Non-swellable hemostatic materials for surgical use

Abstract

In various aspects, the present application provides methods for achieving hemostasis during surgery in a patient in need thereof. The present application contemplates that such methods include applying a flowable and non-swellable composition comprising a hydrophobically- modified chitosan to a surgical site comprising a bleed. In embodiments, the surgical site is at risk of pressure-related injury.

Description

[0001] NON-SWELLABLE HEMOSTATIC MATERIALS FOR SURGICAL USE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 553,787, filed February 15, 2024, the entire contents of which are hereby incorporated by reference in their entirety.

[0004] BACKGROUND

[0005] Various surgical procedures benefit from the use of topical hemostatic agents to staunch active bleeding. For example, hemostatic agents are often employed as adjunct or alternatives to surgical techniques (e.g., electrocautery, vessel ligation, suturing) to manage bleeding from surgical surfaces. Particular conventional use of topical hemostats include diffusing nonanatomic bleeding, bleeding associated with sensitive structures, and bleeding in patients with hemostatic abnormalities.

[0006] Though hemostatic agents can prove beneficial for controlling intraoperative bleeding, reports of adverse events relating to swelling properties of hemostatic devices contribute to uncertainty around their safety, particularly for sites that are at risk of compression- or pressure-related injuries. Accordingly, there remains a need for hemostatic compositions that can be used during surgical procedures without the risk of adverse events. The subject matter of the present application addresses these and other objectives.

[0007] SUMMARY OF THE INVENTION

[0008] In various aspects and embodiments, the present application provides methods for hemostasis during surgery in a patient in need thereof. In embodiments, the methods include applying a flowable and non-swellable composition comprising a hydrophobically-modified chitosan to a surgical site comprising a bleed, and wherein the surgical site is at risk of pressure-related injury. In embodiments, the surgical site is at risk of injury upon compression of tissues. In embodiments, the patient has one or more factors that influence volume of blood loss during surgery. Such factors include those selected from advanced age, higher body mass index, presence of osteoporotic bone, neuromuscular scoliosis, bone metastasis, and use of anti-coagulant therapy.

[0009] In embodiments, the surgery is selected from neurosurgery, orthopedic surgery, brain surgery, ocular surgery, otorhinolaryngological surgery, thoracic surgery, prostate surgery, thyroid surgery, cardiac surgery, vascular surgery, and gynecological surgery.

[0010] In embodiments, the composition is applied to a site selected from one or more of neurological structure, spinal cord, optic chiasm, spinal column, thecal sac, peritoneal sac, blood vessel, nerve, pulmonary artery, superior vena cava, inferior vena cava, brain tissue, bladder, cavernous nerve, muscle, bone, and joint. In embodiments, the surgery site comprises bleeding selected from one or more of subcutaneous and muscular bleeding, bone bleeding, epidural bleeding, and large blood vessel bleeding.

[0011] In embodiments, the surgery is spine surgery. In embodiments, the patient undergoing spine surgery has one or more of disc herniation, spondylosis of the cervical and / or lumbar spine, cervical myelopathy, and spondylolisthesis. In embodiments, the spine surgery is discectomy, laminectomy, laminotomy, lumbar decompression surgery, arthrodesis surgery, or anterior cervical discectomy. In embodiments, the pressure-related injury (to be avoided) is compression of one or more neurological structures, such as thecal sac compression, spinal cord compression, and nerve compression.

[0012] In embodiments, the hydrophobically-modified chitosan comprises hydrocarbon groups attached to the chitosan backbone. In embodiments, the hydrocarbon groups comprise linear hydrocarbon groups independently selected from C8 to C18 hydrocarbon chains, which provide effective hemostatic properties. In embodiments, the hydrophobic groups further comprise Cl to C4 hydrocarbon groups, which provide for a predictable and tunable degradation property. In embodiments, the hydrophobically modified chitosan comprises from 30% to about 80% of monomers retaining a free amine, sufficient for effective tissue adhesive properties. In embodiments, the hydrophobically-modified chitosan further comprises one or more benzenediol groups, and optionally oxidized forms thereof, to tune adhesive and cohesive properties of the composition. In embodiments, the one or more benzenediol groups comprise a catechol moiety, and optionally oxidized forms thereof. In embodiments, at least some of the benzenediol groups are oxidized to the corresponding quinone.

[0013] In embodiments, the flowable composition is a gel or foam.

[0014] Other aspects and embodiments of the disclosure will be apparent from the following detailed description.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 compares hydrophobically modified chitosan with varied catechol substitutions. The vial on the right shows that a 2 wt% composition of a hydrophobically modified chitosan having 8% of chitosan monomer units comprising catechol grafts fails to make a stable gel. On the other hand, the vial on the left having 0.8% of monomer units comprising a catechol graft results in a stable gel.

[0017] Figure 2 shows a UV spectral analysis of a hydrophobically-modified chitosan containing partially oxidized catechol grafts where two shoulders at 280 nm and 330 nm can be seen, which indicate the partial oxidation of the benzenediol groups. The peak at 280 corresponds to un-oxidized catechol. The peak at 330 corresponds to the quinone groups, which are the oxidized catechol groups.

[0018] Figure 3 shows an example of a composition having partial oxidation of catechol groups and that is maintained at a pH of 5.4, and a composition that is titrated to pH of 12.0. The composition at pH of 5.4 provides strong hemostatic properties reflected in the full combination of the gel with the sample blood. On the other hand, the composition at pH 12.0 fails to interact with the blood. In the pH 12 gel, the catechol groups are fully oxidized and the combination of the high pH with the full catechol oxidation results in a structure that fails to react with the blood in the sample. DETAILED DESCRIPTION OF THE INVENTION

[0019] In various aspects and embodiments, the present application contemplates methods for achieving hemostasis during surgical procedures by applying topical hemostat agents to surgical sites having a bleed, where the hemostat does not swell after application to the surgical site (i.e., to the site of the bleed). In embodiments, the present application provides, in part, methods for achieving hemostasis during surgical procedures that avoid complications associated with application of conventional topical hemostatic agents, such as, for example, swelling of the material after application to the bleed and subsequent compression of anatomical structures close to the application site. Swelling and compression can create serious risk during various surgeries dealing with sites close to nerves and blood vessels. For example, compression of nerves and blood vessels during surgery may result in decreased blood flow and subsequent injury. Various symptoms of adverse swelling events at surgical sites include, but are not limited to, pain, paralysis, large nerve and / or blood vessel injury, numbness, and inflammation. In some instances, symptoms of swelling and compression events at surgical sites do not present until the patient is in post-operative recovery period.

[0020] In embodiments, the present application contemplates a topical hemostat agent that is applied to the bleeding surgical site and comprises hydrophobically-modified chitosan comprising hydrocarbon groups attached to the chitosan backbone. In embodiments, the topical hemostat agent is a gel or a foam. In some embodiments, the agent is a hydrogel, such as a syringable hydrogel. In embodiments, the hydrocarbon groups comprise hydrocarbon groups independently selected from linear C8 to C18 hydrocarbon chains, which provide effective hemostatic properties. In embodiments, the hydrophobic groups further comprise hydrocarbon groups independently selected from Cl to C4 hydrocarbon groups (e.g., C2), which provide for a predictable and tunable degradation property. In embodiments, the hydrophobically-modified chitosan further comprises one or more benzenediol groups, and optionally oxidized forms thereof, to tune adhesive and cohesive properties of the composition. In embodiments, the one or more benzenediol groups comprise a catechol moiety, and optionally oxidized forms thereof. In embodiments, at least some of the benzenediol groups are oxidized to the corresponding quinone. In embodiments, the benzenediol groups are partially oxidized, that is, the benzenediol groups are not all fully oxidized to the corresponding quinone.

