June 2001

Aim: LeadDiscovery is a company of industrial scientists dedicated to identifying areas of research with pharmaceutical/biotech potential. We use our experience to help academic scientists or biotech companies highlight this potential. Equally we provide an impartial and non-commission based service to industry identifying field leaders and by suggesting how their research areas can be adapted to product development.

Abstract: The newly created, French based ATIC pharmaceuticals has developed a therapeutic candidate for the effective treatment of cystic fibrosis. Molecules are able to activate chloride conductance in airway cell line, primary human cell cultures and most importantly from cells with CFTR mutations. The therapeutic candidate, MPB-91 is able to activate chloride conductance in cells expressing G551D-CFTR, a mutation that prevents ATP binding and activation. MPB-91 is also able to activate conductance in primary cell cultures taken from patients with the D508 mutation. This molecule is therefore relevant to the majority of cystic fibrosis phenotypes. In addition to increasing chloride conductance, ATIC compounds are able to stimulate the release of secretory leukoprotease inhibitor which acts as an endogenous modulator of neutrophil function. The combined potential to increase hydration and reduce inflammation is considered to represent a near optimal profile for cystic fibrosis candidates. Few molecules in development display this profile and ATIC molecules therefore stand an excellent chance of competing for the $800 million market associated with pharmaceutical sales for the treatment of cystic fibrosis. MPB-91 molecules are ready for toxicological and pharmacokinetic evaluation and industrial partners are sought to expedite these studies and/or subsequent clinical trials.

1. Background: A general overview of cystic fibrosis can be found at the Cystic Fibrosis Foundation web page. This disease is the most common genetically inherited disease afflicting 1 in 2000 babies born of Caucasian or North European descent. Cystic fibrosis is characterized by a plugging up of epithelialized organs most importantly the airways, leading to recurrent respiratory tract infection and hence gradual decline in cardiovascular and pulmonary function. Similar blockages in the pancreas and the intestinal tract lead to poor development and furthermore, plugging of the small intestine often necessitates surgery. Palliative treatments have gradually improved and as a result sufferers enjoy clearer airways and better protection against chronic airway infection as well as more rapid development. Consequently, the median life expectancy of sufferers is now 30 years, however since current treatments do not target the underlying defect, cure is not yet possible.

Great strides in cystic fibrosis research have been made over the past twenty years culminating in the characterization of CFTR (cystic fibrosis transmembrane conductance regulator). This work is the subject of many excellent reviews including the on-line publication authored by Welsh and Smith. Research into the cellular basis of cystic fibrosis took a dramatic upturn in the 1980's when Paul Quinton demonstrated that defective sweat production in cystic fibrosis patients resulted from sweat duct epithelia failing to take up chloride efficiently from the lumen of the glands (Quinton, 1983). Around the same time (1983) Knowles and Boucher reported that chloride movement from epithelial tissue into the airway lumen was diminished and that sodium uptake by the epithelium was enhanced. Reduced chloride transport has now also been demonstrated in the epithelia of the pancreatic ducts in mice with mutant CFTR (Gray et al, 1994) and of the intestines in patients (Baxter et al., 1988). The latter is of significant interest since it suggested that defective intestinal secretion might contribute to intestinal plugging (muconeum ileus) and also poor digestive function. In 1989 Kerem et al and Riordan et al reported the isolation of the gene coding for CFTR and showed that the deletion of three nucleotides from the gene caused the deletion of phenylalanine at position 508 and accounted for the majority of cases of cystic fibrosis (D508). CFTR showed no similarity to known chloride channels but striking homology to the traffic ATPases family of proteins which includes a number of proteins used by bacteria to pump nutrients across their cell membrane and also the drug-resistance protein that ejects chemotherapeutic drugs from cancer cells. These proteins are composed of 2 sequences of transmembrane peptides and 2 nucleotide binding cytoplasmic domains that take up and cleave ATP. The CFTR molecule was predicted to have an additional cytoplasmic domain. Consequently, it was suggested that CFTR was an ATP-driven pump that actively transferred an inducer of chloride transport or a protein that activated chloride channels. However these hypotheses have been superceded by the idea that CFTR is a novel chloride channel. Indeed Zabner et al (1994) showed that gene transfer of CFTR evoked chloride movement and perhaps even more convincingly, Bear et al (1992) showed that the insertion of highly purified CFTR proteins into lipid bilayers containing no other channel-like proteins afforded ion transport.

