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WO2000037421A1 - Nephronine: serie de composes destines au traitement de maladies infectieuses et du cancer - Google Patents

Nephronine: serie de composes destines au traitement de maladies infectieuses et du cancer Download PDF

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WO2000037421A1
WO2000037421A1 PCT/AU1999/001157 AU9901157W WO0037421A1 WO 2000037421 A1 WO2000037421 A1 WO 2000037421A1 AU 9901157 W AU9901157 W AU 9901157W WO 0037421 A1 WO0037421 A1 WO 0037421A1
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cells
nephronin
compound
compounds
groups
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PCT/AU1999/001157
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Iraj Ghadiminejad
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Iraj Ghadiminejad
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Priority to CA002355840A priority Critical patent/CA2355840A1/fr
Priority to AU22704/00A priority patent/AU778532B2/en
Priority to JP2000589493A priority patent/JP2002533317A/ja
Priority to EP99966796A priority patent/EP1150939A4/fr
Publication of WO2000037421A1 publication Critical patent/WO2000037421A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C305/00—Esters of sulfuric acids
    • C07C305/02—Esters of sulfuric acids having oxygen atoms of sulfate groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C305/04—Esters of sulfuric acids having oxygen atoms of sulfate groups bound to acyclic carbon atoms of a carbon skeleton being acyclic and saturated
    • C07C305/08—Dialkylsulfates; Substituted dialkylsulfates
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/225—Polycarboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
    • C07C69/675—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids of saturated hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
    • C07C69/675—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids of saturated hydroxy-carboxylic acids
    • C07C69/704—Citric acid esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl

Definitions

  • Nephronin A series of compounds for treatment of infectious diseases and cancer.
  • the present invention relates novel compounds and their use in treatment and or prevention of disease states.
  • Lipopolysaccharidc (LPS) or endotoxin is the major constituent of the outer membrane of Gram-negative bacteria (5). They cause a variety of pathophysiological effects in human and experimental animals (6). Bacteria release endotoxin when in circulation (7), causing the release of a number of pro-inflammatory cytokines that may lead to septic shock. It is noteworthy that the use of antibiotics results in bacterial death, and release of endotoxin (8; 9). This in turn may exacerbate the problem by overwhelming the immune system, leading to shock (10).
  • Lipid A is the most conserved region of LPS and is responsible for virtually all of the toxic activities of LPS.
  • LPS stimulates a range of cells, most notably macrophages (M0). to synthesise cytokines including intcrlcukin-l ⁇ (IL-l ⁇ ). interlcukin 6 (IL-6) and tumour necrosis factor- ⁇ (TNF- ⁇ ) (1 1-13).
  • cytokines including intcrlcukin-l ⁇ (IL-l ⁇ ).
  • IL-6 interlcukin 6
  • TNF- ⁇ tumour necrosis factor- ⁇
  • Pico-molar concentrations of LPS have been shown to induce TNF- ⁇ production 30-60 minutes after intravenous injection of LPS (14).
