WO2022036335A1

WO2022036335A1 - Organ transplant perfusion solution modifications using bryostatin-1

Info

Publication number: WO2022036335A1
Application number: PCT/US2021/046717
Authority: WO
Inventors: Trevor Castor; J. Steven Alexander; Luke WHITE
Original assignee: Louisiana State University
Current assignee: Louisiana State University
Priority date: 2020-08-09
Filing date: 2021-08-19
Publication date: 2022-02-17
Anticipated expiration: 2023-02-09

Abstract

Solutions and methods for preserving tissues or organs comprising administering to a harvested tissue or a harvested organ a composition comprising bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof and a protein free carrier including Polyethylene glycol-Ethanol-Tween 80 for between 10.0 minutes and 1.0 minutes and washing the tissue or organ with a saline solution after administering the composition, wherein the saline solution is substantially free of bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof, the composition is administered only less than 30 minutes before the tissue or organ is transplanted into a patient in need thereof, and a concentration of bryostatin-1 in an intravenous location of the tissue or organ after washing is below 10-9 M, below 10-10 M, or effectively zero M.

Description

[0001] ORGAN TRANSPLANT PERFUSION SOLUTION MODIFICATIONS USING BRYOSTATIN-1

[0002] CROSS REFERENCE TO RELATED APPLICATIONS/PRIORITY

[0003] The present invention claims priority to United States Provisional Patent Application Number 63063382 filed August 9, 2020, which is incorporated by reference into the present disclosure as if fully restated herein. Any conflict between the incorporated material and the specific teachings of this disclosure shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this disclosure shall be resolved in favor of the latter.

[0004] BACKGROUND

[0005] Leukocyte dependent injury is a significant risk in harvested organs for transplantation. It is desirable to have therapies which reduce neutrophil infiltration into transplanted organs and tissue. For the foregoing reasons, there is a pressing, but seemingly irresolvable need for treating transplanted tissues and organs.

[0006] SUMMARY

[0007] Wherefore, it is an obj ect of the present invention to overcome the above mentioned shortcomings and drawbacks associated with the current technology.

[0008] The presently disclosed invention relates to a novel composition of organ perfusion solution which reduces the concentration of plasma protein for delivering Bryostatin-1 as a transplant stabilizer

[0009] The presently disclosed invention relates to for treating transplantation according to an embodiment of the invention characterized in that it includes washing of organs with Bryostatin-1 or one of its derivatives under conditions which permits vascular selective exposure to these compounds only once, for a short time and then washing off, immediately before implanting the organ into the organ recipient, and not exposing the organ to Bryostatin-1 contemporaneously with organ ‘harvesting’ [0010] The presently disclosed invention relates to solutions and methods for preserving tissues or organs comprising, administering to a harvested tissue or a harvested organ a composition comprising bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof, wherein the composition is administered less than 30 minutes before the tissue or organ is transplanted into a patient in need thereof. According to a further embodiment the method further comprises only administering the composition less than 30 minutes before the tissue or organ is transplanted into a patient in need thereof. According to a further embodiment the method further comprises the composition to the tissue or organ for between 10.0 minutes and 1.0 minutes. According to a further embodiment the method further comprises washing the tissue or organ with a saline solution after administering the composition, wherein the saline solution is substantially free of bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof. According to a further embodiment the composition comprises a protein free carrier. According to a further embodiment the carrier is Polyethylene glycol-Ethanol-Tween 80. According to a further embodiment the composition includes the bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an organ preservation solution at a concentration of between 1.0 to 5000.0 nanograms per milliliter. According to a further embodiment the composition includes the bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an amount of 0.00005% to 0.500% by weight. According to a further embodiment the composition includes the bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an amount in an amount of 0.001 to 0.1% by weight. According to a further embodiment sufficient composition is administered such that an endothelial cell concentration in the tissue or organ is at a level of at least 10'⁶ M. According to a further embodiment the level in the endothelial cell concentration of the organ is at least 10'⁶ M after washing the organ with a saline solution. According to a further embodiment an intravenous level of Byrostatin-1 in the organ after washing is less than 10'⁹ M, less than 10'¹⁰ M, or effectively zero M.

