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WO2017079505A1 - Rnase7 pour le traitement d'une infection bactérienne - Google Patents

Rnase7 pour le traitement d'une infection bactérienne Download PDF

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Publication number
WO2017079505A1
WO2017079505A1 PCT/US2016/060443 US2016060443W WO2017079505A1 WO 2017079505 A1 WO2017079505 A1 WO 2017079505A1 US 2016060443 W US2016060443 W US 2016060443W WO 2017079505 A1 WO2017079505 A1 WO 2017079505A1
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Prior art keywords
rnase
insulin
akt
subject
pi3k
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John David Spencer
Tad Eliot EICHLER
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Nationwide Childrens Hospital Inc
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Nationwide Childrens Hospital Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/11—Exodeoxyribonucleases producing 5'-phosphomonoesters (3.1.11)

Definitions

  • Diabetes mellitus is a systemic disease associated with a deficiency of insulin secretion or action. Diabetes is associated with many complications, including increased infection risk. With diabetes, the most common site of infection is the urinary tract. Urinary tract infection (UTI) is ten times more common in patients with diabetes. In diabetic patients, UTI is more likely to cause acute kidney injury, which increases the risk of chronic kidney disease. Goswami et al., Diabetes Res Clin Pract 53, 181-186 (2001). Complicated UTIs, including emphysematous pyelonephritis, renal abscess, and necrotizing papillitis are also more common.
  • UTI Urinary tract infection
  • Antimicrobial peptides a major component of the innate immune response, are potent antimicrobials that may be developed as novel UTI therapeutics. Recent evidence suggests that AMPs shield the urinary tract from invasive bacterial infection. Becknell et al., Nature reviews Nephrology, 11(11), 642-55 (2015). AMPs have considerable advantages over traditional antibiotics - including broad-spectrum antimicrobial activity at micromolar concentrations, rapid onset of action, limited microbial resistance profiles, and the ability to synergize with conventional antibiotics.
  • RNase 7 Ribonuclease 7
  • the inventors have identified Ribonuclease 7 (RNase 7) as an epithelial-derived AMP that helps maintain urinary tract sterility. Spencer et al, Kidney Int. 83(4), 615-25 (2013). RNase 7 kills Gram-positive and Gram-negative uropathogens at low micromolar concentrations. Spencer et al., Kidney Int 83, 615-625 (2013). It has been stated that, on a per molar basis, RNase 7 is the most potent human AMP. E. Boix, M. V. Nogues, Molecular bioSystems 3, 317-335 (2007).
  • PI3K/AKT pathway is required for a diverse array of cellular activities. Over the last decade, PI3K/AKT signaling has become a focal point in arenas like oncology, neurologic disorders, and diabetes. More recently, PI3K/AKT activity has been recognized to impact microbial pathogenesis and host defense. Williams et al., J Immunol 172, 449-456 (2004). PI3K/AKT is also a key regulator of the insulin-signaling pathway. T. Kondo, C. R. Kahn, J.B.C., 279, 37997-38006 (2004). When insulin binds to its receptor, members of the insulin receptor substrate (IRS) family are activated.
  • IRS insulin receptor substrate
  • IRS activates tyrosine kinase adaptor molecules on the cell membrane that recruit class I PI3K to the receptor complex.
  • PI3K phosphorylates phosphatidylinositol 4,5 bisphosphonate (PIP2) to generate phosphatidylinositol 3,4,5 triphosphate (PIP3) as a second messenger to recruit and activate downstream targets including AKT ( Figure 1). J. A. Deane, D. A. Fruman, Annual review of immunology 22, 563-598 (2004).
  • AKT also referred to as Protein Kinase B, is the key downstream effector of PI3K.
  • the invention provides a method of treating or preventing bacterial infection in a subject having metabolic syndrome, insulin resistance, or diabetes mellitus by administering a therapeutically effective amount of Ribonuclease 7 (RNase 7) and/or a phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) agonist to the subject.
  • RNase 7 Ribonuclease 7
  • PI3K/AKT phosphoinositide 3-kinase/protein kinase B
  • the subject has diabetes mellitus, while in further embodiments the subject has type II diabetes mellitus.
  • RNase 7 is administered to the subject, while in other embodiments a PI3K/AKT agonist is administered to the subject.
  • both RNase 7 and a PI3K/AKT agonist are administered to the subject.
  • the RNase 7 and/or the PI3K/AKT agonist can be administered in a pharmaceutically acceptable carrier.
  • the bacterial infection is a bacterial urinary tract infection, while in further embodiments the RNase 7 and/or the PI3K/AKT agonist are administered by bladder injection.
  • the invention provides a method of treating or preventing bacterial infection in a subject having metabolic syndrome, insulin resistance, or diabetes mellitus and receiving non-insulin anti-diabetic therapy, by administering a therapeutically effective amount of insulin to the subject.
  • the subject has diabetes mellitus, while in other embodiments the subject has type II diabetes mellitus.
  • the bacterial infection is a bacterial urinary tract infection.
  • Figure 1 provides a scheme showing the PI3K/AKT pathway.
  • Phosphatidylinositol-3 kinase PI3K
  • PIP2 phosphatidylinositol 4,5-bisphosphate
  • PIP3 phosphatidylinositol-3,4,5-trisphosphate
  • Akt also termed protein kinase B; mammalian target of rapamycin (mTOR).
