|Title of Invention||
A MEDICAMENT AND A METHOD OF MANUFACTURING THE SAME
|Abstract||A medicament, comprising a therapeutically effective quantity of a glycopeptide antibiotic and antibody specific against the epitope by the peptide having the amino acid sequence of SEQ ID NO:3.|
|Full Text||The present invention is concerned with the treatment of infections of the human or animal body by micro-organisms such as S. aureus, particularly antibiotic resistant strains of the organisms such as MRSA. Provided are medicaments for the treatment of infection by micro-organisms such as S. aureus, methods of manufacture of medicaments for the treatment of infection, pharmaceutical packs containing medicaments for the treatment of infection, and methods of treatment of the human or animal body for infection by said micro-organisms.
Multiple drug resistance (MDR) is an increasing problem amongst gram-positive bacteria (Banergee, S.N. ef a!. 1991, Am. J. Med. 91: 865-895; Shaberg, D.R. e( al., 1991, Am. J. Med. suppl., 88: 72-75; Gaynes, R.P. et al, 1994, Infect. Dis. Clin. Pract., 6: 452-455), particularly in hospitals. In particular, methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CNS), particularly methicillin-resistant CNS, prove problematic, being resistant to all penicillins and cephalosporins. Resistance to other agents such as quinolones is widespread (Malabarta, A. et al., 1997, Eur. J. Med. Chem., 32: 459-478; Lewis, K., 1994, TIBS, 19: 119-123; Traub, W.H. et al, 1996, Chemotherapy, 42: 118-132). Treatment is typically effected using vancomycin or teicoplanin. However, resistance to these agents is spreading and so new therapies are needed. For example, Hubert SK et al., (J Clin Microbiol. 1999 Nov;37(ll):3590-3; PMID: 10523558) discusses S. aureus isolates with reduced susceptibility to vancomycin and teicoplanin. It has also been founithat certain epidemic strains of MRSA, including EMRSA-15 (v.^\-vv.phls.org.uk. Public Health Laboratories, Colmdale, UK), can give rise to sub clones with creased resistance to vancomycin (Bumie J et al, Clui Infect Dis. 2000 Sep;31(3):684-9; PME): 11017816). It was hoped that linezolid would provide a much needed alternative to current therapies, but Tsiaris S. et al. (Lancet. 2001 Jul "
21;358(9277):207-8; PMID: 11476839) have reported a clinical isolate of MRSA resistant to linezolid in a patient being treated with this antibiotic for dialysis associated peritonitis.
In addition administration of large quantities of such antibiotics to patients to effect treatment of infections, especially of infections which display resistance to the antibiotic(s) being administered, can be cytotoxic and nephrotoxic and although helping to save the patient"s life can result in severe side-effects.
The treatment and diagnosis of Staphylococcal infections is discussed widely in the prior art and particularly relevant publications include WO 98/01154, WO 99/50418, and EP 0786519. The causes of antibiotic resistance are also discussed in pubhcations such as Allignet J. (Gene 1997 Nov 20; 202(1-2): 133-8; PMID: 9427556).
Thus there is a clear need for new and enhanced therapies for micro-organism, particularly i^. aureus, infection.
The present inventors have now found that a particular combination of agents, namely an antibody (detailed below) having a novel antigen binding part and a glycopeptide antibiotic (such as vancomycin or teicoplanin), has a very surprising synergistic therapeutic effect -the therapeutic efficacy of the glycopeptide antibiotic is substantially enhanced when compared to its therapeutic efficacy when used alone. This is particularly the case when the micro-organism being treated is resistant to the glycopeptide antibiotic. Importantly, the present invention is able to effect treatment of fully vancomycin resistant and vancomycin intermediately resistant strains of s.. aureus using vancomycin together with the antibody of SEQ ID NO: 2. Nowhere in the prior art is it suggested that this might be achieved. The glycopeptide antibiotics work by affecting the bacterial cell wall. Other antibiotics such as the penicillins also affect the bacterial cell wall. However, the present inventors have found that their efficacy is not improved by using them with the , antibody of SEQ ID NO: 2. in particular, experiments have shown that the efficacy of
flucloxacillin is not improved by using it with the antibody of SEQ ID NO: 2. Bacteria resistant to flucloxacillin did not have their resistance reduced by using the antibody of SEQ ID NO: 2.
According to the present invention there is provided an antibody having the sequence of SEQ ID NO: 2 or an antigen binding fragment thereof. The antibody is encoded by the nucleotide sequence of SEQ ID NO: 1 although of course the nucleotide sequence can be readily modified to e.g. optimise the codons for specific micro-organisms used to synthesize it or for other reasons as desired.
The antibody of SEQ ID NO: 2 (also referred to as "Aurograb" RTM) is a human genetic recombinant antibody. It binds to the immunodominant cell surface antigen, GrfA, a staphylococcal .ABC transporter protein (WO 99/50418; Bumie JP et al. Infect Immun. 2000 Jun;68(6):3200-9; PMID: 10816464). It was originally derived from patients who had recovered from S. aureus septicemias and were producing antibodies to Graf. It has intrinsic anti-staphylococcal activity and shows synergy with vancomycin and other glycopeptide antibiotics, both in vitro and in vivo, against a wide range of s. aureus strains including MRSA and vancomycin-resistant MRSA. The antibacterial activity of the antibody of SEQ ID NO: 2, both alone and in combination with glycopeptide antibiotics, is of significant benefit in the treatment of 5. aureus infections.
Both human plasma-derived immunoglobulin (Ramisse F et al, J Infect Dis. 1993 Oct;168(4):1030-3; PMID: 8376815) and rabbit hyperimmune antiserum raised against GrfA are protective in models of staphylococcal infections (see "Experiments" section below). Human recombinant antibodies specific for GrfA are protective in mouse models of MRSA infection. A particular amino acid sequence of GrfA, the epitome of SEQ ID NO: 3, was found to be frequently targeted by the humoral immune response in patients recovering from S. aureus septicaemia (Bumie JP et al„ 2000 supra).
However, nowhere in the prior art is it suggested that the improved treatment of micro¬organism infections such as S. aureus may be effected using a combination of a glycopeptide antibiotic such as vancomycin, teicoplanin and daptomycin, and an anti-GrfA antibody, particularly not antibody specific to the epitope of SEQ ID NO: 3, and more" particularly not the antibody having the sequence of SEQ ID NO: 2.