[0011] The presently disclosed invention further relates to trans-endothelial migration prevention solutions comprising between one of 10.0 uM and 0.01 uM , 5.0 uM and 0.05 uM, and 1.0 uM and 0.5 uM of Bryostatin-1, Bryostatin-1 analog or a pharmaceutically acceptable salt thereof, and a dosage form comprising a protein free carrier. According to a further embodiment the carrier is Polyethylene glycol-Ethanol- Tween 80. According to a further embodiment the composition includes the bryostatin- 1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an organ preservation solution at a concentration of between 1.0 to 5000.0 nanograms per milliliter. According to a further embodiment the composition includes the bryostatin- 1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an amount of 0.00005% to 0.500% by weight.

[0012] The presently disclosed invention further relates to methods for preserving tissues or organs comprising administering to a harvested tissue or a harvested organ a composition comprising bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof and a protein free carrier including Polyethylene glycol-Ethanol- Tween 80 for between 10.0 minutes and 1.0 minutes and washing the tissue or organ with a saline solution after administering the composition, wherein the saline solution is substantially free of bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof, the composition is administered only less than 30 minutes before the tissue or organ is transplanted into a patient in need thereof, and a concentration of bryostatin-1 in an intravenous location of the tissue or organ after washing is below 10'⁹ M, below 10'¹⁰ M, or effectively zero M.

[0013] Various objects, features, aspects, and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention, along with the accompanying drawings in which like numerals represent like components. The present invention may address one or more of the problems and deficiencies of the current technology discussed above. However, it is contemplated that the invention may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the claimed invention should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS [0015] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate various embodiments of the invention and together with the general description of the invention given above and the detailed description of the drawings given below, serve to explain the principles of the invention. It is to be appreciated that the accompanying drawings are not necessarily to scale since the emphasis is instead placed on illustrating the principles of the invention. The invention will now be described, by way of example, with reference to the accompanying drawings in which:

[0016] Fig. 1 shows the chemical structure of Bryostatin-1, according to the disclosed invention;

[0017] Fig. 2 shows evidence that Bryostatin-1 inhibition of neutrophil emigration in liver (HHSEC) and kidney (HRGEC) endothelium is partially dependent of PKC in kidney but (apparently) not in in hepatic endothelium (n = 4 replicates). Go 6983 : broad PKC inhibitor; GF 109203X: broad PKC inhibitor; Go 6976: PKC a and pi inhibitor;

[0018] Figs. 3A and 3B show Bryostatin-1 inhibition of neutrophil emigration across kidney (HRGEC, iHRGEC) endothelium depends upon Bryostatin-1 transfer from vascular cell membrane (or environment) to neutrophils, with Fig. 3A showing Neutrophil TEM measured by Calcein AM (n = 4 replicates. ** p < 0.01), and Fig. 3B showing Neutrophil migration through 8.0 um inserts coated with fibronectin in the absence of endothelial cells, measured by MPO assay (n = 4 replicates ** p<0.01);

[0019] Figs. 4A and 4B show Bryostatin-1 induced neutrophil release of myeloperoxidase and evidence for a second mechanism of protection, with Fig. 4A showing Bryostatin-1 stimulates neutrophil release of MPO, leading to a decreased signal in the MPO assay (n= 4 replicates), and Fig. 4B showing MPO assay of HRGEC s/iHRGECs on transwells followed by the addition of neutrophils, Bryostatin- 1 pre- treatment results in a decrease of MPO signal in the lower (trans-migrated) chamber (n = 4 replicates, •••• p < 0.0001 ), and Fig. 4C showing a Western blot of MPO on supernatant collected from isolated neutrophils incubated with Bryostatin-1;

[0020] Fig. 5 shows Bryostatin-1 reduces MPO 'cargo' in neutrophils, diminished transport release of myeloperoxidase, MPO assay of HRGEC S/iHRGECs on transwells followed by the addition of neutrophils, Bryostatin-1 pre-treatment results in an increase of MPO signal in the upper (trans-migrated) chamber (n = 4 replicates, p < 0.0001);

[0021] Fig. 6 shows Bryostatin-1 induces 'NETosis' as a mechanism of transplant protection, non-permeabilized photographs of phase (top) and nuclear DAPI staining (bottom) of neutrophils treated with Bryostatin-1 (10'⁷M) demonstrates formation of NETs, inset figure shows higher magnification closeup of extruded NETs seen as elongated figures and non-uniform contours;

