Compositions and methods for the treatment of obstructive sleep apnea - Patent Application 20070123333

Abstract

Disclosed herein is a method that allows for treating sleep apnea by removing fatty tissue and increasing collagen production in a patient's upper airway by injecting an ice slurry containing ingredients such as sterile water, a biocompatible surfactant, e.g., glycerin, and saline into a target area. TIFF2024539656000002.tif124170

Description

[Background technology]

[0001] 2. Background of the Invention Obstructive sleep apnea (OSA) is a chronic disease that leads to excessive daytime sleepiness and cognitive impairment, which increases the risk of car accidents.However, the exact pathophysiology of OSA has not yet been elucidated.Therefore, there is an unmet need for targeted therapy that is a minimally invasive and painless method for treatment in this patient population.Here, we disclose a novel method of treating OSA, which reduces upper airway fat and induces new collagen formation in the tongue by cryolipolysis.

[0002] Obesity is the most important risk factor for obstructive sleep apnea (OSA). Obesity is believed to increase the size of the soft tissue structures of the upper airway through various mechanisms, one of which is the direct deposition of fat in these tissues. Increased upper airway adipose tissue, specifically that deposited in the lateral parapharyngeal fat pad, uvula, and tongue, is believed to play a major role in the pathogenesis of sleep apnea. In subjects with sleep apnea, the volume of pharyngeal wall, tongue, and total soft tissue is reported to be larger.

[0003] The amount of visceral fat in the neck is thought to be involved in the hypertrophy of upper airway soft tissue structures, but may not be the most important factor. Factors other than obesity (in addition to age, sex, craniofacial size, and ethnicity) may be important in mediating the increase in the size of the tongue and the lateral pharyngeal wall. Tongue stiffness has been shown to be decreased in patients with OSA compared to age- and BMI-matched controls. This leads to the increased tongue flaccidity and collapsibility seen in patients with OSA, suggesting that there are fundamental differences in the mechanical properties of tongue tissue in patients with OSA and that these changes are not the result of age or obesity alone. The invention disclosed herein includes a new method of inducing new collagen formation in the tongue to reduce tongue flaccidity and improve obstructive sleep apnea independent of fat loss. Summary of the Invention

[0004] [Brief description of the drawings]

[0005] The patent or application file contains at least one drawing originally executed in color.

[0006] [Figure 1] FIG. 1 shows the coding of the collagen content score. [Figure 2A] Figures 2A-B show tissue temperature and histological images of the tongue base of pig #4 after a series of ice slurry injections. Figure 2A shows tissue temperature recorded by a thermocouple embedded in the needle administering the ice slurry injections. Pig #4 was injected with 20 mL of ice slurry three times sequentially, each time the tissue temperature re-elevated to zero degrees. Figure 2B shows histological images from a biopsy of the tongue base two months after ice slurry treatment. The blue color is trichrome stain highlighting new collagen formation in places where fat had been lost. [Figure 2B] Please see the legend to FIG. 2A. [Figure 2C] Figure 2C shows representative ultrasound images of the injection site at the base of the tongue before (left) and after (right) injection of the slurry. The yellow arrow indicates the hyoid bone, the blue arrow indicates the base of the tongue, the red arrow indicates the tip of the injection needle, and the white arrow represents the injected slurry at the base of the tongue. [Diagram 3] Figures 3A-B show tissue temperature and histological images of the tongue base of pig #5 after a series of ice slurry injections. Figure 3A shows tissue temperature recorded by a thermocouple embedded in a needle. The pig was sequentially injected with 20 ml, 17 ml, and 20 ml of ice slurry three times, each time the tissue temperature re-elevated to zero degrees. Figure 3B shows histological images from a biopsy of the tongue base two months after ice slurry treatment. The blue color in Figure 3B highlights new collagen formation. [Figure 4]Figure 4 shows tissue temperature and histology images of the tongue base of pig #6 after a series of ice slurry injections. Figure 4A shows tissue temperature recorded by a thermocouple embedded in a needle. The pig was sequentially injected with 17 ml, 12 ml, 5 ml, 14 ml, 5 ml, and 7 ml of ice slurry six times, each time the tissue temperature re-elevated to zero. Figure 4B shows histology images from a biopsy of the tongue base two months after ice slurry. The blue color in Figure 4B highlights new collagen formation. [Figure 5-1] FIG. 5 shows collagen area measurements and damage scores following ice slurry injection in pigs #4, #5, and #6. [Figure 5-2] This figure shows a continuation of Figure 5-1. [Figure 6] FIG. 6 shows the safety of ice slurry injection into the base of the tongue. FIG. 6A shows the body weights of animals in the ice slurry and RT control groups at baseline and 8 weeks post-injection. FIG. 6B shows a representative gross view of the tongue, with the biopsy site at the base of the tongue depicted in red. FIG. 6C includes representative histological images of the base of the tongue at an untreated site, a site injected with room temperature (RT) control solution, and a site injected with ice slurry. H&E sections (top row) show adipose tissue (white sections) mixed with myofibers (red sections) in similar ratios between untreated tongue tissue and RT control tissue. The treated panels show a decrease in adipose tissue and an increase in fibrosis (pink wavy areas between dark myofibers within the adipose tissue). Trichrome sections (second row from the top) show newly formed collagen stained light blue. The treated panels (far right column) show copious new collagen deposition. Luxol blue sections (third row from the top) show myelinated fibers stained blue. Treatment panels (far right column) show similar levels of myelinated fibers compared to untreated and RT control sections, demonstrating that myelinated fibers are not altered by slurry injection. Neurofilament immunostained sections (fourth row from the top) show neuronal axons in brown. Neuronal axons were preserved in all treatment groups. Scale bars in these images are 100 μm. [Figure 7]Figures 7A-B show histological images of the cervical fat pad and adjacent salivary glands of mice following injection of ice slurry or room temperature control solution into the anterior cervical fat pad. Figure 7A shows representative histological images of the anterior cervical fat pad and adjacent salivary glands 12 weeks after treatment with slurry or room temperature control solution. Scale bar in Figure 7A is 10 μm. Figure 7B shows the body weights of slurry-treated and control mice at baseline and 12 weeks after injection. [Figure 8] Figures 8A-B show axial MRI imaging of the anterior cervical fat pad after ice slurry injection or room temperature control solution injection. Figure 8A shows axial MRI imaging with outlined regions of interest (red) and fat volume change (blue); scale bar is 1 cm. Figure 8B shows anterior cervical fat volume fraction in slurry-treated and control mice at baseline and 12 weeks after injection. Figure 8C shows the change in anterior cervical fat volume fraction per animal weight in slurry-treated and control mice. Age-matched male New Zealand mice were used in this study; n=13-14 in each group. [Figure 9-1] FIG. 9 shows the change from baseline in anterior cervical fat pad volume fraction per mouse body weight between the ice slurry treatment group and the RT control group. [Figure 9-2] This figure shows a continuation of Figure 9-1. [Figure 10] FIG. 10 shows the approach and route of anterior transcervical injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] definition The following are definitions of terms used herein. The first definition provided for a group or term herein applies to that group or term throughout the specification, unless otherwise indicated, either individually or as part of another group. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0008] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. When used in the claims and / or specification with the term "comprising," the use of the words "a" or "an" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0009] As used herein, the term "about" is used herein to mean approximately, roughly, in the region of, or near. When used in conjunction with a numerical range, the term "about" modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify a numerical value by a variance of 20 percent above and below (high and low) the set forth value.

[0010] As used herein, the term "subject" refers to a vertebrate. In one embodiment, the subject is a mammal or a mammalian species. In one embodiment, the subject is a human. In one embodiment, the subject is a healthy human adult. In other embodiments, the subject is a non-human vertebrate, including, but not limited to, non-human primates, laboratory animals, livestock, race horses, domestic animals, and non-domestic animals. In one embodiment, the term "human subject" refers to a population of healthy human adults.

[0011] As used herein, the term "patient" refers to a human or animal.

[0012] The term "mammal" includes, but is not limited to, a human, a mouse, a rat, a guinea pig, a dog, a cat, a horse, a cow, a pig, or a non-human primate, such as a monkey, a chimpanzee, a baboon, or a rhesus monkey. In one embodiment, the mammal is a human.

[0013] Detailed Description of the Invention There is currently no injectable device capable of reducing upper airway fat or increasing new collagen formation that is completely physiological and non-toxic to surrounding tissues. The present disclosure provides a method that is physiological and biocompatible because the composition used contains components such as sterile water, a biocompatible surfactant, e.g., glycerin, and saline. Furthermore, the composition used in the method disclosed herein is injectable, thus providing a composition that infiltrates the target area. Thus, precise precision regarding the location of the injection is not required, instead, injection in the vicinity of the target area is effective. Furthermore, due to the injectability and infiltrative nature of the slurry, the completion of this procedure takes only a few minutes, whereas other tongue reduction procedures, such as the application of topical cooling to the tongue surface or surgical removal of tongue tissue (which also requires the use of general anesthesia), take several hours. Thus, the disclosed method allows for the treatment of obstructive sleep apnea in a few minutes in a clinic.

[0014] The method disclosed herein does not damage surrounding tissue and is adipose tissue selective. The method disclosed herein has a series of injections and tissue cooling duration that results in collagen increase. Furthermore, the method disclosed herein does not induce scarring or damage to important surrounding tissue. Thus, the present disclosure provides a method for treating sleep apnea by injecting ice slurry into target area to remove adipose tissue and increase collagen production in the upper airway of a patient.

