Patent Grant
9682099
The present disclosure relates generally to compositions and methods for treating joints.
Osteoarthritis (“OA”), the most common form of arthritis, is a type of arthritis that is characterized by degenerative (gradual deterioration of joint) or abnormal changes in bone, cartilage, and synovium of the joints. OA is often characterized by a progressive wearing down of opposing joint surfaces accompanied at times by inflammation resulting in pain, swelling, and stiffness for the patient. OA can occur in one or more joints following trauma to the joint, following an infection of the joint, or simply as a result of aging. Furthermore, there is emerging evidence that abnormal anatomy may contribute to early development of OA. In the US alone, over 25 million people are estimated to have radiographic OA of the knee, with 16 million of those suffering from symptomatic OA. The prevalence of OA is increasing rapidly, in part due to an aging population that is more active, as well as an increasingly obese general population.
Treatment of OA generally involves a combination of exercise or physical therapy, lifestyle modification, and analgesics. Acetaminophen is typically the first line of treatment for OA. For mild to moderate symptoms, effectiveness is similar to non-steroidal anti-inflammatory drugs (“NSAIDs”), such as ibuprofen. For more severe symptoms, NSAIDs may be more effective. However, while more effective, NSAIDs in severe cases are associated with greater side effects such as gastrointestinal bleeding and renal complications. Another class of NSAIDs, COX-2 selective inhibitors (such as Celecoxib), is equally effective as NSAIDs but no safer in terms of side effects. There are several NSAIDs available for topical use, including diclofenac. Typically, they have less systemic side-effects than oral administration and at least some therapeutic effects. While opioid analgesics, such as morphine and fentanyl, improve pain, this benefit is outweighed by frequent adverse events and thus they are not routinely used.
Intra-articular steroid injections are also used in the treatment of OA, and they are very effective at providing pain relief. However, the durability of the pain relief is limited to 4-6 weeks and there are adverse effects that may include collateral cartilage damage. If pain becomes debilitating, joint replacement surgery may be used to improve mobility and quality of life. There is no proven treatment to slow or reverse the disease.
For patients who do not get adequate pain relief from simple pain relievers, like acetaminophen or from exercise and physical therapy, intra-articular injections of hyaluronic acid (HA) provide another treatment option to address symptomatic pain and delay the need for a total joint replacement surgery. It is known that the concentration of native HA is deficient in individuals suffering from OA and therefore joint injections of exogenous HA is believed to replenish these molecules and restore the viscoelastic properties of synovial fluid. It is this property that is responsible for lubricating and cushioning the joints. There is also evidence that HA has biological activity through binding to cell surface receptors and may have a role in mitigating inflammation. Independent of the mechanism of action, pain relief is observed for about six months following a treatment course. A treatment course for HA products on the US market can range from single injection product to others that require 3 to 5 weekly injections to attain this durability of pain relief. In using intra-articular injections, it is well-known that a neutral pH and an isotonic solution are preferred to avoid pain with injection and joint or tissue damage. The desire for a neutral and isotonic solution can limit the type and concentration of drugs that can be injected.
While the above therapies can provide at least a partial relief of OA pain, there are no approved therapies that can slow or halt the progression of the disease in humans. Accordingly, there remains a need for improved methods and compositions for treating OA in joints, and to address the pain and structural degeneration associated with OA.
Provided herein are compositions and methods for treating joint conditions, such as osteoarthritis and/or the pain associated therewith. In one embodiment, a composition for treating joints is provided and includes a first component hyaluronic acid (HA), a second component, glucosamine, and a third component, chondroitin sulfate. The composition can have a pH in the range of about 3-5 and an osmolality in the range of about 330-750 mOSM. The composition can be injectable. It is surprising that the most effective injectable solution for treating joints is both acidic and hypertonic, both of which would be expected to cause pain, irritation, and inflammation. As demonstrated herein, hyaluronic acid combined with glucosamine and chondroitin sulfate overcomes the pain, irritation and inflammation typically associated with an injection of low pH and high osmolality used herein.
The concentration of the components can vary. In one embodiment, glucosamine is present in a concentration of about 0.005-54 mg/mL. In one embodiment, chondroitin sulfate is present in a concentration of about 0.005-54 mg/mL. In one embodiment, hyaluronic acid is present in a concentration of about 3.6-36 mg/mL. In one embodiment, when the components are combined to form a composition, the glucosamine and chondroitin sulfate are present within the composition at a ratio of about 1:1 by weight.
In one exemplary embodiment, the composition includes about 18-20 mg/mL of glucosamine, about 18-20 mg/mL of chondroitin sulfate, about 12-17.5 mg/mL of hyaluronic acid; and the composition has a pH value of 3.5-4 and an osmolality of about 600-650 mOSM.
The composition can also include an additional component, such as a stabilizer. In another embodiment, the additional component can be trehalose.
In other aspects, a method for treating joints is provided and includes injecting a therapeutically effective amount of a formulation into a joint of a subject, where the formulation comprises hyaluronic acid (HA), glucosamine, and chondroitin sulfate. The formulation can have a pH of about 3-5 and an osmolality of about 330-750 mOSM. In an exemplary embodiment, the HA is combined with a mixture of glucosamine and chondroitin sulfate no more than about 60 minutes prior to injection, and more typically, no more than about 30 minutes prior to injection.
The concentration of the components in the formulation used in the method can vary. In one embodiment, glucosamine is present in a concentration of about 0.005-54 mg/mL. In one embodiment, chondroitin sulfate is present in a concentration of about 0.005-54 mg/mL. In one embodiment, hyaluronic acid is present in a concentration of about 3.6-36 mg/mL. In one embodiment, when the components are combined to form a formulation, the glucosamine and chondroitin sulfate are present within the formulation at a ratio of about 1:1 by weight.
In one exemplary embodiment, the formulation includes about 18-20 mg/mL of glucosamine, about 18-20 mg/mL of chondroitin sulfate, about 12-17.5 mg/mL of hyaluronic acid; and the composition has a pH value of 3.5-4 and an osmolality of about 600-650 mOSM.
The formulation can also include an additional component, such as a stabilizer. In another embodiment, the additional component can be trehalose.
In one embodiment, a kit for treating joints is provided and includes a first component comprising hyaluronic acid and a second component comprising a mixture of glucosamine and chondroitin sulfate, and a syringe for injecting a mixture of the first and second components. The mixture of the first and second components can have a pH of about 3-5 and an osmolality of about 330-750 mOSM. The syringe can have various configurations, and in one embodiment the syringe has a first chamber containing the first component, a second container containing the second component, and a plunger configured to displace the second component from the second container into the first container, combine them, and form the formulation.
The kit can also include an additional component, such as a stabilizer. In some embodiments, one or more components can be lyophilized.
The compositions, methods, and kits will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
The appended drawings have been included herein so that the above-recited features, advantages and objects will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate exemplary embodiments and should not be considered to limit the scope.
Certain exemplary embodiments will now be described to provide an overall understanding of the principles of the compositions, formulations and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the compositions and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the present invention is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure.
Reference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” or “an embodiment,” or the like, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments,” “in one embodiment,” or “in an embodiment,” or the like, in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, the particular features, structures, or characteristics illustrated or described in connection with one embodiment may be combined, in whole or in part, with the features structures, or characteristics of one or more other embodiments without limitation. Such modifications and variations are intended to be included within the scope of the present disclosure.
A therapeutically effective amount or effective amount of the composition can be administered to achieve a pharmacological effect. The term “therapeutically effective amount” includes, for example, a prophylactically effective amount. An “effective amount” is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. For example, an effective amount refers to an amount that increases operability, or increases weight bearing load, or decreases pain, or increases growth in the bone and cartilage of one or more joints, or reduces joint distortion, pain, swelling, or stiffness. The effective amount of an agent will be selected by those skilled in the art depending on the particular patient and the disease level. It is understood that “an effective amount” or “a therapeutically effective amount” can vary from subject to subject, due to variation in metabolism of therapeutic agents and/or prokinetic agents, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.