[0022] Fig. 7 is a schematic diagram of a working hypothesis of the mechanism of

Bryostatin-1 protection;

[0023] Fig. 8 is a schematic diagram of the neutrophil transendothelial migration and MPO assay used by the inventors;

[0024] Fig. 9 is a set of four graphs showing Bryostatin-treated endothelial cells attenuate neutrophil TEM;

[0025] Figs. 10 and 11 are schematic diagrams of the Transwell shear ring setup for flow experiment used by the inventors;

[0026] Fig. 12 is a set of four graphs showing Bryostatin-treated endothelial cells inhibit neutrophil TEM under flow;

[0027] Fig. 13 is a schematic diagram of elements of the cold storage experiment used by the inventors;

[0028] Fig. 14 is a set of four graphs that show overnight cold storage Bryostatin- treated endothelial cells inhibits neutrophil TEM; and

[0029] Fig. 15 is a set of four graphs showing an in vivo bridge study tissue analysis.

[0030] DETAILED DESCRIPTION

[0031] The present invention will be understood by reference to the following detailed description, which should be read in conjunction with the appended drawings. It is to be appreciated that the following detailed description of various embodiments is by way of example only and is not meant to limit, in any way, the scope of the present invention. In the summary above, in the following detailed description, in the claims below, and in the accompanying drawings, reference is made to particular features (including method steps) of the present invention. It is to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features, not just those explicitly described. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention or a particular claim, that feature can also be used, to the extent possible, in combination with and/or in the context of other particular aspects and embodiments of the invention, and in the invention generally. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and grammatical equivalents and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures, are used herein to mean that other components, ingredients, steps, etc. are optionally present. For example, an article “comprising” (or “which comprises”) components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes that possibility).

[0032] The term “at least” followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example “at least 1” means 1 or more than 1. The term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40% means 40% or less than 40%. When, in this specification, a range is given as “(a first number) to (a second number)” or “(a first number)-(a second number),” this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100 mm.

[0033] The embodiments set forth the below represent the necessary information to enable those skilled in the art to practice the invention and illustrate the best mode of practicing the invention. For the measurements listed, embodiments including measurements plus or minus the measurement times 5%, 10%, 20%, 50% and 75% are also contemplated. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0034] In addition, the invention does not require that all the advantageous features and all the advantages of any of the embodiments need to be incorporated into every embodiment of the invention.

[0035] Turning now to Figs. 1 - 15, a brief description concerning the various components of the present invention will now be briefly discussed

[0036] The inventors disclose herein novel Bryostatin-1 perfusion methods and solutions which can be used to briefly (e.g., between 45.0 minutes and 5.0 minute, preferably between 30.0 minutes and 1.0 minute, and most preferably less than 5minutes) prewash donor organs substantially immediately (e.g., between 15.0 minutes and 0.1 minutes) before implantation.

[0037] This novel organ treatment method minimizes the amount of time for exposure to Bryostatin-1 and allows for a rapid and more efficacious application of Bryostatin-1 to the surface of the vascular space of the donor organ. This also minimizes the number of hospital locations which might need Bryostatin-1, as only the organ recipient institution would need to keep Bryostatin-1 available. Importantly, the reduction of protein in the treatment solution will maximize Bryostatin-1 effects on immune cells in the donor organs.

[0038] In one embodiment of the current application, Bryostatin- 1 is used as an endothelial cell independent effect, where Bryostatin-1, which has adsorbed onto the vascular surface, is acquired from the endothelial cell which binds to marginating neutrophils and interferes with their penetration and extravasation mediated by induction of netosis, which causes localized apoptosis of neutrophils, effectively preventing their extravasation and/or chemotaxis. This is in addition to the described effects of Bryostatin-1 on endothelial cells where Bryostatin-1 acts to persistently stabilize the monolayer against leukocyte emigration in a neutrophil-independent manner.

