Patent Application
20220265832
The present disclosure provides compositions, pre-filled delivery devices, and methods of using both for delivery of active agents.
For therapeutic and prophylactic treatments to be successful, the active agents need to be maintained at a certain concentration level for a period of time at the location or locations of interest within a subject or patient. Although treatment for diseases and disorders can be easily controlled in a hospital setting, treatment regimens are increasingly being done at home by the patient or a caregiver, or in locations without access to hospital care, including military installations and remote towns and villages. Patient compliance with the therapeutic and prophylactic treatment regimen becomes a significant challenge when the treatments are moved outside a hospital setting due to a number of factors, including complicated storage and preparation protocols or difficult administration requirements. It is estimated that only about half of patients who leave a physician's office with a prescription for a treatment regimen do so as directed.
The most obvious result of non-compliance is that the disease or disorder may not be relieved or cured, and in some case can actually lead to a worsening of the disease or disorder. For example, missed doses or early cessation of antibiotic or antiviral therapy may lead to resistant organisms. If patients administered the therapeutic and prophylactic treatments as directed many nursing home admissions, hospital admissions, physician visits, diagnostic tests, and other unnecessary treatments could be avoided.
For example, in the United States, according to a comprehensive 5-year report generated by the Centers of Disease Control and Prevention, approximately 2.4 million health care visits are diagnosed with outer ear infections (otitis externa or OE) annually. Both the pediatric and adult population are affected by OE, with a higher prevalence recorded in adults (approximately 53% of cases). The global ear infection treatment market is forecasted to have an incremental growth of $2.79 billion by 2023, with the largest market share being attributed to North America and Europe.
The most common cause accounting for approximately 98% of all ear infections is bacterial septicity typically attributable to Pseudomonas aeruginosa or Staphylococcus aureus. Treatment of OE involves patient or caregiver administered topical antibiotic drops multiple times daily for 7-14 days. Compliance with the treatment regimen, however, poses significant challenges for the target population, especially for military personnel, school nurses, nursing homes, prisons, the geriatric population, and for patients with head or hand tremors.
Incorrect application or non-compliance with administration schedule of antibiotics often translates to ineffective drug doses at the infection site and leads to infection persistence, recurrence and development of antibiotic resistant bacterial strains. In elderly, diabetic or immunocompromised patients, OE can spread to the surrounding tissue as necrotizing or malignant otitis externa (MOE), causing bone erosion, cranial nerve deficits, abscesses or even death. MOE has been primarily associated with P. aeruginosa infections and ciprofloxacin-resistant strains have been found in as many as 33% of isolates. Alarmingly, several recent studies have reported MOE rates to be increasing worldwide. Patients diagnosed with MOE currently undergo more than six weeks of bacteria-culture specific oral systemic antibiotic treatment.
Thus, formulations for therapeutic and prophylactic treatment which decrease complicated storage and dosage or administration requirements and increase ease of use are desired for remote and in-home settings.
Disclosed herein are compositions comprising a hydrolyzed tetraethyl orthosilicate (TEOS), at least one macromolecule selected from the group consisting of hyaluronan and silk fibroin, and water or a biocompatible buffer (e.g. citrate, phosphate and acetate). The composition may further comprise at least one active agent, including for example, a drug, protein, enzyme, hormone, polysaccharide, glycoprotein, oligopeptide, steroid, analgesic, anesthetic, vitamin, antimicrobial agent, anti-inflammatory agent, antibody or a combination thereof. In some embodiments, the compositions are thixotropic hydrogels.
Also disclosed herein are methods of manufacturing the compositions comprising obtaining hydrolyzed TEOS, preparing an aqueous solution of the at least one macromolecule in water, wherein the macromolecule is selected from the group consisting of hyaluronan and silk fibroin; and mixing the hydrolyzed TEOS with the aqueous solution of macromolecule. The methods may further comprise adding at least one active agent.
Also disclosed herein are pre-filled delivery devices comprising the composition, kits comprising the composition and a delivery device, methods of treating or preventing a disease or disorder comprising administration of the composition, and methods for the delivery of at least one active agent.
Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.
The present disclosure provides cyto- and bio-compatible compositions comprising a hydrolyzed tetraethyl orthosilicate (TEOS), at least one macromolecule selected from the group consisting of hyaluronan and silk fibroin, and water. In some embodiments, the compositions comprise a hydrolyzed tetraethyl orthosilicate (TEOS), at least one macromolecule selected from the group consisting of hyaluronan and silk fibroin, and a biocompatible buffer. The compositions are thixotropic and exist as gels but liquefy when under shear stress, such as when passed through a syringe nozzle, such that they can be delivered as a liquid or a gel. The compositions allow controlled release of an active agent, thereby dropping the required dosage of the active agent. The observed drug release profile suggests rapid delivery of a therapeutically effective dose and the maintenance of this concentration at the administration site.
The compositions are stable over a broad temperature range including room and physiological temperature, removing the need for temperature dependent handling. These properties overcome many of the challenges which exist with existing gel-based products. For example, Otiprio®, a slow-release formulation based on a thermosensitive liquid-to-gel system that contains 6% ciprofloxacin, was approved by the FDA in March 2018 for the treatment of otitis externa. However, the thermosensitive aspect of this therapeutic requires multiple preparation steps and handling protocols that open the possibility of user error and noncompliance, including: use of multiple components for preparation (first needle for dosing, a catheter for syringe priming, and a second syringe for bilateral applications) and the material needs to be kept cold as inadvertent contact with the physician's hand or prolonged exposure to room temperature would cause the material to gel and affect dosing.
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
As used herein, the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.
As used herein, “treat,” “treating” and the like means a slowing, stopping or reversing of progression of a disease or disorder when provided a composition described herein to an appropriate control subject. The term also means a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the symptoms of the disease or disorder. As such, “treating” means an application or administration of the compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or symptoms of the disease.
