TECHNICAL FIELD

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.

BACKGROUND ART

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 TNFα 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

TNFα are provided by way of example only.

More specifically, 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 .

Symptoms arising from sepsis present a significant challenge in critical care. In the U.S. more than 750,000 people will develop severe sepsis each year and more than 215,000 of these people will die from the condition. Treating patients with symptoms arising from sepsis is estimated to cost hospitals in the US nearly $US 17 billion a year ⁷ . Internationally the incidence of severe sepsis is in the order of over 18 million cases worldwide each year and the number of sepsis patients is expected to increase by 1.0% per year.

¹ Landry DW, Oliver JA. The pathogenesis of vasodilatory shock. N Engl J Med 2001 ;345:588-595

² Martin GS. Sepsis: The Bermuda Triangle of the Pharmaceutical Industry American Thoracic Society 100th International Conference May 21 - 26, 2004, Orlando, FL

³ van Deventer SJ, Bulder HR, ten Cate JW, Aarden LA, Hack CE, Sturk A. Experimental endotaxemia in humans: analysis of cytokine release and coagulation, fibrinolytic and complement pathway. Blood 1990 76: 2520-2526.

⁴ Vervolet MG, Thijs LJ, Hack CE. Derangements of coagulation and fibrinolysis in critically patients with sepsis and septic shock. Semin. Thromb. Hemost. 199824: 33-44.

⁵ Kidokoro A ₁ lba T, Fukunaga M, Yagi Y. Alterations in coagulation and fibrinolysis during sepsis. Shock 1996 5: 223-228.

⁶ Lorente JA, Garcia-Frade LJ, Landin L, et al. Time course of hemostatic abnormalities in sepsis and its relation to outcome. Chest 1993103: 1536-1542

⁷ Aird WC. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood 101: 3765-3777.

As a result of the above health issues surrounding sepsis and other unwanted immune responses, it is desirable to have treatment options available that can at least suppress the immune system from a severe reaction due to sepsis or other immune response related events. It has also been suggested that pharmacological agents may inhibit TNFα cytokine synthesis ⁸ . Given that TNFα may be associated with immune function, the results of this study suggest that it may be possible to influence immune system function by adjusting the cytokine response using such agents.

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 .

Some mycotoxins (not indole diterpenes) have been found previously to have an effect on the immune system.

A sesquiterpenoid mycotoxin, deoxynivalenol, produced by Fusarium species, is known to cause vomiting and weight loss in animals. This mycotoxin has been shown to either suppress or stimulate immune responses depending on dose and duration of exposure in animals. 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.

Experiments have also demonstrated that many 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

⁸ Sato T.O., Keelan, J.A. and Mitchell M.D. (2003) Critical paracrine interactions between TNFα and IL-10 regulate liposaccharide-stimulated human choriodecidual and prostaglandin E2 production, JnI of Imm. VoM 70 pp158-166.

⁹ Latch GCM. Endophytes and ryegrass staggers. In: Lacey J, ed. Trichothecenes and other mycotoxins. New York, NY: John Wiley and Sons.1985, 135-139

¹⁰ Galey FD, Tracy ML, Craigmill AL, Barr BC, Markegard G, Peterson R, and O'Connor M. (1991). Staggers induced by consumption of perennial ryegrass in cattle and sheep from northern California. J. Am. Vet. Med. Assoc. 199(4), 466-470

¹¹ Dombrink-Kurtzman MA, Gomez-Flores R, and Weber RJ. (2000). Activation of rat splenic macrophage and lymphocyte functions by fumonisin B1. Immunopharmacology, 49, 401-409

¹² McLeay LM, Smith BL, and Munday-Finch SC. Tremorgenic mycotoxins paxilline, penitrem and lolitrem B, the non-tremorgenic 31-epilolitrem B and electromyographic activity of the reticulum and rumen of sheep. Res Vet Sci. 1999 Apr. 66(2):119-27

similar to those in neurological diseases occurring in animals experiencing alteration of their immune responses ¹³ ' ¹⁴ ' ¹⁵ ' ¹⁶ .

The functions of 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 TNFα ²¹ . BK channels are involved in lipopolysaccharide (LPS) signalling in macrophages ¹⁹ ' ²² .

BK channels are also known as maxi-K channels. The Slowpoke (SIo) gene encodes the pore- forming α subunit of the channel, the associated β subunits (β1-β4) 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). One patent application, WO 03/105868 suggests a link between BK channel blocking and treatment of glaucoma.