  • TNF- ⁇ and/or IL-1 play an important role in the development of septic shock (10; 15). Further, these cytokines were shown to mimic the whole spectrum of LPS toxicity in animal models (16-18), and that they may have synergistic lethal effect in septic shock (19
  • LBP LPS-binding protein
  • BPI inhibits the ability of LPS to stimulate cells Furthermore. ⁇ hen in contact with intact bacteria. BPI has a bactericidal activity . whereas LBP acts as an opsonin Although the levels of LBP increase from 3-10 ⁇ g/ml to as much as 200 ⁇ g/ml during Gram negative sepsis, the levels of BPI rarely increase above normal
  • LBP seem to function as a lipid transfer protein for the delivery of endotoxin as LPS-LBP complex to CD14
  • CD14 is a 50-55KDa gly cos> lphosphatidy 1 inositol (GPI)-anchored membrane glycoprotcin expressed on myeloid cells (membrane CD 14. mCD14) (27) or a soluble form lacking the GPI anchor (soluble CD 14. sCD14) (28)
  • SRNS steroid responsive nephrotic syndrome
  • Heparan sulphate serves as a receptor for adherence of herpes simplex viruses (HSV). chlamy dia trachomatis. Neisse ⁇ a gonorrhoeae. and indirectly human immunodeficiency virus (37) HSV causes many disease states, including mucosal lesions, encephalitis or disseminated infection in lmmunoco promised host These diverse clinical manifestations reflect the capacity of the virus to infect both epithelial and neuronal cell types (38)
  • Previous studies have shown that the first step in herpes virus infection is attacliment to heparan sulphate molecule on the surface of cells (39-41) Given the fact that Nephronin specifically binds to heparin and heparan sulphate and its ability to neutralise Gram negative endotoxin suggests that Nephronin may be a prime candidate for inhibiting HSV infection and other pathogens that employ similar mechanism for infection Furthermore, recent studies have placed and important role for he
  • the present invention consists in a non-protemaceous compound isolatablc from the urine of patients suffering from steroid responsive nephrotic syndrome, the compound having the following characterising features
  • the present invention consists in a compound of the general formula - CH 2 -(CH 2 ) ⁇ -COO
  • l.m and n may be the same or different and integers of 0 to 10.
  • X is R-CO-0-.
  • the X is R-CO-O- in which R is CH 3 or CH 3 (CH 2 ) ⁇ where f is an integer from 1 to 18. preferably 1 to 8 It is further preferred that R is selected from the group consisting of CH 3 , CH,CH 2 CH 3 (CH 2 ) 2 CH 3 (CH 2 ) 4 and
  • the present invention consists in a composition comprising the compound of the first or second aspects of the present invention and a phanriaceutically acceptable earner
  • the present invention consists in a method of treating, preventing, or reducing the risk of Gram negative septic shock or another disease state involving elevated cytokine levels in a subject, the method comprising administering to the subject an effective amount of the composition of the third aspect of the present invention
  • the present invention consists in a method of treating, preventing, or reducing the risk of viral infection in a subject, the method comprising administering to the subject an effective amount of the composition of the third aspect of the present invention It is preferred that the virus is Herpes virus or HIV In a sixth aspect the present invention consists in the use of the compound of the present invention in medicine
  • the present invention consists in the use of the compound of the present invention in the preparation of a medicament for use in the treatment. pre ⁇ cntion or reducing the risk of Gram negative septic shock or another disease state lm olving ele ⁇ ated cy tokme levels and the preparation of a medicament for use in the treatment, prevention or reducing the risk of viral infection
  • the present invention consists in a method of treating. pre ⁇ enting. or reducing the risk of metastases or angiogenesis in a subject, the method comprising administering to the subject an effective amount of the composition of the third aspect of the present invention
  • the present invention consists in the use of the compound of the present invention in the preparation of a medicament for use in the treatment, prevention or reducing the risk of of metastases or angiogenesis
  • Fig. 1 Dose-dependent inhibition of LPS-induced TNF- ⁇ and IL- ⁇ by four different preparations of compounds of the present invention The indicated amount of each preparation was added to chambers of 24 well culture plates 1ml of PBMC at 1 5 ⁇ 10 6 cells/ml containing lng/ml LPS was then added to each well LPS was added to the cell suspension a few minutes prior to dispensing the cells into the wells of the tissue culture plates The cells were harvested at 24 and 48 post-stimulation and the concentration of the cytokines measured by sandwich ELISA
  • Fig. 2 Dose dependent inhibition of human platelet hcparanase activity by the preparations of Nephronin.
  • Preparations 1-5 refer to preparations of compounds of the present invention with increasing carbon chain length
  • Preparation 6 is a control preparation Blank (Blk) refers to a control for the enzy me reaction.