As used herein, the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of the compositions of the disclosure into a subject by a method or route which results in at least partial localization of the composition to a desired site. The compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.
As used herein, the term “thixotropic” refers to a property of a composition, e.g. a hydrogel, that enables it to flow when subjected to a mechanical force such as a shear stress or when agitated and return to a gel-like form when the mechanical force is removed. Thus, “thixotropy” refers to the time and shear rate dependence of viscosity of a fluid.
As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans and non-human animals. The terms “subject” and “patient” may be used interchangeably herein in reference to a human subject. The term “non-human animals” refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, ayes, etc. The subject may include either adults or juveniles (e.g., children). In one embodiment of the methods and compositions provided herein, the mammal is a human.
A “syringe” is a device comprising a barrel, typically but not necessarily tube-shaped, for injecting/applying or withdrawing a sample in a thin stream, typically through a hollow needle. Samples are injected/applied or withdrawn via pressure, typically from a reciprocating pump (e.g., employing a piston or plunger). A plunger can be linearly pulled and pushed along the inside of the barrel, allowing the syringe to take in and expel liquid or gas through a discharge orifice at the front (open) end of the tube. The open end of the syringe may be fitted with a needle, a nozzle or a tubing to help direct the flow into and out of the barrel. The syringe barrel may be divided into multiple chambers, such that individual components of a composition or sample to be injected mix upon application or injection only.
A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide” and “protein,” are used interchangeably herein.
The term “controlled release” herein refers to any formulation or dosage form that comprises an active drug and which is formulated to release the drug for longer duration of time and to provide a longer duration of pharmacological response after administration of the dosage form than is ordinarily experienced after administration of a corresponding immediate release formulation comprising the same drug in the same amount. Controlled release formulations include, inter alia, those formulations described elsewhere as “prolonged release,” “delayed release,” “sustained release,” “extended release,” “programmed release,” “time release,” and/or “rate controlled” formulations or dosage forms.
The term “hydrogel” herein refers to a specific type of gel in which water-swellable polymeric matrices that can absorb a substantial amount of water in a three-dimensional network of macromolecules held together by covalent or noncovalent crosslinks.
As used herein, the term “fluoroquinolones” refers to a class of antibiotics which exert their antibacterial effects by inhibiting bacterial DNA gyrase and which include a fluorinated quinolone ring system. Exemplary fluoroquinolones include, without limitation, ciprofloxacin, enrofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin), lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, difloxacin, cinofloxacin, pefloxacin, tosufloxacin, temafloxacin, fleroxacin, amifloxacin, binfloxacin, danofloxacin, marbofloxacin, ruflocaxin, and sarafloxacin.
Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
Disclosed herein are compositions comprising a hydrolyzed tetraethyl orthosilicate (TEOS), at least one macromolecule and water. In some embodiments, the compositions comprise a hydrolyzed tetraethyl orthosilicate (TEOS), at least one macromolecule and a biocompatible buffer. The biocompatible buffer includes those approved and commonly in use for parenteral delivery of drugs, examples of which can be found in Pharmaceutical Dosage Form: Parenteral Medications, Volume 1, 2nd Edition, Chapter 5, p. 194, De Luca and Boylan, “Formulation of Small Volume Parenterals”, Table 5: Commonly used additives in Parenteral Products. The biocompatible buffer may include, but is not limited to, those derived from acetic, ascorbic, aspartic, citric, glutaric, gluconic, lactic, malic, succinic, phosphate, tartaric, and carbonic acids, those using amino acids (e.g. histidine), those including amines (e.g. tromethamine (Tris)), and those used for injectables (e.g. normal saline, bacteriostatic water, bacteriostatic sodium chloride solution, dextrose 5% water, Ringer's solution, lactated Ringer's solution) as known in the art. The at least one macromolecule may be selected from the group consisting of hyaluronan and silk fibroin. In some embodiments, the composition is a thixotropic hydrogel.
In some embodiments, the compositions comprise about 0.1% to about 20% (w/v) of the at least one macromolecule, based on volume of hydrolyzed TEOS. The composition may include about 0.1% (w/v) of the at least one macromolecule, about 0.5% (w/v) of the at least one macromolecule, about 1% (w/v) of the at least one macromolecule, about 2% (w/v) of the at least one macromolecule, about 3% (w/v) of the at least one macromolecule, about 4% (w/v) of the at least one macromolecule, about 5% (w/v) of the at least one macromolecule, about 6% (w/v) of the at least one macromolecule, about 7% (w/v) of the at least one macromolecule, about 8% (w/v) of the at least one macromolecule, about 9% (w/v) of the at least one macromolecule, about 10% (w/v) of the at least one macromolecule, about 11% (w/v) of the at least one macromolecule, about 12% (w/v) of the at least one macromolecule, about 13% (w/v) of the at least one macromolecule, about 14% (w/v) of the at least one macromolecule, about 15% (w/v) of the at least one macromolecule, about 16% (w/v) of the at least one macromolecule, about 17% (w/v) of the at least one macromolecule, about 18% (w/v) of the at least one macromolecule, about 19% (w/v) of the at least one macromolecule, or about 20% (w/v) of the at least one macromolecule, based on volume of hydrolyzed TEOS. In select embodiments, the composition comprises about 10% (w/v) of the at least one macromolecule, based on volume of hydrolyzed TEOS. In select embodiments, the composition comprises about 0.5% (w/v) of the at least one macromolecule, based on volume of hydrolyzed TEOS.
