Oral peptide administration

Abstract

The present invention relates to compositions and methods that facilitate the non-invasive administration (e.g., oral administration) of therapeutic proteins or peptides (e.g., insulin) that retain biological activity when absorbed. The compositions of the invention comprise a therapeutic amount of a conjugate, the conjugate comprising a quantum dot and a therapeutically active peptide or protein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from Australian Provisional Patent Application No. 2020 / 900129, filed on 17 January 2020, the entire contents of which are incorporated herein by cross-reference.

[0002] The present invention relates to compositions and methods for administering therapeutic doses of polypeptides and proteins, such as insulin, to a subject via the oral route. [Background technology]

[0003] There are many diseases or conditions in which subjects are unable to produce enough peptides to maintain health.These conditions include, for example, insufficient human growth hormone, which leads to growth and development disorders, and lack of insulin, which leads to diabetes.There are many other examples of conditions that result from impaired peptide production in mammals.

[0004] Type 1 diabetes, also known as insulin-dependent diabetes or juvenile diabetes, is a chronic disease characterized by the inability of the pancreas to produce insulin. Insulin is a hormone produced exclusively by the islet cells of the pancreas to regulate the amount of glucose in the bloodstream. In non-diabetic subjects, when blood glucose levels rise, insulin is secreted into the bloodstream by the pancreas, causing glucose uptake by cells throughout the body, such as the muscle and liver, to be used for energy or stored as glycogen, thus leaving the bloodstream. When blood glucose levels decrease, insulin secretion also decreases.

[0005] The subjects diagnosed with type I diabetes cannot produce insulin, and therefore cannot regulate the amount of glucose in bloodstream.Excessively low and high blood glucose levels can both have serious health consequences.Although the subjects with type I diabetes can limit the fluctuations of blood glucose through non-pharmaceutical interventions, such as monitoring macronutrient intake and limiting the foods that are high in simple carbohydrates, they still need insulin to regulate glucose and take up glucose into cells, especially after eating.

[0006] Type II diabetes generally occurs in adults, especially those who are overweight. In the early stages of the disease, type II diabetes is associated with insulin resistance and increased circulating levels of insulin. A variety of oral and injectable medications are used to treat type II diabetes. Many people with long-term type II diabetes eventually require insulin therapy because insulin production becomes insufficient to regulate whole-body metabolism.

[0007] Diabetics typically monitor their blood glucose levels frequently throughout the day and must either ingest glucose if blood glucose levels are too low or administer insulin parenterally, usually via invasive subcutaneous injection, if blood glucose levels are too high. Insulin is a protein that relies on its tertiary structure for recognition by the insulin receptor and therefore cannot currently be delivered orally. This is because peptides such as insulin found in the gastrointestinal tract are too large to be absorbed and enter systemic cells intact, and the harsh environment of the gastrointestinal tract, with its low pH and proteases, disrupts this structure, rendering insulin ineffective. Proteins also typically cannot be absorbed in the intestine in their intact tertiary structure.

[0008] One approach is to provide formulations of insulin that contain excipients, such as chloroquine, which slow the breakdown of insulin in the stomach, as well as absorption enhancers, antioxidants, and binders. However, such formulations tend to be absorbed slowly in the intestine, resulting in an excessively long duration of action, and in some cases may provide longer insulin activity than commercially available "long-acting" injections. Such a prolonged activity profile may make it more difficult for a subject to maintain consistent blood glucose levels and / or may have a prolonged initial period of activity, making such formulations less able to rapidly treat hyperglycemic episodes.

[0009] Another approach to delivering insulin that avoids the gastrointestinal tract is to formulate insulin in dosage forms such as sprays, chewable gums, or lozenges that can be absorbed transdermally across the oral and nasal membranes. However, such formulations usually require excipients such as buffers, penetration enhancers, and stabilizers to protect the insulin from the environment, such as in microemulsion formulations. Such formulations also usually require refrigeration to ensure insulin integrity. It should also be noted that absorption through the oral and nasal membranes avoids hepatic metabolism.

[0010] Another approach is to tether insulin to nanoparticles for oral delivery. Such nanoparticles have previously been fabricated from biopolymers. In one such example, chitosan functionalized to a quaternary ammonium salt form was used as the core, then coated with hyaluronic acid for absorption through the mucosal layer of the intestinal tract. However, these nanoparticles require insulin release in the intestine for use; instead, they are too large (average size of approximately 120 nm) to allow for self-absorption. In another such example, gold nanoparticles with thiol-containing proteins attached to the gold surface have been described. However, these nanoparticles are most effective as transdermal agents for absorption across the oral and nasal mucosa, with poor intestinal absorption documented. As discussed below, sublingual absorption avoids hepatic first-pass metabolism.

[0011] While the above discussion has focused on insulin as a primary example of a protein or peptide-based outpatient therapy, problems exist in the current state of the art regarding insulin delivery for other proteins and peptides that may be therapeutically beneficial to a subject when formulated for oral administration.

[0012] Therefore, there is a need for compositions and methods that facilitate the noninvasive administration of therapeutic proteins or peptides (e.g., insulin) that maintain biological activity upon absorption. Desirably, the compositions and methods are orally administrable and allow for intestinal absorption and entry into the intestinal vasculature. This may provide a means for overcoming existing problems encountered during oral delivery of proteins and peptides such as insulin. Furthermore, in some specific applications, it may be desirable for such compositions and methods to act primarily on the liver, the primary target for insulin action, which may reduce circulating systemic levels of insulin and limit adverse effects such as hypoglycemia and weight gain. Summary of the Invention

[0013] The present invention aims to alleviate at least one existing deficiency in existing approaches for treating conditions resulting from insufficient endogenous peptide or protein production in a subject, and / or to orally administer proteins and peptides to treat such conditions.

[0014] In a first aspect of the present invention, a composition is provided comprising a therapeutic amount of a conjugate, the composition comprising a quantum dot and a therapeutically effective peptide or protein. The peptide or protein may be less than about 30 kDa in size. It may be 1-25 kDa, 3-25 kDa, 10-20 kDa, 5-30 kDa, or 15-30 kDa, i.e., it may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 kDa. It may have a primary amine group available for attachment to the QD. Each may be selected from the group consisting of insulin, growth hormone, fibroblast growth factor 21 (FGF21), glucagon-like peptide-1 (GLP-1) agonist, GLP-2 receptor agonist, platelet-derived growth factor (PDGF) beta receptor modulator, integrin alpha-4 / beta-7 antagonist, PYY(3-36) analog, vasopressin, interleukin (size less than 30 kDa), enkephalin, endorphin, or any other suitable protein or peptide, and combinations thereof. The insulin may be natural insulin (such as natural insulin from porcine origin), or may be a long-acting analog (such as glargine insulin or detemir insulin), an intermediate-acting analog (such as isophane insulin or intermediate-acting protamine Hagedorn insulin), or a rapid-acting analog (such as insulin aspart, insulin lispro, or insulin glulisine). The GLP-1 agonist can be selected from liraglutide or exenatide, or it can be any other suitable GLP-1 agonist. The GLP-2 agonist can be apraglutide, or it can be any other suitable GLP-2 agonist. The PDGF beta receptor modulator can be BOT191 (also known as fibroferon), or it can be any other suitable PDGF beta receptor modulator.The integrin alpha-4 / beta-7 antagonist can be PN-10943, or it can be any other suitable integrin alpha-4 / beta-7 antagonist. Other suitable peptides and proteins that are less than 30 kDa and contain at least one primary amine group may be known to those skilled in the art and would be suitable for QD conjugation.

[0015] The following options may be used individually or in any suitable combination in combination with either the first or second aspect.

[0016] The composition may be formulated for oral administration.

[0017] The quantum dots may be Ag2S quantum dots. The average diameter of the quantum dots may be about 1 nm to about 20 nm. It may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm. The average diameter of the quantum dots may be less than about 10 nm.

[0018] The conjugate may further comprise a polymer. The polymer may be a biopolymer. The biopolymer may cover at least a portion of the conjugate, or it may cover substantially all of the conjugate. The biopolymer may be selected from the group consisting of heparin, gelatin, hyaluronic acid, chitosan, galactose, glucose, and any combination thereof. The polymer or biopolymer may cover at least a portion of the conjugate. These polymers or biopolymers may protect the protein or peptide of the conjugate during transport through the digestive tract or may specifically target hepatocytes in the liver. The conjugate may accumulate within or on the surface of hepatocytes of a subject after oral administration.