  • Fig. 3 Two preparations of compounds of the present invention were tested for their ability to inhibit HSV infection of human kcratinocytcs
  • the HSV inoculum used contained 15 plaque forming units (PFU) of HSV per cell 5 ⁇ l of each preparation of compound was added to chambers of 12 well tissue culture plate containing the keratinocytes in a final volume of 2ml tissue culture media
  • Fig. 4 Inhibition of axonal spread of HSV 1 to cpide ⁇ nal cells
  • An in vitro model consisting of human dorsal root ganglia (DRG) neurones and autologous epidermal cells in two separate chambers to study anterograde axonal transport of HSV 1 was used 5 ⁇ l of compounds of the present invention was added at the indicated times to the epidermal cell (ECs) side of the chambers in a final volume of 2ml HSV1 inoculum was added to the DRG side of chambers
  • Urine from SRNS patients in relapse, was collected in untreated clean 24-hour collection bottles The urine bottles were then stored at -20°C until used The protein content of urine samples was determined using Multistix SG (Bay cr Diagnostic UK LTD. Basingstokc. Hampshire. UK) Urine samples with protein content of 3+ were used for the purification of the factor
  • the freeze-d ⁇ ed material was then resuspended in 20-50 ml of 0 IM sodium citrate pH 3 5
  • the pH of the sample was adjusted to pH 3 5 with IM citric acid
  • the resultant cloudy solution was centrifuged at 30.000g for 20m ⁇ n at 4°C and the clear supernatant was then loaded onto a Hiload 26/10 S-Sepharosc high perfo ⁇ nance column, previously pre-equilibrated with 0 IM sodium citrate pH 3 5
  • the sample was loaded onto the column at 2ml/m ⁇ n. followed by isocratic clution of unbound material by 0 1 M sodium citrate until the base line was achieved
  • the bound material was eluted with step clution of 10.
  • Nephronin was shown to bind specifically to Heparin and heparan sulphate but not chondroitin and de ⁇ natan sulphate This is demonstrated by the inhibition in migration of these glycosaminoglycans
  • the inhibition in migration of heparin and heparan sulphate is ascribed to specific interaction of Nephronin with these molecules, rather than on the basis of charge, since all these molecules are highly negatively charged
  • Endothelial cells were obtained from human umbilical veins by collagenase (0 1%) digestion as previously described (42) and modified by Klein et al (43)
  • Endothelial cells were grown to confluence on gelatinised cover slips. Once confluence was achieved the cover slips were removed and the cells were washed with phosphate buffered saline (PBS) at 37°C. The cells were then fixed with 4% paraformaldchyde in PBS solution For specific staining of heparan sulphate, the staining procedure was pcrfonned at pH 1.9. Under these conditions all proteinaccous material is positively charged, whilst heparan sulphate moieties remain negatively charged. 5nm gold-conjugated poly-L-lysinc probe particles (Biocell Research
  • a method for detecting Nephronin. was to use a one dimensional cellulose acetate electrophoresis. that is often used in the characterisation of GAGs (47)
  • the data obtained using cellulose acetate electrophoresis must be treated cautiously, since false positive data can be obtained w ith high concentrations of salts, including sodium citrate
  • a factor was isolated from the urine of children with SRNS, that binds to and inhibits the migration of glycosaminogly can. heparin Further studies for the specificity of this factor for binding other GAGs.
  • Nephronin was found to be highly negatively charged based on its tight binding to anion exchange columns (Q-column) at neutral pH As well as the cellulose acetate electrophoresis for its detection, thin lay er chromatography was performed for the identification of various components associated with Nephronin during the purification procedure Staining of this factor for peptides. carbohydrates and lipids at various stages of purification clearly demonstrated that Nephronin was tightly bound to lipids The lipid contamination proved to be a great obstacle in the purification of Nephronin. and for a long period the hepa ⁇ n/hcparan sulphate binding ability of Nephronin was ascribed to the lipid entity
  • Nephronin was then further purified by a number of other chromatographic techniques and the active fractions retained on the basis of their binding to heparin and inhibition of its migration on cellulose acetate electrophoresis
  • Nephronin had a relatively large molecular weight However, under reducing conditions. Nephronin was found to pass through membranes with a cut of IkDa Gel filtration studies under reducing conditions also indicated a molecular size of less than 2KDa
  • Nephronin w as added to confluent endothelial cells
  • the endothelial cells were exposed to Nephronin for one hour as described above
  • the culture medium containing the cells was removed and centrifuged at 1 OOOrpm for 1 Omin
  • the pelleted cells were resuspended in cold PBS and washed twice with cold PBS Try pan blue staining of the cells showed that over 95% of the cells were viable
  • the incubation time was reduced to 15 minutes and the amount of Nephronin added was reduced to 2 5% v/v of the culture volume A considerable reduction in the GAG stain
  • Nephronin was found to prolong coagulation time in much the same way as heparin Furthermore, the anti-coagulant effect of Nephronin was found to be syncrgistic with heparin To further investigate this relationship with heparin. a commercially available chromogenic kit for the measurement of heparin like molecules, based on the acceleration of antithrombin III activity for the inhibition of thrombin formation was used (50. 51) Once again, it was found that Nephronin behaved much the same way as heparin and in synergy with heparin These findings suggest that Nephronin may be able to self-poly me ⁇ sc. since activation of antithrombin III requires at least a pcntasaccha ⁇ de sequence of heparin (52)
  • PBMC isolated from SRNS patients were maintained in RPMI 1640 culture medium containing 10% v/v heat inactivated FCS and 50un ⁇ ts/ml of IL-2 The culture medium was removed at 24. 48 or 72 hour time periods and stored frozen at -70°C until use
  • PBMC peripheral blood cells
  • the culture mediums were then processed analy ticallv for the purification of Nephronin To assess the effect of the Nephronin on unstimulated blood cells.
  • PBMC peripheral blood cells
  • the PBMC were maintained as above except that on these occasions the culture media were supplemented with 10% SRNS patient's plasma
  • PBMC isolated from healthy subjects were cultured as above and supplemented with 10% normal plasma
  • Plasma from SRNS and healthy subjects were prepared by mixing 10 ml of whole blood with 1ml of sterile sodium citrate / citric acid (3 parts 0 IM sodium citrate. 2 parts 0 IM citric acid) as anticoagulant
  • the cells were removed by ccnt ⁇ fugation at 850g for l min
  • the clear plasma was removed and stored in small ahquots at -70°C
  • the pelleted cells ere usually used for other related experiments.
  • Table 3 clearly demonstrates that, lymphocytes isolated from SRNS patients during relapse are capable of producing Nephronin
  • addition of patients plasma, but not plasma from normal subjects, to cultures of ly mphocytes from normal subjects also induced these cells to produce Nephronin
  • Nephronin To test this hypothesis, urine from three children with meningitis and two nonnal children were processed for analytical isolation of Nephronin Nephronin was isolated from urine of two of the meningitis patient who had survived the disease The urine of the patient that did not contain Nephronin did not survive the disease Nephronin was not isolated from the urine of normal children
  • Nephronin Because of the anionic nature of Nephronin and its ability to bind to lipids, it was postulated that this factor might behave as a cationie detergent Furthermore, the fact that Nephronin was also isolated from urine of children with meningitis suggested that this factor is produced in response to infection Consequently. Nephronin was thought to behave similarly to that of the cationie antibiotic, polymyxin
  • Limulus Amocbocytc L satc (LAL). utilises the E coli endotoxin for the activation of a pio-cnzy me which then hydroly scs a substrate to y leld a chromogcnic product
  • LAL Limulus Amocbocytc L satc
  • Nephronin Physicochemical Characterisation of Nephronin
  • Preliminary characterisation of Nephronin suggested the absence of peptide bonds involved in its heparin binding ability, since this activity is not affected by treatment with 6M HC1 and heating at 105 U C for 24h
  • thin layer chromatography and staining for carbohydrates, lipids and ohgosaccha ⁇ de. as well as phenol-sulphuric acid assay for carbohy drate determination were negative Pronase digestion of Nephronin also seems to have no effect on its heparin binding and inhibition of its migration on cellulose acetate electrophoresis
  • NMR Nuclear Magnetic Resonance
  • MS mass spectrometry
  • the factor was subjected to reverse phase column chromatography using a cl 8 column with a liner gradient of 0-100% acetonit ⁇ le. Essentially there were four pooled fractions. These fractions were subjected to mass spectroscopy. The data showed the presence of fatty acids of saturated C 18 and C 16 and an unsaturated C 16 fatty acyl groups. These data were inconclusive for determination of an exact molecular structure, nevertheless these studies indicated that the factor was an ester of citrate esterified with heterogeneous group of fatty acids on the 2-hydroxyl group of the citrate molecule.
  • Nephronin-like compounds were then proposed, and successful synthesis of of these compounds achieved.
  • the synthesis procedure described for the ne compounds of the present invention is not limiting and only serves to demonstrate the synthesis of five different, biologically active compounds.
  • the tube containing the cells were inverted and the centrifuged at 1700rpm for 15min.
  • the cell pellets were resuspended in 10ml of PBS and centrifged as before.
  • the rsulting cell pellet were washed once more with PBS.
  • the lymphocytes were counted using a haemocytometer and the number of cells were adjusted to 1.5xl0 6 cells per ml. Lymphocytes were stimulated with lng/ml of endotoxin (E.coli. scrotype 055:B5. Sigma-Aldrich chemical company) in the presence and absence of the synthcsiscd compounds.
  • PHA Concanavalin A. strcptococcal bacterial membrane protein (M protein) and enterotoxins (supcrantigcns) SpeA and SpeB.
  • Sandwich ELISA for the measurement of mitogen induced cytokines was established. Sandwich ELISA for TNF- ⁇ . and IL-l ⁇ was routinely used for the measurement of these cytokines in the culture supernatant of the activated cells post-stimulation. Cells were harvested at 24. 48 and 72 hours post-stimulation and the culture supcrnatant ' s were used in the determination of the cytokine levels.
  • mice monoclonal and rabbit polyclonal antibodies against human TNF- ⁇ were purchased from Genzyme. USA. supplied by Lab Supply. Australia.
  • IL-6 and interfcron- ⁇ (IFN- ⁇ ) were purchased from Endogcn. USA, supplied by CSL Biosciences.
  • Horseradish peroxidase conjugated anti-rabbit antibodies were purchased from Zymcd. USA.
  • Cytokines used in the standard curves were purchase from Peprotech INC, supplied by- Australian Laboratory Services Ltd.
  • the fry pothesised structure for Nephronin assumes that this compound is surfactant like in structure with specificity towards certain sugar moieties, in particular hcpa ⁇ n/heparan sulphate, hence an anionic molecule that behaves like a cationie detergent Taking this into consideration, one can then visualise Nephronin being able to inhibit LPS
  • R is an alky 1 group
  • the reaction tube was centrifuged as before and a further 10ml of acetic acid was added to the white precipitate and mixed vigorously .
  • the pellet (pre-treated Na3Citratc) from this step is to be used in the synthesis of the compounds of the present invention.
  • the tubes were centrifuged and the clear supernatant was removed.
  • the proposed structure of Nephronin is a platform for synthesising an array of esters of citrate, where different fatty acid molecules are esterified to the hydroxyl group on the citrate molecule. In this instance, essentially five such acids are used in the synthesis procedure to demonstrate the validity of the synthesis and the bioactivity of the products.
  • Phosphorus oxychloride reacts violently with water to fon hydrochloric acid (HC1). To prevent this reaction occurring, the entire procedure was performed in the absence of water, in hexane. The following reaction tubes were set up for the formation of the acid chloride.
  • the tubes were quickly capped under nitrogen and incubated at RT on a roller for 30minutcs. The content of each tube was added to appropriately labelled tubes containing the pre-treated Na ⁇ Citrat. Once again each tube was quickly capped under nitrogen and the content of the tube was mixed vigorously until the entire Na 3 Citratc pellet was fully mixed with the solvent mixture containing the acid chloride of each fatty acid. The tubes were then incubated at RT on a roller over night.
  • control tube was otherwise treated exactly the same as the reaction tubes.
  • the pellets were dissolved in a 10% solution of ammonium hydroxide and the pH of the samples continuously monitored and the pH of the samples adjusted to pH 7.2-7.4. In each case the volume of the samples were adjusted to 20ml with milli-Q water.
  • SRNS also known as Minimal Change Nephrotic Syndrome (MCNS) is the most frequent form of nephrotic syndrome in children, with the peak incidence at 3-5 ears of age
  • MCNS Minimal Change Nephrotic Syndrome
  • SRNS also known as Minimal Change Nephrotic Syndrome
  • MCNS Minimal Change Nephrotic Syndrome
  • kidney has been shown to function both as a size and a charge selective barrier (59. 60). and that the glomerular basement membrane (GBM) and the foot piocesses of the epithelial cells are covered by negatively charged heparan sulphate glycoproteins (61) (62), the reduction m the amonic sites is thought to greatly contribute to loss of albumin in the urine (63)
  • Modified flat-bed cellulose acetate electrophoresis was used for the detection of material in chromatographic fractions that might bind to heparin and inhibit its migration (47) Using this methodology it was first noticed that fractions with little or no protein could potently inhibit migration of heparin Initial analy sis showed the presence of non-estc ⁇ ficd fatty acids, although this activity was not related to the lipid portion To demonstrate the specificity of this compound for heparin.
  • Nephronin This factor was named "Nephronin" to reflect the source and the origin of the material
  • the inhibition of LPS by Nephronin in the LAL assay is thought to be due to chemical modification of the lipid A moiety by Nephronin
  • Further studies showed that Nephronin could agglutinate Gram-negative but not Gram-positive bacteria (Table 2) This suggests that Nephronin binds directly to the LPS molecule on the bacterial cell surface, however the fact that Nephronin with molecular weight less than lkD can agglutinate Gram-negative bacteria, is of particular interest
  • Nephronin The interaction of Nephronin with LPS might prevent the binding of LPS to LBP. thereby inhibiting cytokme production by M0 and other LPS responsive cells
  • Preliminary studies showed that Nephronin inhibited LPS-induced IL-4 and IL-6 by fibroblasts
  • Nephronin ' s ability to bind to heparin and heparan sulphate it was important to establish whether this material had any effect on blood coagulation
  • a commercially available chromogcmc kit for the measurement of heparin like molecules, based on the acceleration of antithrombin III activity for the inhibition of thrombin fo ⁇ nation was used (50.
  • Nephronin seemed to behave in much the same way as heparin and in synergy with heparin These findings further support the notion th ⁇ t Nephronin may self-poly mc ⁇ se, since activation of antithrombin III requires at least a pcntasaccha ⁇ de sequence of heparin (52) Both extrinsic. UPTT and intrinsic. KPTT were performed as directed by the suppliers (Diagnostic Reagents Limited. Thames. Oxon. UK) Nephronin was found to prolong coagulation time in much the same way as heparin Contrary to expectations, the anti-coagulation effects of Nephronin were found to be in synergy with heparin Identification of cells of origin
  • SRNS peripheral blood mononuclear cells
  • PBMC from normal subjects were cultured m media containing 10% SRNS patient s plasma, and the culture supernatant processed as above and the presence of Nephronin determined semi-quantitativ eh
  • Table 3 clearly demonstrates that, lymphocvtes isolated from SRNS patients during relapse are capable of producing Nephronin
  • addition of patients " plasma, but not plasma from nonnal subjects, to cultures of lymphocytes from nomial subjects also induced these cells to produce Nephronin
  • Nephronin alone has an autoc ⁇ nc ability together with its anti-LPS ability suggests that Nephronin is an immune related compound and that the pathogcnesis of SRNS is solely due to lack of immune regulation
  • urine from three children with meningitis and two normal children were processed for anahtical isolation of Nephronin Nephronin was isolated from the urine of two of the meningitis patients who had survi ed the disease The urine of the patient that did not contain Nephronin did not survive
  • Fig 1 shows the inhibition of LPS-induccd TNF- ⁇ and IL-l ⁇ by five different compounds of the present invention Human PBMC were first mixed with lng/ml of LPS.
  • Fig 2 shows the dose dependent inhibition of heparanase activity
  • heparanase activity is shown to be cf particular importance in tumour development and metastasis
  • the ability of the compounds of the present invention to inhibit heparanase activity might suggest that the compounds of the present invention could play an important role in the inhibition of metastasis and inhibition of tumourogemc cells to solid tumours
  • C2C12 normal mouse muscle cell line
  • B35 mouse neuron tumourogenic cell
  • Increasing amounts of the 5 compounds of the present invention prepared were added to the cells.
  • the cells were monitored on a regular basis for any changes to their appearance and whether they separated from the substratum. Shortly after the addition of the compounds of the present invention, the cells changed shape very quickly giving a round spherical appearance, irrespective of the amount of the compound added to the cells, although they remained bound to the culture plates. After 2h incubation with the compounds of the present invention, the cells separated from the substratum forming large clumps. The cells were incubated in media containing the compounds of the present invention for five days.
  • ⁇ nteropathogenic E colt carry virulence factors in the fonn of adhesins including plasmid-encoded fimb ⁇ ae and a chromosomally bora adhesin
  • adherent E colt alter epithelial cell morphology and the cytoskeleton of host cells (in this model a T84 epithelial cell line) as a mechanism to allow effective attachment and thus colonisation
  • Infection of confluent T84 cells w ith ⁇ P ⁇ C usually results in bacterial adherence, measured by a bacterial binding assay
  • HSV Herpes Simplex Virus
  • Herpes simplex viruses establish lifelong latent infections in the sensory neuron of the host dorsal root ganglia (DRG) where they undergo periodic reactivations
  • DRG dorsal root ganglia
  • An in vitro model consisting of human DRG neurones and autologous epidermal cells in two separate chambers to study anterograde axonal transport of HSVl was established
  • HSVl infection of the human DRG neurone results in separate axonal transport of glycoproteins and nuclcocapsids, which are likely to assemble into mature vi ⁇ ons before crossing the intercellular gap between axonal termini and epide ⁇ nal cells
  • glycoprotein and nucleocapside antigens are detected in the epidermal cells by lmmunohistochcmistry and confocal microscopy at 20 hours post-infection of DRG (86)
  • Subsequent development of HSVl eytopathic plaques can be observed over the next 48 hours This sy stem
  • HIV virus binds to the cell surface of cells by a virus gly coprotein GP120
  • heparan sulphate as well as other cell surface sugar moieties indirectly play a role in HIV infectivity
  • the compounds of the present invention bind to heparin and heparan sulphate as well other sugar moieties, suggested that the compounds of the present invention might be able to inhibit HIV virus infection of macrophages Alternatively, since the compounds bind to a host of sugar moieties, it might also be able to bind directly to glycoproteins. such as GP120 and inhibit infectivity of cells by the HIV virus
  • Adherent macrophages were prepared from human blood The cells were either pre-treated with preparations of the compounds of the present invention 24h prior to addition of HIV inoculum, or the compounds were added simultaneously with the HIV inoculum The cells were then incubated w ith the HIV inoculum for 4h. prior to being washed and fresh media added The concentration of extracellular p 12 antigen and levels of intracellular HIV DNA measured by quantitative PCR determined the susceptibility of macrophages to HIV infection
  • the data presented here describes a low molecular weight, non-protcmaccous compound that is produced by human lymphocytes when appropriately stimulated
  • the presence of Nephronin in the urine of children w ith nephrotic sy ndromc might suggest that therapeutic use of this compound might lead to induction of Ncphrosis
  • Nephronin was also isolated from the urine of children with bacterial meningitis suggests that Nephronin can be produced by the immune cells in response to infection
  • its presence in the urine of children with SRNS is thought to be due to lack of immune down regulation which results in the observed Ncphrosis
  • the discovery and synthesis of the compounds of the present invention presents a set of new therapeutic agents for the treatment of a number of infections diseases such as sepsis and herpes vims and other viruses that use similar mechanism in their pathogencsis
  • Nephronin potently inhibits production of cytokines such as TNF- ⁇ .