The at least one macromolecule may be hyaluronan, also known as hyaluronic acid. In some embodiments, the hyaluronan may have a molecular weight less than about 20 kDa. The hyaluronan may have a molecular weight less than 15 kDa, less than 12 kDa, less than 10 kDa, less than 8 kDa, less than 5 kDa or less than 1 kDa. The hyaluronan may have a molecule weight greater than 1 kDa, greater than 5 kDa, greater than 8 kDa, greater than 10 kDa, greater than 12 kDa, or greater than 15 kDa. In select embodiments, the hyaluronan has a molecular weight of about 5 kDa. The composition may comprise about 10% (w/v) hyaluronan, based on volume of hydrolyzed TEOS.
The at least one macromolecule may be silk fibroin. In some embodiments, the composition comprises about 0.5% (w/v) silk fibroin, based on volume of hydrolyzed TEOS.
In some embodiments, the composition comprises water and hydrolyzed TEOS at a volume ratio of about 2:1 to about 1:1. In select embodiments the macromolecule is hyaluronan and the volume ratio is about 2:1. In select embodiments, the macromolecule is silk fibroin and the volume ratio is about 1:1.
a) Active Agent
The composition may further comprise at least one active agent. The active agent may be a drug, protein, enzyme, hormone, polysaccharide, glycoprotein, oligopeptide, steroid, vitamin, antibody or a combination thereof, with analgesic, anesthetic, antimicrobial, anti-fungal, or anti-inflammatory pharmacologic effects. Examples of such active agents include: Antibiotics such as ofloxacin (0.3%), finafloxacin (0.3%), ciprofloxacin or ciprofloxacin HCl (0.2-6%), gentamycin sulfate (0.2, 0.3%), neomycin sulfate (0.3%, 0.4%, 0.5%, 2.5%), Penicillin G procaine (10,000 IU), polymyxin B sulfate (5,000 IU or 0.05%), kanamycin sulfate (0.5%), amphomycin calcium (0.5%), orbifloxacin (1.0%), enrofloxacin (0.5%), silver sulfadiazine (1.0%), and florfenicol or acetate florfenicol (1.0%, 1.7%); Anti-inflammatories such as dexamethasone (0.1%), hydrocortisone (1%), hydrocortisone aceponate (0.1%), hydrocortisone acetate (0.2%, 0.5%, 1%) mometasone furoate or mometasone furoate monohydrate (0.1%, 0.2%), betamethasone or betamethasone valerate (0.1%), isoflupredone acetate (0.1%), and prednisolone acetate (0.3%, 0.5%); Antifungals such as clotrimazole (1%), miconazole nitrate (1.5%, 2.3%), thiabendazole (4%), posaconazole (0.1%), and terbinafine (1%, 1.5%); Anesthetics such as tetracaine hydrochloride (0.5%), and procaine; and Analgesics such as aspirin, naproxen, ibuprofen, and acetaminophen.
The antimicrobial agent may include antibacterial, antifungal, antiviral, antiprotozoal, and/or antiparasitic agents. In some embodiments, the antimicrobial agent is an antibiotic. The antibiotic may be selected from the group consisting of (fluoro)quinolones, carbapenems, aminoglycosides, polypeptide antibiotic, phenicols, and derivatives or combinations thereof
In some embodiments, the active agent is selected from the group of quinolones and quinolone-based medicaments, for example (fluoro)quinolones (e.g., ofloxacin, ciprofloxacin, levofloxacin, trovafloxacin). Non-limiting examples of quinolones and fluoroquinolones include
Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid, Gemifloxacin, and Perfloxacin. The quinolone antibiotic may include first generation quinolones (e.g., cinoxacin, cinobac, nalidixic acid), second generation quinolones (e.g., ciprofloxacin, enoxacin, norfloxacin), third generation quinolones (e.g,balofloxacin, levofloxacin, tosufloxacin) fourth generation quinolones (e.g., clinafloxacin, gatifloxacin, moxifloxacin, trovafloxacin) and/or other quinolones (e.g. prulifloxacin).
In some embodiments, the antibiotic is ciprofloxacin. Ciprofloxacin can be present in any pharmaceutically acceptable form, e.g., ciprofloxacin hydrochloride, monohydrate. The composition may comprise less than about 3000 μg/mL ciprofloxacin. The composition may comprise about 10 μg/mL to about 3000 μg/mL ciprofloxacin, about 10 μg/mL to about 2500 μg/mL ciprofloxacin, about 10 μg/mL to about 2000 μg/mL ciprofloxacin, about 10 μg/mL to about 1500 μg/mL ciprofloxacin, about 10 μg/mL to about 1000 μg/mL ciprofloxacin, about 10 μg/mL to about 750 μg/mL ciprofloxacin, about 10 μg/mL to about 500 μg/mL ciprofloxacin, about 10 μg/mL to about 250 μg/mL ciprofloxacin, about 10 μg/mL to about 100 μg/mL ciprofloxacin, about 100 μg/mL to about 3000 μg/mL ciprofloxacin, about 100 μg/mL to about 2500 μg/mL ciprofloxacin, about 100 μg/mL to about 2000 μg/mL ciprofloxacin, about 100 μg/mL to about 1500 μg/mL ciprofloxacin, about 100 μg/mL to about 1000 μg/mL ciprofloxacin, about 100 μg/mL to about 750 μg/mL ciprofloxacin, about 100 μg/mL to about 500 μg/mL ciprofloxacin, about 100 μg/mL to about 250 μg/mL ciprofloxacin, about 250 μg/mL to about 3000 μg/mL ciprofloxacin, about 250 μg/mL to about 2500 μg/mL ciprofloxacin, about 250 μg/mL to about 2000 μg/mL ciprofloxacin, about 250 μg/mL to about 1500 μg/mL ciprofloxacin, about 250 μg/mL to about 1000 μg/mL ciprofloxacin, about 250 μg/mL to about 750 μg/mL ciprofloxacin, about 250 μg/mL to about 500 μg/mL ciprofloxacin, about 500 μg/mL to about 3000 μg/mL ciprofloxacin, about 500 μg/mL to about 2500 μg/mL ciprofloxacin, about 500 μg/mL to about 2000 μg/mL ciprofloxacin, about 500 μg/mL to about 1500 μg/mL ciprofloxacin, about 500 μg/mL to about 1000 μg/mL ciprofloxacin, about 500 μg/mL to about 750 μg/mL ciprofloxacin, about 750 μg/mL to about 3000 μg/mL ciprofloxacin, about 750 μg/mL to about 2500 μg/mL ciprofloxacin, about 750 μg/mL to about 2000 μg/mL ciprofloxacin, about 750 μg/mL to about 1500 μg/mL ciprofloxacin, about 750 μg/mL to about 1000 μg/mL ciprofloxacin, about 1000 μg/mL to about 3000 μg/mL ciprofloxacin, about 1000 μg/mL to about 2500 μg/mL ciprofloxacin, about 1000 μg/mL to about 2000 μg/mL ciprofloxacin, about 1000 μg/mL to about 1500 μg/mL ciprofloxacin, about 1500 μg/mL to about 3000 μg/mL ciprofloxacin, about 1500 μg/mL to about 2500 μg/mL ciprofloxacin, about 1500 μg/mL to about 2000 μg/mL ciprofloxacin, about 2000 μg/mL to about 3000 μg/mL ciprofloxacin, about 2000 μg/mL to about 2500 μg/mL ciprofloxacin, or about 2500 μg/mL to about 3000 μg/mL ciprofloxacin. In some embodiments, the composition comprises about 30 μg/mL to about 3000 μg/mL ciprofloxacin.
Carbapenems are members of the beta lactam class of antibiotics, which kill bacteria by binding to penicillin-binding proteins, thus inhibiting bacterial cell wall synthesis. carbapenems possess the broadest spectrum of activity of the beta lactam class and greatest potency against Gram-positive and Gram-negative bacteria. Carbapenems include, without limitation, imipenem, meropenem, ertapenem, doripenem, panipenem, tebipenem, and biapenem.
In some embodiments, the antibiotic is imipenem. The composition may comprise less than about 10,000 μg/mL imipenem. The composition may comprise about 50 μg/mL to about 10,000 μg/mL imipenem, about 100 μg/mL to about 10,000 μg/mL imipenem, about 500 μg/mL to about 10,000 μg/mL imipenem, about 1,000 μg/mL to about 10,000 μg/mL imipenem, about 2,000 μg/mL to about 10,000 μg/mL imipenem, about 3,000 μg/mL to about 10,000 μg/mL imipenem, about 4,000 μg/mL to about 10,000 μg/mL imipenem, about 5,000 μg/mL to about 10,000 μg/mL imipenem, about 6,000 μg/mL to about 10,000 μg/mL imipenem, about 7,000 μg/mL to about 10,000 μg/mL imipenem, about 8,000 μg/mL to about 10,000 μg/mL imipenem, about 9,000 μg/mL to about 10,000 μg/mL imipenem, about 50 μg/mL to about 9,000 μg/mL imipenem, about 100 μg/mL to about 9,000 μg/mL imipenem, about 500 μg/mL to about 9,000 μg/mL imipenem, about 1,000 μg/mL to about 9,000 μg/mL imipenem, about 2,000 μg/mL to about 9,000 μg/mL imipenem, about 3,000 μg/mL to about 9,000 μg/mL imipenem, about 4,000 μg/mL to about 9,000 μg/mL imipenem, about 5,000 μg/mL to about 9,000 μg/mL imipenem, about 6,000 μg/mL to about 9,000 μg/mL imipenem, about 7,000 μg/mL to about 9,000 μg/mL imipenem, about 8,000 μg/mL to about 9,000 μg/mL imipenem, about 50 μg/mL to about 8,000 μg/mL imipenem, about 100 μg/mL to about 8,000 μg/mL imipenem, about 500 μg/mL to about 8,000 μg/mL imipenem, about 1,000 μg/mL to about 8,000 μg/mL imipenem, about 2,000 μg/mL to about 8,000 μg/mL imipenem, about 3,000 μg/mL to about 8,000 μg/mL imipenem, about 4,000 μg/mL to about 8,000 μg/mL imipenem, about 5,000 μg/mL to about 8,000 μg/mL imipenem, about 6,000 μg/mL to about 8,000 μg/mL imipenem, about 7,000 μg/mL to about 8,000 μg/mL imipenem, about 50 μg/mL to about 7,000 μg/mL imipenem, about 100 μg/mL to about 7,000 μg/mL imipenem, about 500 μg/mL to about 7,000 μg/mL imipenem, about 1,000 μg/mL to about 7,000 μg/mL imipenem, about 2,000 μg/mL to about 7,000 μg/mL imipenem, about 3,000 μg/mL to about 7,000 μg/mL imipenem, about 4,000 μg/mL to about 7,000 μg/mL imipenem, about 5,000 μg/mL to about 7,000 μg/mL imipenem, about 6,000 μg/mL to about 7,000 μg/mL imipenem, about 50 μg/mL to about 6,000 μg/mL imipenem, about 100 μg/mL to about 6,000 μg/mL imipenem, about 500 μg/mL to about 6,000 μg/mL imipenem, about 1,000 μg/mL to about 6,000 μg/mL imipenem, about 2,000 μg/mL to about 6,000 μg/mL imipenem, about 3,000 μg/mL to about 6,000 μg/mL imipenem, about 4,000 μg/mL to about 6,000 μg/mL imipenem, about 5,000 μg/mL to about 6,000 μg/mL imipenem, about 50 μg/mL to about 5,000 μg/mL imipenem, about 100 μg/mL to about 5,000 μg/mL imipenem, about 500 μg/mL to about 5,000 μg/mL imipenem, about 1,000 μg/mL to about 5,000 μg/mL imipenem, about 2,000 μg/mL to about 5,000 μg/mL imipenem, about 3,000 μg/mL to about 5,000 μg/mL imipenem, about 4,000 μg/mL to about 5,000 μg/mL imipenem, about 50 μg/mL to about 4,000 μg/mL imipenem, about 100 μg/mL to about 4,000 μg/mL imipenem, about 500 μg/mL to about 4,000 μg/mL imipenem, about 1,000 μg/mL to about 4,000 μg/mL imipenem, about 2,000 μg/mL to about 4,000 μg/mL imipenem, about 3,000 μg/mL to about 4,000 μg/mL imipenem, about 50 μg/mL to about 3,000 μg/mL imipenem, about 100 μg/mL to about 3,000 μg/mL imipenem, about 500 μg/mL to about 3,000 μg/mL imipenem, about 1,000 μg/mL to about 3,000 μg/mL imipenem, about 2,000 μg/mL to about 3,000 μg/mL imipenem, about 50 μg/mL to about 2,000 μg/mL imipenem, about 100 μg/mL to about 2,000 μg/mL imipenem, about 500 μg/mL to about 2,000 μg/mL imipenem, about 1,000 μg/mL to about 2,000 μg/mL imipenem, about 50 μg/mL to about 1,000 μg/mL imipenem, about 100 μg/mL to about 1,000 μg/mL imipenem, about 500 μg/mL to about 1,000 μg/mL imipenem, about 50 μg/mL to about 500 μg/mL imipenem, about 100 μg/mL to about 500 μg/mL imipenem, or about 50 μg/mL to about 100 μg/mL imipenem. In some embodiments, the composition comprises about 100 μg/mL to about 10,000 μg/mL imipenem.
