WO2008038022A1

WO2008038022A1 - Test for h. pylori infection

Info

Publication number: WO2008038022A1
Application number: PCT/GB2007/003703
Authority: WO
Inventors: Kevin Peter Moore; Alireza Mani
Original assignee: UCL Business Ltd
Current assignee: UCL Business Ltd
Priority date: 2006-09-28
Filing date: 2007-09-28
Publication date: 2008-04-03
Anticipated expiration: 2009-03-28
Also published as: EP2078200A1; GB0619166D0

Abstract

The invention relates to detection of urinary 3-nitro-hydroxyphenylacetic acid as a biomarker for H. pylori infection. Assay kits and devices for detection of urinary 3-nitro-hydroxyphenylacetic acid are also described.

Description

Test for H. pylori infection

Field of the invention

The invention relates to a novel biomarker for Helicobacter pylori infection in human subjects. More specifically, the invention relates to the identification of urinary 3-nitro-hydroxyphenylacetic acid as a marker for H. pylori infection and to a test for H. pylori infection based on detection of 3-nitro-hydroxyphenylacetic acid in urine.

Background to the invention

Helicobacter pylori infection is one of the most common chronic bacterial infections of human populations worldwide. H. pylori infection occurs in approximately 30% of the UK population and is recognised as the major cause of gastritis and peptic ulcer disease. Infection also increases the risk of gastric carcinoma in chronic carriers by 4-5 fold.

Currently available tests for diagnosis of H. pylori infection include endoscopy and gastric biopsy, involving attendance at hospital, or C¹³-urea breath analysis (Perri

F. et al. , Am J Gastroenterol. (2002) 97, 2756-62) . Both of these tests are labour intensive and relatively expensive. Serological screening and tests on faecal samples are also available. However, serological tests remain positive even after successful treatment of the infection, and tests on faeces are impractical. Accordingly, there is a clear need for a simple screening test that is either diagnostic, or which identifies patients likely to benefit from more invasive diagnostic tests, or which can be used to monitor whether treatment has been successful in eradicating H. pylori infection. There has recently been described a new metabolic pathway by which circulating or salivary derived p- hydroxyphenylacetic acid, a product of tyrosine metabolism, is nitrated by reactive nitrogen species to form 3-nitro-p- hydroxyphenylacetic acid (3-nitro-HPA) , which is excreted in urine (Mani A. R. et al. , Biochem J. (2003) 374, 521-527). 3-nitro-HPA can also be formed by a second pathway involving metabolism of nitrotyrosine, which is itself formed by direct nitration of tyrosine. It has been determined, however, that the majority of urinary 3-nitro-HPA is formed by nitration of salivary or gastric p-hydroxyphenylacetic acid, which is then absorbed and excreted in urine, rather than by nitration of endogenous circulating p- hydroxyphenylacetic acid, or the alternative pathway via nitrotyrosine metabolism (Pannala A. S. et al., Free Radic Biol Med. (2006) 15; 41, 896-901) .

Summary of the invention

Initially it was postulated that urinary 3-nitro-HPA would serve as a general time-integrated biomarker of the formation of reactive nitrogen species in vivo, based on the erroneous assumption that the majority of urinary 3-nitro- HPA is derived from nitrotyrosine metabolism. The inventors now know that nitrotyrosine metabolism only accounts for a minor fraction of 3-nitro-HPA excreted in the urine, and that the vast majority is formed by nitration of para- hydroxyphenylacetic acid in the stomach. In fact, the present inventors have unexpectedly found that the nitration of salivary or gastric p-hydroxyphenylacetic acid is markedly up-regulated in the stomach of patients with Helicobacter pylori infection and this is reflected in elevated levels of 3-nitro-HPA in the urine of infected patients, as compared to "normal" individuals not infected with H. pylori. Urinary 3-nitro-HPA thus provides a useful specific biomarker for the presence of H. pylori infection. Measurement of urinary 3-nitro-HPA can form the basis of a simple and sensitive assay for H. pylori infection which avoids many of the drawbacks of the currently available tests.

