FIELD OF THE INVENTION

This application relates generally to the field of treatment or prevention of “open” vascular surgical interventions with administration of a macrocyclic triene immunosuppressive compound in order to reduce or eliminate stenotic and/or vascular cancer conditions.

BACKGROUND OF THE INVENTION

Occlusive vascular diseases are a leading cause of mortality and morbidity worldwide. Recently use of drug eluting stents and drug coated balloons for minimally invasive percutaneous endovascular interventions have reduced the frequency of stenotic situations. However unlike the technical advances of using drug eluting stents and drug coated balloons, there are no approved clinical options for preventing surgery induced intimal hyperplasia in “open” vasculature intervention. (Chen, et al., Biomacromolecules, (18)2205-2213, 2017) Open surgery is still the only solution for cases considered unsuitable for the percutaneous approach, including coronary bypass surgery, carotid endarterectomy, autologous arteriovenous fistula grafting procedures for hemodialysis patients, stenosis after kidney transplant and for treating various types of complications following endovascular surgery.

Limited flow stenotic conditions continue to be a major cause of surgical failure in these cases. A further complication in such surgically manipulated vessels is the induced occurrence of angiosarcoma or cutaneous squamous cell carcinoma at the surgery site. (Oskrochi, et al., Eur J Vase Endovasc Surg, (51)127-133, 2015; Jennings, etal., Cancer, (62)11:2436-2444, 1988; Costa, et al., Case Rept Transplant, 2426859, 2017)

There are approximately 400,000 open vascular procedures annually in the United States (Jim et al., J Vase Surg (55)5:1394- 1400, 2012) Open procedures result in up to 50% occluding (restenose) depending on the type of surgical intervention. An example of open intervention is that required for hemodialysis treatment (HD). HD is driven by chronic kidney disease that continues to be a global public health issue. In the United States approximately 750,000 patients per year are affected by end stage renal disease (ESRD) which is increasing by 5% per year. (https://pharm.ucsf.edu/kidney/need/statistics, accessed 05/08/2019) ESRD is total and permanent kidney failure and is treated with a kidney transplant or dialysis. In 2016, 71% of all U.S. ESRD cases were receiving HD therapy. (https://www.kidney.org/news/newsroom/factsheets/End- Stage-Renal-Disease-in-the-US, accessed 05/08/2019) The process of HD requires frequent blood access and the arteriovenous fistula (AVF) created through open surgery has emerged as the preferred blood access method. The AVF utilizes an artery surgically connected directly to a vein, usually in the arm which allows blood to be withdrawn and returned on a regular basis. The AVF is not without significant restenotic and graft failure problems in both the U.S.A, and Europe. (Gallieni, et al., Semin Intervent Radiol (26)96-105, 2009) The current treatment of such flow limited vessels is usually by use of balloon angioplasty, stenting or new graft formation. These retreatment procedures can be compromised because of the complicated nature of the surgically modified vessels which can include unnatural orientations and small sizes. Additionally, besides patient inconveniences, there are increased patient surgical risk factors, frequent need for blood thinning drugs over a period of time and increased costs.

Initial attempts at treatment means to mitigate or reduce vessel restenosis focused primarily on two areas: placement of perivascular (surrounding the outside) drug-free medical devices including steel alloy meshes, nitinol meshes and Dacron® sheaths to hold or reinforce the graft, or the systemic delivery of various vasoactive compounds. In the case of medical devices, the surgical procedures were prone to induce increased vessel injury and results demonstrated low or contradictory efficacy. (Mylonaki, et al., Biomaterials (128)56-68, 2017; Rescigno, et al., Thorac Cardiovasc Surg, (63)4:292-297, 2015) While systemic drug delivery resulted in only modest promise, there was still the potential for adverse systemic toxicity. (Schomig, et al., Circulation (112)2759-2761, 2005; Davies, et al., EurJ Vasc Endovasc Surg (42)519-529, 2011)

To overcome negative systemic drug effects, the direct targeted application of neat pharmaceutical agents has been attempted. (Chaudhary, et al., J Control Release, (233)174-180, 2016) These direct drug applications have also resulted in poor overall outcome due to fast drug washout and clearance and/or drug migration when used without a mechanism to retain the drug at the site where needed. Resulting attempts to overcome washout effects necessitated much higher drug loading and possible local toxicities. Together those findings suggested that a drug and drug carrier of some type was needed to facilitate both placement and retention of drug at the surgical site to provide control and sustained drug delivery over the course of treatment.