Aminoglycosides are a class of antibiotics derived at least part from a saccharide or polysaccharide and having the empirical formula CmHoNpOq (where m, n, p, and q are appropriate integers). For instance, the aminoglycosides are oligosaccharides consisting of an aminocyclohexanol moiety glycosidically linked to other amino sugars. Aminoglycosides include, without limitation, amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin(s), fradiomycin, gentamicin, ispamicin, kanamycin, micronomicin, neomycin, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, streptonicozid, and tobramycin.
Polypeptide antibiotics are a chemically diverse class of antibiotics containing non-protein polypeptide chains. The polypeptide antibiotics may be from either of the two classes: non-ribosomally synthesized peptides and ribosomally synthesized (natural) peptides. Polypeptide antibiotics include, without limitation, actinomycin, bacitracins, and polymyxins (e.g. colistin and polymyxin B), Bleomycin, gramicidins, and glycopeptides.
Phenicols are derived from dichloroacetic acid with two other parts: an aromatic nucleus with an alkyl group in the para position and an aminopropanediol chain. Phenicols block peptide elongation by binding to the peptidyltansferase centre of the 70S ribosome. Phenicols include, without limitation, chloramphenicol, thiamphenicol, and florfenicol.
The composition may further comprise at least one pharmaceutically acceptable excipient including carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. In those embodiments in which the compositions comprise a biocompatible buffer, an additional buffer is unlikely to be included in the composition, unless such a combination buffer is known in art, such a citrate-histidine. Such carriers, adjuvants, and other excipients will be compatible with the environment in the hydrogel compositions.
b) Methods of Making
Also disclosed herein are methods of manufacturing the compositions described herein. The methods may comprise obtaining hydrolyzed TEOS, preparing an aqueous solution of the at least one macromolecule in water, and mixing the hydrolyzed TEOS with the aqueous solution of macromolecule. Hydrolyzed TEOS may be obtained by incubating TEOS under acidic conditions. In some embodiments, TEOS is incubated with acetic acid to yield hydrolyzed TEOS.
The hydrolyzed TEOS and the aqueous solution of macromolecule is mixed at a volume ratio which allows gel formation. In some embodiments, the volume ratio between about 1:1 and about 2:1. The macromolecule is selected from the group consisting of hyaluronan and silk fibroin. In some embodiments, the macromolecule is hyaluronan and the volume ratio is about 2:1. In some embodiments, the macromolecule is silk fibroin and the volume ratio is about 1:1.
The method may further comprise adding at least one active agent. In some embodiments, the at least one active agent is added to the hydrolyzed TEOS or the aqueous solution of the at least one macromolecule. In some embodiments, the at least one active agent is added to the composition following mixing the hydrolyzed TEOS with the aqueous solution of macromolecule.
Descriptions of the macromolecule, active agents, and relative amounts thereof, set forth above in connection with the inventive composition are also applicable to the method of manufacturing the compositions.
a) Prefilled Delivery Device
The disclosure also provides a pre-filled delivery device comprising the compositions described herein. Descriptions of the macromolecules, active agents, and relative amounts thereof, set forth above in connection with the inventive composition are also applicable to the method of manufacturing the compositions.
A pre-filled delivery device is a device which is filled with the composition prior to distribution to the end user that administers the composition. In some embodiments, the drug delivery device has separate chambers for the storage for the hydrolyzed TEOS and the macromolecule. In this embodiment, the hydrolyzed TEOS and the macromolecule are mixed just prior or concomitantly with administration.
The drug delivery device may include any device configured to allow administration or delivery of a liquid or gel composition. The drug delivery device may include, without limitation, an autoinjector, a pen, a dermal patch, an eye/ear dropper, a dropper bottle, a syringe, a pump, a wound dressing (e.g., gauze or bandage), or a transdermal patch or implant.