[0039] This approach is believed to be a completely novel formulation of Bryostatin-1, which is currently taught in the art to be used for perfusion into donor organs in a protein solution, such as the UW solution. This Bryostatin-1 plus low / no-protein solution allows for much greater surface of the Bryostatin-1 binding to the donor organ rather than binding to the plasma proteins, which are present in most organ perfusion solutions. This higher binding solution will greatly enhance the amount of drug delivered to the organ immediately before implantation and minimize both the amount of Bryostatin-1 which needs to be used as a therapeutic dose, minimizing cost and reducing overall exposure of the recipient to Bryostatin-1. Furthermore, this approach also utilizes the evidence that the timing for Bryostatin-1 drug delivery is much more effective acutely when given as a ‘backtable’ flush’ minutes before implantation rather than its use as an additive instilled into the donor organ at the time of organ collection and then throughout cold-storage and transportation. This is significant because it enormously simplifies the way in which Bryostatin-1 can be used. This also reduces the requirement for the organ procurement site to have Bryostatin-1, which might be difficult to keep available / accessible at all points of organ collection. This is a huge competitive advantage in that Bryostatin-1 might only be needed to be maintained at implantation centers.

[0040] Bryostatin-1 was used in an endothelial cell-contact independent mode to locally inhibit neutrophil emigration / action in liver and kidney endothelial models (Fig. 2). Using cell models (both transformed and non-transformed cells) the inventors were able to demonstrate for the first time that in addition to Bryostatin-T s potent effects on endothelial cells, adsorbed Bryostatin-1 is also transferred in a contact-dependent manner from endothelial cell and/or environmental surfaces to neutrophils, where it still appears to impair leukocyte emigration across endothelial monolayers towards a chemotactic gradient of LTB4, an inflammatory signal. Strikingly, this inhibition of neutrophil trans-endothelial migration takes place even in the absence of an endothelial monolayers (Fig. 3). Therefore, while the inventors previously described an endothelial-dependent mechanism of Bryostatin-1, the inventors have now identified a second set of mechanisms which are neutrophil-dependent, but endothelial independent. The inventors further describe emigration studies that have relied on myeloperoxidase within neutrophils as a marker of neutrophil abundance.

[0041] Bryostatin-1 triggers acute neutrophil myeloperoxidase exocytosis. As an important control, the inventors evaluated whether the apparent decrease in neutrophil emigration observed with Bryostatin-1 might potentially reflect changes in the neutrophil content of myeloperoxidase under these conditions. Fig. 4 shows that Bryostatin-1 treatment of neutrophils causes a potent release of myeloperoxidase from neutrophils. Fig. 4A shows that myeloperoxidase content of neutrophils was similar in control or leukotriene B4 stimulated kidney endothelial models. However, the content of myeloperoxidase in Bryostatin-1 or Bryostatin-1 +LTB4 treated monolayers was significantly diminished. This shows that at least part of the benefit of Bryostatin-1 in kidney transplant models must reflect the shedding of myeloperoxidase from the neutrophil before the neutrophil can migrate as well as preventing migration into injured tissues. Therefore, at least part of the protection seen in the reduction of neutrophil signal in emigration studies (Fig. 4B) reflects the exocytosis of neutrophil myeloperoxidase. This finding is confirmed in Fig. 4C, which shows a western blot for the presence of myeloperoxidase (MPO) in the supernatants from control, LTB4 (10‘⁷ M treated) or Bryostatin-1 (10'⁷ M) treated neutrophils. MPO is dramatically released only in the presence of Bryostatin-1.

[0042] Neutrophil contact with Bryostatin-1 coated surfaces prevents leukocyte retention of MPO. Fig. 5 shows that in models of neutrophil emigration, MPO appears to be left behind in the abluminal chamber when these chambers had been treated and washed extensively with buffer to remove non-adsorbed Bryostatin-1 (both normal kidney and developed kidney endothelial cell lines were used). This finding shows that the MPO content of the neutrophils which migrate is diminished in addition to suppressing their migration. Because MPO is an essential component of tissue injury in the setting of transplantation, the observation that Bryostatin-1 both reduces emigration as well as the MPO ‘cargo’ of the remaining migrated neutrophils, the inventors now recognize the therapeutic value of applying Bryostatin-1 as a brief ‘back table’ setting flush additive, compared to requiring Bryostatin-1 to be administered in advance of implantation. This finding is striking in that it demonstrates a dramatically different clinical application of Bryostatin-1, such that Bryostatin-1 may only be required at right before implantation, not at the time of collection. That is to say, cold storage injury may ‘prime’ tissues for injury by neutrophils which can be controlled by a brief perfusion with Bryostatin-1. This represents further benefit in that the organ does not have to be exposed to Bryostatin-1 for an extended period of time, nor does the organ carry an appreciable amount of Bryostatin-1 back into the patient to enter into the patient’s bloodstream.

