WO2003006035A1

WO2003006035A1 - Anti-emetic compositions comprising mycobacterial material

Info

Publication number: WO2003006035A1
Application number: PCT/GB2002/003124
Authority: WO
Inventors: Graham Arthur William Rook; Christopher Alan Lowry; Stafford Louis Lightman
Original assignee: Stanford Rook Limited
Priority date: 2001-07-10
Filing date: 2002-07-08
Publication date: 2003-01-23

Abstract

This invention relates to the control and/or treatment of emesis in an individual using compositions which activate the Th1 immune response, for example compositions which comprise mycobacterial material. Compositions and methods of use are provided.

Description

ANTI-EMETIC COMPOSITIONS COMPRISING MYCOBACTERIAL MATERIAL

This invention relates to the control and/or treatment of emesis using therapeutic compositions which generate a Thl- dominated immune response.

Emesis, which is typically characterized as vomiting, is a reflex which is developed to different degrees in different species, which allows an animal to rid itself of ingested toxins or poisons. The reflex can be elicited either by direct neuronal connections from visceral afferent fibres, especially those from the gastrointestinal tract, or from humoral factors .

The present inventors have shown that the generation of a Thl dominated immune response in an individual, for example by mycobacterial material, produces anti-emetic effects and agents and compositions which generate such responses are therefore useful in the treatment of emesis, nausea and vomiting.

Without being in any way limited to a particular mechanism, it is believed that the observed anti-emetic effects relate to the discovery that specific areas of the brain can be activated in a highly selective manner by Thl dominated immune responses, i.e. responses which are mediated predominantly by Thl lymphocytes, using novel neural pathways. These novel pathways relay signals of inflammation from the periphery to highly selective areas of the brain. Such brain activation leads to the modulation of functions of the central nervous system (CNS) , including the emetic reflex. Agents which elicit Thl dominated patterns of immune response, in particular mycobacteria such as M. vaccae, are therefore found to be useful in the treatment of emesis.

The present invention thus provides preparations comprising activators of Thl lymphocytes, for example, preparations and material from mycobacteria such as M. vaccae, for treating emesis, methods of treatment of emesis involving such activators and the use of such activators in such methods of treatment .

One aspect of the present invention provides the use of an activator of Thl lymphocytes in the manufacture of a medicament for use in a method of treating emesis.

Preferably the activator of Thl lymphocytes is mycobacterial material, for example M. vaccae material.

Emesis includes nausea and vomiting.

Another aspect of the present invention provides a method of treatment of emesis, the method comprising administering to a patient an effective amount of a therapeutic composition comprising an activator of Thl lymphocytes as disclosed herein.

A further aspect of the present invention provides a method of selectively activating a defined area of the brain and spinal cord of an emetic individual, comprising the step of stimulating a peripheral sensory afferent nerve.

A peripheral sensory afferent nerve may be stimulated in such methods by administering to a patient an effective amount of a therapeutic composition comprising a Thl lymphocyte activator, such as mycobacterial material .

Afferent nerves stimulated by methods according to the present invention may be associated with the vagus nerve, the dorsal root ganglia or other neural pathway.

A Thl activator is an agent which elicits, generates, induces or produces an immune response in an individual which has a major Thl component, i.e. the Thl component may be detected, determined or discerned using the methods described herein. More preferably, such an agent elicits an immune response which is predominantly or substantially Thl. It will be appreciated^' that complete polarization of an- immune response in a mammal into either Thl or Th2 is virtually impossible, so a minor proportion of any immune response which is elicited by a Thl activator will be Th2.

Suitable Thl lymphocyte activators may include antigens to which an individual or patient already produces an immune response which includes a major Thl component. Preferred Thl lymophocyte activators for use in accordance with the present invention include mycobacterial material, for example M. vaccae material .

The presence of an immune response in a patient with a major Thl component may be determined by means of a delayed hypersensitivity skin-test response after intradermal injection. In a delayed hypersensitivity response to soluble antigen there is swelling and induration that peaks at 48-72 hours .

Alternatively the existence of a major Thl component in a patient ' s immune response may be determined by culturing peripheral blood mononuclear cells from the patient in vi tro with the antigen, and determining or measuring the production of Thl cytokines, for example, using enzyme-linked immunoabsorbent assay or by reverse transcriptase polymerase chain reaction. A suitable indicator of a Thl response is release of interferon gamma (IFNy) .