  • Figures 4A-4D provide images showing the kidney collecting tubules express insulin receptor and RNase 7. Human kidney labeled for (a,b) insulin receptor, (c,d) RNase 7, and cell-specific markers.
  • AE-1 anion exchanger- 1; RNase 7, Ribonuclease 7; V-ATPase, vacuolar-type H+ adenosine triphosphatase.
  • FIG. 5 provides a graph showing insulin induces RNASE7 mRNA expression over time.
  • Primary human urothelial cells (HUC) and human renal epithelial cells (HRC) were cultured in insulin-free media and treated with recombinant human insulin (1 ⁇ / ⁇ ).
  • Quantitative real-time PCR shows insulin-induced RNASE7 expression over time.
  • the asterisk * indicates significant P values of ⁇ 0.05, compared with time zero control for each respective cell type, as determined by 1-way ANOVA with Tukey test.
  • ANOVA analysis of variance
  • HRC human renal epithelial cells
  • HUC human urothelial cells
  • PCR polymerase chain reaction.
  • Figures 6A-6D provide graphs and images showing insulin induces urothelial PI3K/AKT activity and RNase 7 peptide production.
  • Primary (a,b) HUC and (c,d) HRC were cultured in insulin-free media and stimulated with recombinant human insulin (1 ⁇ / ⁇ ) for the given time points. Cells were also pretreated with wortmannin (500 nmol/1) for 1 hour prior to insulin treatment.
  • RNase 7 was not detected by Western blot in the renal epithelial cells.
  • FIG. 7A-7C provide graphs showing PI3K/AKT regulates RNase 7 expression.
  • Human 5637 urothelial cells were transfected with plasmids encoding wt-AKT, DN-AKT, or m/p-AKT.
  • (b) Quantitative real-time PCR evaluated RNASE7 mRNA expression in transfected cells. Results are derived from 3 independent experiments where cells were transfected in quadruplicate (n 3; columns represent means +SEM).
  • ELISAs measured RNase 7 concentrations in the transfected cell culture media (columns represent means +SEM).
  • the asterisk * indicates significant P values of ⁇ 0.05, for the indicated pairwise comparison, as determined by the 1-way ANOVA with Tukey test.
  • ANOVA analysis of variance
  • DN-AKT dominant negative AKT
  • ELISA enzyme-linked immunosorbent assay
  • HA hemagglutinin
  • m/p-AKT membrane targeted AKT
  • pAKT phospho-AKT
  • PCR polymerase chain reaction
  • PI3K AKT phosphatidylinositide 3-kinase signaling pathway
  • wt-AKT wild-type AKT.
  • Figures 8A-8C provide graphs showing insulin-induced RNase 7 suppresses UPEC growth and shields the urothelium from UPEC.
  • UPEC growth was measured by changes in turbidity using the absorbance at 600 nm (OD 6 oo)- Addition of anti-RNase 7 antibody (solid black line) neutralized the antimicrobial activity of RNase 7, resulting in increased bacterial growth,
  • the asterisk * indicates significant P values of ⁇ 0.05, for the indicated pairwise comparison, as determined by the 1- way ANOVA with Tukey test.
  • the asterisk * indicates significant P values of ⁇ 0.05, for the indicated pairwise comparison, as determined by the 1-way ANOVA with Tukey test.
  • ANOVA analysis of variance; CFU, colony forming unit; LDH, lactate dehydrogenase; RNase 7, Ribonuclease 7; UPEC, uropathogenic E. coli.
  • Figures 9A and 9B provide images showing HlyA-producing UPEC isolates dephosphorylate urothelial AKT to suppress RNase 7.
  • Human 5637 urothelial cells were challenged with UPEC strains UTI89 and UTI89DhlyA for the indicated times. Shown are representative Western blots of cell lysates probed with antibodies specific for pAKT (ser473), RNase 7, and GAPDH.
  • GAPDH glyceraldehyde 3-phosphate dehydrogenase
  • HlyA oc-hemolysin
  • pAKT phospho-AKT
  • UPEC uropathogenic E. coli.
  • FIGs lOA-lOC provide graphs and images showing that RNase 7 overexpression shields urothelial cells from uropathogenic E. Coli.
  • Human urothelial cells UROtsa
  • UROtsa Human urothelial cells
  • A Representative Western blot of infected cell lysates confirms RNase 7 overexpression.
  • B/C Confluent UROtsa cells infected with empty vector or RNase 7 were incubated with 10 5 uropathogenic E. coli (UPEC, strain UTI-89).
  • Figures 11A and 11B provide graphs and images showing that silencing RNase 7 promotes uropathogenic E. Coli binding to human urothelial cells. RNase 7 was knocked- down in human 5637 urothelial cells (UROtsa) using siRNA.
  • UROtsa human 5637 urothelial cells
  • the present invention provides a method of treating or preventing bacterial infection in a subject having metabolic syndrome, insulin resistance, or diabetes mellitus.
  • the method includes administering a therapeutically effective amount of RNAse 7 and/or a PI3K/AKT agonist to the subject.
  • Also described is a method of treating or preventing bacterial infection in a subject having metabolic syndrome, insulin resistance, or diabetes mellitus and receiving non-insulin anti-diabetic therapy by administering a therapeutically effective amount of insulin to the subject.
  • diagnosis can encompass determining the likelihood that a subject will develop a disease, or the existence or nature of disease in a subject.