In some embodiments, the device is a syringe. A pre-filled syringe typically includes a containment container forming part of a syringe body, a plunger, and either an attached hypodermic needle or such features to allow a needle to be attached by the user prior to administration. In some embodiments, the device is a wound or incision dressing.
The device may contain a single dose of the composition or multiple doses which can be measured out and administered over time.
b) Method of Treating or Preventing a Disease or Disorder
The present disclosure further provides methods for treating or preventing a disease or disorder in a subject in need thereof. The methods comprise administering an effective amount of the composition described herein to a subject in need thereof. In some embodiments, the composition is administered in a liquid form. In some embodiments, the composition is administered as a gel. Descriptions of the macromolecules, active agents, and relative amounts thereof, set forth above in connection with the inventive composition are also applicable to the method of manufacturing the compositions.
The disease or disorder may comprise infection, inflammation, pain, irritation, loss of tissue or tissue damage, or a combination thereof. The disease or disorder may include, but is not limited to, eye infections, ear infections, puncture wounds, sutures, abrasions, lacerations, burns, radiation skin damage, and skin and soft-tissue infections. In select embodiments, the disease or disorder is an ear infection.
The route and regimen of administration will vary depending upon the nature and location of the disease or disorder and is to be determined by the skilled practitioner. The composition may be administered topically, intramuscularly, subcutaneously, intrathecally, vaginally, rectally, or transdermally. In some embodiments, the composition is administered to the skin, at least one eye, at least one ear, a wound, a tissue abrasion or loss, a burn, a suture or cut, or the mouth or nasal cavity.
The subject may be a human, a non-human primate, a member of the family Canidae, a member of the family Felidae, a member of the family Equidae, a member of the family Leporid, a member of the family Bovidae, a member of the family Suidae, a member of the family Cervidae, a member of the family Macropodidae, or a member of the family Ursidae.
An “effective amount” of the compositions disclosed herein is an amount that is delivered to a subject, either in a single dose or as part of a series, which is effective for treating or preventing the disease or disorder. The specific dose level may depend upon a variety of factors including the activity of the peptide, composition or vaccine, the age, body weight, general health, and diet of the subject, time of administration, and route of administration.
c) Method for the Delivery of at least One Active Agent
The composition of the invention may suitably be used for the delivery of at least one active agent, where controlled release of active agent is desired. Provided herein are methods for the delivery of at least one active agent. The methods comprise providing at least one active agent in a thixotropic hydrogel comprising a hydrolyzed tetraethyl orthosilicate (TEOS), at least one macromolecule selected from the group consisting of hyaluronan and silk fibroin, and water.
The active agent may be a drug, protein, enzyme, hormone, polysaccharide, glycoprotein, oligopeptide, steroid, analgesic, anesthetic, vitamin, antimicrobial agent, anti-inflammatory agent, antibody or a combination thereof.
The antimicrobial agent may include antibacterial, antifungal, antiviral, antiprotozoal, and/or antiparasitic agents. In some embodiments, the antimicrobial agent is an antibiotic. The antibiotic may be selected from the group consisting of (fluoro)quinolones, carbapenems, aminoglycosides, polypeptide antibiotic, phenicols, and derivatives or combinations thereof.
Descriptions of the macromolecules, active agents, and relative amounts thereof, set forth above in connection with the inventive composition are also applicable to the method for the delivery of at least one active agent. 4. KITS
Also within the scope of the present disclosure are kits that include compositions described herein. In some embodiments, the kits comprise the composition described herein and a delivery device. The drug delivery device may include any device configured to allow administration or delivery of a liquid or gel composition. The drug delivery device may include, without limitation, an autoinjector, a pen, a dermal patch, an eye/ear dropper, a dropper bottle, a syringe, a pump, a wound dressing, or a transdermal patch or implant. Descriptions of delivery devices set forth above is also applicable to the kits.
Individual member components of the kits may be physically packaged together or separately. The components of the kit may be provided in bulk packages (e.g., multi-use packages) or single-use packages. The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the compositions, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
It is understood that the disclosed kits can be employed in connection with the disclosed methods. The kit may further contain additional containers or devices for use with the methods disclosed herein. The kits optionally may provide additional components such wound dressings (gauze, adhesive bandages and the like), cotton swabs or wipes, and cleaning or antibiotic wipes.
The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.
Gel Formation. TEOS was hydrolyzed with 0.15 M Acetic Acid (HOAc) for 1.5 hours at a 1:9 (v/v/) ratio. Hydrolyzed TEOS (hTEOS) was then combined with HA5 solution (10% w/v) or SF solution (0.5%), respectively in different ratios. The solutions were vortexed and the pH was adjusted to ˜2 with 3.0 N HOAc. After 3 hours, the pH was raised to 8.5 with 1.5 N NH4OH. All solutions formed gels when left unstirred overnight at room temperature. The antibiotic agents were added (as solid or liquid) to the TEOS or macromolecule solution and dissolved/mixed in prior to gel formation, or alternatively, was added as a liquid directly to the gel, followed homogenization by vortexing, up and down pipetting or shaking.
Rheological characterization. All hydrogels were characterized within the materials pseudolinear viscoelastic range with a 1.00 mm gap, at 20° C. unless otherwise specified. Oscillatory strain sweeps for thixotropy investigation were conducted with 8 mm parallel plate fixtures within a strain range of 1-250% and an angular frequency of 10 rad/sec. The wait time between the cycles was 30 sec. For temperature dependent hydrogel behavior evaluation, samples were loaded onto the 20 mm parallel plate fixtures (to provide a larger temperature exchange surface), equilibrated to 4° C. then subjected to a temperature ramp of 5° C./min up to 40° C., at a stain rate of 0.4% and an angular frequency of 10 rad/sec. Swelling tests were performed with an active force adjustment protocol by loading the hydrogels onto the 8 mm fixtures, equilibrating them at 37° C. and pre-loading them dry with a 0.1N axial compression force 120 seconds, then adding PBS and monitoring the gap distance for a total of 30 min (1800 sec).