[0015] The methods and disclosures herein provide many unexpected results. In one embodiment, the disclosed ice slurry can be injected from an anterior transcervical approach (as shown in FIG. 10). This is advantageous because the fatty tongue tissue is located approximately 2-4 cm from the site of the patient's neck where the needle is inserted, allowing easy access to this injection target. In another embodiment, the ice slurry may be injected via other routes, for example, from the superior surface of the tongue. In another embodiment, ultrasound may be used to identify the patient's anatomy and the location of the injection. Ultrasound also allows visualization of the location of the ice spheres after injection. Ultrasound guidance may increase the safety of such injections.

[0016] In one embodiment, the method disclosed herein provides the advantage of allowing as much as 60 mL of ice slurry to be injected into the target site. In a preferred embodiment, 60 mL of ice slurry is injected in a series of 5 mL, 10 mL, or 20 mL volumes. The structure of the tissue at the base of the tongue, which is a mixture of fat and muscle tissue, allows the slurry to infiltrate and spread through the tissue. This is in contrast to the injection of slurry into other denser tissues, which may result in ice balls forming at the site of injection. This amount of slurry that can be injected allows for a distributed treatment of the entire base of the tongue.

[0017] In one embodiment, the method disclosed herein provides the advantage of not damaging other tissues in the tongue. In a preferred embodiment, the method disclosed herein does not damage the nerves located in the tongue. This is due in part to the injection of ice slurry into the center of the tongue. In one embodiment, the method disclosed herein provides the advantage of having an easy injection route that reduces patient discomfort. By accessing the injection site through the patient's neck, the physician can easily and quickly inject ice slurry without causing patient nausea.

[0018] Non-Limiting Aspects of the Subject Matter An embodiment of the present invention provides an injectable ice slurry that can be used to treat obstructive sleep apnea by reducing upper airway adipose tissue and / or increasing collagen formation in the tongue. Such a slurry can target and destroy desired tissue through heat extracted from adjacent tissue during melting of the ice portion of the slurry.

[0019] In one non-limiting embodiment, the ice slurry is injected around the upper airway targeted for the treatment of obstructive sleep apnea. In one embodiment, the injection of ice slurry around the upper airway reduces upper airway adipose tissue. In one embodiment, the slurry is injected through a needle inserted via an anterior transcervical approach. In one embodiment, the slurry is injected without puncturing the surface of the tongue. In one embodiment, the slurry is injected through a needle inserted into the tongue of the patient under the jaw of the patient.

[0020] Fat hypertrophy around the upper airway is a newly proposed contributing factor to obstructive sleep apnea (OSA), specifically upper airway obstruction in obese patients. OSA causes substantial morbidity and mortality. It has been reported that fat can be selectively targeted and removed by tissue cooling, which has led to a popular non-invasive alternative to liposuction. Recently, we developed a biocompatible injectable ice slurry composed of saline and glycerin as a novel method to remove subcutaneous fat. Fat deposits in the neck result in increased neck circumference that shows a strong correlation with the severity of apnea in patients. New Zealand obese (NZO) mice have an increased volume of neck fat around the upper airway and have been used as a translational model of OSA. This study demonstrated the safety and efficacy of injectable ice slurry in selectively destroying neck fat deposits in NZO mice.

[0021] In certain aspects, the sterile ice slurry reduces upper airway adipose tissue. In some embodiments, the ice slurry comprises sterile water and ice particles. In one embodiment, the ice particles in the sterile ice slurry have a maximum cross-sectional diameter of less than 2 millimeters.

[0022] The sterile ice slurry is cooled to a predetermined temperature. In one embodiment, the predetermined temperature is between -10°C and 0°C. In one embodiment, the desired tissue region includes upper airway adipose tissue. In one embodiment, the upper airway adipose tissue is an anterior cervical fat pad. In one embodiment, injection of the sterile ice slurry reduces the volume of the anterior cervical fat pad.

[0023] In another embodiment, the upper airway adipose tissue is tongue fat. In a further embodiment, the tongue fat is the base of the tongue adipose tissue. In one embodiment, the injectable slurry comprises a plurality of sterile ice particles and one or more freezing point depressants. The freezing point depressant can also change the viscosity of the slurry, prevent the ice particles from clumping together, increase the thermal conductivity of the liquid phase, and otherwise improve the performance of the slurry. In one embodiment, the injection of the sterile ice slurry reduces the volume of tongue fat.

[0024] The size of the ice particles can be adjusted to ensure that the slurry can be injected into a subject through a needle or catheter. The slurry is injectable when all or a majority of the ice particles (e.g., more than about 50% by number, more than about 75% by number, more than about 80% by number, more than about 90% by number, more than about 95% by number, more than about 99% by number, etc.) have a maximum cross-sectional dimension (i.e., the maximum distance between any two points on the surface of the ice particles) that is less than half the inner diameter of the container (e.g., needle, cannula, catheter, tube, etc.) used. For example, when the slurry is injected using a catheter with an inner diameter of 3 mm, the ice particles will preferably have a maximum cross-sectional dimension that is less than or equal to about 5 1.5 mm. In some embodiments, the ice particles have an average maximum cross-sectional dimension of 1 mm or less.

