Macrocyclic triene immunosuppressive compounds for perivascular medical treatment of vascular access fistula, graft and other vascular diseases

Abstract

The present application generally relates to the field of treating or preventing "open" vascular surgical interventions to reduce or eliminate stenosis and / or vascular cancer conditions by administering macrocyclic triene immunosuppressive compounds.

Description

Technical Field

[0001] The present application relates generally to the field of treatment or prevention of "open" vascular surgical interventions to reduce or eliminate stenosis and / or vascular cancer conditions by administering macrocyclic triene immunosuppressive compounds. Background Art

[0002] Occlusive vascular disease is the leading cause of mortality and morbidity worldwide. Recently, minimally invasive percutaneous endovascular intervention using drug eluting stents and drug-coated balloons has reduced the frequency of stenosis. However, unlike the technological advances using drug eluting stents and drug-coated balloons, there is no approved clinical option for preventing surgically induced intimal hyperplasia in "open" vascular system intervention (Chen et al. Biomacromolecules, (18) 2205-2213, 2017). Open surgery remains the only solution for cases that are considered unsuitable for percutaneous methods, including coronary artery bypass surgery, carotid endarterectomy, autologous arteriovenous fistula transplantation for hemodialysis patients, stenosis after renal transplantation, and various types of complications after endovascular surgery.

[0003] In these cases, limited flow stenosis remains the leading cause of surgical failure. Another complication in such surgically manipulated vessels is the induction of angiosarcoma or cutaneous squamous cell carcinoma at the surgical site (Oskrochi et al., Eur J Vasc Endovasc Surg, (51) 127-133, 2015; Jennings et al., Cancer, (62) 11: 2436-2444, 1988; Costa et al., Case Rept Transplant, 2426859, 2017).

[0004] There are approximately 400,000 open vascular surgeries performed each year in the United States (Jim et al., J Vasc Surg (55) 5: 1394-1400, 2012), and open surgery results in up to 50% occlusion (restenosis), depending on the type of surgical intervention. An example of an open intervention is the intervention required for hemodialysis therapy (HD). HD is driven by chronic kidney disease, which remains a global public health problem. In the United States, approximately 750,000 patients are affected by end-stage renal disease (ESRD) each year, which increases by 5% each year. (https: / / pharm.ucsf.edu / kidney / need / statistics, accessed on 05 / 08 / 2019). ESRD is complete and permanent kidney failure and is treated with kidney transplantation or dialysis. In 2016, 71% of all US ESRD cases were being treated with HD (https: / / www.kidney.org / news / newsroom / factsheets / End-Stage-Renal-Disease-in-the-US, accessed on 05 / 08 / 2019). The process of HD requires frequent blood access, and arteriovenous fistulas (AVFs) created by open surgery have become the preferred blood access method. AVFs utilize an artery that is directly connected to a vein by surgery, usually in the arm, which allows regular withdrawal and return of blood. AVFs are not without significant restenosis and graft failure problems in both the United States and Europe. (Gallieni et al., Semin Intervent Radiol (26) 96-105, 200 9). Current treatments for such flow-restricted vessels are typically created using balloon angioplasty, stenting, or new grafts. These retreatment procedures may be compromised due to the complex nature of the surgically modified vessels, which may include unnatural orientations and small sizes. Furthermore, in addition to the patient inconvenience, there is the added risk factor of surgery for the patient, the frequent need for blood thinning medications over a period of time, and the increased cost.

[0005] Initial attempts to mitigate or reduce restenosis have focused on two areas: placement of perivascular (around the outside) non-drug containing medical devices, including steel alloy mesh, nitinol mesh, and Sheaths to hold or enhance grafts, or to deliver various vasoactive compounds systemically. In the case of medical devices, surgery is prone to cause increased vascular damage, and the results show low or contradictory efficacy. (Mylonaki, et al., Biomaterials (128) 56-68, 2017; Rescigno, et al., Thorac Cardiovasc Surg, (63) 4: 292-297, 2015). Although systemic drug delivery has only modest promise, there is still the possibility of adverse systemic toxicity. ( et al., Circulation (112) 2759-2761, 2005; Davies et al., Eur J Vasc Endovasc Surg (42) 519-529, 2011)

[0006] To overcome negative systemic drug effects, direct targeted application of pure agents has been attempted. (Chaudhary et al., J Control Release, (233) 174-180, 2016). When used without a mechanism to retain the drug at the desired site, these direct drug applications also lead to poor overall results due to rapid drug elution and clearance and / or drug migration. The resulting attempts to overcome the washout effect require much higher drug loads and possible local toxicity. Together, these findings suggest that some type of drug and drug carrier is needed to facilitate placement and retention of the drug at the surgical site to provide controlled and sustained drug delivery during treatment.

[0007] Subsequent development work has investigated various drug-loaded perivascular systems that are intended to provide IH therapy at the surgical site. A suggested classification of these systems has recently been provided: gel, gel+particles, mesh, sheath, cuff, wrap, and matrix. These authors also compared the characteristics of each system. (Mylonaki, op. cit.). The comparison considered ease of administration, potential for repeated administration, site-specific localization, mechanical support, and sustained drug release. These authors concluded that a system that optimizes all requirements has not yet been developed.