[0019] The composition may be for administration to a subject who may have been diagnosed with a condition associated with insufficient endogenous peptide production. The condition may be type I or type II diabetes, or another condition associated with insufficient endogenous peptide production. The composition may be for administration to a subject who may have been diagnosed with a condition requiring treatment with an exogenous (i.e., non-naturally occurring) protein or peptide. The condition may be diabetic kidney disease, liver fibrosis, nonalcoholic steatohepatitis (NASH), renal fibrosis, celiac disease, inflammatory bowel disease (IBD), or other conditions associated with insufficient endogenous peptide production. D) ulcerative colitis or another gastrointestinal disease, which may be treated or treatable by delivering proteins or peptides to the liver, small intestine, kidney, pancreas or other gastrointestinal organs or tissues.

[0020] The composition may further comprise a pharmaceutically acceptable excipient.

[0021] In a second aspect of the present invention, there is provided a method of treating hyperglycemia in a subject in need thereof, the method comprising administering to the subject a therapeutic amount of a conjugate comprising a quantum dot and insulin.

[0022] In a third aspect of the present invention, there is provided a method of treating insufficient endogenous peptide production in a subject in need thereof, the method comprising administering to the subject a therapeutic amount of a conjugate comprising a quantum dot and a protein or peptide effective to replace the insufficient endogenous peptide.

[0023] In a fourth aspect of the present invention, there is provided a method of treating a subject suffering from a condition, wherein the condition is treatable by administration of a therapeutic exogenous peptide or protein, the method comprising administering to the subject a therapeutic amount of a conjugate comprising a quantum dot and a therapeutic exogenous peptide or protein.

[0024] The following options may be used individually or in any suitable combination, in combination with either the second, third or fourth.

[0025] The conjugate can be orally administered to the subject.The subject may be diagnosed with diabetes or another condition that causes an insufficient amount of endogenous peptide or protein in the subject's tissue.The diabetes that is treated in the subject can be type I diabetes or type II diabetes.

[0026] The quantum dots may be Ag2S quantum dots. The average diameter of the quantum dots is from about 5 nm to about 20 nm, or it may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm.

[0027] The conjugate may further comprise a polymer. The polymer may be a biopolymer. The biopolymer may at least partially coat the conjugate or may substantially coat the conjugate. The biopolymer may be selected from the group consisting of heparin, gelatin, hyaluronic acid, chitosan, galactose, glucose, and any combination thereof.

[0028] In a fifth aspect of the present invention, there is provided a method for delivering a peptide or protein to an organ of a subject, the method comprising orally administering to the subject a conjugate comprising a quantum dot and a peptide or protein, wherein the organ is selected from the liver, pancreas, small intestine or kidney.

[0029] The following options may be used in conjunction with the fifth aspect, either individually or in any suitable combination.

[0030] The quantum dots may be Ag2S quantum dots, which may have a diameter of about 5 nm to about 20 nm.

[0031] The conjugate may optionally include a polymer. The polymer may be a biopolymer. The biopolymer may be selected from the group consisting of gelatin, chitosan, galactose, glucose, and any combination thereof.

[0032] In a sixth aspect of the present invention, there is provided a method of lowering blood glucose in a subject, the method comprising orally administering to the subject a conjugate comprising quantum dots and insulin as described herein, wherein the quantum dots are Ag2S quantum dots of about 5 nm to about 20 nm, and the conjugate is metabolized on or bound to the surface of hepatocytes, thereby releasing insulin into the bloodstream of the subject.

[0033] In a seventh aspect of the present invention, there is provided a use of a conjugate comprising quantum dots and insulin as described herein for the manufacture of a medicament for the treatment of type I and type II diabetes.The medicament may be formulated for oral administration.The medicament may further comprise a polymer or biopolymer.

[0034] In the eighth aspect of the present invention, there is provided a use of a conjugate comprising a quantum dot and a protein or peptide in the preparation of a pharmaceutical.The pharmaceutical can be for treating insufficient endogenous production of a peptide or protein in a subject, or it can be for providing a therapeutically active exogenous peptide or protein.The pharmaceutical can be formulated for oral administration.The pharmaceutical can further comprise a polymer or biopolymer. [Brief explanation of the drawings]

[0035] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0036] [Figure 1]Schematic diagrams of the method for synthesizing and characterizing water-soluble Ag2S quantum dots of the present invention are shown. Specifically, (a) a schematic showing the production of cyclohexane-soluble QDs; (b) NIR II emission at 1175 nm with 658 nm excitation; (c) FTIR spectrum showing dodecane C-H chains on the surface of the QDs; (d) TEM image of the QDs showing their arrangement, size, and lattice structure; (e) a schematic of the phase transfer of COOH-capped QDs using a representative sample of 1 mM QDs in organic solvents and water; (f) FTIR spectrum showing OH functional groups with C=O, CO, and OH fingerprints; (g) TEM image of monodispersed QDs in water. [Figure 2] The changes in protein corona and synthetic surface topology on quantum dots are shown. Specifically, (a) FTIR of water-soluble QDs, (b-d) protein corona formation after 24 hours of incubation with (b) RPMI medium, (c) mouse serum, and (d) BSA. The protein corona shows the formation of amine functional groups (amines I and II). (f and g) Synthetic polymer deposition on the QD surface topology. The gray insert box shows the raw FTIR spectrum, and the gray arrows indicate the determinant peaks identified by the OPUS wizard. Conjugation of biopolymers to QDs was performed using click chemistry (EDC / NHS coupling). Both QD-heparin (e) and QD-gelatin (f) showed FTIR spectra similar to those of the raw biopolymer. The black arrows indicate the determinant peaks identified by the OPUS wizard, which correspond to the peaks shown in the insert. The peaks were confirmed to be amines I, II, and III. [Figure 3]The life cycle of Ag2S quantum dots after oral gavage is shown. Specifically, (a) time-lapse images of QD-488 after injection into the small intestinal lumen of an anesthetized mouse. After injection, the small intestine exhibits strong 488 fluorescence relative to the blood vessels (t = 0–3 min). At 5–6 min, the blood vessels exhibit 488 fluorescence relative to the surrounding tissue, and a gradual decrease in 488 fluorescence is observed in the small intestine up to 10 min post-injection. (b) The biodistribution of 3H-QDs was measured 30 min, 2 h, and 24 h after gavage of 100 μl of 1 mM QDs. 30 min after gavage, the liver showed accumulation of 60% of the ingested radioactive dose, with minimal expression in other organs. 2 h after gavage, the liver showed expression of 40% of the ingested radioactive dose. Over 24 h after gavage, fecal material showed 80% of the ingested radioactive dose, with minimal expression observed in organs. (c) Liver function tests were performed using AST and ALT assays and blood samples collected 24 hours after gavage. (d) H&E stained tissue samples from the liver, kidney, small intestine, and spleen showed no immune cell infiltration or cell necrosis. [Figure 4] Graph shows the blood glucose level of healthy rodents that are administered glucose gavage 30 minutes after being treated with either oral gavage of quantum dot-insulin conjugates equivalent to 20 IU / kg of insulin or intraperitoneal injection of active insulin equivalent to 0.1 IU / kg of insulin, and compared with a control group that does not receive any insulin therapy.Then, each subject is monitored for blood glucose for 90 minutes after receiving glucose gavage.The graph shows that both oral gavage of conjugates and intraperitoneal injection reduce the blood glucose of rodent subjects by approximately the same amount, and reduce the blood glucose spikes seen in the control group. [Figure 5]Blood glucose levels are shown for 3-month-old healthy C57BL / 6 mice (n = 5) that received either a sham subcutaneous injection of saline (control), a subcutaneous injection of insulin (SC-INS, 2 IU / kg), or oral gavage of oral insulin (QD-INS, 20 IU / kg) 30 min before the oral glucose tolerance test (oGTT). The oGTT was performed using a bolus oral gavage of glucose (2 g / kg), and blood was sampled via tail snip at -15, 0, 15, 30, 45, 60, and 90 min. Data in this figure show the mean ± SD and area under the curve (AUC). [Figure 6] This figure shows the pharmacological biodistribution of C-radiolabeled insulin when administered by subcutaneous injection or oral gavage to 3-month-old healthy C57BL / 6 mice (n = 3). This was done either 0.5 or 2.0 h before euthanasia and isolation of whole blood and whole organs (liver, kidney, spleen, and small intestine). Data are presented in this figure as the percentage of radiolabeled insulin relative to the administered dose. Data are shown as the mean ± SD. [Figure 7]Figure 5 shows the pharmacodynamic (PD) and pharmacokinetic (PK) effects of subcutaneous and oral insulin in 3-month-old healthy C57BL / 6 mice (n = 3). These mice were given either a sham subcutaneous injection of saline (control), a subcutaneous injection of insulin (SC-INS), or oral gavage of insulin (QD-INS) 0.5 h before an oral glucose tolerance test (oGTT). As shown in Figure 5, insulin treatment promoted a reduction in the AUC of the GTT. PD data were collected using 0.5, 1, and 2 IU / kg SC-INS or 10, 20, 30, 40, 50, and 100 IU / kg QD-INS. Data show the percentage effect size (%) on the oGTT AUC relative to 2 IU / kg SC-INS (i.e., relative to the effect size shown by SC-INS in Figure 4). PK / PD data were collected using data generated by oGTTs performed 0.5, 1.0, and 2.0 hours after administration of sham, 2 IU / kg SC-INS, or 20 IU / kg QD-INS. Data represent the percentage reduction in oGTT AUC, and data points represent the mean ± SD. [Figure 8] Figure 1 shows an insulin tolerance test in 4-month-old NOD / Scid mice (n=3) using subcutaneous (SC-INS) and oral (QD-INS) insulin. Mice showed the development of diabetes with increasing blood glucose concentrations. NOD mice with blood glucose levels below 30 mg / dl were treated with SC-INS (1 IU / kg) or QD-INS (25 IU / kg) at time 0, and mice with blood glucose levels above 31 mg / dl were treated with 4 IU / kg SC-INS or 100 IU / kg QD-INS. Blood glucose samples were collected at -15, 0, 15, 30, 45, and 60 minutes. The data in this figure show the change in blood glucose levels relative to the initial blood glucose concentration. Data points represent the mean ± SD. [Figure 9] Compared to the therapeutic dose of QG-insulin (25.6 ng / ml), in vivo toxicity of Ag2S QDs in 3-4 month old WT C57BL / 6J mice occurs at 256 μg / ml. [Figure 10]The effects of QD-liraglutide conjugates on (a) blood glucose levels and (b) body weight in 15-month-old WT C57BL / 6J mice are shown. Blood glucose levels (Figure 10a) were collected 30 minutes after intraperitoneal injection of 250 μg / kg liraglutide or 2 hours after oral gavage of QD-liraglutide at either 1250 μg / kg or 2500 μg / kg. Body weight (Figure 10b) was measured after three weekly treatments with oral QD-liraglutide at 2500 μg / kg. [Figure 11] Distribution of a 100,000 DPM dose of oral 14C-labeled metformin, 14C-labeled QD-metformin conjugate (NP-metformin), and 14C-labeled QD (NP) in 3-4 month old WT C57BL / 6J mice (n = 3 per treatment group) over 24 hours post-administration.

