atherosclerotic plaques in the vessels of the graft.

Obtaining gaseous compositions

The carbon monoxide composition may be a gaseous carbon monoxide composition. Compressed or pressurized gas suitable for the methods of the present invention can be obtained from any commercial supplier and in any type of container suitable for storing compressed gas. For example, compressed or pressurized gases can be obtained from any supplier of compressed gases for medical use, such as oxygen. A pressurized gas, including carbon monoxide, used in the methods of the invention can be obtained provided that all such gases of the desired final composition (e.g., CO and O ₂ , and optionally N ₂ , He, and / or CO ₂ ) are mixed together in the same tank. If necessary, the methods according to the invention can be carried out using several containers containing individual gases. For example, you can use a single container that contains carbon monoxide with or without other gases and the contents of which are optionally mixed with room air or with the contents of other containers, for example, containers containing oxygen, nitrogen, carbon dioxide, compressed air, or any other suitable gas or mixtures thereof.

Gaseous compositions administered to a patient in accordance with the present invention typically contain 0-79 wt.% Nitrogen, from about 21 to 100 wt.% Oxygen and from about 0.0000001 to 0.3 wt.% (Which corresponds to about 0.001 ppm (i.e. 1 ppb) to 3000 ppm) carbon monoxide. Preferably, the nitrogen content in the gaseous composition is about 79 wt.%, The oxygen content is about 21 wt.%, And the carbon monoxide content is from about 0.0001 to 0.25 wt.%. The carbon monoxide content is preferably at least about 0.001 wt.%, For example at least about 0.005, 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0, 08, 0.10, 0.15, 0.20, 0.22 or 0.24 wt.%. Preferred ranges of carbon monoxide include from about 0.001 to 0.24, from about 0.005 to 0.22, from about 0.010 to 0.20, and from about 0.015 to 0.1% by weight. It should be noted that gaseous carbon monoxide compositions containing carbon monoxide in concentrations exceeding 0.3% (e.g. 1% or higher), depending on the application, can be used for short periods of time (e.g. for one or more breaths ) They are especially suitable during ex vivo use when there is no risk of carbon monoxide poisoning. In cases where this gas is used to form a gaseous medium for culturing cells in vitro, it may also contain carbon monoxide, helping to maintain the pH of the medium. This carbon monoxide may be present, for example, in an amount of 1-10, typically 5 wt.%.

A gaseous carbon monoxide composition can be used to create an atmosphere that contains carbon monoxide gas. You can create an atmosphere that includes appropriate levels of carbon monoxide, for example, by using a container containing a pressurized gas including carbon monoxide gas, and releasing this pressurized gas from the tank into a chamber or space to form an atmosphere that includes gas carbon monoxide inside a given chamber or space. Alternatively, gases can be released into the apparatus to achieve maximum concentration in the respirator or in the breathing tube so as to create an atmosphere containing carbon monoxide gas in the respirator or breathing tube and to provide the patient, who is the only person in the room, a substantial level of oxide carbon.

The level of carbon monoxide in the atmosphere or in the ventilation system can be measured or controlled using any method known in the art. Such methods include electrochemical detection, gas chromatography, radioisotope reading, infrared absorption, colorimetry, and electrochemical methods based on the use of selective membranes (see, for example, Sunderman et al., Clin. Chem. 28: 2026-2032, 1982; Ingi et al., Neuron 16: 835-842, 1996). Using, for example, gas chromatography and radioisotope readings, carbon monoxide can be detected in fractions per million. In addition, it is known that sub-ppm ranges of carbon monoxide can be measured in biological tissue using a gas sensor for the mid-infrared spectrum (see, for example, Morimoto et al., Am. J. Physiol. Heart. Circ Physiol 280: H482-H488, 2001). Carbon monoxide sensors and gas detection devices are widely available from many commercial sources.

Obtaining liquid compositions

The carbon monoxide composition may also be presented as a liquid carbon monoxide composition. The liquid can be incorporated into the carbon monoxide composition by any method known in the art for causing gases to dissolve in liquids. For example, a liquid can be placed in a so-called “CO ₂ incubator” and exposed to a continuous stream of carbon monoxide until the desired concentration of carbon monoxide is reached in the liquid. As another example, carbon monoxide can be bubbled directly into the liquid until the desired carbon monoxide concentration is reached in the liquid. The amount of carbon monoxide that can be dissolved in an aqueous solution increases with decreasing temperature. As another example, an appropriate liquid can be passed through tubes that allow gas to diffuse when these tubes pass through a gas medium containing carbon monoxide (for example, using a device such as an oxygenator with an artificial membrane). Carbon monoxide diffuses into a liquid to create a liquid carbon monoxide composition.

The fluid may be any fluid known to those skilled in the art that is suitable for administration to patients (see, for example, Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994) or for preserving organs, tissues or ex cells vivo. In general, the liquid should be an aqueous solution. Examples of solutions include phosphate buffered saline (PBS), Celsior ^™ , Perfadex ^™ , Collins, citrate and Wisconsin University (UW) (Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University) Press, 1994). Liquid compositions may include carbon monoxide in concentrations ranging from about 0.0001 to 0.0044 g CO / 100 g liquid, at least 0.0002, 0.0004, 0.0006, 0.0008, 0.0010 , 0.0013, 0.0014, 0.0015, 0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028, 0.0030, 0 , 0032, 0.0035, 0.0037, 0.0040, or 0.0042 g CO / 100 g medium. Preferred ranges include, for example, from about 0.0010 to 0.0030 g CO / 100 g of liquid, from about 0.0015 to 0.0026 g of CO / 100 g of liquid, or from about 0.0018 to 0.0024 g CO / 100 g liquid. The saturation point of water at 0 ° C is about 0.0044 g of CO / 100 g of medium.

Any suitable liquid can be saturated with carbon monoxide to a specific concentration using a diffusion apparatus. Alternatively, you can use pre-prepared solutions, the quality of which is controlled by the content of a certain level of carbon monoxide. Exact dose control can be accomplished by measuring with a gas permeable membrane impervious to liquid connected to a carbon monoxide analyzer. Solutions can be saturated to the required effective concentrations and maintained at these levels. Incorporation of helium inert gas into liquid and gaseous compositions can improve the delivery of carbon monoxide to certain tissues of any organ.

Treatment of Patients with Carbon Monoxide Compositions

The present invention contemplates the use of carbon monoxide compositions for treating donors, recipients, organs, tissues, groups of cells, and / or individual cells at any stage of the preparation, storage and transplantation process. An organ, tissue, cell population, or individual cells can be obtained from a donor treated with an ex vivo carbon monoxide composition in accordance with the present invention and transplanted to a recipient thereof. Alternatively or in addition, an organ, tissue, a collection of cells, or individual cells can be processed in situ while they are still in the donor. Optionally, the carbon monoxide composition can be administered to the recipient before, during and / or after surgery: for example, after the blood supply to the blood of the recipient is restored in the organ. The carbon monoxide composition may also be administered to the donor prior to or during an operation to produce an organ, tissue, cell population, or individual cells.

Organs, tissues, groups of cells, and / or isolated cells can be obtained from a donor and transplanted using any method known to specialists in this field (see, for example, Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press ( 1994)). It will be apparent to skilled practitioners that the methods of preparation and transplantation may vary, depending on many circumstances, such as the type of organ, tissue or cell and the characteristics of the donor.

In addition, the present invention implies that the methods described herein can be used in relation to organs, tissue, groups of cells or isolated cells ex vivo, for example, artificial organs such as an artificial liver, kidney or pancreas (see, for example, Sambanis et al ., Cytotechnology 15: 351-363, 1994). Organs, tissues, or cells (or groups of cells) can be treated with carbon monoxide either before they are placed in the specified apparatus, or during their stay in this apparatus, or in both cases. Alternatively or in addition, carbon monoxide may be administered to the donor animal before removal of an organ, tissue, cell population or individual cells for use in this apparatus.

Alternatively or in addition, the cell can be cultured as described below and transplanted to the recipient.

The patient can be treated with the carbon monoxide composition using any method known in the art for administering gases and / or liquids to patients. The present invention contemplates the systemic administration to patients of liquid or gaseous carbon monoxide compositions (e.g., by inhalation and / or oral administration), and topical administration of the compositions to patient organs or tissues in situ (e.g., by oral administration, insufflation and / or administration in abdominal cavity).

Carbon Monoxide Delivery

Gaseous carbon monoxide compositions can be delivered to a patient systemically, for example, to a patient undergoing transplantation or in need thereof. Gaseous carbon monoxide compositions are usually administered by inhalation through the oral or nasal passages into the lungs, where the carbon monoxide administered is readily absorbed by the patient’s blood stream. The concentration of the active compound (CO) used in the therapeutic gaseous composition depends on the rate of absorption, distribution, inactivation and excretion (mainly through respiration) of carbon monoxide, as well as other factors known to those skilled in the art. In addition, it should be borne in mind that for any individual patient, a specific administration schedule can be adjusted over time in accordance with individual need and at the discretion of the introducing or supervising specialist, and that the concentration range set forth here is only approximate, and it is not intended to limit its volume or the effects of the claimed invention. The present invention contemplates acute, subacute or continuous administration of carbon monoxide, depending, for example, on the severity or duration of a dysfunction in a patient. Carbon monoxide can be delivered to the patient over a period of time (including indefinite), sufficient to treat the condition and the implementation of a pharmacological or biological effect.

The following are examples of some methods and devices that can be used to administer gaseous carbon monoxide compositions to patients.

Breathing apparatus

Carbon monoxide (concentration may vary) can be purchased mixed with air or other oxygen-containing gas in a standard compressed gas tank (e.g. 21% O ₂ , 79% N ₂ ). It is inert, and the concentrations that are needed to implement the methods according to the invention are much lower than the ignition region (10% in air). Under stationary conditions, the gas should supposedly be delivered by bedside, where it should be mixed with oxygen or with room air in the mixer to the desired concentration. The patient should inhale the gas mixture using a breathing apparatus, which should regulate the gas flow rate based on the comfort and needs of the patient. This is determined using pulmonary schedules (i.e., respiratory rate, tidal volume, etc.). In the delivery system, it is possible to create devices that protect the patient from unwanted receipt exceeding the required level of carbon monoxide. The patient’s carbon monoxide level can be monitored by analyzing (1) carboxyhemoglobin (COHb), which can be measured in venous blood, and (2) exhaled carbon monoxide from an opening on the left side of the breathing apparatus. The effects of carbon monoxide can be controlled based on the patient’s health and based on the meter reading. If necessary, carbon monoxide can be "washed" out of the patient by switching to 100% O ₂ inhalation. Carbon monoxide is not absorbed; therefore, everything that is inhaled must be exhaled at the same time, with the exception of a small fraction of a percent, which turns into CO ₂ . Carbon monoxide can also be mixed with any amount of O ₂ to provide therapeutic delivery of carbon monoxide without subsequent hypoxic conditions.

Mask and tent

The carbon monoxide gas mixture is prepared as described above, which allows the patient to be inhaled using a mask or tent. The respirable concentration may vary and may be “washed out” by simply switching to 100% inhalation of O ₂ . The operation to control the level of carbon monoxide is supposed to be carried out on the mask or near it, or in the tent using a safe mechanism that could prevent the inhalation of an excessively high concentration of carbon monoxide.

Portable inhaler

Compressed carbon monoxide can be provided with a portable device for inhaling and inhaling a controlled dose, for example, in order to be able to periodically treat a patient who is not in a stationary condition. Containers can be filled with different concentrations of carbon monoxide. The device can be presented in the form of a simple, small reservoir (for example, less than 5 kg) with respectively diluted CO and a two-position valve and a tube from which the patient draws in a stream of CO in accordance with the standard scheme or as necessary.

Intravenous artificial lung

An artificial lung (probe device for gas exchange in the blood), designed to deliver O ₂ and remove CO ₂ , can be used to deliver carbon monoxide. The implantable catheter remains in one of the large veins and must be able to deliver carbon monoxide at a given concentration for systemic delivery, or to a local site. The delivery may be local delivery of high concentration carbon monoxide over a short period of time to a specific site (this high concentration should be rapidly diluted in the bloodstream), or it may represent a relatively longer systemic effect of a reduced concentration of carbon monoxide (see, for example, Hattler et al., Artif. Organs 18 (11): 806-812, 1994; and Golob. et al, ASAIO J. 47 (5): 432-437, 2001).

Pressure chamber for creating normal pressure (Normábaric chamber)

In some cases, it would be desirable to apply carbon monoxide to the entire patient. The patient must be inside a sealed chamber, which must be filled with carbon monoxide (the level of which should not endanger the patient or whose level is an acceptable degree of risk that is safe for the exposed outsider). At the end of the exposure, the chamber should be cleaned with air (for example, 21% O ₂ , 79% N ₂ ), and the samples should be analyzed in a carbon monoxide analyzer to ensure that no carbon monoxide remains before allowing the patient to leave this exposure system.

Aqueous solutions

The present invention also considers that for systemic delivery to a patient, for example, for oral delivery and / or by injection into his body, for example, intravenous, intraarterial, intraperitoneal and / or subcutaneous, aqueous solutions containing carbon monoxide can be created.

The storage buffers and the culture medium can be saturated to a predetermined concentration with carbon monoxide using a gas diffusion apparatus or pre-prepared stock solutions that are subjected to quality control for a given level of carbon monoxide. Precise dosing control is carried out by measuring with a membrane permeable to gas and impermeable to liquid, connected to a carbon monoxide analyzer. Buffers and solutions can be saturated to the required effective concentrations and kept at that level. Regarding the procedures required to perfuse an organ, tissue, or cell preparation, pre-prepared saturated solutions can be on hand to maintain carbon monoxide levels. If the level of carbon monoxide is reduced, instead of solutions in which the concentration of carbon monoxide is reduced, fresh solutions can be added. After receiving the organ, tissue or cells, they can be stored in the specified solution for transportation in an airtight container. The presence of an inert helium gas makes carbon monoxide absorption more efficient.