CFTR is localized to the fluid producing cells of airway submucosal glands, notably the serous components of the secretory tubules as well as a subpopulation of cells in ducts (Engelhardt et al, 1992) and this is probably the main reason why cystic fibrosis mucous is thickened and accumulates in the lungs of sufferers. Consequently, chronic Pseudomonas aeruginosa infection occurs in 75-90% of patients and this is the foremost factor in pulmonary function decline and early mortality. Cystic fibrosis therapies should therefore focus on improving mucous hydration. More recent studies have suggested that CFTR plays an active as well as a passive role in microbial defense. Firstly, P. aeruginosa is internalized by epithelial cells and channel defects may reduce microbial elimination through this pathway (see Pier, 2000). Secondly the etiology of reduced pulmonary function arising from chronic airway infection involves neutrophil infiltration and the consequent release of neutrophil protease. These enzymes are normally countered by endogenous antiproteases such as secretory leukoprotease inhibitor (SLPI; Birrer, 1995) which are released by the serous cells lining submucous glands. Therapies able to increase antiproteases secretion as well as mucous hydration would represent an improved treatment of cystic fibrosis.

Following the discovery of CFTR, considerable hope was attached to the development of gene therapies, however realizing this concept has been fraught with difficulties and progress has been slow (see Alton & Kitson, 2000). As an alternative, much work has been undertaken to understand its regulation since the activation of existing channels could overcome the cystic fibrosis defect. Research activity can be divided into the study of channel trafficking and activity. The most common mutation, D508 causes a defect in trafficking so that CFTR does not reach the plasma membrane and is instead degraded (Zeitlin et al, 2000). This defect can be treated by fatty acids that increase membrane cycling of CFTR. However, even when trafficking defects have been overcome, D508 CFTR activation remains attenuated (Wang et al, 2000). Therefore therapies able to enhance the activation as well as trafficking of D508 CFTR are likely to have considerable therapeutic benefit. Such molecules are also likely to be of use in other cystic fibrosis genotypes characterized by altered ATP binding to nucleotide binding domains such as G551D (see Logan et al, 1994). This mutation causes reduced activation with no reported alterations in trafficking. In summary, therapeutic approaches to the activation of CFTR have targeted:

 

• Intracellular messenger pathways such as phosphodiesterase inhibitors (McPherson et al, 1986; Drumm et al, 1991) which increase cAMP levels and hence protein kinase A activity and phosphatase inhibitors (Becq et al, 1993). Targeting such crucial biochemical pathways is however associated with a significant side-effect risk.

• Overcoming the trafficking defect through fatty acid molecules (Zeitlin et al, 2000) or anthracyclines (Maitra et al, 2001).

• Direct activation of CFTR by:

2. Project Profile - benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel: Synthesis of the benzo[c]quinolizinium series of compounds is fully described by Becq et al (1999). Four members of this series were selected for testing in a variety of models, the lead MPB-07 and its inactive congener, MPB-04 are represented in panel to the left. Optimization of MPB-07 has produced the therapeutic candidate, MPB-91 which is also shown.

Activation of CFTR in transfected CHO cells: CFTR conductance was assayed directly through whole cell current recording and indirectly through the measurement of ¹²⁵I efflux. MPB-07 (250mM) caused a rapid increase in iodide efflux an effect mirrored by an increase in whole cell currents. Activity was blocked by 100mM glibenclamide confirming that conductance was due to the opening of CFTR. A structure-function study was initiated and it was found that the nature of the group at position 6 determined functional activity as the amide, MPB-04 was inactive. Similar results were obtained from whole-cell patch clamp studies.

Activation of CFTR in human primary cultures: MPB-07 generated a dose dependent increase (EC50=10-50mM) in short-circuit current generated by monolayers of human nasal epithelial cells. The maximum response was similar in magnitude to that generated by cpt-cAMP. This increase in short-circuit current was blocked by glibenclamide demonstrating its dependence on chloride secretion. It is of importance that MPB-07 was active at the apical surface suggesting that not only is this molecule able to stimulate epithelial transport, but it is likely to be active when given locally.

Activation of CFTR in human tracheal gland cell lines and SLPI release: MPB-07 (250mM) evoked single chloride channel activity in human MM39 tracheal gland. This was accompanied at even lower concentrations by a large increase in SLPI release. Thus, not only is MPB-07 likely to rehydrate mucous but it should also increase the level of endogenous anti-inflammatory factors in the mucous.

Effect of MPB-07 on mucous secretion: At 500mM, MPB-07 caused a 46% increase in mucous secretion from rat submandibular acinar cells. This level of secretion was minimal compared to isoproterenol which caused a 10-fold increase in secretion.  Thus, although MPB-07 is likely to rehydrate mucous it is unlikely to cause a large increase in absolute volumes.

Mechanism of action: MPB-07 was unable to increase intracellular levels of cAMP, neither was it able to stimulate a wide range of phosphatases. This is important since it supports the concept that MPB-07 is a direct activator of CFTR and is therefore likely to avoid the toxic and non-specific effects associated with indirect activation of chloride conductance.

Recent development of MPB molecules: Important recent development of the MPB-07 series have been made to generate molecules with impressive therapeutic activity. Most recently MPB-91 has been identified as an activator of CFTR in the human airway cell line, Calu-3 (EC50=47 mM).