[0037] definition The following definitions are provided to enable those skilled in the art to better understand the invention disclosed herein, and are intended to be general and not to limit the scope of the invention to only these terms or definitions.

[0038] The term " blood glucose " as used herein refers to the amount of glucose circulating in the blood of the circulatory system of the subject.Therefore, the related term " lower blood glucose " or " reduce blood glucose " etc. should be understood to refer to the less glucose circulating in the blood of the subject compared to an earlier time, which may be before the administration of treatment.Similarly, the term " raise blood glucose " or " increase blood glucose " etc. should be understood to refer to the more glucose circulating in the blood of the subject compared to an earlier time.

[0039] Terms such as "hyperglycemia" and "hyperglycemic" refer to when a subject's blood glucose level is higher than the normal range of blood glucose in a healthy subject. Similarly, terms such as "hypoglycemia" or "hypoglycemic" refer to when a subject's blood glucose level is lower than the normal range of blood glucose in a healthy subject.

[0040] The term "quantum dot," as used herein, refers to nanoparticle materials having an average size distribution of less than about 20 nm. Such quantum dots also generally exhibit optical and / or electronic properties that differ from larger sized particles formed from the same material.

[0041] The term "conjugate" as used herein refers to an arrangement in which two or more species and / or structures are associated.For example, the quantum dots of the present invention can be associated with insulin protein chains to form quantum dot-insulin conjugates.This association can take any suitable chemical form, as long as two or more conjugate elements are found in close proximity.

[0042] The term "polymer," as used herein, refers to a molecule or macromolecule formed by linking monomers with covalent bonds. The monomers may be the same or different. The monomers may be formed into repeating subunits of monomers (e.g., polyethylene) or may have a non-repeating sequence (e.g., proteins). The term "polymer" is understood to encompass polymers of both synthetic and biological origin.

[0043] The term "biopolymer," as used herein, refers to a polymer chain of biological origin, whereby the polymer is produced by a living system and / or whereby the polymer chain is formed from monomer moieties that have a biological origin.

[0044] The terms "peptide" or "polypeptide," as used herein, refer to a short chain of amino acids joined in a sequence by peptide bonds, typically about 2 to about 50 amino acids in length. The term "protein," as used herein, refers to an amino acid chain longer than a peptide, i.e., about 51 or more amino acids in length.

[0045] The term "oral," in reference to administration of the compositions of the present invention, refers to delivery to the mouth of the subject to which the composition is administered, and it is expected that most, if not all, of the composition will be absorbed in the digestive tract. The term "oral" in the context of the present invention is not intended to refer to transdermal absorption across mucous membranes.

[0046] The term "administration" or variations including, but not limited to, "administer" or "administering," as used herein, refers to providing a therapeutic composition to a subject.

[0047] The terms "treat," "treatment," "therapy," "therapeutic," and the like, as used herein, refer to administering a composition that ameliorates or reduces the symptoms of an adverse medical condition in a subject to which the composition is administered. Thus, in the context of the present invention, the effect of administering a "therapeutic conjugate" includes reducing at least one of the symptoms of type 1 diabetes, such as lowering blood glucose in a hyperglycemic subject, which may be administered as a curative or preventative therapy.

[0048] The term "subject," as used herein, refers to any human or non-human animal to be treated. Thus, the compositions of the present invention may be suitable for human treatment, or for veterinary treatment of non-human animals, including companion animals such as cats and dogs, or farm animals such as pigs, horses, sheep, and cattle.

[0049] The term "hydrodynamic diameter," as used herein, refers to the degree of arrangement of water molecules close to the surface when the conjugate is dispersed in water. It is defined as the diameter of a perfect solid sphere that exhibits the same hydrodynamic friction as the conjugate when dispersed in water. In other words, because the surface of the conjugates described herein has an electrostatic charge, water molecules adjacent to the surface will be arranged and associated with the surface by electrostatic forces to provide an essentially defined water layer shell, and thus a particle with a hydrodynamic diameter equivalent to a solid particle without such water association.

[0050] As used herein, the term "comprising" means "including." Derivatives of the term "comprising," such as "comprise" and "comprises," have correspondingly different meanings. As used herein, the terms "comprising" and "including" are non-exclusive. As used herein, the terms "including" and "comprising" do not imply that a particular integer represents a major portion of a whole.

[0051] As used herein, the term "consisting essentially of" means "excluding other additional components that are intentionally added" or "only the elements listed below are intended to be present." Additional components that are in the defined composition or device but are not intentionally present are permitted. DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention relates to compositions for providing therapeutic peptides and proteins to a subject in need thereof. The compositions may be used in methods for treating insufficient endogenous peptide production in a subject, or in methods for delivering therapeutic exogenous peptides or proteins to a subject. The subject may suffer from a condition in which the level of at least one endogenous peptide is insufficient and requires replacement therapy. A well-known, non-limiting example of a condition in which a subject has insufficient endogenous protein production is type I diabetes. Thus, in one embodiment, the present invention is directed to lowering blood glucose in a subject and methods for lowering blood glucose in a subject. The subject may suffer from type I or type II diabetes, and the subject is unable to produce insulin and therefore requires therapy to maintain normal blood glucose levels. The subject may be hyperglycemic and require therapeutic doses of insulin to lower blood glucose levels. The subject may suffer from another condition in which the level of at least one endogenous peptide is insufficient and requires replacement therapy, an example of which is type I diabetes. The subject may be suffering from a condition requiring delivery of a therapeutic exogenous peptide or protein, such as, for example, PDGF receptor beta modulators in the treatment of diabetic nephropathy, liver fibrosis, nonalcoholic steatohepatitis (NASH) and renal fibrosis, GLP-2 receptor agonists in the treatment of celiac disease and other gastrointestinal diseases, or integrin alpha-4 / beta-7 antagonists in the treatment of inflammatory bowel disease (IBD) and ulcerative colitis. The compositions of the present invention can advantageously deliver therapeutic endogenous or exogenous peptides and proteins directly to organs or cells affected by the condition and / or organs capable of releasing the peptide or protein, in a process similar to the natural process for distributing endogenous or exogenous peptides and proteins.

