Final ReportSummary - KINACEPT (Novel anti-inflammatory compounds for autoimmune diseases)
KINACEPT sets out to investigate the clinical application of novel inhibitors of the MAP kinase family. These are compounds of diverse clinical potential but, to date, limited application due to apparent complexity in the biochemistry of the target in the context of disease.
Our project has had the following key goals:
- Are their novel indications for our compounds, and if so, which models and test systems would demonstrate this utility most convincingly?
- If there are novel indications, how can we identify patients most likely to benefit from the use of our compounds?
- Which of these compounds, and with which general selectivity would be most useful?
To reach these goals, the partners delivered the following key results.
New indications
The team focused on novel indications found that human nephritis is specifically reliant on p38alpha and that this disease is, therefore a straightforward and specific unmet need for which a trial with our lead substances can be rationalised.
The team focusing on arthritis demonstrated that there is only one animal model that recapitulates central aspects for advanced human arthritis and that this, therefore, is the appropriate model for validating the compounds.
The team focusing on IBD observed both positive and negative effects of the imidazole derived p38 targeted substances in human tissue biopsy and concluded that the sum of these effects is unlikely to be beneficial.
The team focusing on the use of macrolide-based inhibitors, in contrast, observed very clear positive effects of the substance in human Crohn's disease lesion biopsy material. These data show that one compound class is at least worthy of progression in this indication.
Synovo demonstrated the relevance of certain additional classes of p38 inhibitor to cancer and has now prepared two molecules for the clinic.
Patient stratification and selection
- Blood and hair can be used to determine patient status, however, biopsy tissue remains the ideal medium.
- In IBD disease types, biopsy was preferred and appeared more predictive.
- In arthritis, the presence of tertiary lymph nodes or aggregates in the arthritic synovium is considered to be the best selection criterion
- Cytokines were less intrinsically predictive, however, relative effect on TNF vs. IL-10 was considered most important.
Compound selection
As of the end of P2, the following compounds were slated for clinical trial:
SYD003 - pancreatic cancer
SYD004 - cancer and or advanced rheumatoid arthritis
SYD005 - lung cancer
SYD001 - Crohn's disease, and or rheumatoid arthritis.
Project context and objectives:
Autoimmune diseases have varied aetiology but many elements in common, notably a dependence on specific cytokines to mediate pro-inflammatory signals.
In recent years, the ability to selectively suppress cytokines via protein receptor molecules or antibodies has demonstrated that specific cytokine suppression can dramatically improve signs of disease in both rheumatoid arthritis and inflammatory bowel diseases. However, for reasons of cost and long-term loss of efficacy, small molecule drugs are preferred for chronic therapy.
The applicants have developed a new class compounds that suppress the production of the cytokine TNFalpha via inhibition of the signalling kinase known as p38 alpha. By phosphorylating its target, p38 kinase contributes to the perpetuation of pro-inflammatory signals emanating from outside the cell. Inhibition of this signal causes a reduction in cytokine and specifically TNFalpha release.
Like many drug targets, it now appears that p38 kinase contributes to inflammatory states in different ways under different conditions. In certain patients, the isoforms of p38 kinase that are over expressed vary, and in others, the overall level of p38 expression varies.
Given this variation in the target expression, it is not surprising that responses to p38 kinase inhibitors in clinical trials has been varied leading to the requirement for relatively large patient collectives in order to see effects.
Large collectives of this kind are not within the scope of small companies and so, instead, we are seeking to take a more informed and selective approach to patient screening before entering clinical trials.
Having established a variety of lead molecules, we wish to characterise and optimise these compounds using models based on human explant tissues. Compounds that are effective in modulating inflammatory signals in explant tissues will then be preferred candidates for trial.
Tissues will be simultaneously profiled for their expression of the target and related markers. Data from the tissues will be compared with data obtained from the peripheral blood of the same patients taken at the same time. From these data, we intend to establish the likely cytokine and p38 expression patterns that may associate with efficacy in our drug candidates.
Thus, the flow of logic in our proposal may be summarised thus:
- Biological drugs show that TNFalpha suppression is an effective way to suppress RA and IBD.
- Not all patients respond to this therapy, this means that p38 alpha suppression may also not be effective in all patients
- P38 alpha expression is highly variable in patients.
- Thus, we seek to characterise lesions, and peripheral blood for response to the inhibitors, and expression of the target and related proteins.
- From these data, we intend to select a compound and a patient stratification system for the clinical trial of our compounds
These data will be deployed at two levels:
1. clinical trial design - a stratification protocol for the selection of patients based on either biopsy or blood samples that is most likely to respond to p38 kinase inhibitors;
2. selection within our drug candidate set for the analogy most likely to bring about patient benefit.
Our objectives in this project therefore fall into three groups:
Chemistry
- Completion of analogs with isoform specificity
- Scale-up methodology for toxicology and formulation.
Cell biology and assay design
- Verification target specificity, non-toxicity and stability in the candidate compounds
- Validation of a human diagnostic system for detecting p38 responsive patients.
Immunology and tissue screening
- Sampling blood and biopsy material from a variety of patients
- Correlating plasma markers and biopsy response to the compounds.
Thus the three strands of the project are tightly interwoven in so far as the in vitro analysis of explants and murine models will be used in parallel to select compounds. In parallel, the population genetics will be focused on identifying a patient sub-group in which p38 activation is potentially most relevant, and in which a clinical trial of a candidate compound may be most fruitful.
Project results:
WP 1 - Population genetics
Tasks 1.1 1.2 1.3 Expression profiling of existing patient tissue collections
We analysed mRNA expression of p38 MAPK isoforms in PBMCs tissue biopsy and hair to examine inter-individual and intra-individual variability of enzyme expression. These data show that hair or blood are interchangeable for purposes of determining systemic expression of these enzymes. Due to the relative rarity of using hair, continued sampling was on-going at the time of reporting.
Task 1.4 Differential p38 MAPK isoform expression in stimulated PBMCs
We analysed the specific p38 MAPK isoforms in human peripherial blood mononuclear cells (PBMCs) on RNA level and protein level. For mRNA expression analysis we used isoform specific TaqMan gene expression assays on a 7900HT fast real time PCR system.
In the protein expression part we first optimised the PBMC protein lysis and the Western Blot protocols to work with the isoform specific antibodies. The specificity of the new antibodies were confirmed with recombinant proteins in vitro and with PBMC lysates in vivo. Unfortunately, with these isoform specific antibodies it was not possible to account for phosphorylation state. As protein yields from PBMCs were too low for immunoprecipitation. Instead, we found the proteome profiler human phospho MAPK array from R&D systems as an alternative method with reasonable efficiency.
Protein extracts pooled from four individuals were analyzed by the phospho MAPK array. In PBMCs from healthy individuals p38a and p38? isoforms are activated upon stimulation. Because this array was established for cell lines, we first had to optimise the assay protocol and conditions to use this array with tissue extracts. For determination of activated p38 MAPK isoforms we used pooled protein lysates from different individuals.
In conclusion, we are able to detect all p38 MAPK isoforms in PBMCs on a protein or RNA level, with p38 MAPK alpha showing the strongest expression. On the protein level, p38 MAPK alpha and gamma are activated in PBMCs upon stimulation.
Variability of p38 MAPK isoform expression in inflamed colon tissue of patients with Crohn's disease
We are able to detect all p38 MAPK isoforms in colon tissue of patients on the RNA level, with p38 MAPK alpha and delta showing the strongest expression. This is in contrast to PBMCs where isoform gamma was the next most abundant. There was no difference in isoform expression between inflamed (N = 6) and non inflamed (N = 10) tissue of patients with Crohn's disease.
The protein data correlated very well with the mRNA expression data. In inflamed colon tissue of patients with high inflammation (CDAI > 150; N = 3), we detected all p38 MAPK isoforms with the protein array, with p38 MAPK delta and alpha showing the strongest expression and activation. So also p38 delta could also play a role in inflammation. In addition, ERK and JNK are activated in inflammation in the intestine, confirming a known crosstalk of different MAP kinases in inflammation. These data suggest that combined activity toward p38 and JNK isoforms could be desirable if a compound were to be used more generally.
The correlation of isoform expression between RNA and protein level was such that protein phosphorylation appears to provide the more reliable indicator of inflammation. In the above pancel, the p38 alpha and delta are highlighted in a red box. With the differential for p38 alpha being most clear.
Task 1.5 Luminex of biopsy tissues
A key objective in identifying the appropriate disease indications and models of these diseases is the presence of the target as a limiting factor in a lesion. To examine this in arthritis, samples were obtained from animal models and processed to determine the impact of compounds on them.
Mice were induced to express rheumatic disease to bovine collagen (mouse collagen induced arthritis) using two protocols. The active protocol (FCA / collagen sensitisation) and passive (antibody induced). Paws were taken at termination and subjected to Luminex cytokine assays. Comparison of the two systems produced similar cytokine profiles apart from IL12(p40) which was raised in the active protocol in 30 % of subjects. IL12(p40) or IL12B, is produced by macrophages presenting antigen to Th cells to differentiate into the Th1 disease inducing phenotype. This means that in the paws of those mice induced with the active protocol that there are some that have active T-cell differentiation within the joints, unlike the passive protocol. The use of this cytokine in human tissues may thus differentiate between different subsets of patients.
Treatment with dexamethasone revealed a very different profile of activity between the two protocols, raising the anti-inflammatory cytokines IL4 and IL10, whilst suppressing IL12(p40), in the active protocol only. Effects were very different in the passive protocol, with stimulation of IL2, IL1a and IL12(p70) (IL12A). This indicates that the different subtypes of disease, or stage of disease, may respond differently to pharmacological intervention.
Task 1.6 and 1.7 Comparison of PBMCs from healthy donors and patients with Crohn's disease and effect of p38 inhibitors of the imidazole class and their analogs
The Crohn's disease activity index (CDAI) is a research tool used to quantify the symptoms of patients with Crohn's disease. Disease activity was defined as a value of greater than 150. Remission of Crohn's disease is defined by a score under 150.
To determine the utility of using blood cells from Crohn's disease patients, we conducted a study on both the level of cytokine production in these patients and the effect of a model compound on the production of these cytokines. The data show that the model compound inhibits cytokine production generally, but is less effective on RNA levels than cytokine release, and is again less effective in patient material compared with that from healthy subjects.
These observations lead one to focus on where response is present at both the protein and mRNA level, notably IL-6.
Samples from patients with both high and low disease activity were studied in detail for the correlation of plasma parameters with disease parameters. There was no difference between Crohn's patients with high (red) and low (black) inflammation detectable. Remarkable differences between patients with crohn's disease and healthy individuals were observed for IL-1ß and IL-6 release. The inhibitory effect of SB neu on IL-1ß release was weaker in patients with Crohn's disease compared to healthy individuals. Inhibition of IL-6 realease by SB neu was highly variable in patients with Crohn's disease. As IL-6 is a key marker in inflammatory bowel diseases (IBD) it looks like p38 inhibition is not able to control / dominate IL-6 release in all IBD patients, but, rather, as suspected, the effects are most prominent in only a sub-set of patients.
These data suggest that one may be able to use the IL-6 response in patient blood immune cells as a predictive marker of potential response.
Most marker genes were inhibited with the most divergent results stemming from non-Crohn's (non-MC) patients. These data suggest that the non-Crohn's disease patients are, unfortunately, likely to be an excessively variable group for clinical studies, whereas, the Crohn's group appears to be relatively uniform by comparison.
The main conclusions from this work were that IL-6 is the most dynamic marker and one that most easily differentiates patients and reflects activity of compound. However, the next most dynamic is IL-10 which is an anti-inflammatory cytokine. SB neu inhibits IL-10 expression to a greater extent in patients with Crohn's disease, and leads one to question whether this is associated with an anti-inflammatory or a pro-inflammatory effect. As IBD is thought to be provoked by a deficiency in IL-10, these data suggest it will be difficult to persuade physicians to use a p38 inhibitor in this indication.
Task 1.8 Patient stratification strategy
In P1, we established an assay, in which PBMCs are stimulated whether with 10ng/ml human recombinant TNFalpha or 1 µg/ml E.coli derived lipopolysaccharide (LPS). The aim is to determine p38MAPK isoform expression and activation, as well as p38 MAPK inhibitor responsiveness of patients in parallel.
P38 MAPK isoform expression are analysed by TaqMan assays on RNA level and by a human phospho MAPK array from R&D Systems on protein level. This protein array detects the activation of 26 different MAPKs, kinases upstream / downstream of p38 MAPK or p38 substrates, including also all p38 MAPK isoforms.
In a third step, p38 MAPK inhibitors of the consortium are evaluated in the assay. Compounds are assessed through their potential to inhibit cytokine release. The cytokines TNF-alpha, IL-1beta and IL-6 are measured in an enzyme-linked immunosorbent assay (ELISA) from R&D Systems. Additionally, mRNA expression of selected marker genes (TNF-alpha, IL-1alpha, IL-1beta, IL-6, IL-10, IL-23a and 18SrRNA) is analyzed with TaqMan assays.
After several time courses, we found the right time points for measurement:
(1) measurement of cytokine secretion after eight hours;
(2) measurement of mRNA expression after two hours;
(3) measurement of isoform expression / p38 MAPK activation / phosphorylation after 30 minutes.
WP 2 Immunology and screening
Task 2.1 Novel indications, the role of p38 and its ligands in glomerulonephritis
p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic glomerulonephritis and which of its four isoforms is preferentially involved in kidney inflammation is largely unknown. Given the ligands available within KINACEPT for this target, we thus examined expression and activation of p38MAPK isoforms during anti- glomerular basement membrane (GBM) induced nephritis In addition, p38a conditional knockout mice (MxCre-p38aD/D) were used to examine the role of p38a in anti- GBM induced nephritis. Interestingly, p38a was the only isoform activated in renal tissue of anti- GBM induced nephritis. Both wild type and MxCre- p38áD/D mice developed acute renal failure over time but proteinuria was lower in MxCre- p38áD/D mice. Histological examinations revealed a reduced neutrophil and monocyte influx and less tubular damage in MxCre- p38áD/D mice, whereas glomerular crescent formation and renal fibrosis was similar. Moreover, the levels of pro- and anti-inflammatory cytokines such as TNF, IL-1 and IL-10 were similar. IL-12 and IL-18 were even up-regulated in MxCre- p38áD/D mice. In contrast, we could detect strong down-regulation of chemotactic cytokines in the kidneys of MxCre- p38D/D mice such as CCL-2 and -4. In conclusion, p38a is the primary p38MAPK isoform expressed in anti-GBM induced nephritis and selectively affects inflammatory cell influx, tubular damage and proteinuria. Full protection from nephritis is however not achieved as acute renal failure and structural damage still occurs.
Selective activation of p38a during anti- GBM induced nephritis
To determine whether p38MAPK signalling is active during crescentic glomerulonephritis, we performed an established anti-GBM model and evaluated gene and protein expression during the disease course. In parallel we transferred the model and surrounding technology to partners Synovo.
To run the model, we immunised mice with sheep IgG and then challenged the sensitised mice with sheep anti-GBM antibodies in conjunction with a single TNFa injection. To demonstrate the functionality of our model, we performed histological analysis during different time points. Injection of anti- GBM antibodies causes glomerular inflammation, glomerular crescent formation and tubular damage during the course of disease. We then analyzed expression of pro-inflammatory (TNFa, IL-1, IL-8) and anti-inflammatory (IL-10, TGFß1) cytokines by quantitative real-time PCR (qPCR) to gain insight into the molecular changes during the inflammatory response. TNFa and IL-1 mRNA was up regulated during the first 7 days of disease. In contrast, IL-8 was found to be over-expressed during later stages of disease. We also found up-regulation of the counter-regulatory cytokine IL-10 during anti-GBM induced nephritis. Interestingly, TGFß1 was over-expressed at late stages of anti- GBM induced nephritis, which might correlate to crescent formation occurring at the same time.
Task 2.2 Biochemical effect of compound effect - Effects of macrocyclic inhibitors
In addition to compounds based on the SB-Neu structure generated through the collaboration with Tübingen, Synovo's chemists have developed an alternative scaffold that has broad spectrum cytokine suppression characteristics. The groups set out to determine how the mechanism operated in vivo and in vitro and to validate its activity in various models. The prototype compound for these studies was CSY 0073 which is also known as SYD001 or Synovo development compound 1.
Task 2.4 CSY0073 attenuates immune dysregulation of lamina propria derived CD11b cells
As CSY0073 acts as an immunomodulatory agent, experiments were carried out to further define whether CSY0073 inhibited release of pro-inflammatory cytokines from colon macrophages directly. LPS induced a significant increase in the release of effector cytokines in both naïve and TNBS-treated mice. Exposure to CSY0073 inhibited the release of all cytokines measured from TNBS-treated animals, while it had no effect on IL-12 and IL-23 release from colon macrophages obtained from naïve mice.
Task 2.5 Anti-Inflammatory effects of CSY0073 in the DSS model of colitis
We have then assessed whether CSY0073 exerted protection against colitis development in models of Th2-mediated disease. For this purpose we used the DSS model of colitis, a model of colon inflammation that shows similarities with human UC. In this experimental setting, we have first induced a mild colitis by administering mice with 2 % DSS in drinking water for 2 weeks. CSY0073 and azythromycin were administered at the dose of 12.5 µMol/kg daily. Azythromycin and CSY0073 were equally effective in attenuating wasting disease caused by DSS and this effect was maintained also by CSY0073 (n = 8 - 10; #p < 0.05 versus vehicle; *p < 0.05 versus DSS). Moreover, both azythromycin and its derivative CSY0073 reduced the diarrhoea score (n = 8 - 10; #p < 0.05 versus naïve; *p < 0.05 versus DSS). Since CSY0073 was as effective as azythromycin in reducing symptoms in mild colitis, we have then tested whether this compound would also be effective in attenuating more severe form of colitis induced by administering mice with 5 % DSS in drinking water. Treating mice with CSY0073, 45 µMol/kg, significantly attenuated the wasting and the fecal score, as well as the severity of the histology score. Finally, to investigate whether protection afforded by CSY0073 in the model was comparable to that afforded by agent used in the treatment of IBD, we have challenged mice administered 5 % DSS with CSY0073 (15 µmol/kg) and sulphasalazine (200 µmol/kg). Results demonstrate that 15 µmol/kg CSY0073 was equally effective than 200 µmol/kg sulphasalazine in attenuating clinical scores and histopathology features of colitis.
Administration of CSY0073 and Azithromycin (12.5 µMol/kg, i.p.) protects against the development of DSS-induced colitis in mice (2 % in drinking water). The severity of DSS-induced inflammation (weight loss and diarrhea score) is reduced by CSY0073 and Azithromycin. Data represent the mean ± SE of 8 - 12 mice per group. (n = 8 - 10; #p < 0.05 versus naïve; *p < 0.05 versus DSS). CSY0073, 45 µMol/kg, attenuates signs of colitis induced by DSS including weight loss and the fecal score. Treating with CSY0073 attenuates colon inflammatory infiltration of the mucosa and sub-mucosa. Data represent the mean ± SE of 8-10 mice per group (#p < 0.05 versus naïve). Histopathologic analysis of colon samples obtained from mice sacrificed 12 days after DSS application began. DSS administration causes inflammatory infiltration in the colon mucosa.
Anti-inflammatory activity of CSY0073 compares with that of sulphasalazine in the DSS model of colitis. Mice were administered 5 % DSS alone or in combination with CSY0073 at the dose of 15 µmol/kg or sulphasalazine 200 µg/kg for 5 days.
Task 2.5 Anti-Inflammatory effects of CSY0073 in the TNBS model of colitis
Colon inflammation that develops in mice administered TNBS is thought to be a model of Th1-mediated disease with dense infiltrations of lymphocytes / macrophages in the lamina propria and thickening of the colon wall. In order to assess whether CSY0073 would exert immune-modulatory activity, mice administered TNBS were treated with CSY0073 for five days. CSY0073 effectively attenuated colitis development as measured by assessing local and systemic signs of inflammation. The effect exerted by CSY0073 was dose-dependent. Administering TNBS-treated mice with CSY0073 at the dose of 45 mMol/kg protected against the development of wasting disease measured by the weight loss and the diarrhea score and reduced the macroscopic score of colitis; moreover, it attenuated colon inflammation measured by assessing the histology score: as illustrated in panel D, all animals treated with TNBS developed mild to severe colitis that was attenuated or reversed by administration of CSY0073. In contrast, the lower dose (30 mMol/kg) had no effect on wasting disease, appearance and severity of diarrhea measured as a fecal-score and macroscopic and histology score (n = 8-10, #p < 0.05 versus naïve; *p < 0.05 versus TNBS group). As only the higher dose (45 mMol/kg) of CSY0073 was effective in reducing inflammation in all TNBS-treated mice, we used this dose for mechanistic investigations. Compared with colons of mice given vehicle alone (upper left), colons obtained from mice administered TNBS showed an extensive cellular infiltrate, submucosal edema and large areas of epithelial erosion (upper right) that were reduced by CSY0073 treatment (lower). Furthermore, CSY0073 significantly reduced colonic expression of inflammatory and immune mediators including TNFa, INFg and IL-2 and colonic MPO caused by TNBS (n=5; #p< 0.05 versus naïve; *p < 0.05 versus TNBS).
Early administration of CSY0073 (45 µMol/kg p.o.) protects against the development of TNBS-induced colitis in mice. Colitis was induced by intrarectal instillation of 1 mg of TNBS per mouse, and animals were killed five days after TNBS administration. CSY0073 was administered intraperitoneally daily for five days, starting at the same time of intrarectal instillation of TNBS. The severity of TNBS-induced inflammation (weight loss and fecal score) is reduced by CSY0073 administration. Data represent the mean ± SE of 8 - 10 mice per group. (#p < 0.05 versus naïve; *p < 0.05 versus TNBS). CSY0073 reduces local signs of inflammation and inhibits the increase of macroscopic- microscopic- score induced by intrarectal instillation of TNBS. Data represent the mean ± SE of 8 - 10 mice per group. (#p < 0.05 versus TNBS plus vehicle group). Histologic analysis of colon samples obtained from mice sacrificed five days after TNBS. TNBS administration causes colon wall thickening and massive inflammatory infiltration in the lamina propria. Administering TNBS mice with CSY0073 attenuates colon thickening and inflammatory infiltration of the mucosa and submucosa. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of expression of inflammatory cytokines (INF?, TNFa and IL-2) and Myeloperoxidase (MPO) activity in colons obtained five days after administration of TNBS alone or in combination with CSY0073. Data represent the mean ± SE of six mice per group. (#p < 0.05 versus naïve; *p<0.05 versus TNBS).
Task 2.5 CSY0073 attenuates signs of collagen-induced arthritis
NF-kB activation and subsequent cytokine production play a role in rheumatoid arthritis as well as in animal models of inflammatory arthritis. To explore the possibility that CSY0073 NF- B suppression might have therapeutic efficacy in arthritis, we have tested its effect in the murine CIA model. DBA/1J mice were immunised with type II collagen and treated with CSY0073 (in food, 25 µMol/day) beginning on day 21.
Task 2.6 CSY0073 exerts counter-regulatory activities on NF-kB signalling
Because CSY0073 administration reduced immune-cellular infiltration in the lamina propria of Balb/c mice, experiments were carried out to further define the immune compartment mediating CSY0073 actions in vitro. For this purpose, we tested the effect of CSY0073 at different doses (10, 1 and 0.1 mM) on TNFa and PGE2 release from mouse spleen monocytes stimulated with LPS (1 µg/ml) for 24 hours. Whilst exposure to LPS increased both TNFa and PGE2 release from mouse spleen monocytes, CSY0073, 10 and 1 mM, causes a robust attenuation of this effect (n=5 experiments; #p < 0.05 versus naïve; *p < 0.05 versus LPS group).
As NF-kB is a key transcription factor for the expression of inflammatory genes, to further investigate the mechanisms modulating the anti-inflammatory effects of CSY0073 in comparison to azythromycin, RAW264.7 cells were treated with LPS (1 µg/ml) alone or in combination with CSY0073 (10 µM) or azythromycin (10 µMol/kg). The Western blot analysis revealed that LPS induced a nuclear translocation of the p65 subunit of NF-kB. Azythromycin significantly reduced the NF-kB activation and, similarly, CSY0073 reduced LPS- induced NF-kB translocation in RAW264.7 cells (n=3 experiments; #p < 0.05 versus naïve; *p < 0.05 versus LPS group). We have then assessed the effect of CSY0073 on NF-?B DNA binding activity in LPS-activated RAW264.7 cells, a macrophage cell line of murine origin. Cotreatment with CSY0073 caused a robust attenuation of NF-?B DNA binding caused by LPS. Similarly results were observed with azitromycin. These results suggest that CSY0073 counter-regulates cytokines release by suppressing binding of NF-?B NF-kB responsive elements in the promoter of responsive genes.
Task 2.4 Effects of CSY 0073 in Colon biopsies from IBD patients
Colon biopsies were obtained from 6 patients (5 men, mean age 39.4 ± 6.5 years) undergoing colonscopy for clinical staging of their disease. All patients were affected by Crohn's disease with a colonic localisation. Three of them had also ileal involvement. All subjects were taking active drugs: budesonide (four patients) and azathioprine (two patients). None of the patients had previous surgery. Each patient had an active disease at the macroscopic examination. Samples were taken from non-ulcered mucosa. Written consent was obtained from each patient. Biopsy specimens were gently washed, three times in RPMI with 3 % of penicillin / streptomycin and cultured on 40 uM mesh filters over a culture dish in 12 well tissue culture plates in complete RPMI medium. They were cultured in the presence or absence of LPS (100 µg/ml) alone or in combination with CSY0073 (100 µM), (3 hour of pre-treatment before LPS stimulation) and incubated at 37 °C with 5 % CO2. After 16 h, culture supernatants was removed, while biopsies were processed for RNA extraction.
Data analysis
All values will be express as mean ± SE of experiments. The variation between data sets will test with the unpaired data and ANOVA, for more of two groups.
CSY 0073 reduced the expression of inflammatory genes in colon biopsies activated by LPS
CSY 0073 (100 µM) was added to colon biopsies obtained from IBD patients, for 3h, before LPS stimulation (100 µg/ml) for 16 h. At the end of incubation, the mRNA was extracted for the quantification of (A) tumour necrosis factor (TNF)-a; (B) interleukin (IL)-8; (C) Rantes and (D) macrophage inflammatory protein 3 alpha (MIP-3a) (*p < 0.05 versus untreated biopsies ; **p < 0.05 versus LPS treated biopsies; n = 6).
In the ex vivo experiments, we found that exposure to CSY0073 effectively decreased the expression of TNFa, Rantes, MIP-3a and IL-8 induced by LPS. This observation may have a clinical readout and we feel a clinical trial is warranted..
WP 2 - Immune characterisation
Task 2.2 Cell based assays for activation of p38 and inhibition of TNFa production
P38 MAP kinase sensitivity to p38 inhibitors may vary between patients, explaining the variation in response seen in the rheumatoid clinic. In addition, patient responses in the degree of p38 activation to agonists may also differ. A simple cell based screen of p38 MAP kinase sensitivity to agonists and their inhibition is required for the assessment of these phenomena. The simplest system would be to sample blood, stimulate, and analyse. If achievable, this system would be an improvement on systems requiring the purification of cells by removing this step, retaining the proportional and total cell populations within the blood (and thus their interactions), and also removing the artificiality of isolated cell systems.
Whole blood was taken from healthy volunteers and stimulated with lipopolysaccharide (LPS) or phytohaemaggluttinin (PHA). LPS stimulates the TLRx receptor of blood monocytes to activate the MAPkinase pathway and induce gene expression of TNFa. PHA stimulates the T-cell receptor to induce T-cell TNFa synthesis, this is susceptible to p38 MAPkinase inhibition.
Using human whole blood cultured over 5 days, 2mL in 24-well plates with varying concentrations of PHA, has determined the feasibility of this approach and the dose range for the screen. Wide variety of cytokines were analysed by Luminex and found to be stimulated (IFNg, IL1a, IL1B, IL2, IL4, IL6, IL7, IL8, IL10, IL12, IL13, IL17, GCSF, GMCSF, MCP-1, and VEGF). TNFa was chosen as the end point. Miniaturisation to 96-well LPS between 0.15 and 10 µg/mL, and PHA between 0.3 and 10 µg/mL PHA provided a full concentration effect curve from subthreshold to supramaximal stimulation of TNFa synthesis. Analysis of a sample of healthy subjects revealed that there was significant variation in maximal response to the agonists between individuals as well as between the agonists themselves. Repeating the same subject three times gave consistent data between samples. Thus the variation in response appears to be due to inter-subject variability in responses to specific agonists and thus intracellular messenger pathways. Variability in response to p38 MAP kinase inhibition with ML3404 was seen with PHA and not LPS.
There thus appears to be subject differences in p38-i sensitivity specifically related to the type of agonist used. This will require corroboration with a prospective trial.
The whole blood culture assay appears to be a useful assay for the determination of patient sensitivity to p38i action, requiring the minimum of sample preparation prior to culture within a single 96-well plate followed by a standard TNFa ELISA.
Human whole blood in either 24 well or 96 well culture was stimulated with PHA. The degree of TNF synthesis stimulation was similar between the two assays enabling miniaturisation and the performance of dose response and p38 inhibitor responses within the same subject.
Task 2.5 Compound activity in animal models - The role of cell interactions in RA pathology, relevance to RA p38i modelling in vivo
Whilst such cellular cooperation described in the previous section is considered important in RA, examination of RA synovial tissue shows heterogeneity in pathology, in both degree of infiltration and organisation. It may thus be that patients might need to be stratified according to the cellular interactions occurring within the synovium at the time of treatment. The in vitro data presented in this project may indicate that long term effects of p38i on the close cooperation of accessory and lymphocyte cells might be exploitable. However, if these cellular interaction mechanisms of p38i is to be used, patients may need to be stratified according to the cellular organisation of the inflamed synovium.
It is becoming increasingly clear that RA synovial can be broken down into different types. The prospective early arthritis cohort (PEAC) study led by the Centre for Experimental Medicine classifies synovial according to cellular infiltrate and organisation. Only around 10 - 20 % possess tertiary lymphoid structures that indicate the close cellular cooperation that would be required for this disease modifying activity of p38i drugs. Interestingly, literature search of such structures in models of arthritis reveals few reports of lymphoid aggregates in animal models of RA (Hetal et al., reported in WP5) and it is in these models that p38i show global inhibition of disease that resulted in the false validation of p38 as a drug target for RA.
In order to characterise these models with view to calibrating them with the human variability of pathology and characteristics, a panel of tissues has been generated for rat collagen and rat adjuvant arthritis for comparative analysis. As can be seen each of these is rapid, with a fast attack phase followed by resolution and ankylosis. One model however, which has not been investigated for p38i action, is rat pristane arthritis. This disease is reported to be not self limiting like the other models such as collagen arthritis, and has aspects of re-activation. This arthritis is rarely used because of it severity, and stress-intractability of the Ola strain of rat to handling. WHRI has carried out a pilot dose ranging trial designed to address animal welfare issues through severity limits agreed with the Institutional ethics review committee, and to determine if the re-activation process can be retained at lower disease severity, and if so, a tissue panel created to match the other models.
A clinical assessment system was created that tracked each individual joint for each rat over a period of 90 days. The usual method of assessment is a composite score of all joints, with weighting towards measuring the hind paw. It can be seen that the attack phase is rapid, and persists to after day 20, followed by recovery. During this phase, two of the five highest (150 µL) dose reached the humane end points and had to be culled. None of the lower doses reached this point. As second phase can then be seen starting at round day 60. It would appear that the lower dose (75 µL) of mineral may be more effective at this process. However, analysis of individual joint groups shows a complex process. The ankles remain swollen to about day 70. Inflammation in the midpaw is reduced after the attack phase at day 25. However, a matched flare at day 65 occurs for the front wrists and from hind knuckles / digits. Histological analysis of the ankles showed that of those that retained inflammatory activity, organised cell aggregates were evident.
Subject to an archival study, it would appear that if cell-cooperation mechanisms were to be investigated, the hind ankle at day 70 - 80, and the front wrist and hind knuckles / digits at day 65 would be of prime interest.
Task 2.1 and 3.4 Optimisation of pharmacokinetics
The likely dosage form of the compounds will be a capsule for oral administration. With this in mind, we examined the bioavailability of selected substances in studies of pharmacokineticts. The compounds advanced here as candidates are orally bioavailable.
Task 2.6 2.7 and 3.5 Optimisation for toxicology
We up-scaled the synthesis of the various candidates and entered them into a toxicology program to determine any potential risks. In in vitro studies on cancer cell lines, various cells including J774, B16 melanoma and LLC cells were tested without any obvious effects in the range 10 - 50 µM. These data suggested that the compounds are not cytotoxins but rather act as signalling regulators in an intact organ system.
The studies were followed using in vivo toxicology. A key element of toxicology is selecting the dose range in which to determine toxic effect. A common strategy is dose escalation. Using dose escalation studies in mice, it is apparent that there are neither acute nor chronic toxicity effects of the compounds in the pharmacological range.
Task 2.8 Selection of compounds for development
Based on the foregoing, we have selected a range of compounds to enter the first stages of clinical trials.
SYD003 is a potent modulator of cancer development but has no obvious dose limiting toxicity, is orally available, stable in rodents and easy to manufacture and formulate.
SYD004 is active in combination therapy and is a potential follow-up to SYD003. We are currently selecting the best analog, however, data to date suggest that active and safe analogs are available.
SYD005 is highly active in selected models notably LLC and will be advanced for profiling in human cancers.
SYD001 is a macrocyclic cytokine inhibitor and appears highly active in both murine IBD and arthritis models. We are currently seeking a licensing partner for this compound.
Conclusions WP 1, 2, 3:
These data have shown that p38 alpha is the dominant form of the protein in PBMCs, hair, diseased tissue and inflamed tissue.
Following stimulation, both the gamma and delta isoforms are up-regulated in PBMCs and diseased tissue respectively. These data could suggest that the delta isoform is an ideal disease specific target in Crohn's disease.
Similar data have been obtained in glomerulonephritis suggesting that selected orphan indications have potential with the compounds in our collection.
Nonetheless, actual model data obtained with the compounds suggests that the ideal indications for modulators of p38 may be cancer rather than inflammation. Indeed, of the compounds tested the rather less specific macrocyclic inhibitor appeared to be the most generally applicable.
WP 3 - Synthetic chemistry
The synthetic chemistry is being conducted primarily by Synovo and Tuebingen. The Tuebingen focus, along with Inte:ligand is to better understand binding modes to design better ligands. Synovo's focus is to find uses of the existing ligands.
Task 3.1 Structure activity relationships - these were completed in WP1
Task 3.2 Analog synthesis
On the fringes of the hydrophobic region exchanged by Gln 155. We wanted to place a negative charge at the appropriate position with the aim to achieve a rejection with Asp 112. In JNK3 a hydrogen bond to Asn 152 should be formed.
An acidic moiety at the amino pyridine did not result in any potent and selective inhibitors, so we switched to the imidazole nitrogen
a) dimethylaminopyridine, acetic anhydride, reflux
b) KMnO4 , H2O, 60 °C
c) DMF, CDI, 90 min. at RT, 4-Fluorphenylacetonitrile, K-tert-butoxide, 120 °C, 4h 12h at RT
d) HBr (48 %), reflux
e) acetic anhydride, DMAP, reflux
f) isoamylnitrite, NaOMe, Methanol, RT
g) ethanol, reflux
h) DCM, RT
i) alkyl halide, K2CO3, THF or methanol
j) HCl 6N, reflux
k) acid chloride / triethylamine or anhydride / DMAP, THF / DCM 1:1
Compounds: R = CH3, R1= various, R2= COCH3 (or H)
These compounds have been prepared to explore, which moieties the hydrophobic region is tolerating. Compound KS004 was synthesised to find out, if the carbonyl at the amino pyridine is necessary for interactions with the enzyme:
Compounds: R= CH3, R1= CH2COCH3, R2= various
Compounds: R= various, R1= CH3, R2= COCH3
Compounds: R= (CH2)3COOH, R1= various, R2=COCH3
Once found out, that (CH2)3COOH brings a moderate selective inhibition of JNK3 over p38 we diversified the moieties R1.
Task 3.3 Potency and specificity for JNK3, p38 alpha and or delta
This task was largely completed in P1, since that time, additional refinement has taken place as noted above.
Task 3.4 Formulation for oral delivery
In P1, we demonstrated partition to tumours and inflammation associated tissues. In P2, we focused on formulation and notably simulating application conditions in patients. This culminated in the use of capsules in mice to demonstrate pharmacokinetics.
Task 3.5 Upscale for toxicology
Synovo chemists up-scaled the compounds SYD003, SYD004 and CSY0754, 755, 766, and 768 for toxicology investigations.
Potential impact:
There are three possible areas of medico-societal impact of the project:
- arthritis like diseases;
- inflammatory bowel diseases; and
- cancers.
Each of these disease areas is debilitating to the point of preventing productive work, and one that strikes generally from mid-life onwards. Therapy for the conditions using advanced therapies is in the order of EUR 12 000 to 30 000 per year.
Replacement of expensive biological medications with low molecular weight, synthetic alternatives is a means to both improve disease outcomes and reduce the costs of care.
In our project, we have identified three candidate drugs that are well tolerated, novel and strikingly effective in animal models.
We intend to develop these compounds as alternatives to biological therapies.
Should we succeed, these compounds would:
- compete with medicines whose intellectual property rights lie outside Europe;
- provide a tablet alternative to injected biological;
- stand as an alternative to, or means of reducing doses of poorly tolerated chemotherapeutics.
The societal benefits would be apparent in aggregate and in microscales. Drug therapy that reduces hospital stays is a very cost effective means of delivering healthcare. Oral small molecules are the most cost-effective form of therapy.
At the microscale, it is apparent that lower complimentary doses of therapeutics are normally safer and better tolerated than larger doses of single agents. Having more such therapies to choose from will allow such combinations and lower prices through competition.
For SMES and entrepreneurs, the impact of the project is more immediate. Synovo and its partner companies are part of a network of smaller firms that are growing to replace the inftrastructure of the larger pharma enterprises. Leaner, faster, more efficient, these smaller drug discovery companies will be able to supply therapies at a fraction of the current investment levels, if they are given the chance to do so. This means finding ways for smaller companies to reach the market in this field.
At present the market is inaccessible because of barriers to entry that have been in part established by large enterprises and regulatory agencies. These barriers to entry mean that only large well funded firms can reach the market, and this, by definition, locks in high prices for novel medications.
If one were to allow smaller companies to market therapies on a provisional basis, following establishment of safety in patients, there is the potential to both re-construct the healthcare cost economy, and develop a highly innovative sphere of biotechnology in Europe. In such a provisional phase, marketers of exploratory medicines would be re-imbursed only according to the benefits observed, and thus there would be no need for pharmacoeconomic arguments about pricing. These would come later when the benefits are known, and the treatment is established.
Taking such a far-sighted approach to small biotech is one aspect of medium term societal impact that we should consider within SME policy.
