WO2001011371A2

WO2001011371A2 - Diagnosis of inflammation measuring il-8 in urine

Info

Publication number: WO2001011371A2
Application number: PCT/GB2000/002971
Authority: WO
Inventors: Ali Said Assad Taha
Original assignee: Ali Said Assad Taha
Priority date: 1999-08-07
Filing date: 2000-08-07
Publication date: 2001-02-15
Also published as: AU6455000A; WO2001011371A3

Abstract

A method of diagnosis of a medical condition of a mammal comprising detection of the level of Interleukin-8 (IL-8) in excreted urine of the mammal is described. The use of a detected level of Interleukin-8 in excreted urine of a mammal in the diagnosis of a medical condition of the mammal is also described. The method and use are particularly applicable in the diagnosis of inflammatory conditions such as ulcerative colitis, Crohn's disease, arthritis, cholecystitis, appendicitis, and urinary tract infections. The present invention provides a simple and non-invasive method and apparatus for detecting the level of IL-8 in a mammal, and using the detected level to diagnose, or in the diagnosis of, various medical conditions, particularly but not exclusively inflammatory conditions.

Description

"Method and Apparatus for Diagnosis"

This invention relates to a method and apparatus for diagnosis of medical conditions and in particular to a method and apparatus for non-invasive testing for the diagnosis of inflammatory conditions.

Inflammatory conditions can be chronic, such as chronic inflammatory bowel disease, chronic arthritis, or acute, such as appendicitis, colecystitis or acute surgical inflammatory conditions. Diagnosis of any inflammatory condition requires a number of very unpleasant and invasive tests. These tests are expensive and can only be performed in hospital by specialists.

Patients with chronic inflammatory conditions require long term follow up by both primary care physicians and hospital specialists. The natural history of such conditions comprises lull periods and episodes of increased activity. These episodes of increased activity, which may be referred to as 'flare-ups', often require intensive therapy. These episodes requiring more intensive therapy are not always easy to diagnosis. When such an episode is suspected, tests including sigmoidoscopy, colonoscopy, barium enemas, blood tests, radioisotopes, endoscopy, barium meals and histology are ordered. These tests are time consuming, expensive and must be performed by experts, usually in a hospital environment.

Inflammatory conditions are characterised by the accumulation of inflammatory cells including neutrophils in the inflamed tissue. Neutrophils play a major part in acute mucosa inflammation. For example, in active inflammatory bowel disease neutrophil turnover has been shown to be greatly increased. Neutrophils have a potent ability to produce toxic mediators such as reactive oxygen and nitrogen intermediates. Neutrophils also have a potent ability to produce cytokines .

Cytokines play a pivotal role in the pathogenesis of inflammation, and some of them have been used in the treatment of certain diseases. Interleukin 8 (IL-8) is a member of the α chemokine family of chemotactic cytokines. It is a potent chemo-attractant and activator of neutrophils. It is present in a wide variety of diseased states. Neutrophils are prominent producers of IL-8 in actively inflamed tissue. That is, tissue damage results from inflammatory cell infiltration. Inflammatory cell infiltration is facilitated by a number of mediators. The chief mediator is IL-8. Thus, patients with active disease have elevated tissue level of IL-8.

According to one aspect of the present invention, there is provided a method of diagnosis of a medical condition of a mammal comprising detection of the level of Interleukin-8 (IL-8) in excreted urine of the mammal.

The level of IL-8 in the excreted urine provides a direct indicator of the level of IL-8 in the mammal, and this can be used to diagnose, or be used in the diagnosis of, a medical condition of the mammal.

Preferably said method is applicable in the diagnosis of inflammatory conditions. Such conditions include at least ulcerative colitis, Crohn's disease, arthritis, cholecystitis, appendicitis, urinary tract infections, etc.

Said method may have application in the diagnosis of chronic inflammatory conditions, acute inflammatory conditions, and indeed non-specific inflammations and infections.

According to a second aspect of the present invention, there is provided the use of a detected level of Interleukin-8 (IL-8) in the excreted urine of a mammal in the diagnosis of a medical condition of the mammal .

According to a third aspect of the present invention, there is provided an apparatus for measuring the level of IL-8 in excreted urine.

Preferably said apparatus comprises means for immunoassay such as enzyme linked immunosorbent assay (ELISA) . Typically said apparatus is provided in kit form. Said apparatus may also be adapted for use in the laboratory. Said apparatus may be adapted for use by a patient. According to another aspect of the present invention, there is provided a method of detecting the level of IL-8 in the excreted urine of a mammal, which method uses measuring means or apparatus as hereinbefore defined, preferably an ELISA assay, wherein urine is added to the means or apparatus and a detection means determines either the or a relative level of IL-8, possibly in comparison with a standard level.

According to a further aspect of the present invention there is provided a method of diagnosis of a mammal comprising the steps of obtaining a sample of urine excreted by the mammal; testing the level of IL-8 in the urine; and comparing the level obtained against a standard level to determine whether it is significantly abnormal. Preferably said method includes the step of testing the level of IL-8 using immunoassay. More preferably said method includes the step of testing the level of IL-8 using enzyme linked immunoassay.

The present invention, i.e. the measurement of IL-8 in urine, can also be used to complement or as a replacement for measuring blood parameters of inflammation like acute phase substances, e.g. C- reactive protein, but with the advantage of being a non-invasive test.

Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings in which:

Fig 1 is a graphic representation of urinary levels of IL-8 in a control group;

Fig 2 is a graphic comparison of urinary levels of IL-8 in patients with inactive and active ulcerative colitis;

Fig 3 is a graphic comparison of urinary levels of IL-8 in patients with inactive and active Crohn's disease;

Fig 4 is a graphic comparison of urinary levels of IL-8 in patients with active and inactive rheumatoid arthritis; Fig 5 is a graphic comparison of urinary levels of IL-8 in patients with cholecystitis and a control group;

Fig 6 is a graphic comparison of urinary levels of IL-8 in patients with appendicitis and a control group;

Fig 7 is a graphic comparison of urinary levels of IL-8 in patients with urinary tract infection and a control group;

Fig 8 is a graphic comparison of urinary levels of IL-8 in patients with non-specific inflammation and a control group;

Fig 9 is a graphic comparison of urinary levels of IL-8 in children with an active inflammation/infection and a control group; and

Fig 10 is a graphic comparison of urinary levels of IL-8 in children with a viral illness and a control group.

I have found that the level of IL-8 excreted in urine is a diagnostic indicator of the presence or absence of overall disease activity. This has been confirmed by the following.

EXPERIMENTAL STUDIES Urinary Interleukin-8 Levels in patients with inflammatory bowel disease

The study involved adult patients who presented at hospital gastroenterology clinics with altered bowel habits, or for further assessment and follow up of previously diagnosed ulcerative colitis; Crohn's disease; or Irritable Bowel Syndrome (IBS) .

The initial diagnosis of chronic inflammatory bowel disease and its activity was based on clinical assessment and from sigmoidoscopic, colonoscopic, histological, and/or barium studies' findings. Clinical assessment included the frequency of diarrhoea stools, bleeding, pain, urgency, haemoglobin, ESR and CRP levels.

At such clinics, it is routine for patients to give a urine specimen for analysis for protein, blood, sugar etc. For this study, 1ml of each specimen was taken, given a code number, and frozen at -20°C until time of measurement of IL-8. Patients with urinary tract or other systemic infection, renal failure or who were excess consumers of alcohol, cigarettes or diuretics were excluded from the study. Note was taken of any drug therapy for bowel conditions.

DEMOGRAPHY

The urine specimens of the above group were tested for the level of IL-8 present using enzyme linked immunosorbent assay (ELISA) . The specimens were tested blind. That is, the specimens bore no indication of any clinical details, or of the basic diagnosis for the trial participant.

Full details of the ELISA method of the measurement of IL-8 are provided hereinafter.

The raw data of the IL-8 levels obtained is presented in Table 1 hereinafter and is illustrated graphically in Figures 1, 2 and 3.

The results of the assay were then statistically analyzed. The statistical analysis included multiple group comparisons using the Kruskall Wallis test, and, where appropriate, comparison between two groups using the Mann-Whitney test and 95% confidence intervals.

Results for inflammatory bowel testing

Kruskall allis Test, P = 0.001

All participants Control Group Median IL-8 level 9.20 pg/ml. Interquartile range 7.8 to 21.1 pg/ml.

Patients with Active disease Median IL-8 level 75.1 pg/ml. Interquartile range 24.4 to 205 pg/ml.

Patients with Inactive disease Median IL-8 level 20.1 pg/ml. Interquartile range 10 to 43.1 pg/ml.

Sub-Groups (by condition) Ulcerative colitis Patients with Active disease Median IL-8 level 104.3 pg/ml Interquartile range 16.1 to 196.0 pg/ml.

Patients with Inactive disease Median IL-8 level 12.3 pg/ml Interquartile range 6.3 to 26.0 pg/ml.

Crohn's disease Patients with Active disease Median IL-8 level 53.7 pg/ml Interquartile range 26.3 to 246.6 pg/ml

Patients with Inactive disease Median IL-8 level 12.0 pg/ml Interquartile range 8.8 to 37.0 pg/ml.

Mann-Whitney Test

Control versus Active disease P=0.001 95% confidence interval: 15.9, 126.6.

Control versus Inactive disease P=0.13 95% confidence interval: -1.2, 20.5.

Active disease versus Inactive disease P=0.005 95% confidence interval: 8.9, 103.2.

Active ulcerative colitis versus Inactive ulcerative colitis P =0.002 95% confidence interval: 37.5, 127.9.

Active Crohn's disease versus Inactive Crohn's disease P=0.025 95% confidence interval: 6.9,222.0. FURTHER EXPERIMENTAL STUDIES

Urinary Interleukin-8 Levels in Active and Inactive Rheumatoid Arthritis A total of 43 patients were studied. Twenty-one had active arthritis, including 4 men and 17 women, with a median age of 62 years. Twenty-two patients had inactive arthritis, including 9 men and 13 women, with a median age of 56 years.

Interleukin-8 levels in both groups are shown in Figure 4 , and the raw data are presented in the Table 2. Patients with active arthritis have higher levels compared with those with inactive disease.

Urinary Interleukin-8 Levels in Cholecystitis Compared with Controls Fifteen patients with cholecystitis were studied, including 3 men and 12 women, with a median age of 44 years. Twenty patients were in the control group, including 6 men and 14 women, with a median age of 48 years. They presented with non-specific abdominal pain (n=ll) and constipation (n=9) . Interleukin-8 levels in both groups are shown in Figure 5, and the raw data are presented in Table 3. Patients with cholecystitis have higher levels of IL- 8 than the controls. Urinary Interleukin-8 Levels in Appendicitis Compared with Controls Eight patients with appendicitis were studied, including 2 men and 6 women, with a median age of 29 years . Twenty patients were in the control group, including 6 men and 14 women, with a median age of 48 years. They presented with non-specific abdominal pain (n=ll) and constipation (n=9) . Interleukin-8 levels in both groups are shown in Figure 6, and the raw data are presented in Table 4. Patients with appendicitis have higher levels of IL-8 than the controls.

Urinary Interleukin-8 Levels in Urinary Tract Infection Compared with Controls Twelve patients with urinary tract infection were studied, including 4 men and 8 women, with a median age of 33 years. Twenty patients were in the control group, including 6 men and 14 women, with a median age of 48 years. They presented with non-specific abdominal pain (n=ll) and constipation (n=9) . Interleukin-8 levels in both groups are shown in Figure 7, and the raw data are presented in Table 5. Patients with urinary tract infection have higher levels of IL-8 than the controls.

Urinary Interleukin-8 Levels in Nonspecific Inflammation Compared with Controls Twelve patients with nonspecific inflammation were studied, including 2 men and 10 women, with a median age of 64 years . They were diagnosed as having diverticulitis (n=8) , and gastroenteritis (n=4) .

Twenty patients were in the control group, including 6 men and 14 women, with a median age of 48 years. They presented with non-specific abdominal pain (n=ll) and constipation (n=9) . Interleukin-8 levels in both groups are shown in Figure 8, and the raw data are presented in Table 6. Patients with non-specific inflammation have higher levels of IL-8 than the controls.

Interleukin-8 in Children (i) Urinary IL-8 Levels in Active Inflammation/ Infection Compared with Controls Routine urine samples were taken from 12 children with active inflammation/ infection, including 5 boys and 7 girls, with a median age of 6 years. They were diagnosed as having urinary tract infection (n=7) , bacterial meningitis (n=2) , tonsillitis (n=l) , gastroenteritis (n=l) , and appendicular mass (n=l) . The control group consisted of 10 children, including a boy and 9 girls, with a median age of 9 years. They were diagnosed as having nonspecific abdominal pain (n=7) , diabetes (n=2) , and nonspecific headache (n=l) . Interleukin-8 levels in both groups are shown in Figure 9, and the raw data are presented in Table 7. Subjects with active inflammation/ infection have higher levels of IL-8 than the controls.

Interleukin-8 in Children (ii) Urinary IL-8 Levels in Viral Illness Compared with Controls Routine urine samples were taken from 9 children with viral illness, including 5 boys and 4 girls, with a median age of 8 years . They were diagnosed as having viral upper respiratory tract infection. The control group consisted of 10 children, including a boy and 9 girls, with a median age of 9 years. They were diagnosed as having nonspecific abdominal pain (n=7) , diabetes (n=2) , and nonspecific headache (n=l) . Interleukin-8 levels in both groups are shown in Figure 10, and the raw data are presented in Table 8. The levels were similar in the two groups, and no significant differences were found.

ANALYSIS

From the results of the first study, it can been seen that patients with active inflammatory bowel disease have significantly higher levels of urinary IL-8 than patients with inactive disease. Patients with active inflammatory disease have higher levels of urinary IL-8 than the control group. IL-8 levels in the control group are not statistically different from those in patients with inactive inflammatory bowel disease.

When the group of patients with active inflammatory bowel disease are sub-divided into those having active ulcerative colitis and those having active Crohn's disease, it is also seen that both groups have higher levels of urinary IL-8 than those in the respective sub-group with inactive disease. This is illustrated in Fig 2 and Fig 3.

The results of this study show that it is possible to diagnose the likely presence of active inflammatory bowel disease from the urinary level of IL-8.

From the further studies described hereinbefore, it has been shown that the Interleukin-8 levels in the urine were also elevated in active inflammation in a range of conditions, in both adults and children. In adults, these included: - Ulcerative colitis - Crohn's disease - Rheumatoid arthritis - Cholecystitis - Appendicitis

- Urinary tract infection - Nonspecific inflammation/ infection (diverticulitis, gastroenteritis) . Similar results were found in children with active inflammatory processes (with the exception of viral illness causing upper respiratory tract infection) . This is because IL-8 is likely to rise in conditions characterised by neutrophilic and macrophage infiltration.

It is thus clear from the foregoing results that the level of IL-8 in excreted urine is a measure of systemic inflammation. That is, flare-ups in patients known to have e.g. chronic inflammatory bowel disease can be simply and non-invasively diagnosed by determining the level of urinary IL-8 for comparison with the normal level of IL-8 for inactive inflammatory disease, or with IL-8 levels in healthy patients.

This test is comparatively inexpensive, painless and may overt unnecessary invasive testing or perhaps even unnecessary surgical intervention.

Patients who are unwell have to suffer a wide variety of unpleasant and invasive tests. Non-invasive testing methods are particularly attractive for use on paediatric patients. Children presenting with any kind of medical condition are usually easily distressed and non-invasive testing ensures that their distress can also be kept to a minimum.

As previously described this method of diagnosis has application in chronic inflammatory conditions. Examples of such conditions are chronic bowel disease and chronic arthritis.

In addition, this method of diagnosis has application in acute inflammatory conditions where the level of IL-8 can be compared with the expected level in healthy patients. Examples of such conditions are acute surgical inflammation, appendicitis, colecystitis, pancreatitis and non-specific abdominal pain.

This method of diagnosis has wide application particularly in paediatric patients for both inflammatory and non-inflammatory conditions. That is, this method of diagnosis can be used in place of blood tests, barium meals, radioisotope tests, endoscopy or colonoscopy, at least in the first instance. The level of IL-8 indicating the presence of active disease or acute inflammation obviously differs in adults and in paediatric patients.

The results of a test measuring the level of IL-8 in urine are quickly and cheaply obtained using enzyme linked immunosorbent assay. The results have application as a diagnostic tool, or as an adjective aid to diagnosis.

As a particular example, it is frequently the case that acute appendicitis is mis-diagnosed. That is, the histology of a resected appendix is frequently seen to be normal. The level of the IL-8 in urine is a reliable predictor of presence or absence of intra- abdominal inflammatory process, and is available quickly and cheaply using the above described test. The absence of evidence of intra-abdominal inflammatory process would prevent unnecessary surgical intervention.

This test is also of use in the self management of chronic inflammatory conditions. That is, an apparatus for measurement of the urinary level of IL- 8 will be provided in kit form analogous to a pregnancy test. This will allow patients with known chronic inflammatory conditions to monitor the need for medical intervention. That is, patients diagnosed with chronic inflammatory conditions can confirm a suspicion that their disease may have become active simply by testing a sample of their urine to check if the IL-8 level is significantly high.

The test could be conducted in hospital laboratories on urinary samples as a matter of routine. The urinary level of IL-8 would give doctors confidence that surgical intervention or increased drug therapy is in fact warranted and necessary.

The method of diagnosis may be contra- indicated in a small number of conditions including renal failure, or where a patient is known to abuse substances such as alcohol or drugs. The present invention provides a simple and non- invasive method and apparatus for detecting the level of IL-8 in a mammal, and using the detected level to diagnose, or in the diagnosis of, various medical conditions, particularly but not exclusively inflammatory conditions.

TEST DATA

Table 1

Interleukin-8 raw data:

A Controls

B Active ulcerative colitis

C Inactive ulcerative colitis

D Active Crohn's Disease

E Inactive Crohn' s Disease

Table 2

Active Rheumatoid Arthritis:

3 203 70 103 183 13 106 239 227 59 47 93 100 16 17 14

51 404 135 311 30

Inactive Rheumatoid Arthritis:

290 3 10 7 3 5 88 41 7 16 3 68 28 6 24 50 9 138 37 3

6 12

Table 3

Controls :

3 3 22 35 15 3 15 6 3 37 43 11 41 3 3 3 11 10 3 3

Cholecystitis :

176 48 67 87 321 104 1883 41 184 110 438 379 35 107

83 Table 4 Controls : 3 3 22 35 15 3 15 6 3 37 43 11 41 3 3 3 11 10 3 3 Appendicitis: 44 272 74 180 319 430 48 44

Table 5 Controls: 3 3 22 35 15 3 15 6 3 37 43 11 41 3 3 3 11 10 3 3 Urinary tract infection: 200 1594 551 233 1114 210 616 619 352 1597 343 2641

Table 6 Controls: 3 3 22 35 15 3 15 6 3 37 43 11 41 3 3 3 11 10 3 3 Non-specific Inflammation: 10 265 187 88 471 328 68 55 568 194 319 57

Table 7 Controls: 13 15 3 19 5 6 3 7 41 43 Active inflammation/infection: 204 64 244 423 40 1701 102 1243 61 72 245 193

Table 8 Controls: 13 15 3 19 5 6 3 7 41 43 Viral illness: 9 3 7 5 3 3 12 164 60

ELISA Method of Measurement for Cytokines

ELISA plates were passively coated with captures antibody overnight at 4°C. mA/b was made up to 1ml (500μg/ml) and 40μl was added to 5.1 ml PBS pH7.2 (4μg/ml) . This was scaled up as necessary. 50μl was added per well. The plates were shaken to ensure even coverage of wells.

After 0/N incubation at 4°C, the plates were washed once with cold tap water and coating solution was added (lOOμl per well) . They were left for half an hour at room temperature and then flicked out. The plates were air dryed and stored at +4°C or at -20°C.

The ELISA' s use paired antibodies. Capture antibody was used to coat the plates, then biotinylated detection antibody was used to detect the bound std. This was quantified by subsequently introducing streptavdin peroxidase, then measured peroxidase by a colour reaction. The plates were washed before use. The standard and samples were added to the wells in a total of lOOμl and it was ensured that the standards were diluted in a similar medium to the samples. The plates were then sealed using the adhesive plate sealers, and were incubated overnight at 4°C. After the first incubation the plates were washed four times with washing buffer. The stock detection antibody was made up to 1ml (50μg/ml) . Then 5μg of stock was added to 6.5 ml assay buffer and used at 60μg/well. Again the plates were covered with a plate sealer and incubated for 45 minutes, and were then shaken at room temperature . The plates were then washed four times with wash buffer. Then lOOμl of diluted (1:1000) streptavidin (Boehringer) was added to each well at a concentration of 0.2U/ml (lOOμl of 1:1000 dilution of 200U/ml was used). The streptavidin peroxidase was incubated for 20-30 minutes and was shaken at room temperature.