[0053] In one embodiment, the composition of the present invention comprises a conjugate of insulin and quantum dots. The inventors have surprisingly found that such a conjugate can enter the circulatory system of a subject after oral administration and absorption through the intestinal wall. The quantum dots then deliver insulin to hepatocytes via the portal circulation, where insulin activates insulin receptors, and the conjugate is then taken up by hepatocytes by endocytosis for subsequent metabolism and excretion via bile. As will be explained in more detail below with reference to examples, insulin (as an example of an endogenous protein suitable for replacement therapy) can be effectively orally administered to a subject to reduce blood glucose levels, but those skilled in the art will recognize that such compositions and methods as exemplified herein can be extended to other suitable therapeutic peptides, particularly those that benefit from hepatic administration.

[0054] insulin Insulin is a peptide hormone produced and secreted by the beta or islet of Langerhans cells found in the pancreas of healthy subjects. In healthy subjects, insulin secretion usually occurs in response to high blood glucose levels in the subject, and acts to cause the subject's cells to take up glucose from the blood system, and excess glucose is polymerized and stored as glycogen in the liver and muscle, or converted into fatty acids for storage in adipose tissue as fat.

[0055] Structurally, insulin is a dimer of two chains linked via disulfide bonds, and this dimer is referred to herein as the insulin molecule. As will be understood by those skilled in the art, the resulting quaternary structure is important for insulin's activity in binding to insulin receptors embedded in cell membranes, where any disruption of the disulfide bonds or cleavage of the peptide chain is likely to result in little or no activity. Insulin administration is currently the only available therapy for treating type 1 diabetes, and for people with type 2 diabetes, Because insulin is also the primary treatment option, it is important to administer insulin in a manner that maintains the integrity of the quaternary structure of the insulin molecule.

[0056] Generally, the insulin currently used in human therapy is human insulin, produced through recombinant DNA technology. Insulin from other species, such as pigs, has also been used previously to treat humans. However, the amino acid sequence of insulin and the resulting structure of the resulting insulin vary slightly between species. For example, pig-derived insulin differs by one amino acid residue compared to the human sequence, and bovine insulin differs by three amino acid residues compared to the human sequence. These slight differences mean that using insulin from another species is unlikely to have the same effect as insulin produced by cells of the same species. However, cross-species administration can still be effective in therapy. For example, before recombinant DNA technology became available, insulin was routinely harvested from pigs for use in human therapy due to the similarity of the two amino acid sequences. Insulin derivatives are also available with varying lengths of activity. For example, insulin is available as a long-acting analog (e.g., glargine insulin or detemir insulin), an intermediate-acting analog (e.g., isophane insulin or neutral protamine Hagedorn insulin), or a rapid-acting analog (e.g., insulin aspart, insulin lispro, or insulin glulisine).

[0057] Thus, the present invention is not limited to the use of any particular amino acid sequence or species of origin insulin molecule. "Species of origin," as used herein, means that the insulin has the same sequence as that naturally produced by healthy members of the species of origin. The "species of origin" need not be the same as the species of interest. The insulin may be of a sequence identical or similar to the native sequence of the species of interest, or may be of a different species with a quaternary protein structure effective in treating the subject. The amino acid sequence is preferably native (i.e., produced by cells of the species of interest), but may be chemically modified, so long as any such modification does not significantly affect the effectiveness of the insulin in the subject after administration.

[0058] The insulin used in the present invention can be produced by any suitable method.For example, it can be produced, collected and purified by recombinant DNA technology, or can be collected from animal or mammalian cell culture collections that express insulin, or can be produced by chemical synthesis.It can also be produced as a salt.It can be used herein as the pharmaceutically acceptable salt of insulin.It can be a long-acting, intermediate-acting, or rapid-acting insulin derivative.

[0059] quantum dots The conjugates used in the present invention include nanoparticles referred to herein as quantum dots. By definition, quantum dots are nanoparticles that are relatively small, with diameters up to about 50-100 nm, but that exhibit different electronic and / or optical properties (or "optoelectronic" properties, as used interchangeably herein) compared to larger particles or bulk materials formed from the same substance. An example of an optical property of quantum dots that may be observed is photoluminescence, whereby wavelengths in the ultraviolet portion of the electromagnetic spectrum are absorbed by the quantum dot, exciting electrons to higher energy levels, which then break down into lower-energy electron shells and emit quanta of energy observed as wavelengths in the visible portion of the electromagnetic spectrum. An example of an electronic property of quantum dots that may be observed is superconductivity. These optoelectronic properties of quantum dots may depend on the size of the particle and the materials used in their construction. In the context of the present invention, the optoelectronic properties of quantum dots may be used diagnostically to determine the location of conjugates in different tissues of a subject after appropriate administration of compositions containing these conjugates using imaging techniques.

[0060] Quantum dots can be formed from any suitable material. By "suitable," we mean that the quantum dots must comprise a material capable of forming particles small enough to be characterized as quantum dots, exhibiting the optoelectronic properties characteristic of quantum dots, and capable of associating with proteins either natively on the material surface or after functionalization. "Functionalization" refers to the surface being altered after a chemical reaction. Because they are intended for therapeutic administration to a subject, the quantum dots of the present invention must also be non-toxic and well-tolerated by the subject. Because they are intended for the treatment of chronic conditions, they must also be substantially eliminated (i.e., not accumulate in the subject's body, tissues, or cells) soon after therapy. In this context, "substantial elimination" can mean that more than about 75% of the administered quantum dots are eliminated from the subject within a given time. Preferably, about 75% of the quantum dots are eliminated from the subject within 48 hours, 24 hours, or 12 hours after administration.

[0061] They may be core-type quantum dots formed from a single material, such as a chalcogenide (e.g., selenide, sulfide, or telluride) of a non-heavy metal, such as zinc or silver. They may be formed from materials containing or consisting of Ag2S, ZnS, or any other suitable material. They may be formed from Ag2S, which is understood to be non-toxic to mammals. They may also be core-shell quantum dots formed from two different materials, whereby a layer of a higher bandgap superconducting material is coated on the core. The shell layer must be made of a non-toxic material. A common example of a core-shell quantum dot is a shell layer made of CdSe applied to a core containing ZnS, but this layer material is not suitable for use in the present invention due to the presence of cadmium, a known toxic heavy metal. They may also be alloyed quantum dots, whereby two or more semiconductor materials are combined by alloying. Alloyed quantum dots may result in properties different from the bulk properties of either of the constituent materials. Quantum dots may be crystalline. Any of these types of quantum dots may be suitable for use in the present invention.

[0062] It is known that quantum dots can be manufactured, processed, or prepared using several different methods. The quantum dots of the present invention can be produced or developed by any suitable method currently known. For example, they can be produced by colloidal synthesis, plasma synthesis, self-assembly, or electrochemical assembly.

[0063] In the field of nanomaterials, there appears to be some disagreement regarding the acceptable upper size limit for quantum dots, distinct from nanoparticles, in terms of average particle size. Some published literature in this field defines quantum dots as less than 20 nm, others as less than 50 nm, and still others as between 1 nm and 100 nm. Quantum dots of the present invention are generally defined herein as particles having a diameter of less than 20 nm. Thus, each quantum dot disclosed herein may have a diameter of about 1 nm to about 20 nm, or about 1 nm to 5 nm, 5 nm to 10 nm, 10 nm to 20 nm, 5 nm to 15 nm, 1 nm to 15 nm, or 5 nm to 20 nm, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm. As explained in more detail below, this upper size limit is not due to any inherent limitation imposed by the material itself, but rather to limitations inherent in the process of absorption through the intestinal wall and endocytosis in hepatocytes of the liver, which are believed to be steps involved in the therapeutic effects provided by the present invention. Thus, quantum dots with average diameters greater than 20 nm, up to 50 nm, or up to 100 nm, for example, up to 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 50, or 100 nm, may be suitable for use in the present invention.

[0064] The quantum dots may be of any suitable shape. They may be spherical or mostly spherical. They may be irregularly shaped. A population of quantum dots may all have a uniform shape, or multiple shapes may be present in any given population.