[0021] In embodiments, the present application provides methods for hemostasis at bleeding surgical sites that are susceptible to or at risk of injury due to compression of tissues (e.g., nerves and blood vessels). Such surgical sites may include, without limitation, confined bony structures, spinal cord, the optic nerve / chiasm, and foramina of bone, because unsaturated particles (such as those comprised in conventional hemostatic materials) may swell and compress the surrounding tissues. Cardiac locations near the pulmonary artery can also be susceptible to compression, as well as cavernous nerves at risk during prostatectomy procedures.

[0022] In embodiments, the present application provides methods for achieving hemostasis during a surgical procedure in a patient having a factor that influences volume of blood loss during the procedure, such as advanced age (e.g., 50 years of age or more, 60 years of age or more, or 70 years of age of more).

[0023] In embodiments, the present application provides methods for achieving hemostasis during a surgical procedure in a patient having higher body mass index (e.g., 25 or greater, or 30 or greater).

[0024] In embodiments, the present application provides methods for achieving hemostasis during a surgical procedure in a patient having presence of osteoporotic bone. Osteoporosis is a bone disease that develops when bone mineral density and bone mass decreases, or when the quality or structure of bone changes, thus leading to a decrease in bone strength that can increase the risk of broken bones (fractures).

[0025] In embodiments, the present application provides methods for achieving hemostasis during a surgical procedure in a patient having neuromuscular scoliosis. Neuromuscular scoliosis causes irregular curvature of the spine and is associated with disorders of the nerve or muscular systems such as cerebral palsy, spina bifida, and spinal cord injury. In embodiments, the present application provides methods for achieving hemostasis during a surgical procedure in a patient having bone metastasis (i.e., cancer metastasis to bone).

[0026] In embodiments, the present application provides methods for achieving hemostasis during a surgical procedure in a patient making use of anti-coagulant therapy. Such anticoagulant therapies include, but are not limited to, vitamin K antagonists, direct oral anticoagulants, and low molecular weight heparins.

[0027] In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is neurosurgery. Neurosurgery procedures are directed to treating conditions that affect the central and peripheral nervous system, including the brain, spinal cord, and nerves. In embodiments, during a neurosurgery procedure the composition is applied to a site selected from one or more of neurological structure, spinal cord, optic chiasm, spinal column, thecal sac, peritoneal sac, blood vessel, and nerve. Complications related to swelling that may occur during a neurosurgery procedure that makes use of a conventional topical hemostatic agent include, but are not limited to, inflammation, paresis, and focal seizures. Common examples of neurosurgical procedures include, but are not limited to, anterior cervical discectomy, epilepsy neurosurgery, Chiari decompression, craniotomy, and laminectomy. Anterior cervical discectomy is a neurosurgery procedure that most notably treats herniated discs in the neck, where the goal of the procedure is to remove the herniated disc to provide pain relief. Epilepsy neurosurgery is a procedure for those patients having severe epileptic seizures who have not found relief through non-surgical treatments (e g., medications). An epilepsy neurosurgery procedure involves removing or modifying the part of the brain that is producing severe and potentially fatal seizures. Chiari decompression is used to treat Arnold-Chiari malformation, which is characterized by an inability to properly balance. Chiari decompression involves removing a bone in the back of the skull to widen the foramen magnum in order to help improve balance and coordination. A craniotomy is a common neurosurgery procedure that involves removing a part of the skull bone to allow for direct access to the brain, which is used to treat a variety of neurological concerns, including brain strokes. In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is spine surgery. The spine is made up of 33 segments, called vertebrae, which are stacked on top of each other to form the spinal column. In between each vertebra are protective, circular pads of tissue called discs, which cushion the vertebrae during activities such as walking and running. The spinal canal runs through the center of the spinal column. It contains and protects the spinal cord and nerves. Spinal deformity, spinal infections, trauma, spine tumors and some degenerative spine conditions, such as stenosis and herniated disks, may require surgical treatment. During a traditional open spine surgery procedure, an incision is made along the backbone, and muscle and soft tissue is moved aside to gain access to the bones of the spine and the spinal cord. In embodiments, the patient undergoing spine surgery has a herniated spinal disc, and the spine surgery is a discectomy, which is the surgical removal of the damaged portion of a herniated disc in the spine to relieve pressure on a nerve.

[0028] In embodiments, the patient undergoing spine surgery has a build-up of bony overgrowths of the joints within the spinal canal (e.g., in patients having arthritis in the spine), and in some cases, the patient has spondylosis of the cervical and / or lumbar spine. Cervical spondylosis is a general term for age-related wear and tear in the cervical spine (neck) that can lead to neck pain, neck stiffness and other symptoms. Lumbar spondylosis is an age-related degeneration of the vertebrae and discs of the lower back, often called degenerative disc disease and / or osteoarthritis. In embodiments, the spine surgery is a laminectomy or laminotomy, which is a procedure that involves partially removing or removing a small piece of the back part (lamina) of the small bones of the spine (vertebrae) that results in an enlargement of the spinal canal to relieve pressure on the spinal cord or nerves.

[0029] In embodiments, the patient undergoing spine surgery has spinal stenosis, a slipped disc and / or sciatica, a spinal injury, or metastatic spinal cord compression, and the spine surgery is lumbar decompression surgery, which treats compressed nerves in the lower spine. Lumbar decompression surgery generally involves at least one of a procedure selected from a laminectomy, a discectomy, and a spinal fusion (e.g., joining together two or more vertebrae with a section of bone to stabilize and strengthen the spine).

[0030] In embodiments, the patient undergoing spine surgery has a joint fracture and / or arthritis, and the spine surgery is arthrodesis, which is a method of surgical joint ossification used to fuse the bones in a joint. Arthrodesis surgical procedure involves making incisions around the joint and removing all cartilage in the joint so that the bones are directly touching and eventually heal and fuse together to create an immovable joint.

[0031] In embodiments, the patient undergoing spine surgery has cervical myelopathy, and the spine surgery involves anterior or posterior decompression of the area and / or spinal fusion. Cervical myelopathy is a condition describing a compression of the spinal cord at the cervical level of the spinal column resulting in spasticity (sustained muscle contractions), hyperreflexia, pathologic reflexes, digit / hand clumsiness, and / or gait disturbance. The goal of surgery is to increase the canal space, which will lessen / eliminate cord compression. Surgical intervention can involve either an anterior or posterior approach. A posterior approach is preferable in lordosis deformities and when the pathology occurs at the posterior aspect of the canal. One option is a laminectomy (ideally with preoperative lordosis greater than 10 degrees and absence of instability). Anterior cervical discectomy and fusion (ACDF) is performed via an anterior approach and can involve up to 3 disc levels.

[0032] In embodiments, the patient undergoing spine surgery has spondylolisthesis, and the spine surgery involves lumbar decompression and / or spinal fusion. Spondylolisthesis is a condition in which one of the bones in the spine slips forward, resulting in pain in the lower back. Spondylolisthesis occurs more often as patients age due to the bones of the spine weakening with age. It can also be caused by a stress fracture.

[0033] In embodiments, during spine surgery, the composition is applied to the spinal canal and / or anatomic structures within the spinal canal, such as the dura mater, blood vessels, connective tissue, nerves, fat, and pathologic structures, including adhesion (fibrosis), inflammation, and stenotic change. In embodiments, during spine surgery, the composition is applied to one or more of spinal cord, spinal column, thecal sac, nerve, muscle, bone, and joint. A pressure-related injury can occur during spine surgery, where the pressure-related injury is compression of one or more neurological structures, including thecal sac compression, spinal cord compression, and nerve compressions (which is avoided using the compositions of the present disclosure). For example, complications related to swelling that may occur during a spine surgery procedure that makes use of a conventional topical hemostatic agent include, but are not limited to, thecal sac compression, numbness, paralysis, pain, swelling, incontinence, neck pain, spinal cord injury, nerve damage, muscle weakness, cerebrospinal fluid leakage, edema, and tetra paresis.

[0034] In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is orthopedic surgery. Orthopedic surgery involves surgical procedures on the musculoskeletal system that treat, repair, or prevent conditions affecting bones, muscles, joints, tendons and ligaments, cartilage, and soft tissues. Such conditions include, but are not limited to, pain of the joints, muscles, or bones; a tear of the muscles, cartilage, or ligaments; breaks and fractures; arthritis; bursitis; tumors; and congenital malformations. Common orthopedic procedures include making incisions and repairing broken bones or fractures with screws, pins, rods, or plates, or removing damaged ligaments and replacing them with healthy tissue or a graft from a donor, or placing metal or plastic caps on a bone in the joint to repair arthritis damage. In embodiments, during orthopedic surgery, the composition is applied to bleeding surgical sites, such as muscles, joints, nerves, and bones. Complications that may occur during an orthopedic surgical procedure that makes use of a conventional topical hemostatic agent include, but are not limited to, swelling that puts pressure on bony structures and nerves.

[0035] In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is brain surgery. Brain surgery is used to treat a variety of conditions, such as tumors, blood clots, aneurysms, epilepsy, and Parkinson’s disease, involving the brain or areas around it. Common types of brain surgery procedures include, but are not limited to, a craniotomy, a biopsy, deep brain stimulation, neuroendoscopy, posterior fossa decompression, and thrombectomy and cerebral aneurysm repair. In embodiments, during brain surgery, the composition is applied to bleeding surgical sites, such as the optic chiasm or brain tissue.

[0036] In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is ocular surgery. Ocular surgery is used to treat a variety of conditions, such as cataracts, glaucoma, detached retinas, retinal tears, and / or diabetic retinopathy. Common types of ocular surgery procedures include, but are not limited to, blepharoplasty, cataract surgery, corneal transplant, glaucoma surgery, retina surgery, and eye muscle surgery. In embodiments, during ocular surgery, the composition is applied to bleeding surgical sites, such as nerves, muscles, and eye tissue.

[0037] In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is otorhinolaryngological (ENT) surgery. Otolaryngology focuses on the (i) ears (e.g., treatment of hearing loss, ear infections, balance disorders, ear noise, nerve pain, and facial and cranial nerve disorders), (ii) nose (e.g., treatment of the nasal cavity and sinuses, including treatment of allergies, sinusitis, smell disorders, polyps, and nasal obstruction due to deviated septum), (iii) throat (e.g., treatment of diseases of the larynx and upper aerodigestive tract or esophagus, including voice and swallowing disorders), and (iv) head and neck (e.g., treatment of infectious diseases, facial trauma, and facial deformities. In embodiments, during otorhinolaryngological surgery, the composition is applied to bleeding surgical sites, such as muscles, joints, nerves, and bones. Complications that may occur during an otorhinolaryngological surgical procedure that makes use of a conventional topical hemostatic agent include, but are not limited to, swallowing difficulty, nerve damage, paresis, vocal paralysis, and speech disorders.

[0038] In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is thyroid surgery. Thyroid surgery may involve a thyroid resection, which generally falls into one of three categories: (i) total thyroidectomy, which is the removal of the entire thyroid; (ii) thyroid lobectomy (hemithyroidectomy), which is removal of half of the thyroid; and (iii) completion thyroidectomy, which is removal of any remaining thyroid tissue. In embodiments, during thyroid surgery, the composition is applied to bleeding surgical sites, such as arteries, blood vessels, muscles, joints, nerves, and bones. Complications that may occur during a thyroid surgical procedure that makes use of a conventional topical hemostatic agent include, but are not limited to, swallowing difficulty, nerve damage, paresis, vocal paralysis, and speech disorders.

[0039] In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is thoracic surgery. Thoracic surgery involves various surgical operations in the chest, including areas of the heart, lungs, mediastinum, esophagus, trachea, diaphragm, and chest wall. Common thoracic surgical operations include, but are not limited to, heart valve repair, lung tumor removal, and aneurysm repair in the chest. Thoracic surgery can target a variety of conditions, including but not limited to, stretched blood vessels (aneurysms), congenital heart issues, irregular heart rhythms, heart failure, coronary artery disease, heart or lung transplant, heart valve issues, lung cancer, tumors in the esophagus or thymus, swallowing issues (dysphagia), gastroesophageal reflux disease (GERD), Barrett’s esophagus, and hiatal hernias. In embodiments, during thoracic surgery, the composition is applied to bleeding surgical sites, such as arteries, blood vessels, muscles, joints, nerves, bones, the pulmonary artery, the superior vena cava, and the inferior vena cava. Complications that may occur during a thyroid surgical procedure that makes use of a conventional topical hemostatic agent include, but are not limited to, pain, paralysis, nerve damage, neurological deficit or dysfunction, weakness, hypoesthesia, and injury. A patient undergoing thoracic surgery and receiving a conventional topical hemostatic agent may develop paraplegia. A patient undergoing thoracic surgery and receiving a conventional topical hemostatic agent may develop symptoms associated with swelling and compression during a post-operative recovery period.

[0040] In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is prostate surgery. Radical prostatectomy is a surgical procedure used to treat a number of conditions affecting the prostate, including, most commonly, prostate cancer. A prostatectomy may also be recommended for men with severe urinary symptoms and very enlarged prostate glands, which are known as benign prostatic hyperplasia (BPH). Prostatectomy is surgery to remove part or all of the prostate gland, which is situated in the male pelvis, below the urinary bladder, and surrounds the urethra. When performed to treat localized prostate cancer, a radical prostatectomy may also result in removal of the lymph nodes surrounding the prostate gland. Open radical prostatectomy involves making an incision in the lower abdomen to dissect the prostate gland and nearby tissue from surrounding nerves and blood vessels. In embodiments, during prostate surgery, the composition is applied to bleeding surgical sites, such as nerves, blood vessels, muscles, joints, and bones. In embodiments, the surgery is prostatectomy, and the bleed involves a cavernous nerve. Complications that may occur during a prostate surgical procedure that makes use of a conventional topical hemostatic agent include, but are not limited to, pain, nerve damage, and injury.