[0008] Even now, as Seedial comments, there is currently no approved technology or drug delivery device for reducing restenosis after open surgical revascularization (Seedial, SM, Kent, KC, et al. "Local drug delivery to prevent restenosis" Journal of Vascular Surgery, Vol. 57 pp. 1403-1414, 2013). Extraluminal administration of drugs seems promising because drugs administered extravascularly have been shown to diffuse into the vessel wall (Lovich, MA, Brown, L., and Edelman, ER "Drug Clearance and Arterial Uptake After Local Perivascular Delivery to the Rat Carotid Artery" J. Am. Coll. Cardiol., Vol. 29 pp. 1645-50, 1997). It has been suggested that extraluminal injury from surgical revascularization promotes a signaling cascade that activates myofibroblast migration into the neointima, leading to intimal hyperplasia; drugs administered extraluminally will reduce myofibroblast migration and alleviate restenosis (Siow, RCM, and Churchman, AT "Adventitial growth factors signaling and vascular remodeling: potential of perivascular gene transferfrom the outside-in" Cardiovasc. Re., Vol. 75 pp. 659-68, 2007).

[0009] Therefore, there is a need in the prior art to improve the above-mentioned deficiencies in the field of improved drug delivery to specific surgical sites. Loss of vascular patency after open surgery remains a worldwide problem. There is still a lack of approved drugs, devices or drugs / devices for this occlusive disease. The treatment of this condition is a huge unmet need, which requires new solutions to remedy the above-mentioned problems in the related field. Summary of the invention

[0010] The present invention provides a method of treating an open vascular surgical procedure comprising administering an effective amount of a macrocyclic triene immunosuppressive compound by direct perivascular administration, wherein the macrocyclic triene immunosuppressive compound has the following structure:

[0011]

[0012] Where R is C(O)-(CH2) n -X, n is 0, 1 or 2, X is a cyclic hydrocarbon having 3 to 7 carbon atoms, optionally containing one or more unsaturated bonds. Preferably, C(O)-(CH2) n -X has one of the following structures:

[0013]

[0014] Advantageously, the compound is formulated to allow direct perivascular application to a graft vessel during an open vascular surgery procedure, and preferably at the start of the procedure. The compound is stable when administered to a coronary artery. Similarly, the compound is stable when administered to a peripheral artery. More preferably, upon administration, the compound results in excellent vascular preservation and reduced vascular spread. Preferably, open vascular surgery procedures include coronary artery bypass surgery, carotid endarterectomy, arteriovenous fistula transplantation (hemodialysis), stenosis following renal transplantation, and endovascular surgery.

[0015] In another aspect, the present invention provides a method for treating vascular damage, comprising providing a pharmaceutical preparation comprising a macrocyclic triene immunosuppressive compound dissolved in at least one solvent, wherein the pharmaceutical preparation is directly applied to at least one tissue within the site of vascular damage. Preferably, the at least one solvent is an alcohol. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The disclosed subject matter contained herein is best described in conjunction with the accompanying drawings, in which:

[0017] Figure 1 Shown is a comparison of drug stability of different formulations when applied to porcine coronary artery tissue.

[0018] Figure 2 Shown is a comparison of drug stability of different formulations when applied to porcine peripheral artery tissue.

[0019] Figure 3 Shown is a comparison of drug retention of different formulations when applied to porcine coronary artery tissue.

[0020] Figure 4 A representative ex vivo perivascular drug delivery model of the invention is depicted.

[0021] Figure 5 The amount of drug recovered from the artery and ear skin tissue 24 and 48 hours after surgery is shown.

[0022] Figure 6 Examples of preferred perivascular applications of the pharmaceutical preparations of the present invention are shown.

[0023] Figure 7Concentration and amount of a preferred drug formulation in the jugular vein and surrounding tissues versus time are shown; n=2.

[0024] Figure 8 Shown are the femoral vein and artery of a rat prior to perivascular administration of a preferred pharmaceutical formulation.

[0025] Fig. 9 Preferred drug concentration or amount in peripheral veins + arteries and surrounding tissues versus time are shown; n = 2. Data were analyzed by nonlinear regression based on a single-phase decay model (GraphPad Prism 8.0 software program).

[0026] Fig.10 Preferred drug treatment for coronary artery smooth muscle cells, adjusted; IC 50 = 1.8 nM. Data were analyzed by nonlinear regression based on a dose-response model (GraphPad Prism 7.0 software).

[0027] Fig.11 The total drug concentration in whole blood of Sprague Dawley rats is shown as a function of time. The black dashed line represents the minimum sirolimus concentration required for immunosuppression (16-24 ng / mL). Data were plotted using the GraphPad Prism 8.0 software program.

[0028] Fig.12 Porcine coronary artery washout results for CRC-015, sirolimus, and everolimus are shown, n=2.

[0029] Fig.13 A comparison of drug stability in a perivascular model is shown.