[0065] Silver sulfide quantum dots In one embodiment, the quantum dots of the present invention may consist of or include Ag2S. Quantum dots composed of Ag2S are well known in the field of nanoparticles. They have been shown to have low or no mammalian toxicity and may also have near-infrared fluorescence. While they are typically prepared using a self-assembly method that results in Ag2S quantum dots with a hydrophobic coating, the Ag2S quantum dots of the present invention can be fabricated by any suitable method. Such hydrophobic Ag2S quantum dots are typically functionalized to have a hydrophilic coating. This functionalization can be performed using any suitable method that results in a hydrophilic coating. In one example, the hydrophilic reagent used may be a mercapto- or thiol-containing reagent that, when incubated with the hydrophobic quantum dots under suitable conditions, results in a hydrophilic surface chemistry on the Ag2S quantum dots. The surface of the Ag2S quantum dots may be completely or substantially covered with polar functional groups, such as carboxyl, hydroxyl, thiol, or amino functional groups. The functionalization to form a hydrophilic surface on the Ag2S quantum dots typically occurs in a polar solvent. The resulting hydrophilic Ag2S quantum dots are then generally stable, do not aggregate together, and can associate with insulin to form conjugates suitable for therapy.

[0066] Quantum insulin-dot conjugates The present invention includes the use of therapeutic conjugates. All conjugates referred to herein are stable associations formed between quantum dots and at least one insulin molecule. Standard chemistry may be used to conjugate insulin molecules to the surface of quantum dots. In one such example, the therapeutic conjugate may be formed by contacting suitable quantum dots, which may be hydrophilic or hydrophobic, with a coupling agent and insulin in a solvent, then incubating the mixture to form a crude therapeutic quantum dot conjugate. The contact of these three reagents can occur simultaneously, whereby all reagents are added together and incubated, or sequentially, whereby two of the three reagents, such as the quantum dots and the coupling agent, are added together and then allowed to react to completion before the third reagent is added. The crude therapeutic quantum dot conjugate may then, optionally, be purified by any suitable method to remove unreacted reagents. One such method may be a solid-state purification method that performs several cycles of washing with a suitable solvent and then filtering. Alternatively, the conjugate may be separated from the solvent by centrifugation and then used as part of a composition.

[0067] Coupling agents may be used to form amide or ester linker groups between the polar functional groups attached to the surface of the quantum dot and the conjugated insulin molecule, although those skilled in the art will understand that more than one bond may be formed between the quantum dot surface and insulin. In one example, the quantum dot may be functionalized to exhibit carboxyl groups on its surface, which may be linked to either the carboxyl or amino groups of insulin via amide or ester linker groups. Known and usable coupling agents include benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), carbodiimides such as dicylcohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), Any suitable coupling agent may be used, although examples include N-hydrosuccinamide and sulfo-N-hydroxysuccinamide (NHS).

[0068] Proteins such as insulin can be sensitive to environmental conditions such as temperature, shear, pH, salt concentration, and solvent. Depending on the protein, some conditions can cause irreparable damage to the protein's structure, and some can cause denaturation, rendering it ineffective. Therefore, when forming quantum dot conjugates, processing conditions must be carefully considered to ensure that insulin remains effective after conjugation with quantum dots.

[0069] The solvent in which the coupling reaction occurs can be polar or nonpolar, depending on the chemistry of the quantum dot surface and associated reagents. The solvent can be water. The solvent can be a protic polar organic solvent such as methanol, ethanol, butanol, or propanol. The solvent can be an aprotic polar organic solvent such as acetone, acetonitrile, or N,N-dimethylformamide (DMF).

[0070] When the solvent is water, salts may be added or present to approximate biological osmolality and maintain the integrity of the insulin molecule. The conjugation reaction can be carried out in a saline environment, whereby saline is water with dissolved salts such as NaCl and KCl present. A saline environment can estimate a plasma osmolality of approximately 300-312 mOsm / L. A buffer can be added to the water or saline to ensure that the pH of the reaction mixture does not cause denaturation of the insulin during the conjugation reaction. The buffer may maintain a biological pH of approximately pH 6 to approximately pH 8. The buffer may be, for example, a phosphate buffer, Tris buffer, citrate buffer, or glycine buffer, or any other suitable buffer. A buffer is generally not required in the organic solvent phase because hydronium ions are typically absent and therefore pH is not a factor.

[0071] The temperature at which the reaction occurs can be mild in any step involving insulin as a reagent. The temperature can be about 1°C to about 40°C, 1°C to 20°C, 10°C to 30°C, 5°C to 35°C, 20°C to 40°C, 15°C to 25°C, or 25°C to 35°C, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C. It can also be room temperature. The temperature can be consistent throughout the reaction or can vary throughout, such as with a 1-5°C fluctuation between the maximum and minimum temperatures, or a heating gradient with a cooling gradient. To avoid damage to insulin that can be caused by freezing (in hypotonic aqueous solutions) or heat-induced denaturation, respectively, it is expected that the minimum temperature will be greater than about 1°C and the maximum temperature will be less than about 40°C. If the reactions are performed sequentially, the reaction step involving only the quantum dots and coupling agent may occur at higher temperatures, such as 1°C-100°C, or 10°C-50°C, 25°C-75°C, 15°C-85°C, 35°C-65°C, 70°C-90°C, or 50°C-100°C, e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C.

[0072] Each reaction step in the reaction can occur over a period of about 5 minutes to about 10 hours, or about 5 minutes to 5 hours, 1 hour to 6 hours, 2 hours to 8 hours, 5 hours to 10 hours, or 30 minutes to 3 hours, e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours. The reagents may be left for a period of time long enough to ensure complete or substantially complete reaction.

[0073] biopolymers As described in more detail below, the quantum dot-insulin conjugate may also include a polymer. The polymer may be a synthetic polymer or a biopolymer. The biopolymer may be applied to the quantum dot-insulin conjugate so that the conjugate is at least partially coated with the biopolymer, or the biopolymer may be dispersed with the insulin in an amalgam layer. The conjugate may be completely coated with the biopolymer, or they may be substantially coated with the biopolymer. This outer layer containing the biopolymer may act to protect the insulin by extending its shelf life or by forming a physical barrier and reducing degradation after administration. When administered orally by swallowing or as a spray, it may enable or promote absorption across membranes such as the mesenteric membrane. It may enable targeted absorption by desired cell types after absorption by the subject after administration. The role of the biopolymer may be suitable for any one or more of these roles.

[0074] The biopolymer layer may comprise any suitable biopolymer depending on the desired effect, for example, if the desired effect is as a physical barrier, the biopolymer should be capable of forming a water-impermeable, and optionally air-impermeable, layer around the conjugate.

[0075] For example, biopolymers such as gelatin, heparin, or the absence of a biopolymer coating can result in conjugates with relatively small hydrodynamic diameters, thus favoring endocytosis of the conjugates into hepatocytes via clathrin-mediated endocytosis. Furthermore, certain biopolymers can protect insulin-QD conjugates from the acidic environment and digestive enzymes of the gastrointestinal tract. For example, biopolymers such as chitosan, galactose, or glucose, or combinations thereof, can protect the conjugates from gastrointestinal pH and digestive enzymes. These biopolymers, selected for their barrier properties, can also affect the hydrodynamic radius of the conjugates and, therefore, the uptake of these coated conjugates by liver cells. The surface chemistry of QDs, with or without a biopolymer coating, can be analyzed via FTIR to determine which groups are present (see Figure 2).

[0076] The biopolymer layer can be applied to the conjugate by any suitable process. For example, in one such method, biopolymer attachment can be carried out by the formation of an amide bridge between a carboxylic acid group on the QD and a primary amine group on the biopolymer. For example, this reaction can be carried out in the presence of N-hydroxysuccinimide (NHS) or its water-soluble analog (sulfo-NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). This reaction requires a pH change, which can be facilitated by the addition of HCl and NaOH solutions. Optionally, this method can also use an additional linker, such as adipic acid dihydrazide.

[0077] The thickness of the biopolymer layer, excluding interactions at its surface with water molecules, can be about 5 nm to about 25 nm, e.g., about 5 nm to 10 nm, 10 nm to 20 nm, 15 nm to about 25 nm, or 12 nm to 17 nm, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nm. The hydrodynamic diameter of the resulting conjugate, with or without the biopolymer coating, can be about 10 nm to about 100 nm, e.g., about 10 nm to 50 nm, or 20 nm to 40 nm, 25 nm to 75 nm, 50 nm to 100 nm, 30 nm to 80 nm. m, 40 nm to 60 nm, or 60 nm to 100 nm, for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nm.

[0078] Therapy The conjugates described herein comprise quantum dots and insulin, and optionally further comprise biopolymers, and can be used to treat type I or type II diabetes in subjects in need thereof.As mentioned above, insulin is the most effective therapy for insulin-dependent diabetes.These conjugates have been specifically developed by the present inventors as a therapy to provide insulin to diabetic subjects when needed, such as in a curative manner during hyperglycemic events, or in a preventative manner before meals, during meals, or when waking up.