“Treat” or “treatment” refers to any treatment of a disorder or disease associated with bone or cartilage disorder, such as preventing the disorder or disease from occurring in a subject which may be predisposed to the disorder or disease, but has not yet been diagnosed as having the disorder or disease; inhibiting the disorder or disease, e.g., arresting the development of the disorder or disease, relieving the disorder or disease, causing regression of the disorder or disease, relieving a condition caused by the disease or disorder, or stopping the symptoms of the disease or disorder. Thus, as used herein, the term “treat” is used synonymously with the term “prevent.”
“Co-administered” means simultaneous administration in the same formulation or in two different formulations that are combined into one formulation for administration.
The term “subject” as used herein refers to an animal, such as a mammal and particularly a human who can benefit from the present compositions and methods. There is no limitation on the type of animal that could benefit from the present methods. A subject regardless of whether a human or non-human animal may be referred to as an individual, subject, animal, host or recipient. The methods disclosed herein have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry. In some embodiments, the candidate subject is a mammal such as a human or laboratory test animal such as a mouse, rat, rabbit, guinea pig, hamster or avian species such as a poultry bird.
One skilled in the art will appreciate that the term “precipitation,” as used herein, refers to the formation of an insoluble protein in the solution. In contrast to known examples of drugs that are delivered as a suspension (due to the fact that the carrier, e.g., a mineral, ceramic, metal, or polymeric, is insoluble rather than the active protein), an aspect disclosed herein is the active precipitation of the protein immediately prior to delivery of the composition to a patient.
In general, provided herein are compositions and methods for treating joint conditions, such as pain and degeneration associated with osteoarthritis. The compositions and methods utilize hyaluronic acid (“HA”), in combination with glucosamine and chondroitin sulfate in an injectable formulation with a low pH value and a high osmolality. In an exemplary embodiment, the glucosamine is in the form of glucosamine hydrochloride salt, glucosamine sulfate or N-acetyl-glucosamine. The composition can optionally include additional components including a stabilizer, such as trehalose, sucrose, raffinose, glucose, mannitol, sorbitol, erythritol or any combination thereof.
Surprisingly, the combination of HA with glucosamine and chondroitin sulfate overcomes the potential side effects associated with the low pH and high osmolality of the formulation. It is well-known in the art that neutral pH and isotonic solutions are preferred for injections to avoid pain and tissue degeneration. As a result, glucosamine is typically used in injections at a low concentration to allow for a neutral pH. Similarly, chondroitin sulfate is typically also used in injections at a low concentration to allow for an isotonic solution. Unexpectedly, the combination of hyaluronic acid, with glucosamine and chondroitin sulfate, enables the use of an injectable formulation with a low pH and high osmolality without causing any pain or tissue damage.
The composition can be administered to any synovial joint, including the knee, shoulder, hip, ankle, hands, spinal facet, or temporomandibular joint, both for the relief of pain and slowing of disease progression. The composition may include any of several combinations of hyaluronic acid, glucosamine, and chondroitin sulfate that can be delivered into the joint via an injection. Also provided herein are a kit for administration of an injection, and a method of administering the composition with or without the use of a kit.
Hyaluronic Acid
Hyaluronic acid (HA) can have various formulations and can be provided at various concentrations and molecular weights. The terms “hyaluronic acid,” “hyaluronan,” “hyaluronate,” and “HA” are used interchangeably herein to refer to hyaluronic acid or salts of hyaluronic acid, such as the sodium, potassium, magnesium, and calcium salts, among others. These terms are also intended to include not only pure hyaluronic acid solutions, but hyaluronic acid with other trace elements or in various compositions with other elements. The terms “hyaluronic acid,” “hyaluronan,” and “HA” encompass chemical or polymeric or cross-linked derivatives of HA. Examples of chemical modifications which may be made to HA include any reaction of an agent with the four reactive groups of HA, namely the acetamido, carboxyl, hydroxyl, and the reducing end. The HA disclosed herein is intended to include natural formulations (isolated from animal tissue) or synthetic formulations (derived from bacterial fermentation) or combinations thereof. The HA can be provided in liquid form or in a solid formulation that is reconstituted with a diluent to achieve an appropriate concentration.
HA is a glycosaminoglycan (GAG), and in particular HA is an unbranched polysaccharide made up of alternating glucuronic acid and N-acetyl glucosamine units. In liquid form, the HA has viscoelastic properties. HA is also found in the extracellular matrix of cartilage as an important structural component of aggrecan, which makes up the proteoglycan complex. The major function of the proteoglycan complex is to retain water in the cartilage matrix, imparting its characteristic turgidity and mechanical resiliency.
HA not only helps to maintain healthy mechanical properties of cartilage that cushions joints, but it is also a major component of synovial fluid.
HA can be used in the compositions and methods described herein at various molecular weights. Since HA is a polymeric molecule, the HA component can exhibit a range of molecular weights, and almost any average of modal molecular weight formulation of HA can be used in the compositions and methods described herein, including Low Molecular Weight (“LWM”) Hyaluronan (about 500 to 700 kilodaltons (kDa), Medium Molecular Weight (“MMW”) Hyaluronan (700-1000 kDa), and High Molecular Weight (“HMW”) Hyaluronan (1.0-6.0 million daltons (MDa)). In certain exemplary embodiments, the HA has a molecular weight of at least about 700 kDa, and in certain embodiments, the HA is a High Molecular Weight (“HWM”) HA having a molecular weight of at least about 1 MDa. The molecular weight can be, for example, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000 kDa or more, or any range derivable therein. It is expected that chemically modified HA could have very different molecular weights than described above. A cross-linked HA can likewise have much higher molecular weight than noted above. Regardless, these materials are also applicable to the compositions and methods disclosed herein.
HA can be present in solid, lyophilized or liquid form. When in liquid form, solvents can be used to solubilize HA. Solvents can include, but are not limited to, water, saline or other salt solutions, buffer solutions such as phosphate buffered saline, histidine, lactate, succinate, glycine, and glutamate, dextrose, glycerol, as well as combinations thereof.
The concentration of HA present in the formulation can also vary, but in an exemplary embodiment HA is provided at a pharmaceutically effective amount. In one embodiment, the HA has a concentration of at least about 1 mg/mL. In an exemplary embodiment, the HA has a concentration of at least about 5 mg/mL, and more particularly at least about 7 mg/mL, and more particularly at least about 10 mg/mL, and more particularly at least about 12 mg/mL, and in some embodiments the concentration can be at least about 25 mg/mL. In some embodiments, the HA can have a concentration in the range of about 3.6 mg/mL to about 36 mg/mL. In another embodiment, the HA can have a concentration in the range of about 12 mg/mL to about 25 mg/mL. Suitable concentrations of HA include about 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mg, 12 mg/mL, 12.5 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL, 25 mg/mL, 26 mg/mL, 27 mg/mL, 28 mg/mL, 29 mg/mL, 30 mg/mL, 31 mg/mL, 32 mg/mL, 33 mg/mL, 34 mg/mL, 35 mg/mL, 36 mg/mL, 37 mg/mL, 38 mg/mL, 39 mg/mL, 40 mg/mL, 41 mg/mL, 42 mg/mL, 43 mg/mL, 44 mg/mL, 45 mg/mL, 46 mg/mL, 47 mg/mL, 48 mg/mL, 49 mg/mL, 50 mg/mL, 51 mg/mL, 52 mg/mL, 53 mg/mL, 54 mg/mL, 55 mg/mL, 56 mg/mL, 57 mg/mL, 58 mg/mL, 59 mg/l, 60 mg/mL or more or any range derivable therein.
In one embodiment, the first component comprises an HA having a high molecular weight (1 to 6 MDa) having a concentration in the range of about 5-40 mg/mL. One such product is Orthovisc® manufactured by Anika Therapeutics, Inc. of Bedford, Mass. Orthovisc® is a sterile, non-pyrogenic, clear, viscoelastic solution of hyaluronan. Orthovisc® consists of high molecular weight (1.0-2.9 MDa), ultra-pure natural hyaluronan dissolved in physiological saline and having a nominal concentrations between 12.5-17.5 mg/mL. Orthovisc® is isolated through bacterial fermentation. One skilled in the art will recognize that there are companies such as Shiseido and Lifecore who can produce high molecular weight HA through a bacterial fermentation process. Another example of an HA product available in the United States with these characteristics is Euflexxa®.
