TREA

Method of Treating Fish and Crustaceans With a Tellurium Based Immunomodulator

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Information

  • Patent Application
  • 20080076746
  • Publication Number
    20080076746
  • Date Filed
    December 09, 2004
    20 years ago
  • Date Published
    March 27, 2008
    16 years ago
Information
Abstract
A method for the direct or adjunct treatment of bacterial or fungal infections in fish and crustaceans including shell fish which is based on providing an effective amount of an organic tellurium compound in an aqueous environment to which said fish or crustaceans are exposed.
Description

BRIEF DESCRIPTION OF THE DRAWING


FIG. 1 discloses a dose-dependent inhibition of IL-10L by the immunomodulator AS101.



FIG. 2 discloses Serum IL-10L levels as measured prior to stress induction and at different intervals of time following stress induction



FIG. 3 discloses stress induction up-regulates IL-10L and glucose secretion, however, + only IL-10L is inhibited when stressed fish are treated with AS101.



FIG. 4 discloses AS101 treated fish infected with Aeromonas salmonicida show significantly less wounds (p=0.073) than untreated infected fish.



FIG. 5 discloses AS101 treated stressed fish infected with Aeromonas salmonicida show higher survival rates than control fish.





DETAILED DESCRIPTION OF THE INVENTION

The organic tellurium compounds for use in the invention include those of the formula:







or





TeO2or complexes of TeO2  (C)





or





PhTeCl3  (D)





or





TeX4, when X is Cl, Br or F





or





(C6H5)4P+(TeCl3(O2C2H4))—  (E)


wherein t is 1 or 0; u is 1 or 0; v is 1 or 0; R, R1, R2, R3, R4, R5, R6, R7, R8, and R9 are the same or different and are independently selected from the group consisting of hydrogen, hydroxyalkyl of 1 to 5 carbons, hydroxy, alkyl or from 1 to 5 carbon atoms, halogen, haloalkyl of 1 to 5 carbon atoms, carboxy, alkylcarbonylalkyl of 2 to 10 carbons, alkanoyloxy of 1 to 5 carbon atoms, carboxyalkyl of 1 to 5 carbons atoms, acyl, amido, cyano, amidoalkyl of 1 to 5 carbons, N-monoalkylamidoalkyl of 2 to 10 carbons, N,N-dialkylaminoalkyl of 4 to 10 carbons, cyanoalkyl of 1 to 5 carbons alkoxy of 1 to 5 carbon atoms, alkoxyalkyl of 2 to 10 carbon atoms and —COR10 wherein R10 is alkyl of 1 to 5 carbons; and X is halogen; while the ammonium salt is illustrated, it is understood that other pharmaceutically acceptable salts such as K+ are within the scope of the invention. The compounds with the five membered rings are preferred.


As used herein and in the appended claims, the term alkyl of 1 to 5 carbon atoms includes straight and branched chain alkyl groups such as methyl; ethyl; n-propyl; n-butyl, and the like; the term hydroxyalkyl of 1 to 5 carbon atoms includes hydroxymethyl; hydroxyethyl; hydroxy-n-butyl; the term halkoalkyl of 1 to 5 carbon atoms includes chloromethyl; 2-iodoethyl; 4-bromo-n-butyl; iodoethyl; 4-bromo-n-pentyl and the like; the term alkanoyloxy of 1 to 5 carbon atoms includes acetyl, propionyl, butanoyl and the like; the term carboxyalkyl includes carboxymethyl, carboxyethyl, ethylenecarboxy and the like; the term alkylcarbonylalkyl includes methanoylmethyl, ethanoylethyl and the like; the term amidoalkyl includes —CH2CONH2; —CH2CH2CONH2; —CH2CH2CH2CONH2 and the like; the term cyanoalkyl includes


—CH2CN; —CH2CH2CN; —CH2CH2CH2CN and the like; the alkoxy, of 1 to 5 carbon atoms includes methoxy, ethoxy, n-propoxy, n-pentoxy and the like; the terms halo and halogen are used to signify chloro, bromo, iodo and fluoro; the term acyl includes R16CO wherein R16 is H or alkyl of 1 to 5 carbons such as methanoyl, ethanoyl and the like; the term aryl includes phenyl, alkylphenyl and naphthyl; the term N-monoalkylamidoalkyl includes —CH2CH2CONHCH3,


—CH2—CONHCH2CH3; the term N,N-dialkylaminoalkyl includes —CH2CON(CH3)2; CH2CH2CON(CH2—CH3)2. The tellurium based compounds that are preferred include those of the formula:







wherein X is halogen. The preferred halogen species is chloro.