[0079] Without being bound by theory, it is believed that these quantum dot-insulin conjugates are bound to the cell surface and infiltrate into cells in the subject's body, preferably the liver, and more preferably hepatocytes, where insulin can act to lower the subject's blood glucose. Quantum dot-insulin conjugates preferably target the liver, but they may also be found in other organs such as the small intestine immediately after oral administration, or in the kidney, spleen, and pancreas. Quantum dot-insulin conjugates specifically target hepatocytes, thereby reducing systemic hyperinsulinemia and associated adverse effects. It is envisioned that these conjugates can be administered to a subject by any suitable means that allows the conjugate to enter the subject's bloodstream and come into contact with cells that metabolize the conjugate and release insulin. For example, the conjugates can be administered by subcutaneous injection, similar to the common route of administration of insulin known in the art, although oral administration is preferred.

[0080] Oral administration One particularly advantageous and surprising route of administration for the delivery of these conjugates is oral administration. Oral dosage forms of insulin would avoid known problems associated with subcutaneous injection of insulin (and other proteins and peptides), such as poor subject compliance and the accumulation of scar tissue at typical injection sites, making further injections difficult. Oral dosage forms of the composition are expected to improve compliance and reduce the undesirable side effects associated with subcutaneous administration. "Oral administration" means that the subject takes the composition orally by swallowing a composition containing the conjugate for absorption in the digestive tract, rather than absorption across an oral membrane such as the sublingual mucosa.

[0081] A composition for oral administration of insulin comprises a quantum dot-insulin conjugate, optionally with a biopolymer coating. The composition may also contain pharmaceutically acceptable excipients, such as diluents (e.g., lactose, dextrin, silicate, magnesium salt, or calcium salt), binders (e.g., starch, cellulose, cellulose derivatives, or sugar alcohols), disintegrants (e.g., starch, cellulose derivatives, or alginates), flow agents (e.g., colloidal anhydrous silica and other silica compounds), preservatives (e.g., sodium benzoate, EDTA, sorbic acid, or parabens), antioxidants (e.g., BHA, BHT, tocopherol acetate), and lubricants (e.g., steric acids and their salts). Oral dosage forms may be in any suitable physical form, such as tablets, capsules, powders, suspensions, or solutions. As shown in Example 3 below, quantum dot-insulin conjugates can deliver active insulin to a subject's bloodstream without the addition of additional protective excipients, as evidenced by the effect on measured blood glucose after oral administration. Thus, without being bound by theory, it is believed that conjugating insulin to quantum dots disrupts the action of proteases and acids in the digestive tract, reducing pre-absorption degradation of insulin and resulting in delivery of active insulin to the liver of a subject. It is envisioned that the same protective effect applies to other proteins and peptides conjugated with quantum dots.

[0082] It is envisioned that suitable pharmaceutical compositions deliver the conjugates to the intestinal tract of a subject, from where they are absorbed through the lumen of the small intestine and into the capillaries of the small intestine. Thus, the hydrodynamic size of the conjugates comprising quantum dots, insulin, and optionally a biopolymer must be small enough to allow rapid absorption of the conjugates from the intestine into the intestinal capillaries.

[0083] Oral administration, and therefore the intestinal absorption route, is advantageous and superior to other absorption or administration methods, such as injection or transdermal administration, because blood in the small intestinal capillaries travels directly to the liver for first-pass metabolism. Advantageously, the conjugate is preferably metabolized in the liver rather than any other cells or organs of the subject. This is because the liver plays several important roles in maintaining glucose levels, including as a store of glucagon and a site of lipogenesis (i.e., producing fatty acids from excess glucose). Furthermore, insulin release from the liver more closely represents the natural release of insulin in healthy subjects compared with parenteral administration of activated insulin, which is the current preferred therapy.

[0084] Another advantage of oral administration of insulin is that when insulin is conjugated to quantum dots and optionally coated in a biopolymer, it may be further protected from the harsh conditions of the digestive tract by the biopolymer and / or may have improved absorption through the intestinal wall depending on the properties of the biopolymer.

[0085] Another advantage of oral administration is that the conjugate may be prevented or limited from entering the subject's systemic system. As shown in Example 1, most of the uncoated quantum dots described herein are absorbed immediately after administration, accumulate in the liver, and then removed by bile excretion, thereby preventing them from entering the subject's systemic circulatory system. It has also been shown herein that a biopolymer coating, when applied, can affect the distribution of the conjugate in the subject, further increasing its absorption and accumulation by the liver.

[0086] Diabetes treatment The conjugates of the present invention can be used in methods for treating diabetes in a subject, particularly with reference to Example 4 (insulin) or Example 6 (liraglutide). Treatment can be curative or can include administration of insulin to lower blood glucose in a subject who may be hyperglycemic. Treatment can be preventative or prophylactic, in that the diabetic subject may have normal blood glucose levels at the time of administration, but the subject may be expected to become hyperglycemic shortly after administration of the conjugate. For example, the conjugate may be taken orally before or with food that may be expected to raise the subject's blood glucose level, especially if the food is high in carbohydrates. The diabetes being treated can be type I diabetes or type II diabetes.

[0087] Treatment can include administering a composition comprising the conjugate of the present invention, whereby insulin is released into the subject's bloodstream after the liver metabolizes the conjugate. "Metabolism" means that the conjugate is cleaved intracellularly to form at least one insulin molecule and quantum dots, and insulin is released into the bloodstream, and quantum dots are excreted via the biliary system. Administration can be by any suitable route, for example, parenteral administration, for example, subcutaneous injection, oral administration, or transdermal administration. Preferably, the administration route is oral administration. Composition The product may contain pharmaceutically acceptable excipients that are suitable for the route of administration, and therefore depend on the route of administration, for example, excipients that are suitable for oral administration may not be suitable for parenteral administration.

[0088] The dose of insulin or liraglutide provided by the conjugate may be the same as or higher than the dose of insulin or liraglutide provided by conventional parenteral therapy. In humans, the standard dose of insulin is 1 international unit (IU), defined as 0.0347 mg. The dose of insulin administered to a subject may be about 0.1 IU / kg to about 100 IU / kg, or about 0.1 IU / kg to 15 IU / kg, 0.5 IU / kg to 20 IU / kg, 1 IU / kg to 10 IU / kg, 5 IU / kg to 25 IU / kg, 15 IU / kg to 30 IU / kg, 20 IU / kg to 50 IU / kg, 25 IU / kg to 75 IU / kg, 40 IU / kg to 80 IU / kg, 30 IU / kg to 70 IU / kg, 10 IU / kg to 90 IU / kg, or 1 IU / kg. It may be between 99 IU / kg or 2.5 IU / kg and 12 IU / kg, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 IU / kg. The dose of liraglutide administered to a subject may be between 100 μg / kg and about 3000 μg / kg, or between about 200 μg / kg and about 2500 μg / kg, or between about 1250 μg / kg and about 2500 μg / kg, for example, about 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1050, 1100, 1150, 1200, 125 .... It may be 50, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950 or 3000 μg / kg.The dose of oral QD-insulin conjugate required to achieve a therapeutic effect may be the same as or higher than the dose required to achieve a therapeutic effect of subcutaneous insulin (SC-insulin). The ratio of therapeutic doses of SC-insulin to oral QD-insulin may be about 1:1 to about 1:50, or about 1:5 to about 1:40, or about 1:10 to 1:25, or 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1: The saturation may be 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49 or 1:50.

[0089] The desired therapeutic effect of administration of the conjugates described herein is a reduction in blood glucose in a subject. This therapeutic effect may be evident immediately after administration, e.g., about 5 to about 90 minutes after administration, or about 5 to 30, 10 to 25, 10 to 15, 15 to 45, 20 to 50, 30 to 90, 40 to 80, 10 to 75, 60 to 90, 50 to 85, or 30 to 60 minutes after administration, e.g., 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes after administration. This therapeutic effect may be a long-acting effect or a short-acting effect. "Short-acting" means that a therapeutic blood glucose lowering effect is observed in a subject for a period of time up to about 2 hours after administration, or up to about 1.5 hours after administration, or up to about 1 hour after administration. "Long-acting" means that a therapeutic blood glucose lowering effect can be observed in a subject from about 2 hours to about 8 hours after administration, e.g., 2 hours to 4 hours, 4 hours to 8 hours, or 3 hours to 6 hours after administration, e.g., about 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, or 8 hours after administration. The length of observable activity can be affected by several factors, such as the composition and / or thickness or biopolymer coating, the hydrodynamic diameter of the conjugate, the time of administration, the excipients of the administered composition, or a combination of these factors.