[0030] Fig.14 Total drug recovered from arterial and ear skin tissue at 24 and 48 hours is shown. DETAILED DESCRIPTION

[0031] definition

[0032] As used herein, the term "macrocyclic triene immunosuppressive compound" may also include rapamycin (sirolimus), everolimus, zotarolimus, biolipidimus, novolimus, myolimus, temsirolimus, and the rapamycin derivatives described in the present disclosure.

[0033] The work of Lovich, Brown and Edelman has advanced important understandings of the mechanisms of perivascular drug deposition from the perivascular space to the vessel wall (Lovich et al., JACC, (29) 7: 1645-1650, 1997). These authors determined, through in vivo animal studies using radiolabeled model drugs, that although most drug released into the perivascular space is cleared systemically via the extravascular capillaries, most of the drug deposited into the vessels is provided by direct inward diffusion from the source and very little is provided by systemic intravascular means.

[0034] A large number of practical candidate drugs for perivascular delivery have been investigated in both in vitro and in vivo studies. In parallel, efforts to better understand the pathological basis of postoperative vascular remodeling provide guidance for drug selection. It is now recognized that endothelial changes associated with postoperative vascular remodeling and the resulting neointimal structural damage (intimal hyperplasia (IH)) are at the core of venous and arterial graft stenosis (restenosis) and failure.

[0035] IH has been determined to originate from abnormal migration and proliferation of vascular smooth muscle cells (SMCs), accompanied by deposition of extracellular connective tissue matrix, leading to restenosis (Davies et al. J Vasc Surg, (61) 203-216, 2015). In the case of vascular transplantation procedures, within a few hours, the endothelial damage of the surgical procedure triggers a cascade of mechanical and biochemical events, triggering SMC proliferation, which lasts for several days or weeks, accompanied by negative remodeling of the blood vessels (Mitra et al., Immunol Cell Biol, (84) 115-112, 2006). It has been determined that the resulting vascular graft restenosis lesions occur primarily in the area of ​​surgical connection (Mills et al., J Vasc Surg, (17) 195-206, 1993).

[0036] Preclinical in vitro and model animal studies have shown that the compound sirolimus is effective in inhibiting vascular SMC proliferation. These studies have led to a large number of human studies using sirolimus eluting heart stents, which have demonstrated a significant reduction in restenosis after stent implantation (Marx et al., Circ Cardiovasc Interv, (4) 1: 10 4-111, 2011). Oral delivery of sirolimus and everolimus (sirolimus derivatives) also showed SMC inhibitory activity, although with undesirable systemic side effects (Kurdi et al., Br J Clin Pharmacol, (82) 5: 1267-1279, 2015). Similarly, sirolimus has also been shown to inhibit angiosarcoma at subtoxic doses using several human cancer cell lines (Bundscherer et al., Anticancer Res, (30) 4017-4024, 2010).

[0037] These successes of sirolimus have led to several in vitro studies of sirolimus using perivascular delivery. All reported perivascular studies of sirolimus have used additional means to control drug migration and extend drug delivery time to about 2 to 6 weeks. These means include: wraps, gels and various polymers, as well as micron or nanometer-sized particles with different chemical and mechanical complexities. Recently, a mixture of sirolimus combined with hydrophobic cyanoacrylates has also been proposed as a means of retaining the drug on the target at the implant site (Tiansu-Chu et al. Arq Bras Cardiol, (112) 1: 3-10, 2019). It is speculated that the increased hydrophobicity and mechanical strength of the subsequent drug reservoir contribute to this aspect. This method may also suffer from toxicity issues and drug product manufacturing and end-user complexity.

[0038] Sirolimus delivery collagen wraps (US 6,726,923) are currently in clinical trials in the United States conducted by Vascular Therapies, Inc. However, the use of wraps suffers from the need for specialized surgical delivery requirements and may itself cause negative remodeling. Material toxicity, administration complexity, manufacturing complexity, cost, and regulatory status of these materials remain issues for this approach.

[0039] The present invention provides a method for overcoming the above limitations of the prior art for handling open vascular surgical procedures, comprising administering an effective amount of a macrocyclic triene immunosuppressive compound by direct perivascular administration, wherein the macrocyclic triene immunosuppressive compound has the following structure:

[0040]

[0041] Where R is C(O)-(CH2)n -X, n is 0, 1 or 2, X is a cyclic hydrocarbon having 3 to 7 carbon atoms, optionally containing one or more unsaturated bonds. Preferably, C(O)-(CH2) n -X has one of the following structures:

[0042]

[0043]

[0044] Advantageously, the compound is formulated to allow for direct perivascular application to the graft vessel at the onset of surgery.Thus, the macrocyclic triene immunosuppressive compound is administered directly to at least one tissue within the open vessel surgical site.

[0045] It is further provided that for the methods described herein, the macrocyclic triene immunosuppressive compound is administered in the form of a pharmaceutical preparation, wherein the pharmaceutical preparation comprises the macrocyclic triene immunosuppressive compound and at least one solvent. The solvent may preferably be an alcohol, more preferably a polyol, and most preferably polyethylene glycol. In one embodiment of the method, the pharmaceutical preparation consists of the macrocyclic triene immunosuppressive compound described herein and a solvent, wherein the solvent is an alcohol, more preferably a polyol, and most preferably polyethylene glycol.

[0046] One aspect of the present invention relates to a pharmaceutical formulation comprising or consisting of a macrocyclic triene immunosuppressive compound as described herein and a solvent, wherein the solvent is an alcohol, more preferably a polyol, most preferably polyethylene glycol.

[0047] In one embodiment, the compound is administered in combination with at least one compound selected from the group consisting of rapamycin (sirolimus), everolimus, zotarolimus, biolipidimus, novolimus, myolimus, temsirolimus and a rapamycin derivative.

[0048] Example

[0049] Example pharmaceutical preparations:

[0050] The macrocyclic triene immunosuppressive compounds of the present invention have more than one embodiment and can be described as comprising at least one of the following from Table 1:

[0051] Table 1

[0052] Description of CRC-015 Types

[0053]

[0054]

[0055] As referred to herein, CRC-015 is a term intended to encompass the genus and is used to refer to each of the following species from Table 1: CRC-015a, CRC-015b, CRC-015c, CRC-015d, CRC-015e, CRC-015f, CRC-015g, and CRC-015h.

[0056] I. Increased drug solubility

[0057] An example of the superior solubility of CRC-015 in a simple PEG400 delivery vehicle compared to sirolimus is shown in Table 2, which facilitates a small drug delivery volume.

[0058] Table 2

[0059] Drug Solubility Comparison

[0060] Solubility of CRC-015 in PEG400 Solubility of sirolimus in PEG400 ~304 mg / mL ~2.4 mg / mL

[0061] II. CRC-015 Excellent coronary artery stability

[0062] Stability studies were performed on porcine coronary artery tissue using CRC-015, sirolimus, everolimus or BA9. These compounds were prepared in ethanol (200 proof) respectively. The porcine coronary artery vessels were cut open and cut into 1×1 cm pieces. Each drug preparation was applied to the center of the endothelial surface of the tissue section and the ethanol was dried. The tissue samples were placed in 30 mL vials together with 150 μL of 0.05% NaN3 saline solution to keep the tissue moist and free of microorganisms. The vials were capped and placed in a 37°C water bath under static conditions. At 4 or 8 days, the tissue was removed and placed in a clean vial and extracted with methanol.

[0063] The residual drug was measured by HPLC using an external standard. The estimated half-life of the drug was calculated by linear regression (Microsoft Excel 2010 software program). The stability of CRC-015 on / in coronary artery tissue increased in Figure 1 and as demonstrated in Table 3 below.

[0064] Table 3

[0065] Drug half-life estimated in porcine coronary artery tissue

[0066] drug Half-life (days) CRC-015 16.4 Sirolimus 4.1 Everolimus 4.3 BA9 4.7

[0067] III. Excellent peripheral artery stability of CRC-015

[0068] In a similar manner as described above in II., stability studies and half-life calculations were performed on porcine peripheral artery tissue using CRC-015, sirolimus or everolimus. The increased stability of CRC-015 on / in peripheral artery tissue was determined in the following Figure 2 And Table 4 proves.

[0069] Table 4

[0070] Estimated drug half-life in porcine peripheral arterial tissue

[0071] drug Half-life (days) CRC-015 22 Sirolimus 3.6 Everolimus 3.4

[0072] IV. Excellent retention performance of CRC-015 in blood vessels

[0073] Elution studies in 25% ethanol / saline (w / w) were performed to measure the drugs released from porcine coronary vessels after topical application. CRC-015, sirolimus and everolimus were prepared in ethanol (200proof) respectively. Porcine coronary vessels were cut into 1 cm slices. Each drug formulation was applied to the outer surface of the tissue slices and ethanol was dried. The tissue was placed in a vial containing 25% ethanol / saline (w / w) warmed at 37°C and then oscillated at 100 SPM (strokes per minute). At the specified time point, the elution medium was removed for analysis. Then fresh elution medium was added to each sample and the study was continued.

[0074] The measurement of eluted drug was performed by HPLC using an external standard. The results were compared as Figure 3 CRC-015 showed a slower release rate from tissue immersed in the elution solution.

[0075] V. Decreased vascular drug diffusion

[0076] The initial ex vivo perivascular drug delivery model was developed as an alternative to in vivo testing to reduce the number of animals used for testing in accordance with U.S. government recommendations (OLAW, 2015, National Institutes of Health, Office of Animal Welfare, https: / / olaw.nih.gov / policies-laws / phs-policy.htm, as accessed April 17, 2019). Using this model, a single donor animal can contribute to multiple test conditions and replicates.

[0077] This method utilizes test drugs placed on porcine peripheral artery segments, which are kept in intimate contact with porcine ear tissue after removal of ear tissue cartilage. The goal is to mimic in vivo perivascular delivery using drugs deposited on cylindrical vessels that are in intimate contact with a semi-planar flexible tissue bed and tissue cover, such as Figure 4 As shown. In this way, drug stability and migration can be studied and optimized before multiple animal in vivo testing. At 24 and 48 hour time points, tissues were collected, rinsed with saline and extracted with methanol. Comparative measurements of residual drug in blood vessels and tissues were performed by HPLC using external standards. Comparative results and drug stability ( Figure 5 ). These results correlate well with the retention study results outlined in IV. above and further suggest that CRC-015 reduces drug diffusion from blood vessels to tissues compared to sirolimus.

[0078] VI. Perivascular CRC-015 Uptake in Vivo

[0079] Based on the comparative findings of CRC-015 drug diffusion and sirolimus drug diffusion in the perivascular model, in vivo studies in rats were performed. The initial study focused on jugular vein CRC-015 perivascular delivery, and the second study focused on peripheral arterial + femoral vein CRC-015 perivascular drug delivery. The goal was to measure the elution, clearance, and vascular content of perivascularly applied CRC-015 and compare the results with published studies using sirolimus.

[0080] Jugular Vein Studies:

[0081] Non-fasted male Wistar rats were allowed to drink water ad libitum. Prior to surgery, the rats were anesthetized with isoflurane and then shaved under the neck. A vertical incision was made to expose the right external jugular vein. Using a calibrated 10-μL pipette, 5 μL of CRC-015 in PEG400 was placed directly on the jugular vein twice to deliver a 1 mg CRC-015 dose. It appears that PEG diffuses rapidly, resulting in drug precipitation. The precipitated CRC-015 was observed to be deposited on the surface of the vein in the form of a white deposit ( Figure 6 ).

[0082] The incision was then closed with 3 absorbable sutures. The jugular vein and surrounding tissues were collected at weeks 2 and 4. The jugular vein was flushed with water and wiped with a moistened lint-free cloth to remove any traces of drug on the tissue surface. The tissue was extracted with acetonitrile and the solution was tested by HPLC. The residual CRC-015 tissue concentration and the amount remaining in the surrounding tissue are shown in Figure 7 middle.

[0083] Peripheral artery + femoral vein study:

[0084] Nonfasted male Sprague Dawley rats were allowed to drink water ad libitum. Prior to surgery, the rats were anesthetized with isoflurane and then shaved. An incision was made to expose the right femoral artery / vein. Figure 8 An example of the femoral vein + artery before application is shown in .

[0085] Using a calibrated 100-μL pipette, 11 μL of CRC-015 in PEG400 was placed directly on the femoral artery + vein to deliver a 3mg CRC-015 dose. The incision was then sutured with 3 absorbable sutures. Whole blood was obtained before specific time points and tissue collection. Whole blood was treated with heparin (45 units / 1mL whole blood), quickly frozen on dry ice and stored at -80°C until analysis. Femoral artery + vein and surrounding tissues were collected at 2, 4 and 6 weeks. The tissue was extracted with acetonitrile and the solution was tested by HPLC. The HPLC results showed the effective concentration of CRC-015 combined with vein + artery, as well as the significant remaining reservoir drug contained in the surrounding tissues at each time point, as shown by in vitro CRC-015 arterial smooth muscle IC 50 It is capable of delivering therapeutic amounts of drug as determined by inhibition studies.

[0086] Fig. 9 The obtained nonlinear regression curves are shown. In a separate study, the minimum CRC-015 drug concentration required to achieve IC50 inhibition in human coronary artery smooth muscle cells was 1.8 nM ( Fig.10 ). By extrapolation from the data and curves shown, the time required for venous + arterial tissue concentrations to reach 2 nM would be 21.5 weeks.

[0087] Whole blood analysis was performed during the second study period starting from above. Blood was collected at designated time points and analyzed by LC / MS. The relationship between total drug concentration in whole blood and time is shown in Fig.11 All samples were below the 16-24 ng / mL sirolimus concentrations typically accepted for immunosuppression.

[0088] The potential of delivering site-specific therapeutic drug levels to preventively treat open surgical occlusive diseases without the use of mechanical wraps, collars, sleeves or complex polymer formulations is very attractive. Compared with the current leading candidate sirolimus and other sirolimus derivatives, CRC-015 has been shown to have unique and excellent properties for treating the loss of patency caused by various "open" surgeries. It was found that the delivery of CRC-015 to blood vessels was extended beyond the 4-6 weeks reported in several sirolimus research studies. It is believed that the extended drug delivery time will lead to excellent patient results and reduce or eliminate the need for postoperative intervention. The unique CRC-015 reservoir did not show signs of toxicity to surrounding tissues, and it was found that systemic blood drug levels were lower than the levels that caused immunosuppression.

[0089] VII. Washout and Stability Studies from Porcine Coronary Artery Tissue in a Perivascular Model

[0090] Elution studies in 25% ethanol / saline (w / w) were performed to measure the drug released from porcine coronary vessels after topical application. CRC-015, sirolimus and everolimus were prepared in ethanol (200 proof) respectively and applied to the outside of porcine coronary vessels. Once applied, the ethanol was dried. The tissue was placed in a vial containing 25% ethanol / saline (w / w) warmed at 37°C and then shaken at 100 SPM (strokes per minute). Aliquots of certain time points were taken out and refilled. Samples were analyzed by HPLC.

[0091] Porcine peripheral artery tissue and ear tissue were used to simulate the perivascular model. Figure 4 As shown, the drug formulation was first applied to a peripheral arterial vessel that was located on top of a portion of a saran wrap. Pig ear tissue was then placed on the sample. The Saran wrap was folded to encapsulate the tissue. The sample was then incubated at 37°C for 24 or 48 hours.

[0092] VIII. Chromatographic conditions

[0093] HPLC analysis was performed using an Agilent high performance liquid chromatography (HPLC) 1290 Infinity series coupled to a diode array detector G4212B (SN DEAA300117). The HPLC was equipped with a high performance autosampler G1329B (SN DEABE00184), a thermostat G1330B (SN DEBAK00182), a thermostat column chamber G1316A (SN DEAAK00266) and a quaternary pump G1311B (SN DEAAB00113). The HPLC method parameters are shown in Table 5 and Table 6, respectively.

[0094] Table 5

[0095] HPLC chromatographic conditions of CRC-015

[0096]

[0097] Table 6

[0098] HPLC Chromatographic Conditions of Sirolimus

[0099]

[0100] IX. Washout Procedure on Porcine Coronary Arteries

[0101] To examine the effects of certain formulations of the invention on porcine coronary artery samples, the samples were each prepared as follows:

[0102] CRC-015 formulation preparation: Weigh 3.189 mg of CRC-015 into a 2-mL HPLC vial. Use 1000 μL Ethanol (200 proof) was pipetted into the vial, 100.64 mg (127 μL), to give a final concentration of 25.1 mg / mL.

[0103] Preparation of Sirolimus: Weigh 3.118 mg of Sirolimus into a 2-mL HPLC vial. Use 1000 μL Ethanol (200 proof) was pipetted into the vial, 98.40 mg (125 μL), to give a final concentration of 24.9 mg / mL.

[0104] Preparation of Everolimus: Weigh 3.141 mg of Everolimus into a 2-mL HPLC vial. Use 1000 μL Ethanol (200 proof) was pipetted into the vial, 99.021 mg (126 μL), to give a final concentration of 24.9 mg / mL.

[0105] Preparation of elution medium: weigh 4.5 g of sodium chloride into 500 mL Prepare saline by adding 495.5 g of HPLC water to a 500 mL bottle. Then cap the bottle and shake vigorously. Prepare 25% ethanol / saline elution medium in a 100 mL bottle. Add 75.0 grams of saline to the 100 mL bottle containing ethanol. Cap the bottle and shake vigorously.

[0106] Elution Method: Porcine coronary artery vessels were obtained and dissected from hearts obtained from a butcher shop. The coronary arteries were snap frozen with dry ice and then stored at -80°C. When ready for testing, the arteries were removed from the freezer and allowed to thaw at room temperature. Any fat and muscle tissue were carefully removed using scissors and forceps. The vessels were rinsed several times with saline containing 0.05% sodium azide.

[0107] The blood vessels were then cut into 1 cm sections. 10 μL 5 μL of formulation (approximately 125 μg of drug) was carefully added by syringe onto the outer portion of the tissue. The ethanol was allowed to evaporate at room temperature for 15-20 minutes.

[0108] The tissue was then placed in a 7 mL glass vial. Carefully pipette 2 mL of elution medium pre-warmed to 37° C. into each vial. Cap each vial and place in a 37° C. incubator with gentle shaking at 100 SPM (strokes per minute).

[0109] At certain time points, the elution medium was removed and fresh 2 mL of medium (at 37°C) was slowly added to the vial.

[0110] The elution medium was directly analyzed by HPLC according to the parameters listed in the above section (Example VIII.) describing the chromatographic conditions. After 23 hours, the elution medium was removed and the skin was extracted with 2 mL of methanol, vortexed for 1 minute and centrifuged at 4500 rpm for 6 minutes. The samples were analyzed by HPLC according to the parameters listed in the above section (Example VIII.) describing the chromatographic conditions. The concentrations were calculated from the previous standard curve.

[0111] X. Stability Assessment of Formulations for Perivascular Drug Delivery

[0112] CRC-015 formulation preparation: 7.852 mg of CRC-015 was weighed into a 2-mL HPLC vial. 1000 μL PEG400 (175.253 mg (155 μL)) was pipetted into the vial to give a final concentration of 50.6 mg / mL. This formulation was tested in triplicate by weighing 5 μL into a 2-mL HPLC vial and adding 0.995 μL of methanol. The samples were analyzed by HPLC to give an average of 219 μg CRC-015 with a standard deviation of 10 μg.

[0113] Preparation of Sirolimus: Weigh 6.272 mg of Sirolimus into a 2-mL HPLC vial. Use 100 μL Use a syringe to add 342 μL of ethanol (200 proof) to the vial. Gently vortex the vial until the drug is dissolved in the solvent. Use 1000 μL Pipette PEG400 into the vial, 141.380 mg (125 μL). Place the vial without cap into a 30 mL bottle connected to a rotary evaporator. Place the solution under house vacuum for 1 hour to remove the ethanol and give a final concentration of 48.1 mg / mL with 3% ethanol in the final formulation.

[0114] The formulations were tested in triplicate by weighing 5 μL into a 2-mL HPLC vial and adding 0.995 μL of methanol. The samples were analyzed by HPLC to give an average of 230 μg of sirolimus with a standard deviation of 5 μg.

