Abstract:
There is disclosed a method for vascularizing tissue, a method for improving vascularization of ischemic tissue, and a method for improving the viability of grafted tissue or tissue survival in gradient ischemia of heart, brain or skin, comprising administering locally to the grafted or ischemic tissue or the site of the graft an effective amount of a compound comprising an alk-1-enyl glycerol derivative.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This patent application is a continuation-in-part of U.S. patent application 08/285,153 filed 03 Aug. 1994, now abandoned. 
    
    
     TECHNICAL FIELD OF THE INVENTION 
     The present invention provides a method for using angiogenic lipid formulations to improve tissue vascularization and promote viability of grafted or ischemic tissue. 
     BACKGROUND OF THE INVENTION 
     A group of angiogenic lipid compounds were disclosed to have pharmacologic activity in stimulating angiogenesis, or promoting the directed growth of blood vessels. The genus of such angiogenic lipids was described in U.S. patent application 08/285,153 filed 03 Aug. 1994, the disclosure of which is incorporated by reference herein. Angiogenesis is the process by which new blood vessels are formed and it is also know as neovascularization. 
     Tissue grafts are used in a variety of surgical reconstructions. Vascularization is critical to their success, as by definition, grafts have no blood supply of their own. Therefore, grafts are completely dependent on the recipient bed and adjacent tissues for revascularization. If the grafted tissue is too thick or the revascularization process inadequate, the graft will be either partially or entirely lost. Consequently, not only may the reconstructive result be jeopardized, but prolonged hospitalization, septic complications, and patient death may also occur. 
     Angiogenesis is a complex multi-step process regulated by a delicately balanced combination of both stimulatory and inhibitory factors (Folkman and Klagsburn, Science 235:442-447, 1987). These principally peptide factors have been isolated from various tissues and cell types within the body (Folkman and Klagsburn, Science 235:442-447, 1987; and Schultz and Grant, Eye 5:170-180, 1991). The omentum has been used to augment focal and localized blood delivery (DeRenzi and Boeri, Berl. klin. Woch. 40:773-775, 1903; and Wilkie, Br. Med. J. 2:1103-1106, 1911). Omental transposition can result in increased blood flow to specific vascularly compromised anatomic sites and tissues, and such surgical techniques have yielded successful revascularization of ischemic or otherwise hypovascular structures (Vineberg, Dis. Chest 54:315-322, 1968; and Goldsmith, J. Neurosurg. 66:152-153, 1987). In addition, the omentum is also believed to possess intrinsic angiogenic potential capable of initiating sustained tissue vascularization. Omental lipid extracts have demonstrated angiogenic properties in several experimental settings (Goldsmith et al., Surg. Gyn. &amp; Obs. 162:579-583, 1986; and Nottebaert et al., J. Orthop. Res. 7:157-169, 1989). 
     It is believed that enhanced vascularization of tissue grafts can simultaneously result in increased clinical success and diminished graft failure-associated morbidity. Therefore there is a need in the art to improve vascularization of tissues, grafted tissues and ischemic tissue. The present invention provides surprising results for a group of lipid compounds that can be therapeutically effective for tissue vascularization. 
     SUMMARY OF THE INVENTION 
     The present invention provides a method for vascularizing tissue, comprising administering to the grafted tissue or the site of the graft an effective amount of a compound comprising an alk-1-enyl glycerol derivative having the formula: ##STR1## wherein R is a straight or branched C 1-24  alkyl or a straight or branched C 1-24  acyl and X and Y each is H or an acyl group having from 2 to 26 carbon atoms. Preferably, R is C 14  or C 16  acyl, X is an acyl group and Y is H. Most preferably, R is a C 16  monounsaturated acyl. Preferably the tissue is grafted from self, from a donor, or a synthetic living skin such as skin replacements made from cultured fibroblasts (e.g., Graftskin® from Organogenesis/Sandoz or Dermagraft® from Advanced Tissue Sciences). Preferably the compound further comprises an immunosuppressively effective amount of an immunosuppressive compound. Examples of immunosuppressive compounds are cyclosporin A, cyclosporin G, mycophenolic acid morpholinoethylester, rapamycin, FK506, brequinar, misorabine, and glucocorticosteroids. 
     The present invention further provides a method for improving vascularization of ischemic tissue, comprising administering to the ischemic tissue or the site of the ischemia an effective amount of a compound comprising an alk-1-enyl glycerol derivative having the formula: ##STR2## wherein R is a straight or branched C 1-24  alkyl or a straight or branched C 1-24  acyl and X and Y each is H or an acyl group having from 2 to 26 carbon atoms. Preferably, R is C 14  or C 16  acyl, X is an acyl group and Y is H. Most preferably, R is a C 16  monounsaturated acyl. Preferably, the ischemic tissue is selected from the group consisting of myocardium, brain, liver, bowel, kidney, muscle and skin. 
     The present invention further provides a method for improving the viability of grafted tissue or flap survival in gradient ischemia, comprising administering locally to the grafted tissue or the site of the graft an effective amount of a compound comprising an alk-1-enyl glycerol derivative having the formula: ##STR3## wherein R is a straight or branched C 1-24  alkyl or a straight or branched C 1-24  acyl and X and Y each is H or an acyl group having from 2 to 26 carbon atoms. Preferably, R is C 14  or C 16  acyl, X is an acyl group and Y is H. Most preferably, R is a C 16  monounsaturated acyl. Preferably the tissue is grafted from self or a donor. Preferably, the gradient ischemia is in heart, brain or skin. Preferably, in the case of homograft or xenograft, the compound further comprises an immunosuppressively effective amount of an immunosuppressive compound. Examples of immunosuppressive compounds are cyclosporin A, cyclosporin G, mycophenolic acid morpholinoethylester, rapamycin, FK506, brequinar, misorabine, and glucocorticosteroids. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 illustrates a graph showing the percent full-thickness graft survival at 14 days engraftment treated with the preferred compound MG-FUE (monoglyceride fatty unsaturated ether having Y and X as H and R is C 16  acyl monounsaturated) in phosphate buffered saline (PBS). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     We tested the hypothesis that MG-FUE will enhance graft vascularization, leading to increased graft viability. Such enhanced tissue vascularization will allow for better reconstructive results and patient outcomes, expanded clinical application of composite graft reconstructive techniques and improved vascularization of ischemic tissue. We have isolated and purified a specific omental lipid-derivative and subjected it to a series of biochemical alterations. 
     The resultant semi-synthetic compound MG-FUE dramatically stimulated neovascularization in the rabbit cornea as a model of angiogenesis. We have studied the angiogenic potential of the lipid compound, monoglyceride-fatty unsaturated ether (MG-FUE), in an accepted and reproducible rabbit composite graft (Vedder et al., Plast. Reconst. Surg. 93:1035-1040, 1994; and Hammond et al., Plast. Reconstr. Surg. 91:316-21, 1993) to further examine its effect on tissue vascularization. 
     Synthesis of MG-FUE 
     Bovine heart phosphatidylcholine (30% plasmalogen), denoted as PC, was obtained either by purification, or directly from Sigma. One gram of PC was dissolved in 100 ml of chloroform and subjected to alkaline methanolysis by addition of 100 ml of 0.5N methanolic NaOH (final was 0.25N NaOH), gently mixed and allowed to react for 45 minutes at 25° C. The reaction was stopped by the addition of 50 ml of 1N acetic acid. The reaction mixture was transferred to a 500 ml sepratory funnel, mixed and inverted and then allowed to partition over 1 hr. The lower phase containing the lipid was drawn off and dried using a roto-evaporator at room temperature or with slight warming to 37° C. 
     The resultant dried lipid, containing the lysoplasmenyl PC, LPPC, was brought up in 6.0 mls of 2-propanol:hexane:water (55:40:5) and applied to a preparative column (5×100 cm) packed with iatrobeads® (Iatron, Tokyo), which are a porous silica gel of about 10 uM diameter. The column was first pre-equilibrated by running at 5.0 mls/min over 120 minutes using the gradient: 2-propanol:hexane:water (55:25:20) to (55:40:5). The column was washed for an additional 30 minutes at (55:40:5), and the sample applied in a 10 ml sample loop. 