  • diagnosis also encompasses determining the severity and probable outcome of disease or episode of disease or prospect of recovery, which is generally referred to as prognosis).
  • treatment refers to obtaining a desired pharmacologic or physiologic effect.
  • the effect may be therapeutic in terms of a partial or complete cure for a disease or an adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and can include inhibiting the disease or condition, i.e. , arresting its development; and relieving the disease, i.e. , causing regression of the disease.
  • the term "preventing” includes either preventing the onset of a clinically evident disease (e.g., bacterial infection) altogether or preventing the onset of a preclinically evident stage of disease (e.g., bacterial infection) in a subject.
  • Preventative treatment can be particularly useful in subjects identified as having an elevated risk of developing a bacterial infection.
  • An elevated risk represents an above-average risk that a subject will develop a bacterial infection. Examples of elevated risk include exposure to bacterial infection, residence in a hospital, or a pre-existing condition that increases the risk of developing a bacterial infection such as diabetes or immunosuppression.
  • terapéuticaally effective and “pharmacologically effective” are intended to qualify the amount of an agent which will achieve the goal of improvement in disease severity and the frequency of incidence over treatment of each agent by itself, while avoiding adverse side effects typically associated with alternative therapies.
  • the effectiveness of treatment may be measured by evaluating a reduction in symptoms.
  • polypeptide and "peptide” as used herein, are used interchangeably and refer to a polymer of amino acids. These terms do not connote a specific length of a polymer of amino acids. Thus, for example, the terms oligopeptide, protein, and enzyme are included within the definition of polypeptide or peptide, whether produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. This term also includes polypeptides that have been modified or derivatized, such as by glycosylation, acetylation, phosphorylation, and the like.
  • subject and “patient” can be used interchangeably herein, and generally refer to a mammal, including, but not limited to, primates, including simians and humans, equines (e.g. , horses), canines (e.g. , dogs), felines, various domesticated livestock (e.g. , ungulates, such as swine, pigs, goats, sheep, and the like), as well as domesticated pets and animals maintained in zoos.
  • livestock e.g. , ungulates, such as swine, pigs, goats, sheep, and the like
  • Treatment and evaluation of human subjects is of particular interest. Human subjects can be various ages, such as a child (under 18 years), adult (18 to 59 years) or elderly (60 years or older) human subject.
  • the present invention provides a method of treating or preventing bacterial infection in a subject having metabolic syndrome, insulin resistance, or diabetes mellitus by administering a therapeutically effective amount of ribonuclease 7 (RNAse 7) and/or a phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) agonist to the subject.
  • RNAse 7 ribonuclease 7
  • PI3K/AKT phosphoinositide 3-kinase/protein kinase B
  • the methods of treating or preventing bacterial infection can be used against a variety of pathogenic bacteria including both gram-positive and gram-negative bacteria.
  • the bacterial infection is an infection of the urinary tract, an infection of the gastrointestinal tract, an infection of the respiratory tract, like, for example rhinitis, tonsillitis, pharyngitis, bronchitis, pneumonia, an infection of the inner organs, like, for example, nephritis, hepatitis, peritonitis, endocarditis, meningitis, osteomyelitis, an infection of the eyes, the ears as well as a cutaneous and a subcutaneous infection, diarrhea, skin disorders, toxic shock syndrome, bacteraemia, sepsis, and tuberculosis.
  • Preferred sites for treatment or prevention of bacterial infection using the claimed methods include treatment of bacterial infection of the urinary tract, skin and soft tissue, and respiratory tract.
  • the bacteria causing the bacterial infection are selected from the genus Staphylococcus, Streptococcus, Pseudomonas, Escherichia, Salmonella, Helicobacter, Neisseria, Campylobacter, Chlamydia, Clostridium, Vibrio, Treponema, Mycobacterium, Klebsiella, Actinomyces, Bacterioides, Bordetella, Borrelia, Brucella, Corynebacterium, Diplococcus, Enterobacter, Fusobacterium, Leptospira, Listeria, Pasteurella, Proteus, Rickettsia, Shigella, Sphaerophorus, Yersinia, or combinations thereof.
  • Examples of bacterial which cause urinary tract infection include Escherichia coli, Pseudomonas, Enterococcus, Enterobacter, Klebsiella, or Proteus mirabilis. The majority (80-85%) of bacterial urinary tract infections are caused by E. coli.
  • bacteria which cause bacterial respiratory tract infections include, but not limited to, Chlamydia species (e.g., Chlamydia pneumoniae), Klebsiella species, Haemophilus influenzae, Legionallaceae family, mycobacteria (e.g., Mycobacterium tuberculosis), Pasteur ellaceae family, Pseudomonas species, Staphylococcus (e.g., methicillin resistant Staphylococcus aureus and Staphylococcus pyrogenes), Streptococcus (e.g., Streptococcus enteritidis, Streptococcus Fasciae, and Streptococcus pneumoniae).
  • Chlamydia species e.g., Chlamydia pneumoniae
  • Klebsiella species Haemophilus influenzae, Legionallaceae family
  • mycobacteria e.g., Mycobacterium tuberculosis
  • bacteria which cause intestinal infections include, but not limited to Bacteroidaceae family, Campylobacter species, Chlamydia species (e.g., Chlamydia pneumoniae), Clostridium, Enterobacteriaceae family (e.g., Citrobacter species, Edwardsiella, Enterobacter aerogenes, Escherichia coli, Klebsiella species, Salmonella species, and Shigella flexneri), Gardinella family, Listeria species, Pasteur ellaceae family, Pseudomonas species, Streptococcus (e.g., Streptococcus enteritidis, Streptococcus Fasciae, and Streptococcus pneumoniae), Helicobacter Family.
