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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0684—Cells of the urinary tract or kidneys
  • C12N5/0686—Kidney cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
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• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • C—CHEMISTRY; METALLURGY
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  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/02—Drug screening
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the present invention relates to cystic diseases. More specifically, the invention relates to the use of a culture of cells that form cysts in vitro, to a method of screening for agents which can treat such diseases, and to pharmacological treatments of the diseases.
• the cytoskeleton plays an important role in the growth, division, and migration of eukaryotic cells. Changes in cellular morphology, the repositioning of internal organelles, and cellular migration all depend on complex networks of protein filaments that traverse the cytoplasm.
• microtubules fall into three main categories according to their size: microtubules, intermediate filaments, and microfilaments. Both microtubules and microfilaments are made of globular subunits which can quickly polymerize and depolymerize in the cell resulting in movement and morphological changes. Intermediate filaments are made of fibrous protein subunits and tend to be more stable with longer half-lives than most microtubules and microfilaments.
• Microtubules are formed by a dimer of tubulin proteins which take on a helical shape to form filaments.
• microfilaments comprise actin proteins which agglutinate together to form elongated filaments.
• the cytoskeleton is also made up of many other components for linking the filaments to each other or to the plasma membrane. Many cytoplasmic components can influence the rate of filament polymerization or depolymerization. Also, drugs have been discovered which affect the rate of filament polymerization and lead to either abnormal accumulations of protein filaments or unpolymerized globular subunits.
• Taxol, colchicine, vinblastine, cytochalasin-B, and cytochalasin-D are all well known disruptors of cytoskeletal development. Taxol inhibits depolymerization of the microtubule filaments, while vinblastine and colchicine inhibit microtubule polymerization. Griseofulvin is another drug that interferes with microtubule function, although the mechanism of this interference is not yet established. Cytochalasin-B and cytochalasin-D are inhibitors of microfilament networks. Diseases Involving Cyst Formation
• cysts which contain either semi-solid or fluid material.
• the contents of a cyst sometimes derive from normally retained fluid (e.g. a sebaceous cyst can contain fluid from a blocked sebaceous gland) or from a parasitic infection.
• Benign cysts can occur in the ovary, spleen, lungs, kidney and liver, where they are often congenital.
• Some congenital cysts result from fetal malformations and developmental failure while others are direct results of a disease state.
• PTD polycystic kidney diseases
• PKD can be inherited in autosomal dominant (ADPKD) or autosomal recessive
• ADPKD is the most common dominantly inherited kidney disease of humans, while ARPKD occurs relatively rarely. Clinically, ADPKD represents a major cause of chronic renal failure in humans and accounts for 10% of all patients requiring chronic dialysis or renal transplantation. Currently, 500,000 Americans and 5 million people worldwide are estimated to be afflicted with PKD. In the U.S. this represents an annual health care cost of nearly one billion dollars.
• PKD probably begins in utero in most patients with the kidneys increasing in size and ultimately showing signs of disease in the fourth or fifth decade of life. At present there are methods of detecting PKD in utero. Approximately 25% of patients do not have a family history consistent with ADPKD, suggesting either that the genes responsible have a high mutation rate or that other environmental factors are at work.
• cyst formation relating to PKD
• researchers have employed both in vitro and animal models.
• Previous in vivo and organ culture studies have implicated both accelerated renal epithelial cell growth and electrolyte transport abnormalities in the genesis of these cysts.
• Monolayer cultures of both normal and polycystic human kidneys have similar limited life spans and can be passaged using either serum free or supplemented media. Virtually no cysts or hemicysts have ever been observed in these cultures.
• MDCK and LLCPK are two immortalized normal renal cell lines that spontaneously form hemicysts and cysts in culture. Cyst formation has also been reported in cultures of normal human thyroid, mammary and lung cells.
• Normal and polycystic kidney epithelial cells, embedded in collagen can be induced to form cysts when stimulated with serum, forskolin, or epidermal growth factor.
• conditions enabling the differential expression of normal and polycystic kidney phenotypes using disbursed cells in culture has not been achieved.
• In vivo experiments have been performed on a mouse strain (CPK) which is homozygous for a gene that causes a murine polycystic kidney disease.
• CPK mouse strain
• This murine model arose as a spontaneous mutation of the C57BL 6J strain housed at the Jackson laboratories, and newborn pups rapidly develop a severe form of polycystic kidney disease. Numerous proximal cysts are found in the kidney cortex of these CPK mice at birth.
• mice become visibly lethargic due to azotemia around day 20-25 after birth and usually die of uremia and kidney failure before the 28th day.
• kidney tubules a type of abnormalities in normal kidney tubules have been implicated in the genesis and progression of cysts in PKD: (1 ) renal tubules begin to enlarge, forming a cyst, (2) additional cells line the nephron wall, and (3) a net change in renal fluid handling occurs allowing fluid to accumulate inside the cyst cavity.