Glucosamine
Glucosamine (C6H13NO5) (“GlcN”) or its derivatives can also be included in the formulation to enhance synthesis of key components of cartilage and synovial fluid by feeding both reactions necessary for the production of hyaluronan as well as for proteoglycans. GlcN is an amino sugar carrying four hydroxyl groups and an amine group, and it is a prominent precursor in the biochemical synthesis of glycosylated proteins and lipids. GlcN is a naturally occurring molecule that has nutritive and effector functions. As used herein, “glucosamine” includes glucosamine salts, such as glucosamine hydrochloride and glucosamine sulfate, as well as non-salt forms such as N-acetylglucosamine. In one embodiment, glucosamine hydrochloride is included in the formulation.
GlcN is compatible with and promotes stem cell growth and differentiation of mesenchymal stem cells to form chondrocytes. GlcN can have a role in tissue development and repair, such as cartilage growth and development, in general. It is used as a nutritional supplement to combat the symptoms of OA, and has been shown slow cartilage destruction in clinical studies.
GlcN can be used in the compositions and methods disclosed herein as various different salts, or in non-salt forms such as N-acetyl-glucosamine. Exemplary GlcN used herein can be glucosamine hydrochloride, glucosamine sulfate or N-acetyl-glucosamine. The concentration range is discussed below.
Glucosamine can be present in solid, liquid, or lyophilized form. A person skilled in the art will appreciate that, while lyophilized glucosamine is particularly useful in exemplary embodiments, liquid glucosamine can also be used in the composition. For example, glucosamine can be obtained in powder form and mixed with a solvent, such as water, to form a solution. The solution can be combined with additional components to form a mixture. In an exemplary embodiment the glucosamine is lyophilized to allow for increased stability of the injectable composition.
The concentration of the GlcN used in the compositions, formulations and methods described herein can vary. A suitable local concentration can be at least about 180 mg/mL, about 170 mg/mL, about 160 mg/mL, about 150 mg/mL, about 140 mg/mL, about 130 mg/mL, about 120 mg/mL, about 110 mg/mL, about 100 mg/mL, about 90 mg/mL, about 80 mg/mL, about 70 mg/mL, about 60 mg/mL, about 50 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, 30 mg/mL, 28 mg/mL, 25 mg/mL, 22 mg/mL, 20 mg/mL, about 19 mg/mL, about 18.5 mg/mL, about 18 mg/mL, about 17.5 mg/mL, about 17 mg/mL, about 16 mg/mL, about 15 mg/mL, about 12 mg/mL, about 10 mg/mL, about 5.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.8 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.018 mg/mL, about 0.0018 mg/mL, about 0.00018 mg/mL, or so on. In some embodiments, a high dose of GlcN (e.g., about 180 mg/mL, about 170 mg/mL, about 160 mg/mL, about 150 mg/mL, about 140 mg/mL, about 130 mg/mL, about 120 mg/mL, about 110 mg/mL, about 100 mg/mL, about 90 mg/mL, about 80 mg/mL, about 70 mg/mL, about 60 mg/mL, about 50 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, 30 mg/mL, 28 mg/mL, 25 mg/mL, 22 mg/mL, 20 mg/mL, about 19 mg/mL, about 18.5 mg/mL, about 18 mg/mL) is used. In some embodiments, a low dose of GlcN (e.g., about 5.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.8 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.018 mg/mL, about 0.0018 mg/mL, about 0.00018 mg/mL or lower) is used. A person skilled in the art can determine a suitable local concentration of GlcN practicing methods known in the pharmaceutics art, and that determination will govern the nature and composition of the GlcN composition of interest to obtain the desired concentration of GlcN.
Chondroitin Sulfate
Chondroitin sulfate (CS), which is an essential component of cartilage, is composed of an alternating sequence of sulfated and/or unsulfated D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues linked through alternating β(1,3) and β(1,4) bonds. These compounds each have a polymeric structure consisting mainly of about 40 to 100 times repetition of the disaccharide units. CS can be used in the inventive formulation at various molecular weights and concentrations. CS can be isolated from bovine or marine sources. A chondroitin chain can have over 100 individual sugars, each of which can be sulfated in variable positions and quantities. Chondroitin-4 sulfate, also carbon 4 of the N-acetylgalactosamine (GalNAc) sugar, is found in nasal and tracheal cartilages of bovines and porcines. It is also found in the bones, flesh, blood, skin, umbilical cord, and urine of these animals. Chondroitin-6 sulfate, also carbon 6 of the GalNAc sugar, has been isolated from the skin, umbilical cord, and cardiac valves of these animals. Chondroitin-6 sulfate has the same composition, but slightly different physical properties from chondroitin-4 sulfate. Chondroitin sulfate is involved in the binding of collagen and is also directly involved in the retention of moisture. These are both properties that aid the healing process. A person skilled in the art will appreciate that the terms “chondroitin sulfate,” “CS,” “chondroitin,” “chondroitin sulfuric acid,” and “chonsurid” are used interchangeably herein and also encompass chemical or isomeric or cross-linked derivatives throughout this application.
CS can be present in the compositions and methods disclosed herein at various molecular weights. In certain exemplary embodiments the molecular weight is in the range about 5 to 1,000 kDa, in the range of about 6 to 500 kDa, in the range of about 7 to 300 kDa, in the range of about 8 to 200 kDa, in the range of about 9 to 100 kDa, or typically in the range of about 10 to 80 kDa.
CS can be present in solid, liquid, or lyophilized form. A person skilled in the art will appreciate that, while lyophilized CS is particularly useful in exemplary embodiments, liquid CS can also be used in the composition. For example, CS can be obtained in powder form and mixed with a solvent, such as water, to form a solution. The solution can be combined with additional components to form a mixture. Thus, while liquid, non-lyophilized CS compositions can be used with the present invention, in an exemplary embodiment, the CS is lyophilized to allow for increased stability of the injectable composition.
CS can be used in the compositions and methods described herein at various concentrations. The concentration of CS in the composition can also vary, but in an exemplary embodiment CS is provided at a pharmaceutically effective amount. A suitable local concentration can be at least about 80 mg/mL, about 75 mg/mL, about 70 mg/mL, about 65 mg/mL, about 60 mg/mL, about 55 mg/mL, about 50 mg/mL, about 45 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, 30 mg/mL, 28 mg/mL, 25 mg/mL, 22 mg/mL, 20 mg/mL, about 19 mg/mL, about 18.5 mg/mL, about 18 mg/mL, about 17.5 mg/mL, about 17 mg/mL, about 16 mg/mL, about 15 mg/mL, about 12 mg/mL, about 10 mg/mL, about 5.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.8 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.018 mg/mL, about 0.1 mg/mL, about 0.05 mg/mL, about 0.005 mg/mL, or so on. In some embodiments, a high dose of CS (e.g., about 80 mg/mL, about 75 mg/mL, about 70 mg/mL, about 65 mg/mL, about 60 mg/mL, about 55 mg/mL, about 50 mg/mL, about 45 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, 30 mg/mL, 28 mg/mL, 25 mg/mL, 22 mg/mL, 20 mg/mL, about 19 mg/mL, about 18.5 mg/mL, about 18 mg/mL) is used. In some embodiments, a low dose of CS (e.g., about 5.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.8 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.018 mg/mL, about 0.1 mg/mL, about 0.05 mg/mL, about 0.005 mg/mL or lower) is used. A person skilled in the art can determine a suitable local concentration of CS practicing methods known in the pharmaceutics art, and that determination will govern the nature and composition of the CS composition of interest to obtain the desired concentration of CS.
Additional Components
In an exemplary embodiment, at least one additional component can be added to the compositions and methods disclosed herein. A person skilled in the art will appreciate that the present compositions and methods can include various other joint treatment components, including, for example, amino acids, proteins, saccharides, disaccharides, polysaccharides, lipids, nucleic acids, buffers, surfactants, and mixtures thereof. Other useful components can include steroids, anti-inflammatory agents, non-steroidal anti-inflammatory agents, analgesics, cells, antibiotics, antimicrobial agents, anti-inflammatory agents, growth factors, growth factor fragments, small-molecule wound healing stimulants, hormones, cytokines, peptides, antibodies, enzymes, isolated cells, platelets, immunosuppressants, nucleic acids, cell types, viruses, virus particles, essential nutrients or vitamins, and combinations thereof.