Recently, 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.

¹³ Campbell IL, Abraham CR, Masliah E, Kemper P, lnglis JD, Oldstone MBA, and Mucker L. (1993). Neurologic disease induced in transgenic mice by cerebral over expression of interleukin-6. Proc. Natl. Acad. Sci. USA, 90, 10061-10065

¹⁴ Schwender S, Hein A, lmrich H, Czub S, and Domes R. (1999). Modulation of acute encephalomyelitis in D-irradiated rats by transfer of naϊve lymphocyte subsets before infection. J. NeuroVirology, 5, 249- 257.

¹⁵ Lassmann S, Kincaid C, Asensio VC, and Campbell IL. (2001 ). Induction of type 1 immune pathology in the brain following immunization without central nervous system autoantigen in transgenic mice with astrocyte-targeted expression of IL-12. J. Immunol. 167, 5485-5493.

¹⁶ Sredni-Kenigsbuch D. (2002). TH1/TH2 cytokines in the central nervous system. Int. J. Neurosci. 112(6), 665-793

¹⁷ Cahalan MD and Chandy KG. (1997). Ion channels in the immune system as targets for immunosuppression. Curr. Op. Biotech., 8, 749-756

¹⁸ Blunck R, Scheel O, Muller M, Brandenberg K, Seitzer U and Seydel U. (2001 ). New insights into endotoxin-induced activation of macrophages: Involvement of a K ⁺ Channel in Transmembrane signalling. J. Immunol. 166, 1009-1015

¹⁹ Chandy KG, Cahalan M, Pennington M, Norton RS, Wulff H, and Gutman GA. (2001). Potassium Channels in T lymphocytes: toxins to therapeutic immunosuppressant. Toxicon, 39, 1269-1276

²⁰ Froh M, Thurman RG, and Wheeler MD. (2002). Molecular evidence for a glycine-gated chloride channel in macrophages and leukocytes. Am. J. Physiol. Gastrolintest Liver Physiol. 283 (4), G856-863

²¹ Beeton C, Wulff H, Barbaria J, Clot-Faybesse O, Pennington M, Bernard D, Cahalan MD, Chandy KG, and Beraud. (2001a). Selective blockade of T lymphocyte K ⁺ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc. Natl. Acad. Sci. USA, 98(24), 13942-13947

²² Seydel U, Shceel O, Muller M, Brandenburg K, and Blunck R. (2001). A K ⁺ channel is involved in LPS signalling. J. Endotoxin Res. 7(3), 243-247

It is therefore an object of the present invention to provide an alternative method to inhibit the immune system or at least to provide the public with a useful choice.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country. It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non- specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION It has been found by the inventors that indole diterpene compounds inhibit an immune response associated with cytokines.

For the purposes of this specification the term '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.

The term '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.

The term 'moiety' refers to part of a molecule.

According to one aspect of the present invention there is provided the use of 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:

²³ NZ527607

infection, sepsis, allergies, avian flu, transplant rejection, anaphylactic shock, pain prevention, reducing inflammation, and combinations thereof.

In one embodiment, the infection immune response reaction may be associated with premature birth. According to a further aspect of the present invention there is provided 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.

According to a further aspect of the present invention there is provided 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.

According to a further aspect of the present invention there is provided 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.

According to a further aspect of the present invention there is provided 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.

Preferably, the indole diterpene compound or compounds are selected from the group as shown below in Table 1 :

Table 1:

Pennigritrem Secopenitrem B

which includes compounds selected from: lolitrem B = 31 α, 35β stereochemistry, 31-epi-lolitrem B = 31 β, 35β stereochemistry, lolitrem F = 31 α, 35α stereochemistry, 31-epi-lolitrem F = 31 β, 35α stereochemistry;

which includes compounds selected from: lolitrem E = 31 α, 35β stereochemistry where R = H or acetate, lolitrem L = 31 α, 35α stereochemistry where R = H or acetate;

which includes compounds selected from: lolitrem A = 31 α, 35β stereochemistry, and lolitrem G = 31 cc, 35α stereochemistry;

which includes compounds selected from: lolitriol = 31 α, 35β stereochemistry where R-i = H or acetate and R ₂ = H, lolitrem N = 31 α, 35α stereochemistry where R ₁ =H or acetate and R ₂ =H, Lolitrem J = 31 α, 35β stereochemistry where R-i = H or acetate and R ₂ = acetate;

which includes lolitrem H = 31 α, 35β stereochemistry where R = H or acetate;

which includes lolitrem M = 31 α, 35β stereochemistry;

which includes compounds selected from the group consisting of: 30-desoxylolitrem B-30α-ol 31 α, 35β stereochemistry, 30-desoxy-31-epi-lolitrem B-30α-ol = 31 β, 35β stereochemistry;

which includes 30-desoxylolitrem B-30-ene = 35β stereochemistry;