According to a first aspect of the invention there is provided a method of testing for Helicobacter pylori infection in a human subject which comprises testing a sample comprising urine from the subject for the presence of 3-nitro-hydroxyphenylacetic acid, wherein the presence of an elevated level of 3-nitro-hydroxyphenylacetic acid, compared to control subjects who do not have Helicobacter pylori infection, is taken as an indication that the subject is infected with Helicobacter pylori.

In one embodiment the method of the invention may be based on detection of urinary 3-nitro-hydroxyphenylacetic acid produced by nitration of endogenous salivary or gastric p-hydroxyphenylacetic acid to form 3-nitro- hydroxyphenylacetic acid which is then excreted in the urine. The inventors have observed that the nitration of endogenous salivary or gastric p-hydroxyphenylacetic acid is markedly up-regulated in individuals infected with H. pylori. The inventors postulate that nitration of endogenous salivary or gastric p-hydroxyphenylacetic acid is caused by reactive nitrogen species formed during H. pylori infection. Hence, an elevated level of urinary 3-nitro- hydroxyphenylacetic acid provides a biomarker for H. pylori infection. The invention further provides kits and devices for detecting urinary 3-nitro-hydroxyphenylacetic acid which can be used in the method of the invention.

Brief description of the drawings

Figure 1 illustrates the biochemical pathways for the formation of 3-nitro-HPA in human subjects. Tyrosine can be nitrated by reactive nitrogen species (RNS) such as peroxynitrite to form nitrotyrosine. Nitrotyrosine is metabolised to several products. The major urinary metabolite of nitrotyrosine is 3-nitro-HPA (designated NHPA in the Figure) . The majority of urinary 3-nitro-HPA is formed by direct nitration of salivary/gastric p- hydroxyphenylacetic acid (PHPA) , which is itself formed as a major product of tyrosine metabolism.

Figure 2 illustrates urinary excretion of 3-nitro-HPA (μg) in both normal healthy volunteers (n=16) and patients known to be infected with H. pylori (n=12) measured over a 24hr period for three consecutive days. Day 1 was on a low nitrate diet, day 2 on a high nitrate diet and day 3 a high nitrate diet with gastric acid suppressed by omeprazole. It can be clearly seen that there is a marked elevation of the urinary excretion of 3-nitro-HPA (NHPA) in patients with H. pylori infection compared to healthy controls.

Detailed description of the invention

The invention provides a method of testing for the presence of Helicobacter pylori infection in human subjects which involve testing for 3-nitro-hydroxyphenylacetic acid (3-nitro-HPA) in a sample comprising urine from the subject under test, wherein urinary 3-nitrohydroxyphenylacetic acid is a biomarker for infection with Helicobacter pylori. 3- nitro-HPA is generated by nitration of endogenous salivary or gastric p-hydroxyphenylacetic acid in the presence of reactive nitrogen species which form during H. pylori infection. Hence, this method requires only the collection of a suitable urine sample which can be tested for the presence of 3-nitro-HPA as a biomarker for H. pylori infection.

The method of the invention can be carried out in vitro on a sample (also referred to herein as the "test sample") which must comprise urine from the subject under test. In certain embodiments additional components may be added to the subject's urine to form the test sample. Such additional components may include (but are not limited to) buffers, diluents, preservatives, stabilisers or other components required for long term storage. In other embodiments the test sample may consist only of urine from the test subject. The latter would typically be preferred in the case of simple point-of-care testing. A urine sample may be tested immediately after it is given (or tested midstream) or, if required, the test sample comprising or consisting of urine from the test subject may be stored for a period of time and/or concentrated prior to testing, or re-testing. In particular, the urine may be frozen and thawed or freeze-dried and reconstituted prior to testing.

Testing for the presence of 3-nitro-HPA in the test sample may be carried out using any suitable technique, the invention is not intended to be limited with respect to the methodology used to detect/measure 3-nitro-HPA in the test sample.

Suitable techniques for detecting/measuring 3-nitro-HPA include, but are not limited to, analytical techniques such - S -

as gas chromatography mass spectrometry (GCMS) as described in the accompanying example, LCMS or spectrophotometry. Such techniques may involve derivatisation of 3-nitro-HPA present in the test sample prior to the analysis. In other embodiments of the invention detection of 3- nitro-HPA may be based on a specific binding assay. In particular, 3-nitro-HPA may be detected using an immunological technique, for example an immunoassay using an antibody specific for 3-nitro-HPA, or a fragment thereof which retains specific binding capability. In this context the term "specific" refers to immunological specificity. The general features of immunoassays, for example ELISA, radioimmunoassays, immunochromatographic assays and the like, are well known to those skilled in the art (see Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996) . Immunoassays for the detection of small molecules such as 3-nitro-HPA generally require the use of at least one antibody (or antibody fragment) that exhibits specific immunological reactivity with the molecule of interest. Depending on the format of the assay the antibody may be immobilised on a solid support . A sample to be tested for the presence of the molecule of interest is brought into contact with the antibody and if the molecule of interest is present in the sample it will immunologically react with the antibody to form complexes which may then be detected or quantitatively measured by any suitable means, e.g. based on colorimetric, fluorescent or radioactive labelling of the complex. Binding agents other than antibodies and fragments thereof can also be used if they display binding specificity for 3- nitro-HPA. Antibodies specific for 3-nitro-HPA may be raised using standard techniques available in the art. One approach which may be used in order to raise antibodies to a small immunogen, such as 3-nitro-HPA, is based on coupling of the small immunogen (e.g. 3-nitro-HPA) to a carrier protein which facilitates generation of antibodies specific for the small immunogen. Suitable carrier proteins include lysozyme, keyhole limpet haemocyanin, purified protein derivative or albumin etc. 3-nitro-4 hydroxyphenylacetic acid can be coupled to a carrier protein using carbodiimide and hydroxysuccinimide. The resulting conjugate may then be used as a challenging antigen in a suitable host animal in order to raise antibodies immunologically specific for 3- nitro-HPA. Immunoassays specific for 3-nitro-HPA may also be based on the use of antibody fragments which retain immunological specificity for 3-nitro-HPA. Suitable antibody fragments which may be used include, inter alia, F(ab')₂ fragments, scAbs, Fv and scFv fragments etc. Ideally antibodies or antibody fragments specific for 3-nitro-HPA should exhibit little or no cross-reactivity with p-hydroxyphenylacetic acid (PHPA) .

Depending on the format of the assay in which they are to be used, antibodies or antibodies specific for 3-nitro- HPA may be labelled to facilitate detection. Suitable detectable label include, for example, radiolabels, enzymatic labels, including enzymatic labels which permit colorimetric detection (e.g. horseradish peroxidase, etc), redox labels or particulate labels (e.g. colloidal gold) . The format of the binding assay is not limited and could include, for example, assays performed in standard microtiter plates, test strips, lateral flow devices, dipsticks and the like, the latter being generally preferred for point-of-care testing or home testing kits. Binding assays, including immunoassays, may be performed in a competitive format, or sandwich format or can be based on direct detection of a labelled binding agent (e.g. labelled antibody or antibody fragment) bound to 3-nitro-HPA.

In other embodiments of the invention, testing for urinary 3-nitro-HPA may be based on a specific chemical reactions with 3-nitro-HPA. Again such an assay could take the form of a test strip or dipstick test suitable for point-of-care or home testing, or may be carried out remotely using specific reagent kits .