Cell assays. Primary adult normal dermal fibroblasts (HDFa) (PCS-201-012, ATCC, Manassas, VA) were used to assess the cytocompatibility of the thixogels. Cells were seeded in a 96-well plate at a density of 105 cells/well in 100 μl in serum-free fibroblast conditioned media (ATCC, Manassas, VA) and inbubated for 24 hours. Subsequently, the media was removed and replaced with 50 μl of thixogel (different formulations) and 50 μl of fresh media followed by incubation for 24 h at 37° C./5% CO2. Cellular viability was quantified with a Cell-Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) and detected via absorbance at 450 nm with a microplate reader.
In in vitro assays, tetraethyl orthosilicate (TEOS) hydrogels loaded with gentamycin effectively inhibited the growth of P. aeruginosa and S. aureus. These materials exhibited thixotropy which enabled them to liquefy under shear stress but gel once the stress was removed (
By hydrolyzing TEOS under acidic conditions and mixing it in different ratios with water or aqueous solutions of macromolecules or large molecular weight polymers, such as polyethylene glycol, hyaluronic acid and silk fibroin, optically transparent hydrogels that liquefy under shear stress or agitation but become gels under static conditions were formed. Mechanistically, it is believed the gels form through the polymerization of a low amount of silicate intermediate obtained by TEOS hydrolysis and concomitant entrapment of the macromolecule/large molecular weight polymer within the polymerized TEOS network.
Since thixogels based solely on aqueous TEOS were cytotoxic to primary cells, several thixogel formulations (HA5—hyaluronan MW 5 kDa/TEOS; HA5ABS—chemically modified HA5 with 4-aminobutyl triethoxysilane/TEOS; SFL—silk fibroin low ratio/TEOS; SFL—silk fibroin high ratio/TEOS; PEG10—polyethylene glycol MW 10 kDa/TEOS) were screened for their effect on primary human dermal fibroblast (
Formulations with alternative silanes, including N,N′-bis-[(3-triethoxysilylpropyl)aminocarbonyl]polyethylene oxide (7-10 EO), bis(3-triethoxysilylpropyl)polyethylene oxide (25-30 EO), N-((6)-aminohexyl)aminomethyl triethoxysilane, and 4-aminobutyltriethoxysilane yielded gels that were not thixotropic. 4-Aminobutyl triethoxysilane (ABS) or N-((6)-aminohexyl)aminomethyl triethoxysilane (AHAM) used by themselves or chemically conjugated to HA did not yield cytocompatible gels.
The outer ear canal and ear drum are lined with epithelial tissue similar to skin. EpiDermTM, a validated and regulatory body accepted in vitro skin model representative of in vivo outcomes of chemical, pharmaceutical and skin care testing, was used to assess the biocompatibility of HA5 and SFL thixogels. EpiDerm™ consists of normal human-derived epidermal keratinocytes cultured to form organized basal, spinous and granular layers, and a multilayered stratum corneum containing intracellular lamellar lipid layers arranged in patterns analogous to those found in the human epidermis. Two separate material biocompatibility aspects were evaluated: skin irritability and skin corrosion.
For skin irritability testing, a standardized protocol (OECD TG 439) was used in which pre-conditioned EpiDerm™ human skin samples (surface area of 0.63 cm2) were treated with 30 μL test substance for 60 min, followed by removal of test substance and tissue washing. Subsequently, the tissue viability was assessed with MTT after a 42-hour post-incubation period. Skin irritation is defined by the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (UN GHS) as the production of reversible damage to the skin following the application of a test substance. A test substance was predicted to have skin irritation potential if the mean relative tissue viability of three individual tissues exposed to it was reduced below 50% of the mean viability of the negative controls (NC). The results indicate that both HA5 and SFL are non-irritant (
Similarly, for skin corrosion assessments HA5 and SFL thixogels were tested according to standardized protocol OECD TG 431. Skin corrosion is defined by the UN GHS as the production of irreversible tissue damage in the skin following the application of a test material. The test included treating the EpiDerm™ tissue samples with 50 μl of test material for 3 minutes and 1 hour, respectively. A material was classified as definitely corrosive if the relative tissue viability after 3 minutes treatment with a test material was decreased below 50%. However, materials classified non-corrosive after the 3-minute treatment (viability ≥50%) will be re-classified as corrosive if the relative tissue viability after the 1-hour treatment with the test material was decreased below 15%. For the 3-minute exposure, HA5 and SFL treated tissues showed a slight decrease in mean sample viability compared to the NC (TTEST: HA5 and SFL, respectively, versus NC, p<0.01), but there was no statistical difference between the mean viability of samples treated with HA5 or SFL (TTEST: HA5 versus SFL p=0.61) (
For biological evaluations the thixogels were subjected to syringe driven sterile filtration. To reproduce the final material properties as accurately as possible, the thixotropic behavior of sterile filtered HA5 and SFL formulations was investigated rheologically with a Discovery HR-2 hybrid rheometer/dynamic mechanical analyzer (TA Instruments). Both HA5 and SFL thixogels transitioned between gel-sol states as a function of stress over the three cycles tested (
Swelling is a major adverse event typically associated with hydrogels. The thixogels were rheologically tested by measuring the gap change between rheometer fixtures in response to swelling. Over the course of the 30 minutes while immersed in phosphate buffered saline at 37° C. (conditions mimicking the ear environment), the volumes of the thixogels increased by 0.23±0.19% for HA5 and by 1.72±0.43% for SFL. These results indicate that the swelling of both thixogels was negligible and showed that a product based on either of these formulations would not cause swelling related adverse effects.