Ensuing development work has investigated a variety of drug-carrying, perivascular systems envisioned to provide IH treatment at surgical sites. A suggested classification of these systems has recently been provided: Gel, Gel + particles, Mesh, Sheath, Cuff, Wrap and Matrix. These authors compared the properties of each system as well. (Mylonaki, op. cit.) This comparison took into account the ease of administration, possibility of repeat administration, site specific localization, mechanical support and sustaining the drug release. The concluding opinion of these authors was that a system optimizing all requirements has not yet been developed.

Even now as then commented by Seedial, there are currently no approved techniques or drug delivery devices for reducing restenosis after open surgical revascularizations (Seedial, S.M., Kent, K.C., and et. al. “Local drug delivery to prevent restenosis” Journal of Vascular Surgery, Vol. 57 pp. 1403-1414, 2013). Applying a drug extraluminally appears promising since drug applied outside of the vessel has been shown to diffuse into the blood vessel wall (Lovich, M.A., Brown, L., and Edelman, E.R. “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 argued that extraluminal injury from surgical revascularization promotes signal cascade that activates migration of myofibroblasts into the neointima resulting in intimal hyperplasia; drug applied extraluminally would reduce the migration of myofibroblasts and lessen restenosis (Siow, R.C.M., and Churchman, A.T. “Adventitial growth factor signaling and vascular remodeling: potential of perivascular gene transfer from the outside-in” Cardiovasc. Re., Vol. 75 pp. 659-68, 2007).

Thus, there is a need in the state of the art to improve upon the above described deficiencies in the field of improved drug delivery at the specific site of surgery. Loss of vascular patency after open surgery continues to be a world-wide problem. There continues to be a lack of an approved drug, device or drug/device specific to this occlusive disease. Treatment of this condition is a large unmet need which requires a novel solution to remedy the aforementioned problems in the relevant art.

SUMMARY OF THE INVENTION

The present invention provides for a method of treating open vascular surgical manipulations 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 3-7 carbons, optionally containing one or more unsaturated bonds. Preferably, C(O)-(CH2)n-X has one of the following structures:

With advantage the compound is formulated to allow for direct perivascular application directly to graft vessels during open vascular surgical manipulations and if preferred upon initiation of surgery. The compound is stabile when administered to a coronary artery. Similarly, the compound is stabile when administered to a peripheral artery. More preferably, the compound, once administered, results in superior vessel retention and reduced vessel diffusion. Preferably, open vascular surgical manipulations include coronary bypass surgery, carotid endarterectomy, arteriovenous fistula grafting (hemodialysis), stenosis after kidney transplant and endovascular surgery.

In another aspect, the present invention provides a method of treating a vascular injury comprising providing a drug formulation comprising a macrocyclic triene immunosuppressive compound dissolved in at least one solvent, wherein the drug formulation is applied directly to at least one tissue within the vascular injury site. Preferably, the at least one solvent is an alcohol.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1 shows drug stability comparison across different formulations when applied to porcine coronary arterial tissue. Figure 2 shows drug stability comparison across different formulations when applied to porcine peripheral arterial tissue.

Figure 3 shows drug retention comparison across different formulations when applied to porcine coronary arterial tissue.

Figure 4 depicts a representative ex vivo perivascular drug delivery model of the present invention.

Figure 5 shows amounts of drug recovery from arterial and ear skin tissues at 24 and 48 hours post-surgery.

Figure 6 shows a example of a preferred perivascular application of the drug formulations of the present invention.

Figure 7 shows concentrations and amounts of the preferred drug formulation in the jugular vein and surrounding tissues versus time; n=2.

Figure 8 shows rat femoral vein and artery prior to perivascular application of the preferred drug formulation.

Figure 9 shows preferred drug concentration or amounts in peripheral vein + artery and surrounding tissues vs time; n=2. Data analyzed by nonlinear regression (GraphPad Prism 8.0 software program) based on the one phase decay model.

Figure 10 shows preferred drug treatment on coronary artery smooth muscle cells, adjusted; ICso = 1.8 nM. Data analyzed by nonlinear regression based on the dose response model (GraphPad Prism 7.0 software). Figure 11 shows total drug concentration in Sprague Dawley rat whole blood vs time. Black dotted line represents minimum sirolimus concentration required for immunosuppression (16-24 ng/mL). Data plotted using GraphPad Prism 8.0 software program.

Figure 12 shows porcine coronary artery elution results with CRC-015, sirolimus and everolimus, n=2.

Figure 13 shows drug stability comparisons in perivascular model.

Figure 14 shows total drug recovered from arterial and ear skin tissues at 24- and 48-hours.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term “macrocyclic triene immunosuppressive compound” may also include rapamycin (sirolimus), everolimus, zotarolimus, biolimus, novolimus, myolimus, temsirolimus and the rapamycin derivatives described in this disclosure.

An important understanding of the mechanism of perivascular drug deposition from the perivascular space to the blood vessel wall has been advanced by the work of Lovich, Brown and Edelman. (Lovich et al., JACC, (29)7: 1645-1650, 1997) These authors determined by using utilizing in vivo animal studies with radiolabeled model drugs that, while a large portion of drug released into the perivascular space is cleared systemically through extravascular capillaries, most of the drug deposited into the vessel is supplied by direct inward diffusion from the source and little from systemic endovascular means.

A large variety of actual drug candidates for perivascular delivery have been investigated in both in vitro and in vivo studies. Concomitantly, investigative efforts to better understand the pathological basis of vascular remodeling following surgical procedures have provided guidance for drug selection. It is now recognized that endothelial changes associated with post-surgical vessel remodeling and possible resulting neointimal structural lesion (intimal hyperplasia (IH)) occurrence is central to both vein and arterial graft vessel narrowing (restenosis) and failure).

IH has been determined to originate from the abnormal migration and proliferation of vascular smooth muscle cells (SMC) with the deposition of extracellular connective tissue matrix resulting in restenosis. (Davies, et al., J Vase Surg, (61)203-216, 2015) In the case of vascular grafting procedures, within hours, endothelial disruption by the surgical process initiates a mechanical and biochemical event cascade triggering SMC proliferation that continues for days or weeks with negative remodeling of the vessel. (Mitra, et al., Immunol Cell Biol, (84)115-112, 2006) It has been determined that resulting vascular graft restenotic lesions largely occur at the surgically connected regions. (Mills, et al., J Vase Surg, (17)195-206, 1993)

Preclinical in vitro and model animal studies have shown that the compound sirolimus is effective in inhibiting vascular SMC proliferation. These investigations led to a large number of human studies utilizing sirolimus eluting cardiac stents that have demonstrated marked reduction of restenosis after stent implantation. (Marx, et al., Circ Cardiovasc /c ’, (4) 1 : l 04- 1 1 1 , 2011) Oral delivery of sirolimus and everolimus (sirolimus derivative) has also shown SMC inhibiting activities, albeit with undesirable systemic side effects. (Kurdi, et al., Br J Clin Pharmacol, (82)5: 1267-1279, 2015) Likewise sirolimus has also been shown using several human cancer cell lines to inhibit angiosarcoma at subtoxic doses. (Bundscherer, et al., Anticancer Res, (30)4017- 4024, 2010)

These successes with sirolimus suggested and have resulted in several in vitro investigations utilizing perivascularly delivered sirolimus. All reported sirolimus perivascular studies have utilized an additional means to control drug migration and to extend drug delivery time to approximately 2 to 6 weeks. These means have included: wraps, gels and a variety of polymers, as well as micro- or nano-sized particles of varying chemical and mechanical complexities. Recently a mixture of sirolimus combined with hydrophobic cyanoacrylate(s) applied at the graft site has also been proposed as a means to retain the drug on target. (Tiansu- Chu, et al. Arq Bras Cardiol, (112)1:3-10, 2019) Presumably the increased hydrophobic and mechanical strength of the ensuing drug depot aids in this regard. This approach can also suffer from toxicity concerns as well as drug product manufacturing and end user complexities.

A sirolimus delivering collagen wrap (US 6,726,923) is currently in U.S. clinical trials being conducted by Vascular Therapies, Inc. The use of wraps, however, suffer from requiring specialized surgical delivery requirements and can themselves induce negative remodeling. Material toxicities, administration complexities, manufacturing complexities, cost and regulatory status of these materials remain as issues to this approach.

