Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • 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
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/10—Drugs for disorders of the urinary system of the bladder
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/06—Gastro-intestinal diseases
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/34—Genitourinary disorders
  • G01N2800/348—Urinary tract infections

Definitions

• the invention relates to characterization of the role of Tamm-Horsfall protein (THP) in interstitial cystitis. In patients with interstitial cystitis, the Tamm-Horsfall protein shows abnormal sialyation.
• the invention provides methods for diagnosing, inhibiting and monitoring the course of cystitis.
• the invention also provides pharmaceutical compositions comprising sialyated Tamm-Horsfall protein.
• Mucus is critical in regulating epithelial permeability of the bladder, particularly to low molecular weight solutes. 10"12 ' 25 ' 26 Additional studies have demonstrated that IC patients have significant impairment of epithelial permeability regulation 13"16 that leads to a movement of small cations (potassium) diffusing into the membrane and interstitium of the bladder 1"3 ' 18 . This diffusion initiates a cascade of nerve depolarization, muscle depolarization, and generation of symptoms of urgency, frequency, pain, and incontinence 1"3 ' 8 ' 17 ' 18 ' 27 ' 28 .
• THP Tamm-Horsfall protein
• interstitial cystitis was regarded as a rare disease whose symptoms and progression were difficult or impossible to control. More recent evidence has shown that IC is a relatively common disorder in both women and men, and that most cases can be treated successfully.
• disorder of the lower urinary tract and specifically, to the inhibition of interstitial cystitis, diagnosis of interstitial cystitis, and reducing the symptoms (including treatment) of interstitial cystitis in vivo are described.
• the present invention provides methods for inhibiting Interstitial Cystitis and its symptoms in a subject.
• the method comprises administration of an effective amount of a Tamm-Horsfall protein to the subject, so as to inhibit Interstitial Cystitis and its symptoms in the subject.
• the present invention provides a method for reducing symptoms of Interstitial Cystitis in a subject.
• the method comprises administration of an effective amount of Tamm-Horsfall protein to the subject.
• the invention also provides a method for repairing a mucin layer of bladder in the subject by increasing the levels of Tamm-Horsfall protein in a subject.
• the levels of Tamm- Horsfall protein are increased in a subject by administering an effective amount of Tamm-Horsfall protein to the subject.
• Also provided by the invention is a method for treating a disease associated with decreased levels of Tamm-Horsfall protein.
• the method comprises increasing the levels of Tamm-Horsfall protein in a subject by administering an effective amount of Tamm- Horsfall protein so as to treat the disease associated with reduced levels of Tamm- Horsfall protein.
• the method comprises quantitatively determining in the urine from the subject, the levels of Tamm-Horsfall protein and comparing the amount of Tamm-Horsfall protein so determined to the amount in a sample from a normal subject. The decrease in the amount of Tamm-Horsfall protein in the subject compared to the normal subject is indicative of Interstitial Cystitis.
• the invention also provides a method for monitoring the course of Interstitial Cystitis in a subject which comprises quantitatively determining in a first sample of a urine from the subject the levels of Tamm-Horsfall protein and comparing the amount so determined with the amount present in a second sample from the subject, such samples being taken at different points in time, a difference in the levels of Tamm-Horsfall protein determined being indicative of the course of Interstitial Cystitis.
• the invention further provides a method for screening for agents that modulate production of Tamm-Horsfall protein.
• the method comprises contacting Tamm-Horsfall genes in Tamm-Horsfall positive cells with a molecule of interest and then determining whether the contact results in increased Tamm-Horsfall production. An increase in Tamm-Horsfall production is indicative that the molecule modulates production of Tamm-Horsfall genes.
• Also provided in this invention is a method for screening for agents that modulate production of Tamm-Horsfall protein comprising contacting Tamm-Horsfall protein in Tamm-Horsfall positive cells with a molecule of interest and determining whether the contact results in increased Tamm-Horsfall production, an increased Tamm-Horsfall production being indicative that the molecule modulates production of Tamm-Horsfall protein.
• the invention further provides a method for screening for agents that modulate sialyation of Tamm-Horsfall protein.
• the method comprises contacting Tamm-Horsfall protein in Tamm-Horsfall positive cells with a molecule of interest and determining whether the contact results in increased sialyation of Tamm-Horsfall protein.
• An increase in sialyation of Tamm-Horsfall protein is indicative of modulation of sialyation of Tamm-Horsfall protein.
• Also provided in this invention is a pharmaceutical composition
• a pharmaceutical composition comprising Tamm Horsfall protein and a pharmaceutically acceptable carrier.
• the invention further provides a kit comprising the pharmaceutical composition that comprises the Tamm-Horsfall protein and a pharmaceutically acceptable carrier.
• Figure 2 Representative tracing of rat urodynamic studies.
• A CMG showing smooth voiding contractions of a normal bladder during slow infusion of saline (40 ⁇ l/60 sec).
• B CMG showing smooth voiding contractions of a normal bladder during slow infusion of saline (40 ⁇ l/60 sec).
• a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c.
• a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b.
• reducing and “reducing the symptoms of,” “reducing interstitial cystitis,” and “reducing the symptoms of interstitial cystitis” refer to lowering, lessening and relieving of any one or more of urinary urgency and frequency, and/or pelvic pain.
• the patient may determine if interstitial cystitis symptoms are reduced.
• reducing interstitial cystitis may be determined by the physician's evaluation.
• reducing interstitial cystitis may be determined from comparing a PUF scale score to a previous PUF scale score.
• reducing interstitial cystitis is reducing symptoms in patients whose symptoms indicate, and are similar to, interstitial cystitis.
• the term "known therapeutic compound” refers to a compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
• the term "therapeutic" when made in reference to a compound refers to a compound that is capable of reducing, delaying, or eliminating one or more undesirable pathologic effects in a subject.
• interstitial cystitis and "IC” refers to a progressive disorder of the lower urinary tract that causes the symptoms of urinary frequency, urgency, and/or pelvic pain in a wide variety of patterns of presentation.
• An example of a recent review is Parsons, Clin Obstet Gynecol, 45(l):242-249 (2002).
• urinary frequency refers to the number of urination times per day.
• urinary urgency refers to an inability to delay urination.
• pellet pain refers to pain in the pelvic region of genital and non-genital origin and of organic or psychogenic aetiology.
• urine As used herein, “urinate,” “urination,” “urinating,” “void” and “voiding” refers to release of urine from the bladder to the outside of the body.
• urine refers to a liquid waste product filtered from the blood by the kidneys, stored in the bladder and expelled from the body through the urethra by the act of urinating.
• oral and “by oral administration” refers to the introduction of a pharmaceutical composition into a subject by way of the oral cavity (e.g. in aqueous liquid or solid form).
• oral agent refers to a compound that can be administered by way of the oral cavity (e.g. in aqueous liquid or solid form).
• still refers to one or more of the following; to drop in, to pour in drop by drop, to impart gradually, to infuse slowly, to cause to be imbibed, (e.g. infuse slowly an intravesical solution).
• intravesical refers to inside the bladder.
• intraavesical instillation refers to solutions that are administered directly into the bladder.
• instillation is via catheterization.
• intravesical solution refers to a treatment that can be administered to the bladder.
• intravesical therapy is a combination of an oral and an intravesical agent. It is not intended that the present invention be limited to a combination of an oral and an intravesical agent.
• intravesical therapy is an intravesical agent.
• intravesical therapy is a combination of intravesical agents.
• extravesical refers to outside the bladder.
• cystoscopic examination and “cystoscopy” refers to an examination that uses a cytoscope.
• cystoscope refers to an endoscopic instrument to visualize the lower urinary tract, which includes the bladder and the urethra.
• urethra refers to a tube draining the urine to the outside.
• bladedder refers to a hollow muscular organ that stores urine until it is excreted from the body.
• the terms "subject” and "patient” refer to any animal, such as a mammal. Mammals, include but are not limited to, humans, murines, simians, felines, canines, equines, bovines, porcines, ovines, caprines, rabbits, mammalian farm am ' mals, mammalian sport animals, and mammalian pets. In many embodiments, the hosts will be humans. In one embodiment, a patient has one or more of urinary urgency, urinary frequency, pelvic pain, recurrent urinary tract infections, dyspareunia, overactive bladder, dry, etc.).
• urinary tract infections refers to a condition that includes an inflamed urethra and painful urination.
• a urinary tract infection is caused by bacteria.
• a urinary tract infection is not caused by bacteria.
• recurrent urinary tract infections refers to frequent episodes of urinary tract infections.
• dispareunia refers to pain during intercourse.
• overactive bladder refers to a sudden involuntary contraction of the muscular wall of the bladder causing urinary urgency, an immediate unstoppable need to urinate and a form of urinary incontinence.
• urinary incontinence refers to the unintentional loss of urine and inability to control urination or prevent its leakage.
• urinary continence refers to a general ability to control urination.
• catheter refers to a tube passed through the body for draining fluids or injecting them into body cavities. It may be made of elastic, elastic web, rubber, glass, metal, or plastic.
• catheterization refers to the insertion of a slender tube through the urethra or through the anterior abdominal wall into the bladder, urinary reservoir, or urinary conduit to allow urine drainage.
• catheterized refers to the collection of a specimen by a catheterization.
• sample and “specimen” are used in their broadest sense and encompass samples or specimens obtained from any source.
• biological samples refers to samples or specimens obtained from ' animals (including humans), and encompasses cells, fluids, solids, tissues, and gases.
• Biological samples include tissues (e.g., biopsy material), urine, cells, mucous, blood, and blood products such as plasma, serum and the like.
• tissues e.g., biopsy material
• urine e.g., urine
• cells e.g., mucous, blood
• blood products such as plasma, serum and the like.
• these examples are not to be construed as limiting the types of samples that find use with the present invention.
• Urine cytology refers to an examination of a urine sample that is processed in the laboratory and examined under the microscope by a pathologist who looks for the presence of abnormal cells.
• urinary dysfunction and “urinary tract dysfunction” refers to abnormal urination, patterns or bladder habits, including wetting, dribbling and other urination control problems.
• esthesia refers to a loss of feeling or inability to feel pain.
• local anesthesia refers to a method of pain prevention in a small area of the body.
• phrases “pharmaceutically acceptable salts”, “a pharmaceutically acceptable salt thereof or “pharmaceutically accepted complex” for the purposes of this application are equivalent and refer to derivatives prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.
• lower urinary epithelial dysfunction refers to disorders with positive potassium sensitivity tests (e.g. IC, prostatitis and the like).
• urinary dysfunction refers to abnormal urination, patterns or bladder habits, including wetting, dribbling and other urination control problems.
• sialyation is the modification of glycoproteins with sialic acid.
• Sialic acid is also known as N-acetylneuraminic acid (N-acetymeuraminate).
• N-acetymeuraminate N-acetymeuraminate
• Sialic acid is often found as a terminal residue of oligosaccharide chains of glycoproteins.
• Sialic acid imparts negative charge to glycoproteins.
• Tamm-Horsfall protein is sialyated.
• repairing is restoring to original or close to original condition.
• repairing the mucin layer is restoring the mucin layer of
• repairing the mucin layer is restoring the function of the mucin layer of Interstitial Cystitis patients to mat or normal patients.
• the function of the mucin layer is restored to 50% to that of normal patients.
• the function of the mucin layer is restored to 75% to that of normal patients.
• the function of the mucin layer is restored to 100% to that of normal patients.
• level of a protein is the amount of protein present.
• the level of Tamm-Horsfall protein in a subject is the amount of Tamm- i Horsfall protein present in the urine sample taken from the subject.
• the level of sialyated Tamm-Horsfall protein in a subject is the amount of sialyated Tamm-Horsfall protein present in the urine sample taken from the subject.
• M molar
• mM millimolar
• ⁇ M micromolar
• nM nanomolar
• mol molecular weight
• mmol millimoles
• ⁇ mol micromoles
• nmol nanomoles
• g grams); mg (milligrams); ⁇ g (micrograms); pg (picograms)
• L liters
• mL milliliters
• ml milliliters
• ⁇ L microliters
• cm centimeters
• mm millimeters
• ⁇ m micrometers
• nm nanometers
• the present invention provides a method for inhibiting Interstitial Cystitis and its symptoms in a subject.
• the method comprises administering to the subject an effective amount of Tamm-Horsfall protein.
• the invention further provides a method for increasing the levels of Tamm-Horsfall protein in a subject.
• the method comprises administering to the subject an effective amount of Tamm-Horsfall protein.
• THP may be increased from about 0.1 to 200mg/L urine, about 0.5 to 200mg/L urine, about 1 to 200mg/L urine, about 5 to 200mg/L urine, about 10 to 200mg/L urine, about 25 to 200mg/L urine, about 50 to 200mg/L urine, about 75 to 200mg/L urine, about 100 to 200mg/L urine, about 125 to 200mg/L urine, about 150 to 200mg/L urine, about 175 to 200mg/L urine, about 0.1 to 150mg/L urine, about 0.5 to 150mg/L urine, about 1 to 150mg/L urine, about 5 to 150mg/L urine, about 10 to 150mg/L urine, about 15 to 150mg/L urine, about 20 to 150mg/L urine, about 25 to 150mg/L urine, about 50 to 150mg/L urine, about 75 to 150mg/L urine, about 100 to 150mg/L urine, about 120 to 150mg/L urine, about 130
• increasing the levels of Tamm-Horsfall protein in a subject repairs the mucin layer of the bladder.
• increasing the amounts of Tamm-Horsfall protein in a subject treats diseases that are associated with decreased levels of Tamm-Horsfall protein.
• the Tamm-Horsfall protein administered to a subject is sialyated.
• Sialyation of Tamm-Horsfall protein increases the negative charge of Tamm-Horsfall protein, this making it more anionic. This results in entrapment of cations in the urine, thus inhibiting Interstitial Cystitis or reducing the symptoms of Interstitial Cystitis.
• the amount of sialyation of Tamm-Horsfall protein may be vary such that the Tamm- Horsfall protein may be sialyated at wild-type levels or at greater than wild-type levels.
• a reduced dosage of sialyated Tamm-Horsfall protein may be administered to treat Interstitial Cystitis and its symptoms.
• a person skilled in the art would determine the effective dosage of sialyated Tamm-Horsfall protein that may be administered.
• sialyation of Tamm-Horsfall protein may be increased from about 50 to 3000 pM/ ⁇ g THP, about 100 to 3000 pM/ ⁇ g THP, about 200 to 3000 pM/ ⁇ g THP, about 300 to 3000 pM/ ⁇ g THP, about 400 to 3000 pM/ ⁇ g THP, about 500 to 3000 pM/ ⁇ g THP, about 600 to 3000 pM/ ⁇ g THP, about 700 to 3000 pM/ ⁇ g THP, about 800 to 3000 pM/ ⁇ g THP, about 900 to 3000 pM/ ⁇ g THP, about 1000 to 3000 pM/ ⁇ g THP, about 1200 to 3000 pM/ ⁇ g THP, about 1400 to 3000 pM/ ⁇ g THP, about 1600 to 3000 pM/ ⁇ g THP, about 1800 to 3000 pM/ ⁇ g THP, about 2000 to 3000 pM/ ⁇ g THP, about 2
• increasing the total amount of Tamm-Horsfall protein in a subject inhibits Interstitial Cystitis and its symptoms.
• an increase in the total amount of the Tamm-Horsfall protein may inhibit or reduce the symptoms of Interstitial Cystitis in a subject.
• a person skilled in the art would determine the effective dosage of the Tamm-Horsfall protein that may be administered.
• an effective amount of Tamm-Horsfall protein or sialyated Tamm-Horsfall protein administered to a subject in order to inhibit or reduce symptoms of Interstitial Cystitis is about 0.1 to 200 mg/day, 0.1 to 150 mg/day, 0.1 to 100 mg/day, about 0.5 to 5 mg/day, about 5 to 50 mg/day, about 5 to 10 mg/day, about 10 to 15 mg/day, about 15 to 20 mg/day, about 20 to 25 mg/day, about 25 to 30 mg/day, about 30 to 35 mg/day, about 35 to 40 mg/day, about 40 to 45 mg/day, about 45 to 50 mg/day, about 50 to 55 mg/day, about 55 to 60 mg/day, about 60 to 65 mg/day, about 65 to 70 mg/day, about 70 to 75 mg/day, about 75 to 80 mg/day, about 80 to 85 mg/day, about 85 to 90 mg/day, about 90 to 95 mg/day, about 95 to 100 mg/day, about 2 to 10 mg/
• dosage range will vary depending on the intensity and duration of the Interstitial Cystitis symptoms. Further, it would be clear to one skilled in the art that dosage range will vary depending on the age, sex, height and/or weight of the subject and the stage at which Interstitial Cystitis is diagnosed.