• the present invention provides methods of producing cell cultures which form cysts in vitro, methods of using such cell cultures to screen for agents or compounds that have anti- cystic activity, and methods of treating subjects who have a cyst or who have a disease which causes cysts.
• kidney cells from a human afflicted with a polycystic kidney disease such as Autosomal Dominant Polycystic Kidney Disease
• kidney cells from a human afflicted with a polycystic kidney disease such as Autosomal Dominant Polycystic Kidney Disease
• an agent or compound which has anti-cystic activity Once such a compound has been identified, it can be used to treat a cyst or cystic disease.
• agents which affect the proper functioning of the cytoskeleton or which affect the transport of membrane proteins to the cell membrane can be used to treat cysts and cystic diseases.
• agents include compounds which depolymerize microtubules, such as colchicine, vinblastine, vincristine, and nocodazole, as well as compounds which stabilize microtubule structures, such as taxol and derivatives thereof.
• Also included in the list of compounds which can be used to treat cysts and cystic disease are compounds which depolymerize actin microfilaments, such as cytochalasin B and cytochalasin D, those which stabilize actin microfilaments, such as phalloidin, and some, such as griseofulvin, which interfere with microtubule function by an unknown mechanism.
• the present invention comprises a method of producing a culture of cells which form cysts in vitro by forming a suspension of cells that form cysts in vivo in a medium suitable for their maintenance.
• a solid phase which substantially prevents the formation of a layer of these cells on the surface of the solid phase
• the cells form cysts in vitro.
• the solid phase is coated with a material such as agarose that prevents cells from forming a monolayer on the surface of the solid phase.
• One particular embodiment involves the culturing of cells from a subject with a polycystic kidney disease on an agarose coated solid phase, wherein the apical surfaces of the in vitro cysts formed by such cells face the lumens of these cysts.
• a method in which cells that form cysts in vivo are also cultured in vitro on a solid phase without the formation of a layer of such cells on the surface of the solid phase.
• these cells are cultured in a medium comprising an agent or compound to be tested for its ability to inhibit cyst formation.
• an agent or compound to be tested for its ability to inhibit cyst formation By detecting the extent to which the cells form cysts in culture, the effectiveness of the agent or compound in treating cysts or cystic diseases can be determined.
• a method of treating a mammal having a cyst or a cystic disease is disclosed.
• a mammal having a cyst or cystic disease is first identified.
• identification could be by being diagnosed by a physician.
• a pharmacologically effective anti- cystic amount of an agent or compound that interferes with the normal metabolism or functioning of a cytoskeletal component is administered to the mammal in a pharmacologically acceptable carrier.
• the compound can be one that specifically binds to microtubules or to actin and which either causes the depolymerization of these components or which prevents such depolymerization.
• another method of treating a mammal having a cyst or a cystic disease is provided.
• a mammal having a cyst or ' a cystic disease is again identified, after which a pharmacologically effective anti-cystic amount of a compound that inhibits the delivery of membrane proteins to the membrane of a cell of the mammal is administered in a pharmacologically acceptable carrier.
• the compound brefeldin A can be used to treat such a mammal.
• the present invention also includes the use of an agent that interferes with the normal functioning of a cytoskeletal component or an agent that inhibits the delivery of membrane proteins to the membrane of a cell in the manufacture of a medicament for the treatment of a mammal having a cystic disease.
• an agent can comprise, for example, any of the previously mentioned agents that interfere with the functioning of a cytoskeletal component or with the delivery of membrane proteins to cell membranes.
• Figure 2 shows a graph of the effects of weekly taxol or methotrexate treatment on the survival of polycystic cpk mice.
• Untreated and methotrexate (MTX) treated polycystic cpk mice all died before 30 days of age. Most polycystic cpk mice treated with weekly taxol survive more than 60 days.
• the dotted line starting at day 120 indicates the number and age of apparently healthy taxol treated polycystic mice that were sacrificed for histology studies. Pairs of mice were sacrificed at 120 days, 182 days and 210 days of age. These mice all remained active until sacrifice and attained adult size and weight, despite having palpably enlarged kidneys.
• Figure 3 shows the effects of taxol or methotrexate treatment on the growth and survival of heterozygous and cpk mice.
• the body weights of untreated, taxol treated and methotrexate treated C57BL 6J mice between day 10 and day 45 after birth is shown in A.
• the body weights of untreated, taxol treated and methotrexate treated cpk mice are shown in B. Results in each group are the mean of 6 animals. Untreated and methotrexate treated cpk mice died between days 26 and 28. Taxol treated cpk mice live for an average of 45 ⁇ 2 days.
