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  Pharmaceutical Patents  

 

Title:  Preventing transfusion related complications in a recipient of a blood transfusion
United States Patent: 
7,648,699
Issued: 
January 19, 2010

Inventors:
 Goodrich; Raymond P. (Lakewood, CO), Li; Junzhi (Arvada, CO)
Assignee:
  CaridianBCT Biotechnologies, LLC (Lakewood, CO)
Appl. No.:
 11/469,186
Filed:
 August 31, 2006


 

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Abstract

This invention is directed toward a process for reducing transfusion related complications in a recipient of an allogeneic blood transfusion by adding to the blood to be transfused a photosensitizer comprising riboflavin, irradiating the blood and riboflavin with light, transfusing the irradiated blood into a recipient, and reducing a transfusion related complication by the recipient to cells in the donor blood. The invention is also directed towards a process for preventing rejection of a donor organ by a recipient comprising the steps of transfusing the recipient of the donor organ with treated platelets; and transplanting the donor organ into the recipient.

Description of the Invention

FIELD OF THE INVENTION

This invention is directed to methods of preventing transfusion related complications in a recipient of allogeneic donor blood.

BACKGROUND

Whole blood collected from volunteer donors for transfusion into recipients is typically separated into components: red blood cells, white blood cells, platelets, plasma and plasma proteins, using apheresis or other known methods. Each of these separated blood components may be stored individually for later use and are used to treat a multiplicity of specific conditions and disease states. For example, the red blood cell component is used to treat anemia, the concentrated platelet component is used to control bleeding, and the plasma protein component is used frequently as a source of Clotting Factor VIII for the treatment of hemophilia.

In cell separation procedures, there is unusually some small percentage of other types of cells which are carried over into a separated blood component. When contaminating cells are carried over into a separated component of cells in a high enough percentage to cause some undesired effect, the contaminating cells are considered to be undesirable. White blood cells, which may transmit infections such as HIV and CMV also cause other transfusion-related complications such as transfusion-associated Graft vs. Host Disease (TA-GVHD), alloimmunization and microchimerism.

TA-GVHD is a disease produced by the reaction of immunocompetent T lymphocytes of the donor that are histoincompatible with the cells of the recipient into which they have been transplanted. Recipients develop skin rashes, fever, diarrhea, weight loss, hepatosplenomegaly and aplasia of the bone marrow. The donor lymphocytes infiltrate the skin, gastrointestinal tract and liver. Three weeks following transfusion 84% of patients who develop TA-GVHD die.

Alloimmunization describes an immune response provoked in a recipient by an alloantigen from a donor of the same species. Alloantigens include blood group substances (A, B, O) on erythrocytes and histocompatibility antigens.

Chimerism, or microchimerism refers to the small numbers of donor cells found in the recipient's body outside the region of the organ transplant. It is believed that the presence of these cells may contribute to the long term development of autoimmune diseases in the transfusion recipient.

Human Leukocyte Antigen (HLA) markers are found on the cell membranes of many different cell types, including white blood cells. HLA is the major histocompatibility complex (MHC I) in humans, and contributes to the recognition of self v. non-self. Recognition by a transfusion recipient's immune system of differences in HLA antigens on the surface of the transfused cells may be the first step in the rejection of transplanted tissues. Therefore, the phenomena of alloimmunization of recipients against HLA markers on donor blood is a major problem in transfusion medicine today. This issue arises in recipients of blood products due to the generation of antibodies against white blood cell HLA antigens in donor blood by the recipient.