  • Bacteroidaceae family Campylobacter species, Chlamydia species (e.g., Chlamydia pneumoniae), Clostridium, Enterobacteriaceae family (e.g., Citrobacter
  • the subject has sepsis caused by the bacterial infection.
  • Sepsis is the result of an overly strong immune response to infection, symptoms of sepsis include fever or low body temperature, rapid breathing, elevated heart rate, confusion, and edema, with early symptoms including fast heart rate, decreased urination, and high blood sugar.
  • Sepsis can be diagnosed using systemic inflammatory response syndrome (SIRS), which is the presence of two or more of the following: abnormal body temperature, heart rate, respiratory rate or blood gas, and white blood cell count.
  • SIRS systemic inflammatory response syndrome
  • Sepsis can also be diagnosed using sequential organ failure assessment score (SOFA score), which is the presence of two of increased breathing rate, change in level of consciousness, and low blood pressure.
  • SOFA score sequential organ failure assessment score
  • the bacterial infection is a bacterial urinary tract infection.
  • a urinary tract infection (UTI), as defined herein, is an infection of any part of the urinary tract.
  • the urinary tract includes the kidneys, the bladder, the urethra, and the ureter. Infection of the urinary tract typically results in a variety of symptoms, depending on the specific site of infection.
  • the urinary tract includes both the upper and lower urinary tract. The kidneys and most of the upper part of the ureters comprise the upper urinary tract, while the distal parts of the ureters, urinary bladder and urethra make up the lower urinary tract.
  • the urinary tract infection is in both the upper and lower urinary tract, while in other embodiments, the urinary tract infection is a lower urinary tract infection or an upper urinary tract infection.
  • a urinary tract infection affecting the lower urinary tract it is also referred to as a bladder infection (cystitis), while a UTI affecting the upper urinary tract it is also referred to as a kidney infection (pyelonephritis).
  • cystitis bladder infection
  • UTI affecting the upper urinary tract it is also referred to as a kidney infection (pyelonephritis).
  • Infection of the kidneys can result in upper back and side pain, high fever, shaking and chills, nausea, and vomiting.
  • Infection of the bladder e.g., cystitis
  • pelvic pressure can result in pelvic pressure, lower abdomen discomfort, frequent and painful urination, and blood in the urine.
  • Infection of the urethra typically can be diagnosed based on a burning sensation associated with urination.
  • One or more of these conditions can indicate a urinary tract infection, though it is preferable to confirm the presence of infection since there are other conditions such as irritation of the urethra, vaginitis, interstitial cystitis, or sexually transmitted diseases that can replicate some of these symptoms.
  • a fever will be present, and possibly other associated symptoms such as shaking and chills as well.
  • Urinary tract infections can be acute or chronic.
  • An acute UTI is typically short term (i.e. , less than one month) and of high intensity, whereas a chronic infection is a longer-term infection (i.e. , lasting at least one month, and up to a number of years).
  • a chronic infection and/or colonization the patient typically has bacteria growing in their bladder but they do not have symptoms typically associated with a urinary tract infection.
  • An acute infection is present when the patient has symptoms such as painful urination or fever.
  • a fever as defined herein, is a body temperature above 100 °F. If an acute infection is present simultaneously with a chronic infection, the effects of the acute infection will dominate those of the chronic infection in terms of overall characterization of the infection, for at least the reason that a chronic infection typically shows few effects.
  • the subject has an increased risk of having a urinary tract infection.
  • An increased risk refers to a higher likelihood or percent possibility of having a urinary tract infection in comparison with a subject who is not at an increased risk.
  • urinary tract infections occur most frequently in boys and girls during the first year of life. The likelihood of a urinary tract infection drops sharply after the first year, but then gradually increases with age. Gender is also a factor, with women having a high rate of UTIs due to physiological differences. The risk of having a UTI increases even further after menopause in women, and in pregnant women. Andriole, V.T., Patterson, T.F., Med. Clin. North. Am. 75, 359-373 (1991).
  • Urinary tract infections can be asymptomatic.
  • Asymptomatic bacteriuria is a colonization of a portion of the urinary tract by bacteria that does not display the symptoms typically seen for a urinary tract infection.
  • the urine samples obtained from a subject with asymptomatic bacteriuria may look infected (as evaluated by dipstick, for example) and will result in bacterial growth if cultured.
  • dipstick for example
  • Some types of subjects will be asymptomatic as a result of a lack of inflammatory response due to immunosuppression (e.g., transplant patients) or lack of sensation of symptoms as a result of, for example, having spinal cord injuries or congenital spinal/neural tube defects.
  • Diabetes mellitus is a serious metabolic disease that is defined by the presence of chronically elevated levels of blood glucose (hyperglycemia). This state of hyperglycemia is the result of a relative or absolute lack of activity of insulin, which is produced and secreted by the ⁇ -cells of the pancreas. Insulin promotes glucose utilization, protein synthesis, and the formation and storage of carbohydrate energy as glycogen. Glucose is stored in the body as glycogen, a form of polymerized glucose, which may be converted back into glucose to meet metabolism requirements. Under normal conditions, insulin is secreted at both a basal rate and at enhanced rates following glucose stimulation, all to maintain metabolic homeostasis by the conversion of glucose into glycogen.
