GENE THERAPY

Despite discovery of the gene in 1989, gene therapy progress remains slow and frustratingly difficult. Practical application of gene therapy as a treatment for CF is limited by problems such as :

[1] Adverse immune responses to vector encoded proteins.

[2] Poor efficacy with vectors applied to the apical surface of airway epithelia.

[3] Transient nature of gene expression.

[4] financial resources

Results from the first gene therapy trial looking at clinical end points was presented at the meeting. This was a multi-centre double blind Phase II study in CF patients using adeno-associated viral [AAV ] vector [1]. 3 doses of tgAAV administered (Pari LC Plus nebuliser) at 4 weekly intervals. Excellent gene transfer was shown. Antibody response occurred both locally and systemically. Encouraging trends in reduction of IL8 and improved lung function were seen. A short term phase II trial to verify these clinical end points is due to start soon.

Study from New York showed that the extent of adenoviral [Ad ] mediated lung transfer correlated inversely to the level of pre-existing anti-Ad neutralising antibodies [2]. All patients had low/undetectable antibody levels at baseline. Following a single administration of Ad CFTR a dose dependant expression of mRNA CFTR was seen within 1 week of vector administration. However after 2nd administration gene expression not seen if antibody level > 40. Pre-existing antibodies thus preclude gene transfer and remain a further problem to be overcome.

Clinical trials are underway using non-viral vectors such as DNA complexes stabilised with polyethylene glycol [PEG]. These complexes are stable in saline for up to 2 years at 4'C. The level of gene transfer was found to be sufficient for physiological correction of both primary and secondary CFTR mediated defects [3] . They also found that these complexes provoked little/no inflammatory response in mouse lung [4]. Others such as PLASMIN complexes encoding CFTR found to produce partial correction of nasal potential difference.

A further study looking at specific DNA complexes called sec-R targeted DNA complexes [sec R = serpin enzyme complex receptors ] showed efficient transfer to airway epithelial cells with no blocking antibody formation and more importantly no loss of efficacy with repeated administration [5].

Other viral systems such as lentiviruses are being targeted. The ability of lentiviruses to transfer genes to non-dividing cells makes them attractive candidates to deliver therapeutic cDNA for CF. Chapel Hill, North Carolina have been developing lentiviral vectors based on the equine infectious anaemia virus [ EIAV ] A hybrid virus has been constructed composed of a central lentiviral core particle packaged in a lipid envelope containing influenza proteins. These influenzae/EIAV hybrid vectors have been delivered to the tracheas of 3 week old mice with significant gene transfer and expression seen in surface epithelial cells[6].

Scientists in Pennsylvania have constructed a similar hybrid vector as above using HIV based vector with Ebola Zaire viral glycoprotein envelope. They found that this hybrid vector was able to transduce mouse airway epithelial cells from the apical side for up to 63 days and >50% of the submucosal glands were still positive at day 180 [7]. Neutralising antibodies were not found in the serum 1 month following intratracheal administration [8]. These studies have now been extended to fetal human tracheas and results so far are promising.

A group from France have found that the compound lactosylated polyethylemine [PEI] can mediate efficient in vitro and in vivo gene transfer into airway epithelial cells [9]. After a single transfection, lactosylated PEI allowed efficient transfer into 33.7% of cells and after 3 transfections 60.8%. This greater gene transfer efficiency may be attributed to greater cellular uptake of lactosylated complexes and to their lower cytotoxicity. This compound continues to be under investigation.

Polarity is thought to confer resistance to adenoviral mediated gene delivery through the apical surface of airway epithelial cells. Hepatocyte growth factor [HGF] alters polarity of the cell and has been shown to increase the efficacy of gene transfer [10].

STOP MUTATIONS

10% of patients carry a premature stop mutation in one or both alleles of the CFTR gene which stops the production of CFTR protein in the cell. Examples of such stop mutations are G542X, R553X, R1162X and W1282X. Recent reports shown that aminoglycoside antibiotics can suppress this allowing CFTR protein to continue [11]. Immunofluorescent staining of intestinal tissues from CF mice revealed that gentamicin treatment reveals CFTR protein at the apical surface of the glands.

2 pilot studies have been carried out in CF patients:

[1] 9 CF patients with stop mutations received gentamicin drops intranasally tds for 14 days [12]. Nasal PD measurements indicated significant repolarisation of the nasal epithelium i.e correction of the Cl transport defect.

[2] 5 CF patients with stop mutations and 5 CF controls received intravenous gentamicin for 1 week with repeated nasal PD measurements [13]. During the treatment period the number of readings in the direction of chloride secretion increased significantly in the stop mutations compared to the controls.

These results confirm that gentamicin treatment can suppress premature stop mutations an airway cells of CF patients and produce partial restoration of CFTR function in vivo.

Significant barriers to therapeutic use of these compounds exists. Drug companies have identified new drugs not related to aminoglycosides which can suppress stop mutations at much lower concentrations. These will hopefully be developed into clinically useful drugs to suppress the premature stop mutations that cause CF.

GENETIC MODIFIERS

Patients with CF display a wide range of disease severity in different organ systems which cannot be attributed solely to mutations in CFTR. This is due to other environmental and non-CFTR genes called genetic modifiers. One strategy for identifying modifiers is to compare gene expression in matched individuals with mild or severe lung disease. A multi-centre study based at Chapel Hill, USA has been initiated to study genetic modifiers for CF lung disease [14]. It involves looking at patients with the same genotype (DF508/ Df508) who have either mild or moderate lung disease.

A modifier gene MBL-2 [ mannose binding lecithin 2 ] may play a role early in the course of lung disease. 2 studies have reported low levels to lead to poorer lung function [15].

Beyond a general requirement for 'severe' mutations there is no correlation between CFTR and risk of developing liver disease. It has been shown that a combination of mutations/variants in all 3 genes -

[1] PI - potease inhibitor

[2] TGFB1 -transforming growth factor gene and

[3] MBL 2 conferred the greatest risk for developing liver disease [16].

Meconium ileus is present in 15 - 20% of CF patients and not entirely predictable by CFTR genotype. The presence of a CF modifier locus CFM1 has been found for MI on chromosome 19 [17].