• cystic diseases can be treated pharmacologically in mammals using compounds that affect the cytoskeleton of mammalian epithelial cells. Included among the compounds that are effective inhibitors of cyst formation are those that interfere with the normal metabolism or function of a cytoskeletal component. Other compounds I have found to have pharmacologically effective anti-cystic activity include those that inhibit the delivery of membrane proteins to the membranes of cyst-forming cells.
• This assay system serves as a basis for identifying additional agents that can inhibit cyst formation within the context of the present invention.
• My exemplary in vitro system provides conditions that enable the differential expression of normal and polycystic phenotypes using dispersed cells in culture. Such a system has not, to my knowledge, heretofore been achieved.
• the exemplary in vitro system of the present invention provides cells that have the unique ability to develop into cysts in unstirred suspension culture. The process of cyst formation in this system closely mimics the formation of the blastocoele cavity in blastocysts, as reported by T. P. Fleming et al. in Ann. Rev. Cell Biol. 4:459-85 (1988).
• a suspension of cells that can produce cysts in vivo is first formed in a medium in which the cells will grow.
• Suitable cells include ovarian cyst cells, breast cyst cells, bronchogenic cyst cells, choledochal cyst cells, colloidal cyst cells, congenital cyst cells, dental cyst cells, epidermoid inclusions, hepatic cyst cells, hydatid cyst cells, lung cyst cells, mediastinal cyst cells, ovarian cyst cells, periapical cyst cells, pericardial cyst cells, in addition to the polycystic kidney disease cells, such as human ADPKD cells or murine CPK cells discussed above.
• the cells are then cultured on a solid phase without formation of a layer of the cells thereon.
• the solid phase is coated with a material that will not allow the cells to form a layer in any substantial amount. I have discovered that an agarose coating is one material effective in preventing cell layer formation.
• Example 1 One example of an in vitro assay system for cyst-formation is provided below as Example 1. As such, this Example, like all Examples provided herein, is provided merely to illustrate certain aspects of the present invention, and not to limit the invention in any way.
• mice having congenital polycystic kidney disease (CPK) and their phenotypically normal littermates were sacrificed by cervical dislocation. Kidneys were harvested aseptically and washed with DMEM/F12 (Sigma Chemical Co., St. Louis, MO) in a 1 :1 mix. Each decapsulated kidney was chopped into cubes (1 mm) and incubated in 5ml of collagenase solution composed of 1 mg ml collagenase type IV (Worthington Biochemical Corporation, Freehold, New Jersey), 1 mg/ml egg white trypsin inhibitor (Worthington) in 1 :1 DMEM/F12.
• collagenase solution composed of 1 mg ml collagenase type IV (Worthington Biochemical Corporation, Freehold, New Jersey), 1 mg/ml egg white trypsin inhibitor (Worthington) in 1 :1 DMEM/F12.
• kidneys were cut into halves and the medulla dissected out to provide tissues for harvesting cells. Tissue samples were then incubated at 39°C for 2-3 hrs and the mixture was shaken vigorously at 15-30 minute intervals to promote dissociation. Next, the preparation was centrifuged at 200xg for 1 minute and the supernatant discarded. The pellets were resuspended in 14 mis 1 :1 DMEM/F12 with or without 10% calf serum (Irvine Scientific, Santa Ana, CA), and larger aggregates were removed by allowing them to settle under gravity for 2 minutes.
• the top 12 mis which contained both single cells and aggregates of up to 10 cells, as assessed by inverted phase-contrast microscopy, were placed in 2ml aliquots in the wells of plastic culture plates (Corning, NY) precoated with 1 % agarose in DMEM:F12 (2 ml in each well of a 6-well plate; 1 ml in each well of a 24-well plate). Cell samples were digested with further incubation for 10 minutes in 0.05% trypsin and 0.53 mM EDTA (Gibco, BRL Gaithesburg, MD) at 37°C. All cultures were plated at a final density of 1-3 x 10 4 cells per well.
• cysts form in both serum-free and serum-containing medium with similar efficiencies and can be maintained in culture for at least six weeks.
• cells from normal C57BI76J mouse kidneys were observed to form aggregates of 10-50 cells.
• no cysts could be observed by 24 hours of culture, or during a prolonged culture to 72 hours.
• kidney cell suspensions from all the studied ages of CPK mice had formed both aggregates and cysts by 24 hours of culture. These cysts exhibit a characteristic spheroid shape roughly 50-100 microns in diameter when viewed with the light microscope.
• cyst size was similar in all age groups but became progressively more frequent, when expressed as cyst frequency per total cell number at 24 hours of culture, in 10, 21 and 28 day old CPK mice.
• Table I shows cyst diameter as mean ⁇ Standard Deviation (SD) for 20 mice at each age.