[0025] One or more freezing point depressants can be mixed with the ice to form a slurry below 0° C. that remains injectable. Suitable freezing point depressants include biocompatible compounds such as salts (e.g., sodium chloride), Ringer's lactate, dextrose, biocompatible surfactants such as glycerol (also known as glycerin or glyceline), other polyols, other sugar alcohols, and / or urea. In particular, biocompatible surfactants such as glycerin are believed to cause the ice particles to shrink and become rounder, and also function as cryoprotectants for cells that are not lipid rich. Other exemplary biocompatible surfactants include sorbitan esters of fatty acids, polyoxyethylene sorbitan monooleate (also known as polysorbate 80 and available under the trade name TWEEN® 80 from Croda Americas LLC, New Castle, Delaware), sorbitan monooleate polyoxyethylene sorbitan monolaurate (also known as polysorbate 80 and available under the trade name TWEEN® 80 from Croda Americas LLC, New Castle, Delaware), lecithin, polyoxyethylene-polyoxypropylene copolymers (available under the trade name PLURONICS® from BASF Corporation, Mount Olive, New Jersey), sorbitan trioleate (available under the trade name SPAN® 85 from Sigma-Aldrich, St. Louis, Missouri), and the like. The injectable slurry can be configured to have a desired temperature and to extract a desired amount of heat per unit of slurry volume or mass. Specifically, the solute (ie, freezing point depressant) concentration dictates the temperature of the slurry, and the ice content of the slurry determines the amount of heat extracted by the slurry.

[0026] For selective disruption slurries that target the relative vulnerability of lipid-rich cells, the slurries are preferably isotonic with respect to the target cells. For example, a slurry containing saline and 20% glycerin could target lipid-rich cells while avoiding acute non-selective necrosis. A broad disruption slurry can achieve lower temperatures and greater disruptive power by increasing solute concentration (e.g., up to 20% w / v saline) to form a hypertonic solution that also disrupts cells through osmotic pressure. As ice melts, solute concentration decreases.

[0027] The injectable slurry can contain varying percentages of ice. For example, the slurry can contain about 10% to about 5% ice by weight, about 10% to about 20% ice by weight, about 20% to about 30% ice by weight, about 30% to about 40% ice by weight, about 40% to about 50% ice by weight, about 50% to about 60% ice by weight, about 60% to about 70% ice by weight, and more than about 50% ice by weight (percentages by volume are similar due to the densities of solid and liquid water).

[0028] Method for preparing a slurry The slurry can be prepared using a variety of methods. Any known method of making an ice slurry is contemplated herein.

[0029] In one embodiment, the slurry is prepared using a commercially available ice slurry generator, such as that available under the trade name MODUPAK™ DEEPCHILL™ from Sunwell Technologies Inc. (Woodbridge, Ontario). Commercially available slurry generators include scraped surface generators, in which small ice crystals are scraped (e.g., by blades, augers, brushes) from a cooled surface and mixed with water, direct contact generators, in which an immiscible primary refrigerant is evaporated to supersaturate the water and form small, smooth crystals, and super cooling generators, in which water is supercooled and discharged through a nozzle into a storage tank.

[0030] Slurry storage and further processing Both the slurries and precursor ice particles described herein can be stable for several years if kept below the freezing point of the solution or ice particles. To prevent ice crystal growth or agglomeration, it is preferable to store the slurry at a temperature below the freezing point and then reheat the slurry to the desired injection temperature.

[0031] Any of the above methods may be performed by a single practitioner at a single time and location, or by one or more practitioners at one or more time and locations. For example, small stable ice particles may be packaged and shipped using standard low-temperature shipping methods and stored in a standard freezer (e.g., at -20°C). The ice particles may be combined with one or more additional additives at the clinic shortly before or immediately prior to injection. As described in more detail herein, the additives may be, for example, a biocompatible solution, may contain a biocompatible surfactant such as glycerin, and may be pre-cooled (e.g., to a temperature close to the desired temperature of the slurry at the time of injection).

[0032] Slurry Delivery The injectable slurry may be introduced using a variety of parenteral delivery systems and techniques, including gravity flow, injection with a syringe, cannula, catheter, tube, and / or pump, etc. The regulating device can regulate the flow rate, volume, and / or pressure of the injected slurry to extract the desired amount of heat from the tissue adjacent the injection site.

[0033] Optionally, imaging techniques such as ultrasound, magnetic resonance, x-ray, etc. can be utilized to confirm proper positioning of the injection device and / or slurry. Specifically, ice is an extremely strong reflector of ultrasound, whereas lipid-rich cells are a poor reflector of ultrasound.

[0034] This method may be repeated one or more times at the same injection site and / or target tissue. Multiple injections may be performed sequentially, overlapping, or in parallel.

[0035] In one embodiment, the sterile ice slurry is delivered to the desired tissue area via a pump and the melted slurry is aspirated from the desired tissue area, hi another embodiment, the ice slurry is injected from a syringe through a needle into the desired target area.

[0036] In certain aspects, the sterile ice slurry increases new collagen formation in the tongue. In other certain aspects, the sterile ice slurry reduces the amount of adipose tissue present in the tongue. In one embodiment, the ice slurry comprises water and ice particles. In one embodiment, the ice particles in the sterile ice slurry have a maximum cross-sectional diameter of less than 2 millimeters.

[0037] The sterile ice slurry is cooled to a predetermined temperature. In one embodiment, the predetermined temperature is between -10°C and 0°C. In one embodiment, the desired tissue area includes the tongue. In one embodiment, the injectable slurry includes a plurality of sterile ice particles and one or more freezing point depressants. The freezing point depressants can also change the viscosity of the slurry, prevent agglomeration of the ice particles, increase the thermal conductivity of the liquid phase, and otherwise improve the performance of the slurry.

[0038] In one embodiment, the ice slurry injection is repeated one or more times for the same injection site and / or target tissue. In a preferred embodiment, the injection is repeated 1-6 times for the same injection site and / or target tissue. In one embodiment, the volume of ice slurry for each injection is 5ml-50ml. In one embodiment, the injection of sterile ice slurry hardens the tongue by increasing collagen production. The injection of sterile ice slurry can also reduce adipose tissue in the tongue. EXAMPLES

[0039] In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic examples described herein are provided to illustrate the compounds and methods provided herein, and should not be construed as limiting the scope thereof in any way.

[0040] Example 1 - Ice slurry induced new collagen formation in tongue biopsy sites and histological staging by a pathologist The animal study was approved by the Massachusetts General Hospital (MGH) IACUC, and animals were housed at MGH in accordance with animal care regulations. A total of six 4-6 month old Yorkshire sows were used in this study. Four Yorkshire pigs in the test group were injected with ice slurry (-6°C) at the base of the tongue, and two control pigs were injected with room temperature "slurry solution." A slurry composed of saline (0.9% sodium chloride) and 10% glycerin was used as previously described. Under general anesthesia, ultrasound (US) guidance was used to pass a 14g needle from the anterior neck into the base of the tongue. Anatomical landmarks in the pig model included the posterior mandible and hyoid bone. The needle was inserted just above the hyoid bone and guided into the left or right base of the tongue, noting tissue displacement as the needle passed perpendicular to the probe. A laryngoscope and a rigid 0-degree laryngeal endoscope were used to confirm needle placement at the base of the tongue. A total of 60 ml of ice slurry or RT control was injected by continuous injection. Tissue temperature at the site of injection was recorded using a thermocouple (Figure 2C). Animals were observed clinically for 2 months after injection for possible adverse effects. At 2 months after treatment, animals were sacrificed and tongue tissue was harvested for gross and histological analysis. Multiple biopsy specimens were collected from the base of the tongue. Biopsies were fixed in 10% formalin, embedded in paraffin, sectioned at 5 μm, and then stained with hematoxylin and eosin (H&E). Trichrome staining was used to investigate collagen deposition. Immunohistochemistry for neurofilament and special luxol blue staining were performed to study nerve axon and myelin structure. A board-certified pathologist blindly assessed the biopsy specimens to evaluate histologic changes in adipose tissue, muscle, nerve, blood vessels, salivary glands, lymphatic tissue, and tongue epithelium.

[0041] A study in four pigs has demonstrated that injection of ice slurry is safe and alters collagen content in the tongue, as shown by examination of gross and histological imaging of the tongue; as well as collagen content scores in the treated side of the tongue compared to the untreated side (see Figures 1-6). The study demonstrated an increase in collagen content in the tongue following ice slurry administration.

[0042] Ice slurry treatments were delivered as follows: Under brief anesthesia with inhaled isoflurane (1–3% with 1–1.5 l / min oxygen), a total of 60 ml of approximately −6°C ice slurry (0.9% sodium chloride with 10% glycerin) or 60 ml of room temperature slurry (0.9% sodium chloride with 10% glycerin) was injected into the pig tongue using a needle-tipped syringe. Pigs received multiple sequential injections of 5–20 ml of ice slurry or room temperature solution per injection. The next injection was delivered when tissue temperature had re-elevated to zero degrees after the previous injection. A 14- or 15-gauge hypodermic needle was used for injection. Twelve different sites at the tongue base were biopsied for histological analysis (see Figure 6B showing the location of the biopsies). A semiquantitative score was used by a board-certified pathologist to blindly quantify collagen content in all 12 biopsies from each pig (Figure 1A).

[0043] Thermocouple recordings demonstrated that tissue temperatures below 0°C for several minutes were achieved, sufficient to obtain selective fat loss (Figure 2A). US imaging was used to confirm the position of the injection needle and the formation of an ice ball at the base of the tongue (Figure 2C).

[0044] As shown in Figures 2A-B and 3A-B, tissue temperatures were recorded by thermocouples embedded in needles 2 months after injection of ice slurry into the tongues of pigs #4 and #5. As shown in Figures 2A-B, tissue temperatures were recorded to test the rate of tissue cooling and the duration of tissue temperatures below 0°C after three injections of 20 ml of ice slurry into the base of the tongue of pig #4. Tissue samples collected from the tongues 2 months after ice slurry treatment were stained with trichrome to highlight collagen in blue, and these samples were semiquantitatively scored for new collagen formation. Thermocouple recordings demonstrated that tissue temperatures below 0°C for several minutes were achieved (Figure 1A), which is sufficient to obtain selective fat loss (7). US imaging was used to confirm the position of the injection needle and ice ball formation at the base of the tongue (Figure 1B). As shown in Figures 3A-B, tissue temperatures were recorded to test the rate of tissue cooling and duration of tissue temperature below 0°C after three sequential injections of 20 ml, 17 ml, and 20 ml of ice slurry into the base of the tongue of pig #5. Tissue samples collected from the tongue at 2 months after ice slurry treatment were stained with trichrome to highlight collagen in blue, and the samples were semiquantitatively scored for new collagen formation. As shown in Figures 4A-B, tissue temperatures were recorded to test the rate of tissue cooling and duration of tissue temperature below 0°C after six sequential injections of 17 ml, 12 ml, 5 ml, 14 ml, 5 ml, and 7 ml of ice slurry into one side of the tongue of pig #6. Tissue samples collected from the tongue at 2 months after ice slurry treatment were stained with trichrome to highlight collagen in blue, and the samples were semiquantitatively scored for new collagen formation.

[0045] The data presented in Figures 2-4 quantitatively show that the tongue tissue of pigs #4 and #5 injected with ice slurry was cooled to below 0°C after every injection by graphing the thermocouple recordings, and the duration that the tissue temperature was below 0°C was extended after each injection (Figures 2A, 3A, 4A). Pig #4 and pig #5 had the longest duration that the tissue temperature was below 0°C, while pig #6 had the shortest duration of tissue temperature below 0°C. The data presented in Figures 2-5 also show new collagen formation with ice slurry injection, indicated by blue highlighting, by semi-quantitative histological grading (Figures 2B, 3B, 4B, 5). The blue color of the trichrome stain indicated collagen. Pig #4 and pig #5 had the most collagen formation, while pig #6 had little to no new collagen formation, indicating that the temperature and duration of cooling were important. The location of collagen formation was at the base of the tongue where the injection was made, indicating that the collagen formation was the result of the slurry injection and not a non-specific reaction.

[0046] Injection of ice slurry into the base of the tongue was performed without complications. There was no evidence of tongue necrosis or airway obstruction following injection in either the treatment or control arms. Animals maintained normal weight gain following treatment, suggesting that feeding, which requires use of the tongue, was not altered (Figure 6A). Gross examination of tongue tissue specimens 2 months after treatment showed no scarring or trauma (Figure 6B). Histological analysis from the base of the tongue showed no damage to surrounding muscle, nerves, or blood vessels. Immunohistochemistry for neurofilament and luxol blue special staining showed normal nerve structure, axons, and myelin. Histological evidence of selective fat loss with new collagen deposition was observed in the tongue tissue (Figure 6C). No signs of inflammation, scarring, or necrosis were observed.

[0047] Example 2 - Ice slurry induced anterior cervical fat pad size changes Experiments in mice demonstrate that injection of ice slurry significantly reduces anterior neck fat in the slurry-injected group compared to control animals, as shown by histological images of the cervical fat pads and adjacent salivary glands (Figure 7) as well as axial MRI imaging of the anterior neck (Figures 8, 9). This experiment demonstrated a reduction in anterior neck fat volume following ice slurry administration that was independent of whole-body body weight changes.

[0048] Twenty-week-old male NZO mice were housed in the Massachusetts General Hospital animal facility according to regulations. Baseline MRI imaging was performed before treatment to obtain adipose tissue volume measurements. Sixteen mice in the test group were injected into the anterior cervical fat pad with 1.0 ml of cold (-3°C to -4.8°C) ice slurry composed of saline (0.9% NaCl) + 10% glycerin. The slurry was made in a sterile blender (HGB150, Waring Commercial, Torrington, CT) while the solution was cooled below the freezing point and injected through a 15-gauge needle. In addition to slurry injection, mice in the treatment group were subjected to local cooling. The anterior neck was placed on a bed of ice slurry for 10 min prior to injection and again for 10 min immediately after slurry injection. Fourteen mice in the control group were injected with the same volume and solution composition at room temperature. The same treatment procedure was repeated 8 weeks later, and MRI imaging was acquired at the end of the study 12 weeks later.