A composition comprising such an activator may be administered to an individual without additional Thl adjuvants to invoke the Thl dominated immune response and produce anti-emetic effects.

In other embodiments, a patient may be deliberately immunized to elicit a response against an antigen. This may be done by administration of an agent that has both antigenicity and inherent Thl adjuvanticity (e.g. whole bacterial vaccines or mycobacterial vaccines, such as M. vaccae) , or by administration of a suitable Thl adjuvant mixed with an antigen. In the latter case, subsequent treatments may involve administration of the antigen without the Thl adjuvant .

It is preferred that Thl dominated immune responses are generated in an individual after pre-immunisation with an antigen, preferably a soluble antigen, in combination with a Thl adjuvant, or an agent which has both antigenic and Thl adjuvant activity. After such pre-immunisation with an antigen in combination with a Thl adjuvant, subsequent immunization may be performed using the antigen without the adjuvant, as described above.

A method of treatment of emesis may therefore include administering to a patient an effective amount of a first therapeutic composition comprising an antigen and a Thl adjuvant and subsequently administering to said patient an effective amount of a second therapeutic composition comprising said antigen.

The second composition may be administered 1 day after the first composition, 2, 3, 4 or 5 days, 1, 2, 3, 4 or 5 weeks after the first composition or 1, 2, 3, 4 , 5 or 6 months after the first composition. Such a method may be part of a course of therapy which comprises periodic administration of the second composition to a patient at regular intervals, (for example, daily, weekly, monthly or annually) or as needed.

Soluble antigens, in particular proteins, are very weak inducers of Thl and tend to induce Th2 immune responses. A Thl response will be evoked in the animal only if an adjuvant material is present which has Thl inducing properties.

The presence of a Thl response can subsequently be demonstrated by, as described above by culturing the spleen, blood or lymph node mononuclear cells in vi tro with the immunising antigen and determining the production of Thl cytokines, for example by ELISA or by RT-PCR.

An antigen for use, either alone or in combination with a Thl adjuvant is capable of eliciting, generating or producing an immune response in an individual which has a major Thl component. Suitable antigens include polypeptides, such as keyhole limpet haemocyanin, carbohydrates, nucleic acids or other organic or inorganic compounds. Suitable Thl adjuvants include cholera toxin, IFN-γ, IL-12, superantigens such as staphylococcal enterotoxin B (Kusnecov et al (1999) J. Neurosci 19 4533-4543) and dehydroepiandrosterone (3β-ol- androstene-17-one or DHEA) . In still other embodiments, activators are used which trigger Thl cells non-specifically to initiate the cascade of downstream Thl mediators that generate a signal via the afferent sensory nerves. Such activators may include ligands ■ for signalling molecules on the surface of Thl lymphocytes, such as CD2, CD3' or CD28. Suitable ligands include monoclonal antibodies, for example antibodies reactive with CD2, anti-CD3 or anti-CD28.

Other suitable activators which may be used to trigger Thl cells non-specifically include lectins, such as Concanavalin A and phytohaemagglutinin, which bind to the carbohydrate moiety of Thl lymphocyte surface signalling molecules. These lectins bind to multiple cell membrane glycolipids and glycoproteins and therefore non-specifically trigger multiple Thl activation pathways .

Lectins and other Thl lymphocyte surface binding molecules such as antibodies may also be used as adjuvants in combination with particular antigens. Such an adjuvant may be administered in vivo in an admixture or composition with an antigen according to a suitable immunisation schedule.

An agent which activates Thl lymphocytes non-specifically may be identified by administering the agent to a non-human mammal, such as a mouse, and determining an increase in levels of a cytokine associated with a Thl response, for example IL- 1, IL-2, IL-12 or IFN-γ.

Alternatively, antigenic material may be added to lymphoid cell cultures in vi tro and changes in the release of cytokines or in the expression of mRNA encoding cytokines can be assayed. Molecules which are downstream mediators released by cells involved in Thl immune responses, for example neuropeptides, are also able to signal to neural tissue. Instead of using an agent, compound or substance which predominantly elicits a Thl immune response (i.e. a Thl lymphocyte activator), an agent which is a downstream mediator of the Thl inflammatory response may also be used to stimulate sensory nerve afferents. Suitable mediators include IL-1, IL-2, IL-12 IL- 17, IL-18, IFN-γ, lymphotoxin (TNFβ) , TNFα, prostaglandins, nitric oxide, urocortin (Bamberger et al J. Clin. Endocrinol . Metab. 83: 708-711), prolactin (Matera and Mori (2000) Ann. NY Acad. Sci. 917:505-513), opiates (Mousa et al (2001) J. Neuroimmunol . 115: 71-78), CGRP, bradykinnin, histamine, substance P (Blum et al (2000) Faseb J. 15: 950-957) and other neurokinins, NGF, BDNF (Barouch et al J. Neuroimmunol. 103:112-121) or somatostatin.