The present invention provides for methods that overcome the above described limitations in the state of the art with respect to treating open vascular surgical manipulations comprising administering an effective amount of a macrocyclic triene immunosuppressive compound by direct perivascular application, wherein the macrocyclic triene immunosuppressive compound has the where R is C(0)-(CH2)n-X, n is 0, 1 or 2, X is a cyclic hydrocarbon having 3-7 carbons, optionally containing one or more unsaturated bonds. Preferably, C(O)-(CH2)n-X has one of the following structures:

With advantage the compound is formulated to allow for direct perivascular application directly to graft vessels upon initiation of surgery. Thereby, the macrocyclic triene immunosuppressive compound is applied directly to at least one tissue within the open vascular surgery site.

It is further provided that for method as described herein the macrocyclic triene immunosuppressive compound is administered in the form of a drug formulation, wherein the drug formulation includes the macrocyclic triene immunosuppressive compound and at least one solvent. The solvent may preferably be an alcohol and more preferably a polyalcohol and most preferably polyethylene glycol. In one embodiment of the method the drug formulation consists of the macrocyclic triene immunosuppressive compound as described herein and a solvent, wherein the solvent is an alcohol and more preferably a polyalcohol and most preferably polyethylene glycol.

One aspect of the present invention is directed to a drug formulation including or consisting of the macrocyclic triene immunosuppressive compound as described herein and a solvent, wherein the solvent is an alcohol and more preferably a polyalcohol and most preferably polyethylene glycol.

In one embodiment, the compound administered in combination with at least one compound selected from the group consisting of rapamycin (sirolimus), everolimus, zotarolimus, biolimus, novolimus, myolimus, temsirolimus and rapamycin derivatives. Examples

Example Drug Formulations:

The macrocyclic triene immunosuppressive compound of the present invention has more than one embodiment and may be described as comprising at least one of the following species from Table 1:

Table 1

Description of CRC-015 species

CRC-015, as referred to herein, is a term meant to encompass a genus and 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.

I. Increased drug solubility

An example of superior CRC-015 solubility compared to sirolimus in a simple PEG400 delivery vehicle facilitating small drug delivery volumes is shown in Table 2.

Table 2

Drug solubility comparison

II. Superior coronary arterial stability of CRC-015

Stability studies were performed on porcine coronary arterial tissue with CRC-015, sirolimus, everolimus or BA9. These compounds were formulated, separately, in ethanol (200 proof). Porcine coronary arterial vessels were spliced open and cut into 1 x 1 cm pieces. Each drug formulation was applied onto the center of the endothelial surface of a tissue section and ethanol was allowed to dry. Tissue samples were placed in 30-mL vials along with 150 pL of 0.05% NaNs in saline to keep tissue moist and microbially free. Vials were capped and placed in a 37 °C water bath under static conditions. At 4 or 8 days, tissue was removed and placed into a clean vial and extracted with methanol. Measurement of residual drug was conducted by HPLC using external standards. Estimated half-life of drugs were calculated by linear regression (Microsoft Excel 2010 software program). Increased CRC-015 stability on/in coronary arterial tissue is demonstrated at FIG. 1 and Table 3 below.

Table 3

Estimated drug half-life on porcine coronary arterial tissue

III. Superior peripheral arterial stability of CRC-015

Stability studies and half-life calculations were performed on porcine peripheral arterial tissue with CRC-015, sirolimus, or everolimus in a manner similar to II. above. Increased stability of CRC-015 on/in peripheral arterial tissue is demonstrated at FIG. 2 and Table 4 below.

Table 4

Estimated drug half-life on porcine peripheral arterial tissue

IV. Superior retention of CRC-015 in vessel

An elution study in 25% ethanol/normal saline (w/w) was performed to measure drug released from porcine coronary arterial vessel after topical application. CRC-015, sirolimus and everolimus was formulated, separately, in ethanol (200 proof). Porcine coronary arterial vessels were cut into 1 cm sections. Each drug formulation was applied onto the exterior surface of the tissue section and ethanol was allowed to dry. Tissue was placed in a vial containing 25% ethanol/normal saline (w/w) warmed at 37 °C and then shaken at 100 spm (strokes per minute). At specified time points the elution medium was removed for analysis. Fresh elution medium was then added to each sample and the study was continued.

Measurement of drug eluted was conducted by HPLC using external standards. Comparison results are shown at FIG. 3. CRC-015 shows slower comparative release from tissue submerged in elution solution.

V. Reduced vessel drug diffusion

An initial ex vivo perivascular drug delivery model was developed as an alternative to in vivo tests to reduce the number of animals used for testing per 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) With this model a single donor animal could contribute to multiple test conditions and repetitions.

The method utilized test drugs placed onto porcine peripheral arterial segments brought and held into close contact with porcine ear tissue after the removal of ear tissue cartilage. The goal was to simulate in vivo perivascular delivery using drug deposited onto a cylindrical vessel lying in close contact to a semi-planar, flexible tissue bed and a tissue covering as illustrated in FIG. 4. In this manner, drug stability and migration could be investigated and optimized before multiple animal in vivo testing. At 24 and 48 hour time points, tissues were collected, rinsed with normal saline and extracted with methanol. Comparative measurement of residual drug in vessel versus tissue was conducted by HPLC using external standards. Comparison results and drug stabilities are shown (FIG. 5). These results correlate well with the results of the retention study outlined in IV. from above and further indicate that CRC-015 has reduced drug diffusion from the vessel to tissue as compared to sirolimus. VI. In vivo perivascular CRC-015 uptake

On the basis of the perivascular model’s comparative findings of CRC-015 drug diffusion versus that of sirolimus, rat in vivo studies were conducted. The initial investigation focused on jugular vein CRC-015 perivascular delivery and the second investigation focused on peripheral artery + femoral vein CRC-015 perivascular drug delivery. The goal was to measure washout, clearance and vessel content of perivascularly applied CRC-015 and compare results to published studies utilizing sirolimus.

Jugular vein investigation:

Unfasted male Wistar rats were allowed water ad libitum. Prior to operation, rats were anesthetized with isoflurane then shaved underneath the neck. A vertical incision was made to expose the right external jugular vein. Using a calibrated 10-pL pipette, 5 pL of CRC-015 in PEG400 was placed directly on the jugular twice to deliver a 1 mg CRC-015 dose. It appears that the PEG quickly diffuses resulting in precipitation of the drug. Precipitated CRC-015 as a white depot on the vein surface was observed (FIG. 6).

The incision was then closed with 3 absorbable sutures. Jugular vein and surrounding tissues were collected at 2 and 4 weeks. Jugular vein was rinsed with water and wiped with moisten lint-fee cloths to remove any traces of drug on the surface of the tissue. Tissues were extracted with acetonitrile and solutions were tested by HPLC. Residual CRC-015 tissue concentrations and amounts remaining in surrounding tissues are shown in FIG. 7.

Peripheral artery + femoral vein investigation:

Unfasted male Sprague Dawley rats were allowed water ad libitum. Prior to operation, rats were anesthetized with isoflurane then shaved. An incision was made to expose the right femoral artery/vein. An example of the femoral vein + artery prior to application is shown in FIG. 8. Using a calibrated 100-pL pipette, 11 pL of CRC-015 in PEG400 was placed directly on the femoral artery + vein to deliver a 3 mg CRC-015 dose. The incision was then closed with 3 absorbable sutures. Whole blood was obtained at specific time points and prior to tissue collection. Whole blood was treated with heparin (45 units/ 1 mb 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. Tissues were extracted with acetonitrile and solutions were tested by HPLC. HPLC results indicated effective concentrations of CRC-015 bound to vein + artery, as well as significant remaining depot drug contained in surrounding tissues at each time point to deliver a therapeutic drug amount as determined by in vitro CRC-015 arterial smooth muscle ICso inhibition studies.

FIG. 9 shows the nonlinear regression curve obtained. In separate studies, the required minimum CRC-015 drug concentration for human coronary artery smooth muscle cells to reach ICso inhibition was 1.8 nM (FIG. 10). By extrapolating from the data and curve shown, the time needed for vein + artery tissue concentration to reach 2 nM would be 21.5 weeks.

Whole blood analysis was conducted during the second investigation from above. Blood was collected at specified time points and analyzed by LC/MS. Total drug concentration in whole blood versus time is shown in FIG. 11. All samples were below the 16-24 ng/mL sirolimus concentrations generally accepted for immunosuppression.

The potential to deliver site-specific therapeutic drug levels without the use of mechanical wraps, collars, sleeves or complicated polymeric formulations to prophylactically treat open surgical occlusive conditions is very attractive. CRC-015 has been shown to have unique and superior properties as compared to the currently leading candidate, sirolimus, as well as other sirolimus derivatives for the treatment of patency loss resulting from various “open” surgical procedures. CRC-015 delivery to the vessel was found to extend beyond the 4-6 weeks reported from several sirolimus investigations. It is believed that extended drug delivery time will result in superior patient outcome and reduced or eliminated need for postsurgical interventions. The unique CRC-015 depot did not show signs of toxicity to the surrounding tissue and the systemic blood drug levels were found to be below that resulting in immunosuppression. VII. Elution and stability study from porcine coronary artery tissue in a perivascular model

An elution study in 25% ethanol/normal saline (w/w) was performed to measure drug released from porcine coronary arterial vessel after topical application. CRC-015, sirolimus and everolimus was formulated, separately, in ethanol (200 proof) and applied on the outer part of porcine coronary arterial vessels. Once applied, ethanol was allowed to dry. Tissue was placed in a vial containing 25% ethanol/normal saline (w/w) warmed at 37 °C and then shaken at 100 spm (strokes per minute). Aliquots at certain time points were removed and replenished. Samples were analyzed by HPLC.

Porcine peripheral arterial tissue and ear tissue were used to mimic a perivascular model. As illustrated in FIG. 4, drug formulation was first applied onto the peripheral arterial vessel which sat on top a section of saran wrap. Porcine ear tissue was then placed over the sample. Saran wrap was folded over to enclose the tissues. The sample was then incubated at 37 °C for 24 or 48 hours.

VIII. Chromatographic conditions

HPLC analyses were performed using Agilent High Performance Liquid Chromatography (HPLC) 1290 Infinity Series coupled to a diode array detector G4212B (SN DEAA300117). The HPLC is equipped with a high performance autosampler G1329B (SN DEABE00184), thermostat G1330B (SN DEBAK00182), thermostatted column compartment G1316A (SN DEAAK00266), and quaternary pump G131 IB (SN DEAAB00113). The HPLC method parameters are shown in Table 5 and Table 6, respectively.

Table 5

HPLC Chromatographic Conditions for CRC-015

Table 6

HPLC Chromatographic Conditions for Sirolimus

IX. Elution procedures on porcine coronary artery

To examine the effects of certain formulations of the present invention on porcine coronary artery samples, the samples were each prepared as follows: CRC-015 Formulation Preparation: CRC-015 was weighed, 3.189 mg, into a 2-mL HPLC vial. Ethanol (200 proof) was added to the vial, 100.64 mg (127 pL), using a 1000 pL Eppendorf® pipette to give a final concentration of 25.1 mg/mL.

Sirolimus Formulation Preparation: Sirolimus was weighed, 3.118 mg, into a 2-mL HPLC vial. Ethanol (200 proof) was added to the vial, 98.40 mg (125 pL), using a 1000 pL Eppendorf® pipette to give a final concentration of 24.9 mg/mL.

Everolimus Formulation Preparation: Everolimus was weighed, 3.141 mg, into a 2-mL HPLC vial. Ethanol (200 proof) was added to the vial, 99.021 mg (126 pL), using a 1000 pL Eppendorf® pipette to give a final concentration of 24.9 mg/mL.

Elution Media Preparation: Normal saline was prepared by weighing 4.5 grams of sodium chloride into a 500 mL Pyrex® bottle. 495.5 grams HPLC water was added to the 500 mL bottle. The bottle was then capped and vigorously shaken. 25% ethanol/ saline elution media was prepared by weighing 25.0 grams of ethanol (200 proof) into a 100 mL Pyrex® bottle. 75.0 grams of normal saline was added to the 100 mL bottle containing ethanol. The bottle was capped and vigorously shaken.

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

Vessels were then cut into 1 cm sections. 5 pL of formulation (approximately 125 pg of drug) was carefully added onto the outer part of the tissue using a 10 pL Hamilton® syringe. Ethanol was allowed to evaporate at room temperature for 15-20 minutes. Tissue was then placed into a 7 mL glass vial. 2 mL elution media, previously warmed to 37 °C, was added carefully to each vial using an Eppendorf® pipette. Each vial was capped and placed in a 37 °C incubator and gently shaken at 100 spm (strokes per minute).