In all embodiments of the assay of the invention, a positive result, i.e. a determination that H. pylori infection is present, is indicated by an elevated level of urinary 3-nitro-HPA as compared to control subjects who are known to be free of H. pylori infection (e.g. subjects negative for H. pylori on the basis of accepted clinical criteria, such as CLO test and/or C¹³ -urea breath test) . The method may involve a quantitative determination of the amount of 3-nitro-HPA present in the test sample from the test subject, the result of which can be compared to a predetermined cut-off value. Other tests may be based on a simple yes/no determination. Test strips, dipstick tests or the like may include an internal standard which would enable the user to immediately determine (e.g. by visual inspection) whether the test result is positive or negative without the need to calculate the absolute amount of 3- nitro-HPA present. The method of the invention may include suitable controls or calibrators to take into account background nonspecific levels of urinary 3-nitro HPA, for example 3-nitro HPA derived from metabolism of nitrotyrosine . All human subjects produce background low levels of urinary 3-nitro HPA by metabolism of nitrotyrosine, irrespective of H. pylori infection status. However, the pathway by which 3- nitro HPA is formed by metabolism of nitrotyrosine is separate from the pathway by which 3-nitro HPA is produced by direct nitration of salivary or gastric p- hydroxyphenylacetic acid (PHPA) . The present inventors have observed that (i) the majority (typically greater than 90%) of urinary 3-nitro HPA is in fact formed by the direct nitration of salivary or gastric PHPA, and (ii) this pathway is markedly up-regulated in H. pylori infection. In contrast, the alternative pathway by which 3-nitro HPA is formed by metabolism of nitrotyrosine (see Fig.l) typically accounts for less than 10% of urinary 3-nitro HPA.

The invention also relates to assay kits or devices for detection of urinary 3-nitro-hydroxyphenylacetic acid which can be used in the method of the invention. In particular, the invention provides assay kits comprising at least one antibody or antibody fragment immunologically specific for 3-nitro-hydroxyphenylacetic acid. Such kits may include further reagents needed to perform an immunoassay for 3- nitro-hydroxyphenylacetic acid, such as for example secondary antibodies . The invention also provides a dipstick device, test strip or lateral flow device for detection of urinary 3- nitro-hydroxyphenylacetic acid. Such dipstick devices, test strips and lateral flow devices may be based on a specific binding assay for detection of 3-nitro-HPA, in which case the dipsticks, test strips or lateral flow device will comprise at least one specific binding reagent specific for 3-nitro-HPA. Suitable specific binding reagents include antibodies or antibody fragments immunologically specific for 3-nitro-HPA. The general features of dipstick tests, test strips and lateral flow devices embodying binding assays specific for a particular analyte are known in the art. Such devices may be based on a competitive binding assay format, or a sandwich binding assay format or even direct detection of a labelled specific binding reagent which binds the analyte of interest, i.e. 3-nitro-HPA.

Clinical utility of the test

The method described herein may be used in a variety of clinical situations. In non-limiting embodiments, any of the methods described herein can be used diagnostically, for example to confirm whether H. pylori infection is present in a patient who presents with symptoms consistent with peptic ulcer disease, including but not limited to gastric irritation, dyspepsia or indigestion, or if H. pylori infection is suspected for any other reason. In this context, it is envisaged that the test may be used as a tool for point-of-care testing, for example in a GP surgery, outpatients clinic or pharmacy, allowing a diagnosis to be made and appropriate treatment to be prescribed in a single consultation. In this context it would be preferred that the methodology used to test for the presence of 3-nitro-HPA provide an immediate indication of a positive result. The invention also contemplates home-testing kits for detection of urinary 3-nitro-HPA.

In other non-limiting embodiments, the method described herein may be used as a screening tool, for example to screen a whole population of test subjects, who may be asymptomatic for H. pylori infection or any associated condition, in order to identify those subjects within the population who do in fact carry an H. pylori infection, or to identify those subjects requiring further investigation with more invasive test procedures in order to confirm the presence of H. pylori. Infected subjects may then be selected for appropriate treatment to eradicate the infection.

In further non-limiting embodiments, the method according to the invention may be used as a tool to monitor the course of an infection with H. pylori, and in particular to monitor response to treatment. A major advantage of the method of the invention as compared to prior art tests for H. pylori infection is that it is non-invasive, requiring only that the test subject provide a sample of urine each time the method is to be carried out. Accordingly, the test may be repeated as often as necessary in order to monitor response to treatment and as a follow-up to check that the infection has not recurred. This can be done with minimal inconvenience and discomfort to the patient. The simple nature of the method is particularly suited to point-of-care and home testing. One important aspect of the invention therefore relates to provision of home testing kits and point-of-care testing kits for detection of urinary 3-nitro- HPA.

