Synthesis and characterization of everolimus cyclic hydrocarbon esters

Abstract

The present application relates generally to the field of synthesis, formulations and methods of perivascular treatment of cyclic ester derivatives of everolimus. # imgabs0 # (I)

Description

Technical Field

[0001] The present application relates generally to the field of synthesis, formulation and perivascular treatment methods of cyclic ester derivatives of everolimus. Background Art

[0002] Occlusive vascular disease is the leading cause of mortality and morbidity worldwide. Recently, minimally invasive percutaneous endovascular interventions using drug eluting stents and drug-coated balloons have reduced the frequency of stenosis. However, unlike the technological advances using drug eluting stents and drug-coated balloons, there are no approved clinical options for preventing surgically induced intimal hyperplasia in "open" vascular system interventions (Chen et al. Biomacromolecules, (18) 2205-2213, 2017). Open surgery remains the only solution for cases that are considered unsuitable for percutaneous approaches, 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 about 400,000 open vascular surgeries in the United States each year (Jim et al., J Vasc Surg (55) 5: 1394-1400, 2012), and open surgery leads to up to 50% occlusion (restenosis), depending on the type of surgical intervention. An example of open intervention is the intervention required for hemodialysis treatment (HD). HD is driven by chronic kidney disease, which remains a global public health problem. In the United States, about 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, 05 / 08 / 2019 access). ESRD is complete and permanent renal failure and is treated with kidney transplantation or dialysis. In 2016, 71% of all U.S. ESRD cases were receiving HD treatment. (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 blood to be drawn and returned regularly. AVFs are not without significant restenosis and graft failure issues 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. In addition, in addition to patient inconvenience, there are also increased patient surgical risk factors, frequent need for blood-thinning medications over a period of time, and increased costs.

[0005] Initial attempts at therapeutic approaches to mitigate or reduce restenosis have focused on two areas: placement of perivascular (surrounding the outside) non-drug-free medical devices, including steel alloy mesh, nitinol mesh, and Dacron® sheaths, to maintain or enhance the graft, or systemic delivery of various vasoactive compounds. In the case of medical devices, surgical interventions are prone to increased vascular injury and results have shown low or conflicting efficacy. (Mylonaki, et al., Biomaterials (128) 56-68, 2017; Rescigno, et al., Thorac Cardiovasc Surg, (63) 4: 292-297, 2015). While systemic drug delivery has only yielded modest promise, there is still the potential for adverse systemic toxicity. (Schömig 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). These direct drug applications also lead to poor overall outcomes due to rapid drug elution and clearance and / or drug migration when used without a mechanism to retain the drug at the site of need. 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 reviewed by Seedial, there are currently no approved technologies or drug delivery devices 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 drug delivery 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 reduce restenosis (Siow, RCM, and Churchman, AT “Adventitial growth factor signaling and vascular remodeling: potential of perivascular gene transfer from the outside-in” Cardiovasc. Re., Vol. 75pp. 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 rapamycin 40-O-cyclic hydrocarbon ester compound having the following structure:

[0011]

[0012] Wherein R is a saturated cyclic hydrocarbon substituent. Preferably, R has one of the following structures:

[0013] , , , and .

[0014] Surprisingly, it has been found that these compounds having a cyclic hydrocarbon substituent at the C40 ethoxy ether position exhibit particularly useful properties when administered in a perivascular manner. The inventors have found that compounds as described above exhibit exceptionally slow drug elution from vascular tissue, indicating very slow drug elution in perivascular treatments. Thus, very sustained perivascular drug release to tissues is promoted, addressing current problems in preventing surgically induced intimal hyperplasia in "open" vasculature interventions.

[0015] In another aspect, the present invention provides a method for treating an open vascular surgical procedure, comprising administering an effective amount of the above-described rapamycin 40-O-cyclic hydrocarbon ester compound, preferably by direct perivascular administration, wherein the compound is preferably formulated to allow direct perivascular administration to a graft vessel at the beginning of the procedure. Preferably, the compound is stable when administered to a coronary artery or a peripheral artery. Most preferably, upon administration, the compound results in excellent vascular preservation and reduced vascular diffusion. Preferably, open vascular surgical procedures include coronary artery bypass surgery, carotid endarterectomy, arteriovenous fistula transplantation (hemodialysis), stenosis following renal transplantation, and endovascular surgery.

[0016] In another aspect, the present invention provides a method for treating vascular damage, comprising providing the above-mentioned compound, wherein the compound is dissolved in at least one solvent to produce a pharmaceutical preparation, wherein the pharmaceutical preparation is directly administered to at least one tissue within the site of vascular damage. Preferably, the at least one solvent comprises or consists of an alcohol. Wherein the alcohol acts as a solubilizing agent.