(End of Table 1)

Preferably, the indole diterpene compound or compounds are selected from at least one compound containing the moiety shown in structure (I):

STRUCTURE (I) or derivatives thereof.

Preferably, derivatives of the compounds listed in Table 1 may be selected from: salts, analogues, isomers, and combinations thereof. It should be appreciated that 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. Preferably, 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, loϋtrem C, lolitrem M, lolitriol, lolitriol acetate, lolitrem N, lolitrem J, lolitrem H, lolitrem K, lolicine A and B, 30-desoxy lolitrem B-30α-ol, 30-desoxy-31-epi-lolitrem B-30α-ol, 30-desoxylolitrem B-30-ene, lolilline, and combinations thereof. In preferred embodiments where the compound is used as a medicament, the compound or compounds may be administered to a human. This should not be seen as limiting as it is envisaged that the compound may also be used as a medicament for the treatment of non- human animals as well. In one embodiment the pharmacologically effective amount may be equivalent to approximately 20 nM to 2 μM 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.

For the purposes of this specification, the term 'cell' or 'cells' may include any cytokine producing cells such as macrophage cells and spleen cells. In one preferred embodiment the cells may be macrophage cells. Preferably the cytokine proteins are selected from those with a pro-inflammatory response. More preferably, the cytokines include: IL-6, TNFα, IL1b, IL-10, IL-12, IFNγ, and combinations thereof.

It is the inventors' experience that 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 TNFα 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 TNFα cytokines play a key role in activating, directing and regulating an immune response. Preferably, the composition may also include pharmaceutically and physiologically acceptable carriers. Preferably, 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.

Preferably, where the composition is for treatment of a patient, it 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. However, other methods of administration may also be employed without limiting the scope of the present invention. Preferably, 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. In one embodiment, 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.

Further applications envisaged for the immune response inhibition treatment include use in drug development and diagnostics i.e. a known inhibition effect is generated from lolitrem compounds which may be used to find new drugs or to test if various physiological effects are altered. The above described applications should not be seen as limiting as it should be appreciated by those skilled in the art that other applications may also be possible.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 shows a graph comparing lolitrem B inhibition of the production of TNFα 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 μM ('L1 P+') and 2 μM ('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 μM ('L1 P+') and 2 μM (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 TNFα 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 Ε20P+'), 100 nM (Ε100P+ ¹ ), 200 nM (Ε200P+ ¹ ), 500 nM (Ε0.5P+ ¹ ), 1 μM (Ε1 P+ ¹ ) and 2 μM (Ε2P+ 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. Concentrations of 31-epi-lolitrem B used in the experiments were 20 nM (labelled as 'E20P+'), 100 nM ('E100P+'), 200 nM ('E200P+'), 500 nM (Ε0.5P+ ¹ ), 1 μM (Ε1P+ ¹ ) and 2 μM (Ε2P+ ¹ ) respectively; Figure 5 shows a graph comparing the effect of lolitrem B on cell proliferation by murine macrophage cells. Lolitrem B was applied at concentrations ranging from 20 nM to 5 μM 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 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; and,

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 μM 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.

BEST MODES FOR CARRYING OUT THE INVENTION

The invention is now described with respect to two experiments investigating aspects of the cytokine effects.

Experiment 1

An experiment is now described showing the effects of lolitrem B and 31-epi-lolitrem B on inhibiting production of cytokines IL-6 and TNFα from macrophage cells. It should be appreciated that these examples 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.

Cell Culture

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 μl/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.

IL-6 and TNFα Cytokine Production

Lolitrem B or 31 -epi-lolitrem B at a range of concentrations were added to cultures of the cells. 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. Then tetramethylbenzidine (TMB) substrate in citric phosphate buffer (pH 5.5) was added and an equal volume of 6 N H ₂ SO ₄ was added to stop the reaction. Absorbance was measured at 450 nm with a Vmax Kinetic Micro-titre Reader and interleukin production quantified using the Softmax™ software. Data were analysed by ANOVA and t test using the Genstat software package.