Local treatment of organs, tissues and isolated cells with carbon monoxide in situ and ex vivo

The present invention provides methods for transplanting an organ (s), tissues, groups of cells and / or isolated cells. These methods may include the step of exposing the organ (s), tissues, group of cells and / or isolated cells in the presence of a carbon monoxide composition before transplantation. Such exposure may occur in situ and / or ex vivo. The organ (s), tissues and / or isolated cells can be exposed in an atmosphere containing carbon monoxide gas, in a liquid carbon monoxide composition, for example, in a liquid perfusion solution, in a storage solution or in a washing solution with dissolved carbon monoxide contained therein, or in both.

Exposure of an organ or tissue in the presence of liquid carbon monoxide compositions can be carried out ex vivo and / or in situ using any method known in the art. For example, exposure can be carried out ex vivo in any chamber or space with sufficient volume to immerse an organ or tissue, in whole or in part, in a carbon monoxide composition. As another example, you can expose this organ in the carbon monoxide composition by placing this organ in any suitable container, which allows the carbon monoxide composition to “wash” the organ placed so that the organ is continuously irrigated with the carbon monoxide composition.

Alternatively, the transplanted organ can be perfused with a carbon monoxide composition. The term "perfusion" is a recognized term and refers to the flow of a fluid, for example, a carbon monoxide composition, through the blood vessels of an organ or tissue. Ex vivo and in situ organ perfusion methods are well known in the art. An organ can be perfused with an ex vivo carbon monoxide composition, for example, using a hypothermic continuous perfusion apparatus (see Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994). Optionally, for in situ and ex vivo perfusions, an organ, before perfusion with a carbon monoxide composition, can be perfused with a wash solution, for example, a carbon monoxide-free UW solution, to remove blood from a donor organ. Such an operation should be carried out in order to avoid competition for carbon monoxide donor hemoglobin. Alternatively, the wash solution may be a carbon monoxide composition. The appropriate liquid can be passed through a tube, which makes gas diffusion possible; this tube passes through an atmosphere containing carbon monoxide (for example, through a chamber such as an oxygenator with an artificial membrane) to create a liquid carbon monoxide composition that can then transfer to an organ (for example, to perfuse an organ by attaching a tube to this organ).

The transplanted organ or tissue can be placed, for example, immersed in a medium or solution that does not contain carbon monoxide, and also placed in a chamber in which the medium or solution containing a gas medium with carbon monoxide is located. Alternatively or in addition, carbon monoxide may be "sparged" into a given medium or solution. In situ aging can be accomplished using any method known in the art, for example, washing or perfusing an organ using a liquid carbon monoxide composition (see Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994).

The present invention contemplates that for any given transplant operation, any or all of the above methods for exposing an organ or tissue to a liquid carbon monoxide composition, for example, rinsing, immersion, or perfusion, can be used.

The present invention also contemplates that a solid or semi-liquid carbon monoxide composition can be provided. For example, a liquid that is a carbon monoxide composition as described above can be formulated as a solid or semi-liquid composition into which an organ or tissue can be embedded or coated with it. Alternatively, the semi-liquid carbon monoxide composition may be poured into the transplanted organ. Solid or semi-liquid compositions can be created, for example, by adding a thickening agent, such as a gelling agent (e.g., collagen or alginate) to the liquid used.

Cell culture

The present invention provides a method of maintaining or culturing an animal cell in vivo. The method may include the steps of providing a container containing a pressurized gas, including nitric oxide gas, producing an animal cell in vitro, and releasing the pressurized gas from said container to form a gas medium that contains carbon monoxide gas. The animal cell is then cultured or simply kept in the presence of a gas medium containing carbon monoxide gas.

The method can be carried out in any chamber or space suitable for creating a gaseous medium, which includes an appropriate level of gas carbon monoxide. Such chambers or areas contain, for example, incubators, mixing cylinders, or any container suitable for culturing or containing cells, bottles whose contents are mixed during rotation, cell culture flasks, Petri dishes, and tubes. For example, you can use a CO ₂ incubator, in which carbon monoxide gas is supplied by a continuous stream from a vessel that contains this gas. As another example, you can use a bottle, the contents of which are mixed during its rotation and which includes carbon monoxide, to create a suitable gas medium inside the mixing bottle.

It is obvious to practitioners that cultivation conditions, such as temperature, can be selected and / or changed depending on the type of cultured cell (see, for example, Cells: A Laboratory Manual, Spector and Leinwand, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1997). For example, the mouse insulinoma βTC3 cell line (DSMZ, Braunschweig, Germany) can be incubated in humidified air containing 5% CO ₂ /95% air at 37 ° C.

The animal cell can be placed, for example, suspended or washed in a liquid medium. This medium can be any medium known to those skilled in the art suitable for culturing, preserving, or washing cells of interest (see, for example, Cells: A Laboratory Manual, Spector and Leinwand, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1997). This type of medium includes, but is not limited to, various buffers, the minimum essential Eagle medium (MEM), the minimum essential Eagle medium modified by Dulbecco / Vogt (DMEM), or the Roswell Park Memorial Institute (RPMI) medium. Such a medium may also include appropriate additives, for example, fetal bovine serum (FBS), individual amino acids, antibiotics and / or vitamins. For example, this medium may be RPMI 1640 medium (Life Technologies, Grand Island, New York) supplemented with 2 mM L-glutamine, 100 U / ml penicillin G, 100 U / ml streptomycin and 10% fetal calf serum (FCS) (Life Technologies). In those embodiments of the present invention in which the cultured cells are in a liquid medium, these cells can be maintained with a carbon monoxide composition by contacting the liquid medium with a sealed carbon monoxide gas, for example carbon monoxide gas discharged from a sealed gas source, in accordance with the methods of the present invention.

In another embodiment of the present invention, the liquid medium itself is a carbon monoxide composition formulated as described above. This medium can be infused with carbon monoxide before or after the addition of cells to the medium.

In addition, the present invention provides that it is possible to create a solid or semi-liquid medium in which the solid or semi-liquid medium is a carbon monoxide composition. For example, a liquid medium, which is a carbon monoxide composition, as described above, can be created as a solid or semi-liquid medium in which cells can be immersed or incorporated. Such an action can be carried out, for example, by adding a gelling agent, such as collagen, alginate or agar, to the medium.

Use of hemokisgenase-1, compounds associated with hemoxygenase-1, as well as other compounds, and treatment

The present invention also provides for the induction or expression of hemoxygenase-1 (HO-1) in conjunction with the introduction of carbon monoxide. HO-1 can be obtained for a patient by inducing or expressing HO-1 in a patient, or by administering exogenous HO-1 directly to a given patient. As used herein, the term “induce” means to cause increased formation of a protein, for example, HO-1, in isolated cells or in cells of a tissue, organ or animal using its own cellular endogenous (eg, non-recombinant) gene that encodes the protein.

HO-1 can be induced in a patient, for example, a donor and / or recipient, using any method known in the art. For example, HO-1 production can be induced using hemin, using iron-containing protoporphyrin, or using cobalt-containing protoporphyrin. A variety of non-heme agents are also potent inducers of HO-1 expression, including heavy metals, cytokines, hormones, nitric oxide, COCl ₂ , endotoxin, and heat shock (Otterbein et al., Am. J. Physiol. Lung Cell Mol. Physiol. 279: L1029-L1037, 2000; Choi et al., Am. J. Respir. Cell Mol. Biol. 15: 9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37: 517-554, 1997; and Tenhunen et al., J. Lab. Clin. Med. 75: 410-421, 1970). HO-1 is also strongly induced by a variety of agents and conditions that cause oxidative stress, including hydrogen peroxide, glutathione-depleting agents, UV radiation and hyperoxia (Choi et al., Am. J. Respir. Cell Mol. Biol. 15: 9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37: 517-554, 1997; and Keyse et al., Proc. Natl. Acad. Sci USA 86: 99-103, 1989). “Pharmaceutical composition comprising an HO-1 inducer” means a pharmaceutical composition comprising any agent capable of inducing HO-1 in a patient, for example, any of the above agents, for example, hemin, iron-containing protoporphyrin, and / or cobalt-containing protoporphyrin.

The expression of HO-1 can be increased by gene transfer. As used herein, the term “express (rudimentary)” means to induce, in isolated cells or in cells of a tissue, organ or animal, by an exogenously introduced gene (eg, a recombinant gene), increased protein formation, eg, HO-1 or ferritin. It is preferable that a representative of the same species (e.g., human, mouse, rat, etc.) act as a recipient of the HO-1 transplant or ferritin, so as to minimize any immune response. Expression can be controlled using a constitutive promoter (e.g., cytomegalovirus promoters) or a tissue-specific promoter (e.g., a milk serum promoter in the case of breast cells or an albumin promoter in the case of liver cells). An appropriate gene therapy vector (eg, retrovirus, adenovirus, adeno-associated virus (AAV), poxvirus (eg, vaccinia virus), human immunodeficiency virus (HIV), dwarf virus (“minute virus”) in mice, hepatitis B virus, influenza virus, herpes simplex virus-1 and lentivirus) encoding HO-1 or ferritin should be administered to the patient orally, by inhalation, or by injection at the appropriate transplant site to treat transplant rejection. Particularly preferred is topical administration directly to the transplanted organ, tissue or donor cells, or to the transplantation site at the recipient. Similarly, plasmid vectors encoding HO-1 or apoferritin, for example, in the form of bare DNA, can be introduced as part of liposomes or microparticles.

In addition, exogenous HO-1 protein can be administered directly to the patient using any method known in the art. Exogenous HO-1 can be directly administered, in addition, or alternatively, to induce patient expression of HO-1, as described above. The HO-1 protein can be administered to a patient, for example, as a liposome, and / or as a fusion protein, for example, as a TAT fusion protein (see, for example, Becker-Hapak et al., Methods 24: 247-256 , 2001).

Alternatively or in addition, any of the products of HO-1 metabolism, for example, bilirubin, biliverdin, iron, and / or ferritin, may be administered to the patient, together with or instead of carbon monoxide, to prevent or treat the disorder. In addition, the present invention contemplates that iron-binding molecules other than ferritin can be administered to a patient, for example, deferoxamine (DFO), dextran-iron, and / or apoferritin. Any of the above compounds may be administered to a patient topically and / or systemically.

The present invention also contemplates administration of nitric oxide (NO) to a patient in an organ (s), tissue (s) and / or isolated cells, together with administration of carbon monoxide, HO-1 and / or compounds associated with HO-1. This method involves supplying an NO donor, recipient, or organ, tissue or cell ex vivo, along with the administration of HO-1 and / or any of the products of heme degradation or all products of heme degradation, for example, CO, biliverdin, bilirubin, iron and ferritin.

As used herein, the term “nitric oxide” or (“NO”) refers to the molecular form of nitric oxide in its gaseous state or dissolved in an aqueous solution. Gaseous compositions containing NO are usually administered by inhalation through the mouth or nasal passages into the lungs, where NO can act directly or is readily absorbed by the patient’s bloodstream. Compressed or pressurized gas, for example, NO (and / or CO, as described in more detail above), suitable for the methods according to the invention, can be obtained from any commercial source and in any type of container suitable for storage of compressed gas. If necessary, the methods according to the invention can be carried out using combined vessels containing individual gases. Alternatively, CO and NO can be combined in one vessel, diluted, if necessary, with an inert gas.

Inhalation NO is commercially available (e.g., INOmax ^™ , INO Therapeutics, Inc., Clinton, NJ). This gas can be purchased, as a rule, in the form of a mixture of 200-800 ppm NO in pure N ₂ gas. The supplied NO gas may be substantially 100% NO or diluted with N ₂ or any other inert gas (e.g. helium) to any desired concentration. It is vital that a certain NO is obtained and stored as a mixture free of any impurity of O ₂ or higher nitrogen oxides, because such higher nitrogen oxides (which can be obtained in the reaction of O ₂ with NO) are potentially harmful to lung tissue . If necessary, the degree of purification of NO, before its introduction to the patient, can be shown in chemiluminescent analysis using known methods. Chemiluminescent NO-NO _x analyzers are commercially available (e.g. Model 14A, Thermo Environmental Instruments, Franklin, MA). A mixture of NO-N ₂ can be mixed with a gas containing O ₂ (for example, 100% O ₂ or air), immediately before inhalation, using, for example, a flow meter, the calibration of which was confirmed earlier using a spirometer. The final concentration of NO in the respirable mixture can be checked using a chemical or chemiluminescent method, well known to specialists in this field (for example, Fontijin et al., Anal. Chem. 42: 575, 1970). Alternatively, the concentrations of NO and NO ₂ can be controlled using an electrochemical analyzer. Any impurities, such as NO ₂ , can be purified as a result of their exposure to solutions of NaOH, Baralim or soda lime. As an additional control, you can also determine the FiO _{2 of the} final gas mixture.

Pharmaceutical compositions containing NO can be administered by any of the methods known in the art for administering gases to patients. Safe and effective methods for administering NO by inhalation are described, for example, in US Pat. No. 5,570,683; U.S. Patent No. 5,904,938; and Frostell et al., Circulation 83: 2038-2047, 1991. Some examples of methods for administering gases (such as CO) to patients are discussed in detail above and can be used to administer NO. Examples of methods and devices that can be used to administer gaseous pharmaceutical compositions, including NO, to patients include a ventilator, face masks and tents, portable inhalers, intravenous breathing simulators (see, for example, Hattker et al., Artif. Organs 18 (11): 806-812, 1994; and Golob et al., ASAIO J., 47 (5): 432-437, 2001), and pressure chambers for creating normal pressure. However, NO properties may allow / require some modifications to these methods. In stationary conditions or in urgent field situations, the introduction of NO gas can be accomplished, for example, by connecting a container with compressed NO gas in N ₂ to a second tank with oxygen or an oxygen / N ₂ mixture (such as air) so that the inhaler creates a mixed gas from two sources. The concentration of NO inhaled to the patient can be maintained at an optimal level by controlling the flow of gas from each source. NO can also be mixed with room air using a slow-mixing mixer (e.g. Bird Blender, Palm Springs, CA). NO can be created from N ₂ and O ₂ (i.e., air) using an electric NO generator. A suitable NO generator is described in US Pat. No. 5,396,882. In addition, NO can be generated periodically in an inhalation device using an NO source, such as compressed NO or an electric NO generator. The use of an inhaler is particularly advantageous if the second compound (for example, a phosphodiesterase inhibitor, which is described in detail below), is administered orally or by inhalation, together with NO.

Preferably, if in a pharmaceutical composition containing NO gas, the concentration of NO in air, in pure oxygen, or in another suitable gas or gas mixture at the time of inhalation is from about 0.1 ppm to 300 ppm, for example, 0.5-290 ppm, 1.0-280 ppm, 5-250 ppm, 10-200 ppm, or 10-100 ppm. A suitable starting dose for NO administered by inhalation may be 20 ppm (see, for example, the advertising insert in the INOmax ^TM product packaging), and this dose can be changed, for example, from 0.1 ppm to 100 ppm, depending on age and the condition of the patient, the particular disease or disorder that is being treated, and other factors that the attending physician may consider appropriate. The present invention provides for emergency, sub-emergency and regular administration of NO. NO can be delivered to the patient over a period of time (including indefinite), sufficient to treat the condition and achieve the intended pharmacological or biological effect. The concentration can be temporarily increased over a short period of time, for example, 5 minutes at 200 ppm NO. This can be done when it is necessary to achieve a direct effect. Preferred periods during which the patient is exposed to NO include at least one hour, for example at least six hours; at least one day; at least one week, two weeks, four weeks, six weeks, eight weeks, ten weeks or twelve weeks; at least one year; at least two years and at least five years. The patient can be exposed to a gaseous atmosphere continuously or intermittently during such periods. Administration of pharmaceutical compositions containing NO (and / or CO) can be accomplished by spontaneous or mechanical ventilation.

When inhaled NO is administered, it is desirable to control the effects caused by NO inhalation. Such monitoring in a particular individual can be used to verify the desired effects and to identify unwanted side effects that may occur. Such monitoring is also useful for adjusting the dose level, duration, and frequency of administration of respirable NO in a given individual.

Gaseous NO can be dissolved in an aqueous solution and used in this form. For example, such a solution should be used to wash an organ, tissue or cell ex vivo, or used to perfuse an organ or tissue in situ. The solution may contain other active agents, such as CO, HO-1, heme, biliverdin, and / or bilirubin.

It would be desirable to prolong the beneficial effect of respirable NO on a given patient. To determine how to prolong the beneficial effect of respirable NO, it is useful to bear in mind that one of the in vivo actions of NO is to activate soluble guanylate cyclase, which stimulates the formation of cGMP. At least several beneficial effects of NO can result from its stimulation of cGMP biosynthesis. Accordingly, a phosphodiesterase inhibitor can be administered together with inhaled NO to inhibit the degradation of cGMP by endogenous phosphodiesterase.

The phosphodiesterase inhibitor can be administered to the patient using any suitable method, including oral, through the mucous membrane, intravenous, intramuscular, subcutaneous or intraperitoneal. Alternatively, the patient may inhale the inhibitor. For inhalation, the phosphodiesterase inhibitor is created in the form of a dry powder or as an aerosol, or as a spray solution having a particle or drop size of less than 10 microns for optimal deposition in the alveoli and can optionally be introduced into a gas containing NO.

A suitable phosphodiesterase inhibitor is Zaprinast ^™ (M&B 22948; 2-o-propoxyphenyl-8-azapurin-6-one; Rhone-Poulenc Rorer, Dahenham Essex, UK). Zaprinast ^™ selectively inhibits cGMP hydrolysis with a minimal effect of the destruction of adenosine cyclomonophosphate in vascular smooth muscle cells (Trapani et al., J. Pharmacol. Exp. Ther. 258: 269, 1991; Harris et al., J. Pharmacol. Exp. Ther. 249 : 394, 1989; Lugnier et al., Biochem. Pharmacol. 35: 1743, 1986; Souness et al., Br. J. Pharmacol. 98: 725, 1989). When using Zaprinast ^™ , in accordance with the present invention, the preferred routes of administration are intravenous or oral. A suitable dosage range may be determined by one of ordinary skill in the art. Zaprinast ^™ stock solution can be prepared in 0.05 N NaOH. The stock solution can be diluted to the desired concentration of Zaprinast with Ringer's lactic acid solution immediately before use.

In practice, the present invention can be carried out with other phosphodiesterase inhibitors. A variety of phosphodiesterase inhibitors are known in the art, including Viagra® (sildenafil citrate) dipyridamole and theophylline. An appropriate route of administration and an appropriate dosage range can be determined by one of ordinary skill in the art.

The introduction of NO together with phosphodiesterase inhibitors can be carried out as follows. NO is injected at 20 ppm in air for 45 minutes at the beginning of a 45-minute period by intravenous infusion over 4 minutes. Zaprinast ^™ is administered at a rate of 1.0 mg per kg body weight, followed by a continuous infusion of 0.004 mg / kg / min over the next 45-minute period. Alternatively, at the beginning of the 45 minute period, dipyridamole is administered over a period of 4 minutes at a rate of 0.15 mg per kg body weight, followed by a continuous infusion of 0.004 mg / kg / min over the next 45 minute period. Zaprinast ^™ or dipyridamole is administered in saline.

In the case of transplantation, the present invention further provides that, along with the methods described herein, other methods can be used to increase the viability of the functionality of the transplant. Such methods include, but are not limited to, immunosuppressive therapy and donor-specific blood transfusion (DST). For example, DST may be administered to a recipient before, during and / or after administration of CO, HO-1, other hemassociated products and / or NO. Such administration, for example, administration of DST, along with the treatment described herein, can be carried out before, during and / or after transplantation.

Example 1. Carbon monoxide protects pancreatic beta cells from apoptosis and improves islet functionality / survival after transplantation

Cell culture

The insulinoma cell line βTC3 of mice (DSMZ, Braunschweig, Germany) was cultured in Dulbecco's modified Eagle medium (DMEM) (Life technologies, Grand Island, NY, USA) with the addition of 2 mM L-glutamine, 100 U / ml penicillin G, 100 U / ml of streptomycin and 10% fetal calf serum (FCS) (Life Technologies) and incubated in a humidified atmosphere of 5% CO ₂ /95% air at 37 ° C. Such a murine β-cell line was obtained from transgenic mice carrying the hybrid insulin promoter tumor antigen of the virus SV 40, which is known to support the signs of differentiated β-cells for approximately 50 passages in culture. These cells produce mature insulin from proinsulin I and II in a manner comparable to the β-cell in vivo method and are induced 30-fold using glucose (Efrat et al., Proc. Natl. Acad. Sci. USA 85: 9037-41, 1988). Compared to other commonly used transformed β-cell lines, such as RIN-m5F and HIT, the levels of insulin secreted by βTC3 cells are close to the level of insulin secretion by normal β-cells. Therefore, these cells are suitable for studying β-cell regulation and gene expression (D'Ambra et al., Endocrinology 726: 2815-22, 1990).

The pancreatic Langerhans islands of C57BL / 6 mice were obtained by isolating islets using equipment at the Joslin Diabetes Center.

Vital crystal violet staining

βTC3 is grown to a concentration of 2 x 10 ⁵ cells (Nunc, Marsh Products, Rochester, NY, USA). Cells are washed once with 500 μl of PBS and stained with 200 μl of 0.05% crystal violet in 20% ethanol for 10 min at RT. Rinse off the crystal violet. To elute the dye from the cells, 100 μl of 50% acetic acid are poured into each well. 50 μl was transferred to a 96-well microtiter plate and read using a microtiter plate reader (EL 340 biokinetic reader, Bio-Tek Instruments) absorbance at 562 nm.

Expressing plasmids

A β-galactosidase expression vector (Clontech Laboratories, Palo Alto, California) is cloned into a pcDNA3 vector (Sato et al., J. Immunol. 166: 4185-4194, 2001). (Invitrogen, Carlsbad, California). 1.0 kb The XhoI-HindIII fragment encoding the full length rat HO-1 cDNA was excised from the prHO-1 vector (Shibahara et al., J. Biochem. 113: 214-218, 1993) and subcloned into the pcDNA3 vector.

Transient Transfections

βTC3 is grown in 16 mm wells to a concentration of 3 x 10 ⁵ cells and transfected after 15-20 hours using lipofectamine plus ^TM reagents (Life Technologies), in accordance with the manufacturer's instructions. Total DNA is kept constant using an empty pcDNA3 vector. The percentage of viable cells is estimated by dividing the percentage of viable cells in each DNA preparation to the number of control transfected cells without apoptotic stimulus (100% survival) (Soares et al., Nature Med. 4: 1073-1077, 1998; Sato et al ., J. Immunol. 166: 4185-4194).

Flow cytometry

ΒTC3 cultures are incubated with recombinant TNF-α (500 or 1000 U / ml) (R&D Systems) for 24 hours, and islet cultures stimulate TNF-α (5000 U / ml) (R&D Systems) and cyclohexamide (CHX) (50 μg / ml) within 48 hours. βTC3 or islets are collected, dispersed, fixed in 70% ethanol and suspended in DNA staining buffer (PBS, pH 7.4, containing 0.1% Triton X-100, 0.1 mM EDTA, 50 μg / ml propidium iodide , 50 mg / ml RNase A). DNA content was analyzed on a FACScan ^™ analyzer equipped with Cell Quest ^™ software (Becton Dickinson, Palo Alto, CA). Cells with a normal DNA content (2n) are evaluated as viable, and cells with a hypoploid DNA content (<2n, designated A °) are evaluated as apoptotic. To exclude debris and non-cellular apoptotic fragments, all particles with a profile of the FL-2 region below that of chicken erythrocyte nuclei were excluded from the analysis.

Cell Processing and Reagents

Recombinant murine TNF-α (R&D Systems) was dissolved in PBS with 1% bovine serum albumin, and this solution was poured into the culture medium (17.5 ng / ml = 500 U) 24 hours after transfection. Caspase-3 inhibitor Z-DEVD-FMK and caspase-8 inhibitor IETD-CHO (Calbiochem, San Diego, California) were dissolved in dimethyl sulfoxide (DMSO, Sigma) and added to the culture medium (10 μM and 1 μM, respectively) after two hours after treatment with TNF-α. Tin-containing protoporphyrin (SnPPIX) (Pophyrin Products, Logan, Utah) was dissolved (10 mM) in 100 mM NaOH and added to the culture medium (50 μM) 6 hours after transfection. The guanylyl cyclase inhibitor 1H [1,2,4] oxadiazolo [4,3-α] quinoxaline-1 (ODQ; Calbiochem) was dissolved in DMSO and added to the culture medium (100 μM) 6 hours after transfection. 8-Bromguanosine-3'-5'-cyclomonophosphate (8-Br-cGMP) (Sigma), an analog of cGMP, was dissolved in water and added to the culture medium (10 μM) 30 minutes before the induction of apoptosis. KT5823 protein kinase G inhibitor (Calbiochem) was dissolved in DMSO and added to the culture medium (1.6 μM) 6 hours after transfection.

Exposure with CO

Cells and islets are exposed with 1% carbon monoxide in compressed air balanced with 5% CO ₂ as described elsewhere (see, for example, Otterbein et al., Nature med. 6: 422-428, 2000). The islands are incubated in RPMI medium pre-saturated with carbon monoxide (4 ° C overnight, 1% CO, 5% CO ₂ ) for two hours at 37 ° C, along with treatment with 1% CO, 5% CO ₂ .

Mice and the induction of diabetes

C57BL / 6 males are purchased from Charles River Laboratories (Wilmington. Massachusetts) and maintained as directed by the NIH. These experiments were approved by the Institutional Animal Care and Use Committee (IACUC). In recipient mice (8 weeks old), diabetes is reproduced by a single intraperitoneal injection (220 mg / kg) of Streptozotocin ^™ (Sigma) dissolved in citrate buffer. Patient mice are transplanted if 2 consecutive measurements of blood glucose levels are obtained in the whole blood sample, stably not exceeding a value of 350 mg / dl.

Isolation

Pancreatic islets of Langerhans (C57BL / 6 mice) presented by the Islet Core Laboratory of the JDRF Center for Islet Transplantation at Harvard Medical School are isolated as previously described (Gotoh et al., Transplantation 40: 437-438, 1985).

Transplantation of syngene marginal islets

250 islands with a diameter of 150-250 microns are obtained manually using a microtome microscope. The islands are transplanted into a kidney capsule as previously described (Kaufman et al., J. Exp. Med. 172: 291-302, 1990). The same number of control and treated animals are transplanted by each of the islet preparations.

Graft Functional Analysis Results

Graft function is defined as the point at which, on the first of three consecutive days, the fasting blood glucose level stably is <200 mg / dl. The primary endpoint of the experiment is defined as the time to reach normoglycemia.

Statistical analysis

Blood glucose data are summarized as mean ± standard deviation for mice receiving untreated or treated islets. The recovery time of islet function is calculated using Kaplan-Meier life expectancy tables, and differences between groups are determined using the logarithmic rank criterion for the three islet preparations processed in this analysis as separate series and the average recovery time, with a 95% confidence interval.

TNF-α induces apoptosis in βTC3 cells

The effect of TNF-α on βTC3 cells was investigated. The following operations are used to obtain the data illustrated in FIGS. 1A-C. Figa: βTC3 cells are treated with increasing concentrations of TNF-α. Viable cells are stained 24 hours after activation by crystal violet. Extinction is measured at 562 nm and normalized to untreated cells. Figv: βTC3 cells are treated with TNF-α, stained with propidium iodide after 24 hours and analyzed for DNA fragmentation (FACScan ^™ ). 1C: βTC3 cells are cotransfected with β-gal expressing vectors (pcDNA3 / β-gal) plus control (pcDNA3). Where indicated, cells are treated with a caspase-3 inhibitor Z-DEVD-FMK (C3-i) or a caspase inhibitor -8 IETD-CHO (C8-i). The gray histograms correspond to untreated β-cells, and the black histograms correspond to β-cells treated with TNF-α for 24 hours. The results are presented as the average for duplicated wells corresponding to one of three representative experiments ± standard deviation.

TNF-α induces a high dose-dependent level of cell death in the βTC3 insulin cell line (Stephens et al., Endocrinology 140: 3219-3227, 1999) (Fig. 1A). DNA fragmentation, demonstrated by staining (Fig. 1B) with propidium iodide (PI), suggests that TNF-α induces β-cell death as a result of apoptosis. TNF-α-mediated apoptosis directly depends on caspase-8 activation and partially depends on caspase-3, which is illustrated by the blocking of caspase-8 by a specific caspase-8 inhibitor (IETD-CHO) that prevents apoptosis (96% inhibition), then how blocking caspase-3 by a specific caspase-3 inhibitor (Z-DEVD-FMK) prevents apoptosis only partially (53% inhibition) (Fig. 1C).

Carbon Monoxide Protects βTc3 Cells

Investigate whether carbon monoxide can protect against β-cell apoptosis (Figure 2A-C). The following operations are used to obtain the data illustrated in FIGS. 2A-C. Figa: Exogenous CO can replace HO-1 in the event that the activity of HO-1 is blocked. ΒTC3 cells are cotransfected with a vector expressing β-gal plus control or an HO-1 expressing vector (Brouard et al., J. Exp. Med. 192: 1015-1026, 2000). If indicated, the enzymatic activity of HO-1 is inhibited by tin-containing protoporphyrin (SnPP). If indicated, β cells are exposed with exogenous carbon monoxide (1%) as previously described (Otterbein et al., Nature Med. 6: 422-428, 2000). Gray histograms correspond to untreated β-cells, and black histograms correspond to β-cells treated with TNF-α. The results obtained are the average of the duplicated wells corresponding to one of the three representative experiments, ± standard deviation. Figv: in the analysis of DNA fragmentation, exogenous carbon monoxide protects β cells from apoptosis. ΒTC3 cells are treated with TNF-α. Immediately after stimulation, βTC3 cells are exposed to exogenous carbon monoxide for 24 hours. Control βTC3 cells are treated in the same manner, but not exposed to carbon monoxide. After 24 hours, the cells are stained with propidium iodide and analyzed for DNA fragmentation in FACScan ^™ . 2C: exogenous carbon monoxide in the absence of HO-1 protects β cells from apoptosis. ΒTC3 cells are transfected with a β-gal expression vector and exposed to exogenous carbon monoxide (Stephens et al., Endocrinology 740: 3219-27, 1999). The gray histograms correspond to untreated β-cells, and the black histograms correspond to β-cells treated as indicated by TNF-α or etoposide or subjected to serum deprivation. The results obtained are the average of repeated wells corresponding to one of three representative experiments, ± standard deviation.

To evaluate whether HO-1 expression will really protect β cells from apoptosis mediated by TNF-α, βTC3 cells are transiently transfected with an HO-1 expression vector and tested for their ability to survive when exposed to TNF-α. Overexpression of HO-1 protects βTC3 from TNF-α-mediated apoptosis (Pileggi et al., Diabetes 50: 1983-1991, 2001) (87% survival versus 33% in control) (Fig. 2A). If the activity of HO-1 is blocked by tin-containing protoporphyrin IX (SnPPIX) (Kappas et al., Hepatology 4: 336-341, 1994), the anti-apoptotic effect is suppressed (Fig. 2A), which suggests that education using HO-1 by at least one of the end products of heme catabolism, i.e. iron, bilirubin and / or CO, is necessary for its anti-apoptotic function.

Assuming that the anti-apoptotic effect of HO-1 could be mediated by carbon monoxide, it was tested whether exposure to exogenous carbon monoxide would replace HO-1 in protecting β cells from apoptosis. When the action of HO-1 is suppressed by SnPPIX, exposure to carbon monoxide suppresses TNF-α-mediated apoptosis to a level similar to that of HO-1 (Fig. 2A). Exposure with exogenous carbon monoxide alone is protective (11.7% of apoptotic cells versus 20.3; in the control without exposure to CO), as shown by DNA fragmentation analysis (Figure 2B). Similarly, β-cell apoptosis induced by etoposide or as a result of depletion of serum is inhibited by exposure to carbon monoxide (FIG. 2C).

Induction in donors and recipients of HO-1 leads to prolonged survival of islet transplant

Find out if HO-1 induction in donors and recipients protects islet graft cells. To obtain the data presented below in Table 1, the following steps are taken. In experiments, a mouse model is used. Islet cell donors are exposed to cobalt-containing protoporphyrin (CoPP) (20 mg / kg) once a day before islet cell isolation. Islet cell transplant recipients are exposed to CoPP (20 mg / kg) once a day on days 1, 3, 5, 7, or CoPP (10 mg / kg) once a day on days 1, 3, 5, 7, 9, 11, 13, 15 and 17. Administration of CoPP induces the expression of hemoxygenase-1 (HO-1).

Table 1 Induction of HO-1 in donors and recipients leads to prolonged survival of islet graft Treatment The number of islets Rejection day Average ± SD Rejection / Total CoPP 20 mg / kg x 5 350-400 17, 33, 33, 48,> 58x2,> 67 x 1 44.85 ± 17.81 4/7 CoPP 10 mg / kg x 10 350-400 30, 30,> 51 x 2 40.5 ± 12.12 2/4 The control 350-400 8,8,15,15,16,22,26 15.71 ± 6.65 7/7

The listing in the "day of rejection" column corresponds to the days until which the islets survive. For example, "> 51x2" means that the islands in 2 recipients still remain viable after 51 days. The rejection period is on average presented in the fourth column. These data indicate that the induction of HO-1 leads to longer islet survival after transplantation.

Exogenous Carbon Monoxide Protects Mouse Islet Cells from Apoptosis

They also find out whether exogenous carbon monoxide protects mouse islet cells from apoptosis (Figure 3). To obtain the data presented in figure 3, use the following methods. Apoptosis is induced in freshly isolated mouse islets (C57BL / 6) by stimulation with TNF-α and cycloheximide (CHX). Immediately after stimulation, the islands are exposed for 24 hours with exogenous carbon monoxide. Control islands are treated in the same manner, but without exposure to carbon monoxide. After 48 hours, cells were analyzed in FACScan ^™ for DNA fragmentation. This experiment was carried out twice with indistinguishable results.

As determined by DNA fragmentation analysis (Figure 3), exposure to carbon monoxide for 24 hours protects the isolated mouse (C57BL / 6) islets of Langerhans from apoptosis mediated by TNF-α plus cycloheximide (CHX) (11.7% apoptotic cells against 20.3% in the control, which do not exhibit with CO).

The antiapoptotic effect of exogenous carbon monoxide is mediated by guanylate cyclase activation and signals from cGMP-dependent protein kinases (cGK)

Determine if carbon monoxide exerts an antiapoptotic effect by activating soluble guanylate cyclase (sGC) and the formation of cGMP (Fig. 4A-C). To obtain the data presented in Fig.4A-C, carry out the following steps. Figa: Anti-apoptotic effect of exogenous carbon monoxide is mediated by the activation of guanylate cyclase. ΒTC3 cells are transfected with vectors expressing β-gal and exposed with exogenous carbon monoxide (1%). Where indicated, βTC3 cells are treated with a guanylyl cyclase ODQ inhibitor. 4B: The cGMP analogue is replaced with carbon monoxide to protect against apoptosis. ΒTC3 cells are transfected with vectors expressing β-gal. Where indicated, βTC3 cells are exposed with exogenous carbon monoxide. Where indicated, βTC3 cells are treated with the cGMP analog of 8-Br-cGMP, but are not exposed to carbon monoxide. 4C: cGMP-dependent protein kinases (cGKs) mediate the antiapoptotic effect of carbon monoxide. ΒTC3 cells are co-transfected with a vector expressing β-gal. Where indicated, βTC3 cells are exposed with exogenous carbon monoxide. Where indicated, cells are treated with KT5823 (KT), a protein kinase G inhibitor. Gray histograms correspond to untreated β-cells, and black histograms correspond to β-cells treated with TNF-α. The results obtained are the average of repeated wells corresponding to one of three representative experiments, ± standard deviation.

Investigate whether carbon monoxide exerts an anti-apoptotic effect by activating soluble guanylate cyclase and the formation of cGMP (as described for fibroblasts) (Petrache et al., Am. J. Physiol. Lung Cell Mol. Physiol. 278: L312-319, 2000). Inhibition of sGC activity by oxadiazolechinooxaline (ODQ) suppresses the anti-apoptotic effect of CO, suggesting that soluble guanylate cyclase is the main mediator for carbon monoxide in this experimental system (Fig. 4A). The cGK activator / cGMP analogue, 8-Br-cGMP, suppresses apoptosis of βTC3 cells to a level similar to that observed for carbon monoxide (Fig. 4B). In addition, cGMP-dependent protein kinases, due to the specific KT5823 inhibitor, suppress the anti-apoptotic effect of exogenous carbon monoxide (Fig. 4C), under the assumption that the anti-apoptotic effect of exogenous carbon monoxide is mediated by the activation of one or more cGMP-dependent protein kinases.

Exogenous carbon monoxide provides protection against apotosis in different experimental designs

Investigate the ability of carbon monoxide to protect β-cells after the induction of apoptosis (Figa-C). To obtain the data presented in Fig.5A-C, carry out the following steps. 5A: one hour of exposure with carbon monoxide is sufficient to prevent apoptosis. ΒTC3 cells are transfected with vectors expressing β-gal. Β-cell apoptosis induces TNF-α. Immediately after TNF-α activation, cells are exposed for various times (0-24 hours) with 1% carbon monoxide. Control βTC3 is treated in the same manner, but without exposure to carbon monoxide. Cell survival is determined 24 hours after application of TNF-α. 5B: carbon monoxide protects β cells after induction of apoptosis. βTC3 is transfected with vectors expressing β-gal. Apoptosis is induced by TNF-α. After different induction times (0.5-12 hours, as indicated), βTC3 is exposed with 1% carbon monoxide (Otterbein et al., Nat. Med. 6: 422-428, 2000). Control βTC3 is treated in the same manner, but without exposure to carbon monoxide. Cell survival is determined 24 hours after application of TNF-α. 5C: preincubation with carbon monoxide prevents β-cell apoptosis. βTC3 is transfected with vectors expressing β-gal, and apoptosis is induced by TNF-α. βTC3 is pre-exposed with 1% carbon monoxide for one hour. Control βTC3 is treated in the same manner, but without exposure to carbon monoxide. 1-6 hours after the end of the preliminary exposure, apoptosis is induced by TNF-α. Gray histograms correspond to untreated β-cells, and black histograms correspond to β-cells treated with TNF-α. The results obtained are the average for repeated wells corresponding to one of three representative experiments, ± standard deviation.

βTC3 is exposed with carbon monoxide for different periods of time (1-24 hours) immediately after adding TNF-α and checking for apoptosis after 24 hours. One hour of exposure to carbon monoxide is sufficient to prevent apoptosis of β-cells (Figa).

To find out whether exposure to nitric oxide can actually block subsequent apoptosis, β-cells were exposed for one hour with CO 0.5-12 hours after apoptosis was induced by TNF-α. Even two hours after TNF-α stimulation, exposure to carbon monoxide is still capable of causing suppression of β-cell apoptosis (Fig. 5B).

In order to find out whether preincubation with carbon monoxide would really protect β cells from apoptosis, βTC3 was exposed with carbon monoxide for 0.5-3 hours before apoptosis was induced. One hour of incubation in the presence of carbon monoxide is enough to prevent β-cell apoptosis (data not shown). In order to evaluate how long this effect lasts, if the time between preincubation and apoptotic stimulus is extended, β-cells were pre-exposed for one hour with CO one to six hours before apoptosis was induced by TNF-α (Fig. 5C). One hour of preincubation with carbon monoxide prevents apoptosis of β-cells stimulated by TNF-α even two to three hours after the end of a one-hour treatment with carbon monoxide. These data indicate that a relatively short-term treatment with carbon monoxide can cause an anti-apoptotic effect and that this anti-apoptotic effect persists for a long period.

Exposure of mouse islands with carbon monoxide increases their survival / functionality after transplantation

In order to determine whether carbon monoxide can enhance the functionality of an islet transplant in vivo, the marginal (critical) number of islands from 250 manually collected islets is transplanted into a syngeneic system, a model without primary function (Berney et al., Transplantation 71: 125-32, 2001 ; Kaufman et al., Diabetes 43: 778-83, 1994). Transplantation of a marginal (eg, suboptimal) number of islets (“marginal amount”) to a syngenic recipient with diabetes delays the return to normoglycemia without rejection or a repeat of an autoimmune disease. When determining the marginal number of islets in the syngeneic system C57 / BL6, it was observed that transplantation of manually collected 500 islets into the recipient's renal capsule leads to a quick return to normoglycemia (1.5 ± 0.5 days (n = 4)), while transplantation of 250 islets leads to a significant delay (14.2 ± 2.94 days (n = 9)). Thus, it was found that 250 islets constitute a marginal amount. Therefore, the use of a marginal amount does not entail rejection or repetition of an autoimmune disease (Berney et al., Transplantation 71: 125-132, 2000).