The plates were again washed four times by wash buffer, then 200μl of substrate was added to each well. The substrate was TMB prepared from stable solutions consisting of: A. Urea-hydrogen peroxide 0.3g per 50ml of 50mM sodium acetate buffer pH 6.0 B. Tetramethyl benzidine 2mg/ml in DMF.

2ml of A and 2ml of B were then added to 20ml of lOOmM sodium acetate buffer, pH 6.0. The plates were left for approximately 20 minutes until the colour developed (with an aim for a final OD of approximately 1) , then 50μl of 2N sulphuric acid was added to quench development of colour. Measurement was taken within 30 minutes of quenching.

ELISA Buffer lOOmM Tris pH 7.2 150 mg/1 2-methylisothizolone (Boehringer) 150 mg/1 Bromo nitro dioxane (Boehringer) 2mg/ml BSA Sigma A3803 Phenol red 300μl 0.5% solution per litre Sodium Chloride 9g/litre EDTA 2mM 4ml/litre of 0.5 Molar stock Tween 20 0.05% (5ml/l of 10% stock) Final pH should be 7.2

Preservative: Polyvinyl pyrollidone 2% BSA 5mg/ml Boehringer preservatives, o.l% of stock EDTAS 5mM

Claims

1. A method of diagnosis of a medical condition of a mammal comprising detection of the level of Interleukin-8 in excreted urine of the mammal.

2. Use of a detected level of Interleukin-8 in the excreted urine of a mammal in the diagnosis of a medical condition of the mammal.

3. A method or use as claimed in Claim 1 or Claim 2 wherein the medical condition is an inflammatory condition.

4. A method or use as claimed in Claim 3 wherein the inflammatory condition is a chronic inflammatory condition, an acute inflammatory condition or a non-specific inflammation or infection.

5. A method or use as claimed in Claim 3 or Claim 4 wherein the condition is one of the group selected from ulcerative colitis, Crohn's disease, arthritis, cholecystitis, appendicitis, urinary tract infections.

6. A method of detecting the level of Interleukin-8 in the excreted urine of a mammal wherein urine is added to means for measuring the level of Interleukin-8.

7. A method as claimed in Claim 6 wherein the measured level of Interleukin-8 is compared with a standard level.

8. A method as claimed in Claim 6 or Claim 7 wherein the measuring means is an immunoassay.

9. A method as claimed in Claim 8 wherein the assay is an enzyme-linked immunosorbent assay.

10. A method of diagnosis of a mammal comprising the steps of obtaining a sample of urine excreted by the mammal; testing the level of Interleukin-8 in the urine; and comparing the level obtained against a standard level to determine whether it is significantly abnormal.

11. A method as claimed in Claim 10 wherein the method further includes the step of testing the level of Interleukin-8 using immunoassay.

12. A method as claimed in Claim 11 which further includes the step of testing the level of Interleukin-8 using enzyme-linked immunoassay.

13. Apparatus for measuring the level of Interleukin-8 in excreted urine of a mammal.

14. Apparatus as claimed in Claim 13 where the said apparatus comprises means for immunoassay.

15. Apparatus as claimed in Claim 15 wherein the immunoassay is an enzyme-linked immunoassay.

16. Apparatus as claimed in any one of Claims 13-15 wherein said apparatus is provided in kit form.

17. Apparatus as claimed in any one of Claims 13-16 wherein the apparatus is adapted for use in a laboratory.

18. Apparatus as claimed in any one of Claims 13-17 wherein the apparatus is adapted for use by a patient.

19. Apparatus as claimed in any one of Claims 13-18 wherein the apparatus is used for measuring the level of Interleukin-8 in excreted urine of a mammal for the diagnosis of an inflammatory condition of the mammal.

20. A non-invasive test for the level of an acute phase substance in the blood of a mammal wherein the level of Interleukin-8 in the urine of the mammal is measured.

21. A test as claimed in Claim 20 wherein the acute phase substance is C-reactive protein.

22. Use of a measurement made by a test as claimed in Claim 20 or Claim 21 in the diagnosis of a medical condition of the mammal.

3. Use as claimed in Claim 22 wherein the medical condition is an inflammatory condition.