[0041] In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is gynecological surgery. Gynecology surgery includes any surgical procedure that involves the organs and structure of the female pelvic region, such as the uterus, ovaries, cervix, fallopian tubes, vagina and vulva. Common gynecological surgical operations include, but are not limited to, hysterectomy, total laparoscopic hysterectomy (TLH), laparoscopic supracervical hysterectomy (LSH), laparoscopic assisted vaginal hysterectomy (LAVH), fibroid removal, laparoscopic myomectomy for fibroids, hysteroscopic myomectomy, ovarian cyst removal, laparoscopic ovarian cystectomy, treating adhesions, laparoscopic lysis of adhesions, diagnostic hysteroscopy, removing uterine polyps, hysteroscopic polypectomy, hysteroscopic endometrial ablation, and hysteroscopic sterilization (essure). Gynecological surgery may be used to treat or prevent a condition such as, but not limited to, endometriosis, fibroids (benign tumors), ovarian cysts, cancer, chronic pelvic pain, pelvic inflammatory disease, uterine prolapse and abnormal bleeding. In embodiments, during gynecological surgery, the composition is applied to bleeding surgical sites, such as nerves, blood vessels, muscles, joints, and bones. Complications that may occur during a gynecological surgical procedure that makes use of a conventional topical hemostatic agent include, but are not limited to, fever, pain, stenosis, and fibrosis. In embodiments, the methods for hemostasis contemplated in the present application include applying the non-swellable hemostat compositions to bleeding surgical sites, where the surgery is cardiac or cardiovascular or vascular surgery. Cardiac or cardiovascular or vascular surgery involves the surgical treatments of pathologies related to the heart, thoracic aorta, and blood vessels carrying blood to and from the heart. For example, cardiac or cardiovascular or vascular surgery may be used to treat or prevent heart attacks and blood clots, address irregular heartbeats, open blocked or narrowed arteries, repair congenital heart problems, and fix damaged or diseased heart valves. Common coronary surgical procedures include, but are not limited to, coronary artery bypass grafting (CABG), heart valve repair or replacement, insertion of a pacemaker or an implantable cardioverter defibrillator (ICD), maze surgery, aneurysm repair, heart transplant, insertion of a ventricular assist device (VAD) or total artificial heart (TAH), and transcatheter structural heart surgery. In embodiments, during cardiac or cardiovascular or vascular surgery, the composition is applied to bleeding surgical sites, such as arteries, blood vessels, nerves, muscles, and bones. In embodiments, during cardiac or cardiovascular surgery, the composition is applied to a bleeding surgical site, such as the pulmonary artery, superior vena cava, and / or inferior vena cava. In embodiments, the surgery is cardiac or vascular surgery, and the bleed involves a large blood vessel at risk of compression or stenosis, optionally wherein the blood vessel is a pulmonary artery or superior vena cava. Complications that may occur during cardiac or cardiovascular or vascular surgical procedures that make use of a conventional topical hemostatic agent include, but are not limited to, necrosis, nerve damage, paralysis, pulmonary artery and periaortic compression, severe stenosis, and superior vena cava syndrome.

[0042] In embodiments, the composition is applied to a site selected from one or more of neurological structure, spinal cord, optic chiasm, spinal column, thecal sac, peritoneal sac, blood vessel, nerve, pulmonary artery, superior vena cava, inferior vena cava, brain tissue, bladder, cavernous nerve, muscle, bone, and joint.

[0043] In embodiments, the surgery site comprises bleeding selected from one or more of subcutaneous and muscular bleeding, bone bleeding, epidural bleeding, and large blood vessel bleeding. In embodiments, the present application contemplates applying a non-swellable hemostatic composition to a surgical site comprising a bleed. In embodiments, the surgical bleed is a scale 1 to 3 bleed (out of scale of 5). In embodiments, the bleed is at least a scale 3 bleed. In embodiments, the bleed is a scale 4 bleed. Generally, the bleeding scale can be defined as: 0 (no bleeding, hemostasis); 1 (minimal bleeding); 2 (mild bleeding); 3 (moderate bleeding); 4 (severe bleeding), and 5 (extreme bleeding). In this context, bleeding scales can be modeled as described in U.S. Patent No. 10,283,015, which is hereby incorporated by reference.

[0044] The non-swellable compositions provided in the present application comprise a hydrophobically-modified chitosan (hm-chitosan) and a solvent. In embodiments, the hydrophobically-modified chitosan comprises hydrocarbon groups attached to the chitosan backbone.

[0045] Chitosan is the common name of the linear, random copolymer that consists of P-(l- 4)-linked D-glucosamine and N-acetyl-D-glucosamine. The molecular structure of chitosan consists of a linear backbone linked with glycosidic bonds. Chitosan is the major component of crustacean shells such as crab, shrimp, krill and crawfish shells. Additionally, chitosan is the second most abundant natural biopolymer after cellulose. Commercial chitosan samples are typically prepared by chemical de-N-acetylation of chitin under alkaline conditions. Depending on the source of the natural chitin (extracted from shells) and its production process, chitosan can differ in size (average molecular weight, Mw) and degree of N- acetylation (%DA). While the poor solubility of chitosan in water and in common organic solvents restricts its applications, reactive amino groups in the chitosan backbone make it possible to chemically conjugate chitosan with various molecules and to modulate its properties.

[0046] The hm-chitosan in some embodiments is prepared using fatty acid anhydride chemistry, resulting in amide bonds with the chitosan polymer and the hydrocarbon chains. See U.S. Patent No. 11,274,194, which is hereby incorporated by reference in its entirety. Amide bonds can generally be susceptible to hydrolysis in solution. Hydrolysis of amide bonds is generally known to occur in the presence of acids, which act as catalysts for the reaction between the amide and water. Amides are also susceptible to alkaline hydrolysis. However, the amide bonds formed between chitosan and fatty acid anhydrides are shelf stable, even in the presence of dilute acids that are required to maintain solubility of the hm- chitosan. Accordingly, the modified chitosans may be prepared using a one-pot synthesis, without the need for harsh reagents, including reducing agents. The materials can be precipitated following the reaction and dried for processing and incorporation into products, including flowable products such as solutions, gels, and foams.

[0047] In embodiments, the hm-chitosan is derived from a deacteylated chitin, which may be derived from one or more of crab, shrimp, krill, and crawfish.

[0048] In embodiments, the hm-chitosan is prepared from a chitosan having a degree of deacetylation of from about 40% to about 90%, such as from about 50% to about 90%, such as from about 60% to about 90%. In embodiments, the degree of deacteylation is about 85%. In embodiments, the degree of substitution of the hydrophobic substituent on the chitosan is from about 1% to about 50% of the available amine groups. As used herein, the term “available amines” means with respect to the individual chitosan monomers, that is, assuming 100% deacetylation for purposes of quantifying graft density. In embodiments, the degree of substitution of the hydrophobic substituent on the chitosan is from about 1% to about 50% of the available amine groups, or from about 20% to about 40% of the available amine groups, or from about 1% to about 5% of the available amine groups.

[0049] In embodiments, the molecular weight of the hm-chitosan ranges from about 40,000 to about 500,000 grams per more, or from about 50,000 to about 250,000 grams per mole, or from about 50,000 to about 100,000 grams per mole. In embodiments, the chitosan is a large molecular weight chitosan, or a medium molecular weight chitosan, or a small molecular weight chitosan. Generally, the molecular weight of the hm-chitosan will range from about 25,000 to about 1,500,000 grams per mole. In embodiments, the molecular weight of the hm-chitosan ranges from about 40,000 to about 500,000 grams per more, or from about 50,000 to about 250,000 grams per mole, or from about 50,000 to about 100,000 grams per mole. In embodiments, the chitosan has a low molecular weight of less than 150,000 Daltons (prior to modification). In other embodiments, the chitosan has a medium molecular weight of about 150,000 to about 350,000 Daltons (prior to modification). In yet other embodiments, the chitosan has a high molecular weight of about 400,000 Daltons or more (prior to modification).

[0050] As used herein, the term “molecular weight” means weight average molecular weight. In some embodiments, the chitosan is a medium molecular weight chitosan. Methods for determining average molecular weight of bio-polymers include low angle laser light scattering (LLS) and Size Exclusion Chromatography (SEC). In performing low angle LLS, a dilute solution of the polysaccharide, typically 2% or less, is placed in the path of a monochromatic laser. Light scattered from the sample hits the detector, which is positioned at a low angle relative to the laser source. Fluctuation in scattered light over time is correlated with the average molecular weight of the polysaccharide in solution. In performing SEC measurements, again a dilute solution of biopolymer, typically 2% or less, is injected into a packed column. The polysaccharide is separated based on the size of the dissolved polymer molecules and compared with a series of standards to derive the molecular weight.