Therapeutic activity: Perhaps the most important observation related to the present dossier is the ability of molecules from the MPB-07 series to activate CFTR in cells bearing cystic fibrosis mutations. Firstly, as can be seen in the inset, MPB-91 was able to stimulate conductance in cells expressing G551D-CFTR, a mutation that prevents ATP binding and activation but has little effect on trafficking. Conductance was increased towards normal levels with an EC50 of 76 mM. At this concentration, MPB-91 has minimal cytotoxicity and it can therefore be considered a therapeutic candidate. Secondly MPB-91 also corrects trafficking defects in the D508 CFTR bearing cell line, IB3-1, and in primary cells cultured from cystic fibrosis patients with the D508 CFTR mutation. MPB-91 can therefore be considered a therapeutic candidate for the treatment of cystic fibrosis patients with the two most common mutations.

3. Patent position: MPB compounds are is protected by WO9805642A1 issued to the CNRS. The CNRS have supported the launch of ATIC pharmaceuticals to develop these and other molecules targeted towards cystic fibrosis.

4. Market size: According to SciClone, developers of CPX, the worldwide annual financial burden of cystic fibrosis is US$3 billion. According to Scripp's 2000 yearbook the market value of cystic fibrosis treatments is US$800 million in worldwide sales.

5. Market Competition - Current and emerging treatments of cystic fibrosis: Pharmacologic treatments of cystic fibrosis currently focus on the use of pancreatic enzyme preparations to compensate for some of the intestinal manifestations of the disease resulting from pancreatic insufficiency. The Dnase, Pulmozyme was the first new drug to be specifically developed for cystic fibrosis and acts by breaking down bronchial mucous thereby reducing airway infection. Previously, the reduction of airway infection largely relied on antibiotic therapy.

For the first time emerging therapies are starting to target the cause of cystic fibrosis rather than treating its symptoms. Accordingly, most of the 40 or so therapies now in development for cystic fibrosis focus on gene therapy reflecting the research communities hopes immediately after the identification of CFTR. A recent overview of the literature describes a new swing in fundamental research towards the identification of pharmacological activators of CFTR (Roomans, 2001) including:

 

• The drug 4-phenylbutyrate (4PBA) which overcomes trafficking disorders, probably by breaking the association between D508 CFTR and a chaperone;

• A number of xanthines, in particular 8-cyclopentyl-1, 3-dipropylxanthine (CPX), which are effective in activating CFTR. The mechanism of this action is unclear but may include direct binding or correction of the trafficking defect, prevention of protein degradation and also activation of channel activity;

• The isoflavone genistein which can activate both wild-type and mutant CFTR, probably through direct binding to the channel;

• Purinergic agonists (ATP and UTP) which can stimulate chloride secretion via a Ca(2+)-dependent chloride channel and in this way compensate for the defect in CFTR

6. Advantage of MPB molecules over current drugs in development for cystic fibrosis: Gene therapies such as GR213487 (Glaxo, Phase II) have yet to live up to expectations largely due to airway penetration problems (see Alton and Kitson, 2000). Other molecules in early development that target trafficking defects are hoped to increase CFTR expression at the plasma membrane, however corrected trafficking on its own is unlikely to represent an optimized therapy. Defective trafficking is not a problem in all patients and even in patients expressing D508 CFTR, overcoming trafficking defects may not be sufficient to restore conductance since D508 CFTR activation remains attenuated (Wang et al, 2000) even when trafficking problem have been overcome.

An alternative target for cystic fibrosis has been the identification of channel activators. Genistein is proposed to act by increasing the open time of phosphorylated CFTR (Al-Nakkash et al, 2001). When combined with cAMP, genistein activated a chloride current in airway cells displaying the D508 defect, an effect further enhanced in the presence of the trafficking modifier, 4PBA (Andersson and Roomans, 2000). However activity was not observed in tissue explants taken from cystic fibrosis patients (Mall et al, 2000) or a mouse model of this disease (Goddard et al, 2000). Furthermore, in addition to promoting increased chloride transport, a conductance block has also been reported (Lansdell et al, 2000; Wang et al, 1998). Considerable research is therefore required before genistein-like molecules can be developed towards a treatment for cystic fibrosis. This class of molecules does not therefore represent significant competition to MPB-07.

The principal advantage of MPB molecules is that they are able to correct both trafficking and activation. This is similar to CPX (SciClone) the leading competitor for MPB molecules. This molecule is reviewed on SciClone's excellent website and is described as a CFTR agonist (Arispe et al, 1998) as well as a modulator of CFTR trafficking. This molecule is already in advanced development (phase II) and data is eagerly awaited to determine its therapeutic profile. In particular it would be of interest to compare the anti-inflammatory activity as well as the hydrating properties of MPB molecules with CPX in view of the observation that MPB-07 releases SLPI.

7. Summary, strategic analysis, and suggested further studies: MPB-91 is suggested as a therapeutic candidate for the treatment of cystic fibrosis. Effects of MPB molecules include an increase in: