WO2011033310A1

WO2011033310A1 - Encapsulated intestinal flora extracted from feces for use in the treatment of gastrointestinal disorders

Info

Publication number: WO2011033310A1
Application number: PCT/GB2010/051561
Authority: WO
Inventors: William John Martin
Original assignee: William John Martin
Priority date: 2009-09-17
Filing date: 2010-09-17
Publication date: 2011-03-24
Also published as: GB0916335D0

Abstract

The present invention relates to medicaments for the prevention and/or treatment of nosocomial infections, and methods of manufacturing such medicaments. The invention also relates to methods of preventing and/or treating iatrogenic diseases. The medicaments and methods are particularly suited to the treatment of nosocomial infections causing diarrhoea and to the rehabilitation of iatrogenic degraded intestinal flora. The invention further relates to medicaments comprising an extract representative of the flora of the digestive tract.

Description

ENCAPSULATED INTESTINAL FLORA EXTRACTED FROM FECES FOR USE IN THE TREATMENT OF GASTROINTESTINAL DISORDERS

The present invention relates to medicaments for the prevention and/or treatment of nosocomial infections, and methods of manufacturing such medicaments. The invention also relates to methods of preventing and/or treating nosocomial infections. The medicaments and methods are particularly suited to the treatment of nosocomial infections causing diarrhoea and to the rehabilitation of iatrogenic degraded intestinal flora.

Iatrogenic diseases and nosocomial infections constitute a significant cause of distress and morbidity amongst patients in hospitals or other care facilities.

Clostridium Difficile (C. Difficile) is one of the major infectious organisms associated with the development of nosocomial infections. In recent years, the incidences of such infection have risen by around 850% in England and Wales, and C. Difficile infections are calculated to have cost 26,208 lives in these territories in the period from 1997 to 2007. In a US study the rate of C. difficile acquisition was estimated to be 13% in patients with hospital stays of up to 2 weeks and 50% in those with hospital stays longer than 4 weeks. Patients who share a room with a C. difficile-positive patient acquire the organism after an estimated hospital stay of 3.2 days, as compared with a hospital stay of 18.9 days for other patients. [Reed and Kemmerly (2009) The Ochsner Journal 9:27-31].

Normally the proliferation of C. Difficile within the digestive tract is prevented by the presence of other anaerobic bacteria that compete for the same environmental niche. However, in cases where the natural composition of the digestive tract flora is disrupted, for instance by antibiotic treatment, chemotherapy or radiotherapy, then the C. Difficile may be able to proliferate without competition. In such cases infection by C. Difficile may give rise to diseases including colitis, toxic megacolon and diarrhoea. Diarrhoea of this sort can be very severe, and can pose a threat to the life of those suffering from this condition.

One method that has been reported to be highly effective in the treatment of nosocomial infections, such C. Difficile infections causing diarrhoea, is faecal transplant therapy. In this treatment (which was first reported in 1958) faecal material from a donor is administered to the digestive tract of a patient suffering from a nosocomial infection. The faecal sample includes constituents (such as bacterial constituents) representative of the digestive tract flora in the donor. These constituents are able to replicate within the digestive tract of the recipient, and thus restore the normal flora and eliminate the infection. Treatments have been reported in which faecal samples have been administered by means of enema, colonoscope, catheter or nasoenteral tube (suitable delivery means being selected depending on the site to which the sample is to be administered). Such treatments have been reported to have a success rate of between 90 and 95%.

Although such treatments have proven to be effective, they are subject to a number of shortcomings. These include risk of transmission of infectious agents from one patient to another. A further disadvantage of those techniques described in the prior art that make use of enteral or nasoenteral tubes for administration is the risk associated with incorrect placement of such tubes. If the tube is incorrectly inserted into the respiratory system, rather than digestive tract, there is a risk that materials may be introduced into the lungs, where they will interfere with breathing and can cause infection. In order to avoid these risks intubation of this sort is normally only undertaken under the guidance of skilled medical personnel, usually employing radioimaging. Finally, even though the procedures may provide therapeutic benefits, they are generally considered distasteful by laboratory technicians, medical practitioners and prospective patients, thus meaning that they will often only be considered as a last resort. Despite these difficulties the techniques have not been substantially altered in the more than 50 years that they have been known.

It is an aim of the present invention to obviate or mitigate at least some of the disadvantages associated with the prior art.

In a first aspect of the invention there is provided the use of an extract representative of the flora of the digestive tract in the manufacture of an encapsulated medicament for the prevention and/or treatment of iatrogenic disease of the digestive tract. This first aspect of the invention also provides an extract representative of the flora of the digestive tract for use as an encapsulated medicament for the prevention and/or treatment of iatrogenic disease of the digestive tract.