[0017] In another aspect, the present invention relates to an implant carrying an effective amount of the above-mentioned rapamycin 40-O-cyclic hydrocarbon ester compound. Preferably, the implant is a patch, a graft, or a suture material.

[0018] In another aspect, the present invention relates to a formulation comprising a rapamycin 40-O-cyclic hydrocarbon ester compound as described herein and at least one solvent, wherein the solvent consists of or comprises at least one solubilizing agent or a mixture of solubilizing agents. The solubilizing agent may be selected from alcohols such as ethanol, propanol, butanol, benzyl alcohol, glycerol, polyethylene glycol and propylene glycol, dimethyl sulfoxide, dimethylformamide, propylene benzyl benzoate, glycol monolaurate, Labrasol®, Kolliphor® and amylene hydrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The disclosed subject matter contained herein is best described in conjunction with the accompanying drawings, in which:

[0020] Figure 1 The main structures of everolimus and its cyclic hydrocarbon esters are shown, as defined by the R groups disclosed in the present invention.

[0021] Figure 2 The HPLC chromatogram of CRC-023 under UV spectrum is shown.

[0022] Figure 3 Results from a porcine coronary artery washout study comparing sirolimus, everolimus, and CRC-023 are presented. DETAILED DESCRIPTION

[0023] definition

[0024] 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.

[0025] A large number of actual 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 have provided 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.

[0026] 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 caused by the surgical procedure triggers a cascade of mechanical and biochemical events that trigger SMC proliferation, which lasts for 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).

[0027] 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).

[0028] These successes of sirolimus suggest and have led to several in vitro studies of sirolimus using perivascular delivery. All reported perivascular studies of sirolimus have utilized 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 approach may also suffer from toxicity issues as well as drug product manufacturing and end-user complexity.

[0029] Sirolimus delivery collagen wraps (US 6,726,923) are currently in clinical trials in the United States by Vascular Therapies, Inc. However, the use of wraps suffers from the need for specialized surgical delivery requirements and can itself cause negative remodeling. Material toxicity, administration complexity, manufacturing complexity, cost, and regulatory status of these materials remain issues for this approach.

[0030] The present invention provides a method for synthesizing certain derivatives by adding several cyclic hydrocarbon ester groups to the hydroxyethyl group of everolimus. These everolimus derivatives are shown in Table 1 below. These compounds have been shown to be very stable in vascular tissue and to be very slowly eluted from the tissue in model studies. This behavior is very advantageous in preventing surgically induced intimal hyperplasia in "open" vascular system interventions.

[0031] Example

[0032] Example drug molecules:

[0033] The macrocyclic triene immunosuppressive compounds of the present invention have more than one embodiment and can be described as comprising the compounds from Table 1 and Figure 1 At least one of the following substances:

[0034] Table 1

[0035] Description of CRC-02X material

[0036]

[0037] CRC-023 ae are cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane ester derivatives of everolimus, respectively.

[0038] These compounds were synthesized and tested by RP-HPLC, UV and high-resolution tandem mass spectrometry, and the results confirmed their respective molecular structures. In addition, CRC-023 was synthesized, purified and precipitated in milligram quantities to produce a white powder. CRC-023 was also characterized using the above analytical techniques, and the results were consistent with the structure of everolimus cyclohexane ester.

[0039] I. Chromatographic conditions

[0040] 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 (SNDEABE00184), a thermostat G1330B (SN DEBAK00182), a thermostat column chamber G1316A (SN DEAAK00266) and a quaternary pump G1311B (SN DEAAB00113). Data analysis was performed using the Agilent Chemstation software for the LC 3D system.

[0041] LCMS analysis was performed using an Agilent high performance liquid chromatograph (HPLC) 1290 Infinity series coupled to a diode array detector G4212B (SN DEAA300734). The HPLC was equipped with an automatic loop injector G1367E (SN DEAAN00634), a thermostat G1330B (SN DEBAK11543), a thermostat column compartment G1316C (DEBAC01381), a degasser G1379B (JP60800486) and a binary pump G1312B (SN DEAGD10783).

[0042] Mass spectrometry data were obtained using an Agilent 6540A quadrupole time-of-flight (QTOF) mass spectrometer (SN US93980202). Data analysis was performed using MassHunter Qualitative Analysis, Rev. B.04.00.

[0043] Table 2 shows the HPLC and mass spectrometry parameters used for the testing.