• the Tamm-Horsfall protein is administered directly in to the urinary tract in a subject.
• the Tamm-Horsfall protein may be administered directly into the urinary tract using a catheter 36"38 (Cecil Textbook of Medicine (1992) J.B. Wyngaarden et al., eds., 19 th ed., W.B. Saunders Co.; and Textbook of Surgery (1991) D. Sabiston, ed., 14 th ed., W.B. Saunders Co.).
• the Tamm-Horsfall protein may be administered directly in to the urinary tract using a time-release system.
• time-release systems include but are not limited to a balloon like device regulated to deliver a drug for 30 days, or a catheter system that is connected to a time release mechanism such as a pump, or a time release capsule inserted into the bladder.
• the Tamm-Horsfall protein administered directly in to the urinary tract of the subject may be sialyated.
• the invention also provides a method for diagnosing Interstitial Cystitis in a subject comprising quantitatively determining in the urine from the subject, the levels of Tamm- Horsfall protein.
• the level of Tamm-Horsfall protein from the subject is compared to level of Tamm-Horsfall protein in a urine sample of a normal subject.
• a decrease in the amount of Tamm-Horsfall protein in the subject compared to that in a normal subject is indicative of Interstitial Cystitis.
• Tamm-Horsfall protein is determined using standard techniques including but not limited to SDS-PAGE analysis, isoelectric focusing (IEF), Western Blot Analysis, High-Performance Liquid Chromatography (HPLC), MALDI mass spectrometry and/or high pH Anion Exchange Chromatography (AEC).
• ISO isoelectric focusing
• HPLC High-Performance Liquid Chromatography
• MALDI mass spectrometry and/or high pH Anion Exchange Chromatography (AEC).
• Also provided by the invention is a method for monitoring the course of Interstitial Cystitis in a subject.
• the method comprises quantitatively determining in a first sample of urine from the subject the levels of Tamm-Horsfall protein and comparing the amount so determined with the amount present in a second sample from the subject.
• the first and second samples are taken at different points in time and the difference in the levels of Tamm-Horsfall protein determined is indicative of the course of Interstitial Cystitis.
• the course of Interstitial Cystitis may be graded and the grading of the diseases is based on the amount of Tamm-Horsfall protein present in a subject's urine sample, wherein the urine samples are taken at different points in time.
• the subject is selected from the group consisting of human, monkey, ape, dog, cat, cow, horse, rabbit, mouse and rat subjects.
• the invention also provides a pharmaceutical composition
• a pharmaceutical composition comprising Tamm Horsfall protein and a pharmaceutically acceptable carrier, known to those skilled in the art.
• the Tamm-Horsfall protein is sialyated.
• the pharmaceutical compositions preferably include suitable carriers and adjuvants which include any material which when combined with the Tamm-Horsfall protein or sialyated Tamm- Horsfall protein, retain the molecule's activity, and is non-reactive with the subject's immune system.
• carriers and adjuvants include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, phosphate buffered saline solution, water, emulsions (e.g. oil/water emulsion), salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances and polyethylene glycol.
• buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, phosphate buffered saline solution, water, emulsions (e.g
• compositions comprising such carriers are formulated by well known conventional methods. Such compositions may also be formulated within various lipid compositions, such as, for example, liposomes as well as in various polymeric compositions, such as polymer microspheres.
• the invention provides a kit comprising the pharmaceutical composition of the invention.
• the invention provides a diagnostic kit for determining if a subject has IC.
• the diagnostic kit may be a colorimetric assay wherein when the urine sample from a subject is tested, (1) the presence, absence or reduced amount of Tamm-Horsfall protein is detected or (2) the presence, absence or reduced amount of Tamm-Horsfall activity is detected or (3) the presence, absence or reduced sialyation of Tamm-Horsfall protein is detected.
• the invention encompasses methods for producing Tamm-Horsfall molecules, derivatives and/or fragments thereof.
• the Tamm-Horsfall protein molecules, derivative and/or fragments thereof may be naturally occurring, recombinant or chemically synthesized. These may be modified by one or more purification tags, including, but not limited to, His6, epitope (e.g., myc, V5, FLAG or soft-epitope), streptavidin, biotin, avidin, tetracysteine, calmodulin-binding protein, elastin-like peptide, fusion protein (e.g., glutathione-S-transferase, maltose binding protein, cellulose-binding domain, thioredoxin, NusA or mistin), chitin-binding domain, GFP, alkaline phosphatase, cutinase, O 6 -alkylguanine alkyltransferase (AGT), or halo tag.
• This method involves growing the host-vector system transfected with a plasmid encoding Tamm-Horsfall, derivatives or fragments thereof, so as to produce the Tamm- Horsfall molecules, derivatives or fragments thereof, in the host and then recovering the Tamm-Horsfall molecules, derivatives or fragments thereof.
• the techniques for assembling and expressing DNA encoding the amino acid sequences corresponding to Tamm-Horsfall protein, derivatives and fragments thereof, e.g. synthesis of oligonucleotides, PCR, transforming cells, constructing vectors, expression systems, and the like are well-established in the art, and most practitioners are familiar with the standard resource materials for specific conditions and procedures.
• the nucleotide sequences encoding the amino acid sequences corresponding to the Tamm-Horsfall protein, derivatives or fragments thereof, may be expressed in a variety of systems known in the art.
• the cDNA may be excised by suitable restriction enzymes and ligated into suitable prokaryotic or eukaryotic expression vectors for such expression.
• Suitable vectors containing the desired gene coding and control sequences employs standard ligation and restriction techniques, which are well understood in the art (see Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.
• Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes
• DNA sequences is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution.
• an excess of restriction enzyme is used to insure complete digestion of the DNA substrate.
• Restriction cleaved fragments may be blunt ended by treating with the large fragment of K coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 min at 2O 0 C to 25°C in 50 mM Tris (pH 7.6) 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 5-10 ⁇ M dNTPs.
• the Klenow fragment fills in at 5' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present.
• selective repair can be performed by supplying only one of the dNTPs, or with selected dNTPs, within the limitations dictated by the nature of the sticky ends.
• the mixture is extracted with phenol/chloroform and ethanol precipitated.
• Treatment under appropriate conditions with Sl nuclease or Bal-31 results in hydrolysis of any single-stranded portion.
• Ligations are performed in 10-50 ⁇ l volumes under the following standard conditions and temperatures using T4 DNA ligase. Ligation protocols are standard (D. Goeddel (ed.) Gene Expression Technology: Methods in Enzymology (1991)).
• vector fragment In vector construction employing "vector fragments", the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in order to remove the 5' phosphate and prevent religation of the vector. Alternatively, religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments.
• BAP bacterial alkaline phosphatase
• CIP calf intestinal alkaline phosphatase
• the recombinant protein may be expressed in a prokaryotic, yeast, insect, plant or mammalian system.
• prokaryotic (bacterial) expression systems are E. coli (e.g. BL21, BL21 (DE3), XLl, XLl Blue, DH5 ⁇ or DHlOB cell strains) and B. subtilis.
• Yeast cells include, but are not limited to, P. pastoris, K. lactis, S. cerevisiae, S. pombe, Y. lipolyt und K. marxianus.
• Suitable mammalian cell lines may be, among others, CHO, HEK 293 BHK, NSO, NSl, SP2/0.
• Insect cell lines may include, for example, Drosophila, Aedes aegypti mosquitoe, Sf21, Sf9, and T.ni cell lines.
• the isolated protein may comprise, depending of the expression system, different posttranslational modifications of amino acids, such as acetate groups, phosphate groups, various lipids and carbohydrates, changed chemical nature of an amino acid (e.g. citrullination) or structural changes, like disulfide bridges.
• Suitable vectors include viral vector systems e.g. ADV, RV, and AAV (RJ. Kaufman "Vectors used for expression in mammalian cells" in Gene Expression Technology, edited byD.V. Goeddel (1991).
• non- vector methods include nonviral physical transfection of DNA into cells; for example, microinjection (DePamphilis et al., BioTechnique 6:662-680 (1988)); liposomal mediated transfection (Feigner et al., Proc. Natl. Acad. Sci. USA. 84:7413-7417 (1987), Feigner and Holm, Focus 11:21-25 (1989) and Feigner et al., Proc. West. Pharmacol. Soc. 32: 115-121 (1989)) and other methods known in the art.
• microinjection DePamphilis et al., BioTechnique 6:662-680 (1988)
• liposomal mediated transfection Feigner et al., Proc. Natl. Acad. Sci. USA. 84:7413-7417 (1987), Feigner and Holm, Focus 11:21-25 (1989) and Feigner et al., Proc. West. Pharmacol. Soc. 32
• the invention provides a method for screening for agents that modulate production of Tamm-Horsfall protein.
• the method comprises contacting Tamm-Horsfall genes in Tamm-Horsfall positive cells with a molecule of interest and subsequently determining whether the contact results in increased Tamm-Horsfall production.
• An increase in Tamm-Horsfall production is indicative that the molecule modulates production of Tamm-Horsfall genes.
• the method comprises contacting Tamm-Horsfall protein in Tamm-Horsfall positive cells with a molecule of interest and then determining whether the contact results in increased Tamm-Horsfall production. An increase in Tamm-Horsfall production is indicative that the molecule modulates production of the Tamm-Horsfall protein.
• Also provided in this invention is a method for screening for agents that modulate sialyation of Tamm-Horsfall protein.
• the method comprises contacting Tamm-Horsfall protein in Tamm-Horsfall positive cells with a molecule of interest and subsequently determining whether the contact results in increased sialyation of Tamm-Horsfall protein.
• An increase sialyation of Tamni-Horsfall protein is indicative of modulation of sialyation of Tamm-Horsfall protein.
• an increase in the sialyation of the Tamm-Horsfall protein is measured by measuring the zeta-potential of the sialyated Tamm-Horsfall protein. Examples of other methods that may be used to detect sialyation of the Tamm-Horsfall protein include but are not limited to IEF, HPLC and/or high pH anion exchange chromatography.
• the agents that modulate sialyation of Tamm- Horsfall protein result in hyper-sialyation of the Tamm-Horsfall protein.
• the term "hyper-sialyation” is sialyation of Tamm-Horsfall protein that is more than that of wild type Tamm-Horsfall protein. For example, if an active Tamm-Horsfall protein terminates in four sialic acid molecules at each sialyation site, a hyper-sialyated Tamm- Horsfall protein may terminate in more than four sialic acid residues at each sialyation site of the Tamm-Horsfall protein. Alternately, a hyper-sialyated Tamm-Horsfall protein may terminate in more than four sialic acid residues at one or more of the sialyation sites of the Tamm-Horsfall protein.
• the screening assay comprises mixing the recombinant-Tamm- Horsfall gene or the Tamm-Horsfall protein with a binding molecule or cellular extract. After mixing under conditions that allow association (direct or indirect) of the Tamm- Horsfall gene or the Tamm-Horsfall protein with the binding molecule or a component of the cellular extract, the mixture is analyzed to determine if the binding molecule/component increased the amount of Tamm-Horsfall protein.
• the increase in the Tamm-Horsfall protein may be due to increased synthesis of Tamm-Horsfall protein from the recombinant Tamm-Horsfall gene, hi another embodiment, the increase in the amount of Tamm-Horsfall protein may be increased stability or reduced degradation of Tamm-Horsfall protein.
• the effect of Tamm-Horsfall binding molecules may be assessed by assaying for the amount of Tamm-Horsfall protein produced, using high-through-put screening methods. Accordingly, molecules that increase the levels of Tamm-Horsfall protein can be identified. Alternatively, targets that increase the levels of Tamm-Horsfall protein can be identified using a yeast two-hybrid system (Fields, S. and Song, O.
• an expression unit encoding a fusion protein made up of one subunit of a two subunit transcription factor and the Tamm-Horsfall protein is introduced and expressed in a yeast cell.
• the cell is further modified to contain (1) an expression unit encoding a detectable marker whose expression requires the two subunit transcription factor for expression and (2) an expression unit that encodes a fusion protein made up of the second subunit of the transcription factor and a cloned segment of DNA.
• the expression results in the interaction of the Tamm-Horsfall protein and the encoded protein. This brings the two subunits of the transcription factor into binding proximity, allowing reconstitution of the transcription factor. This results in the expression of the detectable marker.
• the yeast two-hybrid system is particularly useful in screening a library of cDNA encoding segments for cellular binding partners of Tamm-Horsfall protein. Assaying for Tamm-Horsfall production may be used to assess the effect of the targets on the levels of Tamm-Horsfall protein.
• Tamm-Horsfall proteins which may be used in the above assays include, but are not limited to, an isolated Tamm-Horsfall protein, a fragment of a Tamm-Horsfall protein, a cell that has been altered to express a Tamm-Horsfall protein, or a fraction of a cell that has been altered to express a Tamm-Horsfall protein. Further, the Tamm-Horsfall protein can be the entire Tamm-Horsfall protein or a defined fragment of the Tamm-Horsfall protein. It will be apparent to one of ordinary skill in the art that so long as the Tamm-Horsfall protein can be assayed for agent binding, e.g., by a shift in molecular weight or activity, the present assay can be used.
• the method used to identify whether binding molecule and/or cellular component binds to a Tamm-Horsfall protein will be based primarily on the nature of the Tamm-Horsfall protein used. For example, a gel retardation assay can be used to determine whether an agent binds to Tamm-Horsfall or a fragment thereof. Alternatively, immunodetection and biochip technologies can be adopted for use with the Tamm-Horsfall protein. A skilled artisan can readily employ numerous art-known techniques for determining whether a particular agent increases the amount of Tamm-Horsfall protein produced.
• Binding molecules and cellular components can be further tested for the ability to modulate the Tamm-horsfall protein using a cell-free assay system or a cellular assay system. As the activities of the Tamm-horsfall protein become more defined (for example, activities in addition to modulating Interstitial Cystitis), functional assays based on the identified activity can be employed.
• a compound/molecule is said to agonize Tamm-Horsfall activity when the compound/molecule increases Tamm-Horsfall activity by binding more cations in the urine of an IC patient or when a compound/molecule increases the amount of Tamm-Horsfall protein present in a subject or when a compound/molecule increases the sialyation of Tamm-Horsfall protein in a subject.
• the preferred agonist will selectively agonize Tamm- Horsfall, not affecting any other cellular proteins.
• the preferred agonist will increase Tamm-Horsfall activity and/or levels of Tamm-Horsfall protein and/or sialyation of Tamm-Horsfall protein by more than 50%, more preferably by more than 90%, most preferably more than doubling Tamm-Horsfall activity and/or amount of Tamm-Horsfall protein and/or sialyation of Tamm-Horsfall protein.
• Molecules that are assayed in the above method can be randomly selected or rationally selected or designed.
• a binding molecule is said to be randomly selected when the binding molecule is chosen randomly without considering the specific sequences of the Tamm-Horsfall protein.
• An example of randomly selected binding molecule is the use of a chemical library or a peptide combinatorial library, or a growth broth of an organism or plant extract.
• a binding molecule is said to be rationally selected or designed when the binding molecule is chosen on a nonrandom basis that takes into account the sequence of the target site and/or its conformation in connection with the binding molecule's action.
• Binding molecule can be rationally selected or rationally designed by utilizing the peptide sequences that make up the Tamm-Horsfall protein.
• a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to a fragment of a Tamm-Horsfall protein.
• Peptide agents can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
• the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.
• the binding molecule can be, for example, peptides, small molecules, and vitamin derivatives, as well as carbohydrates.
• a skilled artisan can readily recognize that there is no limit as to the structural nature of the agents used in the present screening method.
• classes of molecules that increase the levels of Tamm- Horsfall protein of the present invention are hormones.
• hormones for example, in patients with Interstitial Cystitis, symptoms of Interstitial Cystitis are reduced during pregnancy.
• estrogen is administered to a subject to stimulate the production of Tamm-Horsfall protein.
• progesterone is administered to the subject to stimulate production of Tamm-Horsfall protein.