In a second aspect there is provided the use of an extract representative of the flora of the digestive tract in the manufacture of an encapsulated medicament for the prevention and/or treatment of nosocomial infections of the digestive tract. This second aspect of the invention also provides an extract representative of the flora of the digestive tract for use as an encapsulated medicament for the prevention and/or treatment of nosocomial infections of the digestive tract. The medicaments manufactured in accordance with these aspects of the invention, or using extracts in the manner described in these aspects, may be referred to as medicaments of the invention.

In a third aspect of the invention there is provided a method of preventing and/or treating iatrogenic diseases of the digestive tract, the method comprising administering to a patient in need of such prevention and/or treatment an encapsulated extract representative of the flora of the digestive tract. In a fourth aspect of the invention there is provided a method of preventing and/or treating nosocomial infections of the digestive tract, the method comprising administering to a patient in need of such prevention and/or treatment an encapsulated extract representative of the flora of the digestive tract.

Methods of treatment in accordance with these aspects of the invention may be referred to as methods of the invention. It will be appreciated that medicaments of the invention (including those in accordance with the various embodiments described below) represent preferred means for practicing the methods of the invention.

Iatrogenic diseases are diseases occurring as a result of medical treatment. Such treatments may include the use of medicinal agents, or surgical techniques. Examples of iatrogenic diseases that may be prevented or treated using the medicaments or methods of the invention are considered in more detail below.

Nosocomial infections are infections acquired in a hospital or other healthcare environment, and are secondary to a patient's original condition. An infection may be classified as nosocomial if it first appears 48 hours or more after admission to the hospital or healthcare environment, or within 30 days after discharge of the patient. Examples of nosocomial infections that may be prevented or treated using the medicaments or methods of the invention are considered in more detail below.

The present invention is based upon the inventor's finding that extracts that are representative of the flora of the digestive tract, and that contain constituents-able to reproduce within the digestive tract and establish a beneficial flora, can be encapsulated to produce medicaments that are able to prevent or treat both iatrogenic diseases and nosocomial infections. Preferably the medicaments of the invention may take the form of prepared medicaments in which a suitable extract is encapsulated. Such encapsulated medicaments may be provided in dosage form. It will be appreciated that one or more of such dosage forms may be used to provide a therapeutically effective amount of the extract (as considered elsewhere in the specification) to a patient. Typically a dosage unit may provide approximately 2-4 grams of dried extract, and preferably approximately 2.5 grams of such an extract. The extract may be prepared from an amount of around 20-200 grams of an initial sample representative of the flora of the digestive tract (such as a stool sample), and more preferably prepared from an amount of around 30-150 grams of such a sample.

The word "encapsulated" or term "encapsulated medicament" as used in the context of the present invention should be taken to mean that the extract is enclosed within a barrier that may subsequently be degraded to allow release of the extract. For the purposes of the present disclosure encapsulated medicaments in accordance with the invention may be taken as encompassing any medicament in which the extract is provided within a degradable barrier, including (but not limited to) medicaments in which the extract is contained within a gelatine capsule, or medicaments in which the extract is provided within a coated table, or medicaments in which the extract is microencapsulated. Examples of encapsulation include single-piece gel encapsulation and two piece gel encapsulation.

In preferred embodiments in which the medicaments of the invention are for oral administration to a patient, they may preferably be formulated in a manner in which the extract is protected from the conditions found in the upper reaches of the digestive tract, for example the stomach. In these areas of the digestive tract high acidity and the presence of digestive enzymes may otherwise damage the bacteria present in the extract. It is important that these bacteria should be maintained in a viable form, in order that they can exert their beneficial effect. Accordingly, it may be preferred that medicaments of the invention are formulated comprising an enteric coating. Such a coating does not significantly degrade in the upper reaches of the digestive tract and allows the bacteria within the extract to be released in a patient's intestines, and the bacteria are thus able to colonise this compartment of the digestive tract. The enteric coating may be any such coating known to those of ordinary skill in the art. Non-limiting examples of such coatings include Eudragit SIOO (Evonik Rohm GmbH, Darmstadt, Germany), cellulose acetate phthalate (CAP), a methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, polyvinyl acetate phthalate (PVAP), or a methyl methacrylate-methacrylic acid copolymer. In some particular embodiments, the coating includes Eudragit SIOO. The coating may include a mixture of one or more of Eudragit SIOO, cellulose acetate phthalate (CAP), a methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, polyvinyl acetate phthalate (PVAP), and a methyl methacrylate-methacrylic acid copolymer. Further examples of enteric coating include: cellulose acetate phthalate (CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers and sodium alginate and stearic acid.