[0090] Other therapeutic proteins and peptides In the description above and in the examples provided below, insulin has been used to demonstrate the effectiveness of conjugating a protein or peptide-based therapeutic with the quantum dots of the present invention to provide a dosage form of the protein or peptide-based therapeutic that maintains efficacy when orally administered to a subject. However, those skilled in the art will readily appreciate that, using the same techniques described herein, other proteins or peptides that are therapeutically effective but not orally bioavailable may also be suitable for conjugation with quantum dots for oral administration. In particular, proteins and peptides that are effective when delivered directly to a subject's liver, small intestine, pancreas, kidneys, or other organs of the digestive tract, but that are not currently orally bioavailable, may also be delivered to a subject in oral dosage forms using the present invention.

[0091] Proteins and peptides suitable for conjugation to the quantum dots described herein can be any therapeutically active protein or peptide up to about 30 kilodaltons (kDa) in size, e.g., about or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 kDa in size. To attach proteins or peptides to the QD surface via EDC / NHS, they also require available primary amine groups. Non-limiting examples of proteins and peptides containing primary amines suitable for conjugation with quantum dots of this size for oral administration as described herein include, for example, insulin (e.g., 51 amino acids; approximately 6 kDa) and its analogs or derivatives, growth hormone (e.g., 191 amino acids; approximately 22 kDa), glucagon-like peptide-1 (GLP), such as liraglutide (30 amino acids; approximately 4 kDa), and exenatide (39 amino acids; approximately 4 kDa). The inhibitor may be a glucagon-like peptide-2 (GLP-1) agonist, a glucagon-like peptide-2 (GLP-2) agonist such as apraglutide (33 amino acids; approximately 4 kDa), a platelet-derived growth factor (PDGF) beta receptor modulator such as BOT191 (fibroferon) (approximately 9 kDa), or an integrin alpha-4 / beta-7 antagonist such as PN-10943, vasopressin, interleukins (less than 30 kDa in size), enkephalins, or endorphins. These therapeutically effective proteins and peptides described above can be used to treat type I or type II diabetes (insulin and GLP-1 agonists), obesity (liraglutide), growth hormone deficiency, particularly in elderly subjects (growth hormone), diabetic nephropathy, liver fibrosis, NASH or renal fibrosis (BOT191), celiac disease and other gastrointestinal disorders (apraglutide), or inflammatory bowel disease or ulcerative colitis (PN-10943). The peptides and proteins described above are merely examples of current therapeutic agents that are not currently orally bioavailable, but which would be advantageous for subjects to be able to self-administer orally. [Example]

[0092] The invention disclosed herein may be better understood with reference to the following examples, which are not intended to be limiting.

[0093] Example 1 - Method for forming quantum dot-insulin conjugates Ag2S quantum dots are prepared by the process described in Figure 1. Essentially, diethyl Silver dithiocarbamate was mixed with 1-dodecanethiol and incubated at 200 °C for 1 h to produce soluble QDs in cyclohexane. The QDs were characterized by near-infrared emission at 1175 nm upon excitation at 658 nm (Figure 1b) and FTIR spectra showing C-H bonds on the surface (Figure 1c). To produce water-soluble QDs, a phase transfer reaction was then performed, in which the QDs were incubated with 3-mercaptopropionic acid in acetone / cyclohexane at room temperature for 1 h (Figure 1e). Further FTIR analysis of these water-soluble QDs revealed bonds indicative of carboxylic acid groups on the surface to which proteins or peptides could be conjugated (Figure 1f).

[0094] Conjugation of QDs and insulin is carried out using EDC / NHS, followed by the association of chitosan with galactose or glucose biopolymers. The QDs are incubated with EDC and NHS at room temperature for 1 hour under mixed conditions at pH 5-6. The pH is then changed to 9-10 using either NaCO3 buffer or NaOH. Insulin is then added to the solution under mixed conditions and incubated for 4-6 hours. The solution is then dialyzed in water for 2 hours, 4 hours, and overnight (16 hours) using 3500 or 10,000 kDa cut-off tubing. The solution is mixed with chitosan and galactose or glucose biopolymers at room temperature for 0.5-4 hours. The solution is then dialyzed in water for 2 hours, 4 hours, and overnight (16 hours) using 3500 or 10,000 kDa cut-off tubing.

[0095] Chitosan with galactose or glucose biopolymers is produced from a mixture of chitosan and galactose or glucose (ratios can be 1:1000 to 1000:1 [ratios can be 1:2]). Chitosan is prepared using 1% acetic acid at 70°C for 1-4 hours and filtered (1 μm filter). Conjugation of chitosan with galactose or glucose occurs using gradual, controlled cooling from 70 to 20°C over 1-2 hours. Purification of the material is performed using ethanol solution (50-100%) and centrifugation (chitosan and galactose or glucose biopolymers form a gel, dissolving the unconjugated material in ethanol).

[0096] Although the above method describes the conjugation of QDs to insulin, it is envisioned in this example that any therapeutic protein or peptide can be used in place of insulin to produce therapeutic QD conjugates for treating various diseases.

[0097] Example 2 - Uptake of insulin-quantum dot conjugates Conjugates of radiolabeled insulin and quantum dots were produced using the method described above, except that radiolabeled insulin was used instead of natural insulin. Two groups of healthy rodents were then orally administered a dose of either the conjugate or an equivalent radiolabeled unconjugated insulin. The distribution of radioactivity was then monitored 30 minutes after administration.

[0098] Approximately 50% of the radioactive label was found in the liver of rodents 30 minutes after oral administration, compared to approximately 1% of unconjugated radiolabeled insulin after 30 minutes, demonstrating that the conjugates of the present invention can deliver insulin to the liver via the oral route more effectively than without conjugation, and that conjugation has a protective effect in maintaining insulin availability in the gastrointestinal tract.

[0099] However, the preferential accumulation of QD-insulin conjugates in the liver is not a function of insulin, but rather a characteristic of the biopolymer-coated QDs. This is illustrated in Figure 3, whereby the QDs do not contain proteins conjugated to them. 3 H-labeled QDs were orally administered to a mouse model, and approximately 6% of the QDs were found in the liver 30 minutes after administration. In other words, any protein or peptide that can be conjugated to Ag2S QDs can be delivered to the liver after oral administration.

[0100] Example 3 - Glucose tolerance test with oral doses of conjugate A group of 30 healthy rodents without diabetic symptoms was obtained and divided into six treatment groups. One group of five rodents received 2 IU / kg insulin via intraperitoneal injection 30 minutes before glucose gavage. One group received 100 IU / kg insulin via oral gavage 30 minutes before glucose gavage. One group of five rodents received a dose of quantum dot-insulin conjugate equivalent to 100 IU / kg insulin 30 minutes before glucose gavage. One group of five rodents received a dose of quantum dot-insulin conjugate equivalent to 10 IU / kg insulin 30 minutes before glucose gavage, and one group of five rodents received glucose gavage alone. Blood glucose in all rodents was then monitored for 90 minutes after glucose gavage.

[0101] As can be seen in Figure 4, rodents in the control group that received no therapy experienced a sharp increase in blood glucose levels 15 minutes after receiving glucose, which gradually decreased over the course of the study. Conversely, neither the 2 IU / kg insulin given by intraperitoneal injection nor the quantum dot-insulin group recorded any increase in blood glucose levels, indicating that both the insulin (currently the preferred therapy) and the quantum dot-insulin conjugates of the present invention are effective in lowering blood glucose levels.

[0102] Most surprising is that the orally administered conjugates exhibited comparable, if not better, hypoglycemic effects than injected insulin, demonstrating that the quantum dot conjugates of the present invention can deliver insulin to a subject's bloodstream via oral administration and that the insulin is absorbed in an effective form that can affect blood glucose levels in subjects receiving oral forms of insulin.

[0103] Further studies were performed on healthy rodents without diabetic symptoms, comparing subcutaneous injections of saline (control), subcutaneous injections of insulin (SC-INS, 2 IU / kg), or oral gavage of oral insulin (QD-INS, 20 IU / kg) 30 min before a glucose tolerance test (oGTT). As shown in Figure 5, oral QD-insulin therapy was as effective in lowering blood glucose levels in mice as injected insulin.

[0104] The pharmacodynamics of QD-insulin conjugates were also investigated using radiolabeled insulin. Three mouse treatment groups were fasted for 4 hours and then administered either oral gavage of insulin (oral INS), subcutaneous injection of insulin (SC-INS), or oral gavage of QD-insulin conjugates (QD-INS) at a dose of 2 IU / kg. As shown in Figure 6, after 30 minutes, the orally administered therapy was primarily located in the small intestine, while the subcutaneously injected insulin was found in the liver and blood. However, after 2 hours, while oral insulin was still located in the intestine, approximately 50% of the radiolabeled insulin for both SC-INS and QD-INS was located in the liver. In fact, the distribution of insulin between SC-INS and QD-INS was nearly identical. This indicates that oral QD-peptide conjugate administration results in a post-administration biodistribution that mimics invasive subcutaneous administration.