Chromosome 19 has been suggested as a possible carrier of a locus for modifier genes in CF. Lewis and Secretor genes (found on Chromosome 19) when present together strengthened the association with PsA colonisation [18]. This represents the first time that modifier genes have been identified and correlated to outcome in CF.

LIVER DISEASE

CFTR is restricted to the biliary epithelium in the liver. Recent findings indicate CFTR modulates reduced glutathione transport and that dysfunction of CFTR causes an imbalance in antioxidant defense system. The GST's (glutathione s-transferases) are liver detoxifying enzymes which play a key role in protection against oxidative stress. Found that one member of the GST family - GSTP1 can influence hepatic status in CF [19]. In 106 paediatric patients the frequency of GSTP1 significantly increased in those with liver disease c.f. those without. In addition amongst the youngest patients aged 6 years, GSTP1 associated with an 8 fold increase in risk of liver disease. Thus identification of GSTP1 may have prognostic value and prompt the start of early treatment in CF patients with increased risk of liver disease.

PSEUDOMONAS VACCINE

Once established, infection with Pseudomonas is impossible to eradicate. A vaccine made from purified hen's eggs immunised with PsA has been developed in Sweden. Data from 7 years of use was presented at the meeting [20]. 10 patients have been enrolled in an ongoing study - following 1st colonisation treated with intravenous antibiotics patients gargle the solution daily. 21 control patients had 1st colonisation treated the same but no solution. Over the 7 year period the treated group have had only 14 positive cultures for PsA (2.9 per 100 months of observation) with 0/10 chronically colonised. In the control group 19% positive PsA cultures with 5/21 chronically colonised. No adverse events seen. Thus chronic colonisation can be postponed after 1st isolation with less positive cultures and reduced need for i.v. antibiotics. A double blind trial is needed however before general recommendations can be given.

HIGH RESOLUTION CT SCANNING

The need for improved outcome measures which can assess early disease and discriminate treatment effects in young patients with normal/mild reduction in lung function is now even more important. Lung function however is only indirectly related to lung structure.

A study from the Netherlands looked at the sensitivity of HRCT scoring and lung function to detect changes in lung disease over time [21]. Annual RFT and biennial HRCT (using 5 scoring systems) have been performed since 1996 on 45 children. Age at time first CT scan was 11.1 years and mean FEV1 74% predicted. For all scoring systems HRCT scores worsened significantly over 2 years whilst RFT's remained unchanged. HRCT is thus more sensitive to monitor onset and progression of lung damage and gives important and complementary information to RFT's for clinical follow up of patients. With stable lung function over a 2 year period progressive structural lung damage seen in children.

A study from Cincinnati confirmed these results [22]. They studied 62 patients aged 6-10years with mild lung disease and 38 patients with normal lung function. Each lobe was graded for a variety of abnormalities (air trapping being earliest change consistently seen, bronchiectasis and mucus plugging). In those with mild lung disease score worsened from 16 - 25 over 2 year period, and in those with normal lung function 17% showed bronchiectasis in 4 or > lobes i.e. progression despite normal lung function.

A group from California looked at a composite scoring system (HRCT and RFT) during the 1 year double blind Pulmozyme/Normal saline trial [23]. 3 outcome variables were assessed [1] FEV1 and FEF25-75 [2] global HRCT score and [3] composite score incorporating both. The largest treatment effect at 12 months were with composite scores [ 35.4%] compared to FEF25-75 [ 13%] and FEV1% pred [ 4.1%]. It appears that a composite score provides a more sensitive outcome measure for discriminating treatment effects.

PULMOZYME

2 studies from Germany looked at the effect of Pulmozyme in patients with mild disease as part of a multicentre BEAT study (Bronchoalveolar lavage in Eradication of Anti-inflammatory Treatment)

[1] Effect on DNA concn in lower airways [24] 48 patients 5-25years, FEV1 >80% predicted. Initial BAL then 29 Rx with
Pulmozyme and 19 controls. BAL repeated after 18 months. Baseline DNA levels not different (but higher than previously reported for infants with CF). After 18 months DNA increased in controls and fell in treated patients. A +ve correlation [p< 0.01] was shown between % neutrophils and DNA levels. Thus CF patients with mild disease have significant amounts of DNA in lower airways and
treatment with DNAse shows reduced DNA load which may have positive effect on clearance of lower airway secretions.

[2] Effect on endobronchial inflammation [25]. Previous studies have suggested Pulmozyme may increase endobronchial
inflammation through release of pro inflammatory cytokines such as IL-8. 67 patients aged 5-32years, FEV1 >80%pred underwent initial BAL. Those with increased % neutrophils randomised to either Rx with Pulmozyme or no Rx for 3 years. Those with normal count neutrophils were followed as control group. Results showed a steady increase in neutrophils over time in all groups but more
pronounced in those not on Pulmozyme. No significant difference in IL-8 levels between the goups i.e. no negative effect on endobronchial inflammation.

An important study from California looked at the effects of air trapping on HRCT during the 1 year Pulmozyme intervention trial in mildly affected patients [26]. The degree of air trapping has been shown to be a sensitive measure of early obstructive lung disease. 8 patients with mean age 11.5 years, FEV1 103% predicted in treated group showed improved air trapping and improved FEF25-75 compared to 14 patients in control group (Normal saline). Pulmozyme thus improves small airway function in patients with normal lung function by reducing mucus plugging with resultant reduced focal air trapping.

TOBI

Multi-centre placebo controlled trial in US looked at eradication of PsA with inhaled TOBI [27]. 98 patients were due to be randomised however the study was stopped early due to evidence of microbiological treatment effect. Results were presented from 21 children aged >6 years. BAL performed at baseline and at 28 days. At day 28 all 8 patients treated with TOBI demonstrated eradication of PsA, compared to 1 out of 13 of the placebo group. By day 28 in TOBI group 8/8 patients had therapeutic concentrations of Tobramycin in BAL. 6/8 patients showed sustained eradication of PsA at day 56. No adverse events documented. Thus early infection with PsA can be cleared in young children with TOBI.

Ongoing multicentre trial in Australia - RCT of bronchoscopy and BAL directed treatment[28] . 83 patients diagnosed through neonatal screening were included in the study. Randomised at <6 months of age to either receive/not receive BAL directed therapy. Treatment in the latter group by clinical observations/ sputumculture. Both groups had initial PsA isolation treated with 2 weeks i.v. Tob and Ceftaz then 2 months TOBI and 1 month Ciprofloxacin. To date 13 have isolated PsA from both arms and all cleared with 2 weeks i.v. antibiotics ie PsA can be eradicated. Study revealed so far that sputum culture may not be specific or sensitive enough to confidently diagnose or exclude lower airway infection. The final results are eagerly awaited.

NON-INVASIVE VENTILATION

The results from the Cochrane Systematic Review from England were presented [29]. 4 short term studies have been published - 2 assessed efficacy as a measure of airway clearance, 2 assessed efficacy as a method of overnight ventilatory support. NIV decreases hypoventilation by attenuating the decrease in tidal volume during sleep in patients with severe lung disease. As with O2 therapy NIV increases nocturnal SaO2 but without the resulting concomitant increase in CO2 seen when patients receive O2 alone. Concluded [1] that there was some evidence that NIV may be used as an adjunct to other airway clearance techniques especially if have difficulty expectorating sputum and [2] it may improve gas exchange to a greater extent than O2. However there is a need for more long term multi-centre randomised controlled trials to validate these findings.

BURKHOlDERIA CEPACIA

It is known that B. cepacia infection contributes significantly to morbidity and mortality in CF. Over 8 different genomovars have been identified. The majority of isolates recovered belong to genomovar III and several distinct 'epidemic'strains have been described.

Manchester, UK looked at 48 patients, 15 with genomovar II [B. multivorans] and 33 with genomovar III to see if there was any difference in clinical outcome between the two groups [30]. They found no statistical difference in clinical outcomes
(RFT/BMI /no. of exacerbations/ days on i.v.therapy) over a 5 year period. There was however a trend to increased requirements and shorter survival for those withgenomovar III. They also found

A similar study from Belfast compared 3 patients with genomovar II, 16 with III and 19 with PsA and found that genomovar III was associated with a more rapid significant decline in lung function compared to the other 2 groups [31]. The median rate of decline of FEV1 was -140mls/yr [ III] compared to -6ml/yr for II and -32ml/yr for PsA. This emphasizes the importance of genomovar identification and the need for strict infection control.

The subject of transient B. cepacia infection was reported from Toronto [32]. During a 32 year period [ 1970-2001] 458 patients tested positive for cepacia. Transient infection was defined as at least 3 consecutive negative samples over >1yr period following the last positive cepacia result. 10% became culture -ve without recurrence (median follow up 11.5years) 36% had recurrence following a period of transient culture. The median duration of the culture -ve period was 2.6 years but 10% were -ve for >8.6years. Younger patients with better lung function were more likely to have transient culture. Age at time of 1st cepacia was significantly lower in those with transient infection. FEV1 was significantly lower in those with acute fatal infection vs chronic infection. It thus remains uncertain at what point, if any, a patient with transient infection can be considered clear and no longer a risk for transmission.

In the USA most transplant centres (and some in UK) will not accept cepacia patients for transplantation due to the propensity for tissue invasion, multidrug resistance and poorer survival figures. A study from California presented results following the use of nebulised aztreonam in pre and post transplanted patients with cepacia [33]. 1gm of aztreonam diluted with 4ml water given bd to 5 patients with cepacia. They showed no difference between the 2 groups [ cepacia vs non cepacia] in terms of outcome (intubation times, length of hospital stay, survival). Longer term studies are needed to validate these results.

SPUTUM INDUCTION

Studies looking at airway inflammation/infection in CF have primarily examined BAL fluid or spontaneously expectorated sputum. Bronchoscopy is an invasive procedure with risks. Expectorated sputum is produced chronically, only in patients with moderate/advanced disease which limits the number of patients that can be studied. Studies in healthy/young patients are thus limited when modifying the inflammatory response in these groups may have the most useful therapeutic benefit.

Sputum induction is a non-invasive technique allowing sampling of the lower airway compartment. 2 techniques [1] inhalation of 3% saline over 12 minutes [2] inhalation of increasing concentrations of hypertonic saline from 0.9 to 3 to 4.5 to 6% over 5minute intervals.

In CF all patients are given pre treatment bronchodilators. Children have had more frequent drop in FEV1/symptoms compared to adults. Studies so far have shown that it can be employed in a range of ages and lung disease severity. Bonchospasm may still occur. The inflammatory profile of induced sputum has been shown to be similar to that of BAL and it is more sensitive than BAL/expectorated sputum in detecting bacterial pathogens.

The results of a multi-centre trial from the USA presented at this meeting validated previous findings showing it to be a safe and useful non invasive technique to assess response to novel antibacterial and anti-inflammatory interventions in CF clinical trials. 50 patients completed the study in 8 centres [34]. Patients were >6years with FEV1 >30%. 3% hypertonic saline was given at 2 minute intervals for 12 minutes pre and post intravenous treatment for an exacerbation. Significant reduction in total WCC/neutrophils/IL-8 and free elastase was seen after treatment along with a significant decline in PsA densty [ >90% reduction after i.v. treatment] A comparison of induced vs expectorated sputum is ongoing.

DIRECT SPUTUM SENSITIVITY TESTING [ DSST]

DSST is a novel test enabling assessment of the efficacy of antibiotic combinations ie synergy testing. Use of this novel test was presented from Southampton, UK [35]. 110 DSST results were reviewed and compared to results from standard culture and sensitivity testing. Synergy was detected for 44% with 69% of these demonstrating multiple synergy. Of the 2 drug combinations tested gentamicin/ceftazidime demonstrated most frequent synergy and for 3 it was tobramycin/ceftazidime/septrin or tobramycin/meropenem/septrin. Septrin most frequently added synergy to 2 drug combinations. Concurrent C&S results were available for 94 samples - 57 demonstrated discordant sensitivity results. DSST showed increased sensitivity to an antibiotic compound in 25 and increased resistance in 28. Prospective studies are needed to see if increased synergy testing and choice of antibiotic leads to improved clinical outcomes.

INFECTION CONTROL/SEGREGATION

A very important study from Manchester, UK looked into airborne dissemination of epidemic resistant PsA [36]. It is known that implementing appropriate cross infection control measures is difficult as potential reservoirs for highly transmissible strains in CF centres are unknown. Room air/staff hands/spirometer annd inanimate environment was sampled. 7/58 air sampling plates were +ve for PsA with -ve results from inanimate environment/ staff hands. Epidemic PsA can thus be isolated from room air in the presence of CF patients who harbour these strains. These findings suggest that recent outbreaks may be due to patient to patient spread by airborne dissemination. Strict infection control measures are thus needed.

Results from a questionnaire survey from Belfast looked at what patients are really doing [37]. The centre has segregation policies and discourages communal activities. 60 patients completed a questionnaire. Only 11 out of 43 who did physio washed their hands after treatment. Only 19/43 sterilised their nebuliser equipment weekly and 7 had never done it. Only 9/46 cleaned their inhaler device. They found the majority of patients continue to participate in communal activities [ both in hospital and community]

STRESS INCONTINENCE

Several studies highlighted the importance of this symptom, its potential implications and resulting social/psychosocial embarrassment. Treatment strategies need to be incorporated into the standard care package for all patients.

A questionnaire from N. Ireland was sent to all female patients [38]. 46/59 responded. Urinary incontinence reported in 14/46 [ 8/14 just when chest bad, 3/14 at any time] Faecal incontinence reported in 5/46. Bladder problems affected ability to cough/perform airway clearance [ 13/14 ] and exercise [ 4/14 ] 10/14 did not seek help due to embarrassment. 4 had sought help and pelvic floor exercises improved their symptoms.

A survey from Toronto, Canada highlighted the prevalence in children as well as adults and in both sexes [39]. 128 children >6years were surveyed (all returned the survey) Mean age 11.7 years. 20% reported some urinary incontinence with stress incontinence in 14% of the whole group (higher than figure for non CF). A gender difference was seen - 18.5% females and 9.4% males reported stress incontinence.

A further study from Southampton, UK [40] looked at children and found that 8/20 reported either urinary or faecal incontinence [ 6 female, 2 male ] Commonly occurred with coughing, sneezing, laughing and all concerned re ability to do RFT's properly due to fear of leakage. 4/8 with urinary incontinence had faecal incontinence. Only 1/8 had discussed with health professional.

HALOLITE ADAPTIVE AEROSOL DELIVERY SYSTEM

· CF patients may use conventional nebulisers for up to 1 hour/day for Rx aimed at preserving lung function. Poor techniques such as nose breathing, talking or inadequate lip seal may compromise the time spent breathing correctly and thus the amount of drug getting to the lung. The Halolite system generates aerosol only when the patient breathes correctly and in addition it provides feedback and signals when the pre-programmed dose has been successfully delivered. 3 posters at the meeting from multicentre studies involving SJUH looked at different aspects of this system :

[1] Compliance [41] - Halolite vs conventional nebuliser True compliance with Halolite 51% cf 27% for nebuliser.

[2] Interaction between aerosol delivery system and bronchodilators [42] - Out of 259 in initial study 189 on Colistin. All on bronchodilator - some with study device others with inhaler. When using bronchodilator with Colistin and Halolite +ve effect seen in maintaining both long and short term FEV1 - baseline 5%, day 28 6.6% and day 182 0.5% With inhaled/ other nebulised group -3 to -7% change at day 182. Changes stat.significant.

[3] Comparison with high output nebuliser system [43] n=259 (113 - Halolite, 126 - neb), median age 17years, FEV1 56% predicted. No difference in efficacy outcomes but higher incidence of chest tightness in Halolite group may be indicative of more successful delivery of antibiotic to the lung. Patients found to take significantly more doses to an acceptable level with the Halolite with improved acceptability of device.

NEW DEFINITION FOR PSEUDOMONAS INFECTION

· A poster from the paediatric unit at SJUH with a new definition for PsA infection will hopefully be utilised to facilitate consistent treatment between centres [44]. Previous definitions may overestimate the prevalence of chronic infection. 4 subgroups :

[1] Chronic - when >50% of preceding 12months in which samples taken were PsA culture +ve.

[2] Intermittent - when <50% of above culture +ve.

[3] Free of PsA - no growth for the previous 12 months having previously been culture +ve.

[4] Never infected - PsA never been cultured.

The intermittent category is very important. Minimum of 3 monthly samples needed. They looked at 146 children - 18% chronic, 34% intermittent, 28% free and 20% never infected and correlated with FEV1, Ps antibodies, Shwachman and Northern score. Chronic infection sig. assoc with a +ve antibody result and never with a -ve antibody result. FEV1 in chronic 65% à 85% in never. Northern Xray score stat. significant - never 5.2 à 8.0 chronic. Shwachman score sig. worse for chronic [ 74.5 ] à never [ 92.7].

CF RELATED DIABETES

With the current median age of survival now at 32 years an increased incidence of CFRD seen in pancreatic insufficient patients. It is known to be associated with progressive decline in nutritional and pulmonary status with subsequent increase in mortality. Diagnosis is based on the annual OGTT.

A study from the UK looked at a new oral treatment for CFRD - repaglinide [45]. This is a novel insulin secretagogue which stimulates 1st phase insulin secretion from pancreatic beta cells. Rapid absorption and elimination mean it can be given on a 'one meal one tablet' basis. Starting dose 0.25 or 0.5mg tds (can increase to max 3mg tds) 5 children previously on insulin commenced on Rx with FU to 6 and 12 months. 4/5 showed good glycaemic control with no adverse effects. 2 reverted back as preferred bd insulin regime. Larger study planned as only concern - toxicology studies indicated prolonged half life in hepatic disease.

A study from Edinburgh looked at providing optimum control during overnight feeding in CFRD [46]. Most centres use intermediate action insulin (peak action 4-8hours), however rapid early calorie absorption may cause high sugar levels and loss of calorific benefit during early feed. A ' Glucowatch' glucose monitoring device worn like a watch provides readings every 20 seconds. 4 patients were monitored during overnight feeding and in all 4 insulin changed to a premixed insulin with improved glycaemic control. They suggest as a minimum to monitor sugars pre feed, 2 hourly and at the end of feed.

A further study from the UK found episodes of hyperglycaemia despite normal OGTT testing highlighting what is the optimum method for investigation of glucose metabolism in CF [47]. It is known that lung function and weight can deteriorate up to 4 years prior to the diagnosis of CFRD. There may thus be clinically significant insulin deficiency present prior to an abnormal OGTT. The subcutaneous Continuous Glucose Monitoring System (CGMS) provided readings over 72 hours in both CF patients without diabetes and matched controls. On OGTT 0 patients had diabetes however using CGMS 42% of patients (but 0% of controls) had at least one glucose measurement >11.1mmol/L ie episodes of random hyperglycaemia can occur with normal OGTT and normal HbA1c.

FATTY ACIDS AND CF

At the Seattle meeting 3 years ago research was presented showing a membrane lipid imbalance with increased arachidonic acid [ AA] and decreased docosahexanoic acid (DHA). Previous studies in the CF mouse showed a beneficial effect with Rx with reversal of CF related pathology in the pancreas.

A study looking at hepatobiliary disease in the CF mouse showed significant amelioration in the progression of liver disease with long term DHA treatment [48].

A study from the UK reported modulation of inflammatory lung disease with a FA rich diet [49]. The fish oil diet fed to CF mice resulted in a significant increase in DHA levels and reduction in AA. It also protected against both alveolar inflammation and neutrophil inflammation into airspaces and had a favourable (but not significant) lowering of inflammatory markers (TNF alpha, IL-6).

A study from France looked at the AA profile and AA/DHA ratio in both serum and membrane phospholipids in those with PI and PS [50]. Found reduced DHA, increased AA and increased ratio in both PS and PI patients but especially in PI patients.

VITAMINS AND CF

A study from Israel looked at the correlation between vitamins A and E and pulmonary exacerbations [51]. Over a 3 year period vitamin levels were retrieved during exacerbations and remissions. An increased number of exacerbations correlated with lower vitamin A and E levels with a further reduction in levels during an acute exacerbation. PI patients showed a statistically significant fall in levels with exacerbations, whilst in PS patients downward trend seen but did not reach statistical significance.

There is known to be an imbalance in the oxidant / antioxidant system in CF in favour of increased oxidative damage. Antioxidants include vitamins A and E, carotenoids and total and reduced glutathione. Results show all antioxidants reduced in CF cf controls whatever age or clinical condition [52]. The deficiency worsens with pulmonary function degradation. Courses of i.v. treatment lead to increased A and E levels. Increased nutritional status was found to modify some (vitamin A) but not all antioxidant markers. FEV1 shown to be the primary factor in the variation of the oxidant / antioxidant system.

Vitamin C is an important antioxidant but little data is available. Plasma concentrations found to inversely correlate with inflammatory markers especially in older patients [53]. A study reported vitamin C content below RDA in 12 - 27% of CF patients. Increased attention is needed to improve the vitamin C content of the diet. RDA can be achieved by the daily consumption of 200ml of fruit juice.

Malabsorption of vitamin D is known to be an important factor associated with the development of bone disease. A study from Toronto showed 69% had low / suboptimal levels [54]. For all patients with normal levels the mean supplementation of vitamin D was 843IU/day. With intervention 2/3 were able to achieve normal levels.

It is now important to achieve target vitamin D levels of 30-60ng/ml. The level should be taken prior to winter [ when have maximum storage ] For levels <30 repletion is indicated - 50,000U orally weekly for 8 weeks. If still <30 à 50,000 U give twice weekly for 8 weeks and if still low UVB phototherapy should be considered.

GROWTH HORMONE TREATMENT

Poor growth / delayed puberty / malnourishment and poor clinical status are some of the risk factors associated with the development of bone disease. 3 studies from the states looked at treatment with human recombinant growth hormone :

24 children aged 5.9 - 15.3 years treated with GH ( 0.3mg/kg/week) for 1 year and results compared to 11 gender and age matched children. Looked at BMC [ bone mineral content ] and TNF alpha (potent inflammatory cytokine) [55]. No differences at baseline but in treated group at 1 year BMC increased significantly from 71.6gm to 203.8gm and TNF alpha 6.5 in treated group and 88.1 in control (significant fall).

Ongoing study looking at effect of GH on pulmonary function, weight and height [56]. Patients less than the 25th% for weight or height randomised to receive GH in either year 1 or year 2. So far 17 patients have completed 1 year or more. Results showed that height, weight, LTM, FEV1 and FVC significantly better in the GH treated group at 12 months. No patient developed any adverse events.

Final study looked at GH treatment in adolescents with CF [57]. 13 patients, aged 13.6 to 15.9 received GH for at least 1 year. Data from treatment year compared to data from year prior to treatment. All patients were Tanner 3 or > at time of GH treatment and all continued to progress in puberty during treatment. Weight improved from 38.4 to 46.9kg, FEV1 1.86 à 2.51 L and height 151 to 160cm all statistically significant.

LUNG FUNCTION IN YOUNGEST PATIENTS

It is important to be able to assess lung function in young patients who are not able to perform spirometry. 10 sites in the USA are due to start a prospective study in 100 infants < 24 months of age [58]. The procedure and technique (raised volume technique) has been developed by Dr Castile (Ohio) and involves a baby box - the baby is mildly sedated with a mask over mouth and nose and an inflatable vest around chest which measures FEV1. So far normative data have been published, equipment and software developed and approved. 10 sites have machines to begin clinical trials. Each subject will undergo lung function testing at enrolment, 1, 6 and 12 months later. Results will be eagerly awaited.

DELAYED DIAGNOSIS IN CF

A study from Southampton looked at 73 children homozygous DF508 and compared those diagnosed early ( <2months) to late (>2months) at 1,3,6 and 10 years [59]. Mean height and weight similar at each age. FEV1 similar at 6 and 10 years in early and late. CXR scores similar at all ages except the late diagnosed at 10years when score significantly increased (7.7 vs 4.4). However the late diagnosed patients needed significantly more treatment to maintain good health - regular iv's 0 vs 19%, regular neb antibiotics 57 vs 79% and use regular inhaled steroids 36 vs 85%. As most studies show no improvement in screened vs controls pharmaco-economic considerations might advance the debate on universal newborn screening.

CFTR IN THE PATHOGENESIS OF CF

Step theory in pathogenesis of lung disease [60] :

[1] Volume depletion - Mucus clearance = key component of normal lung defense and is dependant on adequate ASL (airway surface liquid) ASL is composed of mucus layer + paraciliary layer (PCL). Mucus is cleared in normal lungs by ciliary mechanisms at a rate of 60um/sec. In CF ASL is 1/1000 inch thick ie very reduced volume with rate of clearance of 0um/sec, and PCL is missing. Normal airway maintains PCL volume by blend of Na absorption and Cl secretion. In CF with reduced Cl secretion and increased Na absorption à reduced volume.

[2] Mucus stasis and adhesion - ie the 'velcro' step. Due to reduced volume and lack of clearance the mucus sticks causing an inability to cough it up.

[3] Mucus obstruction/plaque formation - mucus sticks to epithelial cll surface. Plaque forms causing obstruction of airflow.

[4] Mucus infection - Thick secretions form an ideal environment for establishment of Pseudomonas growth and biofilm formation. Infection persistent as plaques not removed.

[5] 'Frustration' step - In thick mucus environment it is difficult for the host defence mechanisms to be activated. The thickened mucus thus impairs neutrophil motility. A study from Chapel Hill, North Carolina found neutrophils moved poorly in thick mucus - 1.9um/min compared to thin mucus 5.1um/min [61]. Also the number of neutrophils most in thin and least in thick mucus.

The answer is to add a proper mix of salt and water to restore surface volume but until able to do this other mechanisms [ antimicrobial/ anti-inflammatory/ ion channel modulators/ improved defense/ volume replenishment etc] continue to be investigated. The following sections on new therapies aim to address some of the above problems.

NEW THERAPIES - ION CHANNEL MODULATORS

MOLI - 1901 - This stimulates a Ca++ dependant alternative chloride channel to bypass CFTR à restoring Cl secretion à rehydrate CF mucus à enhanced MCC.Phase I trial previously showed a single dose nasal inhalation to be well tolerated in 4 CF patients and 4 healthy volunteers. The drug was shown to produce an acute and sustained response by inducing Cl transport across the nasal epithelial cells. The results of a further phase I study looking at safety and pharmacokinetics were presented [62]. Single dose inhalation of up to 4ml at 4 concentrations (0.01, 0.1, 0.3, 0.5mg/ml) given to 16 patients and 16 healthy volunteers. The drug was found to be well tolerated with no adverse events reported. A multi-centre phase II trial of this drug is due to start in 2003.

INS 37217 - A selective P2Y2 agonist that stimulates Cl secretion through non-CFTR mechanism and also inhibits epithelial Na transport. Previous P2Y2 agonists such as UTP and INS 365 have been investigated, but this one shown to be better tolerated and more stable. Results from a multi-centre study were presented on safety and tolerability [63]. Drug administered for 5 days by inhalation in 25 adults and 24 paediatric CF patients. 4 cohorts examined - 10, 20, 40 and 60mg. Measurements included RFT/ symptom assess/ O2 saturation/ examination/ ECG / sputum production and vital signs. So far 2 cohorts have been studied - baseline FEV1 84% ie mild. To date doses up to 40mg well tolerated with no adverse effects. A phase II multi-centre 28 day efficacy study is in planning and enrolment due to start end of 2002.

NEW THERAPIES - INCREASING MCC

ICODEXTRIN - High MWT glucose polymer which can produce colloidal osmotic effect to lower airways and cause increased ASL. Not previously tested in humans before. Study from UK - 10 CF patients (>18years, FEV1 >60%) given drug once weekly for 2 weeks - dose 10ml (200mg/ml) and lung function checked daily [64]. CT scans showed no evidence of pulmonary toxicity. Systemic safety documented - no sig changes in FBC, PV, CRP, LFT's, IL-6, IL-8 [65]. Increased MCC shown by scanning but did not reach statistical significance. Further studies warranted.

DCF 987 - Low MWT dextran. Canadian study given by aerosol 500mg or 1000mg bd for 14 days à safe and non immunogenic when rechallenged [66]. Phase II safety trial currently being planned.

BIOFILMS / QUORUM SENSING

Biofilm = complex bacterial community attached to a surface. There is known to be a regulated process of transition from planktonic (free living cells) to biofilm mode of growth. Chronic pseudomonal infection in CF lungs is an example of a bacterial biofilm [67].

Series of developmental stages in biofilm formation :

1st stage - initial attachment to surface

2nd stage - once attached, moves along surface making cell to cell contact to form microcolonies

3rd stage - microcolonies develop to form much larger macrocolonies with secretion of exopolysaccharide matrix. Currently using different approaches to identify targets involved in functioning of this regulatory cascade.

Biofilms develop properties that are different from planktonic counterparts - [1] distinct 3D architexture, [2] increased drug resistance and [3] production of exopolysaccharide. Recently found that pseudomonal biofilms found in mucus layer of CF are oxygen limited. Pseudomonas responds to this hypoxic environment by switching from non-mucoid to mucoid type.

Communication systems have been discovered (quorum sensing systems) which control bacterial virulence. Pseudomonas has 2 quorum sensing systems - las / rhl which it uses to regulate genes (may regulate over 100 genes). Virulence genes by pseudomonas are activated in response to bacterial density. Recently found that this can be inhibited by both natural and synthetic furanone compounds. Cox 2 inhibitors (eg Rofecoxib) possess a furanone structure. Hypothesized that this could block quorum sesnsing in PsA to reduce virulence factors. Rofecoxib treated cells demonstrated impaired biofilm production and may prove useful in attenuaton of bacterial virulence [68].

Mouse model used to see if furanone compounds could exhibit therapeutic effects on lung function [69]. PsA [ PAO1] embedded in beads mimicking biofilm bacteria found in CF lung. Mice inoculated with bacterial beads and divided into 2 groups - placebo and treated group [C30 - furanone compound ] treated tds for 3 days and evaluated on day 7. Bacterial CFU count lower in Rx groups cf placebo. Lung bacteriology showed significant dose dependant correlation in C30 treated groups. Furanone compounds found to [1] repress PsA quorum sensing [2] attenuate PsA virulence and [3] increase sensitivity of antibiotic treatment [70]. It thus seems to hold promise as novel anti-microbial agent.

MOLECULAR CHAPERONES

Approximately 80% of all cases of CF are due to mutations that affect the folding of CFTR and thus prevent its exit from the ER. Molecular proteins are ubiquitous proteins that play a critical role during protein folding and degradation. Molecular chaperones 'decide' if the protein is sufficiently mature to transit through the secretory pathway or whether it should be targeted for degradation. CFTR has been found to be associated with chaperones Hsc/Hsp 70 and Hdj2 which are responsible for retention of CFTR in the ER. Competition with this interaction by related peptides restores plasme membrane CFTR localisation and channel activity and may provide an approach to therapy for such mutations.

Several components of the chaperone machinery require high concentration of calcium ions in the ER to function optimally.

Thapsigargin = inhibitor of calcium pump in the ER and has been shown to greatly increase functional plasma membrane delivery of CFTR [71] [72].

4-BPA (sodium phenylbutyrate) down regulates Hsc70 and allows CFTR to escape from degradation to provide functional surface expression of CFTR [73].

AMINOGLYCOSIDES AND NEPHROTOXICITY

Aminoglycosides [ AG ] = cornerstone of treatment of chronic PsA infection in CF. Doses and dosing intervals still a matter of controversy. Once daily administration may be more effective and less toxic. Early signs of toxicity = proximal tubular dysfunction à enzymuria and proteinuria.

Study from Germany looked at 34 patients treated with Tobramycin (od vs tds) and Ceftazidime for 14 days [74]. Measured urinary excretion of NAG (n-acetyl- b-glucosaminidase) and a-1-M (alpha-1-microglobulin). Proteinuria was assessed by gel electrophoresis. Results [1] both groups showed acute phase toxicity with increased levels NAG and a-1-M. No significant differences seen between the groups [2] LMW proteins days 0-14 similar in both groups [3] HMW proteins significantly higher in tds group. All changes were completely reversed by day 35 in both groups. For all but one parameter early changes of tubular function did not differ between od and tds dosing groups and 3 weeks later all reversible. The excretion of HMW proteins indicates either glomerular damage or a pre lethal cell lesion of tubular cells. The latter was less pronounced in od group thus once daily dosing seems preferable.

NEW THERAPIES - ANTIMICROBIAL

INHALED PA 1806 - Compound related synthetically to Aztreonam and highly active against a wide variety of gram -ve organisms eg PsA, B. cepacia, Stenotrophomonas. Previous phase I study in healthy volunteers showed a 300mg dose to be well tolerated. Current results from phase II study presented looking at safety, pharmacokinetics and antimicrobial activity of 14 day bd administration [75]. 9 patients, mean age 18.5 years and FEV1 60% predicted. Results showed a reduction in sputum count of PsA >10 fold from baseline to day 14. Found to be safe and well tolerated. Clinical effectiveness needs to be evaluated in phase III studies.

LINEZOLID - New oral antibiotic with efficacy against MRSA. No previous published trials in CF. Study from Adult unit, Leeds looked at the absorption and sputum penetration of Linezolid in 10 adult CF patients [76]. Dose - 600mg bd for 6 doses. Results showed that this dosage led to sputum levels which exceeded MIC 90 of MRSA for almost the whole dosing period. Further clinical trials needed to assess efficacy against MRSA in CF patients.

DENDRIMERIC CHO'S - Shown to have potential for blocking PsA colonisation in the lung by preventing adhesion to the lung epithelial cells. Studies showed that dendrimers killed the bacteria with a potency that was as effective as both rifampicin and tetracycline [77]. Studies continue to investigate their potential as a new therapy for PsA.

MEROPENEM vs CEFTAZIDIME - A study looked at 102 patientd randomised to either treatment with Tobramycin and Ceftazidime or Tobramycin and Meropenem, including 19 patients with cepacia [78]. Meropenem was shown to be associated with a significantly greater improvement in FEV1. Both regimens showed a 3 log reduction in sputum PsA colony count. Meropenem conferred improved clinical benefit in cepacia patients.

DISCOVERY OF BETTER ANTIBIOTICS FOR THE FUTURE - The completion of the Pseudomonas genome sequence in 2000 has opened a new era of research to better understand the survival strategies utilized by this bacteria. A complete collection of pseudomonas genes can now be placed on a microscope slide (termed a microarray). These arrays can be used to compare the genes on pseudomonas sensitive to most antibiotics compared to those resistant to most antibiotics to determine if certain genes are turned on or off at the time that resistance emerges. This type of knowledge will assist scientists in developing novel approaches for designing new antibiotics. At the 2002 progress report 1,800 arrays released [79].

ANTI-INFLAMMATORY TREATMENT IN CF

AZITHROMYCIN (AZT) - Macrolides were found to be responsible for the marked improvement in long term survival of patients with diffuse pan bronchiolitis (DPB) a condition which clinically resembles CF. Why AZT in CF ? - marked activity against respiratory pathogens, anti-biofilm activity, well absorbed, long half life, marked accumulation in the lungs, favourable safety profile in adults and children.

2 other published RCT placebo controlled trials of macrolide treatment in CF :

[1] Australia - 60 adults given AZT 500mg daily for 3 months. Found 3.62% increase in FEV1, reduction in number of exacerbations and improved quality of life.