Thus according to the present invention there is provided a medicament comprising a , therapeutically effective quantity of a glycopeptide antibiotic and antibody specific against /cl?^^""" GrfA, particularly antibody specific against the epitope of SEQ ID NO: 3. Also provided are methods of manufacture of medicaments, pharmaceutical packs and methods of treatment all comprising or using same. The medicament may be for the treatment of is". aureus infection. The temi "treatment" as used herein means any treatment which is designed to cure, alleviate, remove or lessen the symptoms of, or prevent or reduce the possibility of contracting such an infection, and includes prophylaxis.
The GrfA protein, which is an ABC transporter protein, has been isolated and purified from S. aureus, but homologues which perform the same function in other organisms exist and they may be used as the target for therapy. Thus antibody can be prepared against a homolog of GrfA produced by a micro-organism other than S. aureus and may be used together with a glycopeptide antibiotic to effect treatment of infection by the micro¬organism in a patient. Examples of other micro-organisms in which treatment can be effected are other Staphylococci, particularly coagulase negative Staphylococci, such as S. haemolyticus, S. epidennidis and S. saprophyticus. Infection by Enterococci, particularly E.faecalis and E.faecium, can also be treated, as can infection by Corynebacteria such as C. jeikeium and C. xerosis.
The homolog of GrfA. may have a sequence homology of at least 60%, for example at least 70, 80, 85, 90, 95, %, 91, 98, 99 or 99.5% with GrfA. Sequence homologies are . determined using the NCBI BLASTN (nucleotide sequence comparisons) or BLASTP
(polypeptide comparisons) programs, Version 2.1.2, with default parameters. The NCBI BLAST programs is to be found at www.ncbi.nlm.nih.gov/blast/. The temi sequence identity used herein refers to amino acid residues in optimally aligned sequences which match exactly at corresponding relative positions.
In organisms other than S. aureus, particularly other Staphylococci, the epitope (SEQ ID NO: 3) against which antibodies are raised and which are used as the basis for the treatment may remain the same.
Thus according to the present invention there is provided a medicament for the treatment of infection, particularly S. aureus infection, said medicament comprising a therapeutically effective quantity of a glycopeptide antibiotic and the antibody of SEQ ID NO: 2 or an antigen binding fragment thereof
Also provided is a glycopeptide antibiotic and the antibody of SEQ ID NO: 2 or an antigen binding fragment thereof for use in a method of manufacture of a medicament for the treatment of infection.
In effecting a therapy against infection, particularly S. aureus infection, the glycopeptide antibiotic and antibody or antigen binding fragment may be administered to a patient separately or sequentially. Also provided according to the present invention is a pharmaceutical pack comprising a therapeutically effective quantity of a glycopeptide antibiotic and the antibody of SEQ ID NO: 2 or an antigen binding fragment thereof
Also provided according to the present invention is the use of a glycopeptide antibiotic and the antibody of SEQ ID NO: 2 or an antigen binding fragment thereof for the manufacture of a medicament for the treatment of infection, particularly S. aureus infection.
Also provided according to the present invention is a method of treating infection, particularly S. aureus infection, in a patient, comprising the step of administering to said patient a therapeutically effective quantity of a glycopeptide antibiotic and the antibody of SEQ ID NO: 2 or an antigen binding fragment thereof
Glycopeptide antibiotics of particular usefulness in the present invention are vancomycin, teicoplanin and daptomycin, although of course the present invention extends to other members of the family of glycopeptide antibiotics.
The term "antibody" in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combhiing site or paratope. Such molecules are also referred to as "antigen binding fragments" of immunoglobulin molecules.
Illustrative antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab", F(ab")2, scoff and F(v).
The term "antibody combining site" refers to that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) antigen.
As regards the antibody of SEQ ID NO: 2, it may be readily modified to alter its amino acid sequence whilst presenting the same paratope and retaining its antigen binding specificity. Generally speaking, its structure is arranged (amino to carboxy terminal sequence) as a human immunoglobulin variable heavy domain (VH) and variable light chain (VK) covalently joined together by a linker and having a His carboxy-terminal sequence.
Each of the variable regions is comprised of the complement determining regions CDR,, CDRo and CDR,, and they are fundamental in defining the antigen binding specificity of the antibody, i.e. the paratope. The present inventors have found that of these regions, the CDR3 part of the Vh region is the most important in defining antigen specificity. Further teachings about antibodies are widely available in the art, e.g. Harlow, E. and Lane, D., "Using Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1998. With the common general knowledge about the paratope defining portions of an antibody combining site and the above teachings, a person skilled in the art is readily able to modify the sequence of SEQ ID NO: 2 to produce alternative antibodies with the same paratope and which enable one to achieve the same or similar therapeutic effect.
For example an antigen binding fragment of the antibody of SEQ ED NO: 2 may be readily prepared by simply removing one or more of the carboxy-terminal His residues. Other examples of modifications include the grafting of the hypers"ariable (complement determining regions) of the antibody of SEQ ID NO: 2 into variable framework regions different to those of SEQ ID NO: 2 such that the resultant antibody still has the same paratope (see e.g. EP 239400 and suchlike).
By "improved treatment" is meant that a given quantity of a glycopeptide antibiotic has a greater therapeutic effect when administered to a patient together with the antibody of the present invention than when administered alone. Similarly, a desired therapeutic effect can be achieved in a patient with a smaller dosage of a glycopeptide antibiotic when administered to a patient together with the antibody of the present invention than when the glycopeptide antibiotic is administered alone. This is particularly the case when the micro¬organism such as S. aureus is resistant to the glycopeptide antibiotic. This can be extremely useful in reducing any side effects of treatment.
By providing existing glycopeptide antibiotics with a "second wind" and allowing them • to be therapeutically effective against e.g. S. aureus infection, even when the S". aureus is
resistant to them, the present invention provides medicaments and treatments whose safety and efficacy can be readily evaluated and which do not involve new classes of substances -the safety parameters for glycopeptide antibiotics are already well known and can be applied to the present invention. Similarly the use of antibodies as the other active ingredient in the present invention is a relatively simple safety issue. In particular, when the antibody is in the form of a recombinant antibody fragment derived from human antibody sequences, its immunogenicity will be extremely low. In addition the use of treatment regimes in which the antibody is administered over a very short time period (for example a few days) means that there is little opportunity for the patient"s immune system to develop an immune response against it. Thus adverse hypersensitivity-type reactions caused by the production of antibody against the antibody of the present invention and the formation/deposition of immune complexes is very small.