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

Elution media was directly analyzed by HPLC following parameters listed in the above section (Example VIII.) describing the chromatographic conditions. After 23 hours, elution media was removed and skin was extracted with 2 mL methanol, vortexed for 1 minute and centrifuged for 6 minutes at 4500 rpm. Samples were analyzed by HPLC following parameters listed in the above section (Example VIII.) describing the chromatographic conditions. Concentrations were calculated from previous standard curves.

X. Evaluation of formulation stability for perivascular drug delivery

CRC-015 Formulation Preparation: CRC-015 was weighed, 7.852 mg, into a 2-mL HPLC vial. PEG400 was added to the vial, 175.253 mg (155 pL), using a 1000 pL Eppendorf® pipette to give a final concentration of 50.6 mg/mL. This formulation was tested by weighing 5 pL into a 2-mL HPLC vial and adding 0.995 pL methanol, in triplicate. Samples were analyzed by HPLC and resulted in an average of 219 pg CRC-015 with 10 pg standard deviation.

Sirolimus Formulation Preparation: Sirolimus was weighed, 6.272 mg, into a 2-mL HPLC vial. Ethanol (200 proof) was added to the vial, 342 pL, using a 100 pL Hamilton® syringe. The vial was gently vortexed until the drug dissolved into the solvent. PEG400 was added to the vial, 141.380 mg (125 pL), using a 1000 pL Eppendorf® pipette. The vial was placed uncapped into a 30-mL vial which was attached to the rotary evaporator. Solution was under house vacuum for one hour to remove ethanol and give a final concentration of 48.1 mg/mL with 3% ethanol in the final formulation. The formulation was tested by weighing 5 pL into a 2-mL HPLC vial and adding 0.995 pL methanol, in triplicate. Samples were analyzed by HPLC and resulted in an average of 230 pg sirolimus with 5 pg standard deviation.

Sodium Azide in Normal Saline Preparation: Normal saline was prepared by weighing 4.5 grams of sodium chloride into a 500 mL Pyrex® bottle. 95.5 grams HPLC water was added to the 500 mL bottle. The bottle was capped and vigorously shaken.

1% sodium azide/saline solution was prepared by weighing 1.0 gram of sodium azide into a 120 mL bottle. 99.0 grams of normal saline was added to the 120 mL bottle containing sodium azide. The bottle was capped and vigorously shaken.

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

1% and 0.5 % solutions were sterilized by a 0.2 um nylon Fisherbrand™ syringe filter.

Stability Method: Porcine peripheral vessels were dissected from euthanized porcine and quickly stored frozen on dry ice and later in a -80 °C freezer. Porcine ears were obtained from euthanized porcine and quickly stored frozen on dry ice and later in a -20 °C freezer.

Vessels were placed on a glass tray and allowed to thaw. Any muscle or fat tissue was carefully removed using scissors. Vessels were cut to 1 cm length pieces. Ears were placed on a glass tray. Ear skin was carefully splayed from cartilage keeping any vascular tissue with the skin. This skin was then cut into 2 x 5 cm pieces.

Both tissues were placed into a 20-mL beaker containing 1.0% sodium azide in normal saline solution for 15 minutes. Tissues where then rinsed by placing into another 20-mL beaker containing 0.05% sodium azide in normal saline solution for 15 minutes. Peripheral vessel was placed on a 5 x 10 cm saran wrap. 5 pL of drug formulation (approximately 250 pg) was carefully applied on the outer side of the peripheral vessel using a 10 pL Eppendorf® pipette, as shown in FIG. 4. After 5 minutes, ear skin was carefully applied on top of peripheral vessel with skin side facing up. Tissues were then wrapped with the saran wrap as shown in FIG. 4. Samples were carefully placed in a clean 250-mL beaker. The beaker was then placed into a 37 °C water bath.