Other compounds which are based on tellurium and may be used in the practice of the invention include PhTeCl3, TeO2 and TeX4 (C6H5)4 P+(TeCl3 (O2C2H4))— (Z. Naturforsh, 36, 307-312 (1981). Compounds of the following structure are also included:







Other compounds useful for the practice of invention include:







wherein R11, R12, R13 and R14 are independently selected from the group consisting of hydrogen, hydroxy-alkyl of 1-5 carbons atoms, hydroxy and alkyl of 1-5 carbons atoms.


Useful dihydroxy compounds for use in the preparation of compounds of structure A or B, include those of formula I wherein R, R1, R4 and R5 are as shown in the Table:










TABLE








(I)






















R
R1
R4
R5







H
H
H
H



H
Cl
H
H



H
OCH3
H
H



H
COOCH3
H
H



H
H
CN
H



H
CHO
H
H



H
H
COOH
H



H
CH2COOH
H
H



H
H
CH2COOCH3
H



H
I
H
H



H
H
Br
H



H
H
CONH2
H



H
H
CH2OH
H



H
COOH
H
H










Other dihydroxy compounds for use in the preparation of compounds A and B include those of formula II wherein R, R1, R2, R3, R4 and R5 are as shown in the Table:














(II)






















R
R1
R2
R3
R4
R5





H
H
H
H
H
H


H
H
Cl
H
H
H


H
CH2OH
H
H
H
H


H
H
OH
H
H
H


H
H
H
CH3
H
H


H
H
H
CH2Cl
H
H


H
H
H
COOH
H
H


H
H
H
CH2COOH
H
H


H
H
H
CHO
H
H


H
H
H
H
H
CH2CHO


H
H
CONH2
H
H2
CH3


H
H
H
CN
H
H


H
H
H
H
CH2COHN2
H


H
H
H
COOCH3
H3
H


H
H3
OCH3
H
H
H









Other dihydroxy compounds for use in making compound of formula A and B include those of formula III wherein R, R1, R2, R3, R4 and R5 are as shown in the Table.














(III)
























R
R1
R2
R3
R4
R5
R8
R9





H
H
H
H
H
H
H
H


H
H
Cl
H
H
H
H
H


H
H
H
H
Br
H
H
H


H
H
OCH3
H
H
H
H
H


H
H
CONH2
H
H
H
H
H


H
Br
H
H
H
H
H
H


H
H
H
H
CH2COOH
H
H
H


H
H
Cl
Cl
H
H
H
H


H
CH2COOH
H
H
H
H
H
H


H
H
CH3
H
H
H
H
H


H
CH3
H
H
H
H
H
H


H
CH2Cl
H
H
H
H
H
H


H
H
H
I
H
H
H
H


H
CH2CN
H
H
H
H
H
H


H
H
H
H
CH2CH2OH
H
H
H









Additional dihydroxy compounds include those of formula IV wherein R, R1, R2, R3, R4 and R5 are as shown in the Table.














(IV)


























R
R1
R2
R3
R4
R5
R6
R7
R8
R9





H
H
H
H
H
H
H
H
H
H


H
H
Cl
H
H
H
Cl
H
H
H


H
H
Cl
Cl
H
H
H
H
H
H


H
H
CONCH3
H
H
H
Br
H
H
H


H
H
Br
H
H
H
CON(CH3)2
H
H
H


H
H
H
OCH3
H
H
H
H
H
H


H
H
H
H
OCH3
H
H
H
H
H


H
H
H
H
CH2COOH
H
H
H
H
H


H
H
COOH
H
H
H
H
H
H
H


H
CH3
H
H
H
H
H
H
H
H


CH3
H
H
H
H
CH3
H
H
H
H


H
CH2CH3
H
H
H
H
H
Cl
H
H


H
CH2CN
H
H
CH2OH
H
H
H
H
H


H
H
H
I
H
H
H
H
CN
H


H
CH2CH2COOH
H
H
H
H
H
H
H
H


H
H
CHO
H
H
H
H
H
H
H


H
H
H
F
H
H
H
H
H
H









Compounds of the following formula are also included:







herein R15, R16, R17 and R18 are independently selected from halogen, alkyl of 1-5 carbons; aryl, acyl of 1-5 carbon hydroxyalkyl of 1-5 carbons and aminoalkyl of 1-5 carbons may be made by reacting the appropriate di, tri or tetrahalotelluride with the appropriate hydroxy compound which may be of the formula: HO—R19;


wherein R19; is alkyl of 1 to 5 carbons, haloalkyl of 1 to 5 carbons, aryl, alkylaryl, alkylamido of 1 to 5 carbons, alkylcarbonyl of 1 to 5 carbons, cyanoalkyl of 1 to 5 carbons, cyanoalkyl of 1 to 5 carbons, and an alkoxyalkyl of 2 to 10 carbons. Specific examples of R16 include methyl, ethyl, n-propyl, phenyl, tolyl, amidoethyl, cyanomethyl, methyloxymethyl and CH2CH2COOH.


These compounds are described in United States Letters Patent No. 4,761,490 which is incorporated by reference. In addition, TeCl4; TeBr4 and compounds which give in aqueous solution TeO2 preferably in the form of a complex such as for example TeO2 complex with citric acid or ethylene glycol.


The preferred compound is ammonium trichloro (dioxoethylene-O,O′) tellurate.


Treatment of Fish

Tilapia hybrids (Oreochromis niloticus X O. aureus) of 100-200 g in weight were raised in the Fish Immunology & Genetics Laboratory, Bar Ilan University. Carp (Cyprinus carpio) of 200-350 gr were purchased from the “Mevo-Hamah” farm (Israel). The fish were kept in 400 L plastic tanks with re-circulating water system. Temperature and oxygen levels were kept constant at 26±2° C. and 4-6 ppm, respectively. The term “organic tellurium” is defined to mean any tellurium element bonded to an organic moiety, including via atoms that differ from carbon, such as oxygen.


For in-vitro studies, AS101 was supplied as PBS solution, pH 7.4, at 4° C. For in-vivo experiments, AS101 powder was dissolved in the appropriate concentration in re-circulating water.


Cell Culture

PBL (peripheral blood leukocytes) were isolated from heparinized blood on a ficoll paque bed (Amersham Bioscience) and cultured as previously reported (9). For AS101 treatments, AS101 was added directly to the cultures at final concentration of 0.025 to 25 μg/ml, preferably 0.05 to 0.5 μg/ml.


Stress Induction

Fish were subjected to air exposure stress every 60 min for 90 sec during 4 hours (10). The fish were then divided into groups of 3, each separately held in 40 L plastic containers at 28°±2° C.


Western Blot Analysis

PBL were lysed as previously reported (6). Samples were electrophoresed on 12% SDS-PAGE and blotted with anti-human-IL-10 mAb (Santa Cruz Biotechnology, Inc sc-8438) (1:660 dilution) and HRP-conjugate secondary Ab (Jackson Immuno-Research) (1:6600 dilution).


Serum IL-10L Detection

Fish blood was coagulated during 2 hours at room temperature, then centrifuged for 10 min at 1800×g. IL-10L levels were quantified in the collected sera using ELISA (Human Interleukin-10 ELISA Kit, pierce-endogen) according to manufacturer's instructions.


Stress Intensity Control

Blood samples were taken 1 hour after stress induction, sera separated by centrifugation as above and glucose levels were measured using Glucose TRINDER reagent (Sigma) according to manufacturers instructions, in a 96 microwell plate at 490 nm.


Results

AS101 effect on the intracellular IL-10L levels of tilapia cell cultures


AS101 caused significant inhibition of intracellular tilapia IL-10L synthesis. This inhibitory effect was dose-dependant and complete inhibition was achieved with 0.5 μg/ml AS101 (FIG. 1).


Influence of extensive stress on serum IL-10L levels Serum IL-10L levels were measured prior to stress induction and at different intervals of time (1, 5, 6, 8, 16, 20, and 24 Hrs) following stress induction. Significant IL-10L increase (p=0.001) started at 1 hour post stress, peaked on hours 8-16 and underwent significant decrease (p=0.006) afterwards (FIG. 2). AS101 effect on stress induced serum IL-10L and glucose levels


The maximal AS101 effect on IL-10L level (decrease from 719 to 209 pg/ml) was obtained 2 hours post-stressed following bath immersion in a 20-ppm water solution of this compound. AS101 treatment, while affecting IL-10L, had no effect on blood glucose levels which remained high (190 mg/dl±0.12) as in control stressed fish, (FIG. 3)


AS101 effect on stressed fish infected with Aeromonas salmonicida


Stress induced fish that were immersed in soluble AS101 after being exposed to Aeromonas salmonicida had significantly lower number of wounds per fish (FIG. 4) and higher rate of survival (FIG. 5) in contrast to stressed fish infected with the bacteria and not treated with AS101. AS101 inhibits tilapia intracellular IL-10L in vitro in a dose dependant manner. The possibility that the inhibition was due to cell toxicity, was examined Blue staining, and did not show elevated cell death beyond control in all AS101 doses, indicating that AS101 has no which were taken 6 hours after stress induction and bath immersion in different AS101 concentrations showed a significant IL-10L decrease, mainly with AS101 dose of 20 μg/ml which was the most efficient. These results show that AS101 reached the fish blood probably by penetrating through the skin, the gills or the gut epithelia. Interestingly, both IL-10L and glucose, which showed significant blood elevation following stress induction, only IL-10L, was down regulated by the AS101 treatment.