[0105] Example 4 - Insulin tolerance test in a diabetic mouse model The above examples demonstrate the glucose-lowering effect of oral QD-insulin conjugates on a healthy mouse model. Similar experiments were also performed on a NOD / Scid mouse model, which showed the development of type 1 diabetes with increased blood glucose concentrations. Blood glucose was measured using a handheld blood glucose meter using Accu-check proforma strips. Blood was collected by tail vein sampling after tail snip. Subcutaneous (SC) insulin injection Blood glucose was collected at -15, 0, 15, 30, 45, and 60 min after gavage of either SC-insulin (0.1, 1, or 2 IU / kg) or QD-insulin conjugate (10, 20, 30, 40, 50, 75, or 100 IU / kg). As shown in Figure 8, QD-insulin doses approximately 25-fold higher than SC-insulin doses resulted in similar glucose reduction in the diabetic model.

[0106] Example 5 - Method for forming quantum dot-liraglutide conjugates Liraglutide was conjugated to QDs via EDC / NHS coupling, similar to insulin (see Example 1). 1 mM Ag2S QDs were mixed with 1 mM EDC and 1 mM NHS in a reaction vial under heavy mixing for 1 hour. The pH was then changed to 9.0 using 0.5 M NaCO3, and 1-10 mM liraglutide was added to the solution. The solution was mixed overnight, and 10,000 mM WCO was transferred to dialysis tubing and dialyzed against 1 ml of solution per 1 L of MQ for 2, 4, or 16 hours at 4°C in the dark. Polymer attachment to QD-liraglutide was also performed using the same method described above in Example 1.

[0107] Example 6 - Oral glucose tolerance test for liraglutide oGTT was performed in C57 / B16 mice after a 4-hour fast (see Figure 10). Blood glucose was measured using a handheld blood glucose meter with Accu-check proforma strips. Blood was collected by tail vein sampling after tail clipping. Blood glucose was collected at -30, -15, 0, 15, 30, 45, 60, and 90 minutes after an oral bolus of 2 g / kg glucose solution. At 120 minutes, mice received a subcutaneous (SC) liraglutide injection (250 μg / kg in saline), a gavage of QD-liraglutide-CS / GS (2500 μg / kg in water), or no treatment. Pharmacodynamics were determined based on the effect of liraglutide treatment on the area under the curve (AUC) of individual oGTT experiments. For pharmacodynamic measurements, the effects of SC-liraglutide and QD-liraglutide were determined relative to the reduction in AUC.

[0108] Example 7 - Other Therapeutic Agents The above examples are directed to conjugates of insulin and liraglutide, due in part to the ease of measuring the therapeutic activity of these therapeutic agents on blood glucose levels. In other words, insulin provides a good "proof-of-concept" model for determining whether orally administered proteins and peptides in such conjugates are protected and remain viable or capable of conferring therapeutic effects after absorption and intrahepatic transport. However, other proteins or peptides are also expected to remain viable when conjugated to Ag2S QDs and coated with biopolymers.

[0109] To determine whether other proteins or peptides are therapeutically effective after conjugation and oral delivery to the liver, one skilled in the art could (1) devise an assay, (2) measure the physiological effects of the targeted therapeutic peptide or protein or blood component by mass spectrometry, or (3) radiolabel the peptide or protein to determine whether the peptide or protein is delivered to the organ of greatest therapeutic interest (e.g., the liver, pancreas, or small intestine) and how long after administration it occurs. For example, if the peptide or protein is intended to treat a condition associated with inflammation (e.g., celiac disease or inflammatory bowel disease), it may be possible to measure blood markers associated with inflammation (e.g., IL-1β, IL-6, IL-8 (MIP-2), IFNγ, TFNα, etc.).

[0110] One example involves comparing oral administration of a conjugate containing Ag2S QDs and apraglutide (or other GLP-2 agonist) with subcutaneous injection of the same therapeutic peptide in a mouse model of celiac disease, similar to the comparative study described above. Following administration of the inhibitor, treated mice may be given an oral gavage of gluten and various blood markers, such as tTG-IgA, to be compared.

[0111] Another example involves comparing subcutaneous injections of human growth hormone with a therapeutic dose administered as a conjugate of QDs and human growth hormone, similar to the comparative study described above. Human growth hormone can be measured directly by known assays to determine blood levels of the hormone. The effectiveness of the administered hormone can also be determined by measuring IGF-1 (insulin growth factor-1) in the blood and / or by observing and comparing the growth of treated and untreated subjects (such as mice).

[0112] Assays for measuring the effects of other therapeutic peptides and proteins could be developed by those skilled in the art based on their knowledge of the particular disease process and the expected therapeutic effect of the protein or peptide.

Claims

1. 1. A composition comprising a therapeutic amount of a conjugate, the conjugate comprising Ag2S quantum dots, a therapeutically effective peptide or protein, and a polymer, the conjugate being formulated for oral administration.

2. The composition of claim 1 , wherein the peptide or protein is less than 30 kDa in size.

3. 3. The composition of claim 1 or 2, wherein the peptide or protein is selected from the group consisting of insulin, growth hormone, fibroblast growth factor 21 (FGF21), liraglutide, exenatide, apraglutide, fibroferon (BOT191), integrins, PYY(3-36) analogs, vasopressin, interleukins (less than 30 kDa in size), enkephalins, endorphins, and combinations thereof.

4. The Ag ２ 4. The composition of claim 1, wherein the average diameter of the S quantum dots is between 1 nm and 10 nm and / or the average hydrodynamic diameter of the conjugate is between 30 nm and 100 nm.

5. The composition according to any one of claims 1 to 4, wherein the polymer is a biopolymer containing any two or more selected from the group consisting of chitosan, galactose, and glucose.

6. The composition of any one of claims 1 to 5, wherein the polymer or biopolymer covers at least a portion of the conjugate.

7. The composition of any one of claims 1 to 6, wherein the conjugate accumulates within or on the surface of liver cells of a subject after oral administration.

8. The composition of any one of claims 1 to 7, wherein the composition is for administration to a subject diagnosed with a condition associated with insufficient endogenous peptide or protein production.

9. 9. The composition of claim 8, wherein the condition is type I or type II diabetes.

10. 8. The composition of any one of claims 1 to 7, wherein the composition is for administration to a subject diagnosed with a condition requiring administration of a therapeutic exogenous peptide or protein.

11. 11. The composition of claim 10, wherein the condition is selected from diabetic nephropathy, liver fibrosis, non-alcoholic steatohepatitis (NASH), renal fibrosis, celiac disease, inflammatory bowel disease (IBD), ulcerative colitis, or another gastrointestinal disease.

12. The composition of any one of claims 1 to 11, wherein the composition comprises a pharmaceutically acceptable excipient.

13. The composition of any one of claims 1 to 7 for treating hyperglycemia in a subject in need thereof.

14. 8. The composition of any one of claims 1 to 7 for treating insufficient endogenous peptide production in a subject in need thereof, wherein the therapeutically effective peptide or protein is a protein or peptide effective to replace the insufficient endogenous peptide.

15. 8. The composition of any one of claims 1 to 7 for treating a subject suffering from a pathological condition, wherein the condition is treatable by administration of a therapeutic exogenous peptide or protein, and wherein the therapeutically effective peptide or protein is the therapeutic exogenous peptide or protein.

16. 15. The composition of claim 14, wherein the condition is type I or type II diabetes.

17. 8. The composition of any one of claims 1 to 7 for delivering a peptide or protein to an organ of a subject, wherein the organ is selected from the liver, pancreas, small intestine, or kidney.

18. 8. The composition of any one of claims 1 to 7 for lowering blood glucose in a subject, wherein the therapeutically effective peptide or protein is insulin and the conjugate is metabolized in liver cells, thereby releasing the insulin into the bloodstream of the subject.

19. Use of a conjugate comprising Ag 2 S quantum dots, a polymer, and insulin for the manufacture of a medicament, formulated for oral administration, effective in the treatment of type I or type II diabetes.

20. Use of a conjugate comprising Ag 2 S quantum dots, a polymer, and a therapeutically active protein or peptide in the preparation of a pharmaceutical product formulated for oral administration.

21. 21. The use according to claim 20, wherein the medicament is for treating insufficient endogenous production of the peptide or protein in a subject.

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