Stabilizers can be used in the present methods and compositions. Stabilizers can be sugars or derivatives, such as saccharides, disaccharides, modified saccharides, sugar alcohols, or polysaccharides. In an exemplary embodiment, the stabilizer can be tocopherol, tocopherol derivatives, glucose, mannitol, sucrose and/or trehalose.
Buffering agents can also be added to the formulation to control pH. Examples of buffering agents can be any one or more of the following agents, and is not limited to, acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, glycine, lactic acid, phosphoric acid, potassium citrate, potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium citrate, sodium lactate solution, dibasic sodium phosphate, monobasic sodium phosphate, TRIS and sodium carbonate.
Additionally, isotonic agents can be added to control ionic concentration and/or osmotic pressure of the formulation. Examples of isotonic agents can be any one or more of the following agents, and is not limited to, dextrose, sucrose, trehalose, glycerin, mannitol, potassium chloride, sodium chloride.
Common stabilizers or stabilizing excipients used in the pharmaceutical industry are saccharides, disaccharides, modified saccharides, sugar alcohols and polysaccharides. In one embodiment, the formulation or composition includes at least one stabilizer or stabilizing excipient, such as tocopherol, tocopherol derivatives, mannitol, glucose, sucrose and trehalose. The stabilizers can be present in a range of about 0.1-70% by weight; about 0.1-50% by weight, about 0.1-20% by weight; or about 0.5-20% by weight. Alternatively, the excipient can be present at a concentration in a range of about 1 mg/mL to about 700 mg/mL, in a range of about 1 mg/mL to about 500 mg/mL, in a range of about 1 mg/mL to about 200 mg/mL, and more particularly in a range of about 5 mg/mL to about 200 mg/mL.
Tocopherol belongs to a class of chemical compounds that encompass mono, di and trimethyltocols. Many of the tocopherols demonstrate vitamin E activity. Beta, gamma and delta are stereoisomers of alpha-tocopherol. Esters of tocopherol are often used in cosmetic and personal care products. These esters include, tocopheryl acetate, the acetic acid ester of tocopherol; tocopheryl linoleate, the linoleic acid ester of tocopherol; tocopheryl linoleate/oleate, a mixture of linoleic and oleic acid esters of tocopherol; tocopheryl nicotinate, the nicotinic acid ester of tocopherol; and tocopheryl succinate, the succinic acid ester of tocopherol. Potassium ascorbyl tocopheryl phosphate, a salt of both vitamin E (tocopherol) and vitamin C (ascorbic acid) may also be used in cosmetic products.
Other tocopherol-derived ingredients that may be found in cosmetic products include dioleyl tocopheryl methylsilanol, which is the dioleyl ether of tocopheryl acetate monoether with methylsilanetriol, and tocophersolan, which is also called tocopheryl polyethylene glycol 1000 succinate. The addition of succinic acid and an average of 22 ethylene oxide groups to tocopheryl makes tocophersolan a water-soluble form of tocopherol.
Mannitol is also an exemplary stabilizer due to its low hygroscopicity, excellent chemical and physical drug compatibility, better sweetness and relatively slower dissolution kinetics. It also has relatively low aqueous solubility and good dispersibility and often used to enhance formulation stability where other excipients have failed. Mannitol can be used in a wide array of dosage forms, including but not limited to, tablets, capsules, sachets, pastilles, liquids, emulsions, suspensions, ointments, paste, lotions and intravenous solutions.
Mannitol also serves as a matrix forming additive for lyophilization. When used at concentrations up to 10% w/v, mannitol forms an amorphous (non-crystalline) matrix which supports proteins and other biomolecules for freeze drying. It is generally inert and once freeze dried, rehydrates rapidly. Its amorphous structure while frozen prevents it from disrupting proteins while providing channels for water sublimation during processing.
Similar to mannitol, sucrose is also widely used in tablet form for oral delivery due to its sweetness and palatability. Sucrose, is a non-reducing disaccharide (glucose linked by its anomeric carbon to fructose) that is widely used as a lyoprotectant.
Trehalose (α-D-glucopyranosyl α-D-glucopyranoside), a disaccharide known for its antioxidant properties, consists of glucoses. Trehalose widely exists in microorganisms, mushrooms, insects, etc., although in relatively low quantities. Trehalose is a non-reducing saccharide, so that it neither reacts with substances containing amino groups such as amino acids and proteins, induces the amino-carbonyl reaction, nor deteriorates amino acid-containing substances. Thus, trehalose can be used without a fear of causing an unsatisfactory browning and deterioration.
Trehalose is also believed to inhibit the inflammatory cascade, thereby suppressing cytokine production. Trehalose is a unique sugar capable of protecting biomolecules against environmental stress and may inhibit the inflammatory cascade that in turn causes oxidative damage and cytokines production. Trehalose has also been shown to preserve cell viability, during exposure to a range of environmental stress, such as heat shock, dehydration and hypoxia.
Trehalose is also a common food additive because it is a strong antioxidant and sweetener, and it is often used as a stabilizing agent in pharmaceutical preparations. Trehalose, like sucrose, is a non-reducing disaccharide (two glucose molecules linked by the anomeric carbon) that can act as an effective lyoprotectant for the freeze drying of proteins and other biomolecules. During the freeze drying process, proteins can denature as water is removed unless a substitute molecule is available to support the structure of the protein. Trehalose fills the void left by exiting water and prevents this denaturation. When used at concentrations as low as 2% it can effectively protect proteins and other biomolecules.
Useful forms of trehalose can include trehalose dihydrate (TD) which is crystalline, amorphous trehalose (AT) which is a vitreous form, and the anhydrous forms of trehalose, anhydrous amorphous trehalose (AAT) and anhydrous crystalline trehalose (ACT). Powdered anhydrous trehalose may contain AAT and/or ACT. The term “trehalose,” as used herein, refers to any physical form of trehalose including anhydrous, partially hydrated, fully hydrated and mixtures and solutions thereof. The manufacture and use of anhydrous trehalose from TD can be found in International Publication No.: PCT/GB97/00367, the disclosure of which is incorporated into this specification by reference.
The addition of trehalose to the formulation or composition can help stabilize the other components of the composition as well as inhibit damaging inflammatory cascades. Trehalose can be present in liquid, solid, lyophilized or crystalline forms. When present in liquid form, trehalose can be in a buffered solution. Solvents that can be used to solubilize trehalose can include, but are not limited to, water, saline or other salt solutions, buffer solutions such as phosphate buffered saline, histidine, lactate, succinate, glycine, and glutamate, dextrose, glycerol, as well as combinations thereof. Particularly, trehalose can be present in the formulation as a solution.
At least one stabilizer, can be present in the formulation. Solvents that can be used to solubilize the stabilizer can include, but are not limited to, water, saline or other salt solutions, buffer solutions such as phosphate buffered saline, histidine, lactate, succinate, glycine, and glutamate, dextrose, glycerol, as well as combinations thereof. Particularly, the stabilizer can be present in the formulation as a solution.
The concentration of the at least one stabilizer present in the formulation can vary, but in an exemplary embodiment at least one excipient is provided at a pharmaceutically effective amount. In an exemplary embodiment, the at least one stabilizer has a concentration of at least about 1 mg/mL, at least about 5 mg/mL, at least about 50 mg/mL, at least about 100 mg/mL, or in some embodiments the concentration can be at least about 200 mg/mL. Suitable concentrations of at least one stabilizer can include about 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mg, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL, 25 mg/mL, 26 mg/mL, 27 mg/mL, 28 mg/mL, 29 mg/mL, 30 mg/mL, 31 mg/mL, 32 mg/mL, 33 mg/mL, 34 mg/mL, 35 mg/mL, 36 mg/mL, 37 mg/mL, 38 mg/mL, 39 mg/mL, 40 mg/mL, 41 mg/mL, 42 mg/mL, 43 mg/mL, 44 mg/mL, 45 mg/mL, 46 mg/mL, 47 mg/mL, 48 mg/mL, 49 mg/mL, 50 mg/mL, 51 mg/mL, 52 mg/mL, 53 mg/mL, 54 mg/mL, 55 mg/mL, 56 mg/mL, 57 mg/mL, 58 mg/mL, 59 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, 500 mg/mL, 600 mg/mL or more or any range derivable therein. Stabilizers can also be in a concentration in a range of about 0.1-60% by weight; about 0.1-50% by weight, about 0.1-45% by weight; or about 0.1-20% by weight. Other suitable concentrations of at least one excipient can include about 0.1%, 0.5%, 1%, 2.5%, 5%, 6%, 7%, 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or about 60% by weight.