     Fractions were collected over 400 minutes, 2 min/tube, hence 200 tubes. Elution was at a constant flow rate of 3.0 mls/min. Gradient elution was as follows: From T=0 min: 2-propanol:hexane:water (55:40:5) to T=300 min: (55:25:20) to T=400 min: (55:25:20). Purified LPPC eluted between fractions 72-96. These tubes were pooled, dried by roto-evaporation and brought up in a suitable volume of 6 uM Imidazole buffer, pH 7.4 with 2.2 mM CaCl 2 . The lipid suspension was warmed, sonicated and vortexed. A suitable amount of Phospholipase C Bacillus cereus (Boehringer Mannheim) (10,000-50,000 units) was added and the reaction allowed to go until completion (greater than 12 hours). Finally, the resultant MG-FUE was purified from the reaction mixture by a similar HPLC protocol as above, but starting with more nonpolar conditions (e.g., 2-propanol: hexane:water, 35:64:1). The resultant purified MG-FUE was dried by roto-evaporator, brought up in a minimal volume of chloroform and stored at -20° C. Stored in this manner, we observed no degradation over a 12 month period. 
     Compound Genus 
     The compound genus of the present inventive method comprises an alk-1-enyl glycerol derivative having the formula: ##STR4## wherein R is a straight or branched C 1-24  alkyl or a straight or branched C 1-24  acyl and X and Y each is H or an acyl group having from 2 to 26 carbon atoms. Preferably, R is C 14  or C 16  acyl, X is an acyl group and Y is H. Most preferably, R is a C 16  monounsaturated acyl. The preferred embodiment is MG-FUE, which consists of X and Y as a hydrogen and R is C 16  monounsaturated acyl. Preferably, X is an acyl group and Y is H or vica versa, wherein said acyl group comprises 20 carbon atoms (e.g., an arachidonate group). Preferably, X is an acyl group comprising 16 or 18 carbon atoms. 
     In Vivo Experiments 
     We performed a series of experiments using anesthetized New Zealand white rabbits having free dorsal composite grafts consisting of skin, subcutaneous tissue, and the panniculus carnosus muscle elevated and the deep surfaces of the grafts coated with varying doses (N=4-8 per dose) of MG-FUE suspended in phosphate-buffered saline (PBS). The control rabbits were treated with vehicle (PBS) alone (N=14). Grafts were sutured in position and assessed daily for 14 days. Graft viability was quantified as the surface area of clearly viable dermis and expressed as a percentage of total graft surface area. Samples of all grafts were sectioned, prepared with H&amp;E stains, and examined histologically. Areas of full-thickness tissue revascularization were seen at all treatment doses, whereas dermal viability was completely absent in controls. The percentage of viable dermal surface area varied in a dose-dependent fashion (FIG. 1). There was a significant (*p&lt;0.05) increase in tissue viability in grafts treated with either 1.0 mM or 2.0 mM MG-FUE compared to grafts treated with either a lower dose or PBS alone. Examined histologic sections demonstrated successful graft revascularization in the MG-FUE-treated grafts but only partial thickness revascularization of the deeper muscle layers in controls. We observed a dose-dependent angiogenic effect of the genus of lipid compounds that resulted in enhanced graft revascularization and a significant improvement in full-thickness tissue viability. These data serve as a predictive model for improving tissue vascularization and particularly ischemic tissue vascularization. The effect of MG-FUE was dose-dependent. 
     Tissue grafts have no intrinsic blood supply. Tissue grafts are entirely dependent on the recipient bed and adjacent tissues for revascularization. Unless revascularization occurs prior to cell death, the graft will fail. When the coated grafts were elevated and returned to their positions, an interface between the recipient beds and surrounding tissues was created. Consequently recipient beds are directly exposed to the study compound and, are treated by it just as the grafts are. This technique of topical lipid compound application to the deep surface of the graft created a localized microenvironment impregnated with compound or placebo vehicle control. 