Platelets also contain low levels of these HLA antigens because they bud from a megakaryocyte cell (a form of white cell) located primarily in the bone marrow. When a recipient of a whole blood or blood component transfusion generates antibodies against the HLA antigens on the white blood cells of the donor blood cells, a consequence is that these antibodies also lead to recognition and clearance of platelets that carry this same marker. When this occurs, it becomes necessary to HLA match the donor and recipient in order to assure that the recipient receiving the transfusion is able to maintain an adequate number of platelets in circulation. This is often a complicated, expensive and difficult procedure but a necessary one, since rapid clearance of the platelets due to antibody-antigen interaction would otherwise put the recipient at severe risk of bleeding to death. In cases where recipients are very heavily transfused with blood or blood products from multiple donors and antibodies to several different HLA markers are generated, or where no suitable matched donor for platelets is available, death frequently results for those patients who become alloimmunized and sustain a bleed.

Since the problem arises from the presence of white cells in the donated blood products, the elimination of white cells from these products would be expected to reduce the likelihood and frequency of reactions. Gamma irradiation of blood products, which kills the cells but does not remove them from the blood product to be transfused, has not been shown to be able to prevent alloimmunization reactions. It is likely that this is due to the fact that the treated cells are still present and capable of presenting antigens to the recipient's immune system.

Filtration of white blood cells from blood or blood products to be transfused has been shown to be capable of reducing alloimmunization reactions. This has been demonstrated based on an extensive clinical study known as the TRAP study. It was conducted as a multi-institutional study between 1995-1997 and results were subsequently published in the NEJM in 1997 (Trial to Reduce Alloimmunization to Platelets Study Group. Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. N Engl J Med. 1997;337:1861-1869). The data from that study suggested that leukoreduction significantly decreased the likelihood of alloimmunization reactions in patients from 13% for non-leukoreduced, untreated products to 3-5% for leukoreduced products. As a result of this work, platelet products have been routinely filtered by a variety of methods to remove WBC. The remaining levels of alloimmunization that were observed were believed to be due to residual white blood cells that were not filtered out. Even the best WBC filters cannot remove 100% of the white blood cells and those left behind are potentially able to stimulate antibody production against the HLA markers on the remaining cells. A decrease in the occurrence rate from 13% of patients receiving platelets to 3-4% is significant, but still leaves several tens of thousands of cases occurring on an annual basis.

In the same TRAP study, treatment of platelet products with ultraviolet B (UVB) light was evaluated. In the case of the UVB treatment, the results were equivalent to those obtained through leukoreduction. The work was consistent with prior studies that showed that UVB treated platelet products possessed significantly reduced alloimmunization responses (Blundell et al. Transfusion 1996; 36: 296-302). This was believed to be due to changes in white cells induced by UVB that cause them to present their antigens and have those antigens processed differently from non-irradiated cells by the patient's immune system. The result is that antibody generation is significantly suppressed for UVB treated products. Although the results were positive, the UVB treatment described in the TRAP study was not adopted widely, because the UV dose required to suppress the alloimmunization response damaged the platelets to an extent which did not allow the platelets to be stored with adequate maintenance of their efficacy (Grijzenhout et al. Blood 1994; 84: 3524-3531).

Photosensitizers, or compounds which absorb light of a defined wavelength and transfer the absorbed energy to an electron acceptor may be a solution to the above problems, by inactivating undesirable cells contaminating a blood product without damaging the desirable components of blood.

There are many photosensitizer compounds known in the art to be useful for inactivating undesirable cells and/or other infectious particles. Examples of such photosensitizers include porphyrins, psoralens, dyes such as neutral red, methylene blue, acridine, toluidines, flavine (acriflavine hydrochloride) and phenothiazine derivatives, coumarins, quinolones, quinones, anthroquinones and endogenous photosensitizers.

As described above, ways to reduce the risks of transfusion related complications from white blood cells is either to reduce the number of white blood cells transfused into a recipient to an extent that no immune response is generated, and/or to effectively destroy the viability and capacity of any transfused white blood cells to function post transfusion.

What is not known is whether donor cells which have been subjected to pathogen reduction treatment with riboflavin and light have modified HLA surface markers, and therefore will not cause transfusion related complications such as alloimmunization, GVHD and microchimerism in the recipient.