[0044] Table 2

[0045] HPLC and mass spectrometry conditions

[0046]

[0047]

[0048] II. Synthesis of Everolimus Cyclic Hydrocarbon Esters

[0049] The synthesis of various cyclic hydrocarbon ester derivatives of everolimus was performed according to the following procedure.

[0050] 10 mg of everolimus was weighed into a 2-mL HPLC glass vial. 50 μL of dichloromethane was added to the vial and the solution was stirred at ambient temperature until the everolimus was completely dissolved. 1.5 μL of pyridine and 5 μL of phosgene reactant (Table 3) were added to the solution.

[0051] The reaction mixture was stirred at ambient temperature for 10 minutes and then quenched by adding 1 mL of ethyl acetate and 0.4 mL of 1N hydrochloric acid. The organic phase was collected and washed three times with brine. The crude reaction was analyzed by HPLC and high-resolution mass spectrometry using the chromatographic method detailed in Table 1.

[0052] Table 3

[0053] Reactants

[0054]

[0055] III. Large-Scale Synthesis and Purification of Everolimus Cyclohexane Ester (CRC-023)

[0056] 200mg of everolimus was weighed into a 7-mL borosilicate glass vial. The drug was dissolved in 1mL of anhydrous tetrahydrofuran. 30μL of anhydrous pyridine and 100μL of cyclohexane phosgene were added to the vial. A stirring bar was placed in the vial and the reaction mixture was stirred at ambient temperature. After 25 minutes, the reaction mixture was transferred to a 125-mL separatory funnel. The 7-mL borosilicate glass vial reaction vessel was washed with a total of 10mL of ethyl acetate, and the washings were added to the separatory funnel. 6mL of 1N HCl was added to the funnel. The solution was mixed by inverting the separatory funnel three times to mix the phases, and the system was vented after mixing. The aqueous phase was collected in a 30-mL beaker and stripped twice with 5mL of ethyl acetate. The organic layers were combined and washed four times with 30mL of saline until a pH of about 5.5-6.5 was reached as measured by litmus paper.

[0057] Excess water was removed from the organic phase by adding sodium sulfate until the added solids moved freely at the bottom of the beaker after stirring with a stir bar. The solution was decanted into a 100-mL round-bottom flask and concentrated under vacuum via a rotary evaporator in a water bath maintained at 37°C.

[0058] Purification by open column chromatography

[0059] An open glass column (1.75 x 12 in.) was assembled and filled with 100 g of silica and 500 mL of a 40 / 60 ethyl acetate / n-heptane (v / v) solution. The purification method parameters are listed in Table 4.

[0060] Table 4

[0061] Open column chromatography parameters

[0062]

[0063] The crude product in the 100-mL round-bottom flask was carefully loaded onto the post with a pipette, ensuring that the column bed was not disturbed. The round-bottom flask was rinsed three times with about 0.5 mL of mobile phase, and the washing solution was also loaded onto the post. The column cock was opened to allow the sample to enter the column bed. The mobile phase was then carefully added to the post, and the column cock was opened to start purification. About 360 mL of void volume was collected, and fractions containing about 15 mL of solvent were then collected. The presence of the product was monitored by TLC using 60 / 40 ethyl acetate / hexane mobile phase. CRC-023 has a TLC Rf value of about 0.5.

[0064] The various fractions containing CRC-023 determined by TLC were tested by HPLC to verify product purity and the amount of approximate compounds, using everolimus as a standard. Fractions with CRC-023 chromatographic area percentage> 97% were combined into a 1-L round-bottom flask. The solution was concentrated by rotary evaporator under vacuum, with the water bath maintained at 37°C.

[0065] CRC-023 methanol precipitation

[0066] Assuming that 150 mg of CRC-023 is recovered from the purification, the sample from the above portion is reconstituted in 420 μL of methanol. 3.9 mL of ice-cold HPLC-grade water is transferred to a 7-mL borosilicate glass vial with a stir bar, and the vial is placed in an ice bath. Using a 100-μL syringe, the sample dissolved in methanol is slowly added dropwise to the vial water under vigorous stirring. The product precipitated in water is filtered under vacuum through a 15-mL medium porosity sintered glass funnel. The white solid product is quantitatively transferred to a 4-mL borosilicate glass vial using a spatula. Cover the vial with aluminum foil with multiple 1 mm perforations.

[0067] The vial was placed in an oven to dry at 45°C under vacuum at a pressure of -28.0 inches Hg. After a 24 hour drying period, CRC-023 was removed from the oven and allowed to reach ambient temperature. A 200 μg / mL solution of the final product was prepared in acetonitrile and tested by HPLC and high resolution mass spectrometry using the conditions detailed in Table 2. CRC-023 was stored dry at ≤-78°C.