  • diabetes mellitus encompasses several different hyperglycemic states. These states include Type I (insulin-dependent diabetes mellitus or IDDM) and Type II (non-insulin dependent diabetes mellitus or NIDDM) diabetes.
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin dependent diabetes mellitus
  • the hyperglycemia present in individuals with Type I diabetes is associated with deficient, reduced, or nonexistent levels of insulin which are insufficient to maintain blood glucose levels within the physiological range.
  • Treatment of Type I diabetes involves administration of replacement doses of insulin, generally by a parenteral route.
  • the hyperglycemia present in individuals with Type II diabetes is initially associated with normal or elevated levels of insulin; however, these individuals are unable to maintain metabolic homeostasis due to a state of insulin resistance in peripheral tissues and liver and, as the disease advances, due to a progressive deterioration of the pancreatic ⁇ -cells which are responsible for the secretion of insulin.
  • initial therapy of Type II diabetes may be based on diet and lifestyle changes augmented by therapy with oral hypoglycemic agents such as sulfonylureas. Insulin therapy is often required, however, especially in the latter states of the disease, in order to produce some control of hyperglycemia and minimize complications of the disease.
  • Diabetes mellitus is characterized by recurrent or persistent high blood sugar.
  • the presence of diabetes in a subject can therefore be identified by a random venous plasma glucose concentration of >11.1 mmol/1; a fasting plasma glucose concentration >7.0 mmol/1 (whole blood >6.1 mmol/1) and/or a two hour plasma glucose concentration >11.1 mmol/1 two hours after 75 g anhydrous glucose in an oral glucose tolerance test (OGTT).
  • Glycated hemogloblin levels >48 mmol/mol, or >6.5 DCCT% (Diabetes Control and Complications Trial) can also be used to diagnose diabetes mellitus.
  • the method of treating or preventing bacterial infection is used in a subject having metabolic syndrome or insulin resistance.
  • Metabolic syndrome and insulin resistance are two diabetes-associated disorders.
  • Metabolic syndrome is a clustering of at least three of five medical conditions, including abdominal (central) obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density lipoprotein (HDL) levels, and is thought to be caused by an underlying disorder of energy utilization and storage.
  • Type 2 diabetes is considered a complication of metabolic syndrome.
  • Insulin resistance is a pathological condition in which cells fail to respond normally to insulin. Cells senses insulin through insulin receptors, with the signal propagating through the PI3K AKT signaling pathway. When the body produces insulin under conditions of insulin resistance, the cells are resistant to the insulin and are unable to use it as effectively, leading to high blood sugar. Insulin resistance can be measured using the "hyperinsulinemic euglycemic clamp," which measures the amount of glucose necessary to compensate for an increased insulin level without causing hypoglycemia. Insulin resistance plays a key role in the development of type II diabetes mellitus. Insulin treatment and non-insulin anti-diabetic therapy
  • the present invention provides a method of treating or preventing bacterial infection in a subject having diabetes and receiving non-insulin anti-diabetic therapy, by administering a therapeutically effective amount of insulin to the subject. While a variety of alternatives to insulin administration have been identified, the inventors have shown that administration of insulin can be effective for treating or preventing bacterial infection in a subject, and can therefore be beneficial even when the diabetes mellitus is being treated by other methods.
  • Insulin is a peptide hormone produced by beta cells of the pancreatic islets.
  • the structure and amino acid sequence of insulin is known to those skilled in the art.
  • Insulin, as used herein includes insulin, effective insulin fragments, insulin analogs such as Humalog, and insulin produced by various species.
  • bovine insulin differs from human insulin by only 3 amino acids
  • porcine insulin differs from human insulin by only a single amino acid, and both can be used therapeutically in place of human insulin.
  • the insulin can be obtained from an animal source, or can be produced recombinantly.
  • Antidiabetic therapy includes administering a therapeutically effective amount of one or more non-insulin anti-diabetic agents to a subject.
  • non-insulin drugs for treating diabetes include sensitizers, secretagogues, oc-glucosidase inhibitors, and glycosurics.
  • non-insulin drugs for treating diabetes include biguanides, metformin, phenformin, buformin, thiazolidinediones (e.g., rosiglitazone, pioglitazone, and troglitazone), sulfonylureas (e.g., tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glibenclamide, glimepriride, gliclazide, glycopramide, and gliquidone), repaglinide, nateglinide, migitol, acarbose, voglibose, exenatide, liraglutide, taspoglutide, lixisenatide, vildagliptin, sitagliptin, saxagliptin, linagliptin, alogliptin, septagliptin, teneliglipt
  • the method of treating or preventing bacterial infection in a subject having diabetes includes administering a therapeutically effective amount of RNAse 7 to the subject.
  • Ribonuclease 7 is a member of a class of proteins (nucleases) that catalyzes the degradation of RNA into smaller components.
  • RNase 7 is 14.5 kDa protein that is part of the RNase A family of proteins.
  • RNase 7 The amino acid sequence of RNase 7 is described by Harder J, Schroder JM., J.B.C., 277, 46779-46784 (2002), the disclosure of which is incorporated herein by reference.