Thus, a suitable barrier for use to encapsulate extracts, and thus to manufacture the medicaments of the invention, may be one that degrades at a predetermined location in the digestive tract. Merely by way of example, the medicaments of the invention may be encapsulated using a barrier that will degrade, and thus release the extract, after approximately four hours in the digestive tract (enteric coatings do not dissolve in acid conditions, such as the stomach, but dissolve in the more alkaline conditions of the small and large intestine).. It may be expected that an item introduced into the digestive tract will have been carried a distance of approximately two metres within the digestive tract, and so alternatively (or additionally) the medicaments of the invention may be encapsulated using a barrier that will degrade, and thus release the extract, after passing approximately two metres through the digestive tract

One way in which the site at which an encapsulated medicament releases the extract may be determined is by the incorporation or attachment of RFID (radio frequency identification) tags in or to the barrier material. A first signal generated when the tags are close to each other (and so associated with an intact encapsulated medicament) may be distinguished from a second signal generated when the tags are relatively further from one another (indicating that the encapsulated medicament has degraded, allowing the tags to drift apart). Thus the time and location of the degradation of the capsule may be determined by the generation of the second signal. The use of RFID tags of this sort in the manufacture of the medicament of the invention can also provide other advantages, as considered further below.

For the purposes of the present invention, an "extract" should be taken as excluding a sample taken from the digestive tract in its natural form. An extract may be produced by processing such samples, for example to dry and/or enrich the bacteria present in the sample. Examples of suitable processing methods by which extracts may be derived from samples, such as faecal samples, are described elsewhere in the specification.

Extracts suitable for use in accordance with the invention will comprise bacteria that are representative of the flora of the digestive tract. Such extracts may optionally further comprise probiotic substances from the digestive tract, such as lactobacilli and bifidobacteria. Extracts representative of the flora of the digestive tract suitable for use in the medicaments or methods of the invention will be capable, on administration to a patient, of generating or maintaining, within the patient, a digestive tract flora that is not associated with iatrogenic disease, nosocomial infection, or diarrhoea. Such extracts may preferably contain elements which are able to counter C. difficile infection, for example, such extracts may optionally further comprise narrow spectrum antimicrobials which target unwanted bacteria such as C. difficile without markedly effecting advantageous gut bacteria, Examples of such narrow spectrum antimicrobials include thuricin CD. For more information about thuricin CD the reader is referred to International patent application, WO/2009/068656. The medicaments or methods of the invention will utilise a therapeutically effective amount of such an extract. A therapeutically effective amount of an extract will be an amount sufficient to allow the generation or maintenance of a digestive tract flora that is not associated with nosocomial infection or diarrhoea. Such a suitable therapeutically effective amount may contain a therapeutically effective quantity of elements able to control or counter C. difficile infection. This beneficial digestive tract flora may be established through colonisation of the digestive tract, and in particular the intestines and colon, by bacteria present in the extract. A suitable extract may preferably comprise such bacteria in an amount sufficient to "saturate" the intestines of a patient to whom it is administered (i.e. to provide sufficient of the beneficial bacteria present in the extract that pathogenic bacteria are not able to establish or maintain themselves in sufficient quantities to cause nosocomial infection or diarrhoea).

The medicaments or methods of the invention may be used in the prevention and/or treatment of iatrogenic diseases caused by: antibiotic treatment, immunosuppressive therapy, radiotherapy, chemotherapy, or therapy with proton pump inhibitors.

Examples of iatrogenic diseases of the digestive tract include: iatrogenic C. difficile infections; iatrogenic Candida infections; and iatrogenic diarrhoea. These examples represent diseases that may be prevented or treated in preferred embodiments of the medicaments or methods of the invention. It may be particularly preferred that the medicaments or methods of the invention be used in the prevention or treatment of iatrogenic diarrhoea (for example as a result of antibiotic treatment, immunosuppressive therapy, radiotherapy, chemotherapy, or therapy with proton pump inhibitors).

In preferred embodiments the nosocomial infection that is to be prevented and/or treated by the methods of the invention is an infection causing nosocomial diarrhoea. However, the medicaments or methods of the invention may also be used in the prevention and/or treatment of other conditions associated with nosocomial infections such as colitis; toxic megacolon; and thrush. The medicaments or methods of the invention may be used in the prevention or treatment of nosocomial infections of the digestive tract including: Clostridium difficile infections; Candida (such as Candida albicans) infections which may result in "thrush".

Patients undergoing allogeneic bone marrow transplantation (aBMT) for malignancy, primary immunodeficiency, and other genetic disorders may be subject to graft versus host disease (GvHD), which is a significant cause of morbidity and mortality for such patients. It has been found that aBMT patients who undergo sustained suppression of intestinal anaerobic flora experience lower rates of GvHD than patients whose flora are not suppressed. In order to allow the patient's digestive tract flora to be reconstituted in a health manner, they are provided with "human restoration flora" after the treatment causing floral suppression is stopped. It will be appreciated that the medicaments or methods of the invention may be used in this context, and that this is a preferred embodiment of such use for the prevention of iatrogenic disease.

In a highly preferred embodiment of the invention the medicaments are for oral administration. Forms of encapsulated medicaments that are suitable for oral administration will be apparent to those skilled in the art, and these may be utilised in the manufacture of medicaments of the invention. While enteral administration is preferably by means of oral administration, but should also be taken as encompassing other forms of administration such as by suppository or the like. However, if medicaments of the invention are administered by routes other than orally this may limit the extent to which they can access the various compartments of the digestive tract, and thus limit the medicaments' effectiveness. For example, if medicaments of the invention are administered anally (e.g. by means of a suppository) the presence of the splenic flexure, cecocolic junction or ileocecal valve may prevent provision of the encapsulated extract to compartments "upstream" of these structures (such as the small intestine) which may allow nosocomial infection to become established (or remain established) in these compartments.

In a preferred embodiment of the medicaments or methods of the invention, the extract is representative of the flora of the digestive tract of a patient to whom the medicament is to be administered. Such extracts may be derived from samples, such as faecal samples, collected from such a patient. The sample from which the extract is to be derived should preferably be collected prior to the development of any nosocomial infection, so that it is representative of the healthy digestive tract flora of the patient. Such samples may, for example, be collected on entry of an individual into hospital or prior to their arrival in hospital. In the event that samples are collected prior to arrival at hospital then this may provide an opportunity for preparation of the medicaments, and subsequent use of the medicaments for prevention or treatment of a disease, to commence at the earliest possible opportunity.

The use of extracts derived from a patient for subsequent use in the medicaments or methods of the invention to be provided to the same patient (herein referred to, for ease of reference, as autologous samples) provides a number of advantages. One such advantage is that there is no need to screen the sample for the presence of infective agents (such as viruses or other pathogens). If allogeneic samples are to be used such screens may be preferred in order to avoid the risk of transmission of infections from the donor patient or patients (from whom the extract is derived) to the recipient patient (to whom the extract is provided in a medicament or method of the invention). It will be appreciated that such screening may be time consuming and costly, and thus the use of autologous samples provides advantages in terms of increased safety, decreased time of production, and decreased cost. Furthermore, the use of autologous samples to produce extracts for use in the medicaments or methods of the invention may generally reduce the perceived distastefulness of such medicaments or methods to a prospective patient.

It will be appreciated that during the processing of samples to produce extracts, and the encapsulation of such extracts to produce medicaments of the invention, it may generally be desirable to track a sample to ensure that that the medicaments produced are returned to the patient from whom the sample was originally obtained. This may prove complicated, since the various steps involved in manufacture of the medicaments may be undertaken at different sites. In order to facilitate such tracking it may be preferred that suitable "tags" be used to identify a sample, extract and medicine, and allow these to be associated with one another, and with the patient to be treated. In a preferred embodiment this may be achieved by the use of RFID tags. The RFID tags used may be the same tags used to report on the site at which an encapsulated medicament releases its contents (as described above). The skilled man would be familiar with other tags suitable for tracking medicaments of the invention, for example bar codes.

Notwithstanding the preceding paragraphs, there may be circumstances in which it will be preferred to use extracts derived from a patient (or patient) other than those to whom the extract will be provided in a medicament or method of the invention

Examples of such circumstances include situations in which it is desired to produce large amounts of medicaments of the invention, for example in order to allow treatment of an infection afflicting many individuals. In such circumstances the need to produce large amounts of the medicaments in a relatively short time may override the advantages associated with the use of autologous samples. The donors from whom such allogeneic samples and extracts may be derived should be thoroughly screened in order to avoid the risk of transmission of infectious agents to patients receiving treatment with the medicaments or methods of the invention. The digestive system flora present in such samples may then be expanded in order to allow the production of sufficient extract to provide medicaments for multiple patients.

Suitable conditions for expansion of the flora in accordance with either autologous or allogeneic embodiments of the invention are described elsewhere in the specification.

Generally, the medicaments or methods of the invention will make use of extracts that are fully or partially dried. The use of dried extracts provides a number of advantages, including reducing the volume of the extract that is to be administered, and improved acceptability to patients. In addition, dried extracts will not dissolve the gels which may be employed for encapsulation. In a preferred embodiment the extract to be used in the medicaments or methods of the invention is a freeze dried extract.

The invention further comprises dosage forms comprising an extract of the invention. In one embodiment the extract is provided in a substantially dry form, for example a freeze- dried form. In a further embodiment the extract is provided in an aqueous solution, for example water, saline or a buffer solution. In a further embodiment the dosage form is encapsulated, for example encapsulated with an enteric coating.

In a further embodiment of the invention there is provided a pharmaceutical composition comprising an extract of the invention.