[0068] IV. HPLC and mass spectrometry analysis results of everolimus cyclic hydrocarbon esters

[0069] The molecular structure of the everolimus cyclic hydrocarbon ester synthesized according to the above description is as follows Figure 1 Shown: cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane.

[0070] The results of mass spectrometry analysis of everolimus cyclic hydrocarbon esters are shown in Table 5. The molecular weight [M+Na]+ of each compound detected matched the molecular formula of the substance. The detected masses were also within the recommended ±0.003 m / z units of the calculated mass that was considered sufficient to support the molecular formula of the compound.

[0071] Table 5

[0072] Mass spectrometry results of everolimus cyclic hydrocarbon esters

[0073]

[0074] *The observed masses are within the recommended ±0.003 m / z units of the calculated values ​​which are considered adequate to support the molecular formula of the compound.

[0075] The mass fragments detected for the cyclic hydrocarbon ester of everolimus are shown in Table 6. Also shown are the mass fragments of everolimus for comparison. Mass fragments 320, 381, 409, 441, 453, 485, 582 and 614m / z are fragments that do not contain the modified molecular region, so these fragments are detected for everolimus and its cyclic hydrocarbon ester derivatives. Everolimus fragments 389, 651, 686 and 775m / z contain the molecular sites where CRC-023 ae is modified. These fragments do not exist in the MS / MS spectrum of the cyclic hydrocarbon ester derivative of everolimus, and are replaced by the corresponding mass fragments unique to each compound detailed in Table 6. The results of tandem mass spectrometry confirmed the molecular structures of these compounds.

[0076] Table 6

[0077] MS / MS results of everolimus cyclic hydrocarbon esters

[0078]

[0079] V. Results of Large-Scale Synthesis and Purification of Everolimus Cyclohexane Ester (CRC-023)

[0080] The synthesis, purification and methanol precipitation of CRC-023 were completed to obtain 133 mg of white powder. Figure 2 The RP-HPLC chromatogram of CRC-023 at 278 nm shows that the elution time of CRC-023 is 6.5 minutes and the area percentage is 97.8%.

[0081] The crude reaction of everolimus with the respective cyclic phosgene reactants produces cyclopropane (CRC-023a), cyclobutane (CRC-023b), cyclopentane (CRC-023c), cyclohexane (CRC-023d) and cycloheptane (CRC-023e) ester derivatives of everolimus. The RPHPLC retention times of these compounds are greater than everolimus, indicating an increase in lipophilicity. It is also observed that the retention times of these compounds increase with each addition of a carbon constituting the cyclic ring from CRC-023 ae. The UV spectrum of CRC-023 ae shows maximum absorption at 268, 278 and 288 nm, corresponding to the conjugated triene portion unique to sirolimus and its derivatives.

[0082] The MS and MS / MS analytical results of these compounds have confirmed their respective molecular formula and molecular structure. Large-scale (200 mg) synthesis, purification and methanol precipitation of CRC-023 have been carried out to produce 133 mg white powder. Characterize the material by RP-HPLC, UV, MS and MS / MS. Find that CRC-023 has 98.7% HPLC area percentage.

[0083] UV analysis of the material showed that the conjugated triene had maximum absorption at 268, 278, and 288 nm. The molecular weight and mass fragments detected from testing of this compound by high-resolution tandem mass spectrometry were consistent with the structure of CRC-023, confirming its identity as a cyclohexane ester derivative of everolimus.

[0084] In summary, cyclopropane, cyclobutane, cyclopentane, cyclohexane and cycloheptane ester derivatives of everolimus as described in the present invention were synthesized and tested by RP-HPLC, UV and high resolution tandem mass spectrometry, and the results confirmed their respective molecular structures. In addition, CRC-023 was synthesized, purified and precipitated in milligram amounts to produce a white powder. CRC-023 was also characterized using the above-mentioned analytical techniques, and the results were consistent with the structure of everolimus cyclohexane ester.

[0085] VI. Washout Procedure on Porcine Coronary Arteries

[0086] To examine the effects of certain formulations of the invention on porcine coronary artery samples, the samples were each prepared as follows:

[0087] Sirolimus Formulation Preparation: Weigh 3.118 mg of sirolimus into a 2-mL HPLC vial. Use a 1000 μL Eppendorf® pipette to add ethanol (200 proof) to the vial, 98.40 mg (125 μL), to give a final concentration of 24.9 mg / mL.