Lyophilization
Any one or more of the components present in the compositions and methods of the present invention can be lyophilized using various techniques known in the art. Lyophilization is a dehydration process that is typically used to preserve a perishable material, and it works by freezing the material and then reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to the gas phase. Standard lyophilization techniques known in the art can be used to lyophilize any one or more of the components. In an exemplary embodiment, glucosamine and chondroitin sulfate are lyophilized, either together in a mixture, or separately. In another embodiment, the hyaluronic acid is lyophilized. In another embodiment, the glucosamine, chondroitin sulfate and hyaluronic acid are lyophilized, either together in a mixture or separately.
Prior to lyophilization, various solvents can be used to form an aqueous mixture containing the component(s) to be lyophilized. In an exemplary embodiment, the aqueous mixture is prepared by combining water with one or more of the components. The component(s) can be present within the mixture at various amounts, for example in the range of about 0.05 mg/mL to 10 mg/mL rhGDF-5. In an exemplary embodiment, the composition is filter sterilized, such as with a 0.2 μm filter, prior to lyophilization.
In one embodiment, the component(s) can be lyophilized using the following cycle:
Freezing: from ambient temperature to 5° C. in 15 minutes
Primary Drying: set pressure at 50 mTorr
Secondary Drying: set pressure at 75 mTorr
Cycle end: backfill with nitrogen to ˜730 Torr
Variations to the temperatures, times and settings can be made in accordance to practices used by a person of skilled in the art. Variations may include, but are not limited to, cycling temperatures for the freezing cycle, drying temperatures and end cycles. Variations may also include differences in holding times for the freezing, drying and capping/crimping cycles. Variations may also include differences in set pressures for the drying cycles and capping/crimping cycles. In addition, the number of drying cycles may be increased or decreased depending on the machine used or component(s) to be lyophilized.
The addition of a buffering agent can provide for improved solubility and stability of the compounds in lyophilized formulations. Biocompatible buffering agents known in the art can be used, such as glycine; sodium, potassium, or calcium salts of acetate; sodium, potassium, or calcium salts of citrate; sodium, potassium, or calcium salts of lactate; sodium or potassium salts of phosphate, including mono-basic phosphate, di-basic phosphate, tri-basic phosphate and mixtures thereof. The buffering agents can additionally have sugar added to the composition to function as a bulking agent. The pH typically can be controlled within about 2.0 to about 5.0 pH units, and more typically within about 2.5 to about 3.5 pH units.
Formulations
While HA alone can be effective to treat joint conditions, the compositions and formulations described herein provide an improved approach for treating joint conditions.
It is well known in the art that most of the available injectable formulations in the market are isotonic. The pH of the available formulations is either close to the pH of synovial fluid (i.e., pH 7.4) or slightly lower, but not below pH ˜5.5, to allow for optimum stability of the active ingredient, while minimizing possible side effects of non-physiological pH values, such as pain or tissue damage at the injection site. However, the acidic compositions and formulations with high osmolality described herein surprisingly produced unexpected benefits. The disclosed compositions and formulations demonstrated improved efficacy in pain relief and disease modification for treating joint conditions. Further, compositions and formulations did not cause any side effects (such as pain, inflammation or tissue damage at the injection site) that one would have expected for a low pH and/or a high osmolality IA formulation based on the current knowledge. The acidic compositions and formulations also provide an excellent preserved solution against the contaminating action of microorganisms such as bacteria and fungi. Such a preserved solution not only provides an excellent sterile environment, but also makes it easier to produce a commercial kit with longer shelf-life.
In addition, the combination of HA, glucosamine and chondroitin sulfate also addresses the issue of low bioavailability in the arthritic joint from the oral administration of glucosamine and chondroitin sulfate. As shown in the data and examples provided herein, both very low dose of glucosamine and chondroitin sulfate and high dose of glucosamine and chondroitin sulfate can provide equal efficacy in pain relief and disease modification during the treatment.
Accordingly, provided herein is a composition that includes an injectable formulation encompassing HA, glucosamine, and/or chondroitin sulfate.
The concentration of HA present in the formulations can vary, but in an exemplary embodiment HA is provided at a pharmaceutically effective amount. Suitable concentrations of HA include about 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mg, 12 mg/mL, 12.5 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL, 25 mg/mL, 26 mg/mL, 27 mg/mL, 28 mg/mL, 29 mg/mL, 30 mg/mL, 31 mg/mL, 32 mg/mL, 33 mg/mL, 34 mg/mL, 35 mg/mL, 36 mg/mL, 37 mg/mL, 38 mg/mL, 39 mg/mL, 40 mg/mL, 41 mg/mL, 42 mg/mL, 43 mg/mL, 44 mg/mL, 45 mg/mL, 46 mg/mL, 47 mg/mL, 48 mg/mL, 49 mg/mL, 50 mg/mL, 51 mg/mL, 52 mg/mL, 53 mg/mL, 54 mg/mL, 55 mg/mL, 56 mg/mL, 57 mg/mL, 58 mg/mL, 59 mg/l, 60 mg/mL or more or any range derivable therein. In some embodiments, the HA can have a concentration in the range of about 3.6 mg/mL to about 36 mg/mL.
The concentration of the GlcN used in the formulations can also vary. In some embodiments, a high dose of GlcN (e.g., about 180 mg/mL, about 170 mg/mL, about 160 mg/mL, about 150 mg/mL, about 140 mg/mL, about 130 mg/mL, about 120 mg/mL, about 110 mg/mL, about 100 mg/mL, about 90 mg/mL, about 80 mg/mL, about 70 mg/mL, about 60 mg/mL, about 50 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, 30 mg/mL, 28 mg/mL, 25 mg/mL, 22 mg/mL, 20 mg/mL, about 19 mg/mL, about 18.5 mg/mL, about 18 mg/mL) per injection is used. In some embodiments, a low dose of GlcN (e.g., about 5.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.8 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.018 mg/mL, about 0.0018 mg/mL, about 0.00018 mg/mL or lower) per injection is used.
CS can be used in the formulations described herein at various concentrations. In an exemplary embodiment CS is provided at a pharmaceutically effective amount. In some embodiments, a high dose of CS (e.g., about 80 mg/mL, about 75 mg/mL, about 70 mg/mL, about 65 mg/mL, about 60 mg/mL, about 55 mg/mL, about 50 mg/mL, about 45 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, 30 mg/mL, 28 mg/mL, 25 mg/mL, 22 mg/mL, 20 mg/mL, about 19 mg/mL, about 18.5 mg/mL, about 18 mg/mL) per injection is used. In some embodiments, a low dose of CS (e.g., about 5.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.8 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.018 mg/mL, about 0.1 mg/mL, about 0.05 mg/mL, about 0.005 mg/mL or lower) per injection is used.
In some embodiments, the formulations disclosed herein include a pharmaceutically effective amount of HA, a high concentration/dose of GlcN (e.g., about 180 mg/mL, about 170 mg/mL, about 160 mg/mL, about 150 mg/mL, about 140 mg/mL, about 130 mg/mL, about 120 mg/mL, about 110 mg/mL, about 100 mg/mL, about 90 mg/mL, about 80 mg/mL, about 70 mg/mL, about 60 mg/mL, about 50 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, 30 mg/mL, 28 mg/mL, 25 mg/mL, 22 mg/mL, 20 mg/mL, about 19 mg/mL, about 18.5 mg/mL, about 18 mg/mL), and a high concentration/dose of CS (e.g., about 80 mg/mL, about 75 mg/mL, about 70 mg/mL, about 65 mg/mL, about 60 mg/mL, about 55 mg/mL, about 50 mg/mL, about 45 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, 30 mg/mL, 28 mg/mL, 25 mg/mL, 22 mg/mL, 20 mg/mL, about 19 mg/mL, about 18.5 mg/mL, about 18 mg/mL).