A further aspect of the present invention therefore provides the use of an agent which is a downstream mediator of the Thl inflammatory response as described herein in the manufacture of a medicament for use in a method of treating emesis as disclosed herein.

Another aspect of the present invention provides a method of treatment of emesis comprising triggering a peripheral sensory afferent by administering to a patient an effective amount of a therapeutic composition comprising a mediator of the Thl immune response as described herein.

In some embodiments, suitable activators (including both antigens and adjuvants) are non-mycobacterial i.e. they are not made from, consist of, or comprise material from a mycobacterial cell, such as M. vaccae . Whole, killed mycobacterial cells, treated mycobacterial cells, mycobacterial cell extracts, fractions or components and material derived either directly or indirectly from a mycobacterial cell are therefore excluded from these preferred embodiments of the invention.

Other aspects of the present invention therefore relate to uses and methods treatment of emesis involving non- mycobacterial activators of Thl lymphocytes.

Such uses may include the use of a non-mycobacterial activator of Thl lymphocytes in the manufacture of a medicament for use in a method of treating emesis in an individual .

A method of treatment of emesis may include administering to a patient an effective amount of a therapeutic composition comprising a non-mycobacterial activator of Thl lymphocytes as described herein.

A method of selectively activating a defined area of the brain and spinal cord of an individual with emesis may include the step of stimulating a peripheral sensory afferent nerve by administration of non-mycobacterial activator of Thl lymphocytes .

In particularly preferred embodiments, a Thl dominated immune response is generated by mycobacterial material. Preparations of mycobacteria such as M. vaccae are known to be potent inducers of Thl immune responses. Mycobacterial material suitable for the stimulation of sensory afferent nerves through the generation of a Thl dominated immune response may include whole, killed mycobacterial cells, treated mycobacterial cells, mycobacterial cell extracts, fractions or components. A preferred mycobacterium for use in accordance with these embodiments is M. vaccae . Other aspects of the present invention therefore relate to uses and methods of treatment of emesis involving mycobacterial activators of Thl lymphocytes.

Such uses include the use of mycobacterial material in the manufacture of a medicament for use the treatment of emesis in an individual .

A method of treatment of an emesis may include administering to a patient an effective amount of mycobacterial material as described herein.

A method of selectively activating a defined area of the brain and spinal cord of an individual with emesis, may include the step of stimulating a peripheral sensory afferent nerve by administration of a mycobacterial material.

Material suitable for use in mycobacterial preparations may include mycobacterial cells adsorbed onto a support, for example nitrocellulose particles, before administration. Cells may be treated before being used for stimulation, such treatment may include ultrasonic disruption.

Material including fractions, extracts or components of mycobacterial cells may also be used to stimulate neural afferent terminals in the treatment of emesis . Such material may be prepared and/or purified apcording to standard techniques. Suitable mycobacterial material may activate a Thl dominated immune response in an individual to stimulate neural afferents.

A mycobacterium suitable for use in preparations, -compositions and methods of the present invention is M. vaccae . A Mycobacterium vaccae preparation for use in stimulating neural afferent terminals may, for example, include material which can be or include dead cells of M. vaccae . Such cells may be killed, for instance using irradiation, e.g. from ⁶⁰Cobalt at a dose of 2.5 megarads, chemically, or by any other means, although autoclaving is preferred, e.g. at 69kPa for 10 minutes at 115°C-125°C. Autoclaving may yield a more effective preparation than irradiation.

M. vaccae cells may be disrupted prior to administration, for example by ultrasonication.

A suitable material for use in an M. vaccae preparation may comprise whole dead cells or a fraction thereof as described above. Instead of killed cells, other material derived from M. vaccae may be used, in particular an extract or a synthetic molecule which has the requisite activity.

SRL172 is a M. vaccae formulation derived from the strain denoted R877R which was deposited under the Budapest Convention at the National Collection of Type Cultures (NCTC) Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, United Kingdom, on 13 February 1984 under the number NCTC 11659. R877R was originally isolated from mud samples from the Lango district of Central Uganda (Stanford and Paul, (1973) Ann. Soc . Beige Med. Trop . 53: 141-389).

SRL172 is an example of a formulation suitable for use in accordance with the present invention. Other suitable formulations for use in accordance with the present invention may be derived from species and strains of Mycobacteria other than M. vaccae NCTC 11659. An organism can be identified as belonging to Mycobacteria, or more precisely to a species such as M. vaccae by biochemical and antigenic criteria (Bonicke et al . , (1964) Zentr albl . Bakteriol . Parasitenkd. Infection skr. Hyg. Abt . 1, Orig., 192: 133), or by molecular methods such as PCR and restriction enzyme analysis as described by Telenti et al . (1993) J. Clin. Microbiol . 31: 175-178.

Prior to being killed and/or disrupted, M. vaccae cells may be grown on a suitable solid medium. A modified Sauton's liquid medium may be preferred (Boyden et al . , (1955) J. Immunol. 75: 15), solidified with agar, preferably 1.3% agar. After aerobic incubation, generally at 32°C for 10 days, the organisms may be harvested, then weighed and suspended in diluent, ready for administration. Storage, if required before use, may be at 4°C.

Instead of growing the cells on a solid medium, a liquid medium, such as the modified Sauton's medium (Boyden et al . supra) , may be employed, for instance in a fermenter.

The diluent may be unbuffered saline, pyrogen-free .

Preferably, the diluent is borate-bύffered, preferably containing a surfactant such as Tween 80. A suitable borate buffer is: Na₂B₄O₇.10H₂O - 3.63 g, H₃B0₃ - 5.25 g, NaCl - 6.19 g, Tween 80⁷ 0.0005%, distilled water to 1 litre. These diluents are pharmaceutically acceptable.

Mycobacterial preparations may also be used in the formulation of pharmaceutical compositions and medicaments for use in accordance with the present invention.

It is preferred for the present invention that a mycobacterial preparation is administered free or substantially free from non-mycobacterial antigenic or immunoregulatory material. In other words the medicament or composition to be administered may include, or may consist essentially of, mycobacterial preparation, such as dead cells, an extract or derivative thereof, and a pharmaceutically acceptable diluent. A preferred mycobacterium for use in such preparations- is M. vaccae .

Administration of a mycobacterial preparation to the respiratory tract may occur using any suitable formulation, for example, in solution as an aerosol, bound covalently or non-covalently to a semi persistent particulate carrier administered, as a snuff for the upper respiratory tract or as an intra-tracheal injection. Particle size may be used to target appropriate parts of the airways.

Suitable formulations for administration via the gastrointestinal tract include heat-killed organisms in capsules designed to release in the appropriate part of the gut. Alternatively, instead of whole organisms, selected components or extracts may be used. A suitable dose for administration by oral route may be 10⁷ to 10¹⁰ organisms or equivalent.

Emesis from gastrointestinal afferents is triggered by projections to part of the nucleus tractus soli tarius. As with a variety of other complex motor functions regulated by the brain stem, the sequence of muscle excitation and inhibition is controlled by a 'central pattern generator' located in the nucleus tractus solitarius. Information from humoral factors via the area postrema and visceral afferents via the vagus nerve converge at this point. This central pattern generator, like those for motor functions such as swallowing, presumably projects to the various motor nuclei, perhaps through inter- neuronal pathways, to elicit the sequential excitation and inhibition that controls the reflex. (Carpenter (1990) Can J Physiol Pharmacol 68 (2) :230-236) . Activation was observed upon induction of a Thl dominated immune response, by increased c-Fos expression in the ventrolateral and dorsolateral parts of Nucleus Tractus Solitarius (nTS) . These specific sub-nuclei are the primary targets of sensory fibres from the extra-thoracic^' and intra- thoracic trachea, the right main bronchus and the upper right lobe of the lung (Kalia M. and Meulam CM. J. Comp . Neurol . (1980) 193 467-508) . Activation of this region as described may inhibit, reduce or decrease the emetic reflex, either directly by desensitizing emesis signalling pathways or indirectly by activating an as yet uncharacterised inhibitory system.

In order to activate regions of the brain associated with emesis, peripheral afferent nerves may be stimulated by administration of a Thl activator via the respiratory, oral, intra-dermal or subcutaneous route. For example, an activator may be administered to the respiratory tract by conventional methods which may include intra-tracheal injection or inhalation.

A peripheral sensory afferent nerve stimulated in accordance with the present invention may have a terminal located in the skin, respiratory tract or gastrointestinal tract.

Compositions suitable for use in preparations may include cells, compounds or agents such as adjuvants and antigens which are adsorbed onto a support, for example nitrocellulose particles, before administration.

Compounds and agents, either individually or in combination which activate Thl lymphocytes may also be used in the formulation of pharmaceutical compositions and medicaments for use in accordance with the present invention.

Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time- course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

For example, IL-10 injected in to the brain has been shown to oppose behavioural changes caused by sickness (Bluthe et al (1999) Psychoneuroendocrinology 24:301-311) and may be used in combination with activators, agents and compositions of the present invention.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well-known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient .

The precise nature of the carrier or other material will depend on the route of administration of compounds as described herein for use in accordance with the present invention. Various routes of administration are possible, such as via the respiratory or pulmonary tract, the gastro-intestinal tract or by injection, these examples being non-limiting.

Formulations suitable for administration via the respiratory tract include a powder or a solution in aerosol form for inhalation via the nose or mouth, or a snuff for administration to the upper respiratory tract. Such a formulation may be bound covalently or non-covalently to a semi-persistent particulate carrier, or may be adsorbed onto a suitable inert support (for example nitrocellulose) . Particle size may be varied to target appropriate parts of the airways .

Suitable formulations for administration via the gastrointestinal tract include capsules designed to release in the appropriate part of the gut .

A pharmaceutical composition suitable for oral administration may be in. tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

Administration by injection may be cutaneous, subcutaneous, intra-muscular, intra-dermal or intra-tracheal . For injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Suitable diluents, which are pharmaceutically acceptable and may be preferred, have been discussed already above.

It is to be understood that in the context of the present invention, 'treatment' refers to therapy which is designed to ameliorate and/or alleviate the symptoms of a disease or condition, for example by modulation of the immune system, as well as to therapy designed to cure such diseases or conditions .

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figure described below.

Fig 1 shows the expression of the immediate-early gene, c-fos, in neurons of the Locus coeruleus (LC) and in the medial, dorsomedial and ventrolateral subnuclei of the Nucleus of the Tractus Solitarius {NTSm, NTSdm and NTSvl) , as indicated by an increase in c~Fos-immuno-reactive cell nuclei, in response to Thl dominated immune responses generated by M. vaccae (V) and keyhole limpet haemocyanin plus IL12 (K) , relative to a Th2 dominated immune response generated by ovalbumin (0) . Expression of c-fos is an indication of cell activation.

Experimental Immunisation

Adult male Balb/c mice (6-8wk, 21-25g) were group housed at 22°C at a 12 hour light/dark cycle. Groups of 6 to 8 mice were immunised on day 0 and day 14 with :

Group 1) saline (control mice)

Group 2) 10⁸ autoclaved M. vaccae suspended in saline s.c.

Group 3) 50:g Keyhole limpet haemocyanin (KLH) +1 :g IL-12 i.p,

Group 4) ovalbumin (50:g) on alum i.p.

Intra-tracheal Challenge with Particulate Antigen On day 28, mice were anaesthetised with a combination of Hypnorm (Janssen) and Hypnovel (Roche) and following exposure of the ventral surface of the trachea, intratracheal (i.t.) injections (50 :1) were made using a sterile 23 g needle.

Groups 1) and 2) received an intra-tracheal injection of nitrocellulose particles onto which soluble antigen (5:g) from M. vaccae had been adsorbed as described (Abou Zeid et al . 1987 J. Immunol. Methods 98:5-10). Groups 3) and 4) received similar intratracheal injections of nitrocellulose particles bearing KLH (5:g) or ovalbumin (5:g) respectively. Following injection, anaesthetised mice were maintained in a vertical position for 2-3 min to facilitate flow of injectate into the upper airways.

Perfusion and Fixation

Twelve hours later mice were killed rapidly by overdose of halothane; blood samples were collected by cardiac puncture, and brains were fixed for immunohistochemistry by transcardial perfusion of ice-cold 0.05 M phosphate buffered saline followed by ice-cold 4% para formaldehyde. Skin was then removed, and crania opened and post-fixed in the same fixative overnight, rinsed in 0.1 M sodium phosphate buffer, then transferred to 0.1 M sodium phosphate buffer containing 30% sucrose and 0.1% sodium azide for storage.

Immunohistochemistry Double-immunostaining of tissues (for c-Fos and tyrosine hydroxylase or tryptophan hydroxylase) was performed as follows: six alternate sets of 30 mm cryostat sections were prepared and placed in cryoprotectant in 24-well polystyrene tissue culture plates. One set of sections was used for double-immunostaining for c-Fos and tryptophan hydroxylase, another was used for double-immunostaining for c-Fos and tyrosine hydroxylase. Labeling for c-Fos was performed as follows; sections were washed in 0.05 M phosphate buffered saline (PBS) then incubated with freshly prepared 1% H₂0₂ in PBS for 10 min, rinsed with PBS, then PBS containing 0.3 %^' Triton X-100 (PBST) , followed by incubation with anti-c-Fos rabbit polyclonal antibody (ab-5; Oncogene Sciences, Calbiochem, Nottingham, U.K.) diluted 1 :8,000 in PBST containing 0.01% NaN₃ for 16 h. Sections were washed using PBST then incubated with biotinylated swine anti-rabbit antibody (1 :200; DAKO, Cat. No. E0353, Cambridgeshire, U.K.) for 80 min. Sections were washed again using PBST then incubated with ABC reagent (1:200; Vector Laboratories, Burlingame, CA, PK-6101) for 80 min. After washing with PBST, then PBS, sections were incubated with substrate {Vector SG peroxidase substrate, SK- 4700) for 10 mins.

Prior to immuno-histochemical labeling of the same sections for tyrosine hydroxylase or tryptophan hydroxylase, sections were rinsed thoroughly in PBS. Sections were incubated with 1) anti-tyrosine-hydroxylase rabbit polyclonal antibody (1 :8,000; Chemicon, Cat. No. AB152, Harrow, U.K.) or 2) anti- tryptophan hydroxylase sheep polyclonal antibody (1:12,000; Biogenesis, Ltd. , 9260-2505 , Poole,U .K.) in PBST with 0.01% NaN₃ for 16 h. Sections were washed using PBST then incubated with 1) biotinylated swine anti-rabbit antibody (1 :200) , or 2) biotinylated rabbit anti-sheep antibody (1 :200; Vector Laboratories, PK-61 06) for 80 min. Sections were washed again using PBST then incubated with ABC reagent (1 :200; Vector Laboratories, Burlingame, CA, PK-6101 or PK-6106) for 2 h. After washing with PBST and PBS, sections were incubated with substrate (0.05% 3 , 3 ' -diaminobenzidine tetrahydrochloride, Sigma, D-5637) , 0.012% H₂0₂ in PBS for 8 mins . Sections were transferred to gelatin-coated glass slides and mounted with cover-slips using DPX mounting medium (BDH Laboratory Supplies, Poole, England) .

Statistical analysis Comparisons of independent observations were made using ANOVA or Student's t-tests when appropriate, SYSTAT Version 5.0 (SYSTAT for Windows : Statistics, Ver. 5 (1992); SYSTAT Inc., Evanston, IL)

Treatment with M. vaccae induces a predominantly Thl response which resulted in secretion of Interferon gamma (IFNγ), but not interleukin 4 (IL-4) . A potent Thl dominated response to a protein that is not found within mycobacteria (Keyhole limpet haemocyanin (KLH) ) can be evoked by treating with KLH mixed with the Thl-inducing cytokine, IL-12. This is based on the method of Wynn et al . (1995) Nature 376: 594-596, in which a Thl response was evoked with the Th2 antigen Schistosome ova by mixing with the Thl adjuvant IL-12.

KLH and IL-12 were therefore used as inducers of a Thl dominated response to determine whether the activation of the important brain areas was due to signals from the lung that depended upon the presence of mycobacterial components, or whether it was attributable to the predominantly Thl pattern of the immune response, and independent of the presence of mycobacterial components.

Signalling via Vagal Afferents and not via the Blood-Brain Barrier

Antigens were bound to nitrocellulose to ensure that they stayed localised within the lungs. If cytokines are released systemically, there can be direct activation of the brain by circulating cytokines acting at sites where there is no blood- brain barrier.

No increase in c-fos was seen in the area postrema,. indicating that brain activation had not occurred by means of cytokines acting at sites devoid of blood-brain barrier.

However, there was strong activation of the ventrolateral nucleus of the NTS (Fig 1) , which is a principal site of vagal afferent input from the lungs (Kalia and Mesulam, (1980) Comp. Neurol . 193: 467-508) . Thus signalling to the brain from the inflamed lungs had passed via vagal sensory afferents, whether induced by M. vaccae or by KLH.

