Recombinant DNase preparations have been used in the treatment of cystic fibrosis since the end of 1993. This genetic disorder is common, particularly in ethnic groups of northern European extraction, where the frequency of occurrence can be as high as 1 in 2500 live births. A higher than average incidence has also been recorded in southern Europe, as well as in some Jewish and African-American populations.

A number of clinical symptoms characterize cystic fibrosis. Predominant among these is the presence of excess sodium chloride in cystic fibrosis patient sweat. Indeed, measurement of chloride levels in sweat remains the major diagnostic indicator of this disease. Another characteristic is the production of an extremely viscous, custard-like mucus in various body glands/organs that severely compromises their function. Particularly affected are:

• The lungs, in which mucus compromises respiratory function.

• The pancreas, in which the mucus blocks its ducts in 85 per cent of cystic fibrosis patients, causing pancreatic insufficiency. This is chiefly characterized by secretion of greatly reduced levels of digestive enzymes into the small intestine.

• The reproductive tract, in which changes can render males, in particular, subfertile or infertile.

• The liver, in which bile ducts can become clogged.

• The small intestine, which can become obstructed by mucus mixed with digesta.

These clinical features are dominated by those associated with the respiratory tract. The physiological changes induced in the lung of cystic fibrosis sufferers render this tissue susceptible to frequent and recurrent microbial infection, particularly by Pseudomonas species. The presence of microorganisms in the lung attracts immune elements, particularly phagocytic neutrophils. These begin to ingest the microorganisms, and large quantities of DNA are released from damaged microbes and neutrophils at the site of infection. High molecular mass DNA is itself extremely viscous and increases substantially the viscosity of the respiratory mucus.

The genetic basis of this disease was underlined by the finding of a putative cystic fibrosis gene in 1989. Specific mutations in this gene, which resides on human chromosome 7, were linked to the development of cystic fibrosis, and the gene is expressed largely by cells present in sweat glands, the lung, pancreas, intestine and reproductive tract.

Some 70 per cent of all cystic fibrosis patients exhibit a specific three-base-pair deletion in the gene, which results in the loss of a single amino acid (phenylalanine 508) from its final polypeptide product. Other cystic fibrosis patients display various other mutations in the same gene.

The gene product is termed cystic fibrosis transmembrane conductance regulator (CFTR), and it codes for a chloride ion channel. It may also carry out additional (as yet undetermined) functions.

Although therapeutic approaches based upon gene therapy (Chapter 14) may well one day cure cystic fibrosis, current therapeutic intervention focuses upon alleviating cystic fibrosis symptoms, particularly those relating to respiratory function. Improved patient care has increased life expectancy of cystic fibrosis patients to well into their 30s. The major elements of cystic fibrosis management include:

• chest percussion (physically pounding on the chest) in order to help dislodge respiratory tract mucus, rendering the patient better able to expel it;

• antibiotic administration, to control respiratory and other infections;

• pancreatic enzyme replacement;

• attention to nutritional status.

The relatively recent innovation in cystic fibrosis therapy is the use of DNase to reduce the viscosity of respiratory mucus. Scientists had been aware since the 1950s that free DNA concentrations in the lung of cystic fibrosis sufferers were extremely high (3-14 mg mT1). They realized that this could contribute to the mucus viscosity. Pioneering experiments, entailing inhalation of DNase-enriched extracts of bovine pancrease, were undertaken, but both product safety and efficacy were called into question. The observed toxicity was probably due to trypsin, or other contaminants, which were damaging to the underlying lung tissue. The host immune system was also probably neutralizing much of the bovine DNase.

The advent of genetic engineering and improvements in chromatographic methodology facilitated the production of highly purified recombinant human DNase (rhDNase) preparations. Initial in vitro studies proved encouraging: incubation of the enzyme with sputum derived from a cystic fibrosis patient resulted in a significant reduction of the sputum's viscosity Clinical trials also showed the product to be safe and effective, and Genentech received marketing authorization for the product in December 1993, under the tradename Pulmozyme. The annual cost of treatment varies, but often falls between US$10 000 and US$15 000.

Pulmozyme is produced in an engineered CHO cell line harbouring a nucleotide sequence coding for native human DNase. Subsequent to upstream processing, the protein is purified by tangential flow filtration followed by a combination of chromatographic steps. The purified 260 amino acid glycoprotein displays a molecular mass of 37 kDa. It is formulated as an aqueous solution at a concentration of 1.0 mg ml-1, with the addition of calcium chloride and sodium chloride as excipients. The solution, which contains no preservative, displays a final pH of 6.3. It is administered directly into the lungs by inhalation of an aerosol mist generated by a compressed-air-based nebulizer system.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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