After 24 and 48 hours, samples were carefully unwrapped. Tissues were removed using tweezers and transferred into 15-mL vials. Ear tissue was rinsed with 3 mL normal saline solution while peripheral tissue was rinsed with 2 mL normal saline solution. Ear tissue was extracted with 3 mL methanol, vortexed for 1 minute and let sit on bench top for 15 minutes. Peripheral tissue was extracted with 2 mL methanol, vortexed for 1 minute and let sit on bench top for 15 minutes.

Solutions were transferred into 1.7-mL centrifuge tubes and centrifuged for 6 minutes at 14,500 rpms. Extracts were placed in 1.5 mL HPLC vials, capped and analyzed by HPLC. Samples were analyzed by HPLC following parameters listed in the above section describing the chromatographic conditions (Example VIII.) and compared to a previous standard curve.

XI. Elution study results

Elution studies were performed on porcine coronary arterial tissue with CRC-015, sirolimus and everolimus. Drug formulations in ethanol were applied (approximately 125 pg) onto previously frozen “butcher shop” porcine coronary arterial vessel and allowed to dry after 15-20 minutes. An elution study was then performed as described in Example IX. Results are shown in FIG. 12 and Table 7.

Table 7

Percent of Drug Eluted Over Time from Elution Study

XII. Stability study results in perivascular model

Stability studies were performed on CRC-015 and sirolimus sandwiched between porcine peripheral artery and ear skin tissue to mimic a perivascular drug delivery model. Drug formulations were applied (approximately 250 pg) onto porcine peripheral arterial vessels with porcine ear skin placed over as described in Example X. Drug stability and total drug recovery results after 24- and 48-hours incubation at 37 °C are shown in FIG. 13 and 14, respectively, as well as Table 8.

Table 8

Drug Stability on Porcine Peripheral and Ear Tissue over 24 and 48 hours

*CRC-015 Theoretical Amount = 219 iig: Sirolimus Theoretical Amount = 230 iig

In conclusion, these elution studies were performed on porcine coronary arterial vessel with CRC-015, sirolimus or everolimus. Drug formulations in ethanol were applied (approximately 125 pg) onto previously frozen “butcher shop” porcine coronary arterial vessel. Ethanol was allowed to dry after 15-20 minutes and samples were place in vials containing 25% ethanol in normal saline as the elution media. Results show sirolimus and everolimus elute significantly faster than CRC-015 and with most of the drug eluting off the tissue within the first 2 hours of the study. CRC-015 and slowly eluted with most of the drug recovered after extraction. Because CRC-015 has been shown to be more lipophilic than sirolimus, CRC-015 was able to remain onto the tissue in the elution media longer than sirolimus.

The stability of CRC-015 with comparison to sirolimus as a perivascular drug delivery was investigated. CRC-015 was formulated in only PEG 400 while sirolimus was only soluble in PEG400 as an ethanolic solution. Not only did CRC-015 easily dissolve in PEG400 without any assistance of ethanol, it was also observed that higher amounts of drug easily dissolved to give up to a concentration of 155 mg/mL. High concentrations of sirolimus were only achieved by first dissolving in ethanol which is limited due to its solubility in ethanol (50 mg/mL).5

Higher CRC-015 amounts have been found on peripheral arterial tissues rather than on skin tissue, in all of the CRC-015 samples. Sirolimus samples show no preference; higher drug amounts were located either on the skin or peripheral tissue. Because of its lipophilicity characteristic, CRC-015 was shown to adhere to the tissue where the depot was applied, better than sirolimus.

When observing total drug stability, CRC-015 was shown to have an average total recovery of 59% and 52% after 24 and 48 hours respectively, while sirolimus samples had average recoveries of 23% and 14%, respectively. CRC-015 total recovery amounts were found to be significantly higher than when compared to sirolimus. Overall, CRC-015 has been shown to be more stable with better localized adherence than sirolimus after 24- and 48-hours incubation at 37 °C.

The inventions illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the future shown and described or any portion thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions herein disclosed can be resorted by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of these inventions. This includes the generic description of each invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised materials specifically resided therein.

In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled 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 is also to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of ordinary skill in the art upon reviewing the above description. The scope of the invention should therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with 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. It will be apparent to those skilled in the art that numerous modifications and variations of the described examples and embodiments are possible in light of the above teaching. The disclosed examples and embodiments may include some or all of the features disclosed herein. Therefore, it is the intent to cover all such modifications and alternate embodiments as may come within the true scope of this invention.