[0043] Bryostatin-1 adsorbed onto environmental/cellular surface induces ‘NETosis’ as a mechanism of transplant protection. Based on the disclosed findings, which have demonstrated the remarkable ability of Bryostatin-1 to interfere with leukocyte emigration and to trigger the ‘shedding’ of the toxic oxidant generating enzyme myeloperoxidase, the inventors next considered whether such activation also triggers ‘NETosis’ or the emergence of DNA ‘NETs’, a pre-apoptotic activation response which might explain the neutrophil-selective, endothelial independent actions of Bryostatin-1. The inventors again observed that Bryostatin-1 causes dramatic changes in the staining patterns of fluorescently stained neutrophils (using DAPI nuclear staining) (Fig. 6). Therefore, the inventors developed a working understanding of the beneficial effects of Bryostatin-1 in the setting of transplantation are shown in Figure 7.

[0044] The Bryostatin-1 solution involves the use of a protein-free saline solution which employs a Bryostatin-1 -carrier (e.g. Polyethylene glycol -Ethanol-Tween 80). The organ should preferably be perfused shortly before implantation with up to 500ml of Bryostatin-1 solution, including 10'⁶ M Bryostatin-1 in saline including Bryostatin-1 ‘carrier’ (e.g., Polyethylene glycol -Ethanol-Tween 80). In one embodiment, the organ is perfused with Bryostatin-1 solution for 5-10 minutes before implantation. After perfusion, the organ is then immediately washed with saline buffer or saline buffer with protein, and then immediately implanted. The time period from initial organ contact with Bryostatin-1 to organ implantation into the patient is preferably between 30.0 minutes and 1.0 minute, more preferably less than 15 minutes, more preferably less than 10 minutes. The concentration of Bryostatin-1 ‘carrier’ is 0.1-0.5% with the Bryostatin-1 concentration in the solution preferably between 0.01 pM and 0.5 pM, and more preferably equal to 0.1 pM molarity. This solution differs from the perfusion solutions used in previous technology at least in that by the novel solution excluding protein, the novel solution makes the vascular wall/endothelial layer the main target for binding of Bryostatin- 1.

[0045] Unlike in current technology, where a pre-treatment Bryostatin-1 perfusion is administered when the donor organ is collected, this novel procedure only needs to be given to the donor organ substantially immediately before implantation (not at harvesting). This greatly simplifies the number of sites which must maintain Bryostatin-1. Furthermore, by only administering Bryostatin-1 solution minutes before the organ is implanted greatly minimizes the time of organ exposure to Bryostatin-1. As before, the wash-out procedure removes the Bryostatin-1 before implantation so that none, or substantially no Bryostatin-1 is actually transferred to the recipient, substantially reducing the risks of toxic side effects of the chemical, which have included myalgia, fever, flu-like symptoms, fatigue, anemia, transient thrombocytopenia, phlebitis, headache, hypotension, bradycardia, flushing, dyspnea, photophobia and eye pain

[0046] Bryostatin-1, in addition to its effects on endothelial cells via PKC (Protein kinase C), also adsorbs to the surfaces of endothelial cells where it triggers MPO degranulation and neutrophil extracellular traps (NETs) DNA ‘Netosis’ both of which dramatically reduce the capacity for neutrophils to migrate and create injury within post-ischemic transplanted tissues. An important consideration for this effect is that when Bryostatin- 1 is adsorbed to the vascular lining is that it becomes tightly bound and is not readily released even by extensive washing. Therefore, organs which are briefly ‘flushed’ with Bryostatin-1 at the time of implantation (rather than at the time of harvesting) would become resistant to leukocyte infiltration and delayed graft injury/primary nonfunction. Importantly, this effect should not be observed systemically, as the concentration of biologically active and adsorbed, (and subsequently released) Bryostatin-1 would be well below the concentration sufficient to produce a generalized interference with leukocyte motility or activation (i.e., immunosuppression).