Examine whether pre-incubation of carbon monoxide with islet grafts before their transplantation improves their functional characteristics in vivo (Fig. 6A-B). To obtain the data presented in Fig.6A-B, carry out the following steps. Fig. 6A: two hundred and fifty freshly isolated manually harvested islets of C57BL / 6 mice are incubated in a medium pre-saturated with 1% carbon monoxide for two hours at 37 ° C. Control islands are treated in the same way, but without exposure to carbon monoxide. Incubated islets are transplanted into the renal capsule of syngeneic recipients with diabetes, as described previously. After transplantation, the blood glucose level is determined daily. A total of 16 animals were transplanted (8 with previously exposed islands; 8 control). One animal transplanted with previously exposed islets died on day 3 for reasons other than exposure, and was included in the statistical analysis as a censored animal. The primary reference point in these experiments was the first day of normoglycemia. Data are presented as mean ± standard deviation. 6B shows the likelihood of recovery (blood glucose below 200 mg / dl) of animals that received islets previously exposed to carbon monoxide or control islets. * P = 0.001 in comparison with the control.

Based on the fact that, as it turned out, the effects of carbon monoxide treatment are extended over time (Figs. 5A and B), and that the relatively short-term carbon monoxide pretreatment (used before the apoptotic stimulus) is anti-apoptotic (Fig. 5C), it was evaluated whether to improve the preliminary exposure of islands with carbon monoxide island survival and / or their functionality after transplantation. A marginal number of islets is transplanted into the kidney capsule of syngeneic recipients with diabetes. The time required to achieve normoglycemia was significantly reduced (P = 0.0011) after the islands were preincubated for two hours in a medium pre-saturated with carbon monoxide (7 days, 95% confidence interval: 6-8 days) , compared with control islands not previously exposed with carbon monoxide (14 days, 95% confidence interval 12-18 days) (Figure 6). As a result, three different islet preparations are used in these experiments. There is no statistically significant difference in time to reach normoglycemia for the islets of these drugs (P> 0.25).

Exposure to carbon monoxide

In cell culture experiments, 5% CO _{2 is} used for buffering purposes. Before pumping CO at a concentration of 1% (10,000 ppm) in compressed air into the chamber for exposure, it is mixed in compressed stainless steel with compressed air with or without CO ₂ . The flow of the gaseous medium into the chamber for animals made of organic glass at 3.70 ft ² is maintained at 12 L / min, and into the chamber at 1.2 ft ² for cell culture - 2 L / min. The cell culture chamber is humidified and the temperature in it is maintained at 37 ° C. For continuous measurement of CO levels in chambers, a CO analyzer (Interscan, Chatsworth, CA) is used. Through a sampling hole in the upper part of the chambers using a CO analyzer, gas samples are taken at a rate of 1 l / min and analyzed by electrochemical detection with a sensitivity of 10-600 ppm. The concentration level is measured hourly. After the gas medium is balanced in this chamber (for approximately 5 minutes), fluctuations in the concentration of CO are not observed.

The animals are kept in a 3.70 cubic foot organic glass chamber containing> 98% O ₂ or a mixture of 98% O ₂ + CO at a flow rate of 12 L / min. The animals are fed food and water during aging. CO at a concentration of 1% (10,000 ppm) in compressed air is mixed with> 98% O ₂ in a stainless cylinder for mixing before the gas mixture enters the exposure chamber. By varying the CO flow rate in the mixing cylinder, the delivered concentration in the exposure chamber is controlled. Since the flow rate is primarily determined using the O ₂ stream, only the CO stream is changed to obtain different concentrations of carbon monoxide in the chamber. The concentration of O ₂ in the chamber is determined using a gas spectrometer.

Cell isolation technique

The following example illustrates the technique used to isolate islet cells in rats or mice. The contents of one vial of Rat Liberase ^™ (from Boehringer Mannheim / Roche Cat. No. 1815032) was dissolved in 4 ml of a sterile HBSS solution, cooled on ice for 30 minutes, divided into 0.5 ml aliquots and stored at -20 ° C. 33 ml of medium, for example, M199, HBSS or RPMI 1640 without calf serum, is poured into each 0.5 ml aliquot.

Rats are anesthetized with an excess dose of Nembutal. For mice, 3 ml syringes are prepared, filled with 2 ml of Liberase solution and equipped with a 27 g-needle bent at an angle of 90 °. For the operation, 2 pairs of scissors are used; one large pair is used to open the belly and one small pair is used to cut the bile ducts. For cutting the pancreas, two pairs of tweezers are used. One chemostat is used to release the bile duct.

They open the belly and expose, as far as possible, the pancreas, making a v-shaped incision from the very bottom of the belly. The pancreatic duct is released from the clamp (with a hemostat in rats and a small “bulldog” clamp in mice) in its duodenal implant, capturing the surrounding pancreatic tissue without damage. The bile duct is separated from the proximal end. Before the introduction of the cannula, fat is removed, making sure that there is no puncture of the portal vein. This duct is cut out with small scissors one third of the transition, and a cannula is inserted into this duct. The cannula is held in the duct using a weak clamp. Quickly inject a solution of Liberase ^tm . The pancreas is stretched and fully expanded after injection of 6 ml of fluid. In mice, the needle is inserted into the duct as close to the liver as possible, and a Liberase ^™ solution is injected. The rat or mouse is then killed by cutting out the diaphragm and the heart or aorta.

After penetration of Liberase ^tm into the pancreas, it is removed, starting the removal of the intestines, then the stomach and then the spleen. When the pancreas remains attached only to the bile duct, it is excised from the rat. The excised pancreas is placed in a 50 ml conical tube, and the tube is placed in a water bath at 37 ° C. for 30 minutes.

After incubation, 20 ml of medium + NCS is poured into each tube. The rest of the isolation procedure is completed on ice. The tubes are shaken vigorously in the hands for 5-10 seconds to destroy tissue. The resulting islands are washed several times in a clinical centrifuge at 800 rpm (approximately 180xg) for 120 seconds or 1200 rpm (approximately 200xg) for 90 seconds to remove Liberase ^tm . The resulting supernatant is decanted off and 25-35 ml of medium is added and gently shaken (max. About 1/2). The centrifugation step is repeated, followed by 2-3 times washing. The tissue is resuspended in 20 ml of medium, and the resulting suspension is filtered through a reinforced mesh with a hole diameter of 400 μm (Thomas scientific mesh 35 cat. No. 8321-M22) to remove the remaining intact tissue, fat and lymph. An additional 5-10 ml of medium for washing the remaining islets is added to the test tube and filtered through the indicated sieve.

The resulting cells are pelleted by centrifugation at 1200 rpm for 90 seconds. The supernatant is removed, leaving as little media as possible.

To create a gradient, the resulting precipitate was resuspended in 10-15 ml of Histopaque 1077 ^tm (Sigma Cat. No. H 1077) and shaken until a homogeneous suspension was obtained (as well as for washing). Layered 10 ml of medium without NCS, carefully preserving the sharp section between Histopaque ^tm and the existing medium. This medium is slowly added with a pipette along the side of the tube. The created gradient is centrifuged for 20 minutes at 2400 rpm (900 g) at 10 ° C with very slow acceleration and without braking.

After centrifugation using a disposable 10 cm ³ serological pipette (Falcon), an islet layer is collected from the interphase and placed in 50 cm ³ conical tubes. The islands are washed several times to remove Histopaque ^™ by adding 25-35 ml of medium + NCS. Initial centrifugation is carried out at 1200 rpm for 2 minutes, and subsequent centrifugation is carried out for 90 seconds. After 3 washes, the islets are resuspended by pipetting up and down. Every 7-10 ml of islets are manually placed in 60 mm sterile culture dishes.

Manual selection of islet cells is carried out under a microscope using 100 μl of a sterile pipette tip. Each islet is taken separately and carefully to avoid capture of cells of any other tissue. Smooth islands with a diameter of between 50 and 225 μm, as well as round or oval, are selected.

Example II Carbon monoxide suppresses rejection of murine cardiac grafts in rats

Animals

For transplantation into inbred adult male rats, Lewis strains were used as the donor organs of the heart of BALB / c mice (Harlan Sprague-Dawley, Indianopalis, IN). Animals are kept in accordance with the guidelines of the American Association for Laboratory Animal Care, and research protocols have been tested by the Institutional Animal Care and Use Committees of the Beth Israel Deaconess Medical Center.

Surgical model

During all procedures, animals are anesthetized with combined inhalation of methoxyflurane (Pitman-Moore, Mundelain, IL) and pentobarbital (Abbott, North Chicago, IL) at a dose of 30-50 mg / kg ip. A heterotopic heart transplant is performed as previously described (Berk et al., Physiol. Rev. 8: 999-1030, 2001; Petkova et al., J. Biol. Chem. 276: 7932-7936, 2001). Graft viability is assessed by daily palpation. Rejection is diagnosed by the cessation of ventricular contraction and confirmed by histological examination.

Reagents Used

Cobra venom factor (CVF; which blocks complement activation) (Quidel, San Diego, CA) is given ip in day - 1 (60 units / kg) and on day 0 (20 units / kg) in relation to the day of transplantation (day 0). Cyclosporin A (CsA; Novartis, Basel, Switzeland), which blocks the activation of T cells, is administered IM (15 mg / kg) starting from day 0 and then daily until the end of each experiment. Tin-containing protoporphyrin (SnPPIX), cobalt-containing protoporphyrin (CoPPIX) and iron-containing protoporphyrin (FePPIX; Porphyrin Products, Logan, UT) are diluted in 100 mM NaOH to obtain 50 mM stock solution and kept at -70 ° C before use. Lighting is limited if possible. Both SnPPIX and FePPIX in PBS were administered ip (30 μM / kg). FePPIX and SnPPIX are administered to the donor on days -2 and -1 (30 μM / kg), and to the recipient during transplantation (day 0) and then daily (30 μM / kg).

CO exposure

Briefly, CO at a concentration of 1% (10,000 ppm; ppm) in compressed air is mixed in a stainless steel cylinder for mixing with balanced air (21% oxygen), before it enters the exposure chamber. CO concentrations are controlled by varying the flow rate of CO in the mixing cylinder before delivery to the chamber. Since the flow rate is initially determined by the rate of O ₂ , only the CO flow rate is changed, which is delivered in the final concentration to the exposure chamber. A CO analyzer (Interscan Corporation, Chatsworth, CA) is used to continuously measure CO levels in the chamber. Donor transplants are placed in a chamber for exposure with CO 2 days before transplantation. Transplant recipients are placed in the camera for exposure immediately after transplantation and kept in the camera for exposure for 14 (n = 3) or 16 (n = 3) days. The concentration of CO is maintained throughout the time between 250 and 400 ppm. Animals were removed from the chamber daily to assess graft viability and to administer CsA, SnPPIX or FePPIX as described above.

Enzymatic activity HO

The enzymatic activity of HO is measured by the formation of bilirubin in the microsomes of the heart and liver. Animals are sacrificed, and their hearts and liver are washed with ice-cold PBS and frozen at -70 ° C until use. The organs are ground in four volumes of sucrose (250 mM) Tris-HCl (10 mM / L) buffer (pH 7.4) on ice and centrifuged (28000 rpm 3X, 20 min, 4 ° C). The resulting supernatant was centrifuged (105,000 rpm 3X, 60 min, 4 ° C), and the precipitate of the microsomes was resuspended in MgCl ₂ (2 mm) potassium phosphate (100 mm) buffer (pH 7.4) and subjected to ultrasonic treatment on ice . The resulting samples (1 mg protein) are added to the reaction mixture (400 μl) containing rat liver cytosol (2 mg protein), hemin (50 μM), glucose-6-phosphate dehydrogenase (0.25 U) and NADPH (0.8 mm ) for 60 min at 37 ° C in the dark. The resulting bilirubin is extracted with chloroform, and the OD measured at 464-530 nm (extinction coefficient, 40 mm / cm for bilirubin). Enzymatic activity is expressed as picomoles of bilirubin formed per milligram of protein in 60 minutes (pmol / mg / h). Protein concentration is determined by protein analysis by bicinchoninic acid (Pierce, Georgetown). The background is ~ 5 pmol / mg / h. All reagents used in this assay are manufactured by Sigma (St. Louis, MO), unless otherwise indicated. Carboxyhemoglobin levels are measured 2 days after transplantation using a Corning 865 gas blood analyzer (Clinical Chemistry, Massachusetts General Hospital, Boston, MA).

Histomorphometric analysis

The grafts are harvested 3 days after the transplantation operation, enclosed in paraffin, fixed in formalin and sequentially cut (5 μm), completely, from the apex to the base. Ten slices are placed on a glass slide, a total of about 20-25 glass slides. Every fifth glass slide is stained with hematoxylin and eosin (H&E) for histomorphometric analysis. Two images are viewed on each slide under a Nicon Eclipse E600 ^tm microscope (Nikon, Melville, NY) connected to a Hitachi 3-CCD color camera (model HV-C20; Hitachi, Tokyo, Japan) and a powerful Macintosh ^tm 7300/200 computer ( Apple Computer, Cupertino, CA) equipped with IPLab Spectrum digital software (Signal Analytics Corporation, Vienna, VA). For two or three animals, about 50 images of each transplanted heart are viewed per group. Images are analyzed by manually moving the plots, looking at each section infarct and non-infarction areas in the right and left ventricle. Using digital image software, areas corresponding to heart attack and non-infarction tissue are counted as the number of pixels corresponding to these areas. Then calculate the percentage of infarct and non-infarction areas throughout the viewing area. The combined data for each group, expressed as a region in pixels or as a percentage of a heart attack, is analyzed using ANOVA. The results obtained in this way are similar to the results using either pixels or a percentage for a heart attack, but the results are presented using only the percentage of a heart attack (see Table II). Results are expressed as mean = SD.