The temperature dependence of these materials was tested to better understand the behavior of the thixogels at potential storage and shipment conditions (refrigeration at 4° C./39.2° F., room temperature 22° C./71.6° F.), physiological conditions (37° C./98.6° F.) and slightly higher temperatures that might occur during typical shipping/transportation (40° C./104° F.). Both formulations tested maintained their viscoelastic behavior (stay as gels) in the tested temperature range (
These cyto- and biocompatible thixogels showed negligible swelling under physiological conditions and were stable as gels in the 4° C./39.2° F. - 40° C./104° F. range.
Clinically used antibiotics from different classes such as vancomycin (glycopeptide), ciprofloxacin (fluoroquinolone), imipenem (carbapenem) and gentamycin (aminoglycoside) are potent and effective compounds. The Minimum Inhibitory Concentration (MIC) defines the lowest antibiotic concentration that inhibits 99.9% of bacterial growth. The MIC concentrations of vancomycin, ciprofloxacin, imipenem and gentamycin (Table 2) were measured when the antibiotic was delivered in a thixogel.
Gentamycin was found to be undesirable as both gels tested (SF and HA) have a deleterious effect upon potency. Vancomycin was also found to be undesirable as it did not give coverage of both S. aureus (Gram positive) and P. aeruginosa (Gram negative) organisms. Imipenem and ciprofloxacin, however, showed potency against both pathogenic bacteria and neither of the gels had a significant impact upon potency. These tests determined the MIC concentration, whereas the customary dosing of ciprofloxacin (3,000 μg/mL) and imipenem (10,000 μg/mL) in eye and ear drops is 12,000 and 5,000 times higher than the MIC concentrations, respectively.
S. aureus 13709
P. aeruginosa 27853
To understand how the thixogel might affect the rate at which a loaded antibiotic affects S. aureus and P. aeruginosa growth, the kill kinetics of ciprofloxacin was measured, with or without HA and SF gels. Ciprofloxacin is most common active pharmaceutical ingredient in topical ear drops. Growth rates in media only, and in media above HA and SF gels were identical and reached cell densities of 109 CFU/mL within twelve hours, as expected. The gels alone did not inhibit bacterial growth. HA and SF gel-loaded antibiotic containing ciprofloxacin concentrations 100 times lower than the 0.3% found in commercial otic solutions (30 pg/mL or 0.003% w/v), completely inhibited bacterial growth in around four hours for both bacterial strains tested (
The mechanism of drug release ciprofloxacin loaded gels covered with saline was assessed for drug release over a period of 72 hours. Most of the drug was released within 24 hours via diffusion (data fitting best matches the Makoid-Banakar mathematical release model) as shown in
Tetraethyl Orthosilicate (TEOS)-Macromolecule Hydrogels for Delivery of Anti-Inflammatory agents
TEOS macromolecule hydrogels may be made as described above for the delivery of an antibiotic except, an anti-inflammatory agent (0.5-5% hydrocortisone) is added to the TEOS or macromolecule solution and dissolved/mixed in prior to gel formation or is added as a liquid directly to the gel, with homogenization by vortexing, up and down pipetting or shaking. Characteristics of the resulting hydrogel including rheology, swelling and temperature dependence may be determined as described in Examples 1 and 4.
TEOS macromolecule hydrogels may be made as described above for the delivery of an antibiotic except, an anesthetic (0.1-1% tetracaine hydrochloride) is added to the TEOS or macromolecule solution and dissolved/mixed in prior to gel formation or is added as a liquid directly to the gel, with homogenization by vortexing, up and down pipetting or shaking. Characteristics of the resulting hydrogel including rheology, swelling and temperature dependence may be determined as described in Examples 1 and 4.
TEOS macromolecule hydrogels may be made as described above for the delivery of an antibiotic except, an analgesic (10-200 mg/mL antipyrine) is added to the TEOS or macromolecule solution and dissolved/mixed in prior to gel formation or is added as a liquid directly to the gel, with homogenization by vortexing, up and down pipetting or shaking. Characteristics of the resulting hydrogel including rheology, swelling and temperature dependence may be determined as described in Examples 1 and 4.
TEOS macromolecule hydrogels may be made as described above for the delivery of an antibiotic except, an antifungal (1-3% miconazole, 0.5-3% clotrimazole, 2-6% thiabendazole) is added to the TEOS or macromolecule solution and dissolved/mixed in prior to gel formation or is added as a liquid directly to the gel, with homogenization by vortexing, up and down pipetting or shaking. Characteristics of the resulting hydrogel including rheology, swelling and temperature dependence may be determined as described in Examples 1 and 4.
Commonly, antibiotic treatments are combined with other active agents, including anti-inflammatories, antifungals, anesthetics, and/or analgesics. To form a TEOS macromolecule hydrogel comprising more than one active agent, a hydrogel may be made as described above. An antibiotic (ciprofloxacin and imipenem, as described above), an antifungal (1-3% miconazole, 0.5-3% clotrimazole, and/or 2-6% thiabendazole) and an anti-inflammatory agent (0.5-5% hydrocortisone) may be added to either the TEOS or macromolecule solution prior to gel formation. Alternatively, or in addition, some or all of the agents may be added either together or sequentially as a liquid to the gel with homogenization. Characteristics of the resulting hydrogel including rheology, swelling and temperature dependence may be determined as described in Examples 1 and 4.
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.
Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof
This application claims the benefit of U.S. Provisional Application No. 62/891,631, filed Aug. 26, 2019, the entire contents of which are incorporated herein by reference.
This invention was made with government support under R41DC017641 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/039551 | 6/25/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62891631 | Aug 2019 | US |