[0051] In embodiments, the non-swellable composition of the present application includes two or more different classes of natural polymers, for instance the chitosan and a cellulosic, or the chitosan and an alginate, or a mixture of the chitosan, an alginate, and a cellulosic.

[0052] The hydrophobic substituents may comprise at least one hydrocarbon group having from about 8 to about 18 carbon atoms attached to the backbone of the chitosan. In embodiments, the hydrocarbon groups comprise linear hydrocarbon chains independently selected from the range of C8 to C18. In embodiments, the hydrocarbon group comprises one or more of linear C8, CIO, C12, C14, C16, and C18 hydrocarbon chains. In some embodiments the C8 to C 18 group is an alkyl group. In some embodiments, the hydrocarbon group comprises an arylalkyl group. As used herein, the term “arylalkyl group” means a group containing both aromatic and aliphatic structures.

[0053] In embodiments, hydrocarbon groups within the range of C8 to C18 are present from about 0.1 mol% to about 10 mol%, or from about 0.5 mol% to about 3 mol%, of available amines on the chitosan backbone. In embodiments, hydrocarbon groups comprise C8 hydrocarbon chains present at about 0.5 mol% to about 3 mol% of available amines, and in some embodiments at about 1 mol%, of available amines.

[0054] In various embodiments, the hm-chitosan has about 0.1% to about 80% substitution of available amines (i.e.., % of chitosan monomers) by a fatty acid anhydride moiety. In some embodiments, Cl to C4 acyl chains are incorporated at from 10% to 80%, such as from 10% to 60% or 25 to 70%, or 25 to 60% of chitosan monomers. In some embodiments, the Cl to C4 acyl chains are incorporated at about 20% to about 60%, or from about 20% to 50%, or from 20% to 40% of the chitosan monomers. In embodiments, the hydrophobic groups attached to the chitosan backbone comprise Cl to C4 hydrocarbon groups, which are optionally C2 hydrophobic groups, present from about 15 mol% to about 50 mol%, and optionally about 25 mol% to about 45 mol% of available amines (i.e., % of chitosan monomers). These grafts allow for tuning of the desired degradation rate in vivo, where the C2 grafts increase degradation rate. In embodiments, incorporation of these small hydrophobic groups, such as Cl to C4 acyl chains, allows the chitosan to degrade more predictably from lysozyme activity in the body, which is important for creating a material that can be left inside the body after treatment of the wound. More specifically, hydrophobic groups below the length of C6 do not contribute towards improved hemostatic effect. However, hydrophobic modification in the range of Cl to C4 allow for a framework to optimize the degradation of the material inside the body via lysozymes. Particularly in the case of surgical-use hemostats, it is ideal for the hemostatic biomaterial to degrade quickly after achieving hemostasis.

[0055] In some embodiments, the hydrophobically-modified chitosan has two or more (e.g., 2 or 3) different hydrocarbon groups conjugated along the chitosan backbone. These chitosans can provide a greater level of control over how these polymers interact with themselves and with other entities in an aqueous or organic environment. For example, the hydrophobically-modified chitosan may have a population of C6 to C12 hydrocarbon groups, and a population of C14 to C18 hydrocarbon groups. In embodiments, the hydrophobic groups further comprise Cl to C4 hydrocarbon groups. In embodiments, the hydrophobic groups comprise a population of Cl to C4 hydrocarbon moi eties, a population of C6 to C12 hydrocarbon moieties, and a population of C14 to C18 hydrocarbon moieties. In embodiments, the hydrophobically-modified chitosans comprise benzenediol groups, optionally where a portion of the benzenediol groups are oxidized to the corresponding quinone. For example, the benzenediol group can comprise catechol groups. The catechol moi eties can be grafted to the biopolymer using hydrocaffeic acid or L-DOPA reagents, for example. The addition of benzenediol groups and oxidized forms thereof to the hydrophobically modified biopolymer increases the tissue adhesive properties (e.g., mucoadhesive properties). Further, such benzenediol groups added to the hydrophobically- modified biopolymer helps the biopolymers remain in solution and form gels that are easy to use. The following illustrates the chemical structure of chitosan to which benzenediol groups have been added by conjugation to available amines (with mixture of acetyl groups and free amines):

[0056] The following formula illustrates each monomer unit separated by brackets (m monomer comprises catechol moiety; n monomer comprises a free amine; and p monomer comprises acetyl:

[0057]

[0058] The following formula shows a chitosan biopolymer in which a benzenediol substituent has been fully oxidized to its corresponding quinone:

[0059] The following formula illustrates a hydrophobically modified chitosan of the disclosure, having benzenediol substituents, a portion of which are oxidized:

[0060]

[0061] According to this formula, hydrophobic grafts (e.g., C8 as illustrated above) are present with benzenediol and its oxidized form in various ratios and densities. The modified chitosan may also comprise C1-C4 groups (e.g., acetyl) as described elsewhere herein as well as free amines.

[0062] In various embodiments, the hydrophobically-modified chitosan can be described according to the following formula, in which an amount of chitosan monomers having a substituent comprising catechol are represented by an integer m; an amount of chitosan monomers having an oxidized form of catechol are represented by an integer p; an amount of chitosan monomers having a free amine are represented by an integer n; and an amount of chitosan monomers having a hydrophobic substituent are represented by an integer q: In various embodiments, the benzenediol groups and oxidized forms thereof (m and p) are grafted to the biopolymer at a density of from 0.1% to about 15% of biopolymer monomers. For example, the hydrophobically-modified biopolymer can be hm-chitosan and benzenediol groups and oxidized forms thereof (m and p above) are grafted to the hm- chitosan at a density of from about 0.1% to about 10% of chitosan monomers, or from about 0. 1% to about 5% of chitosan monomers, or from about 0.1% to about 2% of chitosan monomers (e.g., about 0.8%). In various embodiments, about 10% to about 90% of the benzenediol groups are oxidized (monomer p in the formula above), or from about 25% to about 75% of the benzenediol groups are oxidized, or from about 30% to about 60% of the benzenediol groups are oxidized to the corresponding quinone. In various embodiments, the ratio of unoxidized benzenediol (e.g., catechol) to oxidized benzenediol (e.g. quinones) is about 1 :2, or about 1 : 1 (e.g., a range of from about 1 :2 to about 1:-1); or in other embodiments, the ratio may be about 2:1, about 3: 1, about 4: 1, or about 5:1 (e.g., a range of about 1 :2 to about 5: 1, or a range of about 1 : 1 to about 5: 1. or a range of about 2: 1 to about 5: 1). As the level of oxidation increases, cohesiveness of the material increases. Similarly, the greater the level of unoxidized molecules the greater adhesiveness of the modified biopolymer. The biopolymer in the foregoing formula may further comprise monomers having acetyl groups as already described.