• the cellular extracts embodied in the methods of the present invention can be, as examples, aqueous extracts of cells or tissues, organic extracts of cells or tissues or partially purified cellular fractions.
• a skilled artisan can readily recognize that there is no limit as to the source of the cellular extract used in the screening method of the present invention.
• the method for determining whether a molecule or a compound causes an increase in the amount of Tamm-Horsfall protein comprises separately contacting each of a plurality of samples to be tested according to any of the methods of the invention.
• the plurality of samples may comprise, more than about 10 or more than about 5 X 10 4 samples.
• the method comprises essentially simultaneously screening the molecules according to any one of the described methods of the invention.
• the screening assays of the present invention for identifying candidate agents can, e.g., detect incorporation of a label, where the label can directly or indirectly provide a detectable signal.
• Various labels may be used, include radioisotopes, fluorescers, chemiluminescers, and the like.
• reagents may be included in the screening assay. These include reagents like salts, detergents, neutral proteins, e.g. albumin, etc., that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The mixture of components is added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 0 C and 4O 0 C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening.
• compositions that are useful in treating IC and/or its symptoms, including pharmaceutical compositions, comprising the THP polypeptides, polynucleotides or other molecules of the invention.
• the compositions may include a buffer, which is selected according to the desired use of the THP polypeptide, polynucleotides or other molecules of the invention, and may also include other substances appropriate to the intended use. Those skilled in the art can readily select an appropriate buffer, a wide variety of which are known in the art, suitable for an intended use.
• the compositions may also include a biodegradable scaffold, matrix or encapsulating material such as liposomes, microspheres, nanospheres and other polymeric substances.
• the composition can comprise a pharmaceutically acceptable carrier or excipient, a variety of which are known in the art and need not be discussed in detail herein.
• Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, Gennaro, A.R. (2003) Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus. 20 th ed., Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7 th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3 rd ed., Amer. Pharmaceutical Assoc, hi some embodiments, the composition comprises a matrix that allowsing for slow release of the composition.
• the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers, and diluents, are readily available to the public.
• pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
• the THP polynucleotides and polypeptides may be obtained from naturally occurring sources or synthetically or recombinantly produced. Where obtained from naturally occurring sources, the source chosen will generally depend on the species from which the protein is to be derived.
• the subject proteins may also be derived by synthesis, such as by synthesizing small fragments of a polypeptide and later linking the small fragments together.
• the subject protein can be more efficiently produced by recombinant techniques, such as by expressing a recombinant gene encoding the protein of interest in a suitable host, whether prokaryotic or eukaryotic, and culturing such host under conditions suitable to produce the protein. If a prokaryotic host is selected for production of the protein, such as E.
• the protein will typically be produced in and purified from the inclusion bodies. If an eukaryotic host is selected for production of the protein, such as CHO cells, the protein may be secreted into the culture medium when its native or a heterologous secretory leader sequence is linked to the polypeptide to be made. Any convenient protein purification procedures may be employed. Suitable protein purification methodologies are described in Guide to Protein Purification, Deuthser ed. (Academic Press, 1990). For example, a lysate may be prepared from the original source and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, and the like.
• the molecules of the invention can be formulated into preparations for delivery by dissolving, suspending or emulsifying them in an aqueous solvent, such as phosphate buffered saline (PBS), or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
• PBS phosphate buffered saline
• nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
• the molecules of the invention can be provided in unit dosage forms, i.e., physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of molecules of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.
• unit dosage forms i.e., physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of molecules of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.
• the specifications for the novel unit dosage forms of the present invention depend on the particular molecule/compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
• An effective amount of the molecules of the invention is administered to the subject at a dosage sufficient to produce a desired result.
• compositions of the instant invention will contain from less than about 1% to about 95% of the active ingredient (molecules of the invention), in some embodiments, about 10% to about 50%.
• Administration can be generally by catheterization and often to a localized area. The frequency of administration will be determined by the care giver based on patient responsiveness. Other effective dosages can be readily determined by one of ordinary skill in the art through trials establishing dose response curves.
• the amount of molecules of the invention to be administered could use readily available information with respect to the amount of agent necessary to have the desired effect.
• the amount of a molecule necessary to increase a level of active subject polypeptide can be calculated from in vitro or in vivo experimentation.
• the amount of agent will, of course, vary depending upon the particular agent used and the condition of the subject being treated, such as the subject's age, the extent of the subject's disease, the subject's weight and the likelihood of any adverse effect, etc.
• the therapeutic agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds or treatment procedures.
• the following methods and excipients are merely exemplary and are in no way limiting.
• Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
• the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
• auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
• composition or formulation to be administered will, in any event, contain a quantity of the therapeutic agent adequate to achieve the desired state in the subject being treated.
• compositions of the invention will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual subject, the site of. delivery of the polypeptide composition, the method of administration, the scheduling of administration, and other factors known to practitioners.
• the effective amount of polypeptide for purposes herein is thus determined by such considerations.
• THP Tamm-Horsfall protein
• the LMW (>100 ⁇ 3500) toxic factor (TF; dialysis product) was prepared by dialyzing 500 ml of a 24-hr urine sample from control subjects in a dialysis bag (MWCO 100) (Spectrum Laboratories, Inc., Collinso Dominguez, CA) against distilled water until chloride ion was no longer detectable in the dialysate (outside) with 0.05 M AgCl solution. At that time, the dialyzed urine was placed in another dialysis membrane (MWCO 3500) and the overnight-dialyzed product ( ⁇ 3500 MW) lyophilized and investigated after rehydration (10 mg/ml) for its ability to induce bladder contractions.
• the toxic factor (>100 ⁇ 3500 MWCO) was tested for cytotoxicity using the CellTiter96 Aqueous One Solution Proliferation AssayTM (Promega Corp, Madison, WI). Rat urothelial target cells and human HBT4 urothelial cells were plated in 96-well tissue culture plates (NunclonTM) in triplicate (50000 cells/well). Cells had been maintained in Ham' s-Ml 99 tissue culture media supplemented with 10% fetal calf serum (FCS) + antibiotics (Pen-Strep).
• FCS fetal calf serum
• cytotoxicity assay cells were harvested and resuspended in DME media (without phenol red indicator) with 1% FCS (assay media) containing 100 ⁇ g of solubilized TF in a volume of lOO ⁇ l/well. Negative control wells were similarly prepared but did not contain any TF.
• TF 2 mg/ml was mixed with THP 2 mg/ml and incubated for 1 hour at room temperature and then sedimented in a centrifuge and the supernatant (lOO ⁇ l) added to triplicate test wells.
• Cytotoxicity levels were compared between groups as follows: TF alone versus control, TF plus THP versus TF alone, and TF plus THP versus control.
• the rats were anesthetized with a subcutaneous injection of urethane (1.2 g/Kg) and a 1 cm incision was made along the centerline of the lower ventral abdomen.
• urethane 1.2 g/Kg
• a 22 gauge (0.7 mm) catheter (Intracath, Becton-Dickinson, OH) was inserted into the bladder dome and sutured in place using a purse string suture with 4.0 tapered prolene suture.
• the bladder was returned to the abdomen, with the line escaping through the incision.
• the muscle wall was sutured together using 4.0 tapered prolene suture and the skin was sutured using 4.0 nylon suture.
• the catheter was then connected to a pressure transducer (UFI, Morro Bay, CA) and in turn connected to an infusion pump (Harvard Apparatus, MA).
• UFI pressure transducer
• MA infusion pump
• the pressure was recorded with the transducer using the program Lab View (National Instruments, TX).
• the bladder was first infused with warm (37 0 C) 0.9% saline or 400 mM KCl (29.8 mg/mL, Abbott Laboratories, IL) at 40 ⁇ L/min (2.4 mL/hr) and at least 20 minutes of stable voiding cycles were recorded during infusion.
• the pressure threshold (PT, pressure at which voiding initializes) and peak pressure (PP, pressure maximum or amplitude of bladder contractions) were recorded. Frequency of contractions and inter-contractile interval were recorded, along with the percentage of non-voiding contractions (% NVC, contractions where the PP is greater than 2 cm H 2 O and less than the PT, thus not resulting in a void).
• rats were infused with the test solution.
• TF could injure the bladder mucosa and facilitate KCl-induced bladder hyperactivity
• one group of animals received an infusion of TF (15 mg/mL).
• THP could attenuate the bladder hyperactivity induced by TF and KCl
• a second group of animals received an infusion of a mixture of THP 10 mg/ml and TF (15 mg/ml) mixed in a ratio of 1 : 1.
• Results were analyzed by Student's t test, employing the Bonferroni correction where more than two variables were involved.
• the TF had a significant cytotoxic effect in cultured rat (p ⁇ 0.01) and human (p ⁇ 0.01) HBT4 bladder epithelial cells relative to the negative control. In the wells containing TF plus THP, there were no detectable levels of cytotoxicity (Table 1).
• THP may function as a urinary protector that prevents the natural byproducts of metabolism from injuring mucus. 4 ' 7
• THP is capable of neutralizing these toxic factors in a cell culture assay. 7
• the NVC represent muscle spasticity or fibrillation, an abnormal reaction to KCl.
• THP blocked the NVC induced by the TF (Table 2). While these are animal and cell culture models, the data do support the concept that these functions of both human TF and THP are operating in the fashion described above in the human bladder.
• THP may exert a protective effect in the bladder.
• THP may operate in the urine by electrostatically binding potentially injurious cationic urine factors that might otherwise injure the urothelium. If THP 's function is inadequate, then the resultant increase in urothelial permeability may allow urinary potassium, a passive player in the pathogenetic process, to penetrate the tissue and activate bladder nerves and muscle.
• the initiating event for IC may be a normal protein metabolite which, if left unchecked or if present in sufficient concentrations, injures the urothelium by electrostatically binding to the mucus, altering the permeability of the epithelium, and resulting in K diffusion and tissue response of nerve depolarization, injury, and inflammation.
• THP a highly anionic urinary protein, electrostatically neutralizes the injurious effects of this toxic urine factor.
• THP appears to play a protective role in the normal urinary tract, and a reduction in its protective effect could initiate the cascade of urothelial injury, increased urothelial permeability, and potassium diffusion that we have hypothesized for the pathogenetic process of IC.
• HBT4 Human cells
• THP Tamm-Horsfall protein
• NVC nonvoiding contractions
• the TF injured the rat urothelium and allowed K to diffuse into the interstitium, where it provoked NVC ("fibrillation" activity); the TF was neutralized by THP. f Compared to NaCl baseline value.
• Tamm-Horsfall protein (THP) from normal urine has been shown to protect against the cytotoxic effects of toxic urinary cations (TF) in vivo and in vitro.
• TF toxic urinary cations
• This study investigated the effect of desialylation on the ability of THP to protect the urothelium in vivo and in vitro from the effects of a urine-derived toxic factor (TF). Desialyation would reduce the electronegativity of the proteins, impairing its effectiveness for attracting the cationic TF.
• Healthy female volunteers (median age 30 years) provided 24-hour pooled urine samples. These subjects were screened for IC symptoms using the Pelvic Pain and Urgency/Frequency Patient Symptom (PUF) Scale. 9 A PUF Score of 0 was required for study entry to ensure that THP and TF were obtained from the urine of healthy individuals who had no evidence of bladder disease or voiding symptoms. Urines were stored at -2O 0 C until they were used for isolating TF and/or THP. Animal studies employed adult male Sprague-Dawley (SD) rats weighing 325-350 g.
• SD Sprague-Dawley
• THP was prepared from urines by the method of Tamm and Horsfall. 6 Briefly, THP was recovered by centrifugation after precipitation in the cold overnight with 0.6M NaCl. The gel-like precipitate was resuspended in 50 ml cold 0.6M NaCl, then reprecipitated by centrifugation. This was repeated three times to increase the purity of the final product which was dissolved in a minimal amount of distilled water, pH 7.4. The solubilized THP was exhaustively dialyzed to remove all traces of salt and then lyophilized. Dry weight of this material was subsequently used to prepare stock solutions of THP (10 mg/ml) dissolved in PBS (in vivo studies) or culture media (cell studies). THP preparations were monitored for purity by PAGE and identification of THP made by Western blot.
• Desialylated THP was prepared by mild acid hydrolysis of THP (10 mg/ml) in 2.5M acetic acid and heating for 3 hr at 82°C.
• the THP hydrolysate was neutralized by filtration-washing, three times with 15 ml PBS, on a Centricon (MWCO 30000) cartridge (Millipore). After each wash the volume was reduced by centrifugation to about 1 ml.
• the recovered desialylated protein >30000 MW) in the last wash (2 ml final volume) was devoid of free sialic acid which appeared in the washes and could be quantitated by DMB derivitization and fluorescent detection to assess the extent of desialylation.
• the hydrolysis resulted in 87.7% loss of sialic acid (16.12 vs 1.99 ⁇ g Neu5Ac/mg protein for the THP-d), coincident with an increase in the electrophoretic migration of the THP.
• the LMW (>100 ⁇ 3500) TF was prepared as described previously. 23 Cytotoxicity Assay
• TFl The TF obtained from 2 different pooled urine samples (TFl, TF2) was tested for cytotoxicity using the CellTiter96 Aqueous One Solution Proliferation AssayTM (Promega Corp, Madison, WI). Rat urothelial target cells and human HTB4 urothelial cells were plated in 96-well tissue culture plates (NunclonTM) in triplicate (50,000 cells/well). Cells had been maintained in Ham's-M199 tissue culture media supplemented with 10% fetal calf serum (FCS) + antibiotics (Pen-Strep).
• FCS fetal calf serum
• cytotoxicity assay cells were harvested from seed flasks, washed free of trypsin- EDTA, and resuspended in DME media (without phenol red indicator) -1% FCS (assay media). An aliquot of the cell suspension was counted in a hemocytometer and diluted with assay media to a concentration of 0.5 x 10 6 cells/ml, from which 100 ⁇ l (5 x 10 4 cells) were added to each well in a 96-well microtiter plate. Test samples (100 ⁇ l) were added to these wells in quadruplicate.
• THP obtained from 4 different normal urines
• THP-d desialylated THP from these same samples
• Results were analyzed by Student's t test, employing the Bonferroni correction where more than two variables were involved.
• TF had a significant cytotoxic effect on HTB4 epithelial cells relative to the media controls (P ⁇ 0.01).
• P ⁇ 0.01 cytotoxic effect on HTB4 epithelial cells relative to the media controls.
• cytotoxicity in the wells containing TF mixed with unmodified THP, there were no detectable levels of cytotoxicity (Table 3).
• IC may be the result of a disruption in the balance of protective factors and potentially pathogenic factors in the lower urinary tract. 3 There is substantial evidence that the relative impermeability of the bladder mucosa is the primary mechanism that protects the bladder wall and that the surface mucus is critical in regulating epithelial permeability. 10 ' 12 Data from a number of investigators indicate that IC is associated with an epithelial dysfunction resulting in abnormal permeability. 1 ' 13 ' 14 ' 16> 17
• cation potassium is the chief urinary toxin to initiate the symptoms and tissue injury of IC. 1 ' 9) 18"21 Potassium is present in relatively high levels in the urine (20 - 120 mEq/1). 18 An abnormally permeable epithelium permits potassium to diffuse into the bladder tissue, where it can depolarize nerves and muscle and produce tissue injury. ' Potassium in the bladder does not provoke symptoms when the epithelial permeability barrier is intact. 1
• urine factors have been investigated for their role in IC pathogenesis. These data show that normal human urine contains LMW cations, or TFs, that cause epithelial cell injury in vitro and in vivo.
• the TFs are similar to protamine sulfate, a cationic compound known to be extremely toxic to the mucus of the epithelium, causing a permeability abnormality. It is likely that these TFs are amines or polypeptides that are the end product of protein metabolism. Potentially, they can bind electrostatically to the anionic transitional cell surface mucus and disrupt the permeability barrier. This disruption permits the cascade of potassium diffusion, sensory nerve stimulation, muscle depolarization, and tissue injury. 1 ' 18
• the difference between a healthy state and the disease state in the lower urinary tract may be a qualitative or quantitative deficiency of a factor that normally protects the epithelium from injury by urinary constituents.
• a protective factor might function by electrostatically attracting the potentially toxic cations before they can damage the bladder surface.
• Epithelial injury then, is initiated when there is an imbalance between the urinary TF and the protective factors.
• THP functions as a urinary protective factor to prevent the TF, which are natural byproducts of metabolism, from injuring the bladder epithelium. 4 ' 23 ' 7
• Our data indicate that these TF are neutralized by THP. 23> 7
• THP exerts a protective effect in the bladder.
• Urinary THP may electrostatically bind cationic urine factors and prevent them from injuring the anionic urothelium.
• THP samples do not all have equal ability to perform this protective function. 7 Consequently, such urine factors may then injure the urothelium, increase urothelial permeability, and allow urinary potassium to penetrate the tissue and stimulate bladder nerves and muscle in certain individuals.
• Such "abnormal" THP may result from aberrant sialylation pathways leading to decreased sialic content of the N-(or O- linked) glycans present on THP.
• THP is a critical urinary anion that protects the epithelium, acting to bind potentially injurious cations in the urine in healthy individuals. If THP does indeed play this protective role in the urine, its ability to do so may be a major factor in the prevention of IC in the normal state. Conversely, a reduction in the protective capacity of THP may be an important factor in the pathogenesis of IC. This concept opens new vistas in the diagnosis and therapy of IC, including determining the susceptibility to IC by detecting the presence of disialyted THP.
• THP a highly anionic urinary protein, electrostatically neutralizes the injurious effects of toxic urine factor. This activity appears to depend on the anionic terminal sialic acid moieties on the THP molecule. THP appears to play a protective role in the normal urinary tract, and a reduction in its protective effect could initiate the cascade of urothelial injury, increased urothelial permeability, and potassium diffusion for the pathogenetic process of IC.
• TF toxic factor
• THP Tamm-Horsfall protein
• THP-d desialylated Tamm Horsf all protein
• n number of wells assayed
• NS not significant.
• NVC nonvoiding contractions
• TF toxic factor
• THP Tamm-Horsfall protein
• THP-d desialylated Tamm Horsfall protein.
• This example demonstrates the measurement of the sialic acid content of THP from the urine of normal subjects versus IC patients.
• the zeta potential of the THP molecule was measured, which would be reduced in IC patients if the sialic acid content is lower.
• Urinary THP concentrations in IC patients versus normal control subjects was also determined to rule out the possibility that IC patients produce lower quantities of THP to account for a reduction of protective activity.
• THP is isolated by the cartridge centxifugation-washing method.
• two 15-ml aliquots of urine are centrifuged sequentially until they result in a 3Ox concentration.
• the protein fraction >30000 MW (top) is then washed by 2 centrifugations with 15 ml distilled water.
• the final material containing the THP (>95%) is brought up to 1 ml volume with water containing .01% azide preservative and stored at 4 0 C until ELISA or sialic acid determination.
• the filtrated urine does not contain any traces of protein by detection with Coomassie® Brilliant Blue-G250 reagent (Bio-Rad Laboratories, Hercules, CA).
• representative THP samples are monitored and characterized for purity by PAGE and Western blot identification. This method allows almost 100% recovery of the protein and partial purification by washing and removes all traces of salt.
• THP samples prior to hydrolysis were shown to have no detectable endogenous sialic acid.
• the dried THP samples are dissolved in 50 ⁇ l of milliQ water and 50 ⁇ l of 7mM DMB (l,2-diamino-4,5-methylenedioxybenzene dihydrochloride) in acetic acid added.
• the samples are warmed to 50°C for 2.5 hr and without any further purification 20 ⁇ l of the derivatized sialic acids are injected for HPLC using a reversed phase Cl 8 column (TosoHaas ODS- 120T).
• a gradient of water: acetonitrile: 50% methanol starting at 79:7:14 then changing to 75:11:14 over 40 min elution time is used which separates the various acetylated species.
• the fluorescence detector is set at Ex.373nm and Em.448nm. Quantitation is accomplished by comparing peak heights to known amounts of purified standards derivatized during the same run. The amount of sialic acid in a 150 ⁇ l hydrolysate of the THP samples is then used to calculate the amount of sialic acid pM/ ⁇ g THP in the normal- and IC-derived THP samples. Differences in the sialic acid content of the IC and normal THP (mean THP/group) are compared by Student's t test.
• THP isolated from urines is assayed by an indirect enzyme-linked immunosorbent assay (ELISA) using 96-well plastic plates (Immulon®, Thermo Electron, Waltham, MA). Test plates are coated with purified THP (Biomedical Technologies, Inc., Stoughton, MA) 100 ng/well by incubating overnight in 0.05 M carbonate coating buffer (pH 9.6) at 4 0 C and then washing with PBS 2x and blocked in PBS + 0.5% BSA-.01% Tween-20 for 1 hr at room temperature. Wells are washed in PBS-.01% Tween-20 (assay buffer, pH 7.4) and used immediately after washing with distilled water or stored after being dried.
• ELISA enzyme-linked immunosorbent assay
• a standard curve is prepared by adding 100 ⁇ l of twofold serial dilutions of a THP standard (2.000-0.015 ng/well) to duplicate wells and immediately adding 100 ⁇ l of a 1/2000 dilution of goat anti-THP (ICN Pharmaceuticals, Inc., Costa Mesa, CA) to each well.
• a THP control, no THP (buffer alone) is prepared as above and mixed with the antibody. Samples are assayed in duplicate, 10 ⁇ l of each sample is added to 90 ⁇ l of assay buffer and 100 ⁇ l of the goat anti-THP added (1:2000) and the plate(s) incubated overnight on a shaker at room temperature.
• the assay plate is washed 3X with assay buffer, then 100 ⁇ l of second antibody, rabbit anti goat-peroxidase (Sigma Chemical Co., St. Louis, MO) in assay buffer at 1:1000 dilution added for 1 hr.
• the plates are washed, and OPD peroxidase substrate (Sigma Chemical Co.) added for exactly 10 min in the dark, and the reaction stopped with dilute HCl.
• the plates are blanked to substrate in the plate reader and read at 450 nm. Average amounts of THP in the duplicate samples are extrapolated from the standard curve and the concentrations recorded as ⁇ g THP/150 ⁇ l (hydrolysate) or mg THP/L urine. THP is also normalized to urinary creatinine. Student's t test is used to determine whether there is any significant difference in the THP concentration in IC patients' vs. normal urines.
• Urines were collected from female IC patients (median age 30 years) who were screened for IC symptoms using the Pelvic Pain and Urgency/Frequency Patient Symptom (PUF) Scale (23). Control urines were obtained from healthy individuals who had no evidence of bladder disease, bladder irritative voiding symptoms or clinical history suggestive of recent urinary tract infection and who had a PUF Score of 0. In accordance with institutional review board policy, informed consent was obtained prior to collection of all samples.
• PUF Pelvic Pain and Urgency/Frequency Patient Symptom
• Urines were collected for two purposes: (1) for quantitative determination of urinary levels of THP in IC patients compared to normal individuals and the determination of the sialic acid content (pM sialic acid/ ⁇ g THP protein) of the THP and (2) to provide larger (mg) amounts of THP for determination of zeta potentials.
• Fresh morning urine voids collected in the UCSD Urology Clinic usually provide ⁇ 30 ml of urine. These can be further processed by a rapid, filtration- washing protocol that can process 1-12 urine samples daily, providing purified THP (>95%) for further characterization by ELISA and sialic acid determinations described below.
• To provide sufficient THP for zeta potential determinations requiring ⁇ 5mg/ml purified THP 24-hour urine samples were obtained from some IC and control subjects.
• THP was isolated from control subjects' and IC patients' urine; subjected to hydrolysis and sialic acid determination, measurement of surface charge properties (zeta potential); and quantitated by ELISA as described in the Supporting Online Material.
• THP concentrations in normal urine (28.2 mg/L) and IC urine (28.8 mg/L) were not significantly different.
• THP was normalized to creatinine, there was no significant difference between the normal and IC patients in urinary THP concentration (76.4 versus 70.0 mg THP/mg creatinine) (Table 6).
• bladder surface mucus plays a critical role in controlling the permeability of the epithelium, principally to small molecules 10"12 ' 25 ' 26 . If the mucus is impaired, it results in a dysfunctional epithelium that allows movement of concentrated urinary solutes into the bladder interstitiurn 1"3 ' 13"16>18 .
• One solute, potassium (K+) is 10- to 40-fold more concentrated in urine than in tissue. If there is a dysfunctional epithelium, K+ moves readily down that gradient into the bladder interstitium, where it can directly depolarize nerves, muscles, and generate the symptoms of frequency, urgency, pain, and urinary incontinence, in any combination " > > > • ' .
• K+ solute, potassium
• TF toxic factor
• THP Tamm-Horsfall protein
• THP manufactured by the renal tubular cells, is a large (85 kD) molecular weight protein that is highly anionic. It is a well-conserved protein present in all vertebrate species 6 ' 5 , but in effect has no known obvious urinary activity. THP may thus be a scavenger of the TF, that it is abnormal in IC patients compared to healthy individuals
• THP is able to detoxify the TF in vivo and in vitro 4 ' 23> 7 .
• IC patients' THP has a lower cytoprotective activity than normal subjects' THP against the known toxic effects of protamine sulfate .
• the sialic acid content of THP imparts a substantial amount of the electronegativity to the molecule.
• THP from IC patients has lower protective activity than THP from normal subjects against another cation, PS, in vitro 7 .
• THP of IC patients is abnormal, allowing the TF in urine to cause lower urinary dysfunctional epithelium (LUDE) 3 and begin the entire IC cascade of neurologic upregulation, muscle hyperactivity, tissue injury, and inflammation.
• LDE urinary dysfunctional epithelium
• THP urinary protective molecule
• THP analogs to replace the defective THP
• developing methods for correcting the THP defect or using strategies such as dietary changes to decrease the concentration of urinary TF.
• dietary changes to decrease the concentration of urinary TF.
• THP Tamm-Horsfall protein
• IC interstitial cystitis
• THP Tamm-Horsfall protein
• IC interstitial cystitis
• N.S. not significant.
• This example was performed to determine whether the toxic factor is capable of changing the permeability characteristics of the intact bladder epithelium in vivo.
• a more reliable cytotoxicity assay was used, less subject to artifact errors caused by manipulating target cells during the wash steps and incubations used in earlier assays, to screen urine fractions for cytotoxicity and to measure the ability of pentosan polysulfate CPTS) to neutralize the cytotoxic activity of the toxic factor or protamine sulfate (PS).
• a new in vitro rat urodynamic model 4 was used to investigate the effect on induced bladder contractions of exposure of the normal bladder epithelium to KCl alone versus urinary toxic factor followed by KCl. This new model allows quantitation of bladder muscle reactivity under experimental conditions. Nonvoiding contractions (NVC) of the bladder represent muscle spasticity or fibrillation that is an abnormal reaction to KCl.
• NVC Nonvoiding contractions
• Protamine sulfate was used as a positive control in both models. PS will bind to the glycosaminoglycans (GAGs) of the mucus and disrupt its permeability regulatory mechanism. 5 This provides a good model for potential disease in the bladder relative to making the epithelium dysfunctional. Urine is likely to contain natural "protamine-like" cations also capable of injuring the epithelium. Consequently, the urinary toxic factor was evaluated and compared to our positive control (PS) for its ability to injure cultured urothelial cells as well as an intact rodent urothelium in vivo.
• PS protamine sulfate
• the LMW (>100 ⁇ 3500) toxic factor was prepared by dialyzing 500 ml of a 24-hr urine sample from control subjects in a dialysis bag (MWCO 100) (Spectrum Laboratories, Inc., Collinso Dominguez, CA) against distilled water until chloride ion was no longer detectable in the dialysate (outside) with 0.05 M AgCl solution. At that time, the dialyzed urine was placed in another dialysis membrane (MWCO 3500) and the overnight- dialyzed product ( ⁇ 3500 MW) lyophilized and investigated after rehydration (10 mg/ml) for its ability to induce bladder contractions.
• the toxic factor (>100 ⁇ 3500 MWCO) was tested for cytotoxicity using the CellTiter96 Aqueous One Solution Proliferation AssayTM (Promega Corp, Madison, WI), 7 ' 8 adapted for use with cultured rat urothelial cells and our test substance, the toxic factor derived from urine by dialysis as described above.
• Rat urothelial target cells isolated from rat epithelium were plated in 96-well tissue culture plates (NunclonTM) in triplicate (50000 cells/well). Cells had been maintained in Ham's-M199 tissue culture media supplemented with 10% fetal calf serum (FCS) + antibiotics (Pen-Strep).
• cytotoxicity assay cells were harvested and resuspended in DME (without phenol red indicator) with 1% FCS (assay media) containing 100 ⁇ g of solubilized toxic factor in a volume of 1 ⁇ l/well. Negative control wells were similarly prepared but did not contain any toxic factor. Positive control wells contained protamine sulfate (PS), 1 mg/ml.
• PS + PPS or toxic factor + PPS PS (2 mg/ml) or toxic factor 2 mg/cc was premixed with an equal volume of PPS (2mg/ml) and incubated for 1 hour, then sedimented in a centrifuge and the supernatant (1 OO ⁇ l) added to triplicate test wells.
• Cytotoxicity levels were compared between groups as follows: positive control (PS) versus negative control; toxic factor alone versus negative control; toxic factor plus PPS versus toxic factor alone; and PS plus PPS versus positive control.
• the procedure was as follows: The rats were anesthetized with a subcutaneous injection of urethane (1.2 g/Kg) and a 1 cm incision made along the centerline of the lower ventral abdomen. Once the bladder was exteriorized, a 22 gauge (0.7 mm) catheter (Intracath, Becton-Dickinson, OH) was inserted into the bladder dome and sutured in place, using a purse string suture with 4.0 tapered prolene suture. The bladder was returned to the abdomen, with the line escaping through the incision. The muscle wall was sutured together using 4.0 tapered prolene suture and the skin was sutured using 4.0 nylon suture.
• the catheter was then connected to a pressure transducer (UFI, Morro Bay, CA) and in turn connected to an infusion pump (Harvard Apparatus, MA).
• UFI pressure transducer
• MA infusion pump
• the pressure was recorded with the transducer using the program Lab View (National Instruments, TX).
• the bladder was first infused with warm 0.9% saline (37 0 C) at 40 ⁇ L/min (2.4 mL/hr) and at least 20 minutes of stable voiding cycles recorded during infusion.
• the pressure threshold (PT, pressure at which voiding initializes) and peak pressure (PP, pressure maximum or amplitude of bladder contractions) were recorded.
• Frequency of contractions and inter-contractile interval (ICI) were recorded, along with the percentage of non-voiding contractions (% NVC, contractions where the PP is greater than 2 cm H 2 O and less than the TP, thus not resulting in a void).
• mice were infused with the test solution.
• Group 1 (positive control) animals received 1 mL of a warm solution (37 0 C) of 30 mg/mL PS (Sigma- Aldrich, St. Louis, MO) for 30 minutes. PS has been shown to significantly injure urothelial permeability. 5
• Group 2 animals received an infusion of toxic factor (15 mg/mL).
• Group 3 animals received an infusion of a mixture of PPS (10 mg/ml) and toxic factor (15 mg/ml).
• Results were analyzed by Student's t test, employing the Bonferroni correction where more than two variables were involved. Results
• Urine also has been shown to contain many secondary factors that are a result of the disease process and include inflammatory mediators, neurotransmitters, growth factors or even an antiproliferative factor. 9 ' 10 The occurrence of these factors in urine from patients is mostly of a secondary nature, produced by the disease and not acting as primary inciting factor. Nevertheless, some of these factors may be involved in tissue reactions and disease progression.
• a rodent urodynamics model 4 was used in which the bladders of healthy rats were exposed to toxic factor or PS, both of which caused significant changes in urodynamic parameters (NVC) relative to potassium.
• NVC urodynamic parameters
• the key point in this model is that an intact epithelium prevents potassium diffusion and secondary muscular contractions; for the muscle to react with spasms (Figure 2B), potassium must diffuse through the epithelium.
• the data obtained using this in vivo model indicate physiologic epithelial damage and muscle reactions in the intact bladder, and help corroborate and substantiate the findings from the in vitro cytotoxicity model. Together, these models will be valuable in screening for toxic factors (cytotoxicity) and then demonstrating they are active in the intact bladder.
• normal human urine contains LMW cations that are capable of injuring the bladder mucosa in vivo, resulting in increased epithelial permeability as indicated by potassium sensitivity.
• PPS can neutralize the dialyzed toxic factor from urine.

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