Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• 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.
• 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.
• drug eluting stents and drug coated balloons there are no approved clinical options for preventing surgery induced intimal hyperplasia in “open” vasculature intervention.
• 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.
• ESRD end stage renal disease
• 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.
• C(O)-(CH2)n-X has one of the following structures:
• 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.
• the compound is stabile when administered to a peripheral artery.
• the compound, once administered, results in superior vessel retention and reduced vessel diffusion.
• open vascular surgical manipulations include coronary bypass surgery, carotid endarterectomy, arteriovenous fistula grafting (hemodialysis), stenosis after kidney transplant and endovascular surgery.
• 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.
• the at least one solvent is an alcohol.
• 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 8 shows rat femoral vein and artery prior to perivascular application of the preferred drug formulation.
• 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.
• macrocyclic triene immunosuppressive compound may also include rapamycin (sirolimus), everolimus, zotarolimus, biolimus, novolimus, myolimus, temsirolimus and the rapamycin derivatives described in this disclosure.
• 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.
• SMC smooth muscle cells
• 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.
• SMC smooth muscle cells
• 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.
• 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)
• 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.
• 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.
• 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 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.
• 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.
• C(O)-(CH2)n-X has one of the following structures:
• the compound is formulated to allow for direct perivascular application directly to graft vessels upon initiation of surgery.
• the macrocyclic triene immunosuppressive compound is applied directly to at least one tissue within the open vascular surgery site.
• 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.
• 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.
• rapamycin sirolimus
• everolimus everolimus
• zotarolimus biolimus
• novolimus myolimus
• temsirolimus rapamycin derivatives.
• 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:
• CRC-015 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.
• 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.
• drug stability and migration could be investigated and optimized before multiple animal in vivo testing.
• 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.
• perivascular model 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.
• 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.
• FIG. 9 shows the nonlinear regression curve obtained.
• the required minimum CRC-015 drug concentration for human coronary artery smooth muscle cells to reach ICso inhibition was 1.8 nM (FIG. 10).
• the time needed for vein + artery tissue concentration to reach 2 nM would be 21.5 weeks.
• 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
• 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.
• HPLC analyses were performed using Agilent High Performance Liquid Chromatography (HPLC) 1290 Infinity Series coupled to a diode array detector G4212B (SN DEAA300117).
• 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.
• 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 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.
• 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.
• 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.
• 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.
• 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.
• CRC-015 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
• CRC-015 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.

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