[0088] Everolimus formulation preparation: Weigh 3.141 mg of everolimus into a 2-mL HPLC vial. Use a 1000 μL Lependorf® pipette to add ethanol (200 proof) to the vial, 99.021 mg (126 μL), to give a final concentration of 24.9 mg / mL.

[0089] CRC-023 Formulation Preparation: 3.223 mg of CRC-023 was weighed into a 2-mL HPLC vial. Using a 1000 μL Sppendorf® pipette, ethanol (200 proof) was added to the vial, 99.753 mg (126 μL), to give a final concentration of 25.5 mg / mL.

[0090] Elution medium preparation: Prepare normal saline by weighing 4.5 grams of sodium chloride into a 500 mL Pyrex® bottle. Add 495.5 grams of HPLC water to the 500 mL bottle. Then cap the bottle and shake vigorously. Prepare 25% ethanol / saline elution medium by weighing 25.0 grams of ethanol (200 proof) into a 100 mL Pyrex® bottle. Add 75.0 grams of normal saline to the 100 mL bottle containing ethanol. Cap the bottle and shake vigorously.

[0091] 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.

[0092] The vessels were then cut into 1 cm sections. 5 μL of formulation (approximately 125 μg of drug) was carefully added onto the outer portion of the tissue using a 10 μL Hamilton® syringe. The ethanol was allowed to evaporate at room temperature for 15-20 minutes.

[0093] The tissues were then placed in 7 mL glass vials. 2 mL of elution medium pre-warmed to 37 °C was carefully added to each vial using an Eppendorf® pipette. Each vial was capped and placed in a 37 °C incubator with gentle shaking at 100 SPM (strokes per minute).

[0094] At certain time points, the elution medium was removed and fresh 2 mL of medium (at 37°C) was slowly added to the vial.

[0095] VII. Results of a porcine coronary artery washout study comparing sirolimus, everolimus, and CRC-023

[0096] Elution studies were performed on porcine coronary artery tissue with sirolimus, everolimus, and CRC-023. 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 VI above. The results are shown in Figure 3 and Table 7.

[0097] Table 8

[0098] Percent of drug eluted over time from washout studies

[0099]

[0100] The results clearly show that sirolimus and everolimus eluted significantly faster than CRC-023, with most of the drug eluting from the tissue within the first 2 hours of the study. In contrast, CRC-023 eluted slowly, with most of the drug being recovered only after extraction. This clearly demonstrates that not only do rapamycin 40-O-cyclic hydrocarbon ester compounds show very favorable elution behavior for perivascular applications, but they also show excellent stability.

[0101] 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.

[0102] 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 thereby 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 such claims are entitled. The disclosures of all articles and references (including patent publications) are incorporated herein by reference.

[0103] 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 rapamycin 40-O-cyclic hydrocarbon ester compound having the following structure: , wherein R is a saturated cyclic hydrocarbon substituent.

2. The rapamycin 40-O-cyclic hydrocarbon ester compound according to claim 1, wherein R is one of the following structures: , , , and .

3. A method of treating an open vascular surgical procedure comprising administering an effective amount of a compound according to claim 1 by direct perivascular administration.

4. The method of claim 3, wherein the compound is formulated to allow direct perivascular application to a graft vessel at the start of surgery.

5. The method of claim 3 or 4, wherein the compound is stable when administered to a coronary artery.

6. The method of claim 3 or 4, wherein the compound is stable when administered to a peripheral artery.

7. The method of any one of claims 3 to 5, wherein the compound upon administration results in superior vascular retention and reduced tissue spread.

8. A preparation particularly for treating vascular damage, comprising: Rapamycin 40-O-cyclic hydrocarbon ester compound having the following structure: , wherein R is a saturated cyclic hydrocarbon substituent, wherein the formulation further comprises at least one solvent, wherein the solvent consists of or comprises at least one solubilizing agent or a mixture of solubilizing agents, wherein the solubilizing agent may be selected from alcohols such as ethanol, propanol, butanol, benzyl alcohol, glycerol, polyethylene glycol and propylene glycol, dimethyl sulfoxide, dimethylformamide, propylene benzyl benzoate, glycol monolaurate, Labrasol®, Kolliphor® and amylene hydrate.

9. The formulation of claim 8, wherein the at least one solubilizing agent is ethanol. 10 . An implant comprising an effective amount of the rapamycin 40-O-cyclic hydrocarbon ester compound of claim 1 .

11. The implant according to claim 10, wherein the implant is selected from the group consisting of a patch, a wound patch, a suture material, a graft.

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