Differences between Thl and Th2-mediated inflammation The two Thl-inducing inflammatory stimuli (M^~. vaccae into the lungs of M. vaccae - treated mice, and KLH into KLH+IL-12- treated mice) gave a similar pattern of activation, which was quite different from that seen in the mice with Th2-dominated inflammation (ovalbumin into the lungs of ovalbumin- immunized mice) (Fig 1) .

The activation of neurones in the brain was similar whether a Thl dominated immune response in the lungs is evoked with M. vaccae antigen injected into the lungs of M. vaccae-pre- treated mice (Figure 1) , or with Keyhole Limpet Haemocyanin (KLH) injected into the lungs of mice pre-treated with KLH+IL- 12. Both of these antigens evoked a Thl-dominated response.

In contrast, ovalbumin injected into the lungs of mice which had been pre-treated with ovalbumin on alum to evoke a strong Th2 dominated pattern of response caused less activation of c- fos in the Locus coeruleus (LC) and in the medial and dorsomedial subnuclei of the Nucleus of the Tractus Solitarius (NTSm and NTSdm) but more activation of the ventrolateral subnucleus (NTSvl) .

Clinical Effects of Thl dominated Immune Responses Phase III clinical trials demonstrate that the administration of mycobacterial material has a significant effect on emesis (i.e. nausea and vomiting) in non-small cell lung cancer (NSCLC) patients.

Heat killed M. vaccae formulation SRL172 was administered to these SCLC patients by intra-dermal injection.

Patients and Methods

SR-ON-12 was a stratified, randomised, comparative, open- label, multi-centre Phase III clinical study undertaken in the UK, Poland, Germany and Austria examining the effect in non- small cell lung cancer (NSCLC) patients of chemotherapy alone versus chemotherapy plus intra-dermal injection of the heat- killed M. vaccae formulation SRL 172.

Following a screening period of up to 28 days, patients who fulfilled the entry criteria and had given written informed consent were stratified by extent of disease as either surgically unresectable stage Ilia, or stage Illb/lV (palliative treatment) . Patients were then randomised to receive either chemotherapy alone (up to six cycles of mitomycin-C, vinblastine, and cisplatin or carboplatin on Day 0 and Weeks 3, 6, 9, 12 and 15), or chemotherapy plus SRL172 (intradermal injection on Day 0 and Weeks 4, 8, 12 and 16 during the treatment phase) .

After the initial treatment phase, patients randomised to receive SRL172 entered a maintenance and follow-up phase, during which they received SRL172 and were clinically assessed every 8 weeks until there was clear evidence of disease progression. Patients randomised to receive chemotherapy alone entered a follow-up phase during which they were clinically assessed every 8 weeks until there was clear evidence of disease progression. After disease progression, patients entered the survival phase of the study during which they were clinically assessed every 12 weeks. Patients randomised to receive SRL172 could continue with SRL172 every 12 weeks during this survival phase.

Data collected during the study were used to evaluate patient quality of life (QOL) , in accordance with the European

Organisation for Research and Treatment of Cancer (EORTC) published, validated, methods QLQ-C30 and QLQ-LC13. QOL was evaluated at baseline and at 6 and 12 weeks on treatment (TX) , every 8 weeks on maintenance treatment (MNT) , and every 12 weeks thereafter. The primary analysis assessed treatment group difference on the global health status scale during the treatment plus maintenance phase using generalized estimating equation analysis of covariance on the intent-to-treat population. This analytical procedure is well known to those skilled in the art of statistical analysis. The remaining scales and single items were secondary outcomes and were analysed similarly with p-values adjusted using the Hochberg- Benjamini procedure for multiple comparisons. Details of this procedure are given in Hochberg Y, Benjamin! Y "More powerful procedures for multiple significance testing" Stat Med 1990 Jul; 9(7):.811-8

SRL172 was formulated as a suspension of lOmg/ml heat killed M. vaccae in borate buffered saline (pH 8) and provided in 3 ml glass vials at a concentration of 10⁹ bacilli per 0.1 ml dose and stored refrigerated at 4°C.

Results The results of treatment with the mycobacterial preparation

SRL172 in combination with conventional chemotherapy, compared to conventional chemotherapy alone, for a range of parameters associated with quality of life (QOL) , including nausea and vomiting (table 4), are shown in Tables 2-4. The data shown was analysed using the Last Observation Carried Forward approach and the secondary parameters were adjusted using the Hochberg-Benjamini procedure. Global Health status was not adjusted in this way, as this was the primary QOL (Quality of Life) variable. There are numerous references to the LOCF approach in Biometrika Volumes 83 - 88.

