Classifications

• • 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
  • A61K31/403—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 condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention relates to an immune response inhibitor. More specifically, the invention relates to methods and uses of indole diterpenoid compounds such as lolitrem compounds to inhibit an immune response by decreasing the level of cytokine production from cytokine producing cells.
• Cytokine proteins are one component of the immune system produced by cells in response to an infection or other trigger. Selected cytokines including IL-6 and TNF ⁇ are understood to direct and regulate an immune response and have a pro-inflammatory effect. It should also be appreciated that other cytokines are known to have effects on the immune system and IL-6 and
• TNF ⁇ are provided by way of example only.
• cytokines are a causal factor 1 in the activation of the inflammatory system and a countervailing response from the anti-inflammatory system. Cytokines are recognised as the primary pathophysiologic event in sepsis events 2 i.e. the presence of a foreign organism in the blood stream. Sepsis may result in an allergic reaction ranging from mild cases such as hayfever, through to extreme cases such as an anaphylactic reaction. Other sepsis events also exist such as organ transplant rejection and infection. Severe sepsis results from the body's systemic over-response to infection. This over-response disrupts homeostasis through an uncontrolled cascade of inflammation, coagulation, and impaired fibrinolysis 3 ' 4 ' 5 ' 6 .
• Indole diterpenes are a group of compounds, many of which have known toxic properties. For the purposes of this discussion, focus is given to lolitrem compounds although it should be appreciated that similar findings may be found for other types of indole diterpene compound such as for example: paxiline, paspaline, janthitrem, aflatrem, and terpendole compounds.
• Lolitrem compounds are unique indole-diterpenoids isolated from perennial ryegrass (Lolium perenne L.) infected with the endophytic fungus Neotyphodium loliP. Endophyte-infected perennial ryegrass is the common pasture species in New Zealand and animals grazing these pastures suffer from tremors and ataxia 10 .
• Another mycotoxin, Fumonisin B1 is a neurotoxic mycotoxin which causes equine leukoencephalomalacia in horses, and produces ataxia, blindness and hyperexcitability. It has been found that Fumonisin B1 is able to activate macrophage function and increase T cell proliferation 11 . ' As a result, there is some knowledge that mycotoxin compounds may have some effect on the immune system however this has not been investigated, proven, or is not necessarily obvious with respect to lolitrem compounds.
• lolitrems can elicit tremogenicity in mice and also affect the brain by causing membrane hyperpolarisation or ion channel changes.
• a long acting but completely reversible response to lolitrem neurotoxins has been reported 12 .
• the mode of lolitrem action and the resulting symptoms such as tremor and ataxia are phenomena
• Ion channels have been widely studied in many cell types, including cells of the immune system such as Kupffer cells, macrophages and T cells 17 ' 18 ' 19 ' 20 .
• Ion channels known to be present in immune cells include voltage gated ion channels (Kv1.3), large conductance potassium channels (BK), small conductance potassium channels (SK), intermediate conductance potassium channels (IK), and ligand gated chloride channels. Blocking of voltage-gated K + channels reduces T cell activation by decreasing the activity of cytokine IL-2 release and TNF ⁇ 21 .
• BK channels are involved in lipopolysaccharide (LPS) signalling in macrophages 19 ' 22 .
• LPS lipopolysaccharide
• BK channels are also known as maxi-K channels.
• the Slowpoke (SIo) gene encodes the pore- forming ⁇ subunit of the channel, the associated ⁇ subunits ( ⁇ 1- ⁇ 4) are responsible for BK channel diversity.
• BK channels are characterised by their activation by Ca ++ ions, voltage dependence and a conductance of 100 to 300 picoSiemens (pS).
• pS picoSiemens
• lolitrem B has been shown to be a potent inhibitor of BK channel function in HEK cells 23 which suggests there may be a link between channel function and an effect on the immune response.
• 'immune response' refers to an alteration in the reactivity of an organism's immune system in response to a trigger such as an antigen. This trigger in turn stimulates the production of cytokine proteins to direct and regulate the immune response.
• cytokine' refers to proteins or biological factors that are released by cells and have specific effects on cell-cell interaction, communication and behaviour of other cells.
• 'moiety' refers to part of a molecule.