[2] Brompton, London - 41 children aged 8 - 18 years treated with AZT 250mg od for 6 months in cross over study. Results showed 5.4% improvement in FEV1and less oral antibiotic use but no reduction in the use of i.v. antibiotics
.
At this meeting results from two centres presented - Denmark, Copenhagen and the US multicentre macrolide trial :

The CF centre in Denmark introduced long term AZT therapy [ 250mg daily ] in 2001 in all patients with PsA infection [80]. Median age of 50 patients 30.1 years with mean follow up time 8 months. During the treatment period a statistically significant increase in FEV1 seen from 2.38 L to 2.45L. The mean change in FEV1 for the 12 months pre treatment was -1.06%. During the treatment period an increase of +1.07% seen [ statistically significant ] Significant increase in BMI seen (21.65 à 21.8). The % of sputum samples containing mucoid colonies reduced from 73.9% to 67%.

US MACROLIDE TRIAL [81] - included patients > 6 years with FEV1 > 30% predicted and chronic infection with PsA. If > 40kg 500mg AZT or placebo given x3/week. If weight < 40kg 250mg or placebo given x3/week. All other routine therapies such as Pulmozyme, TOBI and high dose Brufen continued. 185 patients were randomised à 87 AZT and 98 Placebo. No differences seen in baseline characteristics between the 2 groups.

Results showed AZT associated with improvements in several end points including FEV1, body weight and number of exacerbations. The treatment effect ie difference between AZT and placebo groups in terms of relative change in % predicted FEV1 was 6.2% and in terms of body weight 0.8kg. The improvement in FEV1 was seen in the 1st 28 days and sustained but declined to baseline levels after discontinuation. AZT also impacted the number of pulmonary exacerbations as indicated by a 40% reduction in the number of courses of i.v. antibiotic courses and a 47% reduction in the number of days in hospital. Trends towards improvement in quality of life seen in AZT treated group.

There was a mean reduction in PsA density from baseline to end of treatment period of 0.2log10 CFU [ compared to a 0.3log10 increase in placebo group ]. No significant differences seen in sputum microbiology at baseline and no difference in acquisition of resistant organisms during the treatment period.

In general the drug was well tolerated. Three symptoms occurred more frequently in AZT group - nausea, diarrhoea and wheeze. They were reported as mild or moderate and did not lead to discontinuation of the drug. There was no explanation for the excess wheezing in the treated group.

Suggested mechanism of action of AZT -

? microbiological effect
? anti-inflammatory - probably
? other ? interactions with CFTR
? changes in sputum rheology

With regard to anti-inflammatory role study from US looked at effect of AZT on pro-inflammatory cytokines such as TNF alpha and IL-8 [82]. 30 clinically stable patients treated with AZT / placebo for 3 months. TNF alpha release unchanged in AZT group cf significant increase in placebo group. Similar trend seen with IL-8 but not statistically significant.

Comparison with other clinical trials Pulmozyme vsTOBI vs Macrolide
Relative change FEV1 6% 11% 6%
Reduction in hosp days 17% 37% 47%
Reduction in i.v A/Bi days 22% 32% 39%

Future research questions [1] What is the optimal dose

[2] How long should treat for

[3] Who should be treated

[4] Is AZT the best macrolide

Thus need to look at - additional studies in < 13 years - patients with cepacia / without pseudomonas - prolonged therapy - dose response study.

OTHER ANTI-INFLAMATORIES - UPDATE

INHALED STEROIDS [83] - Cochrane systematic review showed not enough evidence at present from trials to show whether ICS of benefit. Also not enough evidence to show that their regular use does no harm. Study in progress in UK (involving both adult and paediatric units in Leeds). New approach - to stop patients already on ICS and assess following discontinuation. Results should be available next year.

BRUFEN [84] - Previous 4 year clinical trial in patients > 5 years of age with mild disease [ FEV1 > 60 % ] showed decline in lung function, preservation of % IBW and reduced frequency of hospitalisations. The youngest patients were found to benefit the most. However its use has not been widely accepted despite the knowledge that inflammation occurs early in the course of the disease. Probably due to concerns re GI haem, renal failure or lack of additional data re effectiveness. A 2 year placebo controlled trial is currently being conducted in Canada and will provide additional data of effectiveness. Currently looking at the follow up data from the original 84 patients in the 4 year study. Lung function followed over 8 year period following the trial - The change from baseline in FEV1 %predicted [ 5.1% ] was shown to persist - 6.6%. In those aged 5 - 12 years in whom the largest effect seen [ 10.8% ] this was also shown to be maintained at follow up at 10.3%. So far the benefits seem to outweigh the risks, but continued monitoring is required to further determine the efficacy and safety of Brufen in CF.

IL-8 SUPPRESSION [85]- IL-8 is a very potent pro inflammatory cytokine, massively secreted by lung epithelial cells in CF patients. In the absence of any infection IL-8shown to be high at early stage, any subsequent infection making IL-8 production worse. Epithelial cells [ eg IB3 ] from CF lung faithfully reproduce the high IL-8 secretion phenotype. Gene therapy of IB3 cells with AAV CFTR causes change from high secretion to low secretion phenotype. CPX also found to suppress IL -8 secretion.

LTB4 RECEPTOR ANTAGONISTS [86] - LTB4 = major chemoattractant for neutrophils. See increased sputum levels in CF lung. A negative correlation found between sputum levels of LTB4 and pulmonary function. LTB4 comes from the metabolism of arachidonic acid. Link also between LTB4 and IL-8 - LTB$ can induce IL -8 generation. BIIL 284 BS = specific LTB4 receptor antagonist. Phase I study (single dose) in CF completed and results pending. Repeated dosing study with 30 patients ongoing. Phase II programme planned in 600 patients - 300 adults and 300 children. Primary outcome Change in FEV1 and incidence of exacerbations.

REFERENCES

All references unless otherwise stated from Pediatric Pulmonology, Supplement 24,2002.

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[16] Abstract 130, page 223. Friedman, K.J. et al.
[17] Abstract 137, page 226. Zielenski, J. et al.
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[20] Abstract 300, page 281. Kollberg,H. et al.
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[26] Abstract 351, page 298. Robinson, T. et al.
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[70] Abstract 349, page 297. Hentzer, M. et al.
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