[0115] Preparation of sodium azide in saline: weigh 4.5 g of sodium chloride into 500 mL Prepare normal saline in a 500 mL bottle. Add 95.5 g of HPLC water to a 500 mL bottle. Cap the bottle and shake vigorously.

[0116] Prepare 1% sodium azide / saline solution by weighing 1.0 grams of sodium azide into a 120 mL bottle. Add 99.0 grams of normal saline to the 120 mL bottle containing the sodium azide. Cap the bottle and shake vigorously.

[0117] 0.05% sodium azide / saline solution was prepared by diluting 2.5 mL of 1% sodium azide with 47.5 mL of normal saline. The bottle was capped and shaken vigorously.

[0118] 1% and 0.5% solutions were obtained by 0.2 μm nylon Fisherbrand TM Syringe filter sterilization.

[0119] Stability Methods: Porcine peripheral blood vessels were dissected from euthanized pigs and stored rapidly frozen on dry ice and subsequently stored in a -80°C freezer. Porcine ears were obtained from euthanized pigs and stored rapidly frozen on dry ice and subsequently stored in a -20°C freezer.

[0120] Place the blood vessels on a glass tray and allow to thaw. Carefully remove any muscle or fat tissue using scissors. Cut the blood vessels into 1 cm long pieces. Place the ear on a glass tray. Carefully unfold the ear skin from the cartilage, keeping any blood vessel tissue with the skin. Then cut the skin into 2 x 5 cm pieces.

[0121] Both tissues were placed in a 20 mL beaker containing 1.0% sodium azide in saline for 15 minutes. The tissues were then rinsed by placing them in another 20 mL beaker containing 0.05% sodium azide in saline for 15 minutes.

[0122] Place the peripheral blood vessels on a 5 × 10 cm Saran wrap. Use 10 μL 5 μL of drug preparation (about 250 μg) was carefully applied to the outside of the peripheral blood vessel by pipette, as Figure 4 After 5 minutes, carefully apply the ear skin on top of the peripheral blood vessels, with the skin side facing up. Then wrap the tissue with saran wrap, as shown. Figure 4 As shown. Carefully place the sample in a clean 250 mL beaker. Then place the beaker in a 37°C water bath.

[0123] After 24 and 48 hours, carefully open the samples. Remove the tissue using forceps and transfer to a 15-mL vial. Rinse the ear tissue with 3 ml of saline solution and the peripheral tissue with 2 ml of saline solution. Extract the ear tissue with 3 ml of methanol, vortex for 1 minute and let stand on the bench for 15 minutes. Extract the peripheral tissue with 2 ml of methanol, vortex for 1 minute and let stand on the bench for 15 minutes.

[0124] The solution was transferred to a 1.7 mL centrifuge tube and centrifuged at 14,500 rpm for 6 minutes. The extract was placed in a 1.5 mL HPLC vial, capped and analyzed by HPLC. The samples were analyzed by HPLC according to the parameters listed in the above section (Example VIII.) describing the chromatographic conditions and compared with the previous standard curve.

[0125] XI. Elution Study Results

[0126] Elution studies were performed on porcine coronary artery tissue with CRC-015, sirolimus, and everolimus. The drug formulations in ethanol were applied (approximately 125 μg) to previously frozen "butcher shop" porcine coronary vessels and dried after 15-20 minutes. Elution studies were then performed as described in Example IX. The results are shown in Fig.12 and Table 7.

[0127] Table 7

[0128] Percent of drug eluted over time from washout studies

[0129]

[0130] XII. Results of Stability Studies in the Perivascular Model

[0131] Stability studies were performed on CRC-015 and sirolimus sandwiched between porcine peripheral artery and ear skin tissue to simulate a perivascular drug delivery model. The drug formulation (about 250 μg) was applied to the porcine peripheral artery blood vessel and the porcine ear skin was placed as described in Example X. The drug stability and total drug recovery results after incubation at 37°C for 24 hours and 48 hours are shown in Fig.13 and 14 and Table 8.

[0132] Table 8

[0133] Drug stability in porcine peripheral and ear tissues over 24 and 48 hours

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] * CRC-015 theoretical amount = 219 μg; Sirolimus theoretical amount = 230 μg

[0140] In summary, these elution studies were conducted on porcine coronary vessels with CRC-015, sirolimus or everolimus. The drug formulation in ethanol was applied (about 125 μg) to previously frozen "butcher shop" porcine coronary vessels. The ethanol was dried after 15-20 minutes, and the sample was placed in a vial containing a saline solution with 25% ethanol as an elution medium. The results showed that sirolimus and everolimus eluted significantly faster than CRC-015, and most of the drugs were eluted from the tissue within the first 2 hours of the study. CRC-015 eluted slowly, and most of the drugs were recovered after extraction. Because CRC-015 has been shown to be more lipophilic than sirolimus, CRC-015 can be retained on the tissue longer than sirolimus in the elution medium.

[0141] Compared with sirolimus, the stability of CRC-015 as perivascular drug delivery was studied. CRC-015 was formulated only in PEG 400, while sirolimus was dissolved only in PEG400 as an ethanol solution. CRC-015 was not only easily dissolved in PEG400 without any assistance from ethanol, but also observed that a larger amount of the drug was easily dissolved to give a concentration of up to 155 mg / mL. High concentrations of sirolimus were only achieved by first dissolving in ethanol, which was limited by its solubility in ethanol (50 mg / mL).

[0142] In all CRC-015 samples, higher amounts of CRC-015 were found on peripheral arterial tissue rather than on skin tissue. Sirolimus samples showed no preference; higher amounts of drug were located on either skin or peripheral tissue. Due to its lipophilic character, CRC-015 was shown to adhere to the tissue where the reservoir was applied, superior to sirolimus.

[0143] When looking at total drug stability, CRC-015 showed an average total recovery of 59% and 52% after 24 and 48 hours, respectively, while the sirolimus samples had an average recovery of 23% and 14%, respectively. The total recovery of CRC-015 was found to be significantly higher than that of sirolimus. Overall, CRC-015 has been shown to be more stable than sirolimus after incubation at 37°C for 24 and 48 hours, with better topical adhesion.

[0144] The present invention described illustratively herein can be appropriately implemented in the absence of any one or more elements, one or more restrictions not specifically disclosed herein. Therefore, for example, the terms "comprise", "include", "contain", etc. should be understood broadly without limitation. In addition, the terms and expressions used herein have been used as descriptive and non-restrictive terms, and when using these terms and expressions, it is not intended to exclude any equivalents or any part thereof shown and described in the future, and it is recognized that various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that, although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and changes of the present invention disclosed herein can be adopted by those skilled in the art, and these modifications and changes are considered to be within the scope of the present invention disclosed herein. The present invention has been described broadly and generally herein. Each narrower category and sub-genus grouping falling within the generally disclosed scope also forms a part of these inventions. This includes a general description of each invention, with its proviso or negative limitation removing any subject from the genus, regardless of whether the removed material is specifically present therein.

[0145] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also therefore described in terms of any individual member or subgroup of members of the Markush group. It should also be understood that the above description is intended to be illustrative and not restrictive. After reading the above description, many embodiments will be apparent to those of ordinary skill in the art. Therefore, the scope of the present invention should not be determined with reference to the above description, but should be determined with reference to the appended claims and the full scope of equivalents to which these claims are entitled. The disclosures of all articles and references (including patent publications) are incorporated herein by reference.

[0146] It will be apparent to those skilled in the art that, according to the above teachings, many modifications and variations of the described examples and embodiments are possible. The disclosed examples and embodiments may include some or all of the features disclosed herein. Therefore, the present invention is intended to cover all these modifications and alternative embodiments that may fall within the true scope of the present invention.

Claims

1. A method of treating an open vascular surgical procedure comprising administering an effective amount of a macrocyclic triene immunosuppressive compound by direct perivascular application, wherein the macrocyclic triene immunosuppressive compound has the following structure: Where R is C(O)-(CH2) n -X, n is 0, 1 or 2, X is a cyclic hydrocarbon having 3 to 7 carbons optionally containing one or more unsaturated bonds.

2. The method according to claim 1, wherein C(O)-(CH2) n -X has one of the following structures:

3. The method of claim 1, wherein the compound is stable when administered to a coronary artery.

4. The method of claim 1, wherein the compound is stable when administered to a peripheral artery.

5. The method of claim 1, wherein the compound upon administration results in superior vascular retention and reduced vascular spread.

6. The method of claim 1, wherein the compound is administered in combination with at least one compound selected from the group consisting of rapamycin (sirolimus), everolimus, zotarolimus, biolipid, novolimus, myolimus, temsirolimus, and a rapamycin derivative.

7. The method of claim 1, wherein the macrocyclic triene immunosuppressive compound is administered directly to at least one tissue within a vascular open surgery site.

8. The method of claim 1, wherein the macrocyclic triene immunosuppressive compound is administered in the form of a pharmaceutical formulation, wherein the pharmaceutical formulation comprises the macrocyclic triene immunosuppressive compound and at least one solvent.

9. The method of claim 8, wherein the at least one solvent is an alcohol.

10. The method of claim 8, wherein the at least one solvent is polyethylene glycol.

11. The method of claim 8, wherein the pharmaceutical formulation consists of the macrocyclic triene immunosuppressive compound and polyethylene glycol.

12. The method of claim 1, wherein the open vascular surgical procedure includes coronary artery bypass surgery, carotid endarterectomy, arteriovenous fistula transplantation (hemodialysis), treatment of stenosis following renal transplantation, and endovascular surgery.

13. A pharmaceutical preparation consisting of a macrocyclic triene immunosuppressive compound and a solvent, wherein the macrocyclic triene immunosuppressive compound has the following structure: Where R is C(O)-(CH2) n -X, n is 0, 1 or 2, X is a cyclic hydrocarbon having 3 to 7 carbons optionally containing one or more unsaturated bonds, wherein the solvent is a polyol.

14. The pharmaceutical preparation according to claim 13, wherein C(O)-(CH2) n -X has one of the following structures:

15. The pharmaceutical formulation of claim 13, wherein the polyol is polyethylene glycol.

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