• an effective amount of at least one indole diterpene compound or derivatives thereof in the manufacture of a composition to inhibit or decrease an immune response associated with cytokines According to a further aspect of the present invention there is provided the use of a composition that contains an effective amount of at least one indole diterpene compound or derivatives thereof to inhibit or decrease an immune response in vivo associated with cytokines. According to a further aspect of the present invention there is provided the use of a composition that contains an effective amount of at least one indole diterpene compound or derivatives thereof to inhibit or decrease an immune response in vitro associated with cytokines. According to a further aspect of the present invention there is provided the use of a composition that contains an effective amount of at least one indole diterpene compound or derivatives thereof, in the manufacture of a composition for the treatment of afflictions selected from:
• the infection immune response reaction may be associated with premature birth.
• a method to inhibit or decrease an immune response in an animal associated with cytokines by administration of a composition that contains an effective amount of at least one indole diterpene compound or derivatives thereof.
• a method to inhibit or decrease an immune response in vivo associated with cytokines by administration of a composition that contains an effective amount of at least one indole diterpene compound or derivatives thereof.
• a method to inhibit or decrease an immune response in vitro associated with cytokines by administration of a composition that contains an effective amount of at least one indole diterpene compound or derivatives thereof.
• a method of treatment of afflictions selected from: infection, sepsis, allergies, avian flu, transplant rejection, anaphylactic shock, pain prevention, reducing inflammation, and combinations thereof in an animal, by administration of a composition that contains an effective amount of at least one indole diterpene compound or derivatives thereof.
• the indole diterpene compound or compounds are selected from the group as shown below in Table 1 :
• lolitrem A 31 ⁇ , 35 ⁇ stereochemistry
• lolitrem G 31 cc, 35 ⁇ stereochemistry
• the indole diterpene compound or compounds are selected from at least one compound containing the moiety shown in structure (I):
• derivatives of the compounds listed in Table 1 may be selected from: salts, analogues, isomers, and combinations thereof.
• indole diterpene compounds exhibit similar properties. Further, it is the inventors understanding that there may be a link between blocking of channel activity known to occur for at least some indole diterpene compounds and inhibition of an immune response. As a result, it should be appreciated by those skilled in the art that indole diterpene compounds may generally exhibit inhibition of an immune response associated with cytokines.
• the compound may be selected from: lolitrem B, lolitrem A, lolitrem F, 31-epi-lolitrem F, 31-epi-lolitrem B, lolitrem E, lolitrem E acetate, lolitrem L, lolitrem G, lo ⁇ trem C, lolitrem M, lolitriol, lolitriol acetate, lolitrem N, lolitrem J, lolitrem H, lolitrem K, lolicine A and B, 30-desoxy lolitrem B-30 ⁇ -ol, 30-desoxy-31-epi-lolitrem B-30 ⁇ -ol, 30-desoxylolitrem B-30-ene, lolilline, and combinations thereof.
• the compound or compounds may be administered to a human.
• the compound may also be used as a medicament for the treatment of non- human animals as well.
• the pharmacologically effective amount may be equivalent to approximately 20 nM to 2 ⁇ M of compound or compounds. This should not be seen as limiting as it should be appreciated that amounts outside this range may also be used which may achieve similar results to that observed within this range.
• Dose rate may also be influenced by many other factors such as delivery vehicle and patient metabolism. It is the inventors understanding that the inhibition in immune response noted may, at least in part, be attributable to a decrease in the production of cytokines from cells.
• the term 'cell' or 'cells' may include any cytokine producing cells such as macrophage cells and spleen cells.
• the cells may be macrophage cells.
• the cytokine proteins are selected from those with a pro-inflammatory response. More preferably, the cytokines include: IL-6, TNF ⁇ , IL1b, IL-10, IL-12, IFN ⁇ , and combinations thereof.
• the amount of inhibition may vary. For example, it is the inventor's experience that the decrease is from approximately 20% to as much as a 90% reduction in IL-6 and/or TNF ⁇ cytokine production from cells. It should be appreciated by those skilled in the art that this may result in a significantly different immune response reaction as IL-6 and TNF ⁇ cytokines play a key role in activating, directing and regulating an immune response.
• the composition may also include pharmaceutically and physiologically acceptable carriers.
• the pharmaceutically and physiologically acceptable carriers may include components selected from: fillers, excipients, modifiers, humectants, stabilisers, emulsifiers, diluents, and other formulation components such as a use of a lipid vehicle.
• the composition may be administered in a form selected from: an injection, a tablet, a capsule, a suppository, an injection, a suspension, a drink or tonic, a syrup, a powder, an ingredient in solid or liquid foods, a nasal spray, a sublingual wafer, a transdermal patch, a transdermal injection, and combinations thereof.
• the lolitrem compound or compounds may be extracted from: endophyte-infected plants and seeds, fungal cultures, chemical synthesis, heterologous expression systems including bacteria, yeast, fungi, plants and animal cells, and combinations thereof.
• the source may be perennial ryegrass seed from Lolium perenne.
• An advantage of the use of lolitrem compounds is that they appear to have little or no toxic effect on cells. As many existing treatments may have side effects, an alternative compound with no or few side effects may be of considerable value.
• Figure 1 shows a graph comparing lolitrem B inhibition of the production of TNF ⁇ from murine macrophage cells.
• Data represents one of two replicate experiments. Each bar represents data from two replicates ⁇ standard deviation (S. D.) error bar. Black bars represent lolitrem B plus DMSO, grey bars represent DMSO control alone. Concentrations of lolitrem B used in the experiments were 20 nM (labelled as 120P+'), 100 nM ( 1 LI 00P+'), 200 nM ('L200P+'), 500 nM ('L500P+'), 1 ⁇ M ('L1 P+') and 2 ⁇ M ('L2P+') respectively.
• the single bar labelled "control" is for samples not treated with LPS;
• Figure 2 shows a graph comparing lolitrem B inhibition of the production of the cytokine
• IL-6 in murine macrophage cells Data represents one of two replicate experiments. Each bar represents data from two replicates ⁇ standard deviation (S. D.) error bar. Black bars represent lolitrem B plus DMSO; grey bars represent DMSO control. Concentrations of lolitrem B used in the experiments were 20 nM (labelled as ⁇ 20P+'), 100 nM ('L100P+'), 200 nM ('L200P+'), 500 nM ('L0.5P+'), 1 ⁇ M ('L1 P+') and 2 ⁇ M (12P+') respectively. The single bar labelled "control" is for sample not treated with LPS;
• Figure 3 shows a graph comparing 31-epi-lolitrem B inhibition in the production of TNF ⁇ from murine macrophage cells.
• Data represents one of three replicate experiments. Each bar represents data from three replicates ⁇ standard deviation (S. D.) error bar. Black bars represent 31-epi-lolitrem B plus DMSO; grey bars represent DMSO control.
• Concentrations of 31-epi-lolitrem B used in the experiments were 20 nM (labelled as ⁇ 20P+'), 100 nM ( ⁇ 100P+ 1 ), 200 nM ( ⁇ 200P+ 1 ), 500 nM ( ⁇ 0.5P+ 1 ), 1 ⁇ M ( ⁇ 1 P+ 1 ) and 2 ⁇ M ( ⁇ 2P+ 1 ) respectively;
• Figure 4 shows a graph comparing 31-epi-lolitrem B inhibition in the production of IL-6 from murine macrophage cells. Data represents one of three replicate experiments. Each bar represents data from three replicates ⁇ standard deviation (S. D.) error bar. Black bars represent 31-epi-lolitrem B plus DMSO; grey bars represent DMSO control.
• Figure 6 shows a graph comparing the effect of 31-epi-lolitrem B on cell proliferation by murine macrophage cells.
• 31-Epi-Lolitrem B was applied at concentrations ranging from 20 nM to 5 ⁇ M and proliferation indices were measured. The data is presented as proliferation indices relative to controls in the absence of lolitrems and each bar represents one of six replicate experiments.
• Black columns represent data for epi-lolitrem B added as an aqueous solution containing DMSO.
• Grey columns represent control data for DMSO at the same concentrations it was present in the treatment solutions.
• lolitrem B and 31-epi-lolitrem B are illustrative only and that other lolitrem compounds may also have similar effects given the similarity in chemical structure and activity of this chemical group.
