Methods of Screening and Compounds for Adverse Response to an Implant

Abstract

Methods of screening a subject to determine whether or not they are at risk of having an adverse reaction to metal debris (ARMD), also known as adverse local tissue reaction (ALTR), methods of screening compounds for use in preventing or ameliorating such an adverse response and compounds for use in treating a subject.

Claims

1. A method of screening a subject to determine the likelihood of adverse response to metal debris (ARMD) or adverse local tissue reaction (ALTR), said method comprising the steps of: i) contacting in vitro a subject's biological sample with means to detect the presence and/or absence of a marker associated with an adverse response ii) determining whether the subject has the marker associated with an adverse response iii) treating the subject with a therapeutic to ameliorate an adverse response.

2. A method as claimed in claim 1 wherein the marker associated with adverse reaction to metal debris (ARMD) and/or adverse local tissue reaction (ALTR) comprises a HLA genotype associated with an autoimmune inflammatory condition.

3. A method as claimed in claim 2 wherein the inflammatory condition is selected from the group comprising rheumatoid arthritis, coeliac disease and/or Crohn's disease.

4. A method as claimed in claim 2 wherein the marker associated with adverse reaction to metal debris (ARMD) and/or adverse local tissue reaction (ALTR) comprises any one or more of the following: HLA-DRB1*01, DRB1*04, DRB1*10(18, 19), DQA1*05:01/DQB1*02:01, HLA-DRB1*07, HLA-DRB1*0103, HLA-DRB1*04 and HLA-DRB3*0301.(20).

5. A method as claimed in claim 1, 2, 3 or 4 wherein the biological sample comprises a solid and/or fluid sample.

6. A method as claimed in claim 5 wherein the fluid sample comprises one or more of the following: a blood sample, saliva, skin cells or a blood extract sample. A method as claimed in claim 3, wherein the biological sample comprises skin cells from the buccal cavity.

7. A method of screening a therapeutic agent for use in the treatment of adverse reaction to metal debris (ARMD) and/or adverse local tissue reaction (ALTR) response to an implant, said method comprising the steps of contacting the agent with albumin-cobalt metalloprotein to determine if binding occurs.

8. A method of screening as claimed in any one of the previous claims wherein the adverse reaction is ALVAL.

9. A method as claimed in any one of the previous claims for use in screening a subject prior to, during and/or after an implant procedure.

10. A method of treating a subject to prevent an adverse reaction to metal debris (ARMD) and/or adverse local tissue reaction (ALTR) to an implant comprising administering an agent to ameliorate or prevent an adverse reaction.

11. A compound for use in the treatment of a subject to prevent an adverse reaction to metal debris (ARMD) and/or adverse local tissue reaction (ALTR) to an implant.

12. A method of screening for a compound for use in preventing or ameliorating the likelihood of adverse response to metal debris (ARMD) or adverse local tissue reaction (ALTR) in a subject, said method comprising the steps of identifying an compound that interferes with MHC mediated immune response.

Description

[0037] The present invention will now be described, by way of example only, with reference to the accompanying examples and figures, in which:

[0038] FIG. 1 is a table showing the result of experimentation to determine the affinity of certain alleles for ATCUN binder, MMBS binder and non-specific binding;

[0039] FIG. 2 shows the rank affinity for ATCUN and for MMBS;

[0040] FIG. 3 is a table summarising the results of data collected from the data set; and

[0041] FIG. 4 is a table showing the correlation between likelihood of a ALVAL response and Coeliac disease markers.

EXAMPLES

[0042] Excised tissues routinely undergo semiquantitative grading by a consultant histopathologist as previously described.(13, 17) From this pool of patients, three lists were created:

[0043] A group of patients with severe ALVAL with lowest wear rates (group A)

[0044] A group of patients with ALVAL exposed to high wear (group B)

[0045] A group of patients with no ALVAL findings (group C)

[0046] The HLA class I and class II genes of these patients were tested using next generation sequencing to a resolution of six digits.

[0047] The genotypes were compared between groups and with a background population of approximately 8500 patients from a previous study.

[0048] It became clear that certain DQA1/DQB1 combinations were more common in the group A patients compared to the group C and control group.

[0049] Identifying and isolating the group C patients with pain and low volumetric exposure wear showed an abnormal distribution of genetic haplotypes compared to the control population. These haplotypes were the same as those associated with the ALVAL response. This indicated that these patients were: 1. Captured prior to development of lymphocyte infiltration (ALVAL) or 2. that these genes are associated with hyperreactive macrophages, capable of causing adverse clinical sequelae without lymphocyte recruitment.