In some embodiments, the formulations disclosed herein include a pharmaceutically effective amount of HA and a high concentration/dose of CS (e.g., about 80 mg/mL, about 75 mg/mL, about 70 mg/mL, about 65 mg/mL, about 60 mg/mL, about 55 mg/mL, about 50 mg/mL, about 45 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, 30 mg/mL, 28 mg/mL, 25 mg/mL, 22 mg/mL, 20 mg/mL, about 19 mg/mL, about 18.5 mg/mL, about 18 mg/mL).
In some embodiments, the formulations disclosed herein include a pharmaceutically effective amount of HA, a low concentration/dose of GlcN (e.g., about 5.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.8 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.018 mg/mL about 0.0018 mg/mL, about 0.00018 mg/mL or lower), and a low concentration/dose of CS (e.g., about 5.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.8 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.018 mg/mL, about 0.1 mg/mL, about 0.05 mg/mL, about 0.005 mg/mL or lower).
In some embodiments, the formulations disclosed herein include a pharmaceutically effective amount of HA and a low concentration/dose of CS (e.g., about 5.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.8 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.018 mg/mL, about 0.1 mg/mL, about 0.05 mg/mL, about 0.005 mg/mL or lower).
It is desirable that the formulations described herein have a pH of about 3.0 to about 5.0. For example, the formulation has a pH value of about 3.0 to about 4.0, about 3.5 to about 4.5 or about 3.5 to about 5.0. For example, the formulation has a pH value of about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 50. Typically, the formulation has a pH value of about 3.5 to about 4.0.
The compositions and formulations can be used in a method of treating a joint by administering the composition or the formulation to a subject, such as by injection into the body of the subject (e.g., by injection into a joint) as discussed below. The results after injecting the acidic and high osmolality composition are surprisingly good with no evidence of post injection pain or tissue degradation.
In one aspect, a composition for treating a joint condition is disclosed. The composition includes a solution of hyaluronic acid (HA) combined with glucosamine (GlcN) and chondroitin sulfate (CS). In the composition, all or some of the components can be liquid, solid, or lyophilized. In an exemplary embodiment, a composition of GlcN/CS is lyophilized and combined with liquid HA.
The compositions can be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, and powders. The form ultimately used depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for in vivo injection. One useful mode of administration is parenteral (e.g., intra-articular, subcutaneous, intraperitoneal, intramuscular). In one embodiment, the composition is administered by infusion or injection directly into the target area, such as a joint. In another embodiment, the composition can be administered by intramuscular or subcutaneous injection.
In an exemplary embodiment, the components are configured to be combined intraoperatively, i.e., immediately before or during an operation. In one embodiment, the components are combined about 60 minutes or less before injection. In another embodiment, the components are combined about 30 minutes or less prior to injection. The components, when combined, can form a resulting composition having each component present in the composition at various amounts. The amount of each component in the composition can vary.
In one embodiment, solvents can be included in the composition. Solvents that can be used to solubilize one or more of the components include, for example, water, acidic solvents, hydrochloric acid, acetic acid, benzoic acid, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Solvents that can be used to solubilize HA can include, but are not limited to, water, saline or other salt solutions, buffer solutions such as phosphate buffered saline, histidine, lactate, succinate, glycine and glutamate, dextrose, glycerol, and other suitable solvents, as well as combinations thereof. Solvents that can be used to solubilize the GlcN/CS can include water, saline, phosphate-buffered saline, hydrochloric acid, acetic acid, benzoic acid, acidic solvent, and other solvents suitable for solubilization of this solution. The compositions can also include additional components, such as stabilizers, buffers, isotonic agents, or other joint treatment components.
Concentration of the components can affect the pH and osmolality of the resulting composition. For example, in some embodiments, lower concentrations of glucosamine and chondroitin sulfate may be used to avoid a composition being too acidic (such as a pH lower than 2.0). Table 1 lists pH values of exemplary compositions with various concentrations of each component. For this composition, GlcN is in the form of GlcN-HCl salt.
A person skilled in the art will appreciate that the concentrations of each component can be modified to regulate pH and/or the therapeutic outcome. In an exemplary embodiment the concentration of glucosamine can be about 0.005-54, 1.8, 1.8-18, 2.0, 2.0-20, 18, 20, 2.0-40, 20-30, 10-40, or 40 mg/mL. In another embodiment, the concentration of chondroitin sulfate can be about 0.005-54, 1.8, 1.8-18, 2.0, 2.0-20, 18, 20, 2.0-40, 20-30, 10-40, or 40 mg/mL. In another embodiment the concentration of HA can be about 3.6-36, 10-20, 12, 15, or 12-17.5 mg/mL. In yet another embodiment the concentration of glucosamine can be a high concentration, such as in the range of about 18-20 mg/mL, the concentration of chondroitin sulfate can be a high concentration, such as in the range of about 18-20 mg/mL, and the concentration of HA can be in the range of about 12-17.5 mg/mL. In yet another embodiment, the weight ratio of glucosamine to chondroitin sulfate is about 1:1, although other ratios are possible (such as any ratio between about 0.445 to about 444,445).
In one embodiment, the composition can have a concentration having any component concentrations within the ranges listed above. In one embodiment, the resultant composition can have a pH of at least about 3, in the range of about 3 to 8, in the range of about 3 to 5, or typically in the range of about 3 to 4. Further, the composition can have an osmolality in the range of about 300 to 3,000 mOSM (e.g., about 300, about 350, about 400, about 450, about 500, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900 or about 3000 mOSM). In some embodiments, composition can have an osmolality in the range of about 300 to 700 mOSM, in the range of about 350-600 mOSM, or typically in the range of 400-600 mOSM.
The compositions can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises HA and at least one of GlcN or CS and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, hydrochloric acid, acetic acid, benzoic acid, acidic solvent, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the composition.
The components and/or the resulting composition can be sterilized prior to use using various techniques known in the art. Sterile injectable compositions can be prepared by incorporating the active compound(s) in a therapeutically effective or beneficial amount in an appropriate solvent with one or a combination of ingredients, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating a compound(s), such as HA into a sterile vehicle which contains a basic dispersion medium and any required other ingredients. In the case of sterile powders for the preparation of sterile injectable compositions, some methods can include preparation of vacuum dried and freeze-dried components which yield a powder of the composition plus any additional desired ingredients from a previously sterile-filtered composition thereof.
Sterile injectable solutions can be prepared by incorporating the active compound in a therapeutically effective or beneficial amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The volume for each injection may vary according to the size of the joint where the injection occurs. Suitable volume for each injection can be about 0.5 mL to about 10 mL (e.g., about 0.5 mL, about 1.0 mL, about 1.5 mL, about 2 mL, about 2.5 mL, about 3 mL, about 3.5 mL, about 4 mL, about 4.5 mL, about 5 mL, about 5.5 mL, about 6 mL, about 6.5 mL, about 7 mL, about 7.5 mL, about 8 mL, about 8.5 mL, about 9 mL, about 9.5 mL or about 10 mL). In some embodiments, the volume of a single injection for knee, shoulder and hip can be in the range of about 1 ml to about 10 ml. In some embodiments, the volume of a single injection for hand can be in the range of about 500 μl to about 1.5 ml.
Administration/Method
The compositions of this invention can be administered, for in vivo applications, parenterally by injection or by gradual perfusion over time. Administration may be intra-articular, intravenous, intraperitoneal, intramuscular, subcutaneous, intracavity, or transdermal.
Examples of symptoms or diseases, for which the composition and methods disclosed herein can be useful, encompass treating articular disorders, such as arthritis caused by infections, injuries, allergies, metabolic disorders, etc., rheumatoids such as chronic rheumatoid arthritis, and systemic lupus erythematosus; articular disorders accompanied by gout, arthropathy such as osteoarthritis, internal derangement, hydrarthrosis, stiff neck, lumbago, etc. Varying the effects depending on the use of the composition or the types of diseases to be treated, the agent can exert desired prophylactic and alleviative effects, or even therapeutic effects on swelling, pain, inflammation, and destroying of articulations without seriously affecting living bodies. The composition for treating articular disorder can be used to prevent the onset of articulation disorders, as well as to improve, alleviate, and cure the symptoms after their onsets.