In a preferred embodiment, the medicaments or methods of the invention may be used for the prevention of nosocomial infections. In a particularly preferred embodiment they may be used for the prevention of nosocomial diarrhoea. In such an embodiment the medicaments or methods of the invention may be employed prophylactically to prevent a patient developing nosocomial diarrhoea. When used in accordance with this embodiment, the medicaments or methods of the invention will allow the maintenance or re-establishment in the patient's digestive tract of a flora that does not cause nosocomial diarrhoea. In the case of the maintenance of such a flora, there will be no period in which the flora of the patient's digestive tract is altered in a manner that may give rise to nosocomial diarrhoea. In the case of the re-establishment of such a flora, there may be a period in which a flora that would give rise to nosocomial diarrhoea exists, but the delivery of the extract will cause this flora be replaced before any symptoms are observed. If a narrow spectrum antibiotic such as thuricin CD is employed to treat C. difficile infection, the patient's gastrointestinal flora will not be re-established, leaving the way open for re-infection. The medicament can be employed to restore a patient's flora following treatment of C. difficile with a narrow spectrum antibiotic, thus providing the patient with protection against further nosocomial infection.

In another preferred embodiment the medicaments or methods of the invention may be used for the treatment of nosocomial infections. In a particularly preferred embodiment these may be infections associated with nosocomial diarrhoea. In such embodiments the medicaments will be administered to a patient whose digestive tract flora has been altered in a manner that causes nosocomial diarrhoea, but the delivery of the extract will cause a beneficial digestive tract flora that is not associated with nosocomial diarrhoea to be established. The establishment of this beneficial digestive tract flora will thus allow the digestive tract to function such that the nosocomial diarrhoea ends.

The medicaments or methods of the invention may be of use in the prevention and/or treatment of nosocomial diarrhoea arising as a result of any of a number of different causes. It may be preferred that the nosocomial diarrhoea to be prevented and/or treated is associated with antibiotic treatment, but the medicaments or methods of the invention are also applicable to the prevention or treatment of nosocomial diarrhoea caused by chemotherapy or radiotherapy.

The medicaments or methods of the invention are preferably of use in the prevention and/or treatment of nosocomial diarrhoea associated with C. Difficile infection. C. Difficile is the species associated with the most common and most severe forms of nosocomial diarrhoea.

The medicaments or methods of the invention will preferably use a sample representative of the flora of a healthy digestive tract. For the purposes of the present invention a "healthy" digestive tract is one that is not subject to nosocomial diarrhoea, without reference to other health considerations. A healthy digestive tract may be one where C. difficile is present, but is kept in check by other constituents of the digestive tract flora. Although C difficile carriage is uncommon in healthy adults (1 %-3%) it is more common in debilitated patients and antibiotic-treated hospitalised adults (15%-25%), including those who receive antibiotics before surgery. Thus, in the case of an autologous extract derived from a patient, the patient's digestive tract may be considered "healthy" as long as the patient is not suffering from nosocomial diarrhoea or another nosocomial or iatrogenic disease of the digestive tract, even if the patient has other health concerns (such as those causing the patient to be admitted to hospital). Generally, an extract representative of the flora of a patient's healthy digestive tract may be obtained from a sample collected prior to the initiation of any treatment (e.g. antibiotic treatment, radiotherapy or chemotherapy) that may cause nosocomial diarrhoea, or otherwise degrade the gastrointestinal flora and provide conditions in which C. difficile may flourish.

A number of factors that appear to predispose individuals to nosocomial infections are known. Thus the medicaments or methods of the invention may be of particular use in the prevention and/or treatment of nosocomial infections that may otherwise be expected to occur in such individuals. Merely by way of example, the medicaments or methods of the invention may be particularly suitable for use to prevent and/or treat nosocomial infections of individuals selected from the group consisting of: those undergoing (or about to undergo) antibiotic treatment; those undergoing (or about to undergo) radiotherapy (and in particular radiation of the pelvis); those undergoing (or about to undergo) chemotherapy; those undergoing (or about to undergo) immunosuppressive therapy; those undergoing (or about to undergo) treatment with proton pump inhibitors; those that have been subject to recurrent nosocomial infections; patients suffering from renal dysfunction; and the elderly.

Patients entering hospital and who require immediate treatment may provide a faecal smear which can be cultured in the laboratory to provide an enhanced sample for freeze drying that is capable of restoring their intestinal flora after their treatment ceases, in order to prevent the onset of nosocomial infection.

Although it will generally be preferred that the medicaments or methods of the invention be used in the prevention and/or treatment of nosocomial infections of human patients they may also be suitable for use in non-human animals, for example domestic animals such as cats and dogs or farm animals such as cows, sheep and pigs. In such cases the teaching of the present specification may generally be followed, though sources of samples and extracts, and the calculation of therapeutically effective amounts, may be altered as required. Preparation of extracts

Extracts suitable for use in the medicaments or methods of the invention may be prepared from stool samples or from rectal swabs.

In the case of extracts prepared from stool samples, the stool may be fully or partially dissolved in a solvent such as sterile water or saline. The process of dissolution may be aided by homogenisation using techniques such as sonication or shaking. The solvent and any homogenisation technique employed should be selected with a view to maintaining viability of the digestive tract flora within the sample (i.e. to prevent the death of bacteria through toxic effects or bacterial cell lysis). Filtration may then be used to remove solid materials from the solution, thus yielding an extract that is representative of the flora of the digestive tract.