The antibody nfFnjn ]s|0- o aln hng a number of specific advantages - it does not have the Fc portion which can trigger complement deposition and macrophage binding and hence is unlikely to cause inappropriate activation of the complement cascade which can cause inflammation and tissue damage.
By producing the antibodies of the present invention without the use of animal-derived products, the possibility of introducing human or animal infectious disease agents or endogens into patients can be avoided.
Treatment regimes and dosages of the medicaments of the present invention are described below, and modification of them will be readily apparent to a person skilled in the art, who in particular will be aware of treatment regimes used with the glycopeptide antibiotic and will be able to modify them appropriately. For example, simple dose-response assays can be readily used and results of treatment in mammalian models such as murine models can be simply extrapolated across to humans.
The present invention will be further apparent from the following description with reference to the accompanying Figure, which shows by way of example only forms of treatment of S. aureus infection.
Figure 1 shows tie pharmacokinetics of high doses of Aurograb. X-axis is time in minutes. Y-axis is μg/rnl of Aurograb in blood samples.
The experiments detailed below show that Aurograb (i.e. SEQ ID NOs: 1 and 2), the human recombinant antibody fragment of the present invention specific against GrfA., has : intrinsic anti-staphylococcal activity in vitro and in murine staphylococcal infection and that it shows synergistic activity with glycopeptide antibiotics, particularly the broad spectrum glycopeptide antibiotic vancomycin.
The synergistic activity with glycopeptide antibiotics is manifested by:
(1) a reduction in the minimum inhibitory concentration of vancomycin in the presence of Aurograb in vitro and
(2) reduced organ colony counts in murine models of 5. aureus infection, when, after challenge with S. aureus, a single dose of Aurograb (2 mg/kg) is given in conjunction with a single dose of vancomycin (6 mg/kg).
Unless stated otherwise, all procedures detailed herein were performed using standard protocols and following manufacturer"s instructions where applicable. Standard protocols for various techniques including PCR, molecular cloning, manipulation and sequencing, the manufacture of antibodies, epitope mapping and mimotope design, cell culturing and phage display, are described in texts such as McPherson, M.J. et al. (1991, PCR: A practical approach, Oxford University Press, Oxford), Sambrook, J. et al. (1989, Molecular cloning: a laboratory manual. Cold Spring Harbour Laboratory, New York), Huynh and Davies (1985, "DNA Cloning Vol I - A Practical Approach", IRL Press, Oxford, Ed. D.M. Glover), Sanger, F. et al. (1977, PNAS USA 74(12): 5463-5467), Harlow, E. and Lane, D. ("Using Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1998), Jung, G. and Beck-Sickinger, A.G. (1992, Anew. Chem. Int. Ed. Eng., 31: 367-486), Harris, M.A. and Rae, I.F. ("General Techniques of Cell Culture", 1997, .. Cambridge University Press, ISBN 0521 573645), "Phage Display of Peptides and
Proteins: A Laboratory Manual" (Eds. Kay, B.K., Winter, J., and McCafferty, J., Academic Press Inc., 1996, ISBN 0-12-402380-0).
Reagents and equipment useful in, amongst others, the methods detailed herein are available from the likes of Amersham (www..amersham.co.uk), Boeliringer Mannheim (www.boehringer-ingeItheim.com), Clontech (www..clontech.com), Genosys (www..genosys.com), Millipore (www.millipore.com), Novagen www.novagen.com), Perkin Ehner (.www.perkinelmer.com), Pharmacia (www..pharmacia.com), Promega (www.promega.com), Qiagen (www.qiagen.com), Sigma (www.sigma-aldrich.com) and Stratagene (www.stratagene.com).
Where "PMID" reference numbers are given for publications, these are the Plumbed identification numbers allocated to them by the US National Library of Medicine, from which full bibliographic information and abstract.for each publication is available at VAvw.ncbi.nlm.nih.gov. This can also provide direct access to electronic copies of the complete publications, particularly in the case of e.g. PNAS, JBC and MBC publications.
The contents of each of the references discussed herein, including the references cited therein, are herein incorporated by reference in their entirety.
1. Human Study
For this study, Aurograb is given in conjunction with vancomycin to reduce the mortality and/or morbidity of patients with culture-confirmed deep-seated MRSA infection. This study is performed in three parts:
i) A tolerance and dose ranging study in which cohorts of 3 patients with MRSA
infection are given staged escalating doses of Aerograph, starting with a sub-therapeutic •
dose (0.1 mg/kg) and rising to 1 rag/kg b.d., during which each patient is carefully monitored for any adverse side effects.
ii) An interim, open, labelled study in which 5 patients with NIRSA infection are
given the anticipated therapeutic course of Aurograb, namely 1 mg/kg b.d. for 7 days, and carefully monitored for tolerance and pharmacokinetics. A preliminary assessment of efficacy can be made based on historical controls.
iii) A prospective, double blind, randomized phase II trial.
2. Physical, chemical and pharmaceutical properties
3. Formulation, composition, handling & storage
3.1 Formulation & Composition
The complete composition of Aurograb is given in Table 2, below. It consists of lyophilized (fireeze-dried) powder provided at 10 mg per vial. The drug is administered by resuspension of the powder in 5 mL sterile inject able water shortly prior to iv injection. No other solvent should be substituted for water as Aurograb is sensitive to pH fluctuations and has been especially formulated for resuspension in water.
L-arginine and urea are present in order to maintain the pH balance of the formulation and ensure solubility of the Aurograb. Both are present in dosages which are considered safe for clinical intravenous administration. For example, a single dose of Aurograb (37.5 mL for 75 Kg patient body weight) contains 7.5 mmoles arginine. This is 26 times less than the maximum permitted daily dose of L-arginine when administered during the measurement of growl hormone levels in children (ABPI compendium, 1998-99). For urea, a single 35 mL dose of Aurograb contains 1.125 gurea. Again, this is much less than the 150 g urea maximum permitted daily dose of urea (this is the maximum dose indicated for the treatment of acute intracranial pressure via intravenous delivery - ABPI compendium, 1998-99).
The lyophilized drug is stored in glass vials at 4 °C in the dark. Overnight transport at ambient temperatures is acceptable. Avoid prolonged exposure to bright light or excessive humidity. No change in Aurograb activity after storage at 4 °C (3 months) has been observed.