Immunohistology

Transplants are harvested 3 days after transplantation, instantly frozen in liquid nitrogen and stored at -80 ° C. Cryostatic sections are fixed and stained as previously described (Soares et al., Nature Med. 4: 1073, 1998). Rat leukocyte populations are usually analyzed using Ag (LCA, CD45; OX-1), αβ TCR (TCR αβ chains; R73), B cells (CD45RB; OX-33), NK cells (NKR-P1; 3.2. 3), as well as MF (CD68; ED-1), monAT (Serotec, Harlan Bioproducts for Science, Indianopalis, IN) against rat leukocytes. Fibrin / fibrinogen detection is carried out using rabbit polyclonal antibodies against human fibrin / fibrinogen (Dako, Carpinteria, CA). Activation of the graft complement was detected using the anti-rat C1q (The Binding Site, Birmingham, UK), C3 (ED11; Serotec) or monAT C5b-9 (Dako). Rat IgM was detected using mouse monAT MARM-4 against rat IgM (kindly donated by Dr. H. Bazin, University of Louvain, Brussels, Belgium). MonAT matched or purified Ig isotypes are included in each experiment, as is the control for residual endogenous peroxidase activity. Apoptosis detection is carried out using the ApopTag ^™ in situ apoptosis detection kit (Oncor, Gaithersburg, MD), in accordance with the manufacturer's instructions.

Complement Hemolysis Assay (CH50)

The CH50 value is defined as the degree of dilution of rat serum required to obtain 50% of the maximum lysis of AT-sensitive sheep erythrocytes. Briefly, AT-sensitive ram erythrocytes (1 x 10 ⁸ cells / ml; Sigma) are incubated (30 min, 37 ° C) in the presence of rat serum in gelatin on veronal buffer (GVB ⁺⁺ ; Sigma). The cells are centrifuged and the released hemoglobin is measured (λ = 550 nm). The background is measured in the absence of ram red blood cells or in the absence of serum and subtracted from the readings for all samples.

Cellular ELISA

Rat mouse anti-mouse AT serum was measured using an indirect cell ELISA. A mouse endothelial cell line 2F-2B (CRL-2168; American Type Culture Collection (ATCC), Manassas, VA) was used as an antigenic target. Briefly, 2F-2B cells were cultured in DMEM (Life Technologies, Rockville, MD), 10% FCS, 100 U / ml penicillin and 100 μg / ml streptomycin (Life Technologies). Glutaraldehyde-fixed cells 2F-2B were incubated (1 h, 37 ° C in the presence of rat serum diluted sequentially in PBS-0.05 Tween 20 (Sigma), and rat anti-mouse ATs were detected using mouse anti-rat IgM (MARM-4), IgG1 (MARG1-2), IgG2a (Marg2a-1), IgG2b (MARGb-8) or IgG2c (Marg2c-5) (kindly donated by Prof. H. Bazin, University of Louvain, Brussels, Belgium). Mouse anti-rat ATs are detected with using HRP-labeled goat anti-mouse Fab 'depleted in cross-reacting anti-rat Ig (0.1 μg / ml, 1 hr, room temperature; Pierce, Rockford, IL). HRP was detected using by calling ortho-phenyldiamine (Sigma) and H ₂ O ₂ (0.03%) in citrate buffer (pH 4.9). Absorption is measured at λ = 490 nm. The relative amount of anti-graft AT circulating in serum is expressed as OD (λ = 490) taken for one serial dilution in the linear range of this analysis (1: 32-1: 1024).

The binding of rat C3 to murine endothelial cells is measured using a modified 2F-2B murine endothelial cell ELISA method as target antigens (Miyatake et al., J. Immunol. 160: 4114, 1998). Briefly, non-fixed endothelial cells 2F-2B are incubated in the presence of rat serum diluted sequentially in a GVB ⁺⁺ buffer (1 h, 37 ° C). Cells were fixed in PBS, 0.05% glutaraldehyde, and rat C3 pellet was detected using murine monAT to rat C3 (Serotec).

Platelet Aggregation Analysis

2F-2B murine endothelial cells were cultured in 0.2% gelatin (Sigma) coating six-well plates, 88% DMEM (Life Technologies), 10% FCS (FCS), 100 U / ml penicillin and 100 μg / ml streptomycin (Life technologies) ) Fused endothelial cells are either left untreated or treated with CoPPIX (50 μM; 18 h), an HO inducing agent, SnPPIX (50 μM, 18 h), an HO inhibitor, or both with CoPPIX (50 μM, 15 h) and SnPPIX (50 μM, 3 h). Platelet-rich plasma is obtained by centrifugation (290 g, 12 min, 19 ° C) of normal rat plasma in 3.8% sodium citrate. Rat platelets (3-10 ⁸ cells / ml) are suspended in HT-buffer (8.9 mm NaHCO ₃ , 0.8 mm KH ₂ PO ₄ , 5.6 mm dextrose, 2.8 mm solution of KCl, 0, 8 mM MgCl ₂ , 129 mM NaCl, 10 mM HEPES). Platelets are layered on murine endothelial cells, and platelet aggregation analysis is performed as previously described (Kaczmarek et al., J. Biol. Chem. 271: 33116, 1996) using an aggregometer (Chrono-Log, Harestown, PA) and ADP ( 0.5-4 μM) as an agonist.

Cell Extracts and Western Blot Analysis

Endothelial cells were washed in PBS (pH 7.2), harvested by scraping and lysed in Laemmli buffer. Electrophoresis is carried out under denaturing conditions in a 10% polyacrylamide gel. Proteins are transferred onto a polyvinyl difluoridine membrane (Immobilon P; Millipore, Bedford, MA) by electroblotting and detected using rabbit polyclonal ATs against human HO-1 or HO-2 (SressGen, Victoria, Canada) or β-tubulin (Boehringer Mannheim, Mannheim, Germany). Proteins are visualized using HRP conjugated donkey anti-rabbit IgG or goat anti-mouse IgG (Pierce) and ECL analysis (Amersham Life Science, Arlington Heights, IL), according to the manufacturer's instructions.

Transient Transfection and Apoptotic Analysis

The mouse 2F-2B endothelial cell line (ATCC) is transiently transfected as described in another publication (Soares et al., Nature Med / 4: 1073, 1998; Brouard et al., J. Exp. Med. 192: 1015, 2000) . All experiments are carried out 24-48 hours after transfection. Cells transfected with β-galactosidase are detected as described in another publication (Soares et al., Nature Med. 4: 1073, 1998; Brouard et al., J. Exp. Med. 192: 1015, 2000). The percentage of viable cells is determined by estimating the number of cells expressing β-galactosidase that maintain normal morphology, as described in another publication (Soares et al., Nature Med. 4: 1073, 1998; Brouard et al., J. Exp. Med. 192: 1015, 2000). The number of random fields calculated is determined, which is at least 200 viable transfected cells per control well. The percentage of viable cells is normalized for each DNA preparation according to the number of transfected cells counted in the absence of an apoptosis inducing agent (100% survival). All experiments are carried out at least three times with repetition. Actinomycin D (Act. D; Sigma) was dissolved in PBS and added to the culture medium (10 μg / ml) 24 hours after transfection. SnPPIX (Porphyrin Products) was dissolved (10 μM) in 100 mM NaOH and stored at -20 ° C until use. SnPPIX was added to the culture medium (50 μM) 6 hours after transfection. Human recombinant TNF-α (R&D Systems, Minneapolis, MN) was dissolved in PBS, 1% BSA, and added to the culture medium (10-100 ng / ml) 24 hours after transfection.

Exposure of cultured endothelial cells with CO

Cells are exposed with compressed air or with varying concentrations of CO (250 and 10,000 ppm), as described in another publication (Otterbein et al., Nature Med. 6: 422, 2000; and Brouard et al., J. Exp. Med. 192 : 1015, 2000).

Aortic transplant model

Aortic transplantation is performed as described in another publication (Plissonnier et al., Transplantation 60: 414-424, 1995). Briefly, the aorta and the inferior vena cava are cut so that it bleeds after heparinization. After left additional thoracotomy, three or four pairs of intercostal arteries are ligated using a 7-0 nylon suture (Keisei Medical Industrial Co., LTD, Tokyo, Japan), a 2 cm piece of the descending aorta is removed. A graft using standard microsurgery using 9-0 nylon sutures (Ethilon ^™ , Ethicon, Inc., Somerville, New Jersey) is placed between the renal arteries and aortic bifurcation. The native abdominal aorta located on the left at both edges is ligated.

CO at a concentration of 1% (10,000 ppm; ppm) in compressed air is mixed with balanced air (21% oxygen) as previously described (Otterbein et al., Am. J. Physiol. 276 (4 Pt 1): L688- L694, 1999). For this transplantation model, a donor transplant is placed in a CO-chamber two days before transplantation. The recipients are placed in the chamber immediately after transplantation and kept here for 56 days. The concentration of CO is always maintained at 250 ppm.

Adult male (250-350 g) Brown Norway rats (RT1 used as aortic transplant donors, and adult male (250-350 g) Lewis rats (RT1) used as recipients (Charles River Lab. Wilmington, MA). C57BL males / 6, null mice p21 ^{- / -} and p53 ^{- / - were} purchased from the Jackson Laboratory (Bar Harbor, ME). MKK3 null mice ^{(- / -)} were bred as previously described (Lu et al., EMBO J. 18: 1845-1857, 1999.) Mice were allowed to get used to rodent food and ad libitum water for one week.

RT-PCR

RT-PCR is carried out after isolation of the transplanted hearts using the RNA extraction kit in accordance with the manufacturer's instructions (Qiagen, Chatsworth, CA). The primers used for mouse β-actin are: semantic (5'-3 '). CCTGACCGAGCGTGGCTACAGC (SEQ ID NO: 1); antisense (3'-5 '), AGCCTCCAGGGCATCGGAC (SEQ ID NO: 2); and for mouse HO-1: semantic (5'-3 '), TCCCAGACACCGCTCCTCCAG (SEQ ID NO: 3); antisense (3'-5 ') GGATTTGGGGCTGCTGGTTTC (SEQ ID NO: 4).

Enzymatic activity is key to suppressing acute vascular rejection

Murine hearts transplanted with untreated rats are prone to acute vascular rejection 2–3 days after transplantation, according to an observation consistent with previous reports (Soares et al., Nature Med. 4: 1073 1998; and Koyamada et al., Transplantation 65: 1210 , 1998). When treated with cobra venom factor (CVF) plus cyclosporin A (CsA), mouse cardiac grafts are viable for a long time (see Table II below), an observation also consistent with previous reports. In CVF plus CsA treatment, transplant viability is associated with increased regulation of HO-1 expression in transplanted endothelial and smooth muscle cells, as well as in cardiac myocytes (Figure 7). Expression of HO-1 mRNA is detected by RT-PCR 12-14 hours after transplantation, and HO-1 protein 24-72 hours after transplantation (Figure 7). Long-term transplant survival cannot be achieved if SnPPIX, an HO inhibitor, is administered to the donor and then to the recipient despite treatment with CVF plus CsA. Under these conditions, all grafts are rejected on the 3rd – 7th day (Table II). Control treatment with FePPIX, protoporphyrin, which does not inhibit HO activity, does not lead to transplant rejection (Table II).

Table II Inhibition of HO-1 activity by SnPPIX accelerates transplant rejection Treatment Lifetime CVF + CsA > 50 (n = 8) CVF + CsA + FePPIX > 50 (n = 4) CVF + CsA + SnPPIX 3, 4, 5 (n = 2); 6 (n = 4); 7 (n = 2)

To obtain the data of Table II, murine hearts are transplanted into rats treated with CVF plus CsA. Recipient transplants are treated with FePPIX or SnPPIX. SnPPIX treatment induces transplant rejection 3-7 days after transplantation (p <0.0001 compared to rats treated with only CVF plus CsA or CVF plus CsA plus FePPIX). Statistical analysis is performed using Fisher's exact test.

To show that SnPPIX, but not FePPIX, block the function of HO-1 in vivo, the total enzymatic activity of HO is quantified in transplanted and recipient hearts 2 days after transplantation (Fig. 8). Untreated murine hearts, compared to untreated hearts, produce 35.5 ± 4 picomoles of bilirubin per milligram of total protein per hour (pmol / mg / h; FIG. 8). HO activity increases significantly in mouse hearts transplanted into untreated rats (98 ± 7.21 pcmol / mg / h; p = 0.001), rats treated with CVF plus CsA (98.3 ± 7.23 pcmol / mg / h), or rats treated with CVF plus CsA plus FePPIX (77.3 ± 5.51 pmol / mg / h; p = 0.0009) (Fig. 8). According to the data presented, in untreated mouse hearts transplanted into rats treated with CVF plus CsA plus SnPPIX (32.37 ± 7.23 pmol / mg / h), HO activity is inhibited to a baseline level. This corresponds to a highly significant inhibition compared to murine hearts transplanted with untreated rats (p = 0.0009), rats treated with CVF plus CsA (p = 0.0009), or rats treated with CVF plus CsA plus FePPIX (p = 00, 18; Fig. 8). HO activity in the liver of recipients also increases after transplantation, similar to how it occurs during heart transplantation (data not shown). However, this does not apply to the recipient's own heart, in which HO activity does not increase after transplantation (Fig. 8). In transplants transplanted into rats treated with SnPPIX, there is a progressive myocardial infarction, which becomes apparent 2 days after transplantation (data not shown). This is not observed in transplants in control rats treated with FePPIX (data not shown).

It was previously shown that rats that undergo CVF plus CsA-treated mouse heart transplantation produce anti-mouse ATs that are exclusively represented by the IgM isotype (Koyamada et al., Transplantation 65: 1210, 1998). Additional processing of SnPPIX or FePPIX does not affect this AT response (Figure 9). The formation of AT to the graft correlates with complement activation, as shown by C3 deposition in murine endothelial cells (Figure 9). Treatment with SnPPIV or FrPPIX does not affect C3 deposition in murine endothelial cells (Figure 9).

Exogenous CO completely replaces the enzymatic activity of HO-1 in the suppression of acute vascular rejection

All mouse hearts transplanted into rats treated with SnPPIX and exposed to CO (400 ppm; 0.04%) remained viable for a long time (see Table III, below). The used dose of CO (400-500 ppm) approximately corresponds to one twentieth of the lethal dose (data not shown). In rats and mice exposed with CO, adverse reactions were not observed. CO exposure interrupted 14 (n = 3) or 16 (n = 3) days after transplantation does not affect the viability of the graft, i.e. transplants continue to function for> 50 days (Table III).