In the case of extracts prepared from rectal swabs, flora present on the swab may be expanded by culture in suitable conditions. Suitable media and other culture conditions that may be used in the expansion of digestive tissue flora (typically utilising anaerobic culture conditions) will be well known to those skilled in the art. Different culture conditions may be used to allow the expansion of different components present in an extract for use in the medicaments or methods of the invention. If such an embodiment is to be used then the different cultures produced after such expansion may be combined to provide a therapeutically effective extract.

However the extract is prepared, it may be preferred that it is dried prior to its encapsulation. This decreases the volume of the extract and increases its stability, allowing storage before use. The dried extract may be stored either prior to encapsulation (e.g. in suitable vials, or the like) or after encapsulation or microencapsulation. It is preferred that dried extracts be produced by freeze drying.

In order to promote uniform production of suitable extracts and medicaments it may be preferred that an individual from whom an extract will be derived be provided with a standardised diet prior to sample collection. This may assist in removing variations in the consistency and floral content of samples.

In a fifth aspect of the invention there is provided a method of producing a medicament for the prevention and/or treatment of an iatrogenic disease of the digestive tract, the method comprising: i) treating a faecal sample to produce an extract representative of the digestive flora of the individual from whom the faecal sample was obtained;

ii) drying the extract; and

iii) encapsulating the extract to produce an encapsulated medicament.

In a sixth aspect of the invention there is provided a method of producing a medicament for the prevention and/or treatment of a nosocomial infection of the digestive tract, the method comprising:

i) treating a faecal sample to produce an extract representative of the digestive flora of the individual from whom the faecal sample was obtained;

ii) drying the extract; and

iii) encapsulating the extract to produce an encapsulated medicament.

The faecal sample may preferably comprise a stool sample or an anal swab.

The treatment step may optionally comprise one or more of the following: addition of a diluent (such as sterile water or sterile saline); sonication; shaking, filtration; and expansion of bacterial constituents within the sample.

The drying step may preferably comprise freeze drying of the extract. The invention further comprises a method of drying, for example freeze drying, an extract representative of the digestive flora of an individual.

The encapsulation step may preferably comprise packing of the dried extract into an enteric capsule, or formulation of the dried extract into a coated tablet, and should also be taken to encompass microencapsulation.

The various aspects and embodiments of the invention describe have been considered in the context of medicaments or methods for use with human subjects. However, it will be appreciated that these medicaments or methods may also be applicable to use in non- human animals, and hence may be used in veterinary applications.

For the use of the invention it may be required to implement a system to facilitate the different stages of preparation and use of extracts of the invention. Such a system may comprise a software program which allows recording and tracking of samples and extracts and detection systems, such as the use of RFID tags, to track the samples and extracts. A method of implementing such a system is another embodiment of the invention.

The invention will now be further described with reference to the following non-limiting examples with reference to the accompanying drawings, in which:

Figure 1 a shows a scheme for the collection and storage of stool sample for the preparation of extracts representative of the flora of the digestive tract|.

Figure 1 b shows a scheme for the preparation of extracts representative of the flora of the digestive tract from stored samples.

Figure 2 shows a scheme showing allogenic rehabilitation of iatorgenically degraded gastrointestinal flora..

Example 1a

Preparation of medicaments of the invention and treatment of patients therewith - processing of sample before or at treatment

This process is also compared to the health status of the patient, as the patient passes from a healthy state, through the development of an iatrogenic disease, and to healthy state in which the medicaments of the invention have rehabilitated the flora of the patient's digestive tract.

1 . The patient provides a sample in a secure container with saline solution while their intestinal flora is still healthy.

2. The sample and saline solution are homogenised in the sample container on a mechanical shaker.

3. The homogenised sample is attached to the filter unit. The sample is now ready to be filtered.

4. Depending on the design of the apparatus used a vacuum or pressure may be applied to the apparatus, for example a vacuum port of a filter unit, in order to carry out the filtration process.

5. The filtrate container is removed from the filter unit. The sample container and filter unit are disposed of. 6. A lyophilisation membrane, for example an expanded polytetrafluoroethylene membrane (ePTFE),. is placed on the filtrate container and the filtrate is freeze dried yielding a powdered product.

7. The powdered product is dispensed into individual capsules using a capsule filling attachment.

8. The capsules are capped off and the filtrate container and capsule filling attachment are disposed of. An RFID tag is attached to the capsule or the capsules are placed in a container with an RFID tag and other identifying data attached.

9. The capsules are enteric coated and ready to be used by the patient. The RFID tag can be removed before consumption, or may be used to monitor degeneration of the capsule within the patient.

10. If the patient develops an iatrogenic disease the patient can be treated with the capsules to rehabilitatise the flora of the patient's digestive tract.

1 1. The use of opaque containers makes the procedures in the laboratory much less distasteful to the technicians carrying out the processing of the faecal samples.