     The enhanced tissue vascularization and significantly improved graft viability demonstrated in the illustrated in vivo study was due solely to MG-FUE. Full-thickness tissue viability was identified at all MG-FUE doses. No dermal viability was detected in control grafts. All such control grafts suffered late slough implying prolonged ischemia and necrosis of all tissue components, due to failure of timely revascularization. The preferred lipid compound, monoglyceride-fatty unsaturated ether (MG-FUE), enhanced tissue vascularization and significantly improved composite graft tissue full-thickness viability in this in viva predictive model. In the clinical setting, such an approach will improve tissue graft survival and reduce associated morbidity. 
     EXAMPLE 1 
     This example illustrates a set of experiments that saw a dose-response relationship for improving tissue vascularization for the compound MG-FUE. Sterile MG-FUE was suspended in sterile phosphate-buffered saline (PBS). MG-FUE was diluted to final concentrations of 0.01, 0.1, 0.5, 1.0 and 2.0 mM in PBS. The compound was stored in sterile glass vials at room temperature. Approximately one hour before application, MG-FUE was heated to 37° C. in an agitating water bath to promote maximal dissolution. Immediately before application, the heated samples were vigorously vortexed for 2 min. to ensure complete mixing. 
     New Zealand white rabbits (1.5-2 kg) were used for this study. They were maintained under approved standard laboratory animal conditions with appropriate veterinary care. Access to a standard diet of food and water ad libiturn was maintained. 
     The experimental method and surgical procedures of this study were performed in accordance with established N.I.H. guidelines for animal experimentation and with formal approval of the University of Washington Animal Care Committee. Rabbits were anesthetized with an intramuscular dose of ketamine (30 mg/kg) and xylazine (2 mg/kg). A lower extremity peripheral heparin-lock and IV were placed. A single intravenous dose of cefazolin (25 mg/kg) was administered, and a titratable intravenous anesthetic preparation was secured. The entire dorsum of the rabbit extending from the scapular tip to the posterior iliac crest was shaved and a sterile prep with Betadine® solution was performed. Excess solution was removed with normal saline. The rabbit was then draped for sterile surgery. 
     A gridded template was used to mark two 4×4 cm grafts. Attempts were made to maintain an intact tissue bridge of &gt;2 cm between the grafts. A circumferential full-thickness skin incision was made around the template and carried through the subcutaneous tissue. The underlying panniculus carnosus muscle was similarly incised and sharply divided with scissors. Complete dissection of the deep avascular connective tissues allowed each graft to be fully elevated and removed. Both graft and recipient bed kept from desiccating by wrapping the grafts in saline-moistened gauze and placing them directly onto the recipient bed. The tissues were maintained at room temperature. 
     MG-FUE was removed from the 37° C. water bath, vigorously vortexed for 2 min., and 1.0 ml aspirated with a 19-gauge needle. Grafts were unwrapped, and 0.5 ml aliquots of compound were then directly applied to the deep surface of each. Control grafts were treated with equivalent volumes of PBS in an identical fashion. The grafts were anatomically oriented and repositioned, allowing the MG-FUE or PBS control to directly contact the recipient bed as well. The grafts were then sutured into place with 4-0 silk suture in running fashion. The operative wounds were gently cleansed. No dressings were applied. A treatment dose identification card was made and immediate post-operative photography was performed. The IV and heparin lock were discontinued and hemostasis assured. The rabbit was then returned to the vivarium where it was allowed to recover. An initial post-operative check to ensure the rabbit&#39;s well-being took place 30 min after completion of the procedure. 
     Rabbits and grafts were assessed daily for a 2-week post-operative period. During this period, the surface areas of all grafts were repeatedly calculated, and grafts examined for the presence of hematoma, seroma, dehiscence or slough. Seromas were aspirated. All grafts were measured and photographed on days 1 and 14 and intermittently in-between. Graft viability was determined by visual inspection and included assessments of tissue color, capillary refill, bleeding upon needle puncture, and the presence or absence of dermal eschar. Tissue texture was evaluated by direct palpation. Findings were recorded and graft size-matched templates were correspondingly marked to demonstrate progressive eschar or other color change. Final clinical graft viability was quantified on day #14. Using a transparent gridded template, both the total graft size and regions of clinically viable dermis were measured. Graft viability was quantified as the surface area of clearly viable dermis and expressed as a percentage of total graft surface area. Mean percentage graft survival for each treatment was calculated and a dose-response curve plotting percentage graft survival vs. MG-FUE dose was derived. Dose-dependent differences in percentage graft survival were calculated using the unpaired t-test with significance determined at a p-value of &lt;0.05. 