Table III Exogenous CO completely replaces HO-1 in transplant rejection suppression Treatment Life time (days) CVF + CsA + SnPPIX 3, 4, 5 (n = 2); 6 (n = 4); 7 (n = 2) CVF + CsA + SnPPIX + CO > 50 (n = 6)

To obtain the data of Table III, murine hearts are transplanted into rats treated with CVF plus CsA. Where indicated, recipient transplants are treated with SnPPIX and exhibit or not exhibit with CO. Graft rejection observed in rats treated with SnPPIX is suppressed when exposed to exogenous CO (p <0.0001 compared with recipients treated with CVF plus CsA plus SnPPIX). Statistical analysis is performed using Fisher's exact test.

To determine whether exogenous CO inhibits the inhibition of HO-1 enzymatic activity by SnPPIX, which could be assessed by the ability of CO to suppress transplant rejection, we studied whether CO affects the enzymatic activity of HO in hearts transplanted with rats treated with SnPPIX. As shown in FIG. 10, this does not occur. The total enzymatic activity of HO hearts transplanted into rats treated with SnPPIX (32.37 ± 7.23 pmol / mg / h) is not significantly different from the activity of HO hearts transplanted into rats treated with SnPPIX and exposed to CO (43.6 ± 7, 57 pcmol / mg / h; p = 0.1095; Figure 10). Similar results are obtained for the liver and hearts of recipients (Figure 10).

Exogenous CO can replace HO-1 activity in preventing transplant rejection. This can occur due to the mechanism associated with the "loading" as a result of inhalation of exogenous CO in RBC and its subsequent delivery to the graft through blood circulation in an adequate concentration. In accordance with this assumption, if the endogenous activity of HO-1 is inhibited by SnPPIX, exogenous CO should mimic the action of endogenous CO, which is produced if the enzymatic activity of HO-1 is not attenuated. Exposure of a transplant of a recipient with 400 ppm of exogenous CO increases the level of carboxyhemoglobin from 0.5 ± 1.5% to 32.1 ± 6.9 (Figure 10). The fact that transplanted hearts survived in animals exposed to CO, even with these suppressive effects of SnPPIX, may indicate that such a level of CO is sufficient to adequately “charge” RBC, delivering CO to the graft, and to suppress transplant rejection ( Figure 10). Alternatively, carbon monoxide dissolved in plasma can be delivered to the graft and to all body tissues.

We studied the question of whether exogenous CO suppresses the development of myocardial infarction, in which transplant rejection occurs in rats treated with SnPPIX. Transplants were harvested 3 days after transplantation and the area affected by a heart attack was quantified as a percentage. Hearts transplanted with untreated rats were affected by almost complete transmural infarction of the right ventricle (87.1 ± 4.9% of the area of the right ventricle), along with extensive endomyocardial and transmural infarction of the left ventricle (32.0 ± 6.7% of the area of the left ventricle; data not presented). Myocardial infarction testified to the non-viability of a nuclear-free eosinophilic myocardium with interstitial hemorrhage, edema, and neutrophils. Left ventricular infarction has always been endomyocardial with transmural spread, depending on the degree of myocardial infarction, and left ventricular infarction is more diffuse in nature. The percentage area of infarction in both ventricles, as a rule, increases from the top of the heart to its base. Hearts transplanted into rats treated with CVF plus CsA showed only small diffuse, non-transmural infarct areas in the right ventricle (4.5 ± 4.9%), but not in the left (0.7 ± 2.1%) (see Table IV, below). In the hearts transplanted to rats treated with CVF plus CsA plus FePPIX, small diffuse infarcted areas were found in the right ventricle (12.2 ± 9.5%), but not in the left (0.7 ± 1.3%) (Table IV ) These hearts are indistinguishable from hearts transplanted into rats treated with CVF plus CsA without FePPIX treatment (data not shown). Significant transmural right ventricular infarction (26.1 ± 12.7%) with extensive endomyocardial and transmural left ventricular infarction (37.6 ± 15.5%) was found in hearts transplanted into rats treated with CVF plus CsA plus SnPPIX (Table IV ) which are indistinguishable in nature from those for hearts transplanted with untreated rats (data not shown). These lesions were specific to a transplanted heart. In the native hearts of the recipients, no heart attack develops. Expressed as a percentage of the area of infarct lesion in hearts transplanted into rats treated with SnPPIX, they were significantly higher (p <0.001) compared with hearts transplanted into rats treated with CVF plus CsA, as well as with treated or untreated FePPIX (Table IV). Hearts transplanted into rats treated with SnPPIX that were exposed to exogenous CO were very slightly affected by a heart attack in the right (8.4 ± 5.3%) and left (1.8 ± 3.4%) ventricles (Table IV), moreover the nature of the lesion was similar to that in hearts transplanted into rats treated with CVF plus CsA, as well as FePPIX (data not shown). The percent areas affected by heart attack in hearts transplanted into rats treated with SnPPIX exposed to exogenous CO did not differ significantly from the percentages affected by heart attack in hearts transplanted into rats treated with CVF plus CsA, with or without FePPIX her. At the same time, expressed as a percentage of the area of infarction lesion in these hearts, they significantly differ (p <0.001) from those in hearts transplanted with the same treatment, but not exposed to exogenous CO.

Table IV Morphometric analysis Treatment Right ventricle Left ventricle CVF + CsA 4,5 ± 4,9 0.7 ± 2.1 CVF + CsA + FePPIX 12.2 ± 9.5 0.7 ± 1.3 CVF + CsA + SnPPIX 26.1 + 12.7 * 37.6 ± 15.5 * CVF + CsA + SnPPIX + CO 8.4 ± 5.3 1.8 ± 3.4

To obtain the data of Table IV, mouse hearts were transplanted into rats (n = 3 in the group) treated with CVF plus CsA. If indicated, recipient grafts were treated with FePPIX or SnPPIX and exposed with CO. The results are presented as percent of infarcted area. Statistical analysis was performed using ANOVA analysis. An asterisk indicates a significant difference when compared with all other treatments.

Exogenous CO causes suppression of vascular thrombosis and infiltration of monocytes / macrophages, which characterizes acute rejection of blood vessels.

Mouse hearts were transplanted into rats treated with CVF plus CsA. SnPPIX or FePPIX was administered and recipient grafts were exposed with CO (250-400 ppm). 3 days after transplantation, grafts were collected (n = 3 in the group) and stained for rat IgM, rat and mouse complement C1q, rat and mouse P-selectin, rat and mouse fibrin / fibrinogen and rat leukocytes expressing CD45. Mouse hearts transplanted into rats treated with CVF plus CsA, with or without FePPIX, showed extensive intravascular deposition of rat IgM and C1q (data not shown), but did not detect IgG, C3, or C5b-9 (data not shown). HO-2, HO-1 and ferritin were detected in the transplant of endothelial cells and smooth muscles, as well as in myocytes of the heart (data not shown). Only minimal vascular thrombosis or host leukocyte infiltration, usually associated with focal areas of a heart attack, was detected (data not shown). A weak but detectable expression of P-selectin was found in vascular endothelium (data not shown). Hearts transplanted into CVF plus CsA treated rats while inhibiting HO-1 activity with SnPPIX showed similar levels of intravascular IgM and C1q deposition compared to control FePPIX treated rats and did not detect IgG, C3 or C5b-9 (data not presented). Widespread vascular thrombosis of large coronary vessels was found, associated with aggregates of platelets expressing P-selectin and with intravascular fibrin. Blood clots were constantly observed in large coronary vessels at the base of the heart. Platelet aggregates expressing P-selectin were not detected in microvessels (data not shown). Extensive infiltration was detected in the transplant with host neutrophils, as well as CD45 ⁺⁺ leukocytes expressing the CD68 / ED-1 marker of MF monocytes and MHC class II antigens (data not shown). Infiltrating monocytes / MFs found near arterioles and scattered throughout the myocardium were associated with heart attack regions.

Hearts transplanted into rats treated with SnPPIX and exposed with CO were practically indistinguishable from hearts transplanted into rats treated with CVF plus CsA, along with or without FePPIX (data not shown). A similar IgM level and C1q deposition in vessels were found in these hearts when compared with hearts transplanted to recipients treated with SnPPIX but not exposed to CO (data not shown). When CO-exposure, there were no signs of vascular thrombosis, which was detected by the absence of platelet aggregates expressing P-selectin, or intravascular fibrin. P-selectin was detected in the vascular graft endothelium. A certain level of monocyte / MF infiltration was found, associated with small focal areas of myocardial infarction (data not shown).

An increase in HO-1 in endothelial cells inhibits platelet aggregation.

The question was examined whether the lack of platelet aggregation in transplants transplanted into rats exposed to CO was caused or whether platelet aggregation in vitro is suppressed by the expression of HO-1 in endothelial cells. Mouse endothelial cells were exposed with CoPPIX or SnPPIX, respectively, to induce or inhibit HO-activity in these cells. Platelets layered on endothelial cells and tested their ability to aggregate, stimulated by ADP (2 μm). Platelets layered on untreated endothelial cells were normally aggregated upon ADP stimulation (Figure 11). When platelets were exposed to endothelial cells pretreated with SnPPIX, platelet aggregation was enhanced compared to platelets exposed to untreated endothelial cells (Figure 11). This observation indicates that untreated endothelial cells have an initial level of HO activity, which, apparently, can be attributed to the constitutive expression of HO-2 in these cells (Figure 11). When platelets were exposed with endothelial cells pretreated with CoPPIX, platelet aggregation was significantly inhibited compared to platelets exposed with untreated or SnPPIX-treated endothelial cells (Figure 11). This inhibitory effect was inhibited when platelets were exposed with endothelial cells treated with both CoPPIX and SnPPIX (Figure 11). Both CoPPIX and SnPPIX enhanced HO-1 expression in cultured endothelial cells (data not shown). The different effects of these protoporphyrins should be related to the ability of SnPPIX to act as a potent inhibitor of the enzymatic activity of HO-1.

HO-1 generates CO, which inhibits apoptosis of endothelial cells.

One of the main signs that characterizes the rejection of mouse hearts transplanted into rats treated with SnPPIX is extensive apoptosis of endothelial cells and heart myocytes (Fig. 12). Apoptosis does not occur in mouse hearts transplanted into rats treated with FePPIX (FIG. 12). It was ascertained whether endothelial cell apoptosis is inhibited in vitro by this HO-1 ability (Soares et al., Nature Med. 4: 1073-1077, 1998; and Brouard et al., J. Exp. Med. 192: 1015, 2000), or this cytoprotective effect is mediated as a result of the formation of CO. Apoptosis did not occur in mouse hearts transplanted into rats treated with SnPPIX and exposed to CO, which suggests that this is the case (Fig. 12). We studied in vitro, when inhibiting HO-1 activity by SnPPIX, whether exogenous CO will protect endothelial cells from TNF-α-mediated apoptosis. The data presented in Fig.6 suggest that this is so. Overexpression of HO-1 inhibited TNF-α-mediated endothelial cell apoptosis occurring in the presence of actinomycin D (Figure 12). The anti-apoptotic effect of HO-1 is mediated by its enzymatic activity, since exposure to endothelial cells with SnPPIX blocked the anti-apoptotic effect of HO-1 (Figure 12). Upon inhibition of HO-1 activity by SnPPIX, exogenous CO (10,000 ppm) induced suppression of TNF-α-mediated apoptosis, suggesting that HO-1 inhibits endothelial cell apoptosis as a result of CO formation (Figure 12).

Carbon monoxide inhibits the development of transplant-associated atherosclerosis.

Brown Norway rat aortas were transplanted into Brown Norway rats (syngenic) untreated Lewis (allogeneic) or Lewis rats exposed to carbon monoxide (250 ppm; allogeneic, carbon monoxide). Samples were collected 56 days after transplantation and stained with modified elastic tissue-masson trichrome or hematoxylineosine. Brown Norway rat aortic portions were transplanted into rats with arteriosclerotic disturbances that were consistent with those associated with chronic transplant rejection (data not shown). These disorders became noticeable 20-30 days after transplantation and pronounced after 50-60 days (data not shown). For this reason, all analyzes were performed 56 days after transplantation. These disorders were characterized by intimal hyperplasia, loss of medial smooth muscle cells (SMC), and accumulation of leukocytes in the adventitia membrane and were not observed in the aorta of transplant recipients (data not shown). Symptoms of these disorders were not observed if sections of the rat aorta were transplanted to syngeneic recipients. To check whether CO will actually inhibit the development of these disorders, aortic transplants were transplanted to allogeneic recipients, who were then exposed to CO (250 ppm) immediately after transplantation and for the next 56 days. Intimal hyperplasia was suppressed in the aortic grafts of recipients exposed to CO, compared with grafts that were transplanted to recipients exposed to air, because leukocyte accumulation occurred in the adventitia membrane (data not shown). CO did not significantly affect the loss of medial SMC compared with grafts transplanted to untreated recipients (data not shown).

Example III Methods of processing organs and tissues, donor and recipient in the process of transplantation .

The following examples illustrate the methods used in treating a donor, organ, and recipient with carbon monoxide during a transplant operation. During the transplant operation, you can use any one or more of the following methods.

Donor Processing

Before harvesting an organ or tissue, the donor can be inhaled with carbon monoxide (250 ppm) for one hour. Carbon monoxide can be administered in doses ranging from 10 ppm to 1000 ppm over a period of one hour to six hours, or during the entire period from the moment when it becomes possible to process a donor whose brain has lost biological activity (corpse) during removal of this organ. Treatment should begin as soon as possible after the fact that the brain has lost biological activity is established. In some cases, it is desirable to begin treatment before the brain has lost its biological activity.