These results suggest that the protective role of AS101 against Aeromonas salmonicida infections in stressed goldfish involves AS101 mediated IL-10L inhibition, emphasizing it as an effective agent against stress-induced immune suppression.


References which are incorporated into this application:

  • [1] Takashi Y, Teruyuki N. Interaction between the endocrine and immune Systems in Fish. Int Rev Cytol. 220:35-92; 2002
  • [2] Jun Z, Melody S C, Secombes C J. Characterisation, expression and promoter analysis of an interleukin 10 homologue in the pufferfish (Fugu rubripes). 55(5): 325-35; August 2003
  • [3] Sredni B, Caspi R R, Klein A, Kalechman Y, Danziger Y, Ben Ya'akov, M Tamari T, Shalit F, Albeck M. A new immunomodulating compound (AS-101) with potential therapeutic application. Nature. 330(6144): 173-176; November 1987
  • [4] Strassmann G, Kambayashi T, Jacob C O, Sredni B. The immunomodulator AS-101 inhibits IL-10 release and augments TNF alpha and IL-1 alpha release by mouse and human mononuclear phagocytes. Cell. Immunol. 176: 180; 1997.
  • [5] Kalechman Y, Zuloff A, Albeck M, Strassmann G, Sredni B. Role of endogenous cytokines secretion in radioprotection conferred by the immunomodulator ammonium trichloro (dioxyethylene-0-0′) tellurate Blood. 85: 1555; 1995
  • [6] Kalechman Y, Longo D L, Catane R, Shani A, Albeck M, Sredni B. Synergistic anti-tumoral effect of paclitaxel (Taxol)+AS101 in a murine model of B16 melanoma: association with ras-dependent signal-transduction pathways Int. J. cancer. 86: 281; 2000
  • [7] Elenkov I J and Chrousos G P. Stress Hormones, Proinflammatory and antiinflammatory Cytokines, and Autoimmunity. Ann. N.Y. Acad. Sci. 966: 290-303; 2002
  • [8] Akdis C A, Blaser K. Mechanisms of interleukin-10-mediated immunesuppression. 103 (2): 131-6; June 2001
  • [9] Rosenberg-Wiser S, and Avtalion R R. The cells involved in the immune response of fish. III. Culture requirement for phytohemagglutinin stimulated peripheral carp lymphocytes. Devel. Comp. Immunol. 6:693-702; 1982
  • [10] Melamed O, Timan B, Avtalion R R and Noga E J. Design of a stress model in the hybrid bass (Morone saxatilis x Morone Chrysops). Isr. J. Aguac-Bamidgeh. 51(1): 10-16; 1999

Claims
  • 1. A method for the direct or adjunct treatment of bacterial or fungal infections in fish and crustaceans including shell fish which comprises providing an effective amount of an organic tellurium compound in an aqueous environment to which said fish or crustaceans are exposed.
  • 2. A method for the direct or adjunct treatment of bacterial or fungal infections in fish and crustaceans as defined in claim 1 wherein the tellurium compound is selected from compounds of the formula: TeO2or complexes of TeO2  (C)orPhTeCl3  (D)orTeX4, when X is Cl, Br or For(C6H5)4P+(TeCl3(O2C2H4))—  (E)
  • 3. A method for the direct or adjunct treatment of bacterial or fungal infections in fish and crustaceans as defined in claim 1 wherein the tellurium compound is ammonium trichloro (dioxoethylene-O, O′) tellurate.
  • 4. A method for the direct or adjunct treatment of bacterial or fungal infections in fish and crustaceans as defined in claim 1 where the treatment of bacterial or fungal infections in fish and crustaceans is a direct treatment.
  • 5. A method for the direct or adjunct treatment of bacterial or fungal infections in fish and crustaceans as defined in claim 1 where the treatment of bacterial or fungal infections in fish and crustaceans is an adjunct treatment.
  • 6. A method for the direct or adjunct treatment of bacterial or fungal infections in fish and crustaceans as defined in claim 1 where the treatment of bacterial or fungal infections in fish and crustaceans is based on the addition of 0.025 to 25 μg/ml to the aqueous environment of the fish or crustacean.
PCT Information
Filing Document Filing Date Country Kind 371c Date
PCT/IB04/04095 12/9/2004 WO 00 4/20/2007
Provisional Applications (1)
Number Date Country
60529142 Dec 2003 US