• Figure 1 shows the incorporation of 3 H-dThd recorded over the first 24 hours of culture in cells derived from day 10, 21 and 28 postnatal kidneys after treatment as in Example 2. This provides an indication of DNA synthesis in these cells.
• the hollow bars designate normal littermates and solid bars are CPK.
• Panels (a) and (b) show 3 H-dThd incorporation in suspension cultures in (a) serum-free and (b) serum supplemented media.
• Panels (c) and (d) show 3 H-dThd incorporation in monolayer culture in serum-free and supplemented media, respectively.
• the data shown in Figure 1 are mean ⁇ SD, with three wells for each time-point from cells derived from day 10, 21 , and 28 postnatal kidneys.
• the incorporation of 3 H-dThd into macromolecules is given as cpm///g protein.
• the * indicates p ⁇ 0.05 between normal and CPK cells at each indicated time point.
• Example 3 shows a micropuncture technique for further analysis of the cysts formed in the in vitro assay system described herein.
• Intracystic hydrostatic pressure was measured in random cysts suspended in culture medium after 24 hours of incubation. The measurement was directly obtained by a servonulling pressure sensor (Instrumentation for Physiology and Medicine, San Diego, CA) employing a 1 ⁇ m tip diameter glass pipette filled with hypertonic saline (1.2M). The intraluminal hydrostatic pressure from the servonull system was monitored with an electronic pressure transducer (model P23 Gb) and was recorded on a Gould amplifier chart recorder.
• the cysts were also analyzed by light and electron microscopy. Procedures for such techniques are provided in Example 4.
• In vitro cysts derived from day 21 CPK mice were processed for light and transmission electron microscopy.
• type IV collagenase Worthington, Freehold, NJ
• Cell suspensions were separated from debris by sedimentation at unit gravity and decantation. After washing once with DMEM/F12 medium, cells were suspended in DMEM/F12 supplemented with 1.2 gm/l sodium bicarbonate, 1 X insulin, transferrin and selenium (Sigma # 11 184), 1 % lipids supplement (Sigma # L4646) and 1X antibiotic/antimycotic (Sigma # A7292) at a density of 5 x 10 4 cells per ml.
• Cyst cells were fixed in 1 % paraformaldehyde (Sigma Chemical Co.) and 0.5% glutaraldehyde (Sigma) in phosphate buffered saline (pH 7.2), post-fixed with 1 % osmium tetroxide, dehydrated through graded ethanols and embedded in medcast resin (Ted Pella, Inc. Redding, CA). Semi-thin sections O ⁇ m) were stained with toluidine blue for light microscopy and thin sections (50nm) stained with saturated uranyl acetate and Reynold's Lead citrate and examined in a Philips 300 electron microscope.
• the cysts formed in the human ADPKD in vitro system can be seen to have an outer layer one cell thick. These cysts range from 10-50 cells in circumference. Some of the cysts that have been viewed under the scanning electron microscope have a deflated appearance that suggests the structure is hollow rather than a solid aggregate of cells. Such balls of cells can be estimated to be roughly 100 microns in diameter. Interestingly, other scanning electron micrographs reveal cysts having sphere-like structures where a portion of the cell layer has detached. In such cysts, a basement membrane layer can be clearly seen. Transmission electron microscopy demonstrated the presence of short, sparse microvilli on the anti-lumenal surfaces of the cells that lined the walls of these cysts. Transmission electron microscopy reveals that the cells lining the cysts are polarized.
• the basement membrane observed in the cysts derived from the human ADPKD system is not readily detectable in the cysts that are produced in the murine CPK model system. This is reflective of the much less prominent basement membrane present in the murine CPK kidney relative to the human ADPKD kidney in vivo.
• the dispersed human polycystic cells aggregate into clusters of fifty to one hundred cells within four hours. After one day, microscopic lumens can be seen in the clusters of cells that are formed by the polycystic kidney cells. The lumens gradually enlarge and reach a steady state size of 50-200 microns in diameter by day seven.
• the cultured cells of the murine system of Example 1 contained a heterogeneous population of kidney cells, the cells of the cyst lining showed morphological characteristics of epithelial cells, namely the presence of polarized microvilli and tight junctions.
• proximal tubules represent the major site of renal cyst formation at the time of birth, but over the next four weeks the proportion of proximal cysts falls and a majority of the collecting tubules in the distal nephron become cystic.
• Table I demonstrate that increased cyst formation, as assayed after 24 hours in culture, correlated with increasing age of the CPK donor.
• the LLC-PK, subline and MDCK cells which have proximal and distal tubular morphological and biochemical characteristics, respectively, form cysts in suspension culture.
• the cyst polarity is apical-side outermost, in common with the current CPK model.
• type I collagen as a substratum
• the polarity of the cysts from these renal cell lines reverses to mimic the conformation seen in renal epithelia in vivo.
• cysts form in collagen matrices from primary cells derived from both normal human and ADPKD kidneys.
• ADPDK or murine CPK cells in the in vitro system of the present invention provides unexpectedly superior results in testing therapeutic interventions for cystic diseases in mammals, including humans.
• a further advantage of the in vitro assay of renal cyst formation of the present invention is that the process is isolated from the confounding effects of glomerular filtration and uremia found in vivo.
• cyst-forming cells are suspended in a medium to which the agent has been added. These cells are then cultured on a solid phase without formation of a layer of cells on the surface, such as in the assay system described in Example 1. The extent to which the cells actually form cysts can be detected and compared to the extent of cyst formation expected if no agent were added. If the amount of cyst formation is lower than expected, then the agent is a good candidate for therapeutic intervention of cystic diseases. Often, the detected level in the presence of the agent is the complete or near-complete absence of cysts.
• the comparison can be entirely qualitative because the expected level of cyst formation is a much higher level of cyst formation.
• the absence of cysts can serve to indicate therapeutic effectiveness for an agent.
• a control assay should be run in which no agent is added to determine the expected level of cyst formation.
• the comparison can then comprise a comparison of number and/or size of the resulting cysts.
• Example 5 provides assays of a variety of metabolic inhibitors on cyst formation using the murine CPK assay model of Example 1.
• Cytosine arabinoside was added at the final concentration of 20//g/ml to inhibit DNA synthesis; ⁇ -amanitin at 100ng ml to inhibit RNA synthesis; cycloheximide at 40 ⁇ g/ml to inhibit protein synthesis; amiloride at 1 mM to inhibit the renal epithelial Na + -H + exchange; ouabain at 0.5mM to inhibit Na + -K + -ATPase; brefeldin-A at 10//g/ml to inhibit transport of surface proteins; griseofulvin at 350//M to inhibit microtubule function; colchicine at 10 ⁇ M to inhibit microtubule polymerization; and cytochalasin-B and cytochalasin-D at 10 ⁇ M to depolymerize actin filaments. At the concentrations used for all of these inhibitors, cell viability was > 95% at 24 hours of suspension culture, as assessed by the trypan blue exclusion method. Preliminary data had shown that I .
• cyst formation was noted to be either present or absent; if present, the diameters of 20 cysts from a 0.2ml aliquot were measured. In the same samples, 3 H-dThd incorporation was also measured as described above.
• cell clusters were transferred to 15 ml sterile centrifuge tubes (Corning) and washed twice with 10 mis of growth media. After each wash, cell clusters were collected by centrifuging at 200x g for 1 minute and the supernatant discarded.
• Washed cell clusters were resuspended in the original volume of growth medium without inhibitors, placed in agarose coated wells and incubated at 37°C in a moisture saturated incubator in 5% CO 2 and air.
• the effect of metabolic inhibitors on cyst formation and size using the methods of Example 5 are shown in Table II.
• the mean cyst size is reported as ⁇ SD with a measurement of twenty cysts.
• the 3 H-dThd incorporation is reported as ⁇ SD using measurements of three wells.
• the * indicates p ⁇ 0.5 versus control.
• the ⁇ indicates that the cells aggregated into sheets rather than clusters in the presence of these inhibitors.
• the entries listed as "NA" in the table indicate data that is not available.
• One particularly preferred class of compounds for use in the present invention is taxol and derivatives thereof.
• a variety of such derivatives can be made in accordance with methods well known in the art.
• Other inhibitors of microtubules such as colchicine, nocodazole, vincristine and vinblastine are also effective when used in accordance with the present invention.
• Inhibitors of actin such as cytochalasin-B and cytochalasin-D are also effective in preventing cyst formation.
• inhibitors of membrane protein transport such as brefeldin A can also be used in accordance with the present invention.
• ADPKD cells are used in the model. Sodium and proton transports are the principal driving forces responsible for the trans-epithelial movement of water in kidney tubules. Inhibition of cyst formation by amiloride and ouabain in the human model indicate that they play important roles in fluid accumulation within cysts. The inhibition of cyst formation by ouabain and amiloride is reversible; lumens begin to appear in cell aggregates twenty-four hours after removing the inhibitors.
• Inhibitors of microtubules such as taxol (25 ⁇ M), colchicine (10 M), and vinblastine (1 ⁇ M), all reversibly inhibited in vitro cyst formation in both models. These results suggest that functional microtubules are required for cysts to form. The exact mechanisms by which these compounds inhibit cyst formation is not certain. I believe that inhibitors of microtubule functions can be blocking cyst formation by inhibiting the clustered cells from developing a polarized phenotype, or the targeted delivery of cellular proteins necessary for maintaining cellular polarity. It is also possible that some other heretofore unknown mechanism exists.
• cytochalasin-B (10 ⁇ M) and cytochalasin-D (10 ⁇ M) effectively inhibited cyst formation in both models.
• These drugs inhibit the formation of the actin microfilament component of the cytoskeleton.
• dispersed cells aggregated but lumens were not observed during the 24 hour observation period.
• these compounds exhibited insignificant effects on DNA synthesis, reported as 3 H-dThd incorporation in Table II.
• the cytochalasins were added to the murine cyst formation assay, cells clustered into flat sheets of about 50 rounded cells. Removal of the cytochalasins after 24 hours did not result in cyst formation in this model.
• Cyst formation can be reversibly inhibited by any of colchicine, vinblastine, nocodazole or taxol in both murine and human in vitro models.
• Cyst formation can also be treated with agents that affect the microfilament network of a cell, usually by acting on the main component of microfilaments, actin. Cytochalasin B, cytochalasin D and phalloidin are examples of such agents. Other agents which affect cyst formation include a drug which interferes with the transport of membrane proteins to the cell membrane, brefeldin A. Derivatives of taxol have been made in order to produce more effective chemotherapeutic agents for cancer. I believe that some of these derivatives will prove especially effective in connection with the methods of the present invention. Additional derivatives can be made and tested for cyst-treating activity in accordance with the methods described herein or by methods well known to those having ordinary skill in the art.
• Microtubules comprise one of the major components of the cytoskeleton and consist of polarized, dynamically maintained tubular structures consisting of ordered filamentous polymers of ⁇ -tubulin/ ?-tubulin heterodimers.
• the two ends of a microtubule exhibit different rates of addition of tubulin dimers and act as the growing (plus) and the vanishing (minus) end respectively.
• Colchicine and nocodazole both bind stoichiometrically to the same site on the dimeric form of a tubulin heterodimer and subsequent binding of the tubulin-colchicine complex to either end of a microtubule effectively caps the microtubule at the growing end thereof.
• the capped microtubule rapidly disassembles in the presence of colchicine. Binding of vinblastine to tubulin induces the formation of paracrystalline aggregates of free tubulin in a cell. The formation of such aggregates prevents the polymerization of tubulins into microtubules. Therefore, in the presence of vinblastine, preformed microtubules rapidly depolymerize.
• taxol inhibits microtubule disassembly. Taxol, in fact, stabilizes microtubules and promotes microtubule assembly from free tubulin. By preventing the normal disassembly of microtubules, taxol interferes with microtubule functions without disrupting the cytoskeleton.
• microtubules In interphase cells, microtubules have several diverse important physiological functions. As a major component of the cytoskeleton, microtubules contribute to the maintenance of the cellular architecture. In living cells the endoplasmic reticulum extends and retracts processes, forming and dissolving a tubular network along the microtubule cytoskeleton. Depolymerization of the microtubules by nocodazole collapses the endoplasmic reticulum membranes into the nuclear region. The same treatment causes fragmentation and dispersal o the Golgi apparatus from the nuclear region. Upon removal of nocodazole, the Golgi fragments reassemble in the juxtanuclear region.
• microtubules participate in many aspects of membrane vesicles mobility within the cell.
• membrane vesicles leave the Golgi carrying newly synthesized proteins to the cell surface along microtubule tracks.
• vesicles travel via microtubule-based movement to the cell center where fusion with prelysosomal compartments occurs. Depolymerization of microtubules with nocodazole inhibits this process.
• the movement of intermediate vesicles between the endoplasmic reticulum and the Golgi apparatus has also been shown to occur along microtubules.
• brefeldin A blocks the movement of membrane proteins between the endoplasmic reticulum and the Golgi bodies. It is postulated that this agent also affects cyst formation by acting on processes that are microtubule-dependent or dependent on other components of the cytoskeleton. The transport of membrane proteins usually takes place by means of vesicles which are moved along microtubules. One reason for this blockage of movement could be due to some interference with the microtubules along which such vesicles are transported.
• Microtubules have also been found to play important roles in the biogenesis and maintenance of epithelial cell polarity.
• formation of the first polarized epithelium is represented by the formation of the trophectoderm from blastomeres in a process known as compaction.
• Nocodazole allows the development of surface polarity but inhibits the formation of cytoplasmic poles during compaction, while taxol has the opposite effect.
• the functional polarity of epithelial cells is maintained by targeted delivery of the respective secreted and membrane proteins to their proper destinations. The mechanisms that direct this sorting process are not well characterized; however, microtubules are clearly involved.
• cyst formation can be blocked by the addition of 1 mM amiloride or 0.5 mM ouabain, which block hydrogen ion and sodium ion transport, respectively.
• the agents that interfere with the cytoskeleton exert their effects by influencing the polarity of the cell. This could result from disrupting polarity (in the case of nocadazole) or by preventing a change in the polarity of the cell (in the case of taxol).
• drugs affecting the cytoskeleton of a cell can be used to treat cystic diseases in vivo. It has been demonstrated that such drugs can extend the lives of CPK mice. Subjects who suffer from a variety of cystic diseases, including the similar Polycystic Kidney Disease in humans are also believed treatable.
• CPK cpk/cpk
• the homozygous recessive pups are born with mild distention of the proximal renal tubules.
• the kidneys of these mice become grossly enlarged as the result of marked cystic dilatation of the renal collecting ducts.
• death from uremia occurs in the fourth postnatal week.
• the murine defect resembles human autosomal recessive polycystic kidney disease, a disorder which is a common cause of childhood renal failure.
• methotrexate another drug known to have anti-mitotic activity
• Another drug known to have anti-mitotic activity methotrexate
• methotrexate was tested for its ability to treat homozygous cpk mice.
• Six cpk mice were injected intraperitoneally with 15 ⁇ l of methotrexate solution (1.1 mg/ml in water) 10-12 days after birth and once every week thereafter.
• methotrexate-injected mice survived until between 18 and 27 days after birth, with only one mouse living longer than 28 days. Thus, the methotrexate treated mice did not appear to survive substantially longer than untreated controls.
• mice were obtained from the Jackson Laboratories (Bar Harbor, Maine). Homozygous CPK mice were identified by the presence of intra-abdominal distention, palpable at 10 days after birth, and visible in the third and fourth weeks of life. Heterozygous mice, which have a normal kidney phenotype, were identified by the presence of polycystic offspring and were bred to provide the litters used in this study. Animals were kept on a 12 hour light/dark cycle and received Purina Mouse Chow and water acf libitum.
• each offspring of heterozygous CPK mice (C57BL/6J-cpk/+ ) was given 15 ⁇ l of taxol (10mg/ml in DMSO) or 15 ⁇ l DMSO alone intraperitoneally. All juvenile animals were kept with their mother, weighed every other day, and weaned when they were 25 days old. The presence of bilaterally enlarged polycystic kidneys was determined by palpating affected animals weighing 5.5 grams or more. As a result of this treatment, the untreated homozygous CPK mice and those treated with DMSO alone survived until day 25-28 postnatal, while those treated once with taxol survived at least 12-19 days longer.
• the drug taxol prevents the depolymerization of microtubules and thus prevents the proper operation of the microtubule-based mitotic spindles involved in cell division.
• the anti- cancer effect of taxol is based on its ability to inhibit cell division.
• Examples 7 and 8 are provided to show further evidence of the effectiveness of taxol in the in vivo treatment of cysts.
• mice raised and identified as in Example 6 were treated with either 15 ⁇ l of taxol (10mg/ml in DMSO), 15 ul of methotrexate (1.1 mg/ml in saline), or 15 ⁇ l DMSO alone intraperitoneally at 10 to 12 days postnatal. Those mice treated with DMSO alone or with methotrexate died approximately 25 to 28 days postnatal, while those treated once with taxol survived 12-19 days longer.
• Examples 6 and 7 demonstrate that even a single dose of taxol can provide some in vivo therapeutic effect.
• Example 8 shows the results with repeated administrations of taxol.
• taxol Since taxol is eventually metabolized by mammals, having an in vivo half-life of approximately 6-12 hours in humans, a further experiment was conducted in which periodic administrations of taxol were given to homozygous CPK mice. This study is shown below in Example 8.
• Example 8 Effect of Weekly Taxol or Methotrexate on cpk Mice Eleven homozygous cpk mice, raised and identified as in Example 6 were treated with
• mice 15 ⁇ l of 10 mg ml taxol in DMSO through intraperitoneal injection beginning 1 1-14 days after birth. Following the first treatment, the mice were administered with the same treatment once every week thereafter. As controls, 6 cpk mice were treated with 15 ⁇ l intraperitoneal injections of DMSO beginning 10-12 days after birth and then once every week thereafter. The results of this therapeutic regimen are shown in Figure 2. The mice injected with
• Example 9 provides a further demonstration that chemical agents which block in vitro cyst formation also can extend the lifespans of animals that ordinarily succumb to cystic disease.
• the results presented in Table II indicate that griseofulvin inhibits the formation of cysts in the in vitro cpk model of PKD. Although the mechanism of its inhibitory activity is not presently understood, this antifungal agent is known to have a colchicine-like effect on mitosis.
• Example 9 presents the results of a weekly treatment protocol in which young cpk mice were administered with griseofulvin. These results are considered as preliminary only because the test animals continue to survive as of this report. For this reason, the value given below for the average lifespan of cpk mice treated with griseofulvin represents a minimal value. This value increases with every day the remaining animals in this trial continue to survive.
• Example 9 Treatment of cpk Mice with Griseofulvin
• mice 25 ⁇ l of 24 mg ml griseofulvin in DMSO through intraperitoneal injection beginning 10-12 days after birth.
• the mice were administered with the same treatment once every week thereafter.
• Six of the mice have died so far. These deaths occurred at 31 , 34, 35, 41, 45 and 60 days of age.
• the remaining mice are still alive at 54, 63 and 78 days of age. Given these results, it is possible to conclude that the minimum average lifespan of the mice that received periodic administration of griseofulvin in this trial is at least 49 ⁇ 15.8 days.
• Thirteen untreated control cpk mice that were palpably polycystic before 13 days of age lived 33.5 ⁇ 6.3 days.
• Cysts such as those formed in the kidney of a patient with Polycystic Kidney Disease can also be treated in human subjects.
• a subject having a cystic disease or at risk for developing a cystic disease is first identified.
• a subject can be identified by a physician, who diagnoses that subject as having Autosomal Dominant Polycystic Kidney Disease.
• Such a subject is then a candidate for undergoing the present treatment for cystic diseases.
• a pharmaceutically effective dose of a compound or agent which interferes with the functioning of the cytoskeletal components of cells, or an agent which interferes with the transport of proteins to cell membranes is administered either before a cyst has formed, in order to prevent its formation, or after a cyst has already been detected in a subject.
• the compound is administered periodically during the time the patient has or is susceptible to developing a cyst. In this way an effective amount of the compound can be maintained in the subject.
• Taxol can be administered in a variety of ways, including intravenously, intraperitoneally, intramuscularly, or in any other appropriate way known to the art.
• a dose of taxol can be administered in the range of .01 to 500 mg, preferably in the range of 10 to 250 mg m 2 of body surface area (bsa), depending on the method of administration.
• 250 mg of taxol can be infused intravenously into a subject over a 24 hour period in DMSO. This treatment can be repeated every 21 days.
• about 100 mg of taxol in an appropriate pharmaceutical carrier can be injected intravenously every week into an adult subject.
• Example 10 is provided to illustrate one exemplary method of treating a human being having a cystic disease.
• a human subject is diagnosed as having Autosomal Dominant Polycystic Kidney Disease by a physician. That subject is injected intravenously with 150 mg/m 2 of a water-soluble taxol derivative in approximately 10 ml of saline solution. This treatment is repeated weekly.
• vinblastine vinblastine
• vincristine vincristine
• colchicine colchicine
• griseofulvin fullvicin
• Vinblastine sold as Velban (Eli Lilly, Indianapolis, Indiana) can be administered intravenously once per week in an amount between 0.1 and 15 mg/m 2 bsa, preferably between 2 and 12 mg m 2 bsa, dissolved in 10 ml saline solution.
• Vincristine (Eli Lilly, Indianapolis, Indiana) can also be administered intravenously in a glucose or saline solution, in an amount between 0.1 and 5 mg/m 2 , preferably between 1 and 2.5 mg/m 2 , weekly.
• Colchicine (Eli Lilly, Indianapolis, Indiana) can also be administered intravenously in a saline solution weekly in a dosage of between 0.1 to 5 mg, and preferably between 1 and 3 mg, per administration.
• Griseofulvin, sold as Fulvicin (Schering-Plough, New Jersey), can be administered orally in dosages ranging from 125-500 mg daily. Further information regarding these commercially available pharmaceutical preparations can be found in the "Physicians' Desk Reference," published annually by Medical Economics Data of Oradell, New Jersey.
• cystic diseases such as Polycystic Kidney Disease.
• cytochalasin B or cytochalasin D which interfere with the polymerization of actin in microfilaments, can be administered to treat cystic diseases.
• Brefeldin A a drug which interferes with the transport of proteins to a cell membrane, can also be administered.
• Those of skill in the art will know how to choose an appropriate pharmaceutical carrier.
• the carriers, excipients or diluents used in the commercially available products discussed above are appropriate in many instances. Those of skill in the art will also appreciate how to discover an appropriate dosage of such anti-cystic compounds or agents through such routine experiments as conducting animal and human trials.
• cystic diseases other than Polycystic Kidney Disease can also be treated with drugs such as those previously identified.
• a number of other cystic kidney diseases are believed treatable with these drugs.
• Such diseases include acquired polycystic kidney disease, juvenile nephronophthisis medullary cystic disease complex, medullary sponge kidney, renal dysplasia, tuberous sclerosis, von Hippel-Lindau Syndrome, solitary multilocular cysts, pyelocalyceal cysts, pericalyceal lymphangiectasis, and hilar and perinephric pseudocysts.
• Cystic diseases other than those of the kidney can be treated as well, including cysts of the lung, spleen, liver, and ovary.

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