For non-human animals (e.g. pigs) that are used as xenograft donors, a live animal can be treated with relatively high levels of respirable carbon monoxide because the carboxyhemoglobin produced does not reduce the viability and functionality of the organ to be transplanted. For example, you can use levels in excess of 500 ppm (for example, 1000 ppm or higher, up to 10,000 ppm, especially for short periods).

In situ organ treatment

Before removing the organ from the donor, it can be poured with a solution, for example, a buffer or medium without red blood cells, while it is still with the donor. The goal is to water the organ with a solution saturated with carbon monoxide and keep it in a carbon monoxide atmosphere so that the carbon monoxide remains in saturation mode. Watering can take place for a certain time of at least 10 minutes, for example, 1 hour, several hours or longer. In the ideal case, this solution should deliver the highest concentration of carbon monoxide to the cells of the donor organ.

Organ or tissue treatment

An organ or tissue can be pre-stored in a medium that contains carbon monoxide, from the time it is removed from the donor until transplantation to the recipient. This can be done by storing an organ or tissue in a medium containing CO, or by perfusing them with such a medium. Since this occurs preferably ex vivo, and not in the animal’s body, very high concentrations of CO gas can be used (for example, 10,000 ppm to maintain the medium in CO saturation mode).

Recipient Processing

The recipient can be treated with carbon monoxide. Treatment can be started on the day of transplantation at least 30 minutes before surgery. Alternatively, treatment can be started at least 30 minutes prior to reperfusion of the organ in the recipient. It can be continued for at least 30 minutes, for example, 1 hour. Doses of carbon monoxide between 10 ppm and 3000 ppm can be delivered by varying the time, for example minutes or hours, and can be administered on the day of transplantation and the day after transplantation. For example, the recipient can inhale a concentration of carbon monoxide, for example, 3000 ppm, holding his breath for three consecutive 10 seconds. Alternatively, the recipient can inhale, say, 200 ppm for an extended period, such as 20 days. Carboxyhemoglobin concentrations can be used as a leading indicator for the proper administration of carbon monoxide to a patient. Typically, treatment of recipients does not cause them to increase carboxyhemoglobin levels above levels acceptable to a patient in need of transplantation.

Other embodiments of the present invention

It should be borne in mind that although the present invention is described in the application in sufficient detail, the presented description is intended to illustrate the invention and is not limited to the scope of the present description, which are defined by the attached claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (69)

1. The method of organ transplantation, providing
(a) administering to the donor a pharmaceutical composition comprising carbon monoxide;
(b) obtaining from a donor an organ selected from the group consisting of a kidney, liver, heart, skin, small intestine, and pancreas; and
(c) transplanting the indicated organ to the recipient, where the amount of carbon monoxide administered to the donor is an amount sufficient to increase the survival or functionality of the organ in the recipient after transplantation. 2. The method according to claim 1, where the pharmaceutical composition is administered to a living donor or donor after the death of his brain. 3. The method according to claim 1, where the pharmaceutical composition containing carbon monoxide is administered to the donor before or after the death of his brain. 4. The method according to claim 1, where the pharmaceutical composition is a first pharmaceutical composition, and the method further comprises treating the organ in situ at the donor with a second pharmaceutical composition containing carbon monoxide. 5. The method of claim 1, further comprising treating the ex vivo organ prior to the transplantation step with a second pharmaceutical composition comprising carbon monoxide. 6. The method according to claim 1, where the pharmaceutical composition is a first pharmaceutical composition, and the method further comprises the step of administering to the recipient a second pharmaceutical composition containing carbon monoxide, before step (c), during step (c), after step (c) before and during stage (c), before and after stage (c), or before, during and after stage (c). 7. The method according to claim 1, where the organ is a kidney. 8. The method according to claim 1, where the donor and recipient belong to the same species or to different species. 9. A method for transplanting an organ to a recipient, comprising
(a) receiving an organ from a donor,
(b) introducing into the resulting organ a pharmaceutical composition comprising carbon monoxide; and
(c) organ transplantation to the recipient, where the amount of carbon monoxide introduced into the organ in step (b) is an amount sufficient to increase the survival or functionality of the organ in the recipient after transplantation of the organ. 10. The method according to claim 9, where stage (b) is carried out by perfusion of the organ in situ while the organ is located at the donor. 11. The method according to claim 9, where stage (b) is carried out ex vivo. 12. The method according to claim 11, further comprising prior to step (b) the step of introducing to the donor a second pharmaceutical composition comprising carbon monoxide; and organ removal from the donor. 13. The method according to p. 12, according to which the second pharmaceutical composition is administered to a living donor or a donor after the death of his brain. 14. The method of claim 12, wherein the second pharmaceutical composition is administered to the donor before and after the death of his brain. 15. The method according to claim 9, further comprising the step of administering to the recipient a second pharmaceutical composition comprising carbon monoxide, before step (c), during step (c), after step (c), before and during step (c), before and after step (c) or before, during and after step (c). 16. The method according to claim 9, where the organ is a liver, kidney, heart, pancreas, lung, small intestine or skin. 17. The method according to claim 9, according to which the donor and recipient belong to the same species or to different species. 18. The method of organ transplantation, providing
(a) obtaining from a donor an organ selected from the group consisting of a liver, kidney, heart, pancreas, lung, small intestine, or skin;
(b) organ transplantation to the recipient and
(c) administering to the recipient, before, during, or after step (b) a pharmaceutical composition containing enough carbon monoxide to increase the survival of the transplanted organ in the recipient. 19. The method according to p. 18, where the pharmaceutical composition is administered to the recipient within 1-20 days after (b), at least once in the period starting from 21 days after (b), or repeatedly or continuously during the period starting 21 days after (b). 20. The method according to p, where the pharmaceutical composition is administered to the recipient when it is established that the transplanted organ is undergoing or close to undergoing chronic rejection, or in establishing that the transplanted organ is undergoing or close to undergoing acute rejection. 21. The method of claim 18, further comprising, prior to receiving the organ from the donor, the step of administering to the donor a second pharmaceutical composition comprising carbon monoxide. 22. The method according to item 21, where the second pharmaceutical composition is administered to a living donor or donor after the death of his brain. 23. The method of claim 18, further comprising, before step (b), the step of introducing into the body a second pharmaceutical composition comprising carbon monoxide. 24. The method according to item 23, in which the second pharmaceutical composition is introduced into the organ of the donor in situ and / or ex vivo. 25. The method according to p, where the donor and recipient belong to the same species or to different species. 26. A method of increasing the functionality of a donor organ, comprising
(a) receiving an organ from a critical donor, and
(b) exposure of the resulting organ with a pharmaceutical composition containing carbon monoxide. 27. A method of maintaining an isolated animal cell in vitro prior to transplantation, comprising
(a) providing a vessel containing a pressurized gas containing carbon monoxide gas,
(b) obtaining an isolated cell in vitro, wherein the cell is a primary cell,
(c) releasing the pressurized gas from said vessel to form an atmosphere containing carbon monoxide gas, and
(d) maintaining the animal cell in vitro in the presence of an atmosphere containing carbon monoxide gas. 28. A method of maintaining an isolated animal cell in vitro prior to transplantation, comprising
(a) providing a culture medium containing at least 0.0001 g CO / 100 g medium; and
(b) maintaining an isolated cell in said medium. 29. The method according to item 27 or 28, further comprising administering the carbon monoxide composition to the recipient before transplantation, during transplantation, after transplantation, before and after transplantation, or before, during and after transplantation. 30. The method according to item 27 or 28, where the animal cell is a part of the islet of the pancreas, a liver cell, a β-cell of the pancreas, fibroblast, a bone marrow cell, a neural cell or a myocyte cell. 31. The use of an animal cell supported according to the method according to item 27, for transplantation. 32. The use of carbon monoxide to obtain a culture medium for maintaining the animal cells before transplantation to the recipient. 33. The application of clause 32, where the animal cell is obtained from a recipient or from a donor who is not a recipient. 34. The application of clause 32, where the animal cell is obtained from a donor by a method comprising
(a) administering to the donor a composition comprising carbon monoxide;
and
(b) obtaining cells from donor tissue. 35. A method of increasing the survival of an animal cell after its removal from a donor, comprising
(a) administering a pharmaceutical composition comprising carbon monoxide to a living donor or donor after the death of his brain, and
(b) obtaining an isolated cell from the donor, where the amount of carbon monoxide introduced to the donor is sufficient to increase cell survival after it is removed from the donor's body. 36. The method according to clause 35, where the pharmaceutical composition is a sealed gas. 37. The method according to clause 35, further providing
(c) maintaining the cell in vitro in the presence of a second pharmaceutical composition comprising carbon monoxide. 38. The method according to clause 37, where the cell from stage (C) is placed in a liquid medium. 39. The method according to § 38, where stage (c) is carried out by creating a source of pressurized carbon monoxide gas and contacting the liquid medium with carbon monoxide gas released from the source of pressurized carbon monoxide gas. 40. The method according to § 38, where the specified liquid medium includes carbon monoxide. 41. The method according to clause 35, where the cell is a part of the islet of the pancreas, a liver cell, β-cell of the pancreas, fibroblast, bone marrow cell, neural cell or myocyte cell. 42. A method of transplanting an animal cell, comprising
(a) administering to a living donor or donor after the death of his brain a pharmaceutical composition comprising carbon monoxide;
(b) obtaining from the body of the donor an isolated cell;
(c) transplanting the cell to the recipient, where the amount of carbon monoxide introduced to the donor is sufficient to increase cell survival after it is removed from the donor's body. 43. The method according to § 42, where the donor is not a recipient, or the donor and recipient are the same animal. 44. The method of claim 42, further comprising the step of administering to the recipient a second pharmaceutical composition comprising carbon monoxide before the transplantation stage, during the transplantation stage, after the transplantation stage, before and after the transplantation stage, or before, during and after the transplantation stage. 45. The method according to § 42, where the cell is a part of the islet of the pancreas, a liver cell, a β-cell of the pancreas, fibroblast, a bone marrow cell, a neural cell or a myocyte cell. 46. A method of increasing the survival or functioning of an animal cell transplanted to a recipient, comprising
(a) transplanting an animal cell to a recipient; and
(b) inhalation by the recipient of the pharmaceutical composition before, during and / or after the transplantation step, an amount of carbon monoxide sufficient to increase the survival or functioning of the transplanted cell in the recipient. 47. The method according to item 46, where the carbon monoxide gas is supplied in a vessel containing a sealed gas containing carbon monoxide. 48. The method of claim 46, further comprising the step of exposing the cell to the ex vivo carbon monoxide composition prior to the transplantation step. 49. The method according to item 46, where before the stage of transplantation, the animal cell is supported in a liquid medium that contains at least 0.0001 g of carbon monoxide / 100 g of medium. 50. The method according to item 46, where before the stage of transplantation, the cell is maintained in vitro in an atmosphere containing carbon monoxide. 51. The method according to item 46, where the cell is obtained from a recipient or from a donor who is not a recipient. 52. The method according to item 46, where the cell is removed from the body of the donor, before it is transplanted to the recipient; and a pharmaceutical composition comprising carbon monoxide is administered to the donor before and / or during the extraction of the cell from the donor organism. 53. The method according to item 46, where the cell is a part of the islet of the pancreas, a liver cell, a β-cell of the pancreas, a fibroblast cell, a bone marrow cell, a neural cell or a myocyte cell. 54. The use of carbon monoxide gas to prepare a pharmaceutical composition for improving the survival of a transplanted cell in a recipient after transplantation. 55. Set for maintaining an animal cell selected prior to, during or after the cell transplantation to a patient, comprising a vessel containing pressurized gas containing at least 0.001 million ^-1 carbon monoxide and a label describing a method of application of the gas to enhance survival of isolated animal cells before, during or after cell transplantation to the patient. 56. A sterile cell medium for maintaining an isolated animal cell before transplantation, containing (a) nutrients suitable for maintaining the animal cell in culture, and (b) at least about 0.0001 g of carbon monoxide / 100 g of medium. 57. A method of maintaining in vitro an isolated animal cell suitable for transplantation, comprising
(a) providing a vessel containing a pressurized gas containing carbon monoxide gas,
(b) obtaining an isolated animal cell in vitro, where the cell is placed in a medium containing dissolved carbon monoxide,
(c) releasing pressurized gas from said vessel to form an atmosphere containing carbon monoxide gas; and
(d) maintaining the cell in the presence of said atmosphere. 58. The use of carbon monoxide gas to provide an environment for maintaining an animal cell before transplantation to a recipient. 59. The use of an animal cell maintained according to the method of claim 57 for transplantation. 60. The method according to claim 20, where the pharmaceutical composition is administered to the recipient when it is established that the transplanted organ undergoes or is close to undergoing chronic rejection. 61. The use of carbon monoxide in the manufacture of a pharmaceutical composition for treating or preventing chronic rejection in a patient who is or is about to undergo chronic rejection of a transplanted organ or tissue. 62. The use of claim 61, wherein the pharmaceutical composition is in gaseous form. 63. Use according to claim 62, wherein the carbon monoxide gas is present in the composition in an amount of from 0.001 to 3000 million ^-1. 64. The use according to any one of claims 61 to 63, wherein the organ or tissue is a liver, kidney, heart, pancreas, small intestine or skin. 65. A method of treating or preventing chronic rejection in a patient who is or is about to undergo chronic rejection of a transplanted organ or tissue, comprising administering to the patient a pharmaceutical composition containing an effective amount of carbon monoxide. 66. The method of claim 65, wherein the pharmaceutical composition is in gaseous form. 67. The method of claim 66, wherein the carbon monoxide gas is present in the composition in an amount of from 0.001 to 3000 million ^-1. 68. The method according to any one of claims 65-67, wherein the organ or tissue is a liver, kidney, heart, pancreas, small intestine or skin. 69. The method according to claim 19, where the pharmaceutical composition is administered to the recipient at least once during the period starting from 21 days after (b), or repeatedly or continuously during the period starting from 21 days after (b).

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