It is to this second aspect that this invention is directed.

SUMMARY OF THE INVENTION

This invention is directed toward a process for reducing transfusion related complications in a recipient of an allogeneic blood transfusion by adding to the blood to be transfused a photosensitizer comprising riboflavin, irradiating the blood and riboflavin with light, transfusing the irradiated blood into a recipient, and reducing a transfusion related complication by the recipient to cells in the donor blood.

Also claimed is a blood product for transfusion into a recipient comprising inactivated blood or a blood product which has been treated with riboflavin and light. The treated blood or blood product will not cause transfusion related complications in the recipient when transfused.

The invention is also directed towards a process for preventing rejection of a donor organ by a recipient comprising the steps of transfusing the recipient of the donor organ with treated platelets; and transplanting the donor organ into the recipient.

DETAILED DESCRIPTION

Photosensitizers useful in this invention include endogenous photosensitizers. The term "endogenous" means naturally found in a human or mammalian body, either as a result of synthesis by the body or because of ingestion as an essential foodstuff (e.g. vitamins) or formation of metabolites and/or byproducts in vivo. When endogenous photosensitizers are used, particularly when such photosensitizers are not inherently toxic or do not yield toxic photoproducts after photoradiation, no removal or purification step is required after decontamination, and the decontaminated product can be directly administered to a recipient in need of its therapeutic effect.

Examples of such endogenous photosensitizers which may be used in this invention are alloxazines such as 7,8-dimethyl-10-ribityl isoalloxazine (riboflavin), 7,8,10-trimethylisoalloxazine (lumiflavin), 7,8-dimethylalloxazine (lumichrome), isoalloxazine-adenine dinucleotide (flavin adenine dinucleotide [FAD]) and alloxazine mononucleotide (also known as flavin mononucleotide [FMN] and riboflavine-5-phosphate). The term "alloxazine" includes isoalloxazines.

Use of endogenous isoalloxazines as a photosensitizer to pathogen reduce blood and blood components are described in U.S. Pat. Nos. 6,258,577 and 6,277,337 both issued to Goodrich et al., and are herein incorporated by reference to the amount not inconsistent.

The process of using endogenous alloxazine and light to reduce the risks of transfusion related complications from contaminating white blood cells in blood or blood products are shown in FIG. 1 (see Original Patent).

Whole blood to be transfused into a recipient is collected from a donor. If desired, the whole blood may be separated into blood components using any available procedures and/or extracorporeal blood processing machines. 50 .mu.M riboflavin in PBS is added to the whole blood or separated blood components. The blood product and riboflavin are illuminated at a wavelength of between about 290-370 nm for a sufficient amount of time to reduce the number of white blood cells present in the donor blood or blood product to an extent that no immune response to the donor blood is generated by the transfusion recipient, and/or to effectively destroy the viability and capacity of any transfused donor white blood cells to function in the recipient post transfusion. An illumination time of around 8 minutes appears to be satisfactory. The inactivated blood product is ready to be transfused into a donor.

The following examples show that allogeneic and xenogeneic donor cells subjected to a pathogen reduction treatment with riboflavin and light will not cause transfusion related complications in a donor such as alloimmunization, TA-GVHD and microchimerism.

EXAMPLE 1

The intent of this study was to determine whether human peripheral blood mononuclear cells (PBMNCs) treated with riboflavin and light (hereinafter known as treated cells) could be induced to proliferate in vitro when exposed to a growth stimulus, or whether the treated cells were rendered inactive by the treatment, and therefore could not be induced to proliferate. Untreated cells (control) are those human PBMNCs not treated with riboflavin and light.

For this study, PBMNC were obtained from three human donors, with each donor set being split into a treated and untreated subset. Each subset was subsequently tested using the in vitro test methods described below. PBMNC were isolated from platelets obtained from the donors using a standard apheresis procedure on a Trima.RTM. apheresis machine (available from Gambro BCT, Lakewood, Colo., USA). For treatment with riboflavin and light, the cells were added to ABO-matched platelet-poor plasma (PPP), which was then mixed with riboflavin and illuminated according to the procedure shown in FIG. 1.

CD3 is the signaling complex of the T lymphocyte cell receptor. Anti-CD3.sup.+ antibody has been shown to induce proliferation of T cells. CD28 is a low affinity T cell receptor that interacts with B7 (ligand for CD28). CD28 is considered a co-stimulatory receptor because its signals are synergistic with those provided by the CD3 receptor in promoting T cell activation and proliferation. Signals from CD28 to the CD3 receptor also increase the synthesis of many cytokines. Cytokines are produced primarily by lymphocytes in response to a stimulus. Production of cytokines is therefore a measure of white blood cell health.

Preparation of CD3, CD3/CD28 or Control Coated Plates

PBS containing 10 .mu.g/mL of anti-CD3 (NA/LE, Pharmingen), 10 .mu.g/mL anti-CD3 and 4 .mu.g/mL anti-CD28 (NA/LE, Pharmingen) or PBS alone were added to wells (50 .mu.l per well) in a 96 well flat bottom plate. The plates were incubated for at least 90 minutes at room temperature. Following 2 washes of the wells with PBS, 100 .mu.l of RPMI 1640 media containing 5% human AB serum, penicillin and streptomycin was added to all wells and the plates were incubated at room temperature for at least another 60 minutes. Then 100 .mu.l of the treated or untreated cells at 2.times.10.sup.6 cells/ml in RPMI 1640 containing 5% human AB serum, penicillin and streptomycin were added to the wells (replicate 6 wells per group).

1. A. The effect of treatment with riboflavin and light on the ability of PBMNC to proliferate in response to CD3 and CD3/CD28 stimulation.

As shown in FIG. 2 (see Original Patent), anti-CD3 antibody induced significant proliferation of untreated (designated as Control cell+CD3 in FIG. 2) cells in all 3 donors. The combination of anti-CD3 and anti-CD28 antibodies further increased proliferation of untreated (Control cell+CD3/CD28) cells. Both treated (designated as Tx+medium in FIG. 2) and untreated (Control cell+medium) cells present in media alone exhibited minimal proliferation. In contrast, the treated PBMNCs did not proliferate in response to either anti-CD3 (Tx cell+CD3) or anti-CD3/CD28 antibody (Tx cell+CD3/CD28) stimulus.

1. B. The Ability of the Treated or Untreated PBMNC to Produce Cytokines

A comparison of the levels of cytokines present in the supernatants of the wells after 2 days in culture indicated that both anti-CD3 antibody (Control cell+CD3) and anti-CD3/CD28 antibodies (Control cell+CD3/CD28) induced increased cytokine production by the untreated PBMNCs. As shown in FIG. 3 (see Original Patent), higher levels of some cytokines could be detected in the wells containing control cells and medium alone (Control cell+medium). However, the treated cells (Tx cell+CD3; Tx+CD3/CD28 or Tx cell+medium) did not produce cytokines in any of the wells, even in the media control. This data demonstrates that the treated leukocytes are unresponsive in that they do not exhibit any significant proliferation or cytokine production.
 

Claim 1 of 9 Claims

1. A process for reducing transfusion related complications in a human recipient due to human donor cells that may be present in an allogeneic blood transfusion comprising the steps of: adding to the blood to be transfused a photosensitizer consisting essentially of riboflavin at a concentration of 50 .mu.M; irradiating the blood and riboflavin with light at a wavelength between 290-370 nm for around 8 minutes to reduce transfusion related complications in the recipient caused by the donor cells; wherein the transfusion related complications are alloimmunization, Transfusion Associated Graft vs. Host Disease (TA-GvHD) or microchimerism; transfusing the irradiated blood into a recipient; and reducing transfusion related complications in the recipient.

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