The QOL study population consisted of 177 chemotherapy alone and 183 chemotherapy plus SRL172 patients.

Global health status for SRL 172 treated patients was significantly greater than those receiving chemotherapy alone (p=0.038 Treatment phase, p=0.049 Treatment and Maintenance phases) . Significant improvements were demonstrated for nausea and vomiting (p=0.034 TX) in SRL 172 treated patients as well as bodily pain (p=0.08 TX, p=0.016 TX+MNT) , cognitive functioning (p=0.024 TX) , role limitations due to emotional health (p=0.012 TX, p=0.040 TX+MNT) and physical functioning (p=0.034 TX) . Patient demographics (i.e. age, sex, race, duration of diagnosis, histological type, stage of disease, current smoking status, and weight loss over the last three months) were well balanced between treatments at baseline. Of these demographic characteristics, age, sex, stage of disease, current smoking status and weight loss were included in . multivariate models assessing treatment difference, since each of these demographic characteristics was shown statistically to be related to the baseline quality of life scores, in general .

In Tables 1-3 for global health status and functioning parameters (parameters 2 to 7) , a decrease from baseline indicates worsening. For the symptom parameters (parameters 8 to 10), a decrease from baseline indicates improvement. GEE p value in the Tables adjusts for baseline value and baseline age, sex, stage of disease, current smoking status and weight loss over the three months prior to the baseline. For all parameters except global health status, the p-values in Tables 1-3 are the Hochberg-Benjamini p-values.

Treatment groups were homogeneous with respect to baseline quality of life (all p-values 0.100 or greater) .

Although subjects deteriorated in general, those subjects treated with activators of Thl dominated immune responses deteriorated less than those on chemotherapy alone (Tables 1- 3) .

Mean changes from baseline to the end of treatment phase for subjects treated with Thl response activator were statistically better on general health status (the primary parameter) , nausea and vomiting and several other secondary parameters (physical functioning, role limitation due to emotional health, cognitive functioning, and bodily pain) . Evaluating trends in QOL using generalized estimating equations yielded similar positive results.

At the end of the treatment + maintenance phase, there were no differences in mean changes from baseline between the treatment groups. However, evaluating trends in QOL using generalized estimating equations yielded a significant (beneficial) treatment difference for general health status (the primary parameter) role limitations due to emotional health and bodily pain. The subjects entering the maintenance phase were better off and therefore differences at the end of maintenance may be equivocal. Subjects who failed treatment would proceed to survival phase and therefore would not be captured in the treatment plus maintenance phase . These procedures are well known to statisticians.

The experimental data provided herein demonstrate that mycobacterial material and other agents which induce immune responses which have a major Thl component, are capable of activating specific regions of the brain in a highly selective manner by triggering previously unknown neural pathways . Regions activated in this way include areas known to be involved in emesis. The therapeutic benefits of such activation are also demonstrated in the present application.

Mycobacterial material and other Thl activators and methods of selective brain neurone activation by stimulation of a Thl immune response therefore find application in the treatment of emesis and disorders.

TABLE 1

TABLE 2

TABLE 3

Claims

CLAIMS :

1. Use of mycobacterial material in the manufacture of a medicament for use in the treatment of emesis.

2. Use according to claim 1 wherein the medicament stimulates a peripheral sensory afferent nerve to selectively activate a defined area of the brain and spinal cord of an individual .

3. Use according to claim 1 or claim 2 wherein the mycobacterial material comprises whole, killed mycobacterial cells.

4. Use according to claim 3 wherein the mycobacterial cells are ultrasonically disrupted.

5. Use according to any one of claims 1 to 4 wherein the mycobacterial material is M. vaccae material.

6. A method of treating emesis in an individual comprising administering mycobacterial material to said individual .

7. A method according to claim 6 wherein the mycobacterial material stimulates a peripheral sensory afferent nerve to selectively activate a defined area of the brain and spinal cord of an individual .

8. A method according to claim 6 or claim 7 wherein the mycobacterial material comprises whole, killed mycobacterial cells.

9. A method according to claim 8 wherein the mycobacterial cells are ultrasonically disrupted.

10. A method according to any one of claims 6 to 9 wherein the mycobacterial material is M. vaccae material.