     At the conclusion of the post-operative study period, rabbits were euthanized with an intraperitoneal dose of sodium pentothal. The entire dorsal composite panel including both grafts and surrounding skin, subcutaneous tissue, and panniculus carnosus muscle was meticulously dissected in the avascular plane deep to the muscle. Both the undersides of the grafts and their respective recipient beds were clinically assessed for the presence of neovascularization and photographed. Grafts were transversely incised, and full-thickness tissue evaluations for evidence of bleeding indicative of successful revascularization were performed. Samples of all grafts were collected, labeled according to graft site, MG-FUE dose and vehicle, and placed in 10% neutral-buffered formalin. Each was submitted for H&amp;E staining and histologic evaluation for the presence and anatomic localization of newly formed vascular structures. Two rabbits had two grafts apiece treated with either 0.01, 0.1, 0.5, or 1.0 mM MG-FU. Five rabbits had two grafts treated with 2.0 mM MG-FUE in PBS. There were seven PBS control rabbits. 
     Results 
     Areas of full-thickness tissue vascularization were noted at all treatment doses. Dermal viability was absent in PBS control-treated grafts. For those treated with MG-FUE, the percentage of viable dermal surface area varied in a dose-dependent fashion (FIG. 1). There was a significant (*p&lt;0.05) increase in tissue viability in grafts treated with either 1.0 mM or 2.0 mM MG-FUE compared to grafts treated with either a lower dose of MG-FUE or PBS alone. 
     Noteworthy complications (see table below) include one 2.0 mM-treated day 3 rabbit death, two 0.01 mM (PBS)-treated day 10 episodes of graft epidermolysis, one 0.1 mM (PBS) day 3 and one 0.5 mM (PBS)-treated day 7 graft dehiscence, and three PBS control full-thickness graft sloughs during the second week of the 2-week assessment period. All complicated grafts were eliminated from data analysis. 
     
         ______________________________________                             #@ risk                                   # plottedDose (mM)   Rabbit N Graft N  complication                             grafts                                   grafts______________________________________0.1/PBS 2        4        none    0     40.5/PBS 2        4        dehiscence                             1     31.0/PBS 2        4        none    0     42.0/PBS 5        10       rabbit death                             2     8PBS     7        14       slough  3     11______________________________________ 
    
     The majority of grafts remained soft and pale during the early period of clinical observation. Most of these treated grafts progressed to take on a dusky gray/purple hue with viable portions subsequently becoming pink. Convincing evidence of partial graft loss did not manifest until approximately the fifth to seventh post-operative day at which time focal areas of eschar were prominent. Simultaneous to the development discrete eschar, many remaining portions were pink, soft and had brisk capillary refill. Healthy portions of the grafts bled when challenged with needle punctures. 
     Post-mortem examination of the graft undersurfaces revealed many moderately large-caliber vessels embedded within the deep tissues of the grafts. These new vessels appeared to have their origins within the tissues peripheral to the suture lines. Elevation of the 1.0 mM treated graft tissues revealed long large-caliber vessels coursing upward from the recipient beds, penetrating the deep surface of the grafts. Full-thickness areas of tissue viability were identified in all MG-FUE-treated. Dermal viability was completely absent in controls. Non-viable tissue was clearly demarcated by the presence of dark, dry, hard eschar. Viable tissue was thick, edematous and pink. Grafts were sectioned transversely and upon direct compression, blood emanated from the those portions that had been successfully revascularized. Sections demonstrate successful graft revascularization in the MG-FUE-treated grafts but only partial thickness revascularization of the deeper muscle layers in controls.