The invention will be further understood with reference to the following non-limiting experimental examples.

Example 1 - measurement of urinary 3-nitro-HPA as a biomarker for Helicobacter pylori infection

Studies were carried out on 16 normal volunteers and 12 patients with proven E. pylori infection (on the basis of a positive CLO test or a C¹³-urea breath test) . All H. pylori subjects were ambulant outpatients, and none were taking any proton pump inhibitors or antibiotics. Urine was collected over a 24 hour period (results are expressed as a concentration rather than as excretion rate per hour) on 3 successive days. On the first day all subjects consumed a low nitrate diet, on day two they consumed a high nitrate diet, since dietary nitrate can increase salivary nitrite and the nitrating potential of the gastric milieu. On day three subjects continued the high nitrate diet but took omeprazole, a proton pump inhibitor, sufficient to suppress gastric acid secretion as confirmed by measurement of gastric juice pH.

3-nitro-HPA was measured in urine using a gas chromatography mass spectrometry (GCMS) based assay, as described in Mani et al. Biochem. J. (2003) 374, 521-527, and in Dynamic assessment of nitration reactions in vivo. A. R. Mani and K. P Moore. Methods in Enzymology, 2005: 396: 151-159., the contents of which are incorporated herein in their entirety by reference .

As illustrated in Figure 2, urinary 3-nitro-HPA was markedly increased in patients with Helicobacter pylori infection and this was unaffected by alterations in dietary nitrate or gastric pH.

Example 2 - production of 3-nitro-HPA immunogenic conjugate

l-ethyl-3- (3-diτnethylaminopropyl) carbodiimide (EDCI) was pre-reacted with N-hydroxysuccinimide in buffer at pH 4.6, followed by the addition of a 4-fold excess of 3-nitro-p- hydroxyphenylacetic acid. Excess unreacted reagents were reduced with mercaptoethanol , and the resulting solution added to lysozyme in water, and left for 2-3 hours at room temperature . The final product was dialysed against water for 2 days, and then analysed by mass spectrometry. Two molecules of 3-nitro-p-hydroxyphenylacetic acid were coupled to each molecule of lysozyme . This method can be used to generate a conjugate with a protein which can then be used as an immunogen to generate antibodies specific for 3-nitro- p-hydroxyphenylacetic acid.

Claims

1. A method of testing for Helicobacter pylori infection in a human subject which comprises testing a sample comprising urine from the subject for the presence of 3- nitro-hydroxyphenylacetic acid, wherein the presence of an elevated level of 3-nitro-hydroxyphenylacetic acid, compared to control subjects who do not have Helicobacter pylori infection, is taken as an indication that the subject is infected with Helicobacter pylori.

2. A method for diagnosis of Helicobacter pylori infection in a human subject presenting with symptoms or signs suggestive of peptic ulcer disease or for any reason in which Helicobacter pylori infection is suspected, which method comprises testing said human subject for Helicobacter pylori infection using the method of claim 1.

3. A method of screening a population of substantially asymptomatic human subjects to identify those carrying

Helicobacter pylori infection, which method comprises testing said population of human subjects for Helicobacter pylori infection using the method of claim 1.

4. Use of urinary 3-nitro-hydroxyphenylacetic acid as a biomarker for infection with Helicobacter pylori in human subjects.

5. An assay kit for detection of urinary 3-nitro- ° hydroxyphenylacetic acid comprising at least one antibody or antibody fragment immunologically specific for 3-nitro- hydroxyphenylacetic acid.

6. A dipstick, test strip or lateral flow device for detection of urinary 3-nitro-hydroxyphenylacetic acid, comprising at least one antibody or antibody fragment immunologically specific for 3-nitro-hydroxyphenylacetic acid.