[0047] This disclosure is related to solutions and methods of treating transplantation injury in a human, comprising the steps of administering to a harvested organ an effective amount of Bryostatin-1, Bryostatin-1 analog or a pharmaceutically acceptable salt thereof to reduce or prevent induced neutrophil trans endothelial migration, wherein said effective amount of Bryostatin-1, Bryostatin-1 analog or a pharmaceutically acceptable salt thereof is held in a dosage form; and wherein the dosage form comprises protein free carrier. The carrier may be Polyethylene glycol-Ethanol-Tween 80. In a further embodiment, the effective amount of Bryostatin-1, Bryostatin-1 analog or pharmaceutically acceptable salt thereof in an organ preservation solution may be at a concentration of 1.0 to 5000.0 nanograms per milliliter. In a further embodiment the organ is administered Bryostatin-1, Bryostatin-1 analog or a pharmaceutically acceptable salt only within an hour of implanting the organ into the human.

[0048] Bryostatin, or Bryostatin-1 is used in its conventional scientific meaning to encompass Bryostatin-1 or any compound which is based on the Bryostatin structural backbone. As used herein, the term “Bryostatin-1 analog” means a composition having the general formula of Brystatin-1 with substitutions comprising methyl or ethyl groups or halogens and ammonium groups which do not substantially alter the biological activity of the composition. Bryostatin-1 has a chemical formula of C47H68O17 and a chemical structure as shown if Fig. 1.

[0049] Turning next to Figs. 8 - 15, continuing experiments are disclosed. As shown in Fig. 8, the inventors used an in vitro model of trans-endothelial migration to evaluate how neutrophils would attach and migrate across endothelial monolayers in response to a chemoattractant, LTB4.

[0050] As demonstrated by the graphs in Fig. 9, these data show that neutrophils migrate across hepatic and renal glomerular endothelial monolayers (HHSEC, HRGEC) in response to the chemoattractant LTB4 in either non-immortalized or immortalized endothelial cultures (iHHSEC, H4RGEC). Bryostatin-1 (“Bryo”) significantly reduced the migration in all cases to nearly zero. [0051] As shown in Fig. 10, the inventors used a shear ring model of fluid flow to evaluate how neutrophils would attach and migrate across endothelial monolayers under conditions simulating blood flow in vivo.

[0052] As shown in Fig. 11, neutrophils were applied to endothelial monolayers in the presence and absence of Bryostatin-1 and migration evaluated after three hours of simulated flow in vitro.

[0053] As shown in Fig. 12, under conditions simulating blood flow in vivo the inventors found that Bryostatin-1 still reduced the migration of neutrophils across endothelial monolayers derived from liver or renal glomerular endothelial cells in both the normal (HHSEC, HRGEC) and immortalized states (iHHSEC, iHRGEC).

[0054] As shown in Fig. 13, the inventors used a model of organ cold storage solution to evaluate how neutrophils would attach and migrate across endothelial monolayers subjected to conditions simulating cold storage ex vivo.

[0055] As shown in Fig. 14, under conditions simulating 20h of organ cold storage ex vivo the inventors found that Bryostatin-1 still significantly reduced the migration of neutrophils across endothelial monolayers derived from liver or renal glomerular endothelial cells in both the normal (HHSEC, HRGEC) and immortalized states (iHHSEC, iHRGEC).

[0056] It was evidenced in the experiments of Figs. 8 - 14 that Bryostatin-1 inhibits neutrophil TEM: when added prior to PMN (granular leukocyte) exposure; under conditions of flow resembling flow in vivo; when added to organ cold storage solutions.

[0057] The inventors next conducted an in vivo Bridge Study. The hypothesis was that Bryostsatin-1 limits neutrophil transmigration following ischemia reperfusion injury and prevents delayed renal function in a porcine autotransplant model. The porcine kidney autotransplantation model was used to test the hypothesis. Briefly, animals had surgery for graft retrieval followed by a standard graft cold storage time (CST) of 20h. During CST, kidneys were preserved with histidine-tryptophan-ketoglutarate (HTK)- solution either supplemented with 100 nM Bryostatin-1 or an equivalent volume of Placebo (PET formulation). Donor animals then had surgery again for renal autotransplantation and the graft was transplanted orthotopically. Thus, the contralateral kidney was removed to specifically measure the transplanted kidney function.