[0049] Mice were imaged on a Bruker Pharmascan 4.7 Tesla MRI. Axial and coronal rare T1 (rare factor: 4, Tr: 900ms, Te: 13.59ms, matrix: 256x256x16 with 0.156x0.156x0.5mm voxels) and axial Dixon (Fa: 80 degrees, Tr: 4.0 / 4.8ms, Te: 500ms, same geometry) sequences were performed at pre-treatment baseline and 1 month after the second treatment. Timepoint T1 images were registered using a region of interest difference similarity measure based on binary images of the brain sections. Fat was isolated using Dixon images registered using affine transformation parameters obtained from the T1 images. Using skull structures as landmarks, anterior cervical 3D regions of interest for volumetric analysis were manually drawn by blinded observers anterior and lateral to the salivary glands, and isolated adipose tissue obtained from Dixon images was quantified from the regions. Observers performed image analysis blinded using Amira (Thermo Scientific™) and Matlab (The Mathworks) software. Tissue biopsies from the treated cervical areas were obtained at the end of the study for histological analysis.

[0050] Statistical analysis was performed using Prism 8 (GraphPad Software, Inc., La Jolla, CA). A paired two-tailed Student's t-test was used to compare the body weight and neck fat fraction of animals in each group before and after treatment. A multiple Student's t-test with adjusted p-value was used to compare the body weight of the test and control groups before and after treatment. The measured anterior cervical fat pad volume fraction of each animal was divided by the body weight at the time of MRI scan (Appendix 1). A two-tailed Student's t-test was used to compare the change in anterior cervical fat pad volume fraction from baseline per body weight between the treatment and control groups. A P value of less than 0.05 was considered significant. All bar graphs show the mean + SEM.

[0051] As shown in Figures 7A-B and 8A-C, histological analysis of samples taken 12 weeks after treatment showed no scarring, ulceration, or nonspecific damage to tissues surrounding the treatment site, including salivary tissue (Figure 6A). In both control and test mouse groups, body weight at 12 weeks was significantly increased compared to baseline (control group 52.11 x 10-3 kg ± 1.23 x 10-3 kg vs. 58.93 x 10-3 kg ± 1.70 x 10-3 kg at 12 weeks, p<0.01 by paired two-tailed Student's t-test; test group 53.18 x 10-3 kg ± 0.94 x 10-3 kg vs. 59.79 x 10-3 kg ± 1.77 x 10-3 kg at 12 weeks, p<0.01) (Figures 6B and 8). There were no significant differences in body weight between the treatment and control groups at baseline or at 12 weeks.

[0052] As shown in Figure 7C,E using MRI imaging, control mice showed a significant increase in anterior cervical fat pad volume per cervical tissue volume in the region of interest at 12 weeks after the first treatment compared to baseline (44.73% ± 2.00% at baseline vs. 54.35% ± 2.89% at 12 weeks; p < 0.01 by paired two-tailed Student's t-test) (Figure 6C and D and Figure 8). In contrast, mice in the treatment group showed no increase in cervical fat pad volume at 12 weeks compared to baseline (46.94% ± 2.66% at baseline vs. 46.31% ± 2.43% at 12 weeks; p = 0.78 by paired two-tailed Student's t-test) (Figure 6C and D and Figure 8). The difference between the treatment and control groups in anterior cervical fat pad volume fraction change from baseline per body weight was significant (-1.09 / kg ± 0.33 / kg vs. 0.68 / kg ± 0.37 / kg; p < 0.01 by two-tailed Student's t-test) (Figure 6E and Figure 8).

[0053] This data supports that ice slurry injection can be used as a novel, minimally invasive method to reduce excess upper airway adipose tissue and increase collagen production without damage to important surrounding tissues. The procedure can be performed quickly and easily in minutes by inserting a needle via an anterior transcervical approach.

Claims

1. 1. A composition comprising a sterile ice slurry comprising water and ice particles for use in a method for treating obstructive sleep apnea by inducing new collagen formation in the tongue, the method comprising: cooling the composition to a predetermined temperature; and repeatedly injecting the composition into a desired tissue area, the desired tissue area including the tongue. A composition comprising:

2. 2. The composition of claim 1, wherein the predetermined temperature is between -10°C and 0°C.

3. 10. The composition of claim 1, wherein the number of ice slurry injections is 1 to 6 injections.

4. 4. The composition of claim 3, wherein the volume of the ice slurry for each injection is between 5 ml and 50 ml.