The methods of treatment can include directly injecting the compositions into the target area, such as a joint. Injections can be performed as often as daily, weekly, several times a week, bi monthly, several times a month, monthly, or as often as needed as to provide relief of symptoms. In alternative embodiments, the compositions of the invention may be administered intermittently over a period of months. It will be appreciated that administration regimens may be continued for extended periods (e.g., on the order of years) to maintain beneficial therapeutic effects provided by initial treatments. In yet other embodiments, maintenance therapy may be effected following an acute dosing regimen designed to reduce the immediate symptoms of the joint condition, such as osteoarthritis. In most embodiments, however, the compositions of the invention are administered to the patient according to the methods described herein at least until the symptoms of the joint condition, such as OA, are alleviated or reduced. More commonly, the compositions of the invention and methods of the invention are used for a period of time after the symptoms are reduced to a tolerable level or completely eliminated so as to result in an improvement in the physiological structure of the joint by reducing or eliminating the underlying physiological causes of the joint condition.
For intra-articular use, from about 1 to about 30 mg/mL of HA and about 0.018 to about 30 mg/mL of GlcN/CS per joint, depending on the size of the joint and severity of the condition, can be injected. The frequency of subsequent injections into a given joint are spaced to the time of recurrence of symptoms in the joint. Illustratively, dosage levels in humans of the composition can be: knee, about 1 to about 30 mg/mL HA and 0.018 to about 20 mg/mL of GlcN/CS per joint injection; shoulder, about 1 to about 30 mg/mL of HA 0.018 to about 20 mg/mL of GlcN/CS per joint injection; metacarpal or proximal intraphalangeal, about 1 mg/mL to about 30 mg/mL of HA and 0.018 to about 20 mg/mL of GlcN/CS per joint injection; and elbow, about 1 to about 30 mg/mL of HA and 0.018 to about 20 mg/mL of GlcN/CS per joint injection. In some embodiments, the total amount of injection can range from about 1 mg/mL to about 200 mg/mL of HA, about 0.005 mg/mL to about 150 mg/mL of GlcN, and about 0.005 mg/mL to about 150 mg/mL of CS.
It will be understood, however, that the specific dosage level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. The pharmaceutical compositions can be prepared and administered in dose units. Under certain circumstances, however, higher or lower dose units may be appropriate. The administration of the dose unit can be carried out both by single administration of the composition or administration can be performed in several smaller dose units and also by multiple administrations of subdivided doses at specific intervals.
In one embodiment, the HA and glucosamine and chondroitin sulfate can be configured to be combined and administered intra-articularly as part of a surgical procedure involving an articulating joint, either immediately before, during, or immediately after the surgical procedure. The HA and GlcN/CS can be co-administered or simultaneously administered in the same formulation or in two different formulations that are combined via the same route. GlcN is not stable at a low pH and will degrade with time, accordingly it is not desirable to have a formulation including GlcN at a low pH. In a preferred embodiment, the HA and GlcN/CS components can be combined just prior to administration of the HA and GlcN/CS. The combination can occur within seconds, minutes, hours, days or weeks prior to the administration of the composition. In another embodiment it is preferred to combine GlcN and chondroitin sulfate, either in a mixture or separately, with HA less than about 60 minutes (e.g., about 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 minutes) prior to injection. In a typical embodiment, the combination occurs about 30 minutes or less before administration. In some embodiments, the combination occurs about 15 minutes or less before administration. In some embodiments, the combination occurs about 5 minutes or less before administration.
In one embodiment, the medical condition is osteoarthritis (OA) and the composition is administered in a joint space, such as, for example, a knee, shoulder, temporomandibular and carpometacarpal joints, elbow, hip, wrist, ankle, and lumbar zygapophysial (facet) joints in the spine. The viscosupplementation may be accomplished via a single injection or multiple intra-articular injections administered over a period of weeks into the knee or other afflicted joints. For example, a human subject with knee OA may receive one, two, or three injections of about 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 mL or more per knee. One skilled in the art will appreciate that for other joints, the administered volume can be adjusted, based on the size on the joint.
Kit
The methods and compositions encompass kits for treating articular disorders, such as joints. In yet another aspect, a kit is disclosed. The kit can include a first component being a solution of hyaluronic acid (HA), a second component including an amount of glucosamine and chondroitin (GlcN/CS), and a syringe for injecting a mixture of the first component and the second component. Moreover, the syringe can have a first chamber containing the first component, a second container containing the second component and a plunger configured to inject the second component into the first chamber to mix the first and second components.
The kit can be stored at room temperature. Additionally, the kit can include a syringe containing the HA solution in a single chamber, and the GlcN/CS in a second chamber. The components can be present in liquid, solid or lyophilized form. The kit can further include a diluent, such as water, saline, and a buffer, to solubilize one of the components.
The components can be stored separately to increase shelf-life. The individual components can be lyophilized or in solid form in one syringe/cartridge with diluent or a second component in a second syringe/cartridge. In one embodiment, one of the compounds is in lyophilized form or in solid form and the second compound is a solution capable of combining with the lyophilized/solid compound.
Pre-filled dual-chamber syringes and/or cartridges can also be utilized with the components and compositions. Pre-filled dual-chamber syringes enable the sequential administration of two separate compositions with a single syringe push, thereby replacing two syringes with one. The benefits of a single delivery capability include increasing the speed and ease of drug administration; reducing risk of infection by reducing the number of connections; lowering the risk of drug administration or sequence errors, and quicker delivery of compositions requiring combination prior to administration. The dual-chamber syringe can accommodate lyophilized, powder or liquid formulations in the front chamber combined with diluents, saline or buffer in the rear chamber.
A person skilled in the art will appreciate that any dual chamber systems known in the art can be used, and that the chambers can be side-by-side chambers with separate syringe plungers that mix into a single chamber or linear chambers with a single plunger.
Prefilled syringes can contain the exact deliverable dose of desired compounds and diluents. The prefilled syringes can contain volumes from about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, 5 mL, 5.5 mL, 6 mL, 6.5 mL, 7 mL, 7.5 mL, 8 mL, 8.5 mL, 9 mL, 9.5 mL, 10 mL or more or any derivative therein. The volume for injection can vary based on the size of the joint for delivery. The volume of composition used can be varied based on the type of administration including the joint to which it is being administered, and the frequency of administration. One skilled in the art will appreciate that the volume for injection can be varied and optimized based on many other factors.
The dual syringe and/or cartridge can be side-by-side chambers with separate syringe plungers that mix into a single chamber or linear chambers with one plunger. The dual chamber syringe and/or cartridges can also have a stopper or connector in the middle to serve as a barrier between the two chambers. The stopper or connector can be removed to allow mixing or combining of the compounds in the two chambers.
A person skilled in the art will appreciate that any dual chamber systems known in the art can be used, and that the chambers can be side-by-side chambers with separate syringe plungers that mix into a single chamber or linear chambers with a single plunger.
A stock solution of GlcN/CS was prepared containing 8.7 mg/mL of glucosamine (as the hydrochloride salt) and 8.7 mg/mL of chondroitin sulfate. Just prior to intra-articular injection, approximately 103 uL of this stock was combined with 400 uL of Orthovisc® (high molecular weight hyaluronan) having an HA concentration of 15 mg/mL. Inter-syringe mixing of the two solutions was carried out using a three-way stopcock. The resultant solution contained 12 mg/mL HA, 1.8 mg/mL GlcN, and 1.8 mg/mL CS. The solution was aliquoted into smaller syringes for intra-articular injection. Injected volumes were about 50 uL for rat studies.
4 mg of GlcN and 4 mg of CS were lyophilized in a sterile vial. Just prior to injection, 2 mL or 15 mg/mL HA was injected into the lyophilized powder vial and vortexed until complete mixing was achieved. The resultant solution contained 15 mg/mL HA, 2 mg/mL CS and 2 mg/mL GlcN. The solution was aliquoted into smaller syringes for intra-articular injection.
Test Data: Rat MMT Model of Osteoarthritis
Several intra-articular HA/GlcN/CS formulations were tested in the rat medial meniscal tear (MMT) model. In this model, transaction of the medial meniscus results in joint deterioration and reduced weight bearing that mimic human osteoarthritis. This degeneration process occurs over a period of several weeks, with significant degeneration evident at 21 days.
The extent of joint deterioration, as determined primarily by the extent of cartilage lesions formed, is measured using a semi-quantitative histological scoring system. Weight bearing is measured using a commercially available incapacitance apparatus. Weight bearing improvement was determined by area under the curve (AUC) analysis of weekly weight bearing data. This provides a measure of the cumulative effect of the treatment on pain over the course of the experiment.
Beginning one week after MMT surgery, intra-articular injections were given weekly for five weeks, followed by euthanasia at week six. Weight bearing measurements were taken weekly just prior to injection.
HA/GlcN/CS formulations tested in the rat MMT model had the following concentrations:
HA/GlcN/CS=12 mg/mL/0.18 mg/mL/0.18 mg/mL (“the 0.18 mg/mL HA/GlcN/CS group/formulation”)
HA/GlcN/CS=12 mg/mL/1.8 mg/mL/1.8 mg/mL (“the 1.8 mg/mL HA/GlcN/CS group/formulation”)
HA/GlcN/CS=12 mg/mL/18.0 mg/mL/18.0 mg/mL (“the 18.0 mg/mL HA/GlcN/CS group/formulation”)
HA/CS=12 mg/mL/0.18 mg/mL (“the 0.18 mg/mL HA/CS group/formulation”)
HA/CS=12 mg/mL/1.8 mg/mL (“the 1.8 mg/mL HA/CS group/formulation”)
HA/CS=12 mg/mL/18.0 mg/mL (“the 18 mg/mL HA/CS group/formulation”)
HA=12 mg/mL
Untreated Controls
As such, the concentration of HA was held constant at a level that approximates commercially available viscosupplements, while the concentration of glucosamine and chondroitin sulfate was varied. For the present discussion, the 0.18, 1.8, and 18.0 mg/mL designations refer to those concentrations of GlcN and CS in combination with HA. The HA used in the formulations had a molecular weight range specification of 1.0-2.9 million Daltons.
Histology results, provided in
In another measure of cartilage preservation, the 1.8 mg/mL HA/GlcN/CS (“1.8 GCS”) and 18 mg/mL HA/CS (“18 CS”) groups showed improvement in cartilage degeneration score as shown in
A further measure of cartilage preservation is the width of lesions as measured by cartilage depletion.
CFA Model of Inflammatory Arthritis
Because OA is now known to have an inflammatory component, an animal model that mimics inflammatory arthritis was used to test HA/GlcN/CS formulations. Complete Freund's Adjuvant (CFA) comprises an emulsion of killed bacterial cells that is injected systemically or into the joint of a rat to induce severe inflammation. Two CFA studies were conducted, the first using a 200 uL injection of CFA to induce inflammation (CFA #1), the second using an injection volume of 100 uL (CFA #2). Test articles were prepared using the liquid/liquid formulation method described above.
In CFA #1, a single injection of HA/GlcN/CS was given on the fifth day following intra-articular CFA injection, which was sufficient time for the animals to develop substantial inflammation and pain sensitivity. Von Frey filaments were used to assess mechano-allodynia (pain sensation from a normally non-painful stimulus) on days 6, 7, 8, 17, and 21 following CFA injection. The potent NSAID diclofenac was used as a positive control, using a daily oral dose regimen. This protocol was used to test the 18.0 and 1.8 mg/mL HA/GlcN/CS formulations. As with the rat MMT model, the cumulative improvements in pain relief were calculated. As shown in
In CFA #2 the same protocol was followed except for the lower dose of CFA which was intended to induce a less severe inflammatory reaction and subsequently milder pain. Also, daily oral ibuprofen (labeled as “IBR” in
PGPS Model of Episodic Inflammatory Arthritis
The PGPS model of arthritis is used to simulate episodic inflammation of the joint. PGPS (peptidoglycan polysaccharide) is a streptococcal cell wall formulation that is injected directly into the joint of the test subject, resulting in a painful inflammatory response. Unlike the continuous FCA response, the inflammatory pain from PGPS is transient, typically lasting only a few days. Following this “priming” injection to the joint, subsequent systemic injections result in inflammatory flares in the previously injected joint for a period of several days. The “reactivation” inflammatory flares can be repeated multiple times, with the inflammatory pain generally decreasing with subsequent reactivation cycles. Alternatively, the joint can be “reprimed” following the initial reactivation, resulting in a more robust inflammatory response in subsequent reactivations.
As with the rat MMT model, the effect of intra-articular therapies is assessed by static weight bearing measurements. It is also useful to conduct a gait analysis of treated animals to assess the effect of treatments on normal function. Gait analysis may reveal functional improvements that are not detectable by weight bearing measurements. In the rat model, gait analysis is done by assessing the paw print pattern of the animal over a defined walking distance. In the present study, the bottom of the rat paw was colored with blue ink, and the dorsal side of the paw was covered in black ink, just prior to the gait analysis measurement. The animal was induced to walk across a sheet of white paper, and gait abnormalities were gauged by assessing the ink patterns compared to untreated animals. For example, a full blue pawprint indicates no pain, a partial blue pawprint indicates limping and black ink on the paper is evidence of foot dragging.
In the present experiment, two reactivation cycles were induced in male Lewis rats following the initial PGPS intra-articular injection. In the first reactivation cycle (beginning on day 0), oral celecoxib and intra-articular dexamethasone (a corticosteroid) were used as positive controls. Weight bearing and gait analysis scores were recorded on days 1, 4, 7, and 14 following the reactivation injection. On day 15, the animals were reprimed with a second IA injection of PGPA, and the second reactivation cycle was initiated 14 days thereafter. For the second reactivation cycle, daily celecoxib and intra-articular triamcinolone were used as positive controls. Weight bearing and gait analysis scores were recorded on days 1, 2, 3, and 4 following reactivation. For both cycles, a single intra-articular injection of an HA/GlcN/CS formulation was administered two hours prior to PGPS reactivation. The following formulations were tested:
HA=15 mg/mL
HA/CS=15 mg/mL/20 mg/mL (“OV/CS 15/20”)
HA/CS=15 mg/mL/2 mg/mL (“OV/CS 15/2”)
HA/GlcN/CS=15 mg/mL/2 mg/mL/2 mg/mL (“OV/CS/GlcN 15/2/2”)
The liquid/solid formulation method described above was used to prepare the test articles.
Following the first reactivation, only oral celecoxib showed a significant improvement in both weight bearing deficit and gait analysis score on day 1. However, as shown in
Over the first four days following the second reactivation, the HA/CS 15/2 group gave a significant cumulative improvement in gait analysis score compared to HA alone. The graph in
In summary, these data collectively demonstrated that both lower doses of GlcN and CS ranging from 0.018 mg/ml to 0.18 mg/ml and higher doses of GlcN and CS ranging from 18 mg/ml to 20 mg/ml showed great efficacy in treating joint conditions, such as OA.
Allometric scaling can be used to extrapolate human dosing from animal dosing, based on established relationships between metabolism, body size, and weight in different species. It is common practice to scale up dosing from one species to another by using the ratio of animal weights to the ¾ power:
Dose 2=Dose 1[(weight 2)**0.75/(weight 1)**0.75],
where Dose 2=human dose and Dose 1=rat dose. Typical rat and human weights are 0.3 and 70 kg, respectively. For example, to extrapolate a dose of 1 mg in a rat to human dosing, allometric scaling would predict:
Dose 2=(1 mg)*[(70 kg)**0.75/(0.3 kg)**0.75]=59.7 mg
For a glucosamine injection concentration of 0.018 mg/ml in a rat, the dose is 0.0009 mg based on an injection volume of 50 μL. The corresponding human dose using the above calculation would be 0.054 mg.
It is common to use volumes ranging from 1-10 ml of solution for intra-articular injection in humans. As such, the necessary glucosamine concentrations for human dosing, corresponding to 0.018 mg/ml in a rat, would be 0.0054-0.054 mg/ml. Using similar calculations for a rat injection concentration of 18 mg/ml, the corresponding human range would be 5.4-54 mg/ml.
One skilled in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein are expressly incorporated herein by reference in their entirety.
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| Number | Date | Country | |
|---|---|---|---|
| 20160206649 A1 | Jul 2016 | US |