After reconstitution with water, Aurograb must be stored at 4 °C and should be used within 14 hours. Activity will slowly deteriorate if left at room temperature.
3.3 Preparation and administration
Aurograb, provided at 10 mg per vial, is administered by resuspension of the powder in 5 nail of sterile injectable water shortly prior to intravenous (iv) injection. An example human dose is 1 mg/kg b.d. Sterile water is introduced directly into the vials via injection through the rubber cap with a hypodermic syringe/needle.
add half the final volume of water (2.5 ml per vial) and swirl to dissolve filter (if necessary) to remove any undissolved material add the remaining water.
NB. If administration delayed, store at 4 °C (for up to 14 hours).
No other solvent should be substituted for water as Aurograb is sensitive to pH fluctuations and has been especially formulated for resuspension in water.
Aurograb should be administered by slow iv bolus injection, for example through an iv giving set which should be flushed first with saline to remove traces of any other iv fluids and then again after administration of the drug.
No special precautions are required for the clean-up of spills or waste disposal.
Saline for iv use is compatible with reconstituted Aurograb (at a 50:50 dilution). It is less compatible with 20% glucose and sodium bicarbonate buffer, as shown by a reduction in ELISA activity, and therefore if being given via the same iv giving set as these or other fluids, the injection port must be flushed through with saline before and after administratmg the Aurograb.
4. Non-clnical studies
Aurograb shows antibacterial activity, both in vitro and in vivo, against a wide range of S. aureus strains. In addition Aurograb has been shown to exert a synergistic antibacterial effect when used in conjunction with vancomycin.
4.1 Non-Clinical Efficacy Studies: in vitro data
Aurograb shows synergistic activity in combination with vancomycin against a wide range of MRSA strains, including strains of EMRSA with intermediate resistance to vancomycin ("VISA" - vancomycin insensitive MRSA)
These data were prepared using the same MIC methodology routinely applied to antibiotics.
Aims: To demonstrate synergy when used in combination with vancomycin, against
a wide range of epidemic strains of MRSA.
Objective: Synergy is demonstrated by a reduction in the MIC of vancomycin when Aurograb is used in combination.
Epidemic strains of MRSA readily give rise to sub-population of intermediate resistance to vancomycin (i.e. MIC of 4 or 8 μg/ml) by serial passage in nutrient broth with increasing concentrations of vancomycin (Bumie J et al, Clin Infect Dis. 2000 Sep;31(3):684-9; PMID: 11017816). These are probably responsible for many of the treatment failures associated with vancomycin therapy. In this study Aurograb activity was examined against both these VISA and the fully vancomycin-sensitive parent EMRSA-15 strain.
Strains of MRS A were grown overnight in nutrient broth and diluted to give an inoculum of 1x103cells per mL. 100 μl aliquots of culture were placed into flat-bottomed microtitre plates and vancomycin added in the range 0.125 - 250 μg/ml. Cultures were incubated for a further 2 days. The minimum growth-inhibitory concentration (N"lIC) is defined as the highest concentration of antibiotic at which no bacterial growth occurs and is determined by spectrophotometric evaluation of the lack of turbidity (growth) in culture. MICs were determined in the presence of exogenous Aurograb or in the presence of Aurograb formulation buffer (negative control).
Spectrum of Activity: Enhanced antibacterial activity is evident in all strains tested demonstrating a broad spectrum of activity against strains of MRSA.
Synergy: Aurograb has the ability to increase the sensitivity of MRSA to vancomycin, including MRSA with reduced sensitivity to vancomycin.
Further experiments were undertaken to determine the MIC of vancomycin for a number of MRSA strains and the effect of the Aurograb antibody on MIC. The experiments were also performed using flucloxacilhn instead of vancomycin to investigate how the efficacy of penicillin-type antibodies was affected by Aurograb. Results are given in Table 3 a:
Results obtained with flucloxacillin showed that the MIC obtained was > 256 |ig/ml for all EMRSA strains. Flucloxacillin + Aurograb gave the same results and showed no decrease in the IvlIC of flucloxacillin.
Conclusion: Use of Aurograb in combination with vancomycin should both increase therapeutic efficacy and impede the emergence of vancomycin resistant strains. Due to the structure and function of the antibody with respect to the target molecule, similar results can be expected to be achieved using other glycopeptide antibiotics such as teicoplanin, . the resistance mechanisms employed by 5". aureus against them being the same as for
vancomycin and therefore also being a target for the antibody and being susceptible to therapy using it.
4.2 Non-Clinical Efficacy Studies: in vivo data
The murine model of staphylococcal infection is widely used and is routinely used in the assessment of antimicrobial drugs. It is a good predictor of efficacy in infected humans since S. aureus is introduced intravenously to create a bacteraemia, from which the pathogen spreads to other organs, including the kidney, liver and spleen. Hence the nature of the infection (septicaemia) is analogous to the situation in infected patients. Also, the intravenous route of administration of Aurograb, used in the animal models (data given below) is the same route as that typically to be used in patients, improving the chances of comparable pharmacokinetics, drug bio-availability and efficacy.
Data Set 1: Aurograb - intrinsic antibacterial activity against EMRSA-15
Aims: To demonstrate Aurograb is therapeutic when given alone in mice infected with a
vancomycin-sensitive strain of MRS A, the EMRSA-15.
Objective: Anti-staphylococcal activity is demonstrated by a reduction in mortality or a reduction in bacterial load (colony forming units) in kidney, liver or spleen in the presence of Aurograb.
Method: 20 female CD-I mice were given a sub-lethal challenge (1.3 x 10^ cfli) of a methicillin-resistant, vancomycin-sensitive strain of S. aureus (EMRSA-15, Typed Hospital Isolate, Central Manchester Healthcare Trust) in 100 |il iv bolus (all iv injections given via the lateral tail vein). 1 hour later, two groups of 10 mice, received, as 100 |il iv bolus:-
1. formulation buffer (Arginine-Urea), as negative control or
2. Aurograb (2.0 mg/kg) in formulation buffer.
All animals were sacrificed after a further 48 h and viable cfu per gram of organ were detennined.
Anti-bacterial activity - Viable staphylococci were found in the kidney, liver and spleen with preferential localisation in the kidney. Administration of Aurogi^ab resulted in a log (10 fold) reduction in viable organisms found in all three organs. This suggests that Aurograb reduces the viability of S. aureus in experimental infections of mice in the absence of any other exogenous antimicrobial using the murine model of deep-seated infection.
Data Set 2: Aurograb dose ranging study
Aims: Dose ranging study for use of Aurograb versus S. aureus strain EMRSA-15.
Objective: Antibacterial activity is demonstrated by the greater reduction in bacterial load (organ cfu) in kidney, liver or spleen when Aurograb is given, as a single agent, compared to the placebo control. Dose range is determined by comparing cfu(expressed as the log of the cfu per gram of tissue) for different organs at different doses.
Method: 40 female CD-I mice (24-26 g) challenged with iv bolus of S. aureus followed 2 hours later by single iv dose of placebo or Aurograb (2 mg/kg, 1 mg/kg or 20 mg/kg). All mice terminated at 48 hours for culture of kidney, liver and spleen to determine organ viability counts.
" Negative control antibody (WC7) specific to a non-protective epitope of GrfA.
Experiment 1: a 10 to 100 fold reduction in viable bacteria for liver and spleen was achieved in the range 1.0 to 2.0 mg/kg Aurograb. At 0.2 mg/kg Aurograb, clearance from the spleen is still good, but reduced in the liver. The highest dose resulted in a 0.8 log reduction in kidney bacterial counts but no reduction occurred with the lower doses.
Experiment 2; At doses as low as 0.2 mg no reduction in viable staphylococci is evident in the kidneys, but there is evidence of bacterial killing in the liver and spleen.
Conclusion: Administration of Aurograb at 2 mg/kg results in a reduction in viable organisms found in all three organs, but particularly the spleen and liver. Aurograb at a dose of 1 mg/kg also showed significant antibacterial activity in the spleen and liver, but lost activit}" in the kidney. Doses as low as 0.2 mg/kg, still shov/ed antibacterial activity in the spleen, and to a lesser extent, the liver.
Extrapolation to humans: A single dose of 2.0 mg/kg in mice gave an approximately 2.0 log drop in liver and spleen counts and a 0.8 log drop in kidney counts. This equates, on a body weight basis, to 1 mg/kg given twice daily to humans.
Data Set 3: Aurograb antibacterial activity and synergy with vancomycin: sub-lethal
model. Aims: To demonstrate Aurograb is antibacterial when given alone and synergistic when given in conjunction with vancomycin in mice infected with a sublethal dose of ^S*. aureus.
Objective: Antibacterial activity is demonstrated by a reduction in bacterial load (colony forming units) in kidney, liver or spleen in the presence of Aurograb alone. Synergy is demonstrated by a greater reduction in organ colony counts when Aurograb and vancomycin are given together than when either antibacterial is given alone.
Methods: 60 female CD-I mice (22-24 g) were given 1 x 10^ cfu of EMRSA-15 as a 100 \i\ iv bolus. 2 hours Jater, in groups of 10 mice, they received, as a single 100 p,l iv bolus:
Group 1 - 6.0 mg/kg vancomycin + 2.0 mg/kg Aurograb
Group 2 - 6.0 mg/kg vancomycin + 0.2 mg/kg Aurograb
Group 3 - 6.0 mg/kg vancomycin + placebo (formulation buffer)
Group 4 - placebo + 2.0 mg/kg Aurograb
Group 5 - placebo + 0.2 mg/kg Aurograb
Group 6 - placebo + 2.0 mg/kg Aurograb All mice were terminated at 48 hours for culture of kidney, her and spleen.
Results: expressed as log cfa per gram of tissue, unless the majority of mice (>5)
were cleared (ie organs were culture-negative). N/A = not applicable. (Table
Alone: Aurograb administered alone at 2.0 mg/kg (group 4) gave comparable results to vancomycin alone (group 3), each clearing the livers of 8 mice and spleens of 4 and 5 mice respectively, compared to one mouse clearing the liver and spleen in the negative control group (group 6). Renal clearance was better with vancomycin than Aurograb, although none of the mice were cleared of renal infection.
Synergy Mice (group 1) receiving 2.0 mg"lcg of Aurograb plus vancomycin (6 mg/kg) showed complete clearance of staphylococci from the livers of all 10 mice, and enhanced clearance from the spleens, being culture negative in 9 mice, compared to 4-5 being cleared by Aurograb or vancomycin alone and 1 being cleared in the untreated group. Renal colony counts in those receiving combination therapy (group 1) were reduced by an additional 0.5 log compared to mice receiving vancomycin alone (group 3), and reduced by 1.6 log compared to negative control mice (group 6). This demonstrates the synergy between Aurograb and vancomycin.
Data Set 4: Aurograb synergy with vancomycin: lethality model.
Aims: To demonstrate Aurograb is antibacterial when given alone and synergistic when
given in conjunction with vancomycin in mice infected with a lethal dose of S. aureus.
Objective: Antibacterial activity is demonstrated by a reduction in mortality with Aurograb alone. Synergy is demonstrated by a greater reduction in mortality when Aurograb is given in combination with vancomycin, rather than vancomycin alone.
Method: 60 female CD-1 mice (22-24 g) were given S. aureus (a fresh clinical isolate of EMRSA-15) at a dose of 8 x 10"" cfu in a 100 ^il iv bolus. 2 hours later, in groups of 10 mice, they received, as a single 100 ^xl iv bolus:
Group 1 - 4.0 mg/kg vancomycin -r 2.0 mg/kg Aurograb
Group 2-4.0 mg/kg vancomycin
Group 3 - 2.0 mg/kg vancomycin -r- 2.0 mg/kg Aurograb
Group 4-2.0 mg/kg vancomycin
Group 5-2.0 mg/kg Aurograb
Group 6 - placebo
All mice were terminated at 48 hours for culture of kidney, liver and spleen. Mice fatalities prior to the 48 hour cull were not subject to organ colony counts.
Results: expressed as log cfu per gram of tissue, unless the majority of mice (>5)
were dead (Table 8)
Mortality: This strain of 6". aureus, probably because it was a fresh clinical isolate, was more virulent in the mouse model than the previously used laboratory strain of EMRSA-15, and a high degree of mortality was observed. Administration of Aurograb alone (2.0
mg/kg) was associated with a reduction in mortality (4 deaths) compared to the untreated group (7 deaths), comparable to the mortality on vancomycin alone (6 and 5 deaths). This suggests that Aurograb has comparable intrinsic anti-staphylococcal activity}" to vancomycin at 4.0 mg/kg.