Results - Lolitrem B Figures 1 and 2 demonstrate the inhibition of TNFα and IL-6 production in murine macrophage cells by lolitrem B at concentrations ranging from 20 nM to 2 μM in the presence of dimethyl sulfoxide (DMSO) and lipopolysaccharide (LPS). LPS, which stimulates cytokine production, was added to the culture simultaneously with Lolitrem B. Following incubation for 24 hours, concentrations of TNFα and IL-6 were determined. Controls using the DMSO/water solvent without lolitrem B were run for each concentration of lolitrem examined. At each concentration of lolitrem B there were highly significant decreases in the production of both TNFα and IL-6 compared with the control sample.

A further control also shown on Figures 1 and 2 was also completed to indicate the production of both TNFα and IL-6 in absence of LPS to stimulate cytokine production. This control indicated that the cultures responded to LPS stimulant as expected.

Results - 31-Epi-Lolitrem B

A further series of analogous experiments were conducted to determine whether 31 -epi-lolitrem

B, a non-tremorgenic isomer of lolitrem B inhibited the production of IL-6 and TNFα cytokines. Figures 3 and 4 show the results obtained using the same procedures and conditions as for data in Figures 1 and 2. Like for lolitrem B, the production of TNFα and IL-6 was significantly less in the presence of all concentrations of 31 -epi-lolitrem B (20 nM to 2 μM) than in corresponding controls for the DMSO/water solvent. These results show that the inhibition effect is likely to be repeatable in the lolitrem group generally and not specific to one lolitrem compound. The effects are also obviously not limited to tremorgenic isomers only.

Toxicity

A further experiment was conducted to determine the toxicity of lolitrems on murine macrophage cells. This is of importance in drug design i.e. the aim is to produce a specific function (inhibition) and not harm the cells or host. The experiment was completed by means of an MTT assay of cell proliferation. Lolitrem B and 31-epi-lolitrem B were applied at concentrations ranging from 20 nM to 5 μM and proliferation indices were measured.

Results - Toxicity

The results found for toxicity are shown in Figures 5 and 6 as proliferation indices relative to control sample absent of lolitrem compounds.

Concentrations of lolitrem B and 31-epi-lolitrem, even at maximum concentrations measured did not have a significant effect on cell proliferation. These maximum concentrations are respectively 100 and 250 times higher than the concentrations at which these two compounds have a maximum effect on cytokine production. This experiment also tested for an effect of the DMSO/water solvent used to prepare the solutions of lolitrems. There was no discernable effect due to the solvent being present.

It should be appreciated from the above description that, by application of lolitrem compounds to cytokine producing cells, production of IL-6 and TNFα 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).

Experiment 2

A second experiment was undertaken to test the effects using different cytokines and different indole diterpene compounds.

Cell Preparation: Macrophage Cells

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 70μm 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.

Compound Treatment:

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.

An aliquot of the stock solutions was then diluted out by sequential addition of PBS to the concentrations to be used in the trial (20 μM, 2 μM, 0.2 μM compound in 0.2, 0.02, 0.002% DMSO/PBS respectively).

Cell treatment:

Spleen Cells

Cell cultures (270μl) containing 1 μmol LPS were treated with 30 μl of each indole diterpene compound at three different concentrations to give a final concentration of either 2μM, 0.2 μM or 0.02 μM. The cells were incubated at 37°C for 24hrs and the supernatants removed. Where necessary, supernatants were stored at -20°C prior to analysis by ELISA.

Macrophage Cells

Cells (270μl) were treated with 30 μl of each indole diterpene compound at three different concentrations to give a final concentration of either 2 μM, 0.2 μM or 0.02 μM in the presence of 1 μg/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.

Culture Controls The following control cultures were also included:

LPS alone, LPS + 0.2, 0.02 or 0.02% DMSO, 0.2, 0.02 or 0.02% DMSO alone to adjust for DMSO effects.

Cytokine Analysis: Cytokine levels were measured using sandwich ELISA using BD Biosciences Mouse OptEIATM ELISA sets for IL-6, TNFα, IL1 b, IL-10, IL-12, and IFNγ.

Results

Taken as a group, 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.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.