5. 10. The composition of claim 1, wherein the sterile ice slurry further comprises a biocompatible surfactant.

6. 6. The composition of claim 5, wherein the biocompatible surfactant is glycerin.

7. injecting the composition into the desired tissue area; delivering the sterile ice slurry to the desired tissue area via a pump; and Aspirating the thawed slurry from the desired tissue area.

10. The composition of claim 1, comprising:

8. injecting the composition into the desired tissue area; delivering the sterile ice slurry to the desired tissue area via a syringe; and Aspirating the thawed slurry from the desired tissue area.

10. The composition of claim 1, comprising:

9. injecting the composition into the desired tissue area; inserting a catheter having an injection port and an aspiration port into the desired tissue region; delivering the sterile ice slurry to the desired tissue area through the injection port; and Aspirating the melted ice slurry from the desired tissue area through the suction port.

10. The composition of claim 1, comprising:

10. injecting the composition into the desired tissue area; inserting a balloon-based catheter into or around the desired tissue region; and recirculating the sterile ice slurry within the balloon-based catheter.

10. The composition of claim 1, comprising:

11. 10. The composition of claim 1, wherein the ice particles in the sterile ice slurry have a maximum cross-sectional diameter of less than 2 millimeters.

12. 10. The composition of claim 1, wherein the tongue is cooled to below 0°C after injection of the composition.

13. 13. The composition of claim 12, wherein the duration of the below zero tongue temperature is at least 5 minutes.

14. 4. The composition of claim 3, wherein each re-injection is performed when the tissue temperature has risen back to 0°C.

15. 10. The composition of claim 1, wherein injection of the composition induces new collagen formation in the tongue.

16. The composition of claim 1, wherein injection of the composition hardens the tongue.

17. 1. A composition comprising a sterile ice slurry comprising water, ice particles, and glycerin for use in a method for treating obstructive sleep apnea by selectively reducing upper airway adipose tissue, the method comprising: cooling the composition to a predetermined temperature; and injecting the composition into a desired tissue area, wherein the desired tissue area comprises upper airway adipose tissue. A composition comprising:

18. 18. The composition of claim 17, wherein the upper airway adipose tissue is an anterior cervical fat pad.

19. 18. The composition of claim 17, wherein the upper airway adipose tissue is tongue fat.

20. 20. The composition of claim 19, wherein the tongue fat is the base of the tongue adipose tissue.

21. 18. The composition of claim 17, wherein the predetermined temperature is between -10°C and 0°C.

22. 18. The composition of claim 17, wherein the sterile ice slurry further comprises a biocompatible surfactant.

23. 23. The composition of claim 22, wherein the biocompatible surfactant is glycerin.

24. injecting the composition into the desired tissue area; delivering the sterile ice slurry to the desired tissue area via a pump; and Aspirating the thawed slurry from the desired tissue area.

18. The composition of claim 17, comprising:

25. injecting the composition into the desired tissue area; delivering the sterile ice slurry to the desired tissue area via a syringe; and Aspirating the thawed slurry from the desired tissue area.

18. The composition of claim 17, comprising:

26. injecting the composition into the desired tissue area; inserting a catheter having an injection port and an aspiration port into the desired tissue region; delivering the sterile ice slurry to the desired tissue area through the injection port; and Aspirating the melted ice slurry from the desired tissue area through the suction port.

18. The composition of claim 17, comprising:

27. injecting the composition into the desired tissue area; inserting a balloon-based catheter into or around the desired tissue region; and recirculating the sterile ice slurry within the balloon-based catheter.

18. The composition of claim 17, comprising:

28. 18. The composition of claim 17, wherein the ice particles in the sterile ice slurry have a maximum cross-sectional diameter of less than 2 millimeters.

29. 18. The composition of claim 17, wherein the anterior cervical fat pad is cooled after injection of the composition.

30. 18. The method of claim 17, wherein the tongue is cooled after injection of the composition.

31. 18. The composition of claim 17, wherein injection of the composition reduces the volume of the anterior cervical fat pad.

32. 18. The composition of claim 17, wherein injection of the composition reduces the volume of the tongue fat.

33. 30. The composition of claim 29, wherein the temperature of the anterior cervical fat pad is reduced to a temperature of about -10°C to about 0°C.

34. 31. The composition of claim 30, wherein the temperature of the tongue fat is reduced to a temperature of about -10°C to about 0°C.

35. injecting the composition into the desired tissue area; Injecting the sterile ice slurry directly into the tongue or cervical fat pad tissue.

18. The composition of claim 17, comprising:

36. 1. A composition comprising a sterile ice slurry comprising water and ice particles for use in a method for treating obstructive sleep apnea by inducing new collagen formation in the tongue, the method comprising: cooling the composition to a predetermined temperature; and repeatedly injecting the composition into a desired tissue area, the desired tissue area comprising the upper respiratory tract. A composition comprising:

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