[0063] The partial oxidation of the benzenediol groups allows for a large degree of tunability of both the adhesive and cohesive properties of the hydrophobically-modified biopolymer, e.g., in an aqueous hydrogel format. There are two measures of functionality of hemostatic gels: cohesiveness and adhesiveness. The cohesive nature of a hemostatic gel is exemplified by the ability of the gel to maintain integrity while under pressure. For example, in burst pressure tests in which a stream of water is directed at a gel, the higher cohesiveness of the gel results in the higher pressure necessary for the water to break through the gel. On the other hand, the adhesive nature of the gel can be measured in a similar way. The higher the level of adhesiveness, the more pressure that is required to dislodge the gel from a tissue or surface to which the gel is adhered. The most functional hemostatic gels have both higher cohesive values and higher adhesive values. While hydrocarbon chains as hydrophobic grafts also provide a framework to tune or balance adhesive and cohesive properties of resulting hm-chitosan, partial oxidation of benzenediol groups along the backbone provide an additional layer of tunability, independent of the hydrophobic interactions conferred by the hydrophobic groups. For example, non-oxidized benzenediol groups amplify the adhesive properties of the polymer (e.g., mucoadhesive properties), whereas oxidized benzenediol groups amplify the cohesive properties of the polymer. In aggregate, the combination of hydrophobic modification, along with the addition of both non-oxidized and oxidized benzenediol groups onto the polymer backbone, results in a highly tunable hydrogel system for optimized adhesive and cohesive properties, as well as hemostatic action, so as to effectively treat bleeding from injured tissues (including moderate to severe surgical bleeds). Cohesive properties of a gel can be measured based on the elastic modulus. For example, according to various embodiments, the elastic modulus of a hydrogel according to the disclosure ranges from about 50 to about 5,000 pascals. In some embodiments, the elastic modulus is less than about 4000 pascals, or less than about 2000 pascals, or less than about 1000 pascals, or less than about 500 pascals.

[0064] Figure 1 shows a comparison between hydrophobically modified chitosan further comprising 8% catechol substitutions or 0.8% catechol substitutions (with respect to the number of monomers in the biopolymer). As show in Figure 1 , the 8% catechol (right) fails to form a gel matrix and forms a precipitate. The 0.8% vial (left) shows a homogeneous and stable gel. While conjugation of catechol to chitosan has been described as exhibiting good tissue adhesive properties, it was surprising that these properties are exhibited at a very low level of catechol conjugation when added to hydrophobically-modified chitosan. As a result, in one embodiment, the hydrophobically modified biopolymer (e g., chitosan) has benzenediol grafts (including oxidized forms thereof) in a concentration of at least about 0.1%, but less than about 10%, or less than about 8%, or less than about 5%, or less than about 3%, or less than about 2% of the monomer units. In various embodiments, the benzenediol grafts (including oxidized forms thereof) are present in the range of 0.2% to about 2% of polymer monomers, or in the range of about 0.5% to about 1.5%. The properties of partial oxidation of catechol provide significant benefits to the material, specifically, it allows for modulation of adhesive and cohesive properties of HMC- C based on the level of oxidation of the benzenediol groups present in the molecule. The partially oxidized catechol groups on the backbone of the biopolymer are quinone functional groups (i.e., hydroxyls in the benzenediol are oxidized to quinone). The unoxidized hm- chitosan has no color, i.e, the gel is translucent. As oxidation increases, color of the gel ranges from clear to dark orange and has less fluid and hemostatic properties, as seen in Figure 3. The level of oxidation can be monitored using UV spectral analysis, for example, as illustrated in Figure 2.

[0065] In embodiments, a non-swellable composition provided by the present application comprises about 1.0 wt% hydrophobically modified chitosan (i.e. with partially oxidized catechol grafted (about 0.8 mol% basis, medium molecular weight chitosan modified with about 35 mol% C2, and about 1 mol% C8)). In embodiments, the composition further comprises about 0.5 wt% of gelatin granules, about 0.05M acetic acid, about 0.5 wt% ethanol, and / or about 40 ppm EDTA.

[0066] The degree of deacetylation of chitin (to provide a native chitosan for modification) may generally range from about 40%-100%, or in some embodiments, from 50 to 100%, which determines the charge density and which makes the chitosan readily reactive for modification. The charge density of chitosan is an important parameter for its tissue adherent properties. The amount of acetylation can be tuned by adding acetyl groups back to the chitosan, according to this disclosure. Thus, according to embodiments of this disclosure, the modified chitosan will have a free amine at about 40% of its monomers or more, or about 50% of its monomers of more, or at about 60% of its monomers or more. In embodiments, the modified chitosan according to this disclosure comprises a free amine on about 40% to about 75% of it monomers, or from about 50% to about 75%, or from about 55% to about 75% of its monomers. The structure of chitosan (shown deacetylated) is depicted in Formula 1:

[0067]

[0068] In some embodiments, the non-swellable hemostat composition is a topical solution or gel, and may be syringable gel.

[0069] In various embodiments, the non-swellable composition of the present application is formulated as a liquid, gel, or foam. That is, the composition may be a flowable product, and may be suitable for topical application to wounds or bleeds, both for internal and external use. In some embodiments, the composition comprises at least one synthetic polymer. Exemplary synthetic polymers include polythenes, polystyrenes, poly acrylates, polyamides, polyesters, polyurethanes, polysulfides, and polycarbonates. In some embodiments, the synthetic polymer is polyethylene glycol. In some embodiments, the synthetic polymer is polyvinyl alcohol. Inclusion of synthetic polymers can modify the physical properties of the composition, including the viscosity as well as the cohesion of the composition or of the artificial clot or seal created upon use.

[0070] In some embodiments, the non-swellable composition of the present application is formulated in a dilute organic acid. In embodiments, the dilute acid is a weak organic acid. An organic acid is an organic compound with acidic properties. The most common organic acids are the carboxylic acids, whose acidity is associated with their carboxyl group -COOH. For example, the organic acid may have a pKa of from about 2 to about 5, or from about 2.5 to about 4, or from about 3 to about 4, or from about 3.5 to about 4. Exemplary organic acids include lactic acid, acetic acid, formic acid, citric acid, and malic acid. In some embodiments, the organic acid is lactic acid, acetic acid, or citric acid. In some embodiments, the dilute acid is from 0.05M to 0.4M of the organic acid, such as lactic acid. In some embodiments, the polymer is formulated with about 0.05M to about 0.2M lactic acid. In some embodiments, the polymer is formulated with about 0. IM lactic acid. In some embodiments, the composition will have a pH of less than about 5, or less than about 4, or less than about 3. In some embodiments, the pH of the composition is from about 2 to about 4. While these organic acids are suitable for solubilizing the hydrophobically-modified chitosan, they do not substantially catalyze hydrolysis of the amide bond between the chitosan and the hydrophobic graft. In embodiments, the organic acid is present in the composition from about 0.01 M to about 0.4 M, or from about 0.01 M to about 0.3 M, or from about 0.01 M to about 0.2 M, or from about 0.05 M to about 0.4 M, or from about 0.05 M to about 0.3 M. In embodiments, the organic acid is acetic acid and is present in the composition at about 0.05M.

[0071] A hydrophobically-modified chitosan material for incorporation into aqueous or organic solutions or suspensions can be based on a solution of the hm-chitosan that is about 0.1% to about 5.0% by weight relative to the total weight of the composition. In some embodiments, the hm-chitosan is present at about 0.5% to about 4%, or about 0.5% to about 3% of the total weight of the composition, or about 0.5% to about 2% of the total weight of the composition. In some embodiments, the hm-chitosan is about 1.0% to about 5.0% by weight relative to the total weight of the composition, or in some embodiments, about 1.5% to about 5%, or about 2.0% to about 4% of the total weight of the composition. In some embodiments, the hydrophobically-modified chitosan is present at from about 1 wt% to about 3 wt%, or from about 1.5 wt% to about 2.5 wt%.

[0072] A non-swellable composition of the present application may further comprise one or more secondary polymers. In embodiments, such a secondary polymer is selected from gelatin, dextran, pectin, alginate, collagen, polyethylene oxide, gellan gum, and polyvinyl alcohol. In embodiments, the composition comprises polymer granules, and optionally gelatin granules. In embodiments, the composition comprises gelatin granules at 0.1 wt% to about 5 wt%, or from about 0.1 wt% to about 2 wt%, or from about 0.1 wt% to about 1 wt%.