  • RNase 7, as defined herein, includes proteins having the defined amino acid sequence, regardless of the presence of normal biochemical modifications of the protein, such as glycosylation, as well as effective fragments of the RNase 7 protein that retain RNase 7 activity.
  • Peptides can be readily purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; ligand affinity chromatography, and the like. Peptides can also be readily purified through binding of a fusion polypeptide to separation media, followed by cleavage of the fusion polypeptide to release a purified polypeptide.
  • RNase 7 can also be prepared via recombinant techniques well known to those skilled in the art.
  • a polynucleotide sequence coding for an antimicrobial peptide can be constructed by techniques well known in the art. It will further be understood by those skilled in the art that owing to the degeneracy of the genetic code, a sizeable yet definite number of DNA sequences can be constructed to encode peptides having an amino acid sequence corresponding to RNase 7. Once the DNA sequence has been determined, it can be readily synthesized using commercially available DNA synthesis technology. The DNA sequence can then be inserted into any one of many appropriate and commercially available DNA expression vectors through the use of appropriate restriction endonucleases.
  • a variety of expression vectors useful for transforming prokaryotic and eukaryotic cells are well known in the art.
  • the DNA sequences coding for the peptide are inserted in frame and operably linked to transcriptional and translational control regions, such as promoters, which are present in the vector and are functional in the host cell.
  • the DNA sequence coding for the peptide can also be inserted into a system that results in the expression of a fusion protein that contains the RNase 7.
  • U.S. Pat. No. 5,595,887 describes methods of forming a variety of relatively small peptides through expression of a recombinant gene construct coding for a fusion protein that includes a binding protein and one or more copies of the desired target peptide. After expression, the fusion protein is isolated and cleaved using chemical and/or enzymatic methods to produce the desired target peptide.
  • the method of treating or preventing bacterial infection in a subject having diabetes includes administering a therapeutically effective amount of a PI3K/AKT agonist to the subject.
  • a PI3K/AKT agonist that induces RNase 7, a potent AMP, and that this can be effective for treating or preventing bacterial infection.
  • Phosphatidylinositol-3 -kinases are a family of related intracellular signal transducer enzymes capable of phosphorylating phosphatidylinositol, and are involved in cell growth and proliferation.
  • PKT Protein kinase B
  • AKT Protein kinase B
  • PI3K and AKT are both kinases involved in the intracellular signaling pathway important in regulating the cell cycle, and can be activated using PI3K/AKT agonists. More specifically, AKT is the key downstream effector of PI3K. Examples of PI3K/AKT agonists include statins, piaglitazone, bilobalide, and caffeine. Small interfering RNA have also been shown to be effective for activating the PI3K/AKT pathway. Kim et al, Immunol Lett., 134, 47-54 (2010). Administration and Formulation
  • the invention also provides pharmaceutical compositions that can be used for the administration of active agents used in the method of the invention to a subject in need thereof.
  • active agents include RNase 7, PI3k/AKT agonists, and insulin.
  • the method of the invention includes administration of an RNase 7 peptide fragment and a pharmaceutically acceptable carrier.
  • the pharmaceutical acceptable carrier can have many forms, including tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow-release formulations, such as shaped polymeric gels.
  • An oral dosage form may be formulated such that the polypeptide or antibody is released into the intestine after passing through the stomach. Such formulations are described in U.S. Patent No. 6,306,434 and in the references contained therein.
  • Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.
  • the active agents can be formulated for parenteral administration (e.g. , by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative.
  • the pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical compositions suitable for rectal administration can be prepared as unit dose suppositories. Suitable carriers include saline solution and other materials commonly used in the art.
  • the active agent(s) can be administered by bladder injection.
  • active agents can be conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the active agents may take the form of a dry powder composition, for example, a powder mix of a modulator and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.
  • the active agents may be administered via a liquid spray, such as via a plastic bottle atomizer.
  • Active agents can be formulated for transdermal administration.
  • the active agents can also be formulated as an aqueous solution, suspension or dispersion, an aqueous gel, a water-in-oil emulsion, or an oil-in-water emulsion.
  • a transdermal formulation may also be prepared by encapsulation of an active agent within a polymer, such as those described in U.S. Pat. No. 6,365,146.
  • the dosage form may be applied directly to the skin as a lotion, cream, salve, or through use of a patch. Examples of patches that may be used for transdermal administration are described in U.S. Pat. Nos. 5,560,922 and 5,788,983.
  • a pharmaceutical composition may be formulated as a single unit dosage form, or it may be administered in multiple doses.
  • the amount of active agent that is delivered to the subject will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the subject has undergone. Ultimately, the attending physician will decide the amount of active agent with which to treat each individual patient. For example, the attending physician can administer low doses of the active agent of the present invention and observe the patient's response. Larger doses of the active agent may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ng to about 100 mg (preferably about 0.1 ⁇ g to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of active agent per kg body weight
  • Example 1 Insulin and the phosphatidylinositol 3-kinase signaling pathway regulate
  • Diabetes mellitus is a systemic disease associated with a deficiency of insulin production or action.
  • Diabetic patients have an increased susceptibility to infection with the urinary tract being the most common site.
  • RNase 7 Ribonuclease 7
  • RNase 7 is a potent antimicrobial peptide that plays an important role in protecting the urinary tract from bacterial insult. Because the impact of diabetes on RNase 7 expression and function are unknown, the effects of insulin on RNase 7 were investigated using human urine specimens. The urinary RNase 7 concentrations were measured in healthy control patients and insulin- deficient type 1 diabetics before and after starting insulin therapy. Compared with controls, diabetic patients had suppressed urinary RNase 7 concentrations, which increased with insulin.
  • Diabetic patients have low urinary RNase 7 concentrations that increase with insulin therapy
  • Triple labeling immunofluorescence shows that the intercalated cells, identified by positive anion exchanger- 1 (AE-1) or vacuolar-type Hp adenosine triphosphatase (V-ATPase) staining, coexpress IR and RNase 7 (Figure 4).
  • AE-1 positive anion exchanger- 1
  • V-ATPase vacuolar-type Hp adenosine triphosphatase
  • HUC primary human urothelial cells
  • HRC primary human renal epithelial cells
  • RT-PCR Quantitative real-time polymerase chain reaction
  • Insulin induces RNase 7 peptide production via PI3K/AKT
  • AKT regulates urothelial RNase 7 expression
  • PI3K/AKT regulates urothelial RNase 7 expression
  • wt-AKT wild-type AKT
  • PKBa constitutively active membrane targeted-protein kinase Ba
  • DN-AKT the dominant negative PKBa-K170A/T308A/S473A
  • the m/p-AKT mutant contains a myristoylation signal that causes AKT translocation to the plasma membrane resulting in AKT hyperactivation.
  • the DN-AKT mutant which contains the mutations K179A (substitution of alanine for lysine at position 179), T308A (substitution of alanine for threonine at position 308), and S473A (substitution of alanine for serine at position 473), is a kinase-inactive, phosphorylation-deficient PKBa/AKTl construct.
  • Western blot demonstrates that urothelial cells transfected with m/p-AKT had substantially elevated levels of AKT (ser473) phosphorylation, whereas DN-AKT transfected cells had suppressed AKT (ser473) phosphorylation (Figure 7a).
  • Insulin-induced RNase 7 production shields urothelial cells from UPEC
  • UPEC inhibits urothelial PI3K/AKT activity and RNase 7 production
  • bladder cells were challenged with UPEC.
  • the inventors used 5637 urothelial cells because they, like most transformed cells, have greater endogenous PI3K/AKT activity.
  • Previous evidence demonstrates that UPEC strains expressing oc-hemolysin (HlyA) inhibit PI3K/AKT activation in these cells. Wiles et al, Mol. Biol Cell 19, 1427-1438 (2008). Consistent with these studies, Western blot demonstrates that the HlyA producing UPEC strain UTI89 dephosphorylates AKT (Figure 9).
  • FIGS lOA-lOC show that RNase 7 overexpression shields urothelial cells from uropathogenic E. coli.
  • Human urothelial cells (UROtsa) were infected with recombinant retrovirus expressing full-length RNase 7 or empty vector.
  • UROtsa Human urothelial cells
  • A Representative Western blot of infected cell lysates confirms RNase 7 overexpression.
  • B/C Confluent UROtsa cells infected with empty vector or RNase 7 were incubated with 10 5 uropathogenic E. coli (UPEC, strain UTI-89).
  • B The percentage of UPEC adhering to the apical surface of UROtsa cells is markedly reduced when RNase 7 is overexpressed.
  • FIGS 11 A and 11B show that silencing RNase 7 promotes uropathogenic E. coli binding to human urothelial cells.
  • RNase 7 was knocked-down in human 5637 urothelial cells (UROtsa) using siRNA.
  • UROtsa human 5637 urothelial cells
  • B Confluent 5637 cells transfected with RNase 7 or control siRNA were incubated with 10 5 uropathogenic E. coli (UPEC, strain UTI-89). The percentage of UPEC adhering to the apical cell surface significantly increases when RNase 7 is silenced.
  • the asterisk (*) indicates significant p- values ⁇ 0.05, for the indicated pairwise comparisons, as determined by the student's t-test.
  • Page and Malik44 used an experimental model of type 2 diabetes (T2DM) and found that rBD-1 mRNA expression was elevated in diabetic GK rat kidneys. This same research group also demonstrated that prolonged hyperglycemia increased hBD- 1 mRNA and peptide expression in human embryonic kidney 293 cells. Malik et al., Biochem Biophys Res Commun., 353, 318-323 (2007). In the work described herein, human urinary RNase 7 concentrations did not correlate with serum or urinary glucose concentrations.
  • PI3K/AKT regulates RNase 3/eosinophil cationic peptide and murine eosinophil-associated RNase degranulation and secretion from human and murine granulocytes.
  • PI3K/AKT inhibition alters host inflammatory and innate immune responses, increases infection risk, and decreases survival. Wortmannin inhibition of PI3K/AKT increases morbidity and mortality following experimental endotoxemia and polymicrobial sepsis.
  • HlyA-expressing UPEC strains are associated with more severe UTI symptoms and are routinely identified in cases of pyelonephritis and bacteremia.
  • the inventors identify insulin as a PI3K/AKT agonist that induces RNase 7, one of the most potent AMPs in the human urinary tract.
  • additional studies and novel models to evaluate the role of PI3K/AKT activity, insulin signaling, and RNase 7 production on host defense in vivo would be beneficial.