[0050] These combinations were entered into published software to determine which peptides the genetically encoded peptide binding grooves would bind with greater affinity.

[0051] The results indicate that there are certain, relatively common, genotypes which render a patient more susceptible to mounting a clinically adverse, lymphocytic response to metal debris. The alleles which showed the most obvious difference in distribution between patients who developed ALVAL, and those who did not, were found in the DQA1/DQB1 genetic loci. At the outset of this study, we focused on this area of the genome due to the clinical and pathological features that ALVAL shares with a number of inflammatory autoimmune/autoimmune like conditions, such as coeliac disease. The results appear to substantiate our hypothesis that these MHC molecules do indeed play a crucial role in the recruitment of lymphocytes in addition to the non-specific, largely macrophage dominated response to particulate debris.

[0052] The Immunogenetics of ALVAL

[0053] The Applicants focused on the divergence in the cellular response with reference to the development of ALVAL. We focused specifically on ALVAL for two major clinical reasons, the first being that the relationship between ALVAL and blood metal ion testing appears complex and ion concentrations do not appear to be of reliable diagnostic indicators in ruling the condition in or out. The second is that ALVAL appears to be an important, if not the most important, factor in the development of progressive and irreversible soft tissue damage.

[0054] The recruitment of lymphocytes to the area of macrophage driven inflammation lies in the handling and presentation of particulate debris to CD4+T cells by MHC II molecules on the membranes of APCs. Peptide-MHC binding is a prerequisite for T-cell immunogenicity and multiple studies have shown that there is a strong correlation between MHC peptide binding strength and peptide immunogenicity. Peptides that make stable peptide-MHC complexes accumulate on the cell surface and it has been shown that the total number of peptide-MHC complexes is important for T-cell activation.

[0055] When particulate matter is ingested by macrophages, peptide fragments which are produced in the lysosomes compete for the binding grooves of MHC molecules.

[0056] Applicants believe that that the antigen presented by APCs to initiate lymphocyte recruitment (the epitope) is a peptide derived from the breakdown of an albumin-Co metalloprotein. Further MHC II genes which differed in frequency between the ALVAL and non-ALVAL patient groups would show greater relative binding affinities for albumin derived peptides. Therefore, given the protection that younger age and male sex appears to confer, testosterone derived peptides might create competition with albumin for these binding sites.

[0057] Each patient has four potential DQA1/DQB1 combinations. It is therefore not ideal, when one is investigating the effect of a three-dimensional structure, to study only some of the building blocks in isolation i.e. by simply comparing alleleic frequencies of single genes between patient groups. By calculating theoretical binding affinities, produced a quantitative measure of a patient's genetic makeup which went beyond the classical approach of labeling a patient as being homozygous/heterozygous/lacking in an individual allele.

[0058] Applicants discovered that DQ molecules which are particularly suited to bind albumin fragments were significantly associated with the development of ALVAL. Conversely, DQ molecules which bind testosterone fragments with greater affinity were negatively associated with the development of ALVAL. Of particular significance, we believe, was that DQ isoforms in trans had relatively little influence on the statistical modeling compared to the cis combinations. Cis DQ isoforms are more easily formed, more numerous on cell membranes and are thought to be more important for T cell activation.

[0059] Albumin Binding and Antigen Presentation

[0060] Following antigen recognition and lymphocyte activation, chemokine release leads to the development of fluid exudates, with greater concentrations of albumin forming in the joint fluid. It is possible therefore that a vicious cycle is set in motion, with lymphocytes sensitised to an antigen which becomes present in ever greater quantities. An increase in the fraction of protein bound metal would also result in greater amounts of metal exiting the joint via the lymphatic system rather than via directly through the synovial membrane, as smaller solutes and ions are capable of doing.

[0061] Modulating Effects of Testosterone

[0062] Sex hormones play an important role in immune modulation. Furthermore, testosterone is present in the synovial fluid of healthy and arthritic joints in concentrations comparable to those of metal ions produced from low wearing MoM devices. A significant fraction of testosterone is albumin bound, meaning that particles digested by macrophages and dendritic cells are likely to contain varying amounts of Co, Cr, albumin and testosterone derived peptides. Levels of gonadal steroids in synovial fluid show an inverse relationship to age. Clearly, the modulation of the immune response by sex hormones is extremely complex. We suggest however that one of the reasons females and older patients may be more vulnerable to ALVAL might be due to their reduced synovial testosterone concentration and thus reduced competition for MHC binding spaces.