[0073] Other aspects and embodiments of this disclosure will be apparent from the following working examples and claims.

[0074] EXAMPLES Example I: Preparation of HMC having catechol functional groups that are partially oxidized and exhibit cohesive and mucoadhesive properties.

[0075] A hydrophobically modified chitosan (HMC) having catechol functional groups that are partially oxidized was prepared by dissolving 10.0 g of HMC (C2 / 35, C8 / 1) in distilled water. This HMC contains 35% of chitosan monomers having an amide (i.e., not deacetylated) and 1% of chitosan monomers have a C8 hydrocarbon graft. Thus, the HMC has available amines on about 64% of chitosan monomers. Once dissolved, 18.4 ml of IM HCL was added, which increased viscosity and solubilized the HMC. In a separate container, 1.375 g of EDC was dissolved in 500 mL of ethanol and 500 mL water. Once the EDC was dissolved, 0.650 g of hydrocaffeic acid was added to the EDC solution. The EDC- hydrocaffeic acid solution was added to the HMC solution. The resulting solution contained HMC-Catechol, with about 5 mol% catechol (i.e., about 5% of monomers have a catechol substituent). The HMC-Catechol was then partially oxidized by adding 40 ml of 2M NaOH to bring the reaction to a pH of 12.27 for about 10 minutes. In a comparative example, the reaction’s pH was brought to 6.93 (Example 3). The composition where the pH is brought to 12.27 has more oxidized catechol groups than the one brought to 6.93. The reaction was stopped by shocking the solution with 1 L of ethanol.

[0076] The compositions were dried into powders. The powder was reconstituted to form a gel at a pH in the range of 4.5 to 5.5 (i.e., about 5.0). Adding catechol groups, a portion of which are oxidized, is believed to provide a balance between cohesive and mucoadhesive properties of the gel, while maintaining the hemostatic properties of the HMC. As oxidation increases, the cohesive properties of the gel increase. If oxidation proceeds too far, estimated to be beyond about pH 12.0 for more than 10 minutes before ethanol shock, the resulting powder no longer goes into solution.

[0077] Example 2: Hydrogels prepared containing HMC with partially oxidized catechol was shown to be more adhesive than HMC.

[0078] A hydrophobically modified chitosan with partially oxidized catechol was prepared by dissolving 10.0 g of HMC (C2 / 35, C8 / 1) in distilled water. Once dissolved, 21.4 mL of IM HCL was added, which increases viscosity and solubilizes the HMC. In a separate container, 1.376 g of EDC was dissolved in 330 mL of ethanol. Once the EDC was dissolved, 0.650 g of hydrocaffeic acid was added to the EDC solution. The EDC- hydrocaffeic acid solution was added to the HMC solution. The resulting solution contained HMC-Catechol. The HMC -Catechol was then partially oxidized by adding 8 mL of 2M NaOH to bring the reaction to a pH of 6.93. The reaction was stopped by shocking the solution with 1.5 L of ethanol.

[0079] Materials prepared substantially according to this example (prepared as hydrogels) were tested for tissue adhesive properties and material cohesive properties by testing burst pressure. Materials prepared substantially according to this example (prepared as hydrogels) were further tested for hemostatic action in vivo using a liver bleeding test. See, for example, US 11,274,194, which is hereby incorporated by reference in its entirety. The resulting material is more adhesive material than HMC. It was determined that the materials show good properties with respect to tissue adhesion, material cohesion, and hemostatic action.

[0080] Analysis of a HMC-Catechol with partial oxidation (brought to pH 12.3) can be seen in Figure 2, which is a UV spectral analysis where two shoulders at 280 nm and 330 nm can be seen and represent the partial oxidation state of catechol in the composition. The peak at 280 corresponds to un-oxidized catechol groups. The peak at 330 corresponds to the quinone groups, which are the oxidized catechol molecules.

[0081] Example 3: HMC-Catechol gels that are partially oxidized show good interaction with blood in forming gel complexes as compared to gels with fully oxidized catechol.

[0082] Partially oxidized HMC-Catechol solution (essentially as prepared in Example 2) 1% (w / v) was prepared in 0.05M acetic acid in deionized water. The solution was viscous but flowable in nature. The pH of the solution was measured at 5.4. When mixed 50 / 50% (v / v) with citrated bovine whole blood and vortexed for 1 second, the resulting mixture formed a gel which holds its own weight upon vial inversion. See Figure 3 (top). The same stock solution of partially oxidized HMC-Catechol solution, 1 % (w / v), was titrated up to pH 12 by dropwise addition of 1.0 M NaOH. The solution (now containing more oxidized catechol) changed in color to a deep, dark orange, and became non-flowing in nature. This change in color from relatively clear, with a slightly red-to-orange hue, to deep, dark orange color is indicative of full oxidation of the catechol groups attached to the hydrophobically- modified chitosan backbone. When mixed 50 / 50% (v / v) with citrated bovine whole blood and vortexed for 1 second, the resulting mixture did not form a gel. The blood remained freely flowing and separated from the orange gel. The orange gel retained its bulk properties and largely did not interact with the blood, aside from a small layer of diffusion of blood into the surface of the gel. As shown in Figure 3 (bottom), gels with fully oxidized catechol fail to interact with blood and create a gel complex. On the other hand, HMC -Catechol gels that are partially oxidized show good interaction with blood in forming gel complexes, while also displaying good tissue adhesion properties and material cohesive properties.

[0083] Example 4: Non-swellable composition shows efficacy in epidural application.

[0084] The purpose of this example is to evaluate the safety and effectiveness of the non- swellable composition of the present application (“test composition”).

[0085] The test composition of the present example comprises 1.0 wt% hydrophobically modified chitosan (i.e. with partially oxidized catechol grafted (0.8 mol% basis, medium molecular weight chitosan modified with 35 mol% C2, and 1 mol% C8)). The composition further comprises 0.5 wt% of gelatin granules, 0.05M acetic acid, 0.5 wt% ethanol, and 40 ppm EDTA.

[0086] The test composition was applied via epidural in an in vivo ovine model, and local histological response to the composition following lumbar laminectomy and histopathology of tissues from the spinal cord, exiting nerve roots, and operative vertebral elements were assessed.

[0087] Specifically, three ovine underwent lumbar laminectomies at the L3 and L5 levels on Day 0 of study, and operative sites received treatment with the test composition. In each animal, one site was treated ‘clinical case’ where excess test composition not involved in the clot was removed per the instructions for use (IFU), and the second site was treated ‘worst case’ where no excess test composition was removed. Animals were survived for 30-, 60-, or 90-days post-operation and subsequently euthanized. At necropsy, a postmortem laminectomy was performed at the intervening (non-operative, L4) level to allow for perfusion, and use of this level as a control. Tissues intended for histopathology and immunohistochemistry (IHC) analysis were collected and immersion fixed in 10% neutral buffered formalin (NBF). The spinal tissues were trimmed, submitted for decalcification, processed, and embedded in paraffin blocks. The resulting blocks were sectioned via microtome and mounted to glass slides. From each block, one slide was stained with hematoxylin and eosin (H&E), one slide was stained with Masson’s Tri chrome (MT), and one slide was immunohistochemically labeled for detection of Ionized Calcium Binding Adaptor Molecule 1 (IBA-1).

[0088] Microscopic evaluation of the lumbar spine with spinal cord from three (3) ovine treated with the test composition of the present application following laminectomy of the L3 and L5 levels and euthanized at 30, 60, or 90 days demonstrated that all tissue reactions at all operative laminectomy sites were consistent and showed normal progression of healing across timepoints. At the surgical access sites (all tissue dorsal to the epidural space), healing was characterized by fibrosis that tended to be replaced by bridging new bone, and minimal residual inflammatory cells, as well as minimal or mild amounts of residual test composition associated with minimal numbers of macrophages, multinucleated giant cells, and rare lymphocytes. The level of fibrosis was as expected in the surgical model and did not appear to be adversely affected by the test composition.

