WO2007027614A2

WO2007027614A2 - Abnormal glycosylation in tamm-horsfall protein in interstitial cystitis patient

Info

Publication number: WO2007027614A2
Application number: PCT/US2006/033544
Authority: WO
Inventors: C. Lowell Parsons
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2005-08-30
Filing date: 2006-08-30
Publication date: 2007-03-08
Anticipated expiration: 2008-02-29
Also published as: WO2007027614A3; EP1919496A2

Abstract

The invention provides a method for inhibiting Interstitial Cystitis and its symptoms, reducing symptoms of Interstitial Cystitis in a subject and repairing the mucin layer in a subject by administering an effective amount of a Tamm-Horsfall protein to the subject.

Description

ABNORMAL GLYCOSYLATION IN TAMM-HORSFALL PROTEIN IN INTERSTITIAL CYSTITIS PATIENT

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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.

Another series of experiments has shown that urine contains toxic, low molecular weight, cations that are capable of interacting with mucus and injuring its ability to regulate permeability⁴' ^{23> 29}. We hypothesized that these cations do not injure normal urothelium because urine contains protective substances that have affinity for these cations, interfering with their capacity to injure the urinary mucosa. This, we believe, takes place in the fluid phase of urine so that the interaction with the outer layer of mucus is prevented. In effect, the combination of urinary cations and anions is in a homeostatic balance so that toxic urinary components are less likely, if at all, able to injure the urothelium. Collectively, these interactions efficiently sequester potential toxins in urine during the storage phase in the bladder.

The experiments herein show that a molecule that acts as a critical protective factor is the highly anionic Tamm-Horsfall protein ("THP"). THP is a highly glycosylated and highly conserved urinary protein that is present in the urine of all vertebrates.⁵' ⁶ A number of investigators have explored its possible role in the urinary tract, but none have identified a function that would account for its ubiquity across vertebrate species.

In the past, interstitial cystitis (IC) 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. In the present invention, 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.

SUMMARY OF THE INVENTION

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.

Further, 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.

Further provided in this invention is a method for diagnosing Interstitial Cystitis in a subject. 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 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.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Zeta potentials of THP from the urine of normal control subjects and IC patients show no overlap and are significantly different (P < 0.002).

Figure 2. Representative tracing of rat urodynamic studies. A, CMG showing smooth voiding contractions of a normal bladder during slow infusion of saline (40 μl/60 sec). B,

CMG showing muscle reaction during KCl infusion after epithelial injury with toxic factor. As can be seen, multiple small nonvoiding contractions, a type of "fibrillation," occur secondary to KCl. This occurs only when the epithelium loses its normal impermeability,

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In order that the invention herein described may be more fully understood, the following description is set forth.

As used herein, the term "comprising" when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and/or after the recited steps. For example, 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. Furthermore, the term "comprising" when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, 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. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth as used herein, are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters herein are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and without limiting the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters describing the broad scope of the invention are approximations, the numerical values in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains standard deviations that necessarily result from the errors found in the numerical value's testing measurements.

As used herein, "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.

In one embodiment, the patient may determine if interstitial cystitis symptoms are reduced. In one embodiment, reducing interstitial cystitis may be determined by the physician's evaluation. In one embodiment, reducing interstitial cystitis may be determined from comparing a PUF scale score to a previous PUF scale score. In some embodiments, reducing interstitial cystitis is reducing symptoms in patients whose symptoms indicate, and are similar to, interstitial cystitis.

As used herein, 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.

As used herein, 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.

As used herein, "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).

As used herein, "urinary frequency" refers to the number of urination times per day.

As used herein, "urinary urgency" refers to an inability to delay urination. As used herein, "pelvic pain" refers to pain in the pelvic region of genital and non-genital origin and of organic or psychogenic aetiology.

As used herein, "urinate," "urination," "urinating," "void" and "voiding" refers to release of urine from the bladder to the outside of the body.

As used herein, "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.

As used herein, "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).

As used herein, "oral agent" refers to a compound that can be administered by way of the oral cavity (e.g. in aqueous liquid or solid form).

As used herein, "instill," "instilled," "instillation," 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).

As used herein, "intravesical," refers to inside the bladder. As such, "intravesical instillation," "intravesical therapy," "instill," and "instillation" refers to solutions that are administered directly into the bladder. In some embodiments, instillation is via catheterization. Further, "intravesical solution," "intravesical agent," "intravesical therapeutic," and intravesical compound" refers to a treatment that can be administered to the bladder. In one embodiment, 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. For example, in one embodiment, intravesical therapy is an intravesical agent. In another embodiment, intravesical therapy is a combination of intravesical agents. As used herein, "extravesical" refers to outside the bladder.

As used herein, "cystoscopic examination" and "cystoscopy" refers to an examination that uses a cytoscope.

As used herein, "cystoscope" refers to an endoscopic instrument to visualize the lower urinary tract, which includes the bladder and the urethra.

As used herein, "urethra" refers to a tube draining the urine to the outside. As used herein, "bladder" refers to a hollow muscular organ that stores urine until it is excreted from the body.

As used herein, 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.).

As used herein, "urinary tract infections" refers to a condition that includes an inflamed urethra and painful urination. In some embodiments, a urinary tract infection is caused by bacteria. In some embodiments, a urinary tract infection is not caused by bacteria.

As used herein, "recurrent urinary tract infections" refers to frequent episodes of urinary tract infections.

As used herein, "dyspareunia" refers to pain during intercourse. As used herein, "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.

As used herein, "urinary incontinence" refers to the unintentional loss of urine and inability to control urination or prevent its leakage.

As used herein, "urinary continence" refers to a general ability to control urination.

As used herein, "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.

As used herein, "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.

As used herein, "catheterized" refers to the collection of a specimen by a catheterization. The terms "sample" and "specimen" are used in their broadest sense and encompass samples or specimens obtained from any source.

As used herein, the term "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. However, these examples are not to be construed as limiting the types of samples that find use with the present invention.

As used herein, the term "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. As used herein, "urinary dysfunction" and "urinary tract dysfunction" refers to abnormal urination, patterns or bladder habits, including wetting, dribbling and other urination control problems.

As used herein, "anesthesia" refers to a loss of feeling or inability to feel pain.

As used herein, "local anesthesia" refers to a method of pain prevention in a small area of the body.

As used herein, the 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.

As used herein, "lower urinary epithelial dysfunction" refers to disorders with positive potassium sensitivity tests (e.g. IC, prostatitis and the like).

As used herein, "urinary dysfunction" refers to abnormal urination, patterns or bladder habits, including wetting, dribbling and other urination control problems.

As used herein, sialyation is the modification of glycoproteins with sialic acid. Sialic acid is also known as N-acetylneuraminic acid (N-acetymeuraminate). Sialic acid is often found as a terminal residue of oligosaccharide chains of glycoproteins. Sialic acid imparts negative charge to glycoproteins. In one embodiment, Tamm-Horsfall protein is sialyated.

As used herein, "repairing" is restoring to original or close to original condition. In one embodiment of the invention, repairing the mucin layer is restoring the mucin layer of

Interstitial Cystitis patients to that or normal patients. In another embodiment of the invention, repairing the mucin layer is restoring the function of the mucin layer of Interstitial Cystitis patients to mat or normal patients. In a preferred embodiment, the function of the mucin layer is restored to 50% to that of normal patients. In a more preferred embodiment, the function of the mucin layer is restored to 75% to that of normal patients. In the most preferred embodiment, the function of the mucin layer is restored to 100% to that of normal patients.

As used herein, "level" of a protein is the amount of protein present. In one embodiment of the invention, 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. In another embodiment of the invention, 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.

In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); mL (milliliters); ml (milliliters); μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); °C (degrees Centigrade).

METHODS OF THE INVENTION

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. In an embodiment of the invention, the amount of

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 to 150mg/L urine, about 140 to 150mg/L urine, about 0.1 to 100mg/L urine, about 0.5 to 100mg/L urine, about 1 to 100mg/L urine, about 5 to 100mg/L urine, about 10 to 100mg/L urine, about 15 to 100mg/L urine, about 20 to 100mg/L urine, about 25 to 100mg/L urine, about 50 to 100mg/L urine, about 60 to 100mg/L urine, about 70 to 100mg/L urine, about 80 to 100mg/L urine, about 90 to 100mg/L urine, 0.1 to 50mg/L urine, about 1 to 50mg/L urine, about 5 to 50mg/L urine, about 10 to 50mg/L urine, 15 to 50mg/L urine, about 20 to 50mg/L urine, about 25 to 50mg/L urine, about 30 to 50mg/L urine, about 40 to 50mg/L urine, about 45 to 50mg/L urine, about 0.1 to 30mg/L urine, about 1 to 30mg/L urine, about 0.5 to 30mg/L urine, about 5 to 30mg/L urine, about 10 to 30mg/L urine, about 15 to 30mg/L urine, about 20 to 30mg/L urine, or about 25 to 30mg/L urine.

In another embodiment of the invention, increasing the levels of Tamm-Horsfall protein in a subject repairs the mucin layer of the bladder. In a further embodiment of the invention, increasing the amounts of Tamm-Horsfall protein in a subject treats diseases that are associated with decreased levels of Tamm-Horsfall protein.

As discussed in the Examples, in some patients with Interstitial Cystitis, sialyation of Tamm-Horsfall is reduced. In one embodiment of the invention, 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. If the Tamm-Horsfall protein is sialyated at greater than wild-type levels, a reduced dosage of sialyated Tamm-Horsfall protein may be administered to treat Interstitial Cystitis and its symptoms. As discussed infra, a person skilled in the art would determine the effective dosage of sialyated Tamm-Horsfall protein that may be administered. In a further embodiment of the invention, sialyation of Tamm-Horsfall protein may be increased from about 50 to 3000 pM/μg THP, about 100 to 3000 pM/μg THP, about 200 to 3000 pM/μg THP, about 300 to 3000 pM/μg THP, about 400 to 3000 pM/μg THP, about 500 to 3000 pM/μg THP, about 600 to 3000 pM/μg THP, about 700 to 3000 pM/μg THP, about 800 to 3000 pM/μg THP, about 900 to 3000 pM/μg THP, about 1000 to 3000 pM/μg THP, about 1200 to 3000 pM/μg THP, about 1400 to 3000 pM/μg THP, about 1600 to 3000 pM/μg THP, about 1800 to 3000 pM/μg THP, about 2000 to 3000 pM/μg THP, about 2200 to 3000 pM/μg THP, about 2400 to 3000 pM/μg THP, about 2600 to 3000 pM/μg THP, about 2800 to 3000 pM/μg THP, 50 to 2500 pM/μg THP, about 100 to 2500 pM/μg THP, about 200 to 2500 pM/μg THP, about 300 to 2500 pM/μg THP, about 400 to 2500 pM/μg THP, about 500 to 2500 pM/μg THP, about 600 to 2500 pM/μg THP, about 700 to 2500 pM/μg THP, about 800 to 2500 pM/μg THP, about 900 to 2500 pM/μg THP, about 1000 to 2500 pM/μg THP, about 1200 to 2500 pM/μg THP, about 1400 to 2500 pM/μg THP, about 1600 to 2500 pM/μg THP, about 1800 to 2500 pM/μg THP, about 2000 to 2500 pM/μg THP, about 2200 to 2500 pM/μg THP, about 2400 to 2500 pM/μg THP, 50 to 2000 pM/μg THP, about 100 to 2000 pM/μg THP, about 200 to 2000 pM/μg THP, about 300 to 2000 pM/μg THP, about 400 to 2000 pM/μg THP, about 500 to 2000 pM/μg THP, about 600 to 2000 pM/μg THP, about 700 to 2000 pM/μg THP, about 800 to 2000 pM/μg THP, 50 to 1500 pM/μg THP, about 100 to 1500 pM/μg THP, about 200 to 1500 pM/μg THP, about 300 to 1500 pM/μg THP, about 400 to 1500 pM/μg THP, about 500 to 1500 pM/μg THP, about 600 to 1500 pM/μg THP, about 700 to 1500 pM/μg THP, about 800 to 1500 pM/μg THP, about 900 to 1500 pM/μg THP, about 1000 to 1500 pM/μg THP, about 1100 to 1500 pM/μg THP, about 1200 to 1500 pM/μg THP, about 1300 to 1500 pM/μg THP, about 1400 to 1500 pM/μg THP, 50 to 1000 pM/μg THP₅ about 100 to 1000 pM/μg THP, about 200 to 1000 pM/μg THP, about 300 to 1000 pM/μg THP, about 400 to 1000 pM/μg THP, about 500 to 1000 pM/μg THP₅ about 600 to 1000 pM/μg THP₅ about 700 to 1000 ρM7μg THP₅ about 800 to 1000 pM/μg THP₅ or about 900 to 1000 ρM7μg THP.

In another embodiment of the invention, increasing the total amount of Tamm-Horsfall protein in a subject inhibits Interstitial Cystitis and its symptoms. Herein, even if the Tamm-Horsfall protein is, for example partially sialyted, an increase in the total amount of the Tamm-Horsfall protein may inhibit or reduce the symptoms of Interstitial Cystitis in a subject. As discussed infra, a person skilled in the art would determine the effective dosage of the Tamm-Horsfall protein that may be administered.

In a further embodiment of the invention, 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/day, about 0.1 to 4 mg/day, about 0.1 to 0.5 mg/day, about 0.5 to 1.0 mg/day, about 1.0 to 1.5 mg/day, about 1.5 to 2.0 mg/day, about 2.0 to 2.5 mg/day, about 2.5 to 3.0 mg/day, about 3.0 to 3.5 mg/day, about 3.5 to 4.0 mg/day, about 4.0 to 4.5 mg/day, about 4.5 to 5.0 mg/day, about 5.0 to 5.5 mg/day, about 5.5 to 6.0 mg/day, about 6.0 to 6.5 mg/day, about 6.5 to 7.0 mg/day, about 7.0 to 7.5 mg/day, about 7.5 to 8.0 mg/day, about 8.0 to 8.5 mg/day, about 8.5 to 9.0 mg/day, about 9.0 to 9.5 mg/day, about 9.5 to 10.0 mg/day, about 0.1 to 2 mg/day, about 2 to 4 mg/day, about 4 to 6 mg/day, about 6 to 8 mg/day, about 8 to 10 mg/day, about 10 to 12 mg/day, about 12 to 14 mg/day, about 14 to 16 mg/day, about 16 to 18 mg/day, about 18 to 20 mg/day, about 0.5 mg/day, about 2 mg/day, about 10 mg/day, about 0.5 mg/day, about 0.5 to 10 mg/day, or about 0.1 to 20 mg/day. It would be clear to one skilled in the art that 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.

In an embodiment of the invention, 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.). In another embodiment, the Tamm-Horsfall protein may be administered directly in to the urinary tract using a time-release system. Examples of 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. The level of 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).

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. In one embodiment of the invention, 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.

In an embodiment of the invention, 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 comprising Tamm Horsfall protein and a pharmaceutically acceptable carrier, known to those skilled in the art. In one embodiment, 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.