• Murine macrophage RAW 264.7 cells were cultured in 100-mm tissue culture plates using Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine serum with 100 ⁇ l/ml penicillin/streptomycin in an incubator at 5-7% CO 2 and 37°C. Cell numbers and viabilities were assessed by trypan blue (Sigma) dye exclusion using a hemacytometer. Cells were cultured at 5 x 10 5 cells cells/ml in 48-well tissue culture plates. Cultures were exposed to Salmonella typhimurium lipopolysaccharide (Sigma) and incubated for different periods of time. After incubation, the supernatant was harvested and stored at -20 0 C for further analysis.
• Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine serum with 100 ⁇ l/ml penicillin/streptomycin in an incubator at 5-7% CO 2 and 37°C. Cell numbers and viabilities were assessed by trypan blue (Sigma) dye exclusion
• Cytokine ELISA assays were performed as follows. Antibodies against cytokines diluted in Phosphate Buffered Saline (PBS, pH7.2) were coated in 96 well micro-titre plates and incubated overnight at 4°C. Samples and standards were added to the wells and incubated at 37 0 C for 1 hour and then polyclonal antibodies against the cytokine in PBS buffer were added to each well and the plates were incubated at 37°C for a further hour. The plates were then washed and detection antibodies conjugated with horse-radish peroxidase were added.
• PBS Phosphate Buffered Saline
• TMB tetramethylbenzidine
• citric phosphate buffer pH 5.5
• 6 N H 2 SO 4 6 N H 2 SO 4
• Absorbance was measured at 450 nm with a Vmax Kinetic Micro-titre Reader and interleukin production quantified using the SoftmaxTM software. Data were analysed by ANOVA and t test using the Genstat software package.
• lolitrem compounds by application of lolitrem compounds to cytokine producing cells, production of IL-6 and TNF ⁇ cytokines is significantly inhibited. This effect lends itself to various applications where a reduced immune function is desirable. Also, as lolitrem compounds have been found to be non-toxic, it would be expected that minimal side effects are likely therefore making the compounds good candidates for drug design. It should further be appreciated that the examples given for lolitrem B and 31-epi-lolitrem B may equally be found for other lolitrem compounds (both tremorgenic and non-tremorgenic).
• RAW 264.7 cells were cultured in RPMI containing 5% heat deactivated FBS. Cells were then transferred onto a 96 well flat-bottomed plate at 10 6 cells/well.
• Spleen Cells The spleen from a C57BI6 mouse was removed and strained through a 70 ⁇ m nylon cell strainer with 10 ml complete IMDM (5% Foetal calf serum). The cell suspension was centrifuged (1500 rpm, 5 mins) and the supernatant removed. The cell pellet was resuspended in 10 ml clMDM. The clMDM wash was then repeated. Cells were then transferred onto a 96 well flat bottomed plate at 106 cells/well.
• IMDM 5% Foetal calf serum
• a stock solution of the each indole diterpene compound was prepared in 2% DMSO.
• the indole diterpene compounds used were lolitrem B 1 31-epilolitrem B, lolitrem E acetate, lolitrem M, lolitriol acetate, paxilline, 13-desoxypaxilline, paspaline, janthitrem B, alfatrem, terpendole C, and verruculogen.
• Cells (270 ⁇ l) were treated with 30 ⁇ l of each indole diterpene compound at three different concentrations to give a final concentration of either 2 ⁇ M, 0.2 ⁇ M or 0.02 ⁇ M in the presence of 1 ⁇ g/ml LPS.
• the cells were incubated at 37°C for 24hrs and the supernatants removed. Where necessary, supernatants were stored at -20 0 C prior to analysis by ELISA.
• Cytokine levels were measured using sandwich ELISA using BD Biosciences Mouse OptEIATM ELISA sets for IL-6, TNF ⁇ , IL1 b, IL-10, IL-12, and IFN ⁇ .
• the compounds studied which are considered to be representative of the indole diterpene class of compounds, have significant effects on immune system function and these effects are principally anti-inflammatory by reducing the level of production of a range of different cytokines.

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• 2006
  • 2006-04-26 WO PCT/NZ2006/000086 patent/WO2006115423A1/en active Application Filing

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