  • Informed written consent was obtained from all patients. For subjects less than 18 years of age, written parental/guardian consent and patient assent were obtained. The National Children's Hospital (NCH) Institutional Review Board approved this study (IRB 14-00376). Serial urine specimens were collected from patients presenting to the NCH Emergency Department with new-onset TIDM. Follow-up urine specimens were collected at least 30 days after starting insulin therapy at the NCH diabetes clinic. Control urine samples were obtained from children 1 to 18 years of age presenting to the NCH Emergency Department. Control patients had no history of abnormal renal function, structural urinary tract disorders, history of UTI, recurrent infection, or endocrine disorders. They were evaluated for nonfebrile minor medical complaints. Human kidney samples, provided by the Cooperative Human Tissue Network, were obtained from patients undergoing nephrectomy. Tissue samples were free of microscopic signs of disease or inflammation.
  • Sections were labeled for principal cells with a goat polyclonal anti-AQP-2 antibody (1 :500; Santa Cruz Biotechnology, Santa Cruz, CA) and intercalated cells with a chicken polyclonal anti-V- ATPase El subunit antibody (1 :2000; Sigma- Aldrich) or a mouse monoclonal anti-AEl antibody (1:2000) Bacterial strains
  • E. coli strains CFT073, UTI89, and UTI89DhlyA were used.
  • UTI89 is a type I- piliated UPEC strain isolated from a patient with cystitis.
  • CFT073 is a UPEC strain isolated from the blood and urine of a patient with pyelonephritis.
  • UTI89DhlyA has disruption of the hlyA gene.
  • hlyA gene deletion was verified by PCR. The hemolytic activity of each UPEC strain was identified and confirmed by growth on blood agar plates at 37 °C.
  • the 5637 human bladder epithelial cells (ATCC HTB-9; American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT) at 37 °C in 5% CO 2 . After reaching confluence, human 5637 cells were serum starved overnight. Urothelial monolayers were challenged with UPEC using a multiplicity of infection (MOI) of 25 to 30 microbes per host cell. Wiles et al, Mol Biol Cell., 19, 1427- 1438 (2008).
  • MOI multiplicity of infection
  • human bladder urothelial cells were transfected with pCMV5 -hemagglutinin (HA)-PKBa (wt-AKT), pCMV5-HA- membrane targeted-PKBa (m/p-AKT), or pCMV5-HA-AAAHA-PKBa (DN-AKT) (purchased from D. Alessi, University of Dundee, Dundee, UK).
  • the 5637 cells were grown in 24-well plates to 60% confluency.
  • Cells were then transfected with 0.6 ⁇ g/well of wt- AKT, DN-AKT, or m/p-AKT using Lipofectamine LTX according to the manufacturer's instructions (Invitrogen, Grand Island, NY). Transfection efficiency of approximately 75% was determined via phase and fluorescence microscopy 48 hours after transfection with a vector containing an irrelevant protein fused to mCherry. At 48 hours after transfection, media from transfected cells were used for ELISA analysis and protein lysates were isolated for Western blot.
  • Kidneys from 3 patients were cut into 150-mg sections and placed in insulin-free RenaLife basal culture medium with the appropriate inhibitors. Kidneys incubated in medium only served as control kidneys. After 4 hours of incubation at 37 °C, kidneys were homogenized and sodium dedecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) Western blot was performed.
  • SDS-PAGE sodium dedecylsulfate polyacrylamide gel electrophoresis
  • CFT073 was cultured as outlined for the UPEC growth inhibition assay and 5 ml was added to 50 ml of media. When indicated, 1 ml of a polyclonal anti-RNase 7 antibody (Biomatik) was added to each test isolate 30 minutes before UPEC inoculation. After 3 hours of incubation at 37 °C, 10 ml of the incubation mixture was plated on Luria-Bertani agar and the number of CFUs was determined the following day.
  • a polyclonal anti-RNase 7 antibody Biomatik
  • HRCs were cultured in RenaLife basal culture medium with supplemental RenaLife Lifefactors in 96-well plates. Once reaching 80% to 90% confluency, HRCs were cultured overnight in insulin-free RenaLife media. Next, cells were stimulated with insulin for 24 hours and then challenged with 10 3 CFU/ml UPEC (CFT073) for an additional 5 hours. When indicated, 2 ml of a polyclonal anti-RNase 7 antibody (Biomatik) was added 45 minutes prior to UPEC challenge. Per manufacturer's instructions, LDH release into the culture media was measured using the LDH Cytotoxicity Detection Assays (Takara Bio, Kyoto, Japan).
  • cytotoxicity (%) [(experimental value - low control)/(high control - low control)] x 100, where culture media served as the low control and HRCs treated with 1% Triton X-100 served as the high control.

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Abstract

L'invention concerne un procédé de traitement ou de prévention d'une infection bactérienne chez un sujet présentant un syndrome métabolique, une insulinorésistance ou un diabète sucré. Le procédé comprend l'administration d'une quantité thérapeutiquement efficace de RNase 7 et/ou d'un agoniste de PI3K/AKT au sujet. L'invention concerne également un procédé de traitement ou de prévention d'une infection bactérienne chez un sujet présentant un syndrome métabolique, une insulinorésistance ou un diabète sucré et recevant un traitement antidiabétique non insulinique par administration d'une quantité thérapeutiquement efficace d'insuline au patient.
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WO2010008995A2 (fr) * 2008-07-14 2010-01-21 Otonomy, Inc. Compositions et procédés modulant l’apoptose à libération contrôlée pour le traitement de troubles otiques
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