These 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. Other carriers may also include sterile solutions; tablets, including coated tablets and capsules. Typically such carriers contain excipients such as starch, milk, sugar (e.g. sucrose, glucose, maltose), certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. 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.

In one embodiment, the invention provides a kit comprising the pharmaceutical composition of the invention. In another embodiment, 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.

Method for producing Tamm-Horsfall of the invention

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, O⁶-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.

Specifically, construction of 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

(See, e.g. New England Biolabs Product Catalog). In general, about 1 μg of plasmid or

DNA sequences is cleaved by one unit of enzyme in about 20 μl of buffer solution.

Typically, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate.

Incubation times of about one hour to two hours at about 37⁰C are workable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzvmology 65:499-560 (1980).

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 2O⁰C to 25°C in 50 mM Tris (pH 7.6) 50 mM NaCl, 6 mM MgCl₂, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fills in at 5' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present. If desired, 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. After treatment with Klenow, 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 μl 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)).

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.

The recombinant protein may be expressed in a prokaryotic, yeast, insect, plant or mammalian system. Examples of well known prokaryotic (bacterial) expression systems are E. coli (e.g. BL21, BL21 (DE3), XLl, XLl Blue, DH5α 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).

Many methods for inserting functional DNA transgenes into cells are known in the art. For example, 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.

Screening assays of the invention

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.

Further provided in the invention is a method for screening for agents that modulate production of Tamm-Horsfall protein. 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. In one embodiment of the invention, 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.

In one embodiment of the invention, the agents that modulate sialyation of Tamm- Horsfall protein result in hyper-sialyation of the Tamm-Horsfall protein. As used herein, 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.

In another embodiment, 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. In one embodiment, 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. (1989), Nature 340:245-246) or using a binding-capture assay (Harlow, supra). In the yeast two-hybrid system, 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. If the cloned segment of DNA encodes a protein that binds to the Tamm-Horsfall protein, 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.

As used herein, 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. Further, 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. As used herein, 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.

As used herein, 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. For example, 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. In addition, 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.

In one embodiment of the invention, classes of molecules that increase the levels of Tamm- Horsfall protein of the present invention are hormones. For example, in patients with Interstitial Cystitis, symptoms of Interstitial Cystitis are reduced during pregnancy. In a further embodiment of the invention, estrogen is administered to a subject to stimulate the production of Tamm-Horsfall protein. In yet another embodiment of the invention, 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. In one embodiment, the plurality of samples may comprise, more than about 10 or more than about 5 X 10⁴ samples. In another particular embodiment, 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.

A variety of other 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 4⁰C and 4O⁰C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening.

PHARMACEUTICAL COMPOSITIONS

The present invention provides 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. In some instances, 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. Moreover, 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. coli, 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.

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. 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 (for example, a subject polypeptide of the invention) is administered to the subject at a dosage sufficient to produce a desired result.

Typically, the 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.

In order to calculate the amount of molecules of the invention to be administered, those skilled in the art 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.

Regarding pharmaceutical dosage forms, 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. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Gennaro, A.R. (2003) Remington: The Science and

Practice of Pharmacy with Facts and Comparisons: Dragfacts Plus. 20¹¹¹ ed., Lippincott, Williams, & Wilkins. The 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.

The 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.

The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

EXAMPLES

EXAMPLE 1

Normal urine contains low molecular weight (LMW) cations toxic to cultured bladder cells and that Tamm-Horsfall protein (THP), a ubiquitous urinary glycoprotein, protects against this cytotoxic effect. THP may sequester and subsequently neutralize these toxic urinary cations. In this study we tested whether the presence of THP can limit the injury caused by these urinary cations using an in vivo rat bladder injury model.

Materials and Methods

Human and Animal Subjects

Collection of Urine from Healthy Human Volunteers. Informed consent was obtained and 24-hour pooled urine samples were obtained from 3 healthy female volunteers (mean age 30 years). After collection, urines were stored at -20EC until use for isolation of the toxic factor (TF).

Animal subjects. Animal studies employed adult male Sprague-Dawley (SD) rats under a protocol approved by the Veterans Affairs Medical Center Institutional Animal Care and Use Committee. Preparation of the Toxic Factor or Dialyzed Product from Human Subjects

Potential human control 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.

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., Rancho 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.

Cytotoxicity Assay

The toxic factor (>100<3500 MWCO) was tested for cytotoxicity using the CellTiter96 Aqueous One Solution Proliferation Assay™ (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).

For cytotoxicity assay, cells were harvested and resuspended in DME media (without phenol red indicator) with 1% FCS (assay media) containing 100 μg of solubilized TF in a volume of lOOμl/well. Negative control wells were similarly prepared but did not contain any TF. For the wells containing TF plus THP, 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 (lOOμl) added to triplicate test wells. After overnight incubation at 37⁰C, the wells were washed twice with DME alone (200μl/well) and fresh DME added to each well (90μl). Then a novel tetrazolium compound (MTS-Owen's Reagent) combined with an electron coupling reagent (PES) was added (10 μl/well) per kit protocol and absorbance measured in a plate reader after 30 minutes incubation at 490 nm and at 630 ran after blanking the plate to DME media containing the tetrazolium compound. The background at 630 nm was subtracted from the 490 nm reading to determine percentage cytotoxicity relative to the media controls.

Cytotoxicity levels were compared between groups as follows: TF alone versus control, TF plus THP versus TF alone, and TF plus THP versus control.

Evaluation of Bladder Overactivity in Rodents

After a few days of acclimatization, two groups (n = 9 each) of adult male Sprague- Dawley rats (325-350 g) underwent urodynamics evaluation of bladder hyperactivity before and after intravesical infusion of a test substance (TF or TF plus THP) followed by intravesical KCl.⁸ The procedure was as follows.

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. 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). During the continuous filling bladder cystometry, the pressure was recorded with the transducer using the program Lab View (National Instruments, TX). To induce hyperactivity, the bladder was first infused with warm (37⁰C) 0.9% saline or 400 mM KCl (29.8 mg/mL, Abbott Laboratories, IL) at 40 μL/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 H₂O and less than the PT, thus not resulting in a void).

After these baseline measurements, rats were infused with the test solution. To test the hypothesis that TF could injure the bladder mucosa and facilitate KCl-induced bladder hyperactivity, one group of animals received an infusion of TF (15 mg/mL). To test the hypothesis that 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.

In both groups, infusion of the test solution was followed by infusion of 400 mM KCl (29.8 mg/mL, Abbott Laboratories, IL) infusion for 30 minutes⁸ and all urodynamics measurements were repeated.

Statistical Analysis

Results were analyzed by Student's t test, employing the Bonferroni correction where more than two variables were involved.

Results

Results of the in vitro cytotoxicity assays

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).

Results of in vivo rat studies

Infusion of intact rat bladder with NaCl or KCl resulted in normal and comparable numbers of voids and NVC (Table 2). After intravesical TF, KCl infusion resulted in a significant increase in NVC relative to pre-TF values (p < 0.0001). After infusion of TF premixed with THP, KCl irrigation was associated with significantly lower numbers of NVC than those seen after infusion of TF alone (p < 0.0001). There was no significant difference in the numbers of NVC recorded at baseline and the number recorded after infusion of TF plus THP (Table 2).