Example 1 b

Preparation of medicaments of the invention and treatment of patients therewith - processing of sample after contracting iatrogenic disease

An alternative approach to Example 1 a is to take a sample from a patient, tag the sample and store it and then only process the sample if the patient develops an iatrogenic disease. This procedure could be implemented as follows:

1 . The patient, who wears an RFID wristband, supplies a sample, for example a faecal sample, onto a plastic film in a bedpan [Figure 1 a - (1 )].

2. The sample is sealed and placed in an RFID-tagged container [Figure 1 a - (2)-

(3)].

3. The container is capped and placed at a known location in a refrigerator at about 4°C The location of the samples are stored in an electronic location device on the refrigerator exterior. [Figure 1 a - (4)].

4. The sample is stored until required. For example, it is used if the patient develops an iatrogenic disease, such as a C. difficile infection.

5. When required the sample is located using the location device and identified from the RFID tag using an RFID reader.

6. The sample is removed from the refrigerator and a fixed volume of saline or sterile water added [Figure 1 b - (6)]. The volume of water/saline will depend on the size of the sample.

7. The container is shaken and sonicated to homogenise the mixture [Figure 1 b - (7)]. 8. The cap of the container is removed and replaced with a filtering device and the sample filtered into a second container. Air pressure though an inlet valve can be used to facilitate the filtering if required [Figure 1 b - (8), small arrow indicates inlet valve].

9. The second container is sealed with a porous membrane, such as a Lyoguard™ membrane (W. L. Gore & Associates, Inc, Newark, USA) and the sample freeze dried.

10. The resulting solid is encapsulated and placed in an RFID tagged container [Figure 1 b - (10)].

1 1 . The capsules are now available for treating the patient.

This procedure is shown in Figures 1 a and 1 b.

Example 2

Allogenic rehabilitation of latrogenically degraded intestinal flora

As described in the specification there may be circumstances in which it would be preferred to use extracts derived from a patient (or patients) other than from those patients which the extract was derived. This is shown in Figure 3. The procedure is identical to Example 1 a up to step 4 and Example 1 b upto step 8. following which:

(1 ) The individual components of the intestinal flora are cultured in various media to ensure proliferation of all required bacteria [Figure 2 - (1 )].

(2) The cultured intestinal flora components are mixed together in the correct proportions. The resultant mixture may be centrifuged to precipitate solids [Figure 2 - (2)].

(3) The precipitates are transferred to Lyoguard™ trays (W. L. Gore & Associates, Inc, Newark, USA) for freeze drying in large quantities [Figure 2 - (3)].

(4) The powdered product is encapsulated using one of a range of encapsulation technologies, for example microencapsulation using an electrostatic generator [Figure 2 -

(4) ].

(5) The capsules are enteric coated.

(6) The capsules are now available for treating the patient.

This procedure is shown in Figure 3. In Figure 3 the depiction of people indicates the extract is derived from one patient but suitable for treatment of a number of patients

Example 3

Characterization of bacterial content of stool samples before and after freeze drying

A number of normal stool samples were examined to assess the quantitative effects of freeze drying on bacterial numbers. Normal stool samples were examined before and after the freeze drying process to compare bacterial numbers. A stool sample was received from each of five anonymous volunteers. Age and sex were supplied, and none of the volunteers had received antibiotics in the previous 3 months, or undergone any surgical procedures in the previous 12 months. All samples were examined for bacterial pathogens and processed for bacterial counts.

In these studies all culture media supplied by E and O Laboratories, Bonneybridge, Scotland.

(i) Routine culture for pathogens

All samples were subjected to routine bacteriological examination for pathogens. They were cultured as follows¹:

C. difficile agar

XLD (xylose lactose deoxycholate citrate) agar

DC (deoxycholate citrate) agar

Campylobacter agar (Butzler's modification)

Selenite F broth

Butzler,s Campylobacter medium comprises Columbia/brucella agar base, Horse blood (5%) Antibiotic supplement (cyclohexamide, cephazolin, novobiocin, bacitracin, colistin sulphate).

The plates were labeled and inoculated with the faecal sample using a cotton swab which had been dipped into the sample. XLD, DCA and selenite were incubated overnight in air at 35-37°C. Plates were examined at 24 hours and the selenite broth subcultured to XLD which was incubated for 24 hours and examined as previously.

Campylobacter medium was incubated for 48hours in a jar with microaerophillic atmosphere. C. difficile agar was incubated for 48hrs anaerobically.

No pathogens were isolated from any of the five samples and No C. difficle was isolated from any of the five samples.

(ii) Preparation of faecal suspension

A minimum volume of approximately 35ml was required for the freeze drying process, which was prepared as follows:

1 . 10g stool was weighed into a sterile graduated screw-topped container and made up to a volume of 40ml with sterile 0.9% saline. 2. The sample was mixed to a homogenous suspension and filtered through one layer of muslin in a funnel into a fresh sterile container. The muslin was squeezed to collect as much filtrate as possible. Following this the volume was made up to 40ml with sterile saline².

3. The filtrate was mixed, 2ml removed for the bacterial count and the remaining 38ml retained for the freeze drying process.

(iii) Bacterial count

1 in 100, 1 in 10,000 and 1 in 1 ,000,000 dilutions of the filtrate were prepared and a given volume (50μΙ) of each dilution of each faecal specimen was then sampled on a Don Whitley spiral plater (WASP) and applied to the following culture media¹:

Columbia blood agar

MRS medium (De Man, Rogosa, Sharpe agar, a selective medium for lactic acid bacilli.)

Chocolate agar

Sabourards agar

MacConkey agar

Streptococcal selective agar

Anaerobe selective agar

All plates, except the anaerobic medium, were incubated in air at 35-37C for 48 hours. The anaerobic medium was incubated at 35-37C in an anaerobic atmosphere for 72 hours. After incubation all plates showing bacterial growth were counted using a Don Whitley 'Acolyte' image analyzer. Results are given in Table 1.

(iv) Processing of freeze dried material

The faecal suspensions submitted to Biopharma Technoology Limited (Winchester, UK) to undergo freeze drying. This removed all liquid from the sample leaving a pale brown friable material.

The five faecal samples submitted to Biopharma, labeled 1 to 5, generated respectively, 2, 2, 2, 3 and 2 freeze dried samples. It was assumed that the faecal suspension supplied to Biopharma generated two freeze dried samples; there was no information explaining why sample 4 had generated three containers. In light of this it was decided to perform bacterial counts on samples 1 , 2, 3, and 5, and leave sample 4 for other tests, such as determining how much freeze dried material was required to fill a gelatin capsule. (v) Preparation of faecal suspensions from freeze dried samples

Samples were reconstituted in 40ml of sterile saline

(vi) Bacterial count for faecal suspensions derived from freeze-dried sarnies

Samples were reconstituted in 40ml of sterile saline, dilutions prepared and grown on culture medium as described above. After incubation all plates showing bacterial growth were counted using a Don Whitley 'Acolyte' image analyzer. Results are given in Table 2.

Results:

Table 1 : Bacterial count on faecal samples before freeze drying

Table 2: Bacterial count on freeze dried samples

Adjusted bacterial count relates bacterial numbers to the original volume that was freeze dried, that is 2 X 15ml.( ie total volume freeze dried = 30ml) . As this data was not available at the time of processing the final total volume was reconstituted to 40ml. therefore the adjusted count takes into account this.

Table 3: Bacterial count on pre and post treated samples

Conclusions

Generally the anaerobic count decreased by 1 -2 logs following freeze drying but this was dependant upon the sample. The aerobic count increased slightly (based on counts on (Blood agar and MacConkey medium). This would indicate that the aerobic bacteria survived freeze drying better than the anaerobic bacteria. Therefore, freeze drying does not markedly alter the pattern or numbers of bacterial flora present in a faecal sample.

Claims

An encapsulated dosage form comprising an extract representative of the flora of the digestive tract.

An encapsulated dosage form according to Claim 1 wherein the extract is dried.

An encapsulated dosage form according to Claim 2 wherein the extact is freeze dried.

An encapsulated dosage form according to any one of the preceding claims wherein the encapsulated dosage form is enteric coated.

An encapsulated dosage form according to any one of the preceding claims wherein the dosage form further comprises a tag.

An extract representative of the flora of the digestive tract wherein the extract is freeze dried.

The use of an encapsulated dosage form according to any one of Claims 1 to 5 or an extract according to Claim 6 in the manufacture of a medicament for the prevention and/or treatment of iatrogenic disease of the digestive tract.

The use of an encapsulated dosage form according to any one of Claims 1 to 5 or an extract according to Claim 6 in the manufacture of a medicament for the prevention and/or treatment of nosocomial infections of the digestive tract.

The use according to Claim 7 or Claim 8 wherein the extract is representative of the flora of the digestive tract of a patient to whom the medicament is to be administered.

The use according to claim 9, wherein the extract is derived from a sample of the flora of the digestive tract of the patient to whom the medicament is to be administered.

The use according to any one of claims 7 to 10 wherein the medicament is for prevention and/or treatment of diarrhoea.

12. The use according to any one of claims 7 to 10 wherein the medicament is for prevention and/or treatment of diarrhoea colitis or toxic megacolon.

13. The use according to one of claims 7 to 12 wherein the iatrogenic disease or nosocomial infection is associated with antibiotic treatment, chemotherapy, or radiotherapy.

14. The use according to any of claims 8 to 13, wherein the nosocomial infection is a Clostridium Difficile infection.

15. A method of producing an encapsulated medicament according to any one of Claims 2 to 5, comprising

i) treating a faecal sample, obtained from a donor, to produce an extract representative of the digestive flora of the donor from whom the faecal sample was obtained;

ii) drying the extract; and

iii) encapsulating the extract to produce an encapsulated medicament.