Project website:
http://www.actakine.com
Questions and enquiries are welcomed. Please contact
Michael Burnet
Coordinator
Synovo GmbH
Paul Ehrlich Str. 15
72076 Tübingen
Germany
Switch: +49-707-1964325
Direct: +49-707-1600543
Fax: +49-707-1964326
Our project has had the following key goals:
- Are their novel indications for our compounds, and if so, which models and test systems would demonstrate this utility most convincingly?
- If there are novel indications, how can we identify patients most likely to benefit from the use of our compounds?
- Which of these compounds, and with which general selectivity would be most useful?
To reach these goals, the partners delivered the following key results.
New indications
The team focused on novel indications found that human nephritis is specifically reliant on p38alpha and that this disease is, therefore a straightforward and specific unmet need for which a trial with our lead substances can be rationalised.
The team focusing on arthritis demonstrated that there is only one animal model that recapitulates central aspects for advanced human arthritis and that this, therefore, is the appropriate model for validating the compounds.
The team focusing on IBD observed both positive and negative effects of the imidazole derived p38 targeted substances in human tissue biopsy and concluded that the sum of these effects is unlikely to be beneficial.
The team focusing on the use of macrolide-based inhibitors, in contrast, observed very clear positive effects of the substance in human Crohn's disease lesion biopsy material. These data show that one compound class is at least worthy of progression in this indication.
Synovo demonstrated the relevance of certain additional classes of p38 inhibitor to cancer and has now prepared two molecules for the clinic.
Patient stratification and selection
- Blood and hair can be used to determine patient status, however, biopsy tissue remains the ideal medium.
- In IBD disease types, biopsy was preferred and appeared more predictive.
- In arthritis, the presence of tertiary lymph nodes or aggregates in the arthritic synovium is considered to be the best selection criterion
- Cytokines were less intrinsically predictive, however, relative effect on TNF vs. IL-10 was considered most important.
Compound selection
As of the end of P2, the following compounds were slated for clinical trial:
SYD003 - pancreatic cancer
SYD004 - cancer and or advanced rheumatoid arthritis
SYD005 - lung cancer
SYD001 - Crohn's disease, and or rheumatoid arthritis.
Project context and objectives:
Autoimmune diseases have varied aetiology but many elements in common, notably a dependence on specific cytokines to mediate pro-inflammatory signals.
In recent years, the ability to selectively suppress cytokines via protein receptor molecules or antibodies has demonstrated that specific cytokine suppression can dramatically improve signs of disease in both rheumatoid arthritis and inflammatory bowel diseases. However, for reasons of cost and long-term loss of efficacy, small molecule drugs are preferred for chronic therapy.
The applicants have developed a new class compounds that suppress the production of the cytokine TNFalpha via inhibition of the signalling kinase known as p38 alpha. By phosphorylating its target, p38 kinase contributes to the perpetuation of pro-inflammatory signals emanating from outside the cell. Inhibition of this signal causes a reduction in cytokine and specifically TNFalpha release.
Like many drug targets, it now appears that p38 kinase contributes to inflammatory states in different ways under different conditions. In certain patients, the isoforms of p38 kinase that are over expressed vary, and in others, the overall level of p38 expression varies.
Given this variation in the target expression, it is not surprising that responses to p38 kinase inhibitors in clinical trials has been varied leading to the requirement for relatively large patient collectives in order to see effects.
Large collectives of this kind are not within the scope of small companies and so, instead, we are seeking to take a more informed and selective approach to patient screening before entering clinical trials.
Having established a variety of lead molecules, we wish to characterise and optimise these compounds using models based on human explant tissues. Compounds that are effective in modulating inflammatory signals in explant tissues will then be preferred candidates for trial.
Tissues will be simultaneously profiled for their expression of the target and related markers. Data from the tissues will be compared with data obtained from the peripheral blood of the same patients taken at the same time. From these data, we intend to establish the likely cytokine and p38 expression patterns that may associate with efficacy in our drug candidates.
Thus, the flow of logic in our proposal may be summarised thus:
- Biological drugs show that TNFalpha suppression is an effective way to suppress RA and IBD.
- Not all patients respond to this therapy, this means that p38 alpha suppression may also not be effective in all patients
- P38 alpha expression is highly variable in patients.
- Thus, we seek to characterise lesions, and peripheral blood for response to the inhibitors, and expression of the target and related proteins.
- From these data, we intend to select a compound and a patient stratification system for the clinical trial of our compounds
These data will be deployed at two levels:
1. clinical trial design - a stratification protocol for the selection of patients based on either biopsy or blood samples that is most likely to respond to p38 kinase inhibitors;
2. selection within our drug candidate set for the analogy most likely to bring about patient benefit.
Our objectives in this project therefore fall into three groups:
Chemistry
- Completion of analogs with isoform specificity
- Scale-up methodology for toxicology and formulation.
Cell biology and assay design
- Verification target specificity, non-toxicity and stability in the candidate compounds
- Validation of a human diagnostic system for detecting p38 responsive patients.
Immunology and tissue screening
- Sampling blood and biopsy material from a variety of patients
- Correlating plasma markers and biopsy response to the compounds.
Thus the three strands of the project are tightly interwoven in so far as the in vitro analysis of explants and murine models will be used in parallel to select compounds. In parallel, the population genetics will be focused on identifying a patient sub-group in which p38 activation is potentially most relevant, and in which a clinical trial of a candidate compound may be most fruitful.
Project results:
WP 1 - Population genetics
Tasks 1.1 1.2 1.3 Expression profiling of existing patient tissue collections
We analysed mRNA expression of p38 MAPK isoforms in PBMCs tissue biopsy and hair to examine inter-individual and intra-individual variability of enzyme expression. These data show that hair or blood are interchangeable for purposes of determining systemic expression of these enzymes. Due to the relative rarity of using hair, continued sampling was on-going at the time of reporting.
Task 1.4 Differential p38 MAPK isoform expression in stimulated PBMCs
We analysed the specific p38 MAPK isoforms in human peripherial blood mononuclear cells (PBMCs) on RNA level and protein level. For mRNA expression analysis we used isoform specific TaqMan gene expression assays on a 7900HT fast real time PCR system.
In the protein expression part we first optimised the PBMC protein lysis and the Western Blot protocols to work with the isoform specific antibodies. The specificity of the new antibodies were confirmed with recombinant proteins in vitro and with PBMC lysates in vivo. Unfortunately, with these isoform specific antibodies it was not possible to account for phosphorylation state. As protein yields from PBMCs were too low for immunoprecipitation. Instead, we found the proteome profiler human phospho MAPK array from R&D systems as an alternative method with reasonable efficiency.
Protein extracts pooled from four individuals were analyzed by the phospho MAPK array. In PBMCs from healthy individuals p38a and p38? isoforms are activated upon stimulation. Because this array was established for cell lines, we first had to optimise the assay protocol and conditions to use this array with tissue extracts. For determination of activated p38 MAPK isoforms we used pooled protein lysates from different individuals.
In conclusion, we are able to detect all p38 MAPK isoforms in PBMCs on a protein or RNA level, with p38 MAPK alpha showing the strongest expression. On the protein level, p38 MAPK alpha and gamma are activated in PBMCs upon stimulation.
Variability of p38 MAPK isoform expression in inflamed colon tissue of patients with Crohn's disease
We are able to detect all p38 MAPK isoforms in colon tissue of patients on the RNA level, with p38 MAPK alpha and delta showing the strongest expression. This is in contrast to PBMCs where isoform gamma was the next most abundant. There was no difference in isoform expression between inflamed (N = 6) and non inflamed (N = 10) tissue of patients with Crohn's disease.
The protein data correlated very well with the mRNA expression data. In inflamed colon tissue of patients with high inflammation (CDAI > 150; N = 3), we detected all p38 MAPK isoforms with the protein array, with p38 MAPK delta and alpha showing the strongest expression and activation. So also p38 delta could also play a role in inflammation. In addition, ERK and JNK are activated in inflammation in the intestine, confirming a known crosstalk of different MAP kinases in inflammation. These data suggest that combined activity toward p38 and JNK isoforms could be desirable if a compound were to be used more generally.
The correlation of isoform expression between RNA and protein level was such that protein phosphorylation appears to provide the more reliable indicator of inflammation. In the above pancel, the p38 alpha and delta are highlighted in a red box. With the differential for p38 alpha being most clear.
Task 1.5 Luminex of biopsy tissues
A key objective in identifying the appropriate disease indications and models of these diseases is the presence of the target as a limiting factor in a lesion. To examine this in arthritis, samples were obtained from animal models and processed to determine the impact of compounds on them.
Mice were induced to express rheumatic disease to bovine collagen (mouse collagen induced arthritis) using two protocols. The active protocol (FCA / collagen sensitisation) and passive (antibody induced). Paws were taken at termination and subjected to Luminex cytokine assays. Comparison of the two systems produced similar cytokine profiles apart from IL12(p40) which was raised in the active protocol in 30 % of subjects. IL12(p40) or IL12B, is produced by macrophages presenting antigen to Th cells to differentiate into the Th1 disease inducing phenotype. This means that in the paws of those mice induced with the active protocol that there are some that have active T-cell differentiation within the joints, unlike the passive protocol. The use of this cytokine in human tissues may thus differentiate between different subsets of patients.
Treatment with dexamethasone revealed a very different profile of activity between the two protocols, raising the anti-inflammatory cytokines IL4 and IL10, whilst suppressing IL12(p40), in the active protocol only. Effects were very different in the passive protocol, with stimulation of IL2, IL1a and IL12(p70) (IL12A). This indicates that the different subtypes of disease, or stage of disease, may respond differently to pharmacological intervention.
Task 1.6 and 1.7 Comparison of PBMCs from healthy donors and patients with Crohn's disease and effect of p38 inhibitors of the imidazole class and their analogs
The Crohn's disease activity index (CDAI) is a research tool used to quantify the symptoms of patients with Crohn's disease. Disease activity was defined as a value of greater than 150. Remission of Crohn's disease is defined by a score under 150.
To determine the utility of using blood cells from Crohn's disease patients, we conducted a study on both the level of cytokine production in these patients and the effect of a model compound on the production of these cytokines. The data show that the model compound inhibits cytokine production generally, but is less effective on RNA levels than cytokine release, and is again less effective in patient material compared with that from healthy subjects.
These observations lead one to focus on where response is present at both the protein and mRNA level, notably IL-6.
Samples from patients with both high and low disease activity were studied in detail for the correlation of plasma parameters with disease parameters. There was no difference between Crohn's patients with high (red) and low (black) inflammation detectable. Remarkable differences between patients with crohn's disease and healthy individuals were observed for IL-1ß and IL-6 release. The inhibitory effect of SB neu on IL-1ß release was weaker in patients with Crohn's disease compared to healthy individuals. Inhibition of IL-6 realease by SB neu was highly variable in patients with Crohn's disease. As IL-6 is a key marker in inflammatory bowel diseases (IBD) it looks like p38 inhibition is not able to control / dominate IL-6 release in all IBD patients, but, rather, as suspected, the effects are most prominent in only a sub-set of patients.
These data suggest that one may be able to use the IL-6 response in patient blood immune cells as a predictive marker of potential response.
Most marker genes were inhibited with the most divergent results stemming from non-Crohn's (non-MC) patients. These data suggest that the non-Crohn's disease patients are, unfortunately, likely to be an excessively variable group for clinical studies, whereas, the Crohn's group appears to be relatively uniform by comparison.
The main conclusions from this work were that IL-6 is the most dynamic marker and one that most easily differentiates patients and reflects activity of compound. However, the next most dynamic is IL-10 which is an anti-inflammatory cytokine. SB neu inhibits IL-10 expression to a greater extent in patients with Crohn's disease, and leads one to question whether this is associated with an anti-inflammatory or a pro-inflammatory effect. As IBD is thought to be provoked by a deficiency in IL-10, these data suggest it will be difficult to persuade physicians to use a p38 inhibitor in this indication.
Task 1.8 Patient stratification strategy
In P1, we established an assay, in which PBMCs are stimulated whether with 10ng/ml human recombinant TNFalpha or 1 µg/ml E.coli derived lipopolysaccharide (LPS). The aim is to determine p38MAPK isoform expression and activation, as well as p38 MAPK inhibitor responsiveness of patients in parallel.
P38 MAPK isoform expression are analysed by TaqMan assays on RNA level and by a human phospho MAPK array from R&D Systems on protein level. This protein array detects the activation of 26 different MAPKs, kinases upstream / downstream of p38 MAPK or p38 substrates, including also all p38 MAPK isoforms.
In a third step, p38 MAPK inhibitors of the consortium are evaluated in the assay. Compounds are assessed through their potential to inhibit cytokine release. The cytokines TNF-alpha, IL-1beta and IL-6 are measured in an enzyme-linked immunosorbent assay (ELISA) from R&D Systems. Additionally, mRNA expression of selected marker genes (TNF-alpha, IL-1alpha, IL-1beta, IL-6, IL-10, IL-23a and 18SrRNA) is analyzed with TaqMan assays.
After several time courses, we found the right time points for measurement:
(1) measurement of cytokine secretion after eight hours;
(2) measurement of mRNA expression after two hours;
(3) measurement of isoform expression / p38 MAPK activation / phosphorylation after 30 minutes.
WP 2 Immunology and screening
Task 2.1 Novel indications, the role of p38 and its ligands in glomerulonephritis
p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic glomerulonephritis and which of its four isoforms is preferentially involved in kidney inflammation is largely unknown. Given the ligands available within KINACEPT for this target, we thus examined expression and activation of p38MAPK isoforms during anti- glomerular basement membrane (GBM) induced nephritis In addition, p38a conditional knockout mice (MxCre-p38aD/D) were used to examine the role of p38a in anti- GBM induced nephritis. Interestingly, p38a was the only isoform activated in renal tissue of anti- GBM induced nephritis. Both wild type and MxCre- p38áD/D mice developed acute renal failure over time but proteinuria was lower in MxCre- p38áD/D mice. Histological examinations revealed a reduced neutrophil and monocyte influx and less tubular damage in MxCre- p38áD/D mice, whereas glomerular crescent formation and renal fibrosis was similar. Moreover, the levels of pro- and anti-inflammatory cytokines such as TNF, IL-1 and IL-10 were similar. IL-12 and IL-18 were even up-regulated in MxCre- p38áD/D mice. In contrast, we could detect strong down-regulation of chemotactic cytokines in the kidneys of MxCre- p38D/D mice such as CCL-2 and -4. In conclusion, p38a is the primary p38MAPK isoform expressed in anti-GBM induced nephritis and selectively affects inflammatory cell influx, tubular damage and proteinuria. Full protection from nephritis is however not achieved as acute renal failure and structural damage still occurs.
Selective activation of p38a during anti- GBM induced nephritis
To determine whether p38MAPK signalling is active during crescentic glomerulonephritis, we performed an established anti-GBM model and evaluated gene and protein expression during the disease course. In parallel we transferred the model and surrounding technology to partners Synovo.
To run the model, we immunised mice with sheep IgG and then challenged the sensitised mice with sheep anti-GBM antibodies in conjunction with a single TNFa injection. To demonstrate the functionality of our model, we performed histological analysis during different time points. Injection of anti- GBM antibodies causes glomerular inflammation, glomerular crescent formation and tubular damage during the course of disease. We then analyzed expression of pro-inflammatory (TNFa, IL-1, IL-8) and anti-inflammatory (IL-10, TGFß1) cytokines by quantitative real-time PCR (qPCR) to gain insight into the molecular changes during the inflammatory response. TNFa and IL-1 mRNA was up regulated during the first 7 days of disease. In contrast, IL-8 was found to be over-expressed during later stages of disease. We also found up-regulation of the counter-regulatory cytokine IL-10 during anti-GBM induced nephritis. Interestingly, TGFß1 was over-expressed at late stages of anti- GBM induced nephritis, which might correlate to crescent formation occurring at the same time.
Task 2.2 Biochemical effect of compound effect - Effects of macrocyclic inhibitors
In addition to compounds based on the SB-Neu structure generated through the collaboration with Tübingen, Synovo's chemists have developed an alternative scaffold that has broad spectrum cytokine suppression characteristics. The groups set out to determine how the mechanism operated in vivo and in vitro and to validate its activity in various models. The prototype compound for these studies was CSY 0073 which is also known as SYD001 or Synovo development compound 1.
Task 2.4 CSY0073 attenuates immune dysregulation of lamina propria derived CD11b cells
As CSY0073 acts as an immunomodulatory agent, experiments were carried out to further define whether CSY0073 inhibited release of pro-inflammatory cytokines from colon macrophages directly. LPS induced a significant increase in the release of effector cytokines in both naïve and TNBS-treated mice. Exposure to CSY0073 inhibited the release of all cytokines measured from TNBS-treated animals, while it had no effect on IL-12 and IL-23 release from colon macrophages obtained from naïve mice.
Task 2.5 Anti-Inflammatory effects of CSY0073 in the DSS model of colitis
We have then assessed whether CSY0073 exerted protection against colitis development in models of Th2-mediated disease. For this purpose we used the DSS model of colitis, a model of colon inflammation that shows similarities with human UC. In this experimental setting, we have first induced a mild colitis by administering mice with 2 % DSS in drinking water for 2 weeks. CSY0073 and azythromycin were administered at the dose of 12.5 µMol/kg daily. Azythromycin and CSY0073 were equally effective in attenuating wasting disease caused by DSS and this effect was maintained also by CSY0073 (n = 8 - 10; #p < 0.05 versus vehicle; *p < 0.05 versus DSS). Moreover, both azythromycin and its derivative CSY0073 reduced the diarrhoea score (n = 8 - 10; #p < 0.05 versus naïve; *p < 0.05 versus DSS). Since CSY0073 was as effective as azythromycin in reducing symptoms in mild colitis, we have then tested whether this compound would also be effective in attenuating more severe form of colitis induced by administering mice with 5 % DSS in drinking water. Treating mice with CSY0073, 45 µMol/kg, significantly attenuated the wasting and the fecal score, as well as the severity of the histology score. Finally, to investigate whether protection afforded by CSY0073 in the model was comparable to that afforded by agent used in the treatment of IBD, we have challenged mice administered 5 % DSS with CSY0073 (15 µmol/kg) and sulphasalazine (200 µmol/kg). Results demonstrate that 15 µmol/kg CSY0073 was equally effective than 200 µmol/kg sulphasalazine in attenuating clinical scores and histopathology features of colitis.
Administration of CSY0073 and Azithromycin (12.5 µMol/kg, i.p.) protects against the development of DSS-induced colitis in mice (2 % in drinking water). The severity of DSS-induced inflammation (weight loss and diarrhea score) is reduced by CSY0073 and Azithromycin. Data represent the mean ± SE of 8 - 12 mice per group. (n = 8 - 10; #p < 0.05 versus naïve; *p < 0.05 versus DSS). CSY0073, 45 µMol/kg, attenuates signs of colitis induced by DSS including weight loss and the fecal score. Treating with CSY0073 attenuates colon inflammatory infiltration of the mucosa and sub-mucosa. Data represent the mean ± SE of 8-10 mice per group (#p < 0.05 versus naïve). Histopathologic analysis of colon samples obtained from mice sacrificed 12 days after DSS application began. DSS administration causes inflammatory infiltration in the colon mucosa.
Anti-inflammatory activity of CSY0073 compares with that of sulphasalazine in the DSS model of colitis. Mice were administered 5 % DSS alone or in combination with CSY0073 at the dose of 15 µmol/kg or sulphasalazine 200 µg/kg for 5 days.
Task 2.5 Anti-Inflammatory effects of CSY0073 in the TNBS model of colitis
Colon inflammation that develops in mice administered TNBS is thought to be a model of Th1-mediated disease with dense infiltrations of lymphocytes / macrophages in the lamina propria and thickening of the colon wall. In order to assess whether CSY0073 would exert immune-modulatory activity, mice administered TNBS were treated with CSY0073 for five days. CSY0073 effectively attenuated colitis development as measured by assessing local and systemic signs of inflammation. The effect exerted by CSY0073 was dose-dependent. Administering TNBS-treated mice with CSY0073 at the dose of 45 mMol/kg protected against the development of wasting disease measured by the weight loss and the diarrhea score and reduced the macroscopic score of colitis; moreover, it attenuated colon inflammation measured by assessing the histology score: as illustrated in panel D, all animals treated with TNBS developed mild to severe colitis that was attenuated or reversed by administration of CSY0073. In contrast, the lower dose (30 mMol/kg) had no effect on wasting disease, appearance and severity of diarrhea measured as a fecal-score and macroscopic and histology score (n = 8-10, #p < 0.05 versus naïve; *p < 0.05 versus TNBS group). As only the higher dose (45 mMol/kg) of CSY0073 was effective in reducing inflammation in all TNBS-treated mice, we used this dose for mechanistic investigations. Compared with colons of mice given vehicle alone (upper left), colons obtained from mice administered TNBS showed an extensive cellular infiltrate, submucosal edema and large areas of epithelial erosion (upper right) that were reduced by CSY0073 treatment (lower). Furthermore, CSY0073 significantly reduced colonic expression of inflammatory and immune mediators including TNFa, INFg and IL-2 and colonic MPO caused by TNBS (n=5; #p< 0.05 versus naïve; *p < 0.05 versus TNBS).
Early administration of CSY0073 (45 µMol/kg p.o.) protects against the development of TNBS-induced colitis in mice. Colitis was induced by intrarectal instillation of 1 mg of TNBS per mouse, and animals were killed five days after TNBS administration. CSY0073 was administered intraperitoneally daily for five days, starting at the same time of intrarectal instillation of TNBS. The severity of TNBS-induced inflammation (weight loss and fecal score) is reduced by CSY0073 administration. Data represent the mean ± SE of 8 - 10 mice per group. (#p < 0.05 versus naïve; *p < 0.05 versus TNBS). CSY0073 reduces local signs of inflammation and inhibits the increase of macroscopic- microscopic- score induced by intrarectal instillation of TNBS. Data represent the mean ± SE of 8 - 10 mice per group. (#p < 0.05 versus TNBS plus vehicle group). Histologic analysis of colon samples obtained from mice sacrificed five days after TNBS. TNBS administration causes colon wall thickening and massive inflammatory infiltration in the lamina propria. Administering TNBS mice with CSY0073 attenuates colon thickening and inflammatory infiltration of the mucosa and submucosa. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of expression of inflammatory cytokines (INF?, TNFa and IL-2) and Myeloperoxidase (MPO) activity in colons obtained five days after administration of TNBS alone or in combination with CSY0073. Data represent the mean ± SE of six mice per group. (#p < 0.05 versus naïve; *p<0.05 versus TNBS).
Task 2.5 CSY0073 attenuates signs of collagen-induced arthritis
NF-kB activation and subsequent cytokine production play a role in rheumatoid arthritis as well as in animal models of inflammatory arthritis. To explore the possibility that CSY0073 NF- B suppression might have therapeutic efficacy in arthritis, we have tested its effect in the murine CIA model. DBA/1J mice were immunised with type II collagen and treated with CSY0073 (in food, 25 µMol/day) beginning on day 21.
Task 2.6 CSY0073 exerts counter-regulatory activities on NF-kB signalling
Because CSY0073 administration reduced immune-cellular infiltration in the lamina propria of Balb/c mice, experiments were carried out to further define the immune compartment mediating CSY0073 actions in vitro. For this purpose, we tested the effect of CSY0073 at different doses (10, 1 and 0.1 mM) on TNFa and PGE2 release from mouse spleen monocytes stimulated with LPS (1 µg/ml) for 24 hours. Whilst exposure to LPS increased both TNFa and PGE2 release from mouse spleen monocytes, CSY0073, 10 and 1 mM, causes a robust attenuation of this effect (n=5 experiments; #p < 0.05 versus naïve; *p < 0.05 versus LPS group).
As NF-kB is a key transcription factor for the expression of inflammatory genes, to further investigate the mechanisms modulating the anti-inflammatory effects of CSY0073 in comparison to azythromycin, RAW264.7 cells were treated with LPS (1 µg/ml) alone or in combination with CSY0073 (10 µM) or azythromycin (10 µMol/kg). The Western blot analysis revealed that LPS induced a nuclear translocation of the p65 subunit of NF-kB. Azythromycin significantly reduced the NF-kB activation and, similarly, CSY0073 reduced LPS- induced NF-kB translocation in RAW264.7 cells (n=3 experiments; #p < 0.05 versus naïve; *p < 0.05 versus LPS group). We have then assessed the effect of CSY0073 on NF-?B DNA binding activity in LPS-activated RAW264.7 cells, a macrophage cell line of murine origin. Cotreatment with CSY0073 caused a robust attenuation of NF-?B DNA binding caused by LPS. Similarly results were observed with azitromycin. These results suggest that CSY0073 counter-regulates cytokines release by suppressing binding of NF-?B NF-kB responsive elements in the promoter of responsive genes.
Task 2.4 Effects of CSY 0073 in Colon biopsies from IBD patients
Colon biopsies were obtained from 6 patients (5 men, mean age 39.4 ± 6.5 years) undergoing colonscopy for clinical staging of their disease. All patients were affected by Crohn's disease with a colonic localisation. Three of them had also ileal involvement. All subjects were taking active drugs: budesonide (four patients) and azathioprine (two patients). None of the patients had previous surgery. Each patient had an active disease at the macroscopic examination. Samples were taken from non-ulcered mucosa. Written consent was obtained from each patient. Biopsy specimens were gently washed, three times in RPMI with 3 % of penicillin / streptomycin and cultured on 40 uM mesh filters over a culture dish in 12 well tissue culture plates in complete RPMI medium. They were cultured in the presence or absence of LPS (100 µg/ml) alone or in combination with CSY0073 (100 µM), (3 hour of pre-treatment before LPS stimulation) and incubated at 37 °C with 5 % CO2. After 16 h, culture supernatants was removed, while biopsies were processed for RNA extraction.
Data analysis
All values will be express as mean ± SE of experiments. The variation between data sets will test with the unpaired data and ANOVA, for more of two groups.
CSY 0073 reduced the expression of inflammatory genes in colon biopsies activated by LPS
CSY 0073 (100 µM) was added to colon biopsies obtained from IBD patients, for 3h, before LPS stimulation (100 µg/ml) for 16 h. At the end of incubation, the mRNA was extracted for the quantification of (A) tumour necrosis factor (TNF)-a; (B) interleukin (IL)-8; (C) Rantes and (D) macrophage inflammatory protein 3 alpha (MIP-3a) (*p < 0.05 versus untreated biopsies ; **p < 0.05 versus LPS treated biopsies; n = 6).
In the ex vivo experiments, we found that exposure to CSY0073 effectively decreased the expression of TNFa, Rantes, MIP-3a and IL-8 induced by LPS. This observation may have a clinical readout and we feel a clinical trial is warranted..
WP 2 - Immune characterisation
Task 2.2 Cell based assays for activation of p38 and inhibition of TNFa production
P38 MAP kinase sensitivity to p38 inhibitors may vary between patients, explaining the variation in response seen in the rheumatoid clinic. In addition, patient responses in the degree of p38 activation to agonists may also differ. A simple cell based screen of p38 MAP kinase sensitivity to agonists and their inhibition is required for the assessment of these phenomena. The simplest system would be to sample blood, stimulate, and analyse. If achievable, this system would be an improvement on systems requiring the purification of cells by removing this step, retaining the proportional and total cell populations within the blood (and thus their interactions), and also removing the artificiality of isolated cell systems.
Whole blood was taken from healthy volunteers and stimulated with lipopolysaccharide (LPS) or phytohaemaggluttinin (PHA). LPS stimulates the TLRx receptor of blood monocytes to activate the MAPkinase pathway and induce gene expression of TNFa. PHA stimulates the T-cell receptor to induce T-cell TNFa synthesis, this is susceptible to p38 MAPkinase inhibition.
Using human whole blood cultured over 5 days, 2mL in 24-well plates with varying concentrations of PHA, has determined the feasibility of this approach and the dose range for the screen. Wide variety of cytokines were analysed by Luminex and found to be stimulated (IFNg, IL1a, IL1B, IL2, IL4, IL6, IL7, IL8, IL10, IL12, IL13, IL17, GCSF, GMCSF, MCP-1, and VEGF). TNFa was chosen as the end point. Miniaturisation to 96-well LPS between 0.15 and 10 µg/mL, and PHA between 0.3 and 10 µg/mL PHA provided a full concentration effect curve from subthreshold to supramaximal stimulation of TNFa synthesis. Analysis of a sample of healthy subjects revealed that there was significant variation in maximal response to the agonists between individuals as well as between the agonists themselves. Repeating the same subject three times gave consistent data between samples. Thus the variation in response appears to be due to inter-subject variability in responses to specific agonists and thus intracellular messenger pathways. Variability in response to p38 MAP kinase inhibition with ML3404 was seen with PHA and not LPS.
There thus appears to be subject differences in p38-i sensitivity specifically related to the type of agonist used. This will require corroboration with a prospective trial.
The whole blood culture assay appears to be a useful assay for the determination of patient sensitivity to p38i action, requiring the minimum of sample preparation prior to culture within a single 96-well plate followed by a standard TNFa ELISA.
Human whole blood in either 24 well or 96 well culture was stimulated with PHA. The degree of TNF synthesis stimulation was similar between the two assays enabling miniaturisation and the performance of dose response and p38 inhibitor responses within the same subject.
Task 2.5 Compound activity in animal models - The role of cell interactions in RA pathology, relevance to RA p38i modelling in vivo
Whilst such cellular cooperation described in the previous section is considered important in RA, examination of RA synovial tissue shows heterogeneity in pathology, in both degree of infiltration and organisation. It may thus be that patients might need to be stratified according to the cellular interactions occurring within the synovium at the time of treatment. The in vitro data presented in this project may indicate that long term effects of p38i on the close cooperation of accessory and lymphocyte cells might be exploitable. However, if these cellular interaction mechanisms of p38i is to be used, patients may need to be stratified according to the cellular organisation of the inflamed synovium.
It is becoming increasingly clear that RA synovial can be broken down into different types. The prospective early arthritis cohort (PEAC) study led by the Centre for Experimental Medicine classifies synovial according to cellular infiltrate and organisation. Only around 10 - 20 % possess tertiary lymphoid structures that indicate the close cellular cooperation that would be required for this disease modifying activity of p38i drugs. Interestingly, literature search of such structures in models of arthritis reveals few reports of lymphoid aggregates in animal models of RA (Hetal et al., reported in WP5) and it is in these models that p38i show global inhibition of disease that resulted in the false validation of p38 as a drug target for RA.
In order to characterise these models with view to calibrating them with the human variability of pathology and characteristics, a panel of tissues has been generated for rat collagen and rat adjuvant arthritis for comparative analysis. As can be seen each of these is rapid, with a fast attack phase followed by resolution and ankylosis. One model however, which has not been investigated for p38i action, is rat pristane arthritis. This disease is reported to be not self limiting like the other models such as collagen arthritis, and has aspects of re-activation. This arthritis is rarely used because of it severity, and stress-intractability of the Ola strain of rat to handling. WHRI has carried out a pilot dose ranging trial designed to address animal welfare issues through severity limits agreed with the Institutional ethics review committee, and to determine if the re-activation process can be retained at lower disease severity, and if so, a tissue panel created to match the other models.
A clinical assessment system was created that tracked each individual joint for each rat over a period of 90 days. The usual method of assessment is a composite score of all joints, with weighting towards measuring the hind paw. It can be seen that the attack phase is rapid, and persists to after day 20, followed by recovery. During this phase, two of the five highest (150 µL) dose reached the humane end points and had to be culled. None of the lower doses reached this point. As second phase can then be seen starting at round day 60. It would appear that the lower dose (75 µL) of mineral may be more effective at this process. However, analysis of individual joint groups shows a complex process. The ankles remain swollen to about day 70. Inflammation in the midpaw is reduced after the attack phase at day 25. However, a matched flare at day 65 occurs for the front wrists and from hind knuckles / digits. Histological analysis of the ankles showed that of those that retained inflammatory activity, organised cell aggregates were evident.
Subject to an archival study, it would appear that if cell-cooperation mechanisms were to be investigated, the hind ankle at day 70 - 80, and the front wrist and hind knuckles / digits at day 65 would be of prime interest.
Task 2.1 and 3.4 Optimisation of pharmacokinetics
The likely dosage form of the compounds will be a capsule for oral administration. With this in mind, we examined the bioavailability of selected substances in studies of pharmacokineticts. The compounds advanced here as candidates are orally bioavailable.
Task 2.6 2.7 and 3.5 Optimisation for toxicology
We up-scaled the synthesis of the various candidates and entered them into a toxicology program to determine any potential risks. In in vitro studies on cancer cell lines, various cells including J774, B16 melanoma and LLC cells were tested without any obvious effects in the range 10 - 50 µM. These data suggested that the compounds are not cytotoxins but rather act as signalling regulators in an intact organ system.
The studies were followed using in vivo toxicology. A key element of toxicology is selecting the dose range in which to determine toxic effect. A common strategy is dose escalation. Using dose escalation studies in mice, it is apparent that there are neither acute nor chronic toxicity effects of the compounds in the pharmacological range.
Task 2.8 Selection of compounds for development
Based on the foregoing, we have selected a range of compounds to enter the first stages of clinical trials.
SYD003 is a potent modulator of cancer development but has no obvious dose limiting toxicity, is orally available, stable in rodents and easy to manufacture and formulate.
SYD004 is active in combination therapy and is a potential follow-up to SYD003. We are currently selecting the best analog, however, data to date suggest that active and safe analogs are available.
SYD005 is highly active in selected models notably LLC and will be advanced for profiling in human cancers.
SYD001 is a macrocyclic cytokine inhibitor and appears highly active in both murine IBD and arthritis models. We are currently seeking a licensing partner for this compound.
Conclusions WP 1, 2, 3:
These data have shown that p38 alpha is the dominant form of the protein in PBMCs, hair, diseased tissue and inflamed tissue.
Following stimulation, both the gamma and delta isoforms are up-regulated in PBMCs and diseased tissue respectively. These data could suggest that the delta isoform is an ideal disease specific target in Crohn's disease.
Similar data have been obtained in glomerulonephritis suggesting that selected orphan indications have potential with the compounds in our collection.
Nonetheless, actual model data obtained with the compounds suggests that the ideal indications for modulators of p38 may be cancer rather than inflammation. Indeed, of the compounds tested the rather less specific macrocyclic inhibitor appeared to be the most generally applicable.
WP 3 - Synthetic chemistry
The synthetic chemistry is being conducted primarily by Synovo and Tuebingen. The Tuebingen focus, along with Inte:ligand is to better understand binding modes to design better ligands. Synovo's focus is to find uses of the existing ligands.
Task 3.1 Structure activity relationships - these were completed in WP1
Task 3.2 Analog synthesis
On the fringes of the hydrophobic region exchanged by Gln 155. We wanted to place a negative charge at the appropriate position with the aim to achieve a rejection with Asp 112. In JNK3 a hydrogen bond to Asn 152 should be formed.
An acidic moiety at the amino pyridine did not result in any potent and selective inhibitors, so we switched to the imidazole nitrogen
a) dimethylaminopyridine, acetic anhydride, reflux
b) KMnO4 , H2O, 60 °C
c) DMF, CDI, 90 min. at RT, 4-Fluorphenylacetonitrile, K-tert-butoxide, 120 °C, 4h 12h at RT
d) HBr (48 %), reflux
e) acetic anhydride, DMAP, reflux
f) isoamylnitrite, NaOMe, Methanol, RT
g) ethanol, reflux
h) DCM, RT
i) alkyl halide, K2CO3, THF or methanol
j) HCl 6N, reflux
k) acid chloride / triethylamine or anhydride / DMAP, THF / DCM 1:1
Compounds: R = CH3, R1= various, R2= COCH3 (or H)
These compounds have been prepared to explore, which moieties the hydrophobic region is tolerating. Compound KS004 was synthesised to find out, if the carbonyl at the amino pyridine is necessary for interactions with the enzyme:
Compounds: R= CH3, R1= CH2COCH3, R2= various
Compounds: R= various, R1= CH3, R2= COCH3
Compounds: R= (CH2)3COOH, R1= various, R2=COCH3
Once found out, that (CH2)3COOH brings a moderate selective inhibition of JNK3 over p38 we diversified the moieties R1.
Task 3.3 Potency and specificity for JNK3, p38 alpha and or delta
This task was largely completed in P1, since that time, additional refinement has taken place as noted above.
Task 3.4 Formulation for oral delivery
In P1, we demonstrated partition to tumours and inflammation associated tissues. In P2, we focused on formulation and notably simulating application conditions in patients. This culminated in the use of capsules in mice to demonstrate pharmacokinetics.
Task 3.5 Upscale for toxicology
Synovo chemists up-scaled the compounds SYD003, SYD004 and CSY0754, 755, 766, and 768 for toxicology investigations.
Potential impact:
There are three possible areas of medico-societal impact of the project:
- arthritis like diseases;
- inflammatory bowel diseases; and
- cancers.
Each of these disease areas is debilitating to the point of preventing productive work, and one that strikes generally from mid-life onwards. Therapy for the conditions using advanced therapies is in the order of EUR 12 000 to 30 000 per year.
Replacement of expensive biological medications with low molecular weight, synthetic alternatives is a means to both improve disease outcomes and reduce the costs of care.
In our project, we have identified three candidate drugs that are well tolerated, novel and strikingly effective in animal models.
We intend to develop these compounds as alternatives to biological therapies.
Should we succeed, these compounds would:
- compete with medicines whose intellectual property rights lie outside Europe;
- provide a tablet alternative to injected biological;
- stand as an alternative to, or means of reducing doses of poorly tolerated chemotherapeutics.
The societal benefits would be apparent in aggregate and in microscales. Drug therapy that reduces hospital stays is a very cost effective means of delivering healthcare. Oral small molecules are the most cost-effective form of therapy.
At the microscale, it is apparent that lower complimentary doses of therapeutics are normally safer and better tolerated than larger doses of single agents. Having more such therapies to choose from will allow such combinations and lower prices through competition.
For SMES and entrepreneurs, the impact of the project is more immediate. Synovo and its partner companies are part of a network of smaller firms that are growing to replace the inftrastructure of the larger pharma enterprises. Leaner, faster, more efficient, these smaller drug discovery companies will be able to supply therapies at a fraction of the current investment levels, if they are given the chance to do so. This means finding ways for smaller companies to reach the market in this field.
At present the market is inaccessible because of barriers to entry that have been in part established by large enterprises and regulatory agencies. These barriers to entry mean that only large well funded firms can reach the market, and this, by definition, locks in high prices for novel medications.
If one were to allow smaller companies to market therapies on a provisional basis, following establishment of safety in patients, there is the potential to both re-construct the healthcare cost economy, and develop a highly innovative sphere of biotechnology in Europe. In such a provisional phase, marketers of exploratory medicines would be re-imbursed only according to the benefits observed, and thus there would be no need for pharmacoeconomic arguments about pricing. These would come later when the benefits are known, and the treatment is established.
Taking such a far-sighted approach to small biotech is one aspect of medium term societal impact that we should consider within SME policy.
Project website:
http://www.actakine.com
Questions and enquiries are welcomed. Please contact
Michael Burnet
Coordinator
Synovo GmbH
Paul Ehrlich Str. 15
72076 Tübingen
Germany
Switch: +49-707-1964325
Direct: +49-707-1600543
Fax: +49-707-1964326