[0058] The time of reperfusion was 8h and sample collection included cortical renal biopsies (snap frozen and formalin fixed), tissue block samples at the end of the study and blood as well as urine sample

[0059] As shown in Fig. 15, Bryostatin-1 reduced the penetration of neutrophils across kidney vascular layers, reduced ultrastructural and histology damage and reduced IL-8 at 8h of reperfusion. The upper left graph shows that Neutrophils are blocked from entering the kidney. The upper right graph shows that Kidney ultrastructural damage scores are reduced. The bottom left graph shows Plasma IL-8 is reduced at 8 hours. The bottom right graph shows kidney histology score at 8 hours is improved.

[0060] The invention illustratively disclosed herein suitably may explicitly be practiced in the absence of any element which is not specifically disclosed herein. While various embodiments of the present invention have been described in detail, it is apparent that various modifications and alterations of those embodiments will occur to and be readily apparent those skilled in the art. However, it is to be expressly understood that such modifications and alterations are within the scope and spirit of the present invention, as set forth in the appended claims. Further, the invention(s) described herein is capable of other embodiments and of being practiced or of being carried out in various other related ways. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. In addition, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items, while only the terms “consisting of’ and “consisting only of’ are to be construed in the limitative sense.

Claims

Wherefore, I/we claim:

1. A method for preserving tissues or organs comprising: administering to a harvested tissue or a harvested organ a composition comprising bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof; wherein the composition is administered less than 30 minutes before the tissue or organ is transplanted into a patient in need thereof.

2. The method of claim 1 further comprising only administering the composition less than 30 minutes before the tissue or organ is transplanted into a patient in need thereof.

3. The method of claim 1 further comprising administering the composition to the tissue or organ for between 10.0 minutes and 1.0 minutes.

4. The method of claim 1 further comprising washing the tissue or organ with a saline solution after administering the composition, wherein the saline solution is substantially free of bryostatin- 1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof.

5. The method of claim 1 wherein the composition comprises a protein free carrier.

6. The method of claim 5 wherein the carrier is Polyethylene glycol-Ethanol-Tween 80.

7. The method of claim 1 wherein the composition includes the bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an organ preservation solution at a concentration of between 1.0 to 5000.0 nanograms per milliliter.

8. The method of claim 1, wherein the composition includes the bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an amount of 0.00005% to 0.500% by weight.

9. The method of claim 1, wherein the composition includes the bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an amount in an amount of 0.001 to 0.1% by weight.

10. The method of claim 1, wherein sufficient composition is administered such that an endothelial cell concentration in the tissue or organ is at a level of at least 10'⁶ M.

11. The method of claim 8 wherein the level in the endothelial cell concentration of the organ is at least 10'⁶ M after washing the organ with a saline solution.

12. The method of claim 9, wherein an intravenous level of Byrostatin-1 in the organ after washing is less than 10'⁹ M, less than 10'¹⁰ M, or effectively zero M.

13. A trans-endothelial migration prevention solution comprising: between one of 10.0 uM and 0.01 uM , 5.0 uM and 0.05 uM, and 1.0 uM and 0.5 uM of Bryostatin-1, Bryostatin-1 analog or a pharmaceutically acceptable salt thereof, and a dosage form comprising a protein free carrier.

14. The trans-endothelial migration prevention solution of claim 13, wherein the carrier is Polyethylene glycol-Ethanol-Tween 80.

15. The trans-endothelial migration prevention solution of claim 13, wherein the composition includes the bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an organ preservation solution at a concentration of between 1.0 to 5000.0 nanograms per milliliter.

16. The trans-endothelial migration prevention solution of claim 13, wherein the composition includes the bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof in an amount of 0.00005% to 0.500% by weight.

17 A method for preserving tissues or organs comprising: administering to a harvested tissue or a harvested organ a composition comprising bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof and a protein free carrier including Polyethylene glycol-Ethanol-Tween 80 for between 10.0 minutes and 1.0 minutes; and washing the tissue or organ with a saline solution after administering the composition; wherein the saline solution is substantially free of bryostatin-1, or a pharmaceutically acceptable salt, analog, or stereoisomer thereof, the composition is administered only less than 30 minutes before the tissue or organ is transplanted into a patient in need thereof, and a concentration of bryostatin-1 in an intravenous location of the tissue or organ after washing is below 10'⁹ M, below 10'¹⁰ M, or effectively zero M.