Synergy: Mice receiving combination therapy (Aurograb 2.0 mg/kg and vancomycin
4.0 mg/kg) had higher rates of survival than with vancomycin or Aurograb alone
suggesting a synergistic effect with the two drugs.
5. Pharmacokinetics and product metabolism in animals
The pharmacokinetic (PK) characteristics of Aurograb have been studied in mice. A murine model was used because efficacy and dose ranging studies have been performed in this species as were the repeat dose toxicology studies. Mice were given a single high dose as an iv bolus and then examined in depurate for blood levels and organ distribution. Urine samples were also analyzed. Samples were analyzed for functional activity, measured by ELISA (blood samples only) and drug concentration (SDS PAGE/immunoblots).
Results are sbo\\m in Figure 1. Mice given a single intravenous bolus injection of Aurograb (10 mg/kg) showed satisfactory blood levels.
All organs (lungs, brain, liver, spleen, heart, kidney and blood) were cleared by 24 hours.
6.2 Blood Compatibility Study
This study was carried out by Biochemical Pharmacology department at Inveresk Research, Scotland, in order to assess the suitability of Aurograb for intravenous administration.
Blood samples were taken from healthy human volunteers, transferred to tubes containing Aurograb, Aurograb vehicle, saponin or saline and incubated at 37 °C for 1 hour. After centrifugation, the amount of haemoglobin in the supernatant was then assessed to determine haemolysis. According to the American Society for Testing and Materials classification system (ASTM F756-93, 1993) the data indicated that 0.1 mg/nil Aurograb and the Aurograb vehicle were non-haemolytic. This compared with physiological saline which was non-haemolytic, and in total contrast to the positive reference compound (saponin) which was found to be severely haemolytic.
7 Manufacture of Aurograb antibody 7.1 Introduction
Aurograb is produced in E.coli in the form of inclusion bodies. The isolation, solubilisation, denaturation and refolding of inclusion bodies has been previously well documented (see "Antibodies: A Laboratory Manual". Harlow, E. and Lane, D. 1988. Cold Spring Harbor Laboratory Press; "Inclusion bodies and purification of proteins in biologically active forms". Mukhopadliyay, A. 1997. Adv. Biochem. Eng. Biotechnol. ;56:61-109). An example of such a procedure is outhunted in this application. Briefly, inclusion bodies were extracted from the cell mass, solubilised/denatured, refolded and purified using chromatography. Over expression of Aurograb is from the powerful T7 promoter in vector pET29b (Novagen). The cell mass is produced as a quality conformed, pure culture prepared in a sterile fermented (lOOOL) using media free of animal products. The E.coli strain used is genetically disabled (unable to colonise/persist in the environment) and is virulent. Impurities removed during downsfream processing are thus (1) E.coli host cell proteins (HCPs) (2) E.coli progeny (mainly antitoxin) (3) fermentation additives (antibiotics) (4) downsfream processing additives (buffers and NiS04).
Large amounts of contaminants from E.coH and virtually all fermentation media derived contaminants are removed during the process extraction of the protein from inclusion bodies. For this, the cell mass is isolated by centrifugations and broken using a micro fluidizer. The Aurograb inclusion bodies are separated from the cell mass by three gentrification-resuspension- micro fluidizations steps thereby washing the inclusion bodies extensively. Inclusion bodies are solubilised and refolded over a two day period in the presence of copper chloride, which serves as an oxidation catalyst, thus establishing the correct and biologically active structure of the Aurograb molecule. Extensive infiltration is performed to remove small molecules from the refolding reaction and trace residual kanamycin (from the fermentation reaction). Remaining host cell proteins are removed by immobilized nickel-metal affinity chromatography (IMAC) and anion exchange. In particular, antitoxin, which is a significant safety issue for drugs manufactured from E.coli, is efficiently removed during IMAC by the addition of deoxycholic acid. The last traces of E.coli are removed during the anion exchange step. Traces of nickel from the IMAC step are removed using a nickel affinity column. Finally small molecules from the chromatography buffer system are removed using diafiltration against formulation buffer. The bulk product is lyophilised in vials for use in patients. The numbered paragraphs below specifically detail the manufacture of the Aurograb antibody.
7.2 Aurograb manufacturing protocol
Fermentation and downstream processing were performed using a total of nine steps as follows: 1. Fermentation
2. Harvest and isolation of inclusion bodies
4. Concentration and diafiltration
5. Filtrarion and sterile filtration
6. Purification - Immobilised Nickel (Ni2+) metal affinity chromatography (IMAC)
7. Purification - Nickel removal by chelating sepharose chromatography
8. Purification - Anion exchange chromatography
9. Diaflltration and bulk filling
A frozen stock of the Aurograb expressing E. coli (Clone JM109 (DE3)(pSaABC4)) was inoculated into 4 x 500 ml glass conical flasks, to prepare the inoculum prior to transfer to a lOOOL stirred fermenter (MBR). The precultures (supplemented with kanamycm) were incubated at 37 °C with shakmg, for 15-20h, until the preculture 00575= 4.0-8.0, representing 7-8 cell divisions. At production scale, the precultures were transferred into a 1000 L fermenter (MBH) containing 1000 L nutrient broth. The production culture was incubated at 37 °C for a total of 14-18h, representing 7 cell divisions. Approximately 10-13h post inoculation, when the 00573= 5-8, the production culture was induced through the addition of 23.8g IPTG (lOOfiM).
7.4 Harvest and isolation of inclusion bodies
Cells were harvested by centrifugation (4h post induction) at 12,800 g in an Alfa Laval BTPX 205 centrifuge with a flow rate of approx. 400 L/h. The harnessed E.coli cell paste was stored at -50""C in PE bags (Kendro). The down stream processing of 100% cell paste mass from a single fermentation batch having been stored at -50°C constituted a single purification batch. The cell paste was thawed and resuspended in lOOOL lysis buffer. The resuspended bacterial cells were subjected to high pressure homogenisation (APV Gaulin MCI5) at ca. 1000 bar (15,000 psi) with a flowrate ca. 500 L/h. The inclusion bodies were sedimented by centrifrigation at ca. 12.800 g with a flowage ca. 400 L/h. The inclusion bodies were then washed with 400L of lysis buffer, and sediment by centrifugation at ca. 12.800 g with a flowrate ca. 400 L/h, for a total of three times. The washed inclusion bodies were stored as a slurry at -70°C prior to the refolding step. The inclusion bodies were solubihsed by strong agitation for 5-10 minutes in 40L 6M urea/lOOmM Tris pH 12.5 at room temperature.