Discussion

It was previously proposed that IC results from a disruption in the critical balance of protective mechanisms and potentially pathogenic factors in the lower urinary tract. It is recognized that the relative impermeability of the bladder mucosa is the primary protective mechanism of the bladder wall. There is substantial evidence that bladder epithelial permeability is regulated by the surface mucus.^10"12 Data from a number of centers indicate that IC is associated with an epithelial dysfunction that results in abnormal permeability. ' '

Data from earlier studies indicate that the primary urinary toxin that appears to be initiating symptoms and tissue injury is the cation potassium.¹' ^{2> 18~21} Potassium is present in relatively high levels (60 - 130 mEq/L) in the urine; when the epithelium is abnormally permeable, potassium is allowed to diffuse down the gradient from high urine levels to lower tissue levels, where it can depolarize nerves and muscle and, on a chronic basis, injure tissue.¹' ^{2> 18} When the epithelial permeability barrier is intact, potassium does not provoke symptoms.¹

Recent investigations have focused on the possible role of urine factors in the pathogenesis of IC. In earlier studies, it was found that normal human urine contains LMW cations similar to protamine sulfate, a cationic compound known to be extremely toxic to the mucus.⁴ These LMW cations, partially isolated in the toxic factor (TF), are likely to be amines that are the end product of protein metabolism. It has been previously reported that the TF causes epithelial cell injury in vitro.⁴ The cationic TF have the potential for electrostatically binding to the anionic transitional cell surface mucus, disrupting the permeability barrier and allowing the cascade of potassium diffusion, sensory nerve stimulation, and tissue injury to occur.¹' ²' ¹⁸ The presence of a urinary toxic factor has also been proposed by Keay, who isolated an antiproliferative factor that may have a role in epithelial injury.²²

The difference between a healthy state and the disease state may be a qualitative or quantitative deficiency of a protective factor that normally prevents the cations from injuring mucus by electrostatically attracting them before they have a chance to damage the bladder surface. In previous studies, it has been proposed that THP may function as a urinary protector that prevents the natural byproducts of metabolism from injuring mucus.⁴'⁷ We have shown that THP is capable of neutralizing these toxic factors in a cell culture assay. ⁷

We conducted the current study to verify the presence of the urinary toxic factors and to test the ability of THP to neutralize their toxic effect both in vitro and in vivo. To accomplish this, we used two models. First, we isolated a LMW fraction from urine and used an in vitro cytotoxicity assay to determine its cytotoxicity to cultured urothelial cells and to test for potential neutralization of the toxic effects by prior incubation with THP. We found that the urinary TF causes significant cytotoxicity. Exposure of the TF to THP, which is highly anionic, abrogated the toxic effect of this LMW fraction (Table 1).

Second, we used a rodent urodynamics model⁸ in which the bladders of healthy rats are infused with KCl before and after urothelial injury with protamine sulfate (PS) and a marked increase in muscle reactivity is observed in response to KCl after the PS treatment. We substituted TF for PS as the epithelial injury agent in this model. A rat with a healthy urothelium should not react to KCl, and our baseline KCl data confirmed this, showing no significant increase in bladder hyperactivity in animals treated with KCl versus NaCl. When the urothelium was injured with TF, however, we found a marked increase in NVC due to altered urothelial permeability to KCl. The NVC represent muscle spasticity or fibrillation, an abnormal reaction to KCl. We found 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.

The findings from the current study lend support to our hypothesis that 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.

Both our earlier data and the results of the current study support the hypothesis that the initiation of IC may rest in the imbalance of cations and anions in urine, with THP being the primary anion responsible for protecting the epithelium.⁴' ⁷ We have isolated a cationic toxic urinary fraction and shown that it is as injurious as protamine to the epithelial cells of the bladder, resulting in increased permeability and increased sensitivity to potassium.⁴ Further, our data indicate that THP can neutralize both protamine⁷ and the anionic urinary toxic factor.⁴ These findings suggest that THP may operate to bind potentially injurious cations in the urine in healthy individuals. If this is the case, then the ability of THP to perform this function may contribute to the prevention of IC in the normal state. Conversely, the pathogenesis of IC may involve a reduction in the protective capacity of THP.

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.

TABLE 1. Cytotoxicity levels in rat and human cultured urothelial cells after treatment with urinary toxic factor or toxic factor plus THP*

Human cells (HBT4) Rat urothelial cells

Cyto- Cytotoxicity Statistical toxicity Statistical Group N (%) significance N (%) significance

Control (negative) 9 0 - 9 0

TF 9 27.1 p < 0.01t 9 31.1 p < 0.01f

TF + THP 9 0 p < 0.01J 9 0 p < 0.01$ Key: THP, Tamm-Horsfall protein; TF = toxic factor

* We used this cytotoxicity assay to verify the presence of the toxic factor (TF).

Clearly, the urine contained TF and the TF was neutralized by THP. This toxic urine fraction was used in the in vivo rat model. t Compared to control. % Compared to TF alone. There was no significant difference between cytotoxicity levels in the control group and those in the group treated with TF plus THP. TABLE 2. Effect of potassium on rat bladder after treatment with urinary toxic factor or toxic factor plus THP*

Agent infused into rat Statistical bladder N NVC/minute significance

NaCl (baseline) 9 0.2400 ± 0.074 -

KCl (baseline) 4 0.25 ± 0.064 -

Toxic factor 9 1.681 ± 0.11 P < 0. .oooit

Toxic factor plus THP 9 0.2778 ± 0.08462 P < 0, .0001$

Key: THP, Tamm-Horsfall protein; NVC, nonvoiding contractions;

TF = toxic factor

* As can be seen, similar to the cytotoxicity results, 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.

% Compared to TF alone. No significant difference when compared to NaCl baseline value.

EXAMPLE 2

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. 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.

Materials and Methods

Human and Animal Subjects

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 -2O⁰C until they were used for isolating TF and/or THP. Animal studies employed adult male Sprague-Dawley (SD) rats weighing 325-350 g.

Preparation of THP and THP-d

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 (THP-d) 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 μg Neu5Ac/mg protein for the THP-d), coincident with an increase in the electrophoretic migration of the THP.

Preparation of the Toxic Factor from Human Subjects

The LMW (>100<3500) TF was prepared as described previously.²³ Cytotoxicity Assay

The TF obtained from 2 different pooled urine samples (TFl, TF2) was tested for cytotoxicity using the CellTiter96 Aqueous One Solution Proliferation Assay™ (Promega Corp, Madison, WI). Rat urothelial target cells and human HTB4 urothelial cells were plated in 96-well tissue culture plates (Nunclon™) 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).

For 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 10⁶ cells/ml, from which 100 μl (5 x 10⁴ cells) were added to each well in a 96-well microtiter plate. Test samples (100 μl) were added to these wells in quadruplicate. For these assays, THP (obtained from 4 different normal urines) was pooled together, and the desialylated THP from these same samples (THP-d) were mixed with TF and incubated at room temperature for 60 min, then centrifuged before they were added to the wells containing the target cells. Samples of each test material were tested individually for cytotoxicity and tested in checkerboard fashion with the THP, THP-d, and TF preparations. Dose response data was determined beforehand for the TF preparations. The final concentrations of all test samples were adjusted so that they were identical during assay.

After overnight incubation at 37⁰C, the target cells were washed twice with DME alone (200 μl/well) and fresh DME added to each well (90 μl). Then a tetrazolium compound

(MTS-Owen's Reagent) combined with an electron coupling reagent (PES) was added

(10 μl/well) per kit protocol and absorbance measured in a plate reader after 30 min incubation at 490 nm and at 630 nm after blanking the plate to DME media containing the tetrazolium compound. The background at 630 nm was subtracted from the 490 nm reading to determine percentage cytotoxicity relative to the media controls. Evaluation of Bladder Overactivity in Rodents

After a few days of acclimatization, adult male SD rats underwent urodynamics evaluation of bladder hyperactivity before and after intravesical infusion of a test substance⁸ (TF or TF plus unmodified THP or TF plus THP-d) followed by intravesical KCl. The procedure for the in vivo rat study was performed as described previously.²³

After baseline measurements of pressure threshold, peak pressure, frequency of contractions, inter-contractile interval, and percentage of nonvoiding contractions (NVC) were recorded as previously described,²³ rats were infused with the test solution. One group of animals received an infusion of. TF (15 mg/niL). To test the effect of desialylation on the ability of THP to attenuate the bladder hyperactivity induced by TF and KCl, second and third groups of animals received an infusion of a mixture of unmodified THP or THP-d 10 mg/ml and TF (15 mg/ml) mixed in a ratio of 1:1.

Infusion of the test solution was followed by infusion of 400 mM KCl for 30 min⁸ and urodynamics measurements were repeated.

Statistical Analysis

Results

Results of the in vitro cytotoxicity assays

TF had a significant cytotoxic effect on HTB4 epithelial cells relative to the media controls (P < 0.01). In the wells containing TF mixed with unmodified THP, there were no detectable levels of cytotoxicity (Table 3). In the wells containing incubated with THP-d, however, there was significant cytotoxicity (13.4%), approximately the same (P = 0.5) as when TF alone was incubated with the target cells (average 7.4 %) (Table 3).

Results of in vivo rat studies

Infusion of intact rat bladder with NaCl or KCl resulted in normal and comparable numbers of voids and NVC (Table 4). After intravesical TF, KCl infusion resulted in a significant increase in NVC relative to pre-TF values (P < 0.0001). After infusion of TF premixed with unmodified THP, KCl irrigation was associated with significantly lower numbers of NVC than those seen after infusion of TF alone (P < 0.0001). There was no significant difference in the numbers of NVC recorded at baseline and the number recorded after infusion of TF plus unmodified THP (Table 4). After infusion of TF mixed with THP-d, KCl irrigation was associated with a significantly higher NVC rate than that seen after infusion of TF plus unmodified THP (P < 0.0001).

Discussion

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}

Studies indicate that the 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 In recent studies, 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.⁴' ^{23> 7} 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. Such 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. On the basis of earlier data, we have proposed that 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}

In the current study, we isolated a LMW TF fraction from urine, verified its cytotoxicity to cultured urothelial cells, and tested the ability of unmodified and desialylated THP to neutralize the toxic effects in vitro and in vivo. The data indicate that the urinary TF causes significant cytotoxicity. The toxic effect of this LMW fraction was neutralized by exposure to unmodified THP but not by exposure of the TF to THP that had undergone desialylation (Table 3).

Second, the ability of unmodified versus desialylated THP to neutralize the toxic effects of the urinary TF in vivo using was investigated using a rodent urodynamics model.⁸ In our adaptation of this model, the bladders of healthy rats are infused with KCl before and after urothelial injury with urinary TF, and the resulting muscle reactivity (NVC) is quantified. The data confirmed that a rat with a healthy urothelium does not exhibit an increase in NVC in response to intravesical KCl₅ but a rat who has undergone urothelial injury with TF does display a marked increase in NVC in response to KCl because the urothelial permeability has been altered. Exposure to THP appeared to prevent TF from inducing urothelial injury, as would be evidenced by a rise in NVC (Table 4). When TF was exposed to desialylated THP, however, there was a significant rise in NVC, suggesting that desialylated THP lacks the protective effects of unmodified THP.

The data presented herein that 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.²⁴ The results of the current study support the hypothesis that IC may result from the disruption of a balance between protective anionic factors and potentially injurious cations in the urine.⁴' ²³' ⁷ These results demonstrate a cationic toxic urinary fraction that is similar to protamine in its ability to injure the epithelial cells of the bladder, resulting in increased permeability and increased sensitivity to potassium.⁴' ²³' ⁷ THP neutralizes the injurious effects of both protamine⁷ and the cationic urinary toxic factor.⁴'²³ The current study indicates that the protective activity of THP is dependent on sialic acid, a highly anionic part of the THP molecule that most likely imparts the protective activity to the protein.

These findings suggest that 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.

TABLE 3. Cytoprotection of target cells against toxic factor by unmodified versus desialylated Tamm-Horsfall protein*

Average Statistical cytotoxicity significance: test Test substance n (%)t vs. media alone Result

Media 8 0 - -

TF 8 7.4 P < 0.01 Toxic

THP + TF: J

THP + TF 32 0 NS Not Toxic

THP-d + TF 32 13.4 P < 0.01 Toxic

Key: TF = toxic factor; THP = Tamm-Horsfall protein; THP-d = desialylated Tamm Horsf all protein; n = number of wells assayed; NS = not significant. *Target cells were human bladder cells (HTB4)-5 x 10⁴ /well fCytotoxicity of protamine sulfate, a known cytotoxic agent: LD50 = ~20 μg ^Cytotoxicity of THP-d +TF (all) vs. TF (1, 2) not significant (P = 0.5)

TABLE 4. Effect of potassium on rat bladder after treatment with urinary toxic factor, toxic factor plus unmodified Tamm-Horsfall protein, or toxic factor plus desialylated Tamm-Horsfall protein*

Agent infused into rat bladder n NVC/minute P value

NaCl (baseline) 9 0.24 ± 0.074 -

KCl (baseline) 4 0.25 ± 0.064 -

TF 9 1.68 ± 0.11 P < 0.000 If

TF + unmodified THP 14 0.42 ± 0.13 P < 0.0001$

TF + THP-d 6 1.55 ± 0.62 P < 0.0001 §

Key: NVC = nonvoiding contractions; TF = toxic factor; THP = Tamm-Horsfall protein; THP-d = desialylated Tamm Horsfall protein. * As can be seen, similar to the cytotoxicity results, the TF allowed K to diffuse into the interstitium, where it provoked NVC ("fibrillation" activity); the TF effects were abolished by THP. f Compared to NaCl baseline value. / Compared to TF alone. No significant difference when compared to NaCl baseline value. § Compared to TF + unmodified THP.

EXAMPLE 3

This example demonstrates the measurement of the sialic acid content of THP from the urine of normal subjects versus IC patients. To investigate a possibility that would lend additional support to the concept that sialic acid content is reduced in IC versus normal subjects, 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.

Materials and Methods

Isolation of THP from Urines

THP is isolated by the cartridge centxifugation-washing method. For this isolation 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 4⁰C 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). Before use, 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.

Hydrolysis of THP and Sialic Acid Determination

An aliquot (150 μl volume) of the THP recovered from normal (n=29) and IC (n=28) urines by filtration-concentration, is subjected to mild acidic hydrolysis (2.5M acetic acid) by heating at 80°C for 3 hr. The free sialic acid in the hydrolysate is recovered by passing through minicon filters (10000MW cutoff) to remove any protein, then dried with mild heat under vacuum centrifugation and finally sent for sialic acid determinations by the DMB fluorescent detection method (sensitivity pM range). THP samples prior to hydrolysis were shown to have no detectable endogenous sialic acid.

The dried THP samples are dissolved in 50 μl of milliQ water and 50 μl 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 μl 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 μl hydrolysate of the THP samples is then used to calculate the amount of sialic acid pM/μg 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.

ELISA Assay for THP Quantitation

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 4⁰C 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. For quantitation of THP by the indirect ELISA, a standard curve is prepared by adding 100 μl of twofold serial dilutions of a THP standard (2.000-0.015 ng/well) to duplicate wells and immediately adding 100 μl 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 μl of each sample is added to 90 μl of assay buffer and 100 μl 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 μl 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 μg THP/150 μl (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.

THP Zeta Potential (mV) Measurements

Surface charge properties (zeta potential) are measured for THP samples from patients («=6) and controls (n=6). THP (Biomedical Technologies, Inc.) and fetuin and asialofetuin (Sigma Chemical Co.) standards (50 μg) are similarly measured. Zeta potential and size measurements are made with a Zetasizer Nano ZS instrument and a CGS-3 Goniometer system (Malvern Instruments Inc., Southborough, MA) with ancillary software version 4.0. All zeta potential measurements are conducted on rehydrated samples in .01M NaCl buffer, pH 7.0 at 25°C, after centrifugation followed by filtration (.45 μM) to remove any aggregates. Measurements are made in triplicate and the results analyzed by Student's t test for significance.

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.

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/μg 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.

Results

There were 29 normal subjects and 28 IC patients. Sialic acid content was significantly lower in IC patients' THP than in control subjects' THP (224 vs. 1001 pM/μg THP; P < 0.01, t test) (Table 5).

IC patients' THP had a significantly lower zeta potential than control THP (-32.7 ± 1.4 vs. -28.0 ± 2.4 mV; n=6 in each group; P < 0.002, t test), a difference of 14.4% (Figure 1). These results indicate IC patients' THP has significantly less surface charge (electronegativity) .

THP concentrations in normal urine (28.2 mg/L) and IC urine (28.8 mg/L) were not significantly different. When 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).

Discussion

Prior experiments have demonstrated that 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 ^{" > > > •} ' . There is extensive evidence that this diffusion of potassium occurs in IC, and it is a phenomenon that could account for the entire cascade of symptom generation and tissue injury seen in this disease^1"3'⁸' ^{17> 18> 27>28}.

The key question is why this dysfunctional epithelium occurs. It is recognized that the glycosaminoglycans in the healthy epithelial mucus are extremely hydrophilic. This "hydrating effect" traps water at the cell surface and inhibits the movement of molecules across the epithelium¹⁰' ^30"32. In the gastrointestinal tract, this layer of water has been called the "unstirred layer of water"^33"35. When the bladder mucus is exposed to highly charged cationic compounds such as protamine sulfate, the mucus is rendered dysfunctional, resulting in a "leaky epithelium"¹⁰. Because the epithelial mucus is highly anionic, naturally occurring urinary cations potentially could bind to it and alter its ability to hydrate the cell membrane. To test this concept, a low molecular weight cationic compound(s) called toxic factor (TF) was isolated from normal urine. TF and protamine sulfate (highly cationic) injure urothelial cells in vitro and alter epithelial permeability m vivo, resulting in potassium diffusion into the bladder muscle and an increase in nonvoiding bladder muscle contractions ²³' ²⁹.

The next logical question is whether urine contains protective molecules that are anionic and capable of electrostatically attracting TF (and protamine) and neutralizing their toxic effects on the epithelium. It appears to have just such a molecule, Tamm-Horsfall protein (THP). The interaction of THP with cationic urine molecules results in sequestering these potentially toxic ions to the oppositely charged layer surrounding THP. Serum factors that are protein bound to albumen likewise become biologically ineffective; an example is testosterone. A similar activity is proposed for THP; in this case, urinary toxic factors are bound. In both instances, the phenomenon can be mediated by surface charge, or the zeta potential. 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 species⁶' 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 vitro⁴' ^{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 vitro⁷.

The results of this example support our hypothesis that THP is abnormal in IC patients and that the abnormality is a significant reduction in THP sialic acid content. Sialic acid content was significantly lower (approximately 80%) in IC patients than in normal controls. In addition, the zeta potential, which represents the electronegativity of the molecule, was significantly lower in THP from IC patients than in THP from normal control subjects. Such a difference could result in the loss of THP' s ability to sequester the TF, ultimately resulting in mucosal injury and epithelial dysfunction leading to the entire cascade that results in IC.

Because a lower total urinary THP concentration could account for the finding of lower sialic acid content and zeta potential in IC patients' THP, the total THP concentration in the urine from normal subjects and IC patients was determined. There were no differences between the normal and the IC samples regardless of whether THP concentration was determined in terms of mg protein per liter of urine or mg protein per mg creatinine (Table 6).

These findings may represent an important piece of the puzzle of IC pathogenesis. It appears that 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. These data may have important implications not only for understanding of the initial cause of IC but also for knowledge of physiologic role of THP in urine, a role that has not been well understood. With the understanding that a defect in a urinary protective molecule (THP) can lead to substantial problems in the health of the urinary bladder, efforts can be focused on new vistas in IC therapy. These might include creating 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. The finding of a specific abnormality in IC patients' THP, coupled with the evidence that normal THP operates as an important protective factor against bladder epithelial injury, represents a major step in understanding the etiology of IC and developing diagnostic and therapeutic treatments for IC .

Table 5. Sialic acid content in urinary THP from IC patients versus normal controls. THP, Tamm-Horsfall protein; IC, interstitial cystitis.

Sialic acid content Group N (pM/μg THP) P value

Control subjects 27 964

IC patients 25 224 _; < 0.001* * Versus control.

Table 6. Total THP in IC versus normal urine. THP, Tamm-Horsfall protein; IC, interstitial cystitis; N.S., not significant.

Urinary THP mg/L urine mg/mg creatinine

Normal IC Normal IC » = 39 n = 115 P Value n = 25 n = 76 P Value

28.2 28.8 N.S. 76.4 70.0 P = 0.596

EXAMPLE 4

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). In addition, a new in vitro rat urodynamic model⁴ 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.

Protamine sulfate (PS) 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.

The mechanism by which PPS can potentially control the symptoms of IC was also explored. It was determined whether the highly anionic structure of PPS can electrostatically bind to charged toxic cations before they can injure the mucosa. Heparinoids may have similar reactivity in their successful use in management of IC.

Materials and Methods

Human and Animal Subjects

Collection of urine from healthy human volunteers. Informed consent was obtained and 24-hour pooled urine samples obtained from 3 healthy female volunteers, median age 30 years. After collection, urines were stored at -20°C until use. The anonymity of the human subjects was preserved in accordance with institutional policies and the Health Insurance Portability and Accountability Act (HIPAA) of 1996. Animal subjects. Animal studies employed adult male Sprague-Dawley (SD) rats under a protocol approved by the Veterans Affairs Medical Center Institutional Animal Care and Use Committee.

Preparation of the Toxic Factor or Dialyzed Product from Human Subjects

Human control subjects were screened for IC symptoms using the Pelvic Pain and

U Urrggeennccyy//FFrrsequency Patient Symptom (PUF) Scale.⁶ A PUF Score of 0 was required for study entry.

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., Rancho 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.

Cytotoxicity Assay

The toxic factor (>100<3500 MWCO) was tested for cytotoxicity using the CellTiter96 Aqueous One Solution Proliferation Assay™ (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 (Nunclon™) 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).

For cytotoxicity assay, cells were harvested and resuspended in DME (without phenol red indicator) with 1% FCS (assay media) containing 100 μg of solubilized toxic factor in a volume of 1 μl/well. Negative control wells were similarly prepared but did not contain any toxic factor. Positive control wells contained protamine sulfate (PS), 1 mg/ml. For 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 OOμl) added to triplicate test wells.

After overnight incubation at 37°C, the wells were washed twice with DME alone (200 μl/well) and fresh DME added to each well (90 μl). Then a novel tetrazolium compound (MTS-Owen's Reagent) combined with an electron coupling reagent (PES) was added (10 μl/well) per kit protocol and absorbance measured in a plate reader after 30 minutes incubation at 490nm and at 630nm after blanking the plate to DME media containing the tetrazolium compound. The background at 630 nm was subtracted from the 490 nm reading to determine percentage cytotoxicity relative to the media controls.

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.

Evaluation of Bladder Overactivity in Rodents

After a few days of acclimatization, three groups (n = 9 each) of adult male Sprague- Dawley rats (325-350 g) underwent urodynamics evaluation of bladder hyperactivity before and after intravesical infusion of a test substance (PS, urinary toxic factor, or toxic factor plus PPS) followed by intravesical KCl.⁴

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). During the continuous filling bladder cystometry, the pressure was recorded with the transducer using the program Lab View (National Instruments, TX).

To induce hyperactivity, the bladder was first infused with warm 0.9% saline (37⁰C) at 40 μL/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 H₂O and less than the TP, thus not resulting in a void).

After these baseline measurements, rats were infused with the test solution. Group 1 (positive control) animals received 1 mL of a warm solution (37⁰C) of 30 mg/mL PS (Sigma- Aldrich, St. Louis, MO) for 30 minutes. PS has been shown to significantly injure urothelial permeability.⁵ To test the hypothesis that urinary toxic products could injure the bladder mucosa and facilitate KCl-induced hyperactivity, Group 2 animals received an infusion of toxic factor (15 mg/mL). To test the hypothesis that PPS could attenuate the bladder hyperactivity induced by toxic product and KCl, Group 3 animals received an infusion of a mixture of PPS (10 mg/ml) and toxic factor (15 mg/ml).

In all three groups, infusion of the test solution was followed by infusion of 400 mM KCl (29.8 mg/mL, Abbott Laboratories, IL) infusion for 30 minutes⁴ and all urodynamics measurements were repeated.

Statistical Analysis

Results were analyzed by Student's t test, employing the Bonferroni correction where more than two variables were involved. Results

Results of the in vitro cytotoxicity assays

Both PS (the positive control) and the toxic factor had a significant (p O.001) cytotoxic effect in cultured rat bladder epithelial cells relative to the negative control. In the wells containing toxic factor plus PPS, however, cytotoxicity levels were significantly lower than in the wells containing toxic factor alone (p < 0.007). The combination of PS and PPS was associated with significantly lower rates of cytotoxicity than PS alone (p < 0.02) (Table 7).

Results of in vivo rat studies

Infusion of intact rat bladder with sodium or KCl resulted in normal and comparable numbers of voids (1.240 ± 0.1875/min) and NVC (Figure 2A; Table 8). After intravesical PS or toxic factor (Figure 2B), KCl infusion resulted in a significant increase in NVC relative to pre-PS/toxic factor values. After infusion of toxic factor premixed with PPS, KCl irrigation was associated with significantly lower numbers of NVC than those seen after infusion of toxic factor alone. There was no significant difference in the numbers of NVC recorded at baseline and the number recorded after infusion of toxic factor plus PPS (Table 8).

Table 7. Cytotoxicity levels in rat cultured urothelial cells after treatment with urinary toxic factor or toxic factor plus PPS

In this in vitro cytotoxicity assay, data was obtained indicating that a cytotoxic factor is present in urine and has a toxic effect on cultured urothelial cells. Pentosan polysulfate was demonstrated to neutralize the cytotoxic effect of the urinary toxic factor as well as of the positive control protamine sulfate.

Cytotoxicity Statistical

Group N significance

Control (negative; no TF or PS)

Protamine sulfate (positive control) 61.9 ± 27.6 p < 0.001t

Toxic factor 12 26.9 ± 13.9 p < 0.001t

Toxic factor plus PPS 10.9 ± 8.6 p < 0.007$

Protamine sulfate plus PPS p < 0.02§ f Compared to negative control group. ^Compared to toxic factor alone. § Compared to positive control group.

Table 8. Effect of potassium on rat bladder after treatment with urinary toxic factor or toxic factor plus PPS

In this in vivo study, infusion of potassium into the bladder provoked "fibrillation" activity, as indicated by a significant increase in nonvoiding contractions, after the bladder had been treated with either the urinary toxic factor or the known epithelial injury agent protamine sulfate. Potassium did not cause fibrillation in an untreated bladder, suggesting the toxic factor injures the urothelium and allows K to diffuse into the interstitium, where it activates the muscle and nerves. As in the in vitro assay, pentosan polysulfate neutralizes the injurious effect of the urinary toxic factor.

Agent infused into rat Statistical bladder N NVC/minute significance

NaCl (baseline) 9 0.2400 ± 0.074

KCl (baseline) 4 0.25 ± 0.064

Group 1: Protamine sulfate 9 1.733 ± 0.55 p = 0.01t

(positive control)

Group 2: Toxic factor 9 1.681 ± 0.1131 p = 0.0004f

Group 3: Toxic factor plus PPS 9 0.63 ± 0.05 p = 0.0102$ f Compared to NaCl baseline value.

% Compared to Group 2. No significant difference when compared to NaCl baseline value.

Discussion

Normal human urine was investigated to determine whether it contained compounds capable of causing epithelial cell injury, possibly by interfering with the protective function of the mucus lining of the bladder, leading to epithelial injury and loss of the permeability barrier function. This would allow access of urinary solutes to the bladder interstitium, leading to initiation of symptoms and potentially to cell injury. 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.

The results presented herein demonstrate that urine contains LMW cations similar to PS, which has been shown to be a toxic, mucus-damaging substance.⁵ These cations, present in urine, have the potential for electrostatically binding to the anionic transitional cell surface mucus, disrupting the permeability barrier and allowing the cascade of potassium diffusion, sensory nerve stimulation, and tissue injury to occur.

Two models were used. First, a LMW fraction was isolated from urine, and its cytotoxicity determined to cultured urothelial cells, and tested for potential neutralization of the toxic effects by prior incubation with the anionic PPS. The urinary toxic factor was just as capable of injuring the urothelium as PS and exposure of the toxic fraction to PPS neutralized the toxic effect (Table 7).

Second, a rodent urodynamics model⁴ 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. 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. In the urodynamics model,⁴ KCl is infused before and after urothelial injury with PS and a marked increase in muscle reactivity is observed after the PS treatment. A rat with a healthy urothelium should be no more reactive to KCl than to Na+, as demonstrated herein. When the urothelium was injured with toxic factor or PS as a positive control, a marked increase in NVC was found due to altered urothelial permeability to KCl and rapid secondary muscular contraction. In addition, we found PPS blocked the NVC induced by toxic factor (Table 8).

Conclusions

As shown herein, 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. Further, the data indicate that PPS can neutralize the dialyzed toxic factor from urine. These findings suggest that PPS may operate to address pathophysiologic processes of IC both in the epithelium and in urine, assisting in repair/replacement of the epithelium and binding potentially injurious cations in the urine. These data also suggest that IC may be initiated by these naturally occurring urinary cations if not sequestered by other compounds.

REFERENCES

I. Parsons CL, Greenberger M, Gabal L, Bidair M, Barme, G. J Urol 1998; 159: 1862-1867. 2. Parsons CL, Stein PC, Bidair M, Lebow D. Neurourol Urodyn 1994; 13: 515- 520.

3. Parsons CL. Urology 2003; 62: 976-982.

4. Parsons CL, Bautista SL, Stein PC, Zupkas P. J Urol 2000; 164: 1381-1384.

5. Badgett A, Kumar S. Urol Int 1998; 61 : 72-75. 6. Tamm I, Horsfall FL. Proc Soc Exp Biol Med 1950; 74: 108-114.

7. Akiyama A, Stein PC, Houshiar A, Parsons CL. Internat J Urol 2000; 7: 176-183.

8. Chuang YC, Chancellor MB, Seki S, Yoshimura N, Tyagi P, Huang L, et al. Urology 2003; 61: 664-670.

9. Parsons CL, Dell J, Stanford EJ, Bullen M, Kahn BS, Waxell T, et al. Urology 2002; 60: 573-578.

10. Lilly JD, Parsons CL. Surg Gynec Obst 1990; 171: 493-496.

I 1. Parsons CL, Stauffer C, Schmidt JD. Science 1980; 208: 605-607.

12. Parsons CL, Boychuk D, Jones S, Hurst R, and Callahan H. J Urol 1990; 143:

139-142. 13. Parsons CL, Lilly JD, Stein PC. J Urol 1991; 145: 732-735.

14. Buffmgton CAT, Woodworth BE. J Urol 1997; 158: 786-789.

15. Chelsky MJ, Rosen SI, Knight LC, Maurer AH, Hanno PM, Ruggieri MR. J Urol 151: 346-349, 1994.

16. Erickson DR, Herb N, Ordille S, Harmon N, Bhavanandan VP. J Urol 164: 419- 422, 2000.

17. Hohlbrugger G. Sant GR (Ed): Interstitial Cystitis. Philadelphia, Lippincott- Raven Publishers, 1997, pp. 87-92.

18. Parsons CL, Greene RA, Chung M, Stanford EJ, Singh G. J Urol 2005; 173:1182-1185. 19. Daha LK₅ Riedl CR, Lazar D, Hohlbrugger G, Pfluger H. Eur Urol 2005,47:393- 397.

20. Gregoire M, Liandier F, and Naud A, Lacombe L₅ Fradet Y. J Urol 2002; 168: 556-557. 21. Chung MK, Chung RP, Gordon D. JSLS 2005;9:25-9.

22. Keay SK, Szekely Z, Conrads TP, Veenstra TD, Barchi JJ Jr₅ Zhang CO₅ et al. Proc Natl Acad Sci U S A. 2004;101:11803-8.

23. Stein P, Rajasekaran M₅ Parsons CL: Urology 66: 903-907, 2005.

24. Easton RL₅ Patankar MS₅ Clark GF₅ et al: J Biol Chem 275: 21928-21938, 2000. 25. R.E. Hurst, R. Zebrowski, J. Urol. 152, 1641 (1994).

26. J. Cornish, J.C. Nickel, M. Vanderwee, J.W. Costerton, Urol. Res. 18, 263 (1990).

27. N.G. Moss, W. W. Harrington, M.S. Tucker, Am. J. Physiol. 272, R695 (1997).

28. L.K. Daha et al, J. Urol. 170, 807 (2003).

29. M. Rajasekaran, P. Stein, CL. Parsons, Int. J. Urol 13, 409 (2006). 30. CC. Gryte, H.P. Gregor, J. Polymer ScL 14, 1839 (1976).

31. H.P. Gregor, in Charged Gels and Membranes, Part I₁ E. Selegny, Ed. (D. Reidel, Boston, 1976), pp. 235-254.

32. H.P. Gregor, in Biomedical Applications of Polymers, H.P. Gregor, Ed. (Plenum, New York, 2005), pp. 51-56. 33. J.M. Dietschy, V.L. Sallee, F.A. Wilson, Gastroenterology 61, 932 (1971).

34. F.A. Wilson, VX. Sallee, J.M. Dietschy, Science 174, 1031 (1971).

35. H. Westergaard, J.M. Dietschy, J. Clin. Invest. 58, 97 (1976).

36. Shirley SW, Stewart BH, Mirelman S., Urology, 11, 215-20 (1978)

37. Parsons, CL, Housley T₅ Schmidt JD, Lebow D, Br J Urol, 73, 504-7 (1994) 38. Parsons, CL, Urology, 65, 45-8 (2005)

Claims

What is claimed is:

1. A method for inhibiting Interstitial Cystitis and its symptoms in a subject comprising administering an effective amount of a Tamm-Horsfall protein to the subject, so as to inhibit Interstitial Cystitis and its symptoms in the subject.

2. A method for reducing symptoms of Interstitial Cystitis in a subject by inhibiting Interstitial Cystitis in a subject by the method of claim 1, thereby reducing the symptoms of Interstitial Cystitis.

3. A method for increasing the levels of Tamm-Horsfall protein in subject comprising administering to the subject an effective amount to Tamm-Horsfall protein, thereby increasing the levels of Tamm-Horsfall protein.

4. A method for repairing a mucin layer of bladder in the subject by the method of claim 3, so as to repair the mucin layer of the bladder.

5. A method for treating a disease associated with decreased levels of Tamm- Horsfall protein by the method of claim 3, thereby treating the disease.

6. A method for diagnosing Interstitial Cystitis in a subject comprising 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 being indicative of Interstitial Cystitis.

7. 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.

8. The method of claims 1, 2, 3, 4, or 5, wherein the Tamm-Horsfall protein is administered directly in to the urinary tract in a subject.

9. The method of claim 8, wherein the Tamm-Horsfall protein is administered using a catheter.

10. The method of claim 8, wherein the Tamm-Horsfall protein is administered using a time-release system.

11. The method of claim 10, wherein the time-release system is a balloon catheter.

12. The method of claims 1, 2, 3, 4, 5, 6, or 7, wherein the Tamm-Horsfall protein is sialyated.

13. A method for screening for agents that modulate production of Tamm-Horsfall protein comprising: a) contacting Tamm-Horsfall genes in Tamm-Horsfail positive cells with a molecule of interest; and b) determining whether the contact results in increased Tamm-Horsfall production, increased Tamm-Horsfall production being indicative that the molecule modulates production of Tamm-Horsfall genes.

14. A method for screening for agents that modulate production of Tamm-Horsfall protein comprising: a) contacting Tamm-Horsfall protein in Tamm-Horsfall positive cells with a molecule of interest; and b) determining whether the contact results in increased Tamm-Horsfall production, increased Tamm-Horsfall production being indicative that the molecule modulates production of Tamm-Horsfall protein.

15. The method of claims 13 or 14, wherein the agent that modulates production of Tamm-Horsfall protein is a reproductive hormone.

16. The method of claim 15, wherein the hormone is estrogen.

17. The method of claim 15, wherein the hormone is progesterone.

18. A method for screening for agents that modulate sialyation of Tamm-Horsfall protein comprising: a) contacting Tamm-Horsfall protein in Tamm-Horsfall positive cells with a molecule of interest; and b) determining whether the contact results in increased sialyation of Tamm- Horsfall protein, increased sialyation of Tamm-Horsfall protein being indicative of modulation of sialyation of Tamm-Horsfall protein.

19. The method of claim 18, wherein increased sialyation of Tamm-Horsfall protein is measured by measuring the zeta-potential of the Tamm-Horsfall protein.

20. The method of claims 13, 14 or 18, wherein the agent is a small molecule, protein, peptide, or a combination thereof.

21. A screening method according to claims 13, 14 or 18, which comprises separately contacting each of a plurality of samples to be tested.

22. The screening method of claim 21 , wherein the plurality of samples comprises more than about 10⁴ samples.

23. The screening method of claim 21 , wherein the plurality of samples comprises more than about 5 X 10⁴ samples.

24. The method of claims 1, 2, 3, 4, 6 or 7, wherein the subject is selected from the group consisting of human, monkey, ape, dog, cat, cow, horse, rabbit, mouse and rat subjects.

25. A pharmaceutical composition comprising Tamm Horsfall protein and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition of claim 25, wherein the Tamm-Horsfall protein is sialyated.

27. A kit comprising the pharmaceutical composition of claims 25 or 26.