[0089] Further, the amount of residual test composition decreased between each time interval, indicating progressive degradation. The pattern of absorption associated with the test composition was relatively benign and associated with minimal or mild numbers of macrophages, multinucleated giant cells, and rare lymphocytes. Changes related to the test composition were shown to be limited to low grade lymphocyte, macrophage, and multinucleated giant cell infiltrates with no effect on healing, suggesting excellent biocompatibility in the ovine model. In addition, as to tissue response, fibrosis in the epidural space was scored as minimal to mild, with no evidence of excessive fibrosis in reaction to the test composition.

[0090] Significantly, the epidural test composition was detected at the 30- and 60-day time intervals, but there was no evidence of residual test composition in the epidural space at 90 days. Additionally, there was no evidence of ante-mortem compression-related injuries noted in the spinal cord, and there was no test composition detected in the spinal cord or subdural spaces. Overall, treatment of the ovine lumbar spine with the test composition, after laminectomy and a survival period of 30-, 60- or 90-days under the conditions of this study, showed excellent biocompatibility, advanced and nominal healing, and no adverse findings or safety concerns.

Claims

CLAIMSWhat is claimed is:

1. A method for hemostasis during surgery in a patient in need thereof, the method comprising: applying a flowable and non-swellable composition comprising a hydrophobically- modified chitosan to a surgical site comprising a bleed, and wherein the surgical site is at risk of pressure-related injury.

2. The method of claim 1, wherein the surgical site is at risk of injury due to compression of tissues.

3. The method of claim 1 or 2, wherein the surgery is selected from neurosurgery, orthopedic surgery, brain surgery, ocular surgery, otorhinolaryngological surgery, thoracic surgery, prostate surgery, thyroid surgery, cardiac surgery, vascular surgery, and gynecological surgery.

4. The method of any one of claims 1 to 3, wherein the composition is applied to a site selected from one or more of neurological structure, spinal cord, optic chiasm, spinal column, thecal sac, peritoneal sac, blood vessel, nerve, pulmonary artery, superior vena cava, inferior vena cava, brain tissue, bladder, cavernous nerve, muscle, bone, and joint.

5. The method of any one of claims 1 to 4, wherein the surgery site comprises bleeding selected from one or more of subcutaneous and muscular bleeding, bone bleeding, epidural bleeding, and large blood vessel bleeding.

6. The method of any one of claims 3 to 5, wherein the surgery is spine surgery.

7. The method of claim 6, wherein the patient has one or more of disc herniation, spondylosis of the cervical and / or lumbar spine, cervical myelopathy, and spondylolisthesis.

8. The method of claim 6 or 7, wherein the spine surgery is discectomy, laminectomy, laminotomy, lumbar decompression surgery, arthrodesis surgery, or anterior cervical discectomy.

9. The method of any one of claims 6 to 8, wherein the pressure-related injury is compression of one or more neurological structures, optionally thecal sac compression, spinal cord compression, and nerve compression.

10. The method of any one of claims 3 to 5, wherein the surgery is cardiac surgery or vascular surgery, and the bleed involves a large blood vessel at risk of compression or stenosis, optionally wherein the blood vessel is a pulmonary artery or superior vena cava.

11. The method of any one of claims 3 to 5, wherein the surgery is prostatectomy, and the bleed optionally involves a cavernous nerve.

12. The method of any one of claims 1 to 11, wherein the patient has one or more factors that influence volume of blood loss during surgery selected from advanced age, higher body mass index, presence of osteoporotic bone, neuromuscular scoliosis, bone metastasis, and anti -coagulant therapy.

13. The method of any one of claims 1 to 12, wherein the hydrophobically-modified chitosan comprises hydrocarbon groups attached to the chitosan backbone, optionally through amide linkages.

14. The method of any one of claims 1 to 13, wherein the hydrocarbon groups comprise linear hydrocarbon groups independently selected from C8 to C18 hydrocarbon chains.

15. The method of claim 14, wherein the hydrocarbon group comprise one or more of linear C8, CIO, Cl 2, Cl 4, Cl 6, and C18 hydrocarbon chains.

16. The method of any one of claims 13 to 15, wherein hydrocarbon groups within the range of C8 to C18 are present from about 0.1 mol% to about 10 mol% of available amines on the chitosan backbone.

17. The method of claim 16, wherein hydrocarbon groups within the range of C8 to C18 are present from about 0.5 mol% to about 3mol% of available amines on the chitosan backbone.

18. The method of claim 17, wherein the hydrocarbon groups comprise C8 hydrocarbon chains present from about 0.5 mol% to about 3 mol%, and optionally about 1 mol%, of available amines.

19. The method of any one of claims 13 to 18, wherein the hydrophobic groups further comprise Cl to C4 hydrocarbon groups, and optionally C2 hydrophobic groups, present from about 15 mol% to about 50 mol%, and optionally about 25 mol% to about 45 mol% of available amines.

20. The method of any one of claims 1 to 19, wherein the chitosan is a medium molecular weight chitosan.

21. The method of any one of claims 13 to 20, wherein the hydrophobically-modified chitosan further comprises one or more benzenediol groups, and optionally oxidized forms thereof.

22. The method of claim 21, wherein the one or more benzenediol groups comprise a catechol moiety, and optionally oxidized forms thereof.

23. The method of claim 21 or 22, wherein the benzenediol groups, and optionally oxidized forms thereof, are grafted to the hydrophobically-modified chitosan at a density of from 0.1 mol% to about 15 mol% of available amines, or from about 0.1 mol% to about 10 mol% of available amines, or from about 0.1 mol% to about 8 mol% of available amines, or from about 0.1 mol% to about 2 mol% of available amines, or from about 0.1 mol% to about 1 mol% of available amines.

24. The method of any one of claims 21 to 23, wherein at least some of the benzenediol groups are oxidized to the corresponding quinone.

25. The method of claim 24, wherein from about 10% to about 90% of the benzenediol groups are oxidized, or from about 25% to about 75% of the benzenediol groups are oxidized, or from about 30% to about 60% of the benzenediol groups are oxidized to the corresponding quinone.

26. The method of any one of claims 1 to 25, wherein the flowable composition is a gel.

27. The method of claim 26, wherein the composition is formulated in a dilute organic acid, optionally wherein the organic acid has a pKa of from about 2 to about 5.

28. The method of claim 27, wherein the organic acid is acetic acid, lactic acid, formic acid, or malic acid.

29. The method of claim 27 or 28, wherein the organic acid is present in the composition from about 0.01 M to about 0.4 M, and is optionally acetic acid at about 0.05M.

30. The method of any one of claims 1 to 29, wherein the hydrophobically-modified chitosan is present at about 0.1% to about 5% by weight, and optionally 0.5% to about 2%, or about 0.5% to about 1.5%, or about 1.0% by weight.

31. The method of claim 30, wherein the composition further comprises one or more secondary polymers, such as polymers selected from gelatin, dextran, pectin, alginate, collagen, polyethylene oxide, gellan gum, and polyvinyl alcohol.

32. The method of claim 31, wherein the composition comprises polymer granules, and optionally gelatin granules.

33. The method of claim 32, wherein the composition comprises gelatin granules at 0.1 wt% to about 5 wt%, or from about 0.1 wt% to about 2 wt%, or from about 0.1 wt% to about 1 wt%.

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