For refolding the inclusion body solution was made up to 560L with refolding buffer, and CuCl2 H2O was added to a final concentration of 100 uM. The preparation was incubated for 48h at 2-8°C under strong agitation. The inclusion bodies were filtered (Sartorius GF 30", 3.0/0.8μm) and then filter sterilized (Seitz Supradisc SDPEK 1, depth filter).
7.6 Concentration and diafiltration
Concentration and diafiltration steps were carried out using cassettes (15m-) of omega screen channel with a lOkDa cutoff (Pall) housed in a "Centrasette" stainless steel holder (Pall), under the control of a 1000 membrane piston pump (pump quattro). The refolded protein preparation was concentration to 150L, followed by diafiltration with 3000 litres of 10 mM ammonium acetate buffer, pH 9.5. The preparation was adjusted to 6 M urea, 50 mM Tris, 10 mM DC A, 1 M NaCl and made up to a final volume of 300L with RO water. The diafiltered refold was stored over night at 2-8°C. The pH was then adjusted to 8.0 through the addition of HCl.
7.7 Filtration steps and sterile filtration
The diafiltered refold was filtered (Seitz Supradisc SDPEK I, depth filter) and then filter sterilised (Millipore Opticap 10", 0.2^m). The sterile product was stored at 2-8°C.
7.8 Immobilised Ni2+ metal affinity chromatography (EVIAC)
The IMAC step was performed in a BPG300/500 column (Amersham Pharmacia).-Sterilisafion of chromatography columns took place during resin cleaning in place (CIP). IMAC chromatography resin (18L) was initally charged with 2 columns volumes (CV) 100 mM NiS04.6H20. Chromatography punished 5.5 (CV) equilibration buffer (MAC A); 5.5 CV "Wash buffer I (IMAC B); 3.3 CV Wash buffer II (IMAC C); 5.5 CV Elution buffer (IMAC D). Fractions collected from the elated were filter sterilised (Millipore Opticap 10", 0.2μm) and stored overnight at 2-8°C.
7.9 Nickel removal chromatography
Nickel removal chromatography was performed in a BPGl 40/500 column (Amersham Pharmacia) using 2L chelating Sophia’s resin. The column was equilibrated and washed with 5 CV buffer (MAC D) and the Aurograb antibody containing solution was applied, the column was washed with 2CV anion/conditioning buffer (50 mM Tris, 6 M urea pH 8.7) and the elated was retained prior to anion exchange chromatography.
7.10 Anion exchange chromatography
The Anion exchange chromatography step was performed in a BPG3 00/500 column (Amersham Pharmacia) using 18 L resin (Q-Sepharose FF). The column was equilibrated using equilibration buffer and then anion/conditioning buffer. The Aurograb antibody containing solution was applied, washed with 2CV anion/conditioning buffer, and the flow through was filter sterilised (Millipore Opticap 10", Online) and stored overnight at 2-8°C.
7.11 Diafiltration and bulk fixing
The pH of the sterile flow through (containing the Aurograb antibody) was adjusted to 9.5, and was then diafiltered against 10 turnover volumes (TOV) 10 niM ammonium acetate; 0.5 M urea, pH 9.5. There followed another diafiltration step, against 5 TOV 0.5 M urea, 0.2 M arginine pH 9.5. The protein was concentrated to 2mg/ml, filter sterilised (Millipore Opticap 10", 0.2μm) into a Stein bag, and stored at 2-8°C.
7.12 Composition of buffers, media and solutions
Media were monitored for microbial contamination prior to inoculation. For 2.5 L preculture:
Peptone A3 food soy bean (Organotechnie) 67.5 g
Yeast autolysate (KAV; Deutsche Hefewerke) _ 35.0 g
Sodium chloride (Ph.Eur, E.Merck) 12.5 g
Glycerol (Ph.Eur, E.Merck) 75.0 g
Dipotassium hydrogen phosphate (Ph.Eur, E.Merck) 11 -5 g
Potassium dihydrogen phosphate (Ph. Eur, E.Merck) 7.5 g
Magnesium sulfate heptahydrate (Ph.Eur, E.Merck) 1.25 g
Kanamycin sulfate (Sigma) 62.5 mg
ROI water 2.5 litres
The pre-weighed substances in the table above are dissolved in 2.5 L RO I-water. After sterilizations of the broth 10 ml kanamycin sulphate (62.5 mg in 50 ml WFI) was added.
Composition for 1000 L culture:
Peptone from soybean (Organotechnie) 27.0 kg .
Yeast autolysate (KAV; Deutsche Hefewerke) 14.0 kg
Sodium chloride (Ph.Eur, E.Merck) 5.0 kg
Glycerol (Ph.Eur, E.Merck) 30.0 kg
Duodenum hydrogen phosphate (Ph.Eur, E.Merck) 4.6 kg
Sodium dihydrogen phosphate (Ph. Eur, E.Merck) . 3.0 kg
Magnesium sulphate heptahydrate (Ph.Eur, E.Merck) 0.5 kg
Struktol (antifoam agent, Sichler) 600 ml
Kanamycin sulfate (USP, Sigma) 25.0 g
RO I water 1000 litres
pH 7.0 ±0.2
The 1000 L fermenter was sterilised for 30 minutes at 12 TC.
23.8 g IPTG (Sigma) was dissolved in 200 ml WFI and sterile filtered (0.2 μ m).
Cell lysis buffer
Tris (Ph.Eur, E.Merck) 6.06 gL-1EDTA (Ph.Eur, E.Merck) 0.37 gL -1
Potassium chloride (DAB, E.Merck) 7.46 gL ■"
ROI water 1000 litres
pH 8.0 ±0.1
Tris (DAB, E.Merck) 5.96 gL""
Copper II chloride (p.a., E.Merck) 0.018 gL""
RO I water (1000 L stainless steel container) 600 litres
pH 9.0 ±0.1
Ammonium acetate (E.Merck) 0.77 gL""
RO I water 1000 litres
pH 9.0 ±0.1 •
Tris 1.82 kg
urea 108 kg
sodium chloride 17.5 kg
deoxycholic acid, sodium salt 1.30 kg
The composition of the 150 L diafiltered refolding reaction was adjusted with the above reagents.
IMAC chromatography buffers:
Nickel solution: 100 mM NiS04.6 HjO (2CV)
IMAC A/Equilibration buffer: 100 mM Tris; 6 M urea; IM NaCl; 10 mM DCA, pH 8.0
IMAC BAVash buffer: 100 mM Tris; 6 M urea; IM NaCl; 50 mM imidazole; 10 mM
DCA, pH 8.0
IMAC C/Elution buffer I: 100 mM Tris; 6 M urea; IM NaCl; 150 mM imidazole; pH 8.0
MAC D/Elution 11:100 mM Tris; 6 M urea; IM NaCl; 300 mM imidazole pH 8.0 (7 CV)
CIP: 1 M NaOH (3 column volumes, incubation time 1 hour)
Buffers were prepared with Tris (USP, E.Merck), Urea (USP, E.Merck or Riedel de Haen),
NaCl (Ph.Eur, E.Merck), Imidazole (p.a., Fluka), DCA (Deoxycholic acid, sodium salt
monohydrate, Micro select, Fluka), NiS04.6 HjO (p.a., E.Merck)
NP Removal Chromatography buffers:
Equilibration buffer: 100 mM Tris; 6 M urea; 50 mM NaCl; 300 mM imidazole; pH 8.0 (5CV)
Anion exchange chromatography buffers:
Equilibration: 100 mM Tris; 6 M urea; 50 mM NaCl; 300 mM Imidazole pH 8.7 Cff; 1 M NaOH (3CV, incubation time 1 hour)
Diafiltration and Formulation Buffer
For sterilisation and storage, the diafiltration cassettes were flushed with RO water, recalculated for at least 45 minutes with 0.5 M NaOH and stored with 0.1 M NaOH. Diafiltration buffer: 0.5 M urea; 10 mM ammonium acetate pH 9.5 Diafiltration (formulation) buffer: 0.5 M urea; 0.2 M arginine pH 9.5 Diafiltration and concentration (2 cassettes, Omega screen channel, 10 KD, Pall)
I, 8. Determination of Synergistic Effect of Aurograb with Vancomycin
In order to determine the Minimum Inhibitory Concentration (MIC), the Fractional Inhibitory Concentration (FIC) and the FIX (fractional inhibitory index), the following was method was used.
Representatives of each of the most common EMRSA strains, a number of vancomycin resistant subpopulations, and three Linzolid resistant strains were grown overnight in nutrient broth. The cells were diluted in Isosensitest Broth (Oxoid, UK) to give a fmal inoculum of 1x10^ cells per mL. 100 \i\ aliquots of culture were placed into flat-bottomed microtitre plates and vancomycin added in the range 0.125 - 250 |ig/ml. To this was added exogenous Aurograb in the range 0.3 - 25 jig/ml in a checkerboard formation. A negative control series was also prepared containing Aurograb formulation buffer alone. Cultures were incubated for 48 hours at 37 °C.
The minimum inhibitory concentration (MIC) was defined as the lowest concentration of agent at which no bacterial growth occurred and was detemiined by spectrophotometric evaluation of the lack of turbidity (growth) in culture. This optical clearing was equivalent to >99% Staphylococcal death.
MIC values were obtained for vancomycin and Aurograb (RTM) alone and in combination. Fractional inhibitory concentrations (FIC) and FIX values were calculated. Synergistic activity" beween the two agents was defined as an FIX value 4 defined antagonism.
Results of the experiment are given below in Tables 9 and 10. VAN indicates vancomycin. The MIC (Alone) column indicates the minimum inhibitory concentration of the agent when administered alone. The MIC (In combination), column indicates the minimum inhibitory concentration of each drug when used in combination.
As can be seen from Tables 9 and 10, the administration of Aurograb with vancomycin results in a synergistic therapeutic effect being achieved, with MIC levels being substantially reduced, which in turn indicates that therapy of patients can be effected with lower dosages of antibiotics and to greater therapeutic effect, wdth an expected reduction in possible side-effects resulting from the antibiotic. The results indicae that even in cases where a patient is infected with a vancomycin-resistant S. aweiis they may be successfully treated with the antibody of the present invention and its use in combination with antibiotics such as vancomycin may enable effective therapy at previously ineffectual non¬toxic dosage levels of antibiotic.
1. A medicament, comprising a therapeutically effective quantity of a glycopeptide antibiotic and antibody specific against the epitope displayed by the peptide having the amino acid sequence of SEQ ID NO: 3.
2. A method of manufacture of a medicament for the treatment of infection, characterized in the use of a therapeutically effective quantity of 0.2 mg/kg to 2mg/kg of a glycopeptide antibiotic and antibody specific against the epitope displayed by the peptide having the amino acid sequence of SEQ ID NO: 3.
3. The medicament or method of manufacture according to any one of claims 1 and 2, said glycopeptide antibiotic being selected from the group consisting of vancomycin, teicoplanin and daptomycin.
4. The medicament or method of manufacture according to any one of claims 1 and 2 or 3, said infection being of a gram positive bacterium.
5. The medicament or method of manufacture according to claim 4, said bacterium being a staphylococcus.
6. The medicament or method of manufacture according to claim 5, said bacterium being a coagulase-negative staphylococcus.
7. The medicament or method of manufacture according to claim 6, said
staphylococcus being selected from the group consisting of S. haemolyticus, S.
epidermidis and S. saprophyticus.
8. The medicament or method of manufacture according to claim 5, said bacterium
being a coagulase-positive staphylococcus.
9. The medicament or method of manufacture according to claim 8, said
staphylococcus being S. aureus.
10. A medicament or method of manufacture according to claim 4, said bacterium
being selected from the group consisting of Enterococcus sp., Enterococcus faecalis,
Enterococcus faecium, Corynebacterium sp., Corynebacterium jeikeium and
11. A medicament or method of manufacture according to claim 4, said bacterium
being resistant to treatment by said glycopeptide antibiotic alone.
|Indian Patent Application Number||1386/CHENP/2004|
|PG Journal Number||13/2008|
|Date of Filing||21-Jun-2004|
|Name of Patentee||NEUTEC PHARMA PLC|
|Applicant Address||Clinical Sciences Building, Central Manchester Healthcare Trust, Oxford Road, Manchester, M13 9WL,|
|PCT International Classification Number||A61K 39/40|
|PCT International Application Number||PCT/GB2002/005135|
|PCT International Filing date||2002-11-13|