  • IL-l ⁇ it can potentially be used as a therapeutic agent in diseases that involve these cytokines. such as rheumatoid arthritis and similar immune related diseases
  • diseases that involve these cytokines such as rheumatoid arthritis and similar immune related diseases
  • the ability of Nephronin to interact specifically w ith heparin and heparan sulphate indicates a potential use for analogues of Nephronin for the inhibition of tumour grow lit and metastasis
  • Viable Organisms Heat and/or penicillin killed
  • PBMC Peripheral Blood Mononuclcocy tcs
  • PBMC peripheral blood mononuclear cells
  • vascular permeability factor is a T lymphocyte product.

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Abstract

L'invention concerne un composé non protéinique, que l'on peut isoler à partir de l'urine de patients souffrant d'un syndrome néphrotique sensible aux stéroïdes, et qui possède les caractéristiques suivantes: (i) un poids moléculaire inférieur à 1 KDa; (ii) une liaison spécifique à l'héparine et au sulfate d'héparane, mais pas à d'autres glycosaminoglycannes; et (iii) une inhibition de la production de TNF-α et/ou IL-1α induite par un lipopolysaccharide. L'invention concerne également un composé correspondant à la formule générale: (I) dans laquelle l, m et n peuvent être semblables ou différents et représenter des nombres entiers compris entre 0 et 10, X représente R-CO-O-, R-O-PO2-O-, R-O-SO2-O-, R-CO-NH-, R-CO-, R-O- ou un monosaccharide ou un oligosaccharide, notamment des saccharides à substitution amide, et R représente une chaîne carbone, saturée, insaturée, ramifiée ou cyclique, possédant jusqu'à 32 atomes de carbone et contenant éventuellement un ou plusieurs des groupes suivants: hydroxyle, carbonyle, acide carboxylique, amino, phosphate ou sulfate, ou des combinaisons de ces groupes. L'invention concerne également des sels et dérivés de ce composé, acceptables sur le plan pharmacologique.
PCT/AU1999/001157 1998-12-21 1999-12-21 Nephronine: serie de composes destines au traitement de maladies infectieuses et du cancer WO2000037421A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002355840A CA2355840A1 (fr) 1998-12-21 1999-12-21 Nephronine: serie de composes destines au traitement de maladies infectieuses et du cancer
AU22704/00A AU778532B2 (en) 1998-12-21 1999-12-21 Nephronin: a series of compounds for treatment of infectious diseases and cancer
JP2000589493A JP2002533317A (ja) 1998-12-21 1999-12-21 ネフロニン：感染性疾患およびガンの処置のための一連の化合物
EP99966796A EP1150939A4 (fr) 1998-12-21 1999-12-21 Nephronine: serie de composes destines au traitement de maladies infectieuses et du cancer

Applications Claiming Priority (2)

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AUPP7849 1998-12-21
AUPP7849A AUPP784998A0 (en) 1998-12-21 1998-12-21 A low molecular weigh, non-proteinaceous compound that inhibits mitogen induced cytokine production

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CA2355840A1 (fr) 2000-06-29
EP1150939A4 (fr) 2003-03-26
AUPP784998A0 (en) 1999-01-21
EP1150939A1 (fr) 2